Ciona intestinalis AOX to Drosophila was able to overcome the lethality produced by toxins or partial knockdown of complex I or IV. Here, we show that AOX can provide a complete or substantial rescue of a range of phenotypes induced by global or tissue-specific knockdown of different cIV subunits, including integral subunits required for catalysis, as well as peripheral subunits required for multimerization and assembly. AOX was also able to overcome the pupal lethality produced by muscle-specific knockdown of subunit CoVb, although the rescued flies were short lived and had a motility defect. cIV knockdown in neurons was not lethal during development but produced a rapidly progressing locomotor and seizure-sensitivity phenotype, which was substantially alleviated by AOX. Expression of Ndi1 exacerbated the neuronal phenotype produced by cIV knockdown. Ndi1 expressed in place of essential cI subunits produced a distinct residual phenotype of delayed development, bang sensitivity and male sterility. These findings confirm the potential utility of alternative respiratory chain enzymes as tools to combat mitochondrial disease, while indicating important limitations thereof.Mitochondrial dysfunction is a significant factor in human disease, ranging from systemic disorders of childhood to cardiomyopathy, ischaemia and neurodegeneration. Cytochrome oxidase, the terminal enzyme of the mitochondrial respiratory chain, is a frequent target. Lower eukaryotes possess alternative respiratory-chain enzymes that provide non-proton-translocating bypasses for respiratory complexes I  or III + IV [alternative oxidase (AOX)], under conditions of respiratory stress or overload. In previous studies, it was shown that transfer of yeast Ndi1 or  Mitochondrial diseases affecting the respiratory complexes of the oxidative phosphorylation (OXPHOS) system are a diverse collection of pathologies, which can affect almost any tissue, at any age ,2. They Km for its substrate, ubiquinol, than does cIII. In contrast, Ndi1 may be constitutively active, but its role in electron flow under normal physiological conditions may be limited by the tight coupling between cI and cIII in supercomplexes.In order to understand better the pathophysiological mechanisms of mitochondrial disease, and develop a possible strategy for eventual therapy, we have exploited the fact that lower eukaryotes, including plants and many invertebrates, possess an alternative, non-proton-pumping respiratory chain in mitochondria, whose components can act as a bypass of the OXPHOS system under conditions of respiratory stress or overload ,4. AlterDrosophila  is also involved in this process. The other nuclear-coded subunits (Cox6a and Cox7a) are then incorporated to form the fully functional cIV, which also contains a recently described subunit  deficiency in humans has a wide variety of pathological manifestations and a diversity of genetic causes  it is bypassing. Because AOX bypasses two of the three proton-translocating steps, it should not restore ATP production for those substrates whose oxidation supplies electrons directly to ubiquinone via cII or other dehydrogenases. Even if AOX can restore redox balance, mitigate mitochondrial reactive oxygen species (ROS) production –9 and liDrosophila offers a convenient model for human diseases, including those affecting mitochondrial functions. The composition of the OXPHOS complexes, the overall structure and gene content of mtDNA and the energetic physiology of the main organ systems are all similar to those of humans. The fact that AOX expression in the fly is able to confer cyanide resistance and overcome the lethality of partial knockdown of Cox6c or Surf1 raises the question of how far it is able to replace cIV throughout the life cycle. We thus set out to use the fly as a model system to address this issue, focusing on three specific questions relevant to understanding the pathophysiology of mitochondrial disease and the possible future use of AOX in therapy. First, what phenotypes result from deficiencies in different classes of cIV subunits, and can AOX alleviate these phenotypes? Secondly, in which tissues is loss of COX function crucial for producing such phenotypes, and their rescue by AOX? Thirdly, what residual phenotypes result from replacing cIV function with AOX? The many genetic tools available in Drosophila (including effective and specific RNAi in vivo) make it an ideal tool for such a study, in advance of translating the most prominent findings to the mouse, which is less flexible, and also takes much longer and is substantially more expensive to manipulate.In a first step, to validate use of the inducible (upstream activating sequence) UAS-AOX transgenic lines that we created previously, we first conducted an important control to verify that the rescue seen previously was not subject to any confounding, ‘promoter-dilution’ effects from the simultaneous use of transgenes dependent on the same transcription factor. We then proceeded to test the phenotypes generated by knockdown of different cIV subunits in the whole fly and in specific tissues, and the ability of AOX to rescue these phenotypes, focusing on the major differentiated cell-types affected by human mitochondrial disease (muscle and neurons).The results highlight the different phenotypes produced by deficiencies of different subunits, indicate a crucial developmental role for cIV in muscle and confirm COX deficiency as a cause of adult-onset neurodegeneration. AOX expression was able to partially rescue these phenotypes. In contrast, loss of proton-pumping at cI promotes a distinct phenotype of developmental delay, bang sensitivity and male infertility.cyclope gene: see Table tub-AOX), with independent single insertions into non-coding DNA on chromosomes X, 2 and 3 . Expression of these transgenes at the RNA level was 20–50-fold lower than from UAS-AOX driven by da-GAL4  system, which partially rescued the lethality of cyanide treatment or the disruption of COX by knockdown of the Cox6c subunit , and single tub-AOX insertions were able to confer almost as great a time of resistance to cyanide as UAS-AOX driven by da-GAL4 , not only resulted in lethality of about three-quarters of the progeny , which also produced a decrease of about 50% in the enzymatically measured COX activity . Flies rescued from the lethality of Cox5a or Cox5b knockdown by two copies of tub-AOX eclosed with a slight, but statistically non-significant developmental delay . The rescued flies were fertile when mated to wild-type flies of the opposite sex.Knockdown of subunits Cox5a or Cox5b also produced an RU486 dose-dependent lethality Fig. E and F. m) of RU486, failed to develop beyond the larval stage, but four copies of tub-AOX enabled the flies to reach pupal stage, with 12% of the pupae eclosing. The flies were tested for bang sensitivity, a sensorineural phenotype associated with mitochondrial dysfunction, but no bang sensitivity was found .Flies in which Cox5b was knocked down during development by a higher dose  expressed ubiquitously, and subunit Cox7a, also encoded by testis-specific (CG18193) and ubiquitous (CG9603) isogenes, plus an additional, widely expressed isogene (CG34172), prominently expressed in muscle .The nuclear-coded subunits of cIV are believed to confer various regulatory properties on COX, as well as being required for its proper assembly and stability. Deficiency of specific subunits leads to the accumulation of various assembly intermediates and to subtly different phenotypes. Most nuclear-coded subunits of COX in es Table . Two notda-GAL4 to drive simultaneously the high-level expression of UAS-AOX and the knockdown of nuclear-coded COX subunits in the whole developing fly, we delineated the limits of AOX rescue of COX deficiency. Knockdown of the somatic Cox4 isogene produced early larval lethality . Blue native polyacrylamide gel electrophoresis (BNE) in-gel histochemistry confirmed the functional knockdown (≥50% decrease of COX activity) at larval stage L3 in the ‘rescued’ flies . Knockdown of Cox5b produced a similar larval-lethal