Here, we discuss the implications of differences in EC coupling when considering the potential applications of human iPSC-CMs in a number of areas as well as detailing the current understanding of this fundamental process in these cells.Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) hold enormous potential in many fields of cardiovascular research. Overcoming many of the limitations of their embryonic counterparts, the application of iPSC-CMs ranges from facilitating investigation of familial cardiac disease and pharmacological toxicity screening to personalized medicine and autologous cardiac cell therapies. The main factor preventing the full realization of this potential is the limited maturity of iPSC-CMs, which display a number of substantial differences in comparison to adult cardiomyocytes. Excitation–contraction (EC) coupling, a fundamental property of cardiomyocytes, is often described in iPSC-CMs as being more analogous to neonatal than adult cardiomyocytes. With Ca Studies of EC coupling in human iPSC-CMs (hiPSC-CMs) have described substantial differences between their properties and those of adult cardiomyocytes. While understanding these differences will be crucial to fully realizing the potential of hiPSC-CMs, research to date is limited. Furthermore, electrophysiological study of hiPSC-CMs is confounded by the variability of these cells in culture. We have previously shown the markedly increased variability in action potential morphology when hiPSC-CMs are cultured as single cells as compared to in a monolayer. As patch clamping studies necessitate the use of individual cells, these findings raise questions over the usefulness of investigations performed in this manner  is the key link between electrophysiological stimulation and the production of mechanical force as well as a feature that distinguishes cardiomyocytes from other types of muscle cells. Cardiomyocyte depolarization causes Ca2+ entry via voltage-sensitive L-type Ca2+ channels, triggering the release of Ca2+ from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyR), raising [Ca2+]i and causing contraction. Relaxation is achieved by removing the Ca2+ from the cytosol, either by re-sequestering it in the SR via the sarco-endoplasmic reticulum ATP-ase (SERCA), or extruding it into the extracellular space through the Na+-Ca2+ exchanger (NCX) or Ca2+ ATPase pump  through patch clamp techniques have measured substantial peak current densities, ranging from −8 pA/pF  are the Ca2+ release mechanism of the SR, and are closely apposed to L-type Ca2+ channels. RYR2, the cardiac isoform of RyRs, appear diffuse throughout the cytosol on immunostaining with reports of both intense, punctate perinuclear . Due to this stoichiometry, NCX generates a substantial inward current (INCX) that contributes to cell depolarization, pace maker activity and action potential duration  and as such need to be interpreted with caution. In summary, hiPSC-CMs display significantly impaired diastolic Ca2+ removal.Regarding the relative contribution of the two main mechanisms, SR Ca2+ handling in these cells.A key feature of hiPSC-CMs is that they beat spontaneously, making for a convenient means to confirm successful differentiation of stem cells into cardiomyocytes. Unexpectedly, study of this phenomenon has given us a number of insights into the regulation of Ca2+ release identified small Ca2+ wavelets propagating within the cell, the majority of which were too small to trigger a global Ca2+ release (/ΔFCaff)(, averaging at ~0.3 vs. 0.6–0.9 in adult human cells (Bassani et al., 2+ release and limited association with sarcolemmal Ca2+ channels contributes to slow and inefficient rise of [Ca2+]i. hiPSC-CMs are devoid of t-tubules (Gherghiceanu et al., 2+ transients, low fractional release and increased dependence on sarcolemmal Ca2+ flux (Figure The lack of the sophisticated adult myocyte structure in hiPSC-CMs may explain some functional observations. From studies discussed here it is clear that SR Cax Figure . This st2+ extrusion rate, high PLN:SERCA ratio and a negative force-frequency response point to decreased SR function and regulation, associated with a greater dependence on sarcolemmal flux to source and extrude Ca2+. As such, greater structural co-ordination may yield limited benefits without improvements in the underlying function of EC coupling components, and vice-versa.Equally so the opposite can be argued. T-tubule formation and maintenance is an inherently energy-intensive process. Low SERCA expression and Ca2+ handling, and as such it could be argued that it is more efficient to rely on sarcolemmal flux driven by electrochemical gradients than ATP-intensive processes. We and others have shown how using culture techniques to manipulate myocyte structure can result in substantial changes in Ca2+ handling properties (Kaji et al., Standard culture environments place little demand on hiPSC-CMs in terms of Ca2+ handling properties of these cells a number of questions remain, particularly with regards to how well Ca2+ channels and RyRs are coupled and what the dominant sources of Ca2+ are in EC coupling.hiPSC-CMs hold enormous promise in many fields of cardiovascular research. While we have a reasonable understanding of the CaWith many of the end applications of hiPSC-CMs dependent on the ability of these cells to recapitulate adult human cardiomyocyte properties to a satisfactory degree, differences in channel pharmacology, mechanisms of automaticity and structural organization between hiPSC-CMs and adult cardiomyocytes pose challenges to fully realizing the potential of this technology. It is important however to remember that there is no truly definitive understanding of human cardiac physiology. Difficulties in obtaining healthy adult tissue have meant that the field has come to rely on failing hearts, and in particular on rabbit physiology, as a basis of comparison. While this by no means invalidates the comparative studies above nor the concerns raised about hiPSC-CM function, it is essential to consider this limitation when discussing hiPSC-CM properties.From a more practical perspective, the minimal variability shown between different cells lines and different differentiation protocols is reassuring, and gives us greater confidence when comparing and collating the work of multiple groups. Consideration must be given however to culture conditions, with substantial variability in hiPSC-CM electrophysiology apparently dependent on cell density (Du et al., Overall, a more solid understanding of the electrophysiological properties and limits of hiPSC-CMs will be extremely valuable in the interpretation of the effects of novel drugs and making judgments on the ability of these cells to successfully recapitulate human disease.We confirm that CK, LC, and CT meet the criteria for authorship of this review based on the recommendations of the ICMJE.CK is funded by a British Heart Foundation MBBS/PhD studentship FS/13/46/30282.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.


Drosophila melanogaster, in combination with the availability of powerful genetics, make this an attractive model for studying fundamental principles of Notch regulation and function. In this article we present some of the established and emerging tools that are available to monitor and manipulate the Notch pathway in Drosophila and discuss their strengths and weaknesses.Notch signaling involves a highly conserved pathway that mediates communication between neighboring cells. Activation of Notch by its ligands, results in the release of the Notch intracellular domain (NICD), which enters the nucleus and regulates transcription. This pathway has been implicated in many developmental decisions and diseases (including cancers) over the past decades. The simplicity of the Notch pathway in  Activation of the Notch transmembrane receptor by one of its ligands, Delta or Serrate , elicits two proteolytic cleavage events. These events are catalyzed sequentially by an ADAM-family metalloprotease (Kuzbanian/ADAM10) and by the γ-secretase complex . The second cleavage results in the release of the Notch intracellular domain (NICD). The latter enters the nucleus and interacts directly with the DNA-binding protein CSL [CBF-1/Su(H)/LAG-1] and the co-activator Mastermind to promote transcription .Drosophila melanogaster, which has one Notch receptor and two ligands, Delta (Dl) and Serrate (Ser). All show a high level of conservation with their mammalian orthologues Drosophila an extremely attractive model for studying Notch pathway. Indeed, it is worth noting that Notch was first identified in Drosophila and that the subsequent large constellation of studies have generated a powerful resource.The Notch pathway is involved in a multitude of developmental decisions as well as in adult homeostasis and stem cell maintenance. Aberrant Notch activity is also linked to many diseases, including inherited disorders  and many types of cancer  and a minimal hsp70 promoter Gbe-Su(H)-lacZ) is highly responsive to Notch activity in tissues such as the imaginal discs and nervous system, known to have high levels of Grh, and is kept silent by Su(H) mediated repression in the absence of Notch activity. The reporter has been used extensively over the past decade and is able to respond to Notch signaling in a wide variety of tissues. Originally utilizing lacZ as the readout, there are now eGFP, mCherry and Venus-Pest variants of the NRE reporter, increasing the versatility Drosophila tissues and embryos exist, including one that summarizes their use with Notch reporters One drawback of the target gene/reporter approach is that many Notch responsive enhancers also exhibit some degree of tissue specificity. Synthetic reporters have the advantage that they lack some of the tissue constraints. They are also less likely to exhibit indirect effects due to changes in other pathways/gene regulatory networks and so represent a more “pure” readout for Notch pathway activity. 2.2Drosophila HES genes present in the Enhancer of split [E(spl)] locus E(spl)HLH, HES family, genes reside in the locus along with 4 other Notch-responsive genes of the Bearded family E(spl) genes.Amongst the most widely expressed and best-characterized targets of Notch pathway are the E(spl) genes upstream of easily monitored reporters such as lacZ, GFP or RFP (E(spl)mα-RFP, a good indicator of Notch activity during SOP development in the pupal notum that has been used for live imaging E(spl)mß1.5-lacZ, a sensitive Notch activity read-out throughout the wing and leg imaginal discs E(spl)mδ0.5-lacZ, a marker of Notch activity in eye disc photoreceptors E(spl)HLHmγ-GFP, a reporter of Notch activity in post-embryonic neuroblasts A series of different reporters have been generated by cloning the Notch responsive regulatory regions containing the Su(H) binding regions from various P or RFP . The onesingle-minded (sim), a gene expressed in the midline of Drosophila embryos that was one of the first locations where Su(H) mediated repression was detected vestigial (vg), cut and wingless (wg), three genes that are specifically up-regulated by Notch at the dorsal/ventral (d/v) boundary in the wing disc klumpfuss (klu) and for other Notch-regulated genes in the lymph gland , which distinguished the Notch dependent regulation of the vestigial gene from its regulation by other signaling pathways There are, in addition a number of other gene-specific Notch-responsive reporters, which are active at different stages/conditions. These include reporters for 2.3lacZ or other reporters are inserted into the gene of interest, are useful for overcoming the second. In future it is quite likely that additional tools of this type will be available as CRISPR mediated gene engineering  for detailed protocol] in all the cells we have tested . The treatment of cells with the calcium chelators leads to shedding of the Notch ectodomain which renders the residual transmembrane fragment a substrate for γ-secretase cleavage and, hence, results in Notch activation The other approach to monitor Notch activity in cells is to measure activity of endogenous 2.5sc25761) perform best in ChIP One final approach to examine whether genes represent a direct output of Notch activity is to analyze chromatin occupancy by the NICD/Su(H) complex via chromatin immunoprecipitation (ChIP). Although NICD binding is a more direct measure of Notch regulation, our experience has been that commercial antibodies recognizing Su(H) . No equivalent exists for Serrate, although a series of reporters have been generated with regulatory regions from SerrateEither in situ hybridization or enhancer trap gene reporters are possible strategies to analyze expression, although they are not useful for probing the trafficking or subcellular localization. Useful enhancer trap lines include the acZ e.g.  insertioneuralized-lacZ , a very good indicator for the expression pattern of this E3-ligase which regulates Delta activity 35UZ-1fng a lacZ p-element insertion in the fringe gene which recapitulates some aspects of its expression kuz-GFP, a GFP tagged kuzbanian (kuz) transgene generated by BAC recombineering Enhancer traps for several of the regulatory genes also exist. These include www.flyprot.org) Notch-GFP/YFP have proven especially valuable for monitoring the sub-cellular distributions of the protein in vivo, as well as being a tool to assess the overall expression patterns. Additional components are being tagged with fluorescent proteins in a similar fashion including the Notch inhibitor Numb More recently, GFP (and YFP) tags have been introduced into Notch within its entire genomic region either by recombineering of BACs Finally, there are several very well-characterized antibodies recognizing key components of the pathway. These include monoclonal antibodies targeting Notch and Delta 4Over the years many mutant strains that abolish the function of Notch pathway have been generated either with X rays, chemicals (e.g. EMS) or by insertion of transposable elements. Other strategies for perturbing gene functions include the expression of dominant negative forms of a protein. Dominant negative Mastermind (MamDN) is one such example, which has been used extensively in mammals and is also effective in flies A recent, but highly effective, strategy to inhibit Notch function is to use RNA interference (RNAi) to knock down the expression of key pathway components  to consider which step in the pathway you want to target and (ii) to keep in mind that blocking some steps will have more complex effects than others. For example, perturbations to ligand function will have effects on the neighboring cells (i.e. will be non-autonomous); interfering with Su(H) may lead to de-repression of some targets e.g. ; some geses e.g. . The mos5To investigate the functional roles of Notch and its consequences on downstream factors, tools that confer increased pathway activity can be a very valuable resource . Among tUAS driven versions of full length Notch are also available Another effective way to mimic increased Notch pathway activity is to express a constitutively active Su(H), generated by fusing Su(H) to the viral activation domain VP16 lgdnumb and Hairless (H) result in more specific phenotypes of ectopic Notch activity [e.g. numb phenotype has been exploited in the larval CNS where ectopic Notch causes the neural stem cells to over-proliferate giving rise to tumours Abruptex alleles . However, their genetics is complex, so they should be used with caution.Finally, mutations in some genes also result in ectopic pathway activity. Many of these are non-specific because they alter trafficking pathways [e.g. ESCRT, ity e.g. . The num6A powerful genetic approach to test whether genes are likely to contribute to the activity of a particular pathway is to use a modifier assay, as initially pioneered for the receptor tyrosine kinase pathway e.g. see . This inDrosophila eye, whose development can be altered in a variety of ways by changing Notch function while at the same time not affecting the viability to adult flies. One very effective strategy is to over-express the ligand Delta, which causes an overgrowth of the eye. This has been powerfully exploited in screens for genes that synergise with Delta/Notch to cause tumours The other tissue that lends itself to such modifier assays and screens is the 7Drosophila such a valuable model to study the basic principles of Notch signaling, which can enable understanding of its roles in physiological and pathological conditions in humans. Further advances such as CRISPR genome editing, genome-scale analysis and advanced imaging are likely to expand on this repertoire to further facilitate the use of this sophisticated model.This review illustrates the current spectrum of tools that make


Celiac disease (CD) is an immune-mediated enteropathy triggered by the ingestion of gluten in genetically susceptible individuals. Gluten is the major protein component of wheat, rye and barley. The major predisposing genes are the HLA-DQ2 and DQ8 genotypes found in at least 95 % of patients. CD is one of the most common lifelong disorders on a worldwide basis affecting 0.5-1 % of the general population in Western countries. CD is a multifactorial disorder that depends on both genetic and environmental factors for expression.The analysis of factors associated with an epidemic of early-onset CD in Sweden during the 1980-90s indicated a role for infant nutrition, the disease risk being substantially lower in infants introducing small amount of gluten when still breast fed . The protective role of breast feeding has been supported by other case-control studies. As far as weaning, an increased risk of CD has been reported in infants introducing gluten containing food before the age of 4 months. Most of these data investigated only clinically detected cases of CD, leaving unanswered the question whether factors related to infant protect or simply delay the onset of disease. Ongoing European, prospective studies on at-risk infants followed on by serological screening are shedding light on the role of infant nutrition in the development of CD. The Italian Baby Study recently completed a 10-years period of follow-up. The results of this study suggest that genetically predisposed children tend to develop CD autoimmunity early in life, generally before the age of 5 years. Early dietary factors, particularly age at gluten introduction, seem to play a minor role in the development of CD, at least in infants at family risk, questioning the validity of the “window of exposure” hypothesis (lower risk in infants introducing the new antigen between 4 and 6 months). The possible preventive effect of delayed gluten introduction in infants with high HLA risk (double copy of DQB1*02) is a puzzling finding deserving further investigation. In this study, breast-feeding in children at-risk of CD did not seem to exert any clear-cut effect on the disease risk. Another European, prospective and randomized study ( PreventCD) will soon clarify whether small amount of gluten given during breast feeding do indeed protect from CD development.


Partial circulatory support is a new option of treatment for heart-failure patients. The Circulite Synergy Micro-Pump is a partial support device where the inflow cannula is connected to the left atrium and the outflow cannula to the right subclavian artery (RSA). Computational fluid dynamics (CFD) simulations can be used to demonstrate changes in hemodynamics post treatment.To investigate the influence of the outflow cannula geometry on hemodynamics in the aorta and supra-aortic vessels in patients with a Circulite Synergy Micro-Pump.Lumina of the aorta and supra-aortic vessels of nine patients were segmented from computed tomography angiographic (CTA) image data. Computational polyhedral meshes were created to conduct CFD-simulations (STAR CCM+) for diastolic flow conditions . To quantify effect of partial circulatory support, two simulations per case were carried out .Velocity magnitudes, velocities in inferior-superior direction (vz) and wall shear stresses were averaged in the innominate artery and descending aorta.Cases were divided into two groups: Outflow cannula placed orthogonal to the RSA  angle between the cannula and the RSA distal to the anastomosis site smaller than 90° .Geometry of the outflow cannula affected vz of the RSA and the innominate artery significantly (p < 0.05), but not in the descending aorta.Increase of velocity in the descending aorta was similar in both groups .Wall shear stress in the innominate artery did not change in group 1(-6.06%), but increased in group 2(+334.12%). The difference was not significant.Angle between SA and outflow cannula in patients with the Circulite Synergy Micro-Pump causes significant alterations in the subclavian and innominate arteries at diastole, but not in the descending aorta.


Salmo salar). This is an economically important aquaculture species, but no reference genes dedicated for use in miRNA-qPCR methods has been validated for this species. Our aim was, therefore, to identify suitable reference genes for miRNA-qPCR methods in Salmo salar.MicroRNAs (miRNAs) are an abundant class of endogenous small RNA molecules that downregulate gene expression at the post-transcriptional level. They play important roles by regulating genes that control multiple biological processes, and recent years there has been an increased interest in studying miRNA genes and miRNA gene expression. The most common method applied to study gene expression of single genes is quantitative PCR (qPCR). However, before expression of mature miRNAs can be studied robust qPCR methods (miRNA-qPCR) must be developed. This includes identification and validation of suitable reference genes. We are particularly interested in Atlantic salmon  contains supplementary material, which is available to authorized users. MicroRNAs (miRNAs) are an abundant class of endogenous small RNA molecules. They regulate gene expression as part of the miRNA-induced silencing complex (miRISC) at the post-transcriptional level by binding to the mRNA of target genes in a sequence specific manner , 2. MostSalmo salar) [The fundamental role of miRNA genes as major regulators of the expression of protein coding genes and their role as causative mediators of disease , 11 haveo salar) . ValidatAdvances in microarray and sequencing technology (deep sequencing methods) the last decade have led to increased sensitivity in detection of gene expression differences. These high throughput methods may be used to analyse expression of thousands of miRNA genes in parallel  while thqPCR methods, and all other methods that measure relative gene expression differences, depend on some strategy to correct for non-biological sample to sample variation to be able to measure the true biological expression levels of the gene(s) of interest (target genes) in a set of samples. The usual approach to control for the effects of systematic experimental bias and technical variation is to utilize within sample measurements of other genes that are expressed in a constitutive and stable manner, so-called normalization genes or reference genes. Assuming that these reference genes are close to identically expressed in all samples, the differences in expression level of the target genes that are not caused by biological variation may be controlled by use of these endogenous references , 23. ConC. elegans) when investigating expression levels across normal tissues or in certain disease states within some particular tissues (e.g. cancer) [Several other studies have suggested particular miRNA genes that may be suitable as normalization controls in miRNA-qPCR methods. These reference genes have been identified by analyzing sample material from humans and different model species (e.g. mouse or  cancer) , 28, 29.Salmo salar orthologs of these miRNA genes would show similar high stability in Atlantic salmon. Then some biological properties of the Salmo salar miRNA orthologs were considered and those that seemed as less good choices were removed. The remaining miRNA genes that still seemed to be good choices as reference genes in Salmo salar were compared regarding their stability in fifteen tissue samples retrieved from deep sequencing analysis of small RNA libraries. A subset of the miRNA genes, those that showed least variation across the deep sequencing samples, where finally validated by testing their performance in miRNA gene specific qPCR assays followed by identification of most suitable reference genes among the candidates by use of the NormFinder algorithm. These best performing reference genes were also tested in a panel of normal kidney samples vs kidneys samples from individuals with infectious salmon anaemia (ISA) to assure that the selected reference genes were not affected by general immune responses associated with virus disease.We have used a systematic approach where we utilize similar studies in other species, some biological criteria, results from deep sequencing of small RNAs in different tissues and, finally, experimental validation of a set of candidate reference genes by qPCR to identify the most suitable reference genes in Atlantic salmon. Validation studies from other species were first used to select a set of conserved miRNAs that were reported as constitutively expressed , 29–31, Salmo salar). Thus, the initially selected set of genes was first compared to the known miRNA genes in Atlantic salmon [Salmo salar. These were, obviously, not further considered. An overview of the remaining candidate reference genes that were selected for our validation study  is shown in Table A total of 40 conserved miRNAs suggested as reference miRNA genes for qPCR from various validation studies were initially included as putative reference genes  , 29–31. c salmon , 32. ThiSalmo salar[Salmo salar than other teleosts [An important property of any reference gene would be that it may be analysed with a high degree of specificity. The most common miRNA-qPCR methods utilize the mature miRNA sequence to achieve specific amplification of a certain miRNA gene, e.g. the miScript-assays used in our study where the specific amplification of a certain mature miRNA entirely depends on a single forward primer identical in sequence to the mature miRNA. However, a comparison of the mature sequences of the initially selected reference genes to all mature conserved miRNAs discovered in mo salar revealedteleosts . This maSalmo salar[The location of the candidate miRNA genes in the Atlantic salmon genome was also considered as an important biological selection criterion. If two, or more, miRNA genes are located in a gene cluster they are often not expressed independently. Such clustered miRNA genes are most often co-transcribed, they respond and regulate similar gene pathways, and, as a consequence of this, the expression of one parallels the other . Therefomo salar. Thus, eCandidate miRNA genes were compared regarding their stability across nine different tissues by use of deep sequencing data sets from a total of fifteen samples including the twelve samples used in a miRNA gene identification study in Atlantic salmon . The toThe coefficient of variation (CV) of each of the eleven candidate genes, as well as the CV of the two new miRNA genes added (ssa-miR-455-5p and ssa-miR-210-5p), is given in the column titled CV Deep seq. in Table Salmo salar  were analyzed in four samples from each of seven different tissues; liver, kidney, brain, intestine, heart, white muscle and gills. The log Ct-values from all measurements (four samples in each of seven tissues) were transformed to linear values and used as input in NormFinder . NormFinWe have a particular interest in studying miRNA genes and their role in virus disease. Therefore, to ensure that the expression level of any miRNA gene chosen as reference genes are not affected by any general response to virus disease, we investigated the effect of virus infection on the stability of the six best performing reference miRNA genes . We concluded that while expression of the five other miRNAs tested did not seem to be affected by ISA, the analyses of ssa-miR-107-3p indicated that this gene did change expression levels. Consequently, this miRNA gene would not be suitable as a reference gene when studying virus disease and miRNA expression.The comparison of the normalized expression level of ssa-miR-107-3p in the two groups did, however, show a larger difference in expression levels. Normal kidney tissues showed a mean normalized value of 1.42 with a SD of 0.34 while the ISA infected kidney tissues showed a mean normalized value of 3.47 with a SD of 0.7. A simple Our aim was to identify a small set of reference genes that may be applied in studies of different tissues and different conditions that included studies of virus disease. We therefore removed ssa-miR-107-3p from our group of suitable reference genes and performed a new NormFinder analysis using the remaining five best reference genes with stability values less than 1 from our initial test . In addition the sample numbers were increased to eight in each tissue group to make the analysis more robust. The best single and best two gene combination as well as a ranking of the five genes by their stability values is given in Table The best performing single gene was ssa-miR-25-3p  while the best performing two-gene combination was ssa-miR-25-3p and ssa-miR-455-5p . These genes, either ssa-miR-25-3p as single reference genes or in a two-gene combination with ssa-miR-455-5p, were, therefore, the most suitable reference genes for miRNA-qPCR across the conditions tested in our study.Salmo salar. The salmon ortholog of the candidate gene miRNA-183 showed e.g. relatively large amount of variation across tissues while the salmon ortholog of miRNA-107 was e.g. differently expressed in virus infected tissues. Results from Sarma et al. [Our systematic approach led to the identification of reference genes suitable for miRNA expression studies in Atlantic salmon. The majority of the candidate miRNA genes included were initially selected because previous validation studies in vertebrates suggested they were suitable as endogenous controls. However, several of these did not seem to be suitable as reference genes in a et al.  also indWe used results from deep sequencing of several tissue samples to screen which candidate genes may be the more stable ones. Similar approaches have been applied in other validation studies . In addiIn conclusion, we have identified two suitable reference genes for studying miRNA gene expression by qPCR in Atlantic salmon. Ssa-miR-25-3p was the best performing gene and together with ssa-miR-455-5p the best performing two-gene combination. They seem to be suitable as reference genes when studying miRNA expression across normal tissues in Atlantic salmon. They also performed well, with no obvious change in expression, in virus infected tissue. The two reference genes are conserved in vertebrates, even identical in their mature sequence in many aquaculture species, and the qPCR assays (miR-25-3p and miR-455-5p) provides good results without any change in primer sequences in rainbow trout and cod (data not shown). Although these genes need to be tested for their stability it is likely that they may be suitable as reference genes in other teleost species as well. The systematic approach used in our study seems to work well for identification of proper reference genes and may be applied in similar validations of reference genes in other aquaculture species.Salmo salar) individuals, described in Andreassen et al. [Two pre-smolt Atlantic salmon  following the manufacturer’s protocol. RNA concentration and purity were determined using Nanodrop following the manufacturer’s protocol. The concentration of total RNA ranged from 40–900 ng/μl .The miScript assays were used for cDNA synthesis and qPCR as described by the manufacturer (Qiagen). A universal primer (reverse primer), provided with the miScript qPCR kit, was used in combination with our own custom designed forward primer in the qPCR amplification of each of the mature miRNA genes. The forward primers were designed based on the mature sequence of each of the miRNA genes to be amplified . All prit-test was therefore performed to test whether the difference between expression in normal kidney tissue and ISA infected kidney tissues was significant (http://studentsttest.com/).The effect of ILA virus infection on the stability of six selected miRNA genes  was tested in the following manner. Five kidney samples from individuals infected with ILA and five kidney samples from normal individuals were analyzed by qPCR. The results  were normalized using ssa-miR-25-3p as normalization gene (best performing single reference gene). The normalized Ct-values from the five other miRNA genes were used to calculate the mean and standard deviations of their expression level in the normal kidney tissue group and in the ISA kidney tissue group. Comparisons of the mean and the spread of the five miRNA gene’s expression level in the two groups were used to reveal whether any gene was differently expressed. One miRNA (ssa-miR-107-3p) seemed to be differently expressed. A http://www.novocraft.com). The total read numbers were used to normalize the read numbers of the individual mature miRNAs within each sample. The average normalised read numbers of each miRNA gene in the fifteen samples was then estimated along with their standard deviations. Finally, the relative standard deviation  was estimated by dividing the mean normalised read number from each miRNA gene by their standard deviation. The CV was then used as a measurement of relative miRNA gene stability across the tissues included in the deep sequencing.Deep sequencing data from 15 samples  [The data normalization tool NormFinder (oftware)  was usedContains complete dataset of read numbers, normalised read numbers and CV’s of reference genes analysed in different tissue samples from deep sequencing.(XLSX 15 KB)Additional file 1:Contains primer sequences of all forward primers and other parameters relevant to the miRNA-qPCR method applied.(XLSX 12 KB)Additional file 2:Shows comparisons of expression levels of the candidate reference genes in normal vs ISA infected kidney tissue.(XLSX 11 KB)Additional file 3:


The formation of new lymphatic vessels, or lymphangiogenesis, is a natural process involved in tissue repair and in the resolution of inflammatory reactions. In some cancers, this process is exploited by the tumor to promote its growth and its metastatic dissemination. Our group recently identified a role for the CD73-adenosine pathway in tumor angiogenesis. As angiogenesis and lymphangiogenesis are similar processes, often occurring in parallel, we suspect that the CD73-adenosine pathway could have a role in the regulation of lymphangiogenesis during inflammatory reactions and in the tumor microenvironment.To confirm this hypothesis, we used in vivo models of inflammatory lymphangiogenesis induced by the administration of lipopolysaccharide (LPS) or Incomplete Freund's Adjuvant (IFA) in the peritoneal cavity of mice. These two models of peritonitis trigger the formation of new lymphatics in the diaphragm that can be analyzed by flow cytometry. To study tumor lymphangiogenesis, we used a B16F10 melanoma model implanted in the mouse foot pad thereby allowing the analysis of tumor cell dissemination in the popliteal draining lymph node.Using A2A-deficient mice, our results demonstrate that the A2A receptor is required for the proper formation of new lymphatic vessels following LPS and IFA administration. Using multicolor flow cytometry we were able to show that A2A-deficiency impairs both the formation of new lymphatic capillaries from pre-existing vessels and the transdifferentiation of pro-inflammatory myeloid cells into lymphatic endothelial cells. Furthermore, our results also indicate that CD73-derived adenosine has a minor involvement in this process as CD73-deficient mice only display a slight alteration in the recruitment of lymphatic endothelial cell in the LPS-peritonitis model. In the B16 melanoma model, host-A2A deficiency impaired tumor-associated lymphangiogenesis which resulted in a reduced number of tumor cells disseminating to the tumor-draining lymph node.Collectively, our results demonstrate that the A2A receptor promotes inflammatory and tumor-associated lymphangiogenesis.


AGAMOUS (AG) lineage and specify both stamen and carpel identity and floral meristem determinacy. In core eudicots, the AG lineage is further divided into two branches, the euAG and PLE lineages. Functional analyses across flowering plants strongly support the idea that duplicated AG lineage genes have different degrees of subfunctionalization of the C-function. The legume Medicago truncatula contains three C-lineage genes in its genome: two euAG genes (MtAGa and MtAGb) and one PLENA-like gene (MtSHP). This species is therefore a good experimental system to study the effects of gene duplication within the AG subfamily. We have studied the respective functions of each euAG genes in M. truncatula employing expression analyses and reverse genetic approaches. Our results show that the M. truncatula euAG- and PLENA-like genes are an example of subfunctionalization as a result of a change in expression pattern. MtAGa and MtAGb are the only genes showing a full C-function activity, concomitant with their ancestral expression profile, early in the floral meristem, and in the third and fourth floral whorls during floral development. In contrast, MtSHP expression appears late during floral development suggesting it does not contribute significantly to the C-function. Furthermore, the redundant MtAGa and MtAGb paralogs have been retained which provides the overall dosage required to specify the C-function in M. truncatula.C-function MADS-box transcription factors belong to the  Arabidopsis thaliana and Antirrhinum majus. These studies provided a general understanding of floral organ development in higher plants and led to the proposal of the ABCDE model, which postulates that floral organ identity in each whorl is defined by five functions named A, B, C, D and E  PLENA (PLE) SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2) Arabidopsis and FARINELLI (FAR) Antirrhinum.In AG-like sequences show that AG and PLE actually represent paralogous lineages derived from a duplication in a common ancestor early in the history of the core eudicots. This duplication gave rise to the euAG lineage which includes AG and FAR, and the PLENA lineage (PLE), where SHP1/SHP2 and PLENA are placed AG lineage) and the ovule-specific D lineage (AGL11 lineage) Phylogenetic studies using a large data set of AG, FAR and PLE all display very similar expression patterns during the developing male and female reproductive organs AG and PLENA in their respective species produce identical phenotypes characterized by developing flowers with petals in whorl 3 instead of stamens, and sepals in whorl 4 instead of carpels. In addition, the floral meristem is not determinate far mutation only affects male reproductive organs, causing partial male sterility SHP genes are only expressed in the ovules, in the developing pistil and show almost identical expression patterns in developing Arabidopsis fruit SHP and FAR genes do not contribute significantly to the C-function although FAR is expressed at early stages of flower development.AG and PLE, different members of a duplicated gene pair, retained the primary homeotic functions in different lineages. Their respective orthologs have taken completely new roles in fruit dehiscence, as the case of SHP1/SHP2 genes, or contribute redundantly in male reproductive development, as the case of FAR. Representatives of both euAG and PLE lineages have been identified in different core eudicot species and several functional analyses are available for the paralogs from petunia, tomato, and Nicotiana benthamianaThese studies showed a random evolutionary trajectory for gene functions after a duplication event. AGAMOUS subfamily, more recent duplication events appear to have occurred in the euAG lineage AG lineage in some species, as for example Populus trichocarpaCucumis sativusGerbera hybridaLotus japonicusPisum sativum and Medicago truncatula (this study). However, in most cases, functional analyses are lacking. Both phylogenetic and functional data of gene lineages are very important to understand the evolution of gene families In addition to the ancient duplication events observed in the Medicago truncatula (Mt) has three C-lineage genes: two euAG genes (MtAGa and MtAGb) and one PLENA-like gene  and 18°C (night) with a 16 h light/8 h dark photoperiod, in soil:sand (3∶1) irrigated with Hoagland No.1 solution supplemented with oligoelements mtaga mutant allele (previously matag-2) was isolated in a previous screening The M. truncatula population used for the screening of mutants was described in detail http://bioinfo4.noble.org/mutant/). The mtagb allele was identified by PCR screening of a segregating population of approximately 10,000 independent lines, using primers annealing to the MtAGb sequence  with the LFY matrix. The score tool permits localization of the best transcription factor binding sites in DNA fragments.The second intron sequences were obtained by PCR using genomic DNA and the primers MtAGa-intron-F and MtAGa-intron-R for or MtSHP . To predMEGA version 4 MtAGa and MtAGb). The tree was rooted using the Arabidopsis D-class gene SEEDSTICK (STK) sequence. All sequences used in this analysis, with their respective species and accession numbers, are included in The phylogenetic tree was inferred by Neighbor-Joining using Poisson-Corrected amino acid distances. Reliability of internal nodes was assessed using bootstrap with 10000 pseudo-replicates. Tree inference was conducted using MtAGa cDNA (positions 572-813) and a 215 bp fragment of the MtAGb cDNA (positions 558-773) were amplified with the specific primers MtAGadir, MtAGarev, MtAGbdir and MtAGbrev1  according to the manufacturer's instructions.+ membranes , and hybridized with 32P-labeled probes under standard conditions. The probes were generated from the same gene fragments used for Southern blot analysis.For Northern blot analysis total RNA (15 µg) from frozen leaves, roots, stems, flowers and pollinated ovaries (young fruits) was used. RNA electrophoresis was carried out in formaldehyde-agarose gels, transferred to Hybond NrDNaseI of the DNase Treatment and Removal Kit . For first-strand synthesis, total RNA (1 µg) was reverse-transcribed in a 20 µl reaction mixture using the PrimerScript 1st strand cDNA Synthesis Kit . One microliter of RT reaction was used for a Real Time RT-PCR analysis with 300 nM of each primer mixed with the Power SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer's instructions. The reaction was carried out into 96 well-optical reaction plates using an ABI PRISM 7500 Sequence Detection System and appropriate software (Applied Biosystems). The relative levels were determined by the 2 Ct–ΔΔ Method Secret Agent  and MtAGb (338 bp) genes were obtained by PCR using primers  that added restriction sites to both ends of the fragments (MtAGab) was confirmed by sequencing before being introduced into Agrobacterium tumefaciens strain C58/pMP90. Agrobacterium inoculation of M. truncatula leaves was performed as described by pCAPE1 and pCAPE2 derivatives were used as vectors for gene silencing ragments . The ampM. truncatula R108 was performed as described previously MtAG construct was performed using a 215 bp fragment from MtAGb (positions 557–772 from the ATG codon), amplified using primers MtAGb-RNAiD and MtAGb-RNAiR .in situ hybridizations, sections were observed and photographed with a Nikon Eclipse E-600 microscope equipped with a digital camera.After For cryo-SEM, samples were frozen in slush nitrogen and attached to the specimen holder of a CryoTrans 1500 Cryo-Preparation System  interfaced with a JEOL JSM-5410 scanning electron microscope. The samples were then transferred from cryostage to the microscope sample stage, where the condensed surface water was sublimed by controlled warming to -85°C. Afterwards, the sample was transferred again to the cryostage in order to gold coat it by sputtering. Finally the sample was put back on the microscope sample stage to be viewed at an accelerating voltage of 15 KeV.M. truncatula floral apices using a set of MADS-box fragments from different species as a probe AGAMOUS subfamily. One of these clones presents high sequence similarity with the Arabidopsis SHP1 and SHP2 genes Antirrhinum PLENA gene  than in the C-terminal region (68% of identity). MtAGa and MtAGb show respectively 71 and 68% amino acid identity with the euAG clade protein FAR and either one 67% amino acid identity with AG  and mth2-76i7 (GenBank AC153460.24). This gene is organized in seven exons and six introns . MtAGa gene is also organized in seven exons and six introns . We found that MtAGb gene is located on chromosome 8 . We searched in these sequences for the presence of putative LFY binding sites that are characteristic of C-function genes. We included in this study the MtSHP gene and then we have sequenced its first intron of 4281 bp (GenBank KJ470635). We used a bioinformatics tool available at the Morpheus webpage facility (http://biodev.cea.fr/morpheus/Default.aspx) which examines the distribution of individual LFY binding sites. The presence or absence of LFY binding sites identified by this approach appears to be helpful in predicting to what extent a C-clade gene acts as a true C-function gene MtAGb first intron, there is a main single binding site of very high score similar to the profile found for the PLE gene MtAGa, lower affinity sites are present but the binding seems to be compensated through the action of several nearby sites as reported for the sites present in the second intron of AG , was isolated as a case study to demonstrate the utility of the reverse genetic platform in the model legume Medicago truncatulaMtAGa or MtAGb transcripts were nearly undetectable in the corresponding homozygous mutant plants without affecting the expression levels of the corresponding MtAG paralog  experiments. We used a VIGS vector based on PEBV (Pea early browning virus) initially developed to induce gene silencing in PisumM. truncatula cultivars than in P. sativumMtAGab construct to simultaneously silence both genes . Twenty one plants were inoculated with the pCAPE2-MtAGab construct and we analysed the phenotype of 150 flowers. 10% of the MtAGab-VIGS flowers showed homeotic transformations in whorls 3 or 4. Only 1% of the MtAGab-VIGS flowers showed a near complete loss-of-C-function phenotype  and three independent lines were obtained. Only one of these lines (line 5.7) showed floral phenotypes similar to those of single mtag mutants (MtAGa and MtAGb genes in the three RNAi lines. These lines showed a different degree of silencing for each targeted gene (MtAGb levels considerably reduced (∼15% of wild-type level), while MtAGa levels are somewhat decreased (∼90% of wild-type level). Strong silencing of one paralogous gene did not produce homeotic floral defects. However, floral defects in line 5.7 correlate with simultaneous downregulation of both genes (∼40% of wild-type level)  .AG results in the homeotic conversion of sepals and petals into carpels and stamens, respectively Arabidopsis. The coding sequence of these genes was located under the control of the CaMV 35S constitutive promoter. We analysed 75 independent primary transgenic (T1) lines for every construct (35S::MtAGa and 35S::MtAGb plants) and found plants with different floral phenotypes that we classified as mild, medium and strong phenotype  and one member of PLENA (MtSHP) AG homologs from this species to achieve a more thorough understanding of the evolution of the AGAMOUS subfamily in core eudicots.The model legume AG duplicated genes (MtAGa and MtAGb) in M. truncatula, as well as in other legume species . The changes of these genes after duplication may have played an important role in the evolution of floral morphology and ontogeny in legumes.The finding of two eucies see  suggestsAG genes is not only found in legumes. Previous phylogenetic studies have revealed the existence of two members of the euAG-lineage in several core eudicots CUM1 and CUM2 from Cucumis sativus, PTAG1 and PTAG2 from Populus trichocarpa, and GAGA1 and GAGA2 from Gerbera hybrida. These studies are mainly focused on the analyses of their expression patterns and limited functional information is available. Recently, phylogenetic analyses using AG protein sequences from several Asteraceae species, have revealed that some of them, harbor two or three members within the euAG-lineage The presence of two euAG homologs AG subfamily M. truncatula euAG genes could have taken place during the WGD event that predates legume speciation followed by gene rearrangement.MtAGa and MtAGb proteins conserve characteristic domains of the C-lineage members, as the presence of an N-terminal extension preceding the MADS domain AGAMOUS genes, it has been shown that the first and second introns are essential for their correct expression LFY is essential for AG activation and binds to cis-elements located in the second intron MtAGa and MtAGb. Although the binding site landscapes observed for each euAG-paralogous gene are different, experimental analyses have demonstrated that both types of distribution of LFY binding sites are functionally relevant MtSHP was not found to be a likely LFY target and accordingly, MtSHP expression is exclusively detected in the ovules at later stages of floral development. Similarly, SHP or STK, which play late roles in fruit and ovule development, were not found to be LFY targets in ChIP-seq experiments MtAG genes have the same probability to be directly activated by LFY, conferring organ identity on floral meristems that arise after the transition to reproductive development.Within the highly similar gene structure of MtAGa and MtAGb transcripts. It would be desirable to measure the MtAG mRNAs levels in MtAGab-VIGS flowers to validate this hypothesis, but sufficient plant material was not available. However, using stable MtAG::RNAi lines we were able to correlate the amounts of MtAG gene products with partial loss-of-C-function phenotypes. Only simultaneous down-regulation of both genes (below 40% of the wild-type level) produces mild aberrations in stamen and carpel development. These results support the quantitative model of AG activity, which suggests that the different AG functions vary with respect to the amount of gene product required MtAG transcripts could explain the indeterminacy of the floral meristem in MtAGab-VIGS flowers. In each single mtaga and mtagb mutants the amount of the remaining paralog is sufficient to confer almost all the C-function. However, the discrete mutant phenotypes of these plants suggest that a single MtAG gene is not sufficient to maintain the overall dosage of these subcomponents of protein complexes which are needed to specify the C-function in M. truncatula.A typical C-function knock-out phenotype is described as the conversion of stamens to petals, and a flower in place of the fourth whorl, which starts with a whorl of sepals AGAMOUS subfamily genes is closely consistent with their conserved function and subfunctionalization AG lineage genes early in the floral meristem, and in the third and fourth floral whorls is probably the ancestral expression profile of these genes AG and PLE-like genes is observed in Petunia, N. benthamiana and Antirrhinum, at early floral developmental stages. This expression profile correlates with their functional redundancy to promote the C-function of these genes in the cited species A. thaliana, the only C-function factor is AG, because, although the PLE-like proteins (SHP proteins) fully maintain a similar potential activity, they are not physically present in the meristem and primordia cells at the appropriate time. Their functions are rather restricted to regulate specific tissue types after carpel identity has been established. It remains possible that SHP genes redundantly regulate carpel identity with AG because they are expressed in early carpel primordia. The Arabidopsis AG and SHP genes are a clear example of subfunctionalization as a result of a change in expression pattern M. truncatula could be considered an illustrative example, where the expression pattern of the members of euAG or PLENA lineages would be very well correlated with their function. The two euAG genes (MtAGa and MtAGb) are expressed early during floral development, having a critical role in the specification of reproductive organs and floral determinacy. MtSHP expression appears late during floral development after all organs have been specified, suggesting it does not contribute significantly to the different components of the C-function . A role for this gene in fruit morphology and valve margin lignification has been suggested MtSHP in ovule development, associated with its expression in this tissue during floral development.The spatial and temporal expression profile of AG and PLENA-like members. In most cases, the ectopic expression of each sub-clade member gave similar results Antirrhinum, where the presence of an additional glutamine in the K3 helix of FAR protein, causes its inability to induce carpel identity in the first whorl Antirrhinum C-lineage genes largely relies on changes in the protein ability to interact rather than on changes in their expression patterns. The ectopic expression of M. truncatula euAG- and PLENA-like genes Click here for additional data file.Figure S2Southern blot analysis of MtAGa and MtAGb genes and localization of the MtAGb gene on the M. truncatula physical map. (A) Southern blot analysis of MtAGa and MtAGb. The length of the DNA markers (in Kb) is shown on the left margin. (B) MtAGb sequence is anchored in BAC AC153460, on the chromosome 8. Pseudochromosomes are represented by the succession of BACs  separated by gaps . Not all gaps are visible at this resolution. Unmapped BACs are shown on the right as MtChr0. MPM1/2: molecular weight markers.(TIF)Click here for additional data file.Figure S3Molecular characterization of Tnt1 insertion line in the mtagb locus. Genotyping of 20 plants from the Tnt1 insertion population of NF4908 line. (A) PCR results using AGb-F/Tnt1-F primers. (B) PCR results using AGb-F/AGb-R genomic primers. Homozygous mutant plants are indicated with bold. numbers. MPM: molecular weight markers; WT: wild-type control; H2O: distilled water.(TIF)Click here for additional data file.Figure S4qRT-PCR expression analyses of loss-of-function plants. Relative expression of MtAGa or MtAGb genes in flower buds of mtaga and mtagb mutants. The height of the bars for a given gene indicates differences in relative expression levels in floral buds. The wild-type expression value was set to 1.00, and lower values are plotted relative to this value.(TIF)Click here for additional data file.Figure S5Floral phenotype and expression analyses of 35S::RNAi-MtAG plants. (A) Wild-type M. truncatula flower. (B-C) Flowers from 35S::RNAi-MtAG transgenic line 5.7. (D) Wild-type stamens. (E-F) RNAi lines showing unfused staminal tubes and mild homeotic transformations (arrow). (G) Wild-type carpel. (H-I) Multiple unfused carpels showing exposed ovules (arrow). (J) Relative expression of MtAGa or MtAGb genes in flower buds of the three transgenic RNAi lines measured by qRT-PCR. The height of the bars for a given gene indicates differences in relative expression levels in floral buds. The wild-type expression value was set to 1.00, and lower values are plotted relative to this value.(TIF)Click here for additional data file.Table S1Sequences of the C-lineage MADS-box genes from the different species used in the phylogenetic analysis. The GenBank accession numbers are indicated.(DOCX)Click here for additional data file.Table S2Primers used in this work.(DOCX)Click here for additional data file.


A systematic review was conducted to assess the cost-effectiveness of routine varicella and herpes zoster (HZ) vaccination in high-income countries estimated by modelling studies.A PubMed search was performed to identify relevant studies published before October 2013. Studies were included in the review if they (i) evaluated the cost-effectiveness of routine childhood or adolescent varicella vaccination and/or HZ vaccination targeting the elderly, and if they (ii) reported results for high-income countries.A total of 38 model-based studies were identified that fulfilled the inclusion criteria. Routine childhood or adolescent varicella vaccination was cost-effective or cost-saving from a payer perspective and always cost-saving from a societal perspective when ignoring its potential impact on HZ incidence due to reduced or absent exogenous boosting. The inclusion of the potential impact of childhood varicella vaccination on HZ led to net quality-adjusted life-year (QALY) losses or incremental cost-effectiveness ratios exceeding commonly accepted thresholds. Additional HZ vaccination could partially mitigate this effect. Studies focusing only on the evaluation of HZ vaccination reported a wide range of results depending on the selected target age-group and the vaccine price, but most found HZ vaccination to be a cost-effective or marginally cost-effective intervention. Cost-effectiveness of HZ vaccination was strongly dependent on the age at vaccination, the price of the vaccine, the assumed duration of protection and the applied cost per QALY threshold.While HZ vaccination is mostly considered cost-effective, cost-effectiveness of varicella vaccination primarily depends on the in- or exclusion of exogenous boosting in the model. As a consequence, clarification on the role of exogenous boosting is crucial for decision-making regarding varicella vaccination. Primary infection with varicella-zoster virus (VZV) causes varicella (chickenpox), which occurs mainly in childhood , 2. The Live-attenuated monovalent varicella vaccines or combination vaccines against measles, mumps, rubella and varicella (MMRV) licensed for use in children are available in most industrialised countries. As of today there is one HZ vaccine licensed for individuals aged 50 years and older.Routine childhood varicella vaccination is generally recommended in the United States, Australia, Canada, Qatar, Saudi Arabia, Republic of Korea, Taiwan, Uruguay and several countries in Europe including Germany, Greece, Finland and parts of Italy and Spain , 8. A siIn the 1960s, Hope-Simpson hypothesised that after primary infection a re-exposure to wild-type VZV would sub-clinically boost the individual’s VZV-specific immunity, thereby suppressing VZV reactivation and decreasing the probability of developing HZ . Since tOur objective was to assess the cost-effectiveness of routine varicella and HZ vaccination in high-income countries estimated by modelling studies.A PubMed search was performed to identify English- and German-language articles on economic evaluations of varicella and HZ vaccination published before October 2013. The systematic literature search was conducted using the following key words: (varicella OR chickenpox OR herpes zoster OR shingles OR varicella-zoster OR VZV OR “Chickenpox” [MeSH] OR “Herpes Zoster” [MeSH]) AND (vaccination OR vaccine OR vaccinating OR vaccinate OR vaccinated OR immunisation OR immunization OR “Vaccination” [MeSH] OR “Vaccine” [MeSH] OR “Chickenpox Vaccine” [MeSH] OR “Herpes Zoster Vaccine” [MeSH]) AND . Application of the non-MeSH search terms was restricted to titles and abstracts of the PubMed records. In addition, we screened reference lists of all included studies to identify further articles of interest.Titles and abstracts of the obtained search results were screened independently by two reviewers. Full-text versions of all potentially relevant studies were retrieved and assessed according to pre-defined inclusion and exclusion criteria by the same two reviewers. Any disagreements between reviewers on inclusion of particular studies were resolved by consensus. A study was included if it was a full or partial economic evaluation of routine childhood (or adolescent) varicella vaccination or a HZ immunisation scheme targeting the elderly and if the modelling study reported results for a high-income country as specified by the World Bank (high-income OECD-members) . InclusiCritical appraisal of all included studies was undertaken by using the framework for quality assessment of decision-analytic models proposed by Philips et al. . The quaTo improve comparability between studies and across countries, all cost estimates were inflated to 2010 values (latest price year used in included studies) applying country-specific consumer price indices and converted to Euros with the German level of purchasing power using purchasing power parities obtained from the Organisation for Economic Co-operation and Development (OECD) .The reporting of this systematic review was performed in accordance with the PRISMA statement . HoweverThe literature search in PubMed identified 351 articles. After screening the titles and abstracts of these hits, 92 papers were considered for full-text review. 41 papers of the obtained full-text articles met the inclusion criteria. The main reasons for exclusion were incorrect type of study (such as review articles), incorrect intervention or studies assessing the vaccination of specific target groups. Few studies were excluded due to insufficient information on the methods used. Four of the 41 papers that met the inclusion criteria reported on the same modelling study and three of them were therefore excluded. Finally, 38 studies were included in the review. A flow chart of the study selection process and the corresponding results is outlined in Fig. Most of the included studies were of high quality. However, some studies evaluating varicella vaccination did not use a dynamic modelling approach and hence are not able to capture indirect effects of vaccination. Most studies performed probabilistic sensitivity analysis to address the issue of parameter uncertainty, but only a few studies evaluated the impact of structural uncertainties. Several studies also lack detailed descriptions/discussion of the sources of utility weights.We included 23 model-based studies evaluating the cost-effectiveness of routine varicella vaccination. Table 13 studies used a fully dynamic modelling approach in terms of simulating the transmission dynamics of varicella. 10 studies were based on static models. By definition, all dynamic models accounted for herd protection effects. The static model from Israel reported to have included partial herd protection effects by use of an adjustment factor. Apart from herd protection effects, varicella immunisation programmes can induce other population-level effects such as the hypothesised increase in HZ incidence due to a reduced or absent exogenous boosting after varicella vaccine introduction and subsequent decrease in wild virus circulation in the population. Most of the models ignored the close relationship between varicella and HZ. Only four studies modelled the possible impact of routine varicella vaccination on HZ incidence in a population due to a decrease in wild-type VZV circulation.Many studies performed a cost comparison analysis and reported the results as BCRs. Some of these studies claimed to have conducted a cost-benefit analysis but this would require a monetary valuation of health effects. However, the included studies which we classified as cost comparison analyses only considered costs and cost offsets instead of valuing health effects in monetary terms.Most of the studies adopted both a health care payer and a societal perspective. Costs were discounted with a discount rate ranging from 3 to 5 %. The discount rate for health effects ranged from 0 to 5 %. Vaccination costs varied widely across studies. The simulated time horizons ranged from 20 years to a lifetime or an infinite time horizon, but most studies used a period of 30 or 50 years.Aspects related to vaccine characteristics and immunisation strategies considered in the included studies are presented in Table We included 17 model-based studies evaluating the cost-effectiveness of HZ vaccination. The main study characteristics are summarised in Table All studies conducted a cost-utility analysis (CUA). Furthermore, almost half of the studies also performed a cost-effectiveness analysis (CEA). According to respective national guidelines, six studies used different discount rates for costs and health effects. One study used equal discount rates for costs and health effects that changed over time following the current French guidelines: A 4 % discount rate was used for the first 30 years of the model run and afterwards the discount rate was reduced to 2 %. Most studies used a lifetime horizon.Details of the HZ vaccination-related input data are shown in Table A wide range of vaccination costs was applied across the studies. One study  considerone-dose varicella vaccination of young children ranged between 0.30 and 1.94 when taking a health care payer perspective and ignoring a potential impact on HZ. Six studies reported BCRs above 1 or stated that vaccination would lead to cost-savings. Eleven studies reported BCRs below 1 or calculated ICERs. In these studies, costs per life year gained (LYG) ranged between EUR 563 and EUR 40,193. One study reported a BCR of 1 which means that one-dose varicella vaccination was a cost-neutral intervention [The results of economic evaluations of varicella vaccination are summarised in Table rvention . When adfor two dose-vaccination targeting young children ranged between 0.13 and 1.08 when adopting a health care payer perspective and between 0.56 and 3.47 when taking a societal perspective. The study by Bilcke et al. [BCRs e et al.  reportede et al.  showed te et al.  a two-doinclusion of the impact on HZ incidence led to less cost-effective results. Brisson et al. [The n et al.  found ann et al.  found a n et al. , which fn et al.  concludevaccinating young adolescents ranged between 0.36 and 1.94 from a health care payer perspective. ICERs were EUR 15.863 per LYG [BCRs of  per LYG , EUR 26, per LYG  and EUR  per LYG . Compare per LYG , 36, whi per LYG , 34, 37.The results of models assessing the cost-effectiveness of HZ vaccination are shown in Table When taking a societal perspective, one US study  reportedSeveral studies, which included waning of vaccine-induced immunity and reported results for different ages at vaccination identified an U-shaped figure of vaccination age-related ICERs: Cost-effectiveness ratios decreased with increasing age at vaccination up to the age of 60 or 70 years and then increased with further increase in age at vaccination. In addition, many studies reported that cost-effectiveness was highly dependent on the assumed duration of vaccine-induced protection and the price of the vaccine. Particularly, the study by Hornberger et al.  showed hThe majority of the included studies concluded that HZ vaccination would represent a cost-effective strategy. However, some studies came to different conclusions, mostly due to the application of different cost-effectiveness thresholds. The authors of a Dutch study  concludeTwo studies , 33 modeThis systematic review was conducted to summarise the current state of evidence on the cost-effectiveness of varicella and HZ vaccination in high-income countries. To our knowledge, this is the first systematic review covering cost-effectiveness studies of both varicella and HZ vaccination. The major findings are outlined below:When ignoring the potential impact on HZ and adopting a health care payer perspective, universal childhood varicella vaccination was usually a cost-effective or even cost-saving strategy.When switching to a societal perspective, childhood varicella vaccination was found to be a cost-saving intervention.Vaccination of adolescents was found to be a cost-effective or cost-saving strategy. However, it remains unclear if adolescent vaccination is more or less cost-effective than childhood vaccination due to inconsistent study results.Taking the potential impact on HZ into account, it is doubtful that childhood varicella vaccination appears to be cost-effective, at least for several decades.In most studies, HZ vaccination was predicted to be cost-effective or marginally cost-effective.When considering both a payer and a societal perspective, the differences in results between the two perspectives decreased with increasing age at vaccination since indirect costs due to sick leave become less relevant in the elderly population.When waning of vaccine-induced immunity was modelled, cost-effectiveness of HZ vaccination was highly dependent on the age at vaccination. ICERs decreased with increasing age at vaccination up to a certain age, followed by a re-increase of the ICERs (U-shape) for older ages. Most results suggest that the optimal age for HZ vaccination is between 60 and 70 years or around 70 years. Furthermore, cost-effectiveness was dependent on the price of the vaccine, the duration of protection and the assumed cost-effectiveness threshold.Model choice can influence the results to a great extent when evaluating the cost-effectiveness of vaccines. In static models such as decision trees and Markov models the force of infection is constant over time because individuals were not allowed to interact with each other. In contrast, dynamic models account for interactions between individuals and therefore the force of infection depends on the number of susceptible, infectious and recovered individuals in the population. This is why dynamic models can include herd protection effects when evaluating the impact of vaccines.More than half of the studies evaluating varicella vaccination were based on dynamic models and took herd protection effects into account. This is an important requirement to assess the impact of different coverage rates. In several studies the varicella vaccination uptake was found to be one of the most influential parameters. Since the force of infection for HZ is constant by nature, the models assessing only HZ vaccination were kept static.The results of studies evaluating the cost-effectiveness of varicella vaccination were very sensitive to the structural assumption of allowing for exogenous boosting and its consequences on HZ incidence. Varicella vaccination was found to be a cost-effective or cost-saving strategy as long as the potential impact on HZ incidence was ignored. When taking the potential impact on HZ incidence into account, varicella vaccination was unlikely to be cost-effective. However, the study by van Hoek et al.  showed tMany of the studies on varicella vaccination underlined the role of indirect costs for the assessment of cost-effectiveness. When adopting a societal perspective, savings were largely due to the inclusion of indirect costs. A previously published review of cost-effectiveness studies on varicella vaccination  found thWe are aware of four previously published systematic reviews on studies assessing the cost-effectiveness of varicella vaccination –46 and oThiry et al.  concludeOne of the strengths of our systematic review is that it is more comprehensive than previously published review articles. We included economic evaluations of varicella vaccination and HZ vaccination while other reviews have been focused on one of the two vaccines at a time. A combined assessment of the cost-effectiveness of varicella and HZ vaccination programmes gives consideration to the close relationship of both diseases.Our review was focused on models evaluating varicella vaccination in children and adolescents as well as HZ vaccination in the elderly. Studies limited to varicella vaccination of specific target groups such as health care workers or pregnant women were excluded because the current debate is more about whether universal varicella and HZ vaccination is good value for money; still the consideration of specific risk groups can affect the overall cost-effectiveness. Furthermore, we excluded studies which provided no sufficient description of the used methods  implementation of HZ vaccination in the elderly. As a consequence, clarification on the role of exogenous boosting is crucial for decision-making regarding varicella vaccine introduction. Cost-effectiveness of HZ vaccination itself is mainly dependent on the chosen age at vaccination, the price of the vaccine and the magnitude of the cost per QALY threshold.Based on this review we identified several important issues that need to be considered when evaluating the health economic impact of varicella and/or HZ vaccination. Future economic evaluations of varicella vaccination should apply a dynamic modelling approach because only dynamic models can take into account herd protection effects and the potential impact of varicella vaccination on HZ incidence due to reduced or absent exogenous boosting. In contrast, when the analysis is focused exclusively on HZ vaccination, a static model seems to be adequate since no change in the force of infection in HZ will be derived from the introduction of HZ vaccination. In addition, since fatality rates of varicella and HZ are rather low, we believe using life years gained (LYG) as an outcome parameter is not sufficient. Thus, we recommend, as done in most existing studies, to consider health-related quality of life outcome parameters (e.g. QALYs). Moreover, waning of vaccine-induced immunity seems to play an important role for the impact of both vaccines. Hence, we recommend addressing this issue already in the base-case analysis of future evaluations . Furthermore, when considering waning of vaccine-induced immunity, the effects of administering a booster vaccine should be captured in sensitivity analysis because a booster shot might have significant impact on the results.


Post-transcriptional control determines the fate of cellular RNA molecules. Nonsense-mediated decay (NMD) provides quality control of mRNA, targeting faulty cellular transcripts for degradation by multiple nucleases including the RNA exosome. Recent findings have revealed a role for NMD in targeting viral RNA molecules, thereby restricting virus infection. Interestingly, NMD is also linked to immune responses at another level: mutations affecting the NMD or RNA exosome machineries cause chronic activation of defence programmes, resulting in autoimmune phenotypes. Here we place these observations in the context of other links between innate antiviral immunity and type I interferon mediated disease and examine two models: one in which expression or function of pathogen sensors is perturbed and one wherein host-derived RNA molecules with a propensity to activate such sensors accumulate. The flow of genetic information from DNA to RNA to protein is a highly regulated process. This not only allows for gene products to be produced in appropriate amounts, at the right time points and in the correct locales, but also provides quality control. In many instances, post-transcriptional control of mRNA contributes to this regulation. In eukaryotic cells, post-transcriptional events can be divided into those that occur in the cell nucleus and those that take place in the cytoplasm. The former include splicing, capping, and polyadenylation of primary transcripts as well as nuclear export of processed mRNAs. Once in the cytoplasm, mRNAs may localise to specific areas within the cell, are translated and eventually degraded. mRNA translation and half-life vary greatly between transcripts and are controlled by a variety of mechanisms such as the miRNA pathway.Other events in the cytoplasm, rather than controlling the amount of protein product produced from an mRNA, survey mRNA integrity and eliminate faulty transcripts. A well-studied example is the nonsense-mediated decay NMD, see  pathway.NMD is a highly conserved mRNA surveillance pathway and has been reviewed in detail elsewhere How does NMD recognise faulty mRNAs bearing a PTC? NMD depends on protein translation and is triggered by ribosomes terminating translation in an ‘unusual’ position along the mRNA . Such unInterestingly, NMD not only rids the cell of faulty, PTC-containing mRNAs but also regulates expression levels of a number of normal cellular transcripts. Indeed, around 10% of the transcriptome is regulated by NMD Many viruses produce RNAs with atypical features and this is often a result of their compact genome structures. For example, some viruses encode multiple proteins within one polycistronic mRNA molecule et al. further showed that silencing of UPF1 stabilises the SFV genomic RNA, although the molecular features that allow the NMD machinery to recognise viral RNA remain to be determined Arabidopsis thaliana and found that mutations in the gene encoding UPF1 facilitate infection with Potato virus X (PVX) A. thaliana to PVX infection and also enhances Turnip crinkle virus infection of Nicotiana clevelandiiA number of recent studies have validated these predictions. Screening for new virus restriction factors, the Helenius and Voinnet groups identified UPF1 in mammalian cells and plants, respectively et al. found that depletion of UPF1 did not enhance infection of cells with respiratory syncytial virus or Uukuniemi virus, both of which have a negative-sense RNA genome in vivo role in limiting virus replication in mammalian hosts will be important to investigate. A recent study using hepatitis C virus (HCV) found that the viral core protein binds the EJC component WIBG  Taken together, these two studies using viruses with single-stranded, positive-sense RNA genomes show that NMD can detect and degrade some viral RNAs. NMD therefore constitutes a defence mechanism against infection with these viruses. It is interesting to ask if NMD might also have antiviral function against other classes of viruses. It is clear that the genomes of DNA viruses, retroviruses and negative-sense RNA viruses cannot be NMD targets because they are not translated. Indeed, Balistreri It is tempting to speculate that other RNA quality control pathways could also contribute to cellular defence against virus invasion. Related to NMD are non-stop decay and no-go decay Another important question is how NMD integrates with other host defence responses. As discussed in further detail in the next section, cells are equipped to sense infection and to couple this to the induction of antiviral effectors. In mammals, many of these effectors are induced by type I interferon (IFN); this, however, does not appear to be the case for NMD factors, which are constitutively expressed and are not classified as interferon stimulated genes (ISGs) Across taxa, inducible cell-autonomous innate immune responses to infections are typically initiated by germline-encoded receptors often called pattern recognition receptors (PRRs). These receptors are activated by a variety of stimuli, including pathogen-associated molecular patterns (PAMPs), which were originally defined as conserved products of microbial biosynthetic pathways that are normally absent from host cells et al. investigated the role of NMD in the inducible innate immune response in plants et al. showed that the phenotype of SMG7-deficient A. thaliana can be rescued by introducing additional defects in the plant's NBS-LRR pathway In a study published back-to-back with those demonstrating an antiviral function of NMD Tlr7 gene in mice, which results in systemic autoimmunity modelling the clinical symptoms seen in the human autoimmune disease systemic lupus erythematosus (SLE) Theoretically, this observation can be explained in at least two ways. Firstly, NMD might downregulate the expression of NBS-LRRs or of proteins acting downstream of NBS-LRRs in this pathway. Absence of NMD would then result in a lower activation threshold and/or spontaneous signalling of the pathway, due to increased expression of its components. In support of this idea, altered cellular levels of PRRs can cause autoimmunity in mammals, as illustrated by duplication of the et al. showed that NMD plays a key role in downregulating mRNA levels of some NBS-LRRs belonging to the TNL subfamily and that this increases thresholds for activation of the plant effector-triggered immunity (ETI) response et al. went on to show that RPS6, a TNL-subfamily NBS-LRR, can mediate autoimmunity in SMG7-deficient A. thalianaThe first of these scenarios provides an explanation for autoimmunity in NMD-deficient plants. Gloggnitzer via the type I interferon receptor to activate transcription of hundreds of ISGs, many of which restrict virus infection Nucleic acids are potent activators of the innate immune response in mammals and numerous specialised nucleic acid-sensing PRRs and their downstream signalling cascades have been identified However, a growing number of human autoinflammatory and autoimmune disorders are linked to type I IFNs, most notably SLE. Collectively termed type I interferonopathies TMEM173 and IFIH1. Both of these cause inflammatory conditions characterised by elevated type I IFN levels  In addition to ADAR1, predicted to be hypomorphic, have been identified in patients with AGS and cause chronic IFN production in vitro, generally having a greater effect on the editing activity of the ADAR1-p150 isoform in vivo, it is tempting to speculate that non-edited RNAs accumulate in cells when ADAR1 function is impaired and that these RNAs in turn chronically activate PRRs. In line with this idea, cultured ADAR1-deficient cells show evidence of spontaneous type I IFN production and respond more strongly to RIG-I activation Adar1 knockout mouse embryos fail to induce type I IFN if they also lack Mavs, the signalling adaptor for RIG-I and MDA5 ADAR1 belongs to the protein family of adenosine deaminases acting on RNA (ADARs) that bind to double stranded RNA (dsRNA) and then convert adenosine to inosine Finally, we would like to highlight another situation in which accumulation of aberrant cellular RNAs results in chronic type I IFN production and disease. The RNA exosome is a multi-protein complex that degrades RNA in a 3′-to-5′ direction and plays a key role in RNA processing and surveillance pathways such as NMD et al. found evidence that patients with loss-of-function mutations in the SKIV2L gene express elevated levels of type I IFNs in vitro type I IFN response to exogenous RNAs known to activate RIG-I and MDA5 et al. show that type I IFN induction in SKIV2L-depleted cells during ER stress is MAVS-dependent In studying a rare disease called trichohepatoenteric syndrome (THES), Eckard SKIV2L have previously been linked with susceptibility to SLE Interestingly, polymorphisms in et al. found that bacterial infection or exposure to PAMPs reduces NMD efficiency and stabilises mRNAs targeted by NMD, including some TNL transcripts et al. describe that endogenous NMD targets are stabilised in virus-infected plants RNA decay pathways are increasingly recognised not only to control the quantity and quality of proteins produced by cells but also to contribute to host defence against infectious microorganisms, particularly viruses. We have illustrated this concept using the example of NMD. Indeed, in addition to its role in mRNA surveillance, NMD has recently been identified as a novel mechanism for cell-intrinsic virus control IFIH1 and ADAR1. Both of these genes are linked to AGS and encode proteins that either recognise and/or modify RNA. It is therefore becoming increasingly evident that the aetiology of AGS, and perhaps other diseases associated with aberrant type I IFN responses is, at least in some cases, intimately linked to RNA biology and RNA sensing pathways.Aicardi-Goutières syndrome (AGS): a genetically heterogeneous autoimmune disease predominantly affecting the brain and skin, which is characterised by the inappropriate activation of a type I IFN-mediated immune response, and which closely mimics congenital viral infection.Autoimmunity: aberrant immune responses of an organism against its own cells and tissues. In the context of human disease, the term is typically reserved for conditions linked to adaptive B and T cell responses Effector-triggered immunity (ETI): a plant immune response mediated by intracellular receptors, which is activated by the detection of pathogen effector molecules.Nonsense mediated decay (NMD): a highly conserved mRNA surveillance pathway, which targets aberrant transcripts for degradation.Non-stop and no-go decay: surveillance pathways that identify and degrade mRNAs, which lack a stop codon or are bound by stalled ribosomes, respectively. Thus far, these pathways have been primarily characterised in yeast.Pathogen-associated molecular patterns (PAMPs): conserved components of microorganisms that are not normally found in host cells.Pattern recognition receptors (PRRs): germline-encoded receptors of the innate immune system, which recognise PAMPs to trigger immune responses.PAMP-triggered immunity (PTI): the first line of defence against pathogens used by plants, triggered by the recognition of PAMPs by PRRs present on the cell surface.RNA editing: a process by which the nucleotide sequence of an RNA molecule is changed after its transcription, for example by deamination of adenosine to inosine.RNA exosome: a protein complex that degrades RNA in 3′-to-5′ direction and consists of a barrel-shaped catalytic core and accessory proteins that recruit RNA substrates.Systemic lupus erythematosus (SLE): a multisystem autoimmune disorder characterised by the presence of autoantibodies against nucleic acids and type I IFN production.Type I interferons (IFNs): a family of secreted proteins (including IFN-β and multiple subtypes of IFN-α) that function as cytokines and signal via the type I IFN receptor to induce the expression of hundreds of interferon-stimulated genes (ISGs), many of which encode antiviral proteins.NMD deficiency in plants leads to an autoimmune phenotype attributable to increased PRR expression that results in aberrant induction of innate immune responses


MS is the most prevalent autoimmune disorder of the central nervous system, with neurological symptoms caused by inflammation and demyelination. In this study, a lipidomic analysis was performed for the rapid profile of CD4A matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry based approach was used for the analysis of lipid extracts using 9-aminoacridine as matrix. Lipids were analyzed in negative mode and selected species fragmented using MALDI tandem mass spectrometry for their structural assignments.+ T lymphocytes with respect to healthy controls with a significant increase of cardiolipin species in MS samples.The analysis reveals some modifications in the phospholipid pattern of MS CD4+ lipid extracts and suggest how alterations in the lipid metabolism characterized lymphocytes of MS patients.These results demonstrate the feasibility of a MALDI-TOF approach for the analysis of CD4 Alteratogenesis , as thesogenesis . Lymphoc+ T cells highlights a characteristic phospholipid pattern in MS and HC patients  [2 in the serum and cerebrospinal fluid of patients affected by amyotrophic lateral sclerosis was observed [+ T cells revealed an increase in PUFA of the n-6 series as well as in the unsaturation index (UI) in MS with respect to HC. These data well correlate with the results of MALDI-TOF.It is well known that in most mammalian tissues cardiolipin acyl composition is predominantly comprised of 18-carbon unsaturated acyl chains, the vast majority of which is linoleic acid  . In our :2, n-6)  enzymes observed . Moreoveobserved . Our gasMany studies have associated the progression of pathological conditions with changes in the fatty acyl moieties of cardiolipin . Shiftin+ T cells from MS patients. The small number of patients represents a weakness of this preliminary study and may limit a rapid clinical application of our results. Further studies enrolling a larger cohort of patients are needed to confirm our finding and to propose a functional link about the possible role of mitochondria on the regulation of lymphocyte lipid metabolism in MS. In this context, the feasibility of MALDI-TOF/TOF in the rapid screening and characterization of lipid samples makes these results useful for potential scale-up to larger patient populations.Taken together, our data obtained by a lipidomic approach can suggest an altered mitochondrial lipid metabolism in CD4