phenotype that was also partially rescued by UAS-AOX, allowing flies to develop to pupal stage, with ∼5–10% of pupae eclosing as viable flies .Expression of UAS-Ndi1 as a control produced no rescue of Cox4 knockdown . Knockdown of the major isogene for Cox7a (CG9603) also resulted in lethality, but in contrast to the early larval lethality produced by knockdown of core subunits, Cox7a knockdown produced lethality only at the pupal stage . No lethality was produced by Cox5b knockdown using any of five nervous system-specific drivers, two of which are active in all neurons , nor were the progeny flies bang-sensitive . Lethality was also not produced with driver BG57 . However, knockdown using driver G14, which expresses mainly in the thoracic muscles , produced lethality . Co-expression of AOX in the same tissues via UAS-AOX rescued the lethality of Cox5b knockdown by the G14 driver . Flies rescued by UAS-AOX from the muscle-specific lethality of Cox5b knockdown nevertheless manifested a severe locomotor defect  in which COX expression is critical for the completion of development, we employed a set of tissue-specific drivers to knockdown either of the enzymatically crucial subunits Cox5b Fig. A or Cox6Supplementary Material, Fig. S4D). Next, we tested a second elav-GAL4 driver located on chromosome X (Bloomington strain 458). Different crosses were implemented, so as to study females heterozygous for the driver, plus males hemizygous for the driver, or control males lacking the driver completely , as well as any rescue produced by UAS-AOX (or UAS-Ndi1). In general, elav-GAL4 driver strain 458 produced stronger effects than driver strain 8760, and stronger phenotypes for knockdown of Cox4, which was now almost completely lethal , than for Cox5b. Males were also more affected than heterozygous females. Expression of AOX, but not Ndi1, improved all phenotypes. Viable Cox5b-knockdown flies were transferred to fresh vials and their survival tracked over 2 weeks , which gave a similar result  the outcome of similar survival experiments was much milder. Cox4 knockdown again gave a stronger phenotype than that of Cox5b, and was again stronger in males than females. After 2 weeks, some of the flies with Cox4 knocked down by the nrv2-GAL4 driver had died (though this varied between experiments), whereas Cox5b knockdown using the nrv2-driver did not impair survival over 2 weeks at all. Cox4 knockdown by the autosomal elav-GAL4 driver  and by immunocytochemistry and histochemistry . These experiments revealed that expression driven by nrv2-GAL4 driver was indeed much weaker than that driven by elav-GAL4, while the latter gave also a very weak expression in thoracic muscles at pupal stage . Serial sections of the brain and thoracic muscle revealed no gross anatomical defects from Cox4 knockdown using even the strongest elav-GAL4 driver .To account for the inconsistency between the effects of knockdown using different neuron-specific drivers we re-examined the tissue-specificity of these drivers, using UAS-GFP (Stinger or mCD8) as a reporter , which includes pronounced developmental delay, bang sensitivity at room temperature immediately upon eclosion, and male sterility.Neuronal depletion of Cox7a driven by ion Fig. A and seiion Fig. B, both oion Fig. A and B, ion Fig. C, but AOion Fig. C and seiion Fig. D. Howevetub-AOX . Expression of UAS-Ndi1 in place of UAS-AOX did not alleviate the seizure sensitivity of 1levy flies , instead mildly exacerbated it, similar to the observation that Ndi1 expression enhanced the degenerative effects of Cox5b knockdown by elav-GAL4 , we also tested the effects of Cox6a knockdown by RNAi. Ubiquitous knockdown was semi-lethal to females at pupal stage , but the resulting flies were still weak. Knockdown of Cox6a using the muscle-specific driver G14 did not produce the uninflated wings phenotype , whereas elav-GAL4 did so (223 of 237 flies). Co-expression of AOX rescued this phenotype, with only 9 of 129 flies failing to inflate wings at eclosion . The Cox6a knockdown phenotype thus appears to be neuronal and again partially rescued by AOX.Although on of Coxa, we alson of Coxa, we als1levy splice-site mutation in Cox6a was reflected in a more severe biochemical phenotype as detected by BNE in-gel histochemistry  or the cIV assembly factor Surf1, to approximately the same extent as when AOX expression was driven by ubiquitously expressed GAL4 . Although tub-AOX expression of was 3- to 5-fold less in adult females than males , produces early developmental lethality which AOX is unable to compensate , is larval lethal in the absence of AOX, and very close to the amount of knockdown that blocks the completion of metamorphosis even in the presence of AOX . Note that many more flies completed development when Cox5b knockdown was brought about by GeneSwitch, with AOX supplied from just two copies of tub-AOX . This may be explained by the fact that the GeneSwitch driver is dependent on the supply of the inducing drug, so that during metamorphosis, when the flies are not eating, but the drug previously ingested can be metabolized, GeneSwitch driven-RNAi decays.The complete absence of COX activity, e.g. via a null mutation for Cox6c  larval stage, although the abnormal larvae survived a further 2 weeks without further growth. Similar phenotypes have been seen for other manipulations affecting mitochondrial OXPHOS, such as knockdown of mitochondrial transcription factor mtTFB2 , indicating the latter as the critical target. The lethality of Cox5b knockdown driven by G14 was fully rescued by co-expression of AOX, although when knockdown was driven by da-GAL4 the result was still almost complete pupal lethality, suggesting that some COX activity in tissues other than muscle is needed for the completion of development. Moreover, while the developmental block caused by muscle-specific knockdown of Cox5b was rescued by AOX, the eclosed flies were very weak. AOX can thus complement COX deficiency sufficiently to enable muscle development but can only do so incompletely for muscle function, indicating that at least some residual proton-pumping activity at cIII/cIV is essential for full activity of the tissue. This may mirror the prominence of muscle involvement in those human mitochondrial diseases where the primary defect causes only a decrease in the activity of cIV . Thus, the degenerative phenotypes produced by Cox4 knockdown range from semi-lethality with the stronger, X-chromosomal elav-GAL4 driver (strain 458), to no detectable impairment within the first 2 weeks of adult life, for nrv2-GAL4. Autosomal elav-GAL4 (strain 8760), which drives GFP expression less strongly in the adult and pupa , gave an intermediate phenotype. The stronger elav-GAL4-driven phenotype in males is an expected consequence of dosage compensation of an X chromosome-derived promoter. In addition, based on GFP  and AOX expression , as well as cIV knockdown as judged by immunocytochemistry  and histochemical staining , the elav-GAL4 drivers are weakly active also in developing thoracic muscle from the pupal stage. This complicates the interpretation of the organismal phenotypes produced using these drivers. The weakness, inactivity and locomotor dysfunction produced by elav-GAL4-driven Cox4, Cox5b or Cox6a knockdown are similar to the effects produced by muscle-specific driver G14, though vary in degree. They appear to constitute a stable, developmentally determined phenotype distinct from the progressive neurodegeneration exhibited by the Cox6a mutant 1levy, which also resembles the other, more progressive effects of cIV knockdown produced by the elav-GAL4 drivers.The different phenotypes produced by cIV knockdown using pan-neuronal drivers partly reflect their different strengths, as revealed by GFP expression . Importantly, the degree of rescue of the 1levy phenotype was the same , and also worsened the degenerative effects of Cox4 or Cox5b knockdown produced by the elav-GAL4 drivers. Unlike AOX, expressed Ndi1 appears to be constitutively active  developmental delay. In contrast, Ndi1-rescued cI-knockdown flies show a pronounced (3–5 days) developmental delay, bang sensitivity detectable at room temperature immediately after eclosion, and male sterility associated with greatly decreased amounts of sperm, which are also immotile. However, the Ndi1 rescued flies are normally active, indicating unimpaired muscle function, and do not show short lifespan or other signs of neurodegeneration.Drosophila flight muscle, ATP production depends on a combination of glycolysis and α-glycerophosphate oxidation in mitochondria, which donates directly electrons to cIII via CoQ, using an flavin adenine dinucleotide-linked enzyme GPD2  locus  locus ,64, or de) locus . Since Ne) locus .Drosophila  and GAL4 driver lines were obtained from stock centres. G14-GAL4 (tubulin-GeneSwitch (tub-GS) driver  driver  from Dr ine 79.1 , levy1 mt strain , UAS-AOXd AOXF24  were desd CG3683  were froCiona intestinalis AOX under the control of the Drosophila αTub84B promoter were created, using, a modified pGREEN-H-Pelican vector  as previously described  and data analysis were performed as described for Surf1 mRNA in Ref. .Imaging of the head and thorax was carried out by haematoxylin and eosin staining of paraffin sections. For immunocytochemistry, heat- and Triton X-100-treated paraffin sections were incubated with primary antibodies against COXIV , ATP5A  or AOX , with appropriate secondary antibodies (Invitrogen). Slides were imaged by spinning disc confocal microscopy. Staining for COX and SDH activity was carried out on cryosections, essentially as described previously described . P.R. received support from AMMi , from Action Rémy and ANR projects MitOxy and AifInter. Funding to pay the Open Access publication charges for this article was provided by University of Tampere.This work was supported by funding from the


E. coli chemotaxis network after genetically perturbing the information flux between upstream and downstream signaling components. We give strong evidence that this signaling system results in dynamic invariance of the activated response regulator against multiplicative intracellular noise. We further demonstrate that for environmental conditions, for which precision in chemosensing is crucial, the invariant response behavior results in highest chemotactic efficiency. Our results resolve several puzzling features of the chemotaxis pathway that are widely conserved across prokaryotes but so far could not be attributed any functional role.Cellular signaling systems show astonishing precision in their response to external stimuli despite strong fluctuations in the molecular components that determine pathway activity. To control the effects of noise on signaling most efficiently, living cells employ compensatory mechanisms that reach from simple negative feedback loops to robustly designed signaling architectures. Here, we report on a novel control mechanism that allows living cells to keep precision in their signaling characteristics – stationary pathway output, response amplitude, and relaxation time – in the presence of strong intracellular perturbations. The concept relies on the surprising fact that for systems showing perfect adaptation an exponential signal amplification at the receptor level suffices to eliminate slowly varying multiplicative noise. To show this mechanism at work in living systems, we quantified the response dynamics of the  Information processing in living cells is limited by a complex balance between randomizing and correcting intracellular forces E. coliS. cerevisiae. For these and most other cellular signaling systems there exists a strict time scale separation between rapid signal transduction from sensory molecules to the pathway output and comparatively slow changes of the dominant noise sources. Examples of the latter are stochasticity in synthesis and degradation of pathway components, assembly of large protein complexes, and changes in availability of cellular resources, such as ribosomes and RNA polymerases. These noise sources are in general multiplicative, show large standard deviations, and therefore do not allow description by linear noise approximation However, most of the previous work focused on the consequences of cellular noise on the stationary pathway output The flow chart of ction, ,  and a lisymmetry property of dynamical systems that show exact adaptation. Here, symmetry refers to invariance of the dynamic behavior under a continuous change of at least one system parameter. To illustrate this point we introduce a generic two-variable adaptation system,An unexpected change in the response dynamics occurs if the gain function satisfies the differential equation et al., the associated dynamically invariant equations can be easily identified  and (6) by choosing appropriate functions The functions entified . Our appsymmetry property to eliminate slowly varying perturbations from biochemical circuits. Returning to the generic signaling network, Eq. (4), we first ask whether the condition The question arises, whether living systems make use of the aforementioned E. coli chemotaxis pathway  provides a good representation of the adaptation dynamics in E. coli chemotaxis, we measured in vivo the activity of the chemoreceptor associated kinase CheA in response to a step increase in chemoattractant while varying the levels of CheB and CheR , it is not yet clear whether slowly varying multiplicative noise has any detrimental effect on chemotaxis efficiency. At this stage it cannot be decided whether the two different response behaviors shown in Although we found strong evidence that chemosensing in E. coli. Recent experiments in this organism showed that chemoreceptor clusters assemble spontaneously and subsequently grow or shrink on time scales of minutes E. coli a The concept of noise compensation by exponential gain is designed to eliminate effects of slowly varying multiplicative noise on the output of a dynamical system that shows precise adaptation of the output signal. The concept requires a strict time scale separation between slow noise dynamics and fast relaxation of the output signal. In living systems, this concept can be realized by utilizing the natural time scale separation of cellular signaling: almost instantaneous conformation changes at the receptor level, fast signal transduction by phosphorylation of cytosolic proteins, and fast signal termination by feedback control in comparison to slow concentration changes of pathway components. It thereby provides a resource efficient alternative to noise reduction by increasing the abundance of signaling components and it is therefore not surprising that this concept has been likely realized in the highly sensitive chemotaxis pathway of E. coli but could not be assigned a functional role. First, CheB phosphorylation as an indirect measure for the ratio of kinase and phosphatase activity is essential for this mechanism to work and would explain why CheB phosphorylation is found in almost all chemotactic prokaryotes The realization of noise compensation by exponential gain requires special pathway properties that have been observed in E. coli chemotaxis system is just one indication of this selection process, that most likely co-evolved with similar outstanding noise compensatory mechanisms, as presented in this work.Given the long evolutionary time span since radiation of the chemotaxis signaling system among prokaryotes, it can be expected that almost all evolutionary accessible mechanisms to increase the signal-to-noise ratio have been systematically scanned, and selected for their performance and resource efficiency. The outstanding signaling gain of order Escherichia coli K-12 strains VS100 [cheY], VS104 [cheY cheZ)], VS124 [cheB cheY cheZ)], VS127 [cheRcheY cheZ)] and VS149 [cheR cheB cheY cheZ)] used in this work were described before flgMcheY cheZ)] and LL5 [flgMcheR cheB cheY cheZ)] were constructed by the in-frame deletion of flgM in VS104 and VS149, respectively E. coli cells were grown under standard chemotaxis conditions  in a rotary shaker at 34°C to mid-exponential phase Click here for additional data file.


The aim of the present study was to test the validity of the transthoracic electrical bioimpedance method PhysioFlow® to measure stroke volume in patients with chronic anemia. Stroke volume index (SVI), as well as cardiac index (CI) obtained by transthoracic electrical bioimpedance method and doppler echocardiography were compared in healthy subjects (n = 25) and patients with chronic anemia , at rest. While doppler echocardiography was able to detect difference in SVI between the two populations, the Physioflow® failed to detect any difference. Bland & Altman analyses have demonstrated no interchangeability between the two methods to assess CI and SVI in anemic patients and healthy subjects. While doppler echocardiography displayed a good concordance for SVI results with those obtained in the literature for anemic patients, the Physioflow® did not. Finally, in contrast to doppler echocardiography: 1) the CI obtained with the Physioflow® was not correlated with the hemoglobin level and 2) the stroke volume determined by the Physioflow® was highly influenced by body surface area. In conclusion, our findings indicate that the Physioflow® device is inaccurate for the measurement of SVI and CI in patients with chronic anemia and has a poor accuracy for the measurement of these parameters in African healthy subjects. The use of alternate non-invasive techniques such as transthoracic electrical bioimpedance methods (PhysioFlow®) are thus of great interests in developing countries because of the limited economic cost and the facility of use. A growing interest has been devoted to the PhysioFlow® within the past decades. However, very few studies performed on small number of sportsmen or patients with lung diseases have suggested that the PhysioFlow® could provide a clinically acceptable and non-invasive evaluation of the cardiac output, at rest and during exercise, compared to the direct Fick, CO methods –4. In co methods , in anes methods , under c methods  and in p methods . Indeed,Chronic anemic disorders are frequent in developing areas, such as in Africa. Patients with sickle cell anemia (SCA) and other chronic anemic disorders have increased cardiac output (Qc) –11 resulThe aim of the present study was therefore to test the validity of the PhysioFlow® to measure the stroke volume in patients with chronic anemia. Stroke volumes obtained by transthoracic electrical bioimpedance method were compared to those acquired with doppler echocardiography in healthy subjects and patients with chronic anemia (mainly SCA). The study took place at the Academic Hospital of Yopougon , and included 32 adults with chronic anemia  and 25 healthy adults. Subjects with cardiovascular or pulmonary diseases, or smoking habits were excluded from the study. All patients were in steady-state condition at the time of the study: no blood transfusions in the previous three months and absence of acute episodes  at least two months before inclusion into the study. The study was conducted in accordance with the guidelines set by the Declaration of Helsinki and was approved by the Ethics Committee of the Academic Hospital of Yopougon . All patients were advised about the purpose and procedures of the study, and gave their informed written consent. Stroke volume (SV) was determined for each subject simultaneously by transthoracic electrical bioimpedancemeter  and doppler echocardiography , which was considered as the gold standard, after 30 minutes of rest in supine position. Since no specific position is recommended for the hemodynamics measurement by the PhysioFlow®, the position adopted by the subject was the one recommended for doppler echocardiographic analyses: left lateral decubitus with left hand placed under the head and chest free. th and 20th minute (10 seconds recording) were averaged. When the two measurements were not reproducible (difference > 5%), a third measure was done at the 25th min and averaged with the two preceding values. The Physioflow designers did not reveal the exact formula for hemodynamical measurements and SV calculations, and only little information is available in a previous study [For the Physioflow®, after gently skin scraping and as recommended by the manufacturer’s instructions, six electrodes (Skintact FS-50) were used: 2 on the neck, 2 at the xiphisternum and 1 on each side of the chest. The bioimpedance method uses changes in transthoracic impedance during cardiac ejection to calculate SV . Heart rth and 20th min (and 25th min if needed), and averaged, using echocardiography device with 5 MHz transducer. The stroke volume was calculated by the product of aortic annular plane and velocity time integral of aortic flow, as recommended [Doppler echocardiographic measurements were performed at the 15ommended ,22. The The cardiac index (CI = CO/body surface area) and the stroke volume index (SVI = SV/body surface area) were calculated for each subject and each method.Values are expressed as means ± standard deviation (SD). Significance level was defined as p < 0.05. Analyses were conducted using SPSS .The reproducibility of each method to measure SVI has been studied in 10 healthy subjects during 4 consecutive days. Anthropometric and blood pressures remained unchanged during this period. The coefficient of variation (CV) of each method was calculated from the average of the CV determined in the 10 healthy subjects over the 4 experimental days. A two-way analysis of variance (ANOVA) with repeated measures was used to compare the values of SVI between the two methods over the 4-days period. An unpaired t test was used to compare the mean CV between the two methods. The hemodynamic measurements were compared between the two groups, and between the two methods, using an unpaired Student t test. A Pearson test was used to assess the correlation between the CI and SVI values obtained by the two methods. The interchangeability between the two methods (CI and SVI parameters) was evaluated by the Bland-Altman analysis . To test2; 1.60 m2; 1.70 m2; 1.80 m2; 1.90 m2; 2.00 m2; 2.10 m2 and 2.20 m2. These values lead to normal body mass indices between 22 and 25 Kg/m2 and close to the real one of the tested subject. The values were randomly assigned and entered into the software of both devices and the measurements of SV and SVI were realized.Pearson test was performed between the hemoglobin level and the values of CI or SVI obtained with each method. Finally, the impact of fictitious changes in body surface area (BSA) on SV measurement was assessed for the two methods in one representative subject. The fictitious values of height and weight were entered manually into the softwares of the PhysioFlow® and echocardiography device before measurement, to obtain, according to the Dubois’s formula (CATO Software Solutions), fictitious body surface areas (BSA) of 1.50 mA Student ‘t’ test was used to compare the CI and SVI values from the present study with the values obtained in previous studies on healthy subjects and on chronic anemic disorders. The studies selected for comparisons were those where the values  were available.The characteristics of the subjects are reported in the The mean reproducibility of SVI over the 4-days period in 10 healthy subjects was not significantly different between doppler echocardiography and PhysioFlow® methods . The meaAs expected, using doppler echocardiography, patients with chronic anemia had greater CI (p < 0.01) and SVI (p < 0.001) than the group of healthy subjects . In contPearson analysis demonstrated no correlation between SVI determined by doppler echocardiography and SVI determined by the PhysioFlow® . MoreoveThe results of the Bland and Altman analysis are displayed in The The The The The The Our study is the first to describe the performance characteristics of an electrical impedance monitor (Physioflow® device) in a group of healthy African and in a group of African patients with chronic anemia (mainly sickle cell anemia). Our main findings demonstrated an inability of the Physioflow® to detect any difference in SVI between the two populations while standard doppler echocardiography was accurate. Moreover, there was no interchangeability according to the Bland & Altman and correlations analyses between the Physioflow® device and standard doppler echocardiography for the measurements of CI and SVI in anemic patients and healthy subjects. Whereas the standard doppler echocardiography displayed a good concordance for CI and SVI results with those obtained in the literature in anemic patients, the Physioflow did not. Finally, in contrast to standard doppler echocardiography: 1) the CI measured by the Physioflow was not correlated with the hemoglobin level and 2) the stroke volume determined with the Physioflow was highly correlated with BSA. On the whole, our findings indicate that the Physioflow® device is inaccurate for the measurement of SVI and CI in patients with chronic anemia and has a poor accuracy for the measurement of these parameters in African healthy subjects.The Physioflow® used in this study is one of the “second-generation” devices using bioimpedance technology to determine cardiac hemodynamic parameters. Historically, the clinical use of bioimpedance methods (i.e. “first-generation devices”) to measure cardiac hemodynamic parameters was hampered by baseline instability. Indeed, the development of the Physioflow® has included a modified proprietary algorithm, which avoids calculation of baseline impedance. The basic equation for calculating stroke volume has been modified, avoiding the calculation of blood resistivity, the distance between the recording electrodes and the Z0 used in the original equations from Kubicek and Sramek-Bernstein ,6. The validity and accuracy of the Physioflow® has been recently challenged in pediatrics population with and without cardiac diseases ,8. The aOur results support a lack of accuracy of the Physioflow® device to measure CI and SVI in African patients with chronic anemia. We found a poor agreement with doppler echocardiography and no interchangeability between the two devices. In addition, while the values of SVI and CI obtained with doppler echocardiography in chronic anemic patients are close to the ones published in the literature using various standard methods , large dIn conclusion, the accuracy of Physioflow® using its current algorithms does not support its use for clinical monitoring of cardiac hemodynamic parameters in chronic anemic patients. In addition, caution is required regarding its use in the general population due to the large influence of the anthropometric data in the SV calculation.


Strongyloides stercoralis, and Infective Dermatitis associated with HTLV-1 (IDH), appear to be risk factors for the development of HTLV-1 related diseases. We used high-throughput sequencing to map and quantify the insertion sites of the provirus in order to monitor the clonality of the HTLV-1-infected T-cell population (i.e. the number of distinct clones and abundance of each clone). A newly developed biodiversity estimator called “DivE” was used to estimate the total number of clones in the blood. We found that the major determinant of proviral load in all subjects without leukemia/lymphoma was the total number of HTLV-1-infected clones. Nevertheless, the significantly higher proviral load in patients with strongyloidiasis or IDH was due to an increase in the mean clone abundance, not to an increase in the number of infected clones. These patients appear to be less capable of restricting clone abundance than those with HTLV-1 alone. In patients co-infected with Strongyloides there was an increased degree of oligoclonal expansion and a higher rate of turnover (i.e. appearance and disappearance) of HTLV-1-infected clones. In Strongyloides co-infected patients and those with IDH, proliferation of the most abundant HTLV-1+ T-cell clones is independent of the genomic environment of the provirus, in sharp contrast to patients with HTLV-1 infection alone. This implies that new selection forces are driving oligoclonal proliferation in Strongyloides co-infection and IDH. We conclude that strongyloidiasis and IDH increase the risk of development of HTLV-1-associated diseases by increasing the rate of infection of new clones and the abundance of existing HTLV-1+ clones.Human T-lymphotropic Virus-1 (HTLV-1) is a retrovirus that persists lifelong by driving clonal proliferation of infected T-cells. HTLV-1 causes a neuroinflammatory disease and adult T-cell leukemia/lymphoma. Strongyloidiasis, a gastrointestinal infection by the helminth  Strongyloides stercoralis) and Infective Dermatitis associated with HTLV-1 (IDH), a skin inflammation with bacterial infection, appear to increase the risk of developing HTLV-1-related diseases. It is well known that the chance of developing HTLV-1-related diseases increases with the number of cells infected by the virus . It is also known that HTLV-1-infected individuals co-infected by Strongyloides or affected by IDH have a higher proviral load, but the mechanism is still unclear. Consequently, the aim of this study was to test if co-infection increases the total number and/or the abundance (or size) of HTLV-1-infected T-cell clones. We have shown that the significantly increased proviral load in HTLV-1-infected individuals with IDH or strongyloidiasis is due to an increase in the mean clone abundance (bigger clones), not to an increase in the number of infected clones. These patients appear to be less capable of restricting clone abundance than those with HTLV-1 alone.HTLV-1 is a human retrovirus estimated to infect 20 million people world-wide and is causing in a small proportion of the infected individuals an inflammatory disease or a leukemia/lymphoma. HTLV-1 persists lifelong by driving clonal proliferation of infected T-cells. Strongyloidiasis, a gastrointestinal infection by an helminth ( Human T-lymphotropic virus type 1 (HTLV-1) causes adult T-cell leukemia-lymphoma (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). It has been estimated that 10 to 20 million persons live with HTLV-1 infection worldwide. A small proportion  of HTLV-1-infected individuals develop disease, while the majority remain asymptomatic carriers (ACs). Infection occurs via breastfeeding, transfusion of infected cellular blood products, or sexual intercourse. Symptoms usually appear after a long period (years or decades) of clinical latency Strongyloides stercoralis (St) is estimated to infect 50-100 million individuals, mainly in the tropics and subtropics. Most people with strongyloidiasis have mild diarrhea, vague abdominal complaints, pruritus, perianal rash or simply remain asymptomatic. The Strongyloides stercoralis larvae migrate to a range of sites like the lung, liver, kidney, and central nervous system. The larvae can carry bacteria from the colon and cause fatal sepsis and meningitis. A severe form of the disease named strongyloides hyperinfection syndrome, characterized by abundant disseminated parasites, has been described in patients with corticosteroid therapy, severe malnutrition, transplantation, haematological malignancies  and HTLV-1 infection Strongyloides stercoralis infection, a high risk of the strongyloides hyperinfection syndrome, and with relapse after treatment with ivermectin, thiabendazole, or albendazole Strongyloides stercoralis co-infection had a higher HTLV-1 proviral load and a higher Strongyloides stercoralis burden than patients with either infection alone Strongyloides stercoralis co-infected patients has been linked with oligoclonal expansion of HTLV-1 infected T-cells Strongyloides stercoralis co-infection is suspected to be a risk factor for the development of ATLL, but the mechanism is still unclear The helminth Staphylococcus aureus and/or beta-hemolytic Streptococci are commonly cultured from the skin lesions. The average age at disease onset is 2 years. The disease decreases in severity with age and rarely continues until adulthood Infective dermatitis (IDH) is a severe, chronic, relapsing dermatitis associated with HTLV-1. IDH has been reported in several populations with endemic HTLV-1 infection, including South Africa, Jamaica, Trinidad, Brazil, Colombia, Peru and Japan. + T-cell clones regardless of the genomic environment of the provirus, in contrast to infection with HTLV-1 alone, in which selective clonal expansion is favored by specific features of the proviral integration site in that clone.The aim of this study was to identify and quantify the impact of co-infection on HTLV-1 clonality. Because HTLV-1 proviral load and oligoclonality are closely correlated with disease status, we aimed to test the hypothesis that each co-infection increases the risk of HTLV-1-associated diseases by increasing the number or the abundance of HTLV-1-infected T-cell clones. We used a newly developed method to map and quantify thousands of HTLV-1 proviral insertion sites. We demonstrate that co-infections significantly alter the HTLV-1 clonality. Patients with strongyloidiasis or IDH had a higher proviral load and a higher average clone abundance than did asymptomatic HTLV-1 carriers even though the major determinant of proviral load was still the number of clones. The degree of oligoclonality of HTLV-1 was higher, and less stable over time, in patients with strongyloidiasis than in patients with neither co-infection nor ATLL, and there was a higher rate of turnover (i.e. appearance or disappearance) of HTLV-1-infected clones in the co-infected patients. Finally, we show that co-infections drive the proliferation of HTLV-1Strongyloides was significantly higher than the proviral load in asymptomatic carriers . These observations are in accordance with data from previous reports Strongyloides co-infected patients was comparable to those of asymptomatic carriers (6 PBMCs) was higher in patients with IDH and Strongyloides co-infection than in asymptomatic carriers, but no significant difference was observed between asymptomatic carriers and HAM/TSP patients , 3.6 (IDH patients) and 3.0 (Strongyloides co-infected patients) . The distribution of clone abundance is depicted in Figure S1A in Strongyloides stercoralis are shown to illustrate the wide variation in clonality observed in this group of patients despite similar proviral load. The oligoclonality index of the first patient was low and in the range observed in Asymptomatic Carriers and patients with HAM/TSP. The oligoclonality index of the second patient with Strongyloides was greater because of the presence of 3 relatively abundant clones  due to oligoclonal expansion of infected clones . The similarity between the populations of HTLV-1-infected T-cell clones at two consecutive time points is shown in Strongyloides co-infected patients differed more in identity and abundance than the clone populations from two consecutive blood samples in patients with HTLV-1 only  had a genomic environment of the provirus similar to the random distribution; i.e. the environment of the provirus does not determine the abundance of the major clones in co-infected patients.In patients with HTLV-1 alone, we previously reported Strongyloides co-infection or IDH is independent of the genomic environment of the proviral insertion site.We conclude that the abundance of the largest clones in patients with We quantified HTLV-1 clonality in two types of sample in which the infected T-cells present are believed to play a direct role in the pathogenesis of the respective inflammatory disease: CSF from patients with HAM/TSP and skin lesions from patients with IDH. The rationale was two-fold: first, over-representation of a few infected clones in these tissues may indicate immune activation of antigen-specific T-cells. Second, we wished to test whether the selectively expanded clones in these tissues are also abundant in the bloodstream.Strongyloides co-infection and IDH. By comparison with patients infected with HTLV-1 only, Strongyloidiasis stercoralis co-infected patients showed five main differences: i. a higher mean proviral load, ii. a higher mean clone abundance, iii. a higher mean oligoclonality index, iv. a higher clone turnover rate (i.e. a higher rate of appearance and disappearance of clones), and v. a proliferation of the largest clones independent of the genomic environment of the provirus. IDH patients had also a higher mean proviral load, a higher mean clone abundance and a change in the selection forces that favor expansion of the largest HTLV-1 clones. The median oligoclonality index in the IDH group was higher than that in Strongyloides co-infected patients (respectively 0.464 vs 0.460), but the difference from asymptomatic carriers did not reach statistical significance. We emphasize that the mean age of the IDH cohort (14 years) was the lowest in the study ; the mean age of Strongyloides co-infected patients was 44 years and that of asymptomatic carriers was 55 years. It would be interesting to follow the oligoclonality index over time in these IDH patients. The cohorts of co-infected patients in the present study also differed from the non co-infected individuals by geographical origin and ethnicity. It is possible that variation in host or viral genotype also influences HTLV-1 clonality. However, HTLV-1 genetic variation is limited, and the Cosmopolitan subtype 1a  dominates in the Caribbean, South Africa, Peru and Brazil The data show that HTLV-1 clonality is extensively altered in vivo by Strongyloides co-infected patients suggests a higher rate of persistent infectious spread of the virus, increasing the total number of clones generated during the HTLV-1 lifelong infection . To test this hypothesis, further work is needed to quantify the rate of infectious spread within a given patient.The increased divergence between successive time-points in the HTLV-1 clonal composition observed in Strongyloides co-infected patients had on average more abundant clones (i.e. the mean number of cells per clone was higher). These patients seem to be less capable of restricting clone abundance than those with HTLV-1 alone. Consistent with this conclusion, we observed a higher oligoclonality index in Strongyloides co-infected patients. Our observation of oligoclonal expansion in Strongyloides co-infected patients confirms a previous report by Gabet et al 2000 et al 2002 et alet al also reported + T-cells in a patient with both IDH and Strongyloides co-infection, and the authors concluded that IDH might be a co-factor for ATLL. From our observations, it appears that Strongyloides co-infection could be a co-factor for oligoclonal expansion. However, in the samples from the IDH patients in this cohort we did not observe any case with a degree of oligoclonal proliferation  within the ATLL range; and the oligoclonality index of HTLV-1-infected cells in the skin in IDH was significantly lower than that in the blood (see below).The observation that a major determinant of proviral load (in patients without ATLL) was the number of clones has important implications for the understanding of the development of HTLV-1-associated diseases. Why do some patients carry more clones than others? It is likely that the efficiency of the host's immune response to HTLV-1, especially the quality of the HTLV-1-specific CTLs + T-cells induces HTLV-1 Tax expression, T-cell proliferation, and may culminate in the development of ATLL et alStrongyloides infection, in whom active HTLV-1 transcription resolved with anti-helminthic therapy Strongyloides co-infection can induce HTLV-1 transcription via T-cell immune activation. Nevertheless, little is known about the nature of the hypothetical activating signals induced by the co-infection. We considered two possible explanations, which are not mutually exclusive: i. co-infection induces immune activation of the few infected clones that are specific to the co-infecting pathogen ; ii. co-infection favours expansion of all HTLV-1 infected clones (either by inducing non specific immune activation of the HTLV-1 infected clones or by impairing immune surveillance against the HTLV-1 infected cells). To answer this question we compared the HTLV-1 infected T-cells present in the skin lesion from IDH patients with the infected cells from the corresponding blood sample. As a reference point, we also compared the HTLV-1 infected T-cells present in the CSF in HAM/TSP patients with the infected cells from the corresponding blood sample. We observed that the major clone in the CSF can be rare or undetectable in the bloodstream  antigens, but rather the non-specific proliferation of the entire population of infiltrating T-cells. HTLV-1 infection in endemic regions frequently occurs during breast-feeding and so predates infection with Strongyloides or Streptococcus/Staphylococcus. Consequently, it is unlikely that HTLV-1 infection is biased towards T-cells specific to Strongyloides or Streptococcus. It remains possible that chronic antigen stimulation favours the expansion of such T-cells in co-infected subjects. However, since the degree of oligoclonality of infected T-cells observed in patients with infective dermatitis was lower in the skin lesion than in blood, we infer that antigen specificity was a minor contributor to selective clonal expansion in these co-infected individuals. It remains possible that stimulation by Strongyloides antigens contributes to clonal expansion in individuals with Strongyloides co-infection. Our observation that the abundance of the largest clones in patients with Strongyloides co-infection or IDH is independent of the proviral insertion site environment supports the idea that Strongyloides co-infection and IDH change the selection forces that favour expansion of HTLV-1-infected clones.Based on observations in a mouse model, it has been shown that immune activation of HTLV-1-infected CD4et al+FoxP3+ regulatory T-cells is abnormally high in HTLV-1 patients +FoxP3+ T-cells +FoxP3+ regulatory T-cells is increased further in Strongyloides stercoralis co-infected patients + T-cells observed in Strongyloides co-infection. First, an autocrine IL2/IL-2R loop was reported by Satoh et al+FoxP3+ T-cells may impair the host T-cell response to HTLV-1 infection.HTLV-1 infection causes activation and proliferation of the infected T-cells. The HTLV-1 Tax protein activates the canonical NF-κB pathway  and mitotic spread (proliferation of existing infected T-cell clones) thereby increasing the risk of both inflammatory disease and malignant transformation.In summary, co-infection with Strongyloides stercoralis and confirmed negative post anti-helminth treatment. Seventeen individuals with IDH  donated blood and skin lesion samples. These patients had active disease at time of blood sampling and biopsy. Table S1 in We studied 61 individuals  infected with HTLV-1 alone . All individuals attended the clinic at the National Centre for Human Retrovirology , and donated blood samples. Four HAM/TSP patients  also donated samples of cerebrospinal fluid (CSF). Fourteen individuals  infected with HTLV-1 and affected by strongyloidiasis donated blood samples. These patients had microbiology confirmed stool positive samples for All subjects gave fully informed, written consent and all clinical investigations have been conducted according to the principles expressed in the Declaration of Helsinki. This study was approved by the UK National Research Ethics Service (NRES reference 09/H0606/106).′CGGATACCCAGTCTACGTGT, SK44:5′GAGCCGATAACGCGTCCATCG, ActFw:5′TCACCCACACTGTGCCCATCTATGA, ActRev:5′CATCGGAACCGCTCATTGCCGATAG). Three dilutions of DNA were amplified by real time quantitative PCR in a Roche light cycler using SYBR Green 1 Dye incorporation (Roche Applied Science). Standard curves were generated using the rat cell line TARL2 which contains 1 copy per cell of the HTLV-1 provirus 6 PBMCs.DNA was amplified for HTLV-1 DNA (using the Tax sequence-specific primers SK43 and SK44) and for β-actin (as a measure of genomic DNA using ActFw and ActRev primers) (SK43:5i (number of cells per 106 PBMCs) was calculated from the number of sister cells and the measurement of the proviral load as follow:iX is the number of sister cells of the ith clone, D the number of observed clones and PVL the proviral load.We used a newly developed protocol to map and quantify thousands of HTLV-1 proviral insertion sites, as previously described i  was expressed as follow:The relative abundance of a given clone To measure the clonality of the infected cell population, i.e. the non-uniformity of the clone abundance distribution, we used the oligoclonality index Oligoclonality index = 1 indicates perfect monoclonality , while oligoclonality index = 0 indicates perfect polyclonality .vice versa). The former index was subsequently improved to take into consideration the abundance of each clone and named SØrensen abundance-based similarity index. Because this index takes clone abundance into account, populations that contain the same clones but have different clone abundance will have an index value of less than 1. Details of the calculations are given in supplemental data.Quantitative measures of similarity (or overlap) between two populations play an important role in statistical ecology. The first similarity indices developed were based on the presence or absence of species between the two populations. The widely used SØrensen incidence-based similarity index ranges from 0 to 1, with 0 indicating that no clones are shared between the two populations and 1 indicating complete identity . This web-based tool houses sequences of newly inserted elements in vertebrate genomes, together with specific genomic annotations, to facilitate analysis of the environment of the genomic insertion site: see Figure S3 in We used the Integration Site Pipeline and Database (INSIPID) from the Bushman laboratory  (The diversity estimation approach (DivE)  used to estimate the total number of clones (observed and unobserved) involves fitting many mathematical models to species-accumulation data, and to successively smaller nested subsamples thereof. Novel criteria are used to score models in how consistently they can reproduce existing observations from incomplete data. The estimates from the best performing models are aggregated (using the geometric mean) to estimate the number of clones in the circulation. We have shown that, when applied to HTLV-1 infection and other microbiological populations, DivE significantly outperforms several classical ecological estimators of unseen species . Let 6 PBMCs.PVL is the proviral load and S the estimated total number of clones.The average clone abundance was calculated from the proviral load divided by the estimated total number of clones in the blood and expressed as the number of cells per 10Statistical tests were performed using GraphPad Prism and R softwares and were two tailed when possible. The symbol *** was used when p<0.001, ** when p<0.01, * when p<0.05, NS (Non Significant) when p>0.05.CXCR4, NG_011587.1; CCR5, NG_012637.1; IL-2Rα, NG_007403.1; IFN-γ, NG_015840.1; IL-10, NG_012088.1; TGF-β, NG_013364.1; HTLV-1 Tax, NC_001436.1.Text S1The supporting file (PDF)Click here for additional data file.


The mitogen-activated protein kinase (MAPK) pathway is the canonical signaling pathway for many receptor tyrosine kinases, such as the Epidermal Growth Factor Receptor. Downstream of the receptors, this pathway involves the activation of a kinase cascade that culminates in a transcriptional response and affects processes, such as cell migration and adhesion. In addition, the strength and duration of the upstream signal also influence the mode of the cellular response that is switched on. Thus, the same components can in principle coordinate opposite responses, such as proliferation and differentiation. In recent years, it has become evident that MAPK signaling is regulated and fine-tuned by proteins that can bind to several MAPK signaling proteins simultaneously and, thereby, affect their function. These so-called MAPK scaffolding proteins are, thus, important coordinators of the signaling response in cells. In this review, we summarize the recent advances in the research on MAPK/extracellular signal-regulated kinase (ERK) pathway scaffolders. We will not only review the well-known members of the family, such as kinase suppressor of Ras (KSR), but also put a special focus on the function of the recently identified or less studied scaffolders, such as fibroblast growth factor receptor substrate 2, flotillin-1 and mitogen-activated protein kinase organizer 1. This can be established by a sequential phosphorylation of a three-tier kinase cascade, which conveys the signal to the nucleus, where a cellular response can be transcriptionally mounted. Protein kinases catalyze the transfer of a γ-phosphoryl group from ATP to a hydroxyl-group of a serine, threonine or tyrosine residue in the substrate protein. They are named after their substrate specificity, proteins . Since pproteins .i.e., the extracellular signal-regulated kinase (ERK) family, the p38 kinase family and the c-Jun N-terminal kinase (JNK) family. The MAPK cascade itself is shaped by three major constituents that form the so-called “three-tiers”, MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K) and MAPK (A prime example for such a kinase cascade is the mitogen-activated protein kinase (MAPK) pathway, an evolutionarily highly conserved signaling mode that controls fundamental cellular processes, such as proliferation, cellular survival and differentiation. A deregulation of one component in this cascade can result in aberrant signaling events associated with a pathological outcome, e.g., cancerogenesis. Three protein families are central to this pathway, and MAPK . In addiThree different isoforms of the MAP3K Raf exist, namely C-Raf , A-Raf and B-Raf, which provide the first tier in the MAPK cascade. Although Ras is involved in the canonical Raf activation, it is not essential for MAPK signaling. Some signaling pathways, e.g., protein kinase C (PKC) signaling, which can be activated with phorbol myristyl acetate (PMA), result in Raf activation without the requirement for the preceding activation of Ras ,27. SimiUpon its activation, Raf transmits the signal further downstream to MEK1/2 (MAPK/ERK kinases 1 and 2), which are the second tier in the MAPK cascade. MEK1/2  are activated by Raf-mediated phosphorylation at Ser218 and Ser222 (hMEK1) –32. MEK1et al. suggested that functional differences between ERK1 and ERK2 would depend on their expression levels [et al. used a model system for tumor invasiveness to show that depletion of ERK2 impairs invasive migration of the cells. Strikingly, the invasive motility could only be rescued by ERK2, but not by ERK1, again implicating that the two ERK proteins exhibit functional differences [−/− mice are viable and exhibit relatively minor defects, e.g., in thymocyte differentiation [−/− mice already die at the embryonic stage, E8.5 [There are eight isoforms of the MAPK ERK, ERK1-8. In this review, we focus on ERK1 and ERK2, as their function has been extensively characterized and they represent typical components of the canonical MAPK signaling pathway. ERK1/2 are serine/threonine protein kinases  that pren levels . Von Thuferences . Howeverntiation , ERK2−/−ge, E8.5 . This imThe subcellular localization of inactive and active ERK1/2 reflects their broad functional effects. About 10–20 min after the cells are exposed to a growth factor/mitogen, active ERK1/2 translocate to the nucleus, where they induce gene expression and facilitate cell cycle entry. It is still under debate whether ERK translocates to the nucleus as a homodimer or in its monomeric form –39,56–58ERK1/2 do not only enhance gene transcription, but can also act as transcriptional suppressors by phosphorylating the Ets2 repressor factor (ERF) . Furtheret al. described the actin-binding protein, palladin, as a novel substrate for ERK and suggested an anti-migratory function for ERK [In addition to the manifold substrates of ERK in the nucleus, it also has cytosolic substrates and localizes to other subcellular compartments, such as endosomes, via the MP1-p14 complex, and mitochondria, via voltage-dependent anion channel 1, VDAC1 –88. In t for ERK . Another for ERK . Amongst for ERK ,93.in vitro[In vivo, ERK1/2 phosphorylates MEK1 as part of a negative feedback regulation during integrin and growth factor signaling [The MAPK cascade is a multifaceted pathway that is involved in a plethora of cellular processes. A deregulation of this pathway results in pathologies, for instance, in cancerogenesis. Regarding the substrate specificities within this pathway, ERK and MEK are in great contrast to each other. While ERK1/2 are the only described substrates of MEK1/2 so far, ERK1/2 has a myriad of substrates in the cytosol and the nucleus. Therefore, a constant feedback mediated regulation of this pathway is important. There are two different regulatory mechanisms: a positive feedback mechanism, which results in an increase in ERK signaling, and a negative feedback mechanism by which ERK signaling is limited and returned to a basal level. For example, ERK1/2 is able to phosphorylate MEK1 at Thr292 and Thr386 in vitro,95. In vignaling ,96. Receignaling . Anotherignaling . In geneignaling ). Like Eignaling ,101. On ignaling . Aside fignaling –105.The model postulating a free diffusion of signaling components in the cytosol comes along with several disadvantages . The proOne main characteristic of scaffolds is combinatorial inhibition, an effect first described for the ERK/MAPK scaffold kinase suppressor of Ras (KSR) . CombinaThe first MAPK scaffold described was the yeast Ste5p protein. However, in this review, we will focus on MAPK/ERK scaffolds in mammalian cells, and thus, Ste5p, as well as the scaffolds of the JNK MAPK pathway, will not be discussed. For Ste5p and JNK scaffolds, we refer to some recent reviews on these topics –114. SevDrosophila melanogaster and Caenorhabditis elegans as a modifier of activated Ras, KSR1 was found to be a positive regulator of MAPK signaling and represents one of the best characterized MAPK scaffolders so far [D. melanogaster, only a single ksr gene is present, which is essential for the viability of the organism. On the other hand, in mammals and C. elegans, two proteins, KSR1 and KSR2, are present and display a functional redundancy [D. melanogaster, the loss of KSR1 has little effect in C. elegans[−/− mice are modestly glucose intolerant. Although they show a normal response to exogenous insulin, they display a three-fold increase in serum insulin levels in response to glucose challenge [Initially identified in genetic screens in s so far –123. In dundancy ,124,125.. elegans–123. Sim. elegans,127, sug. elegans,129 and . elegans. Further. elegans and KSR1. elegans. The ksrhallenge , demonsthallenge . All thiDrosophila KSR was shown to bind D-Raf , but thtivation . Finallytivation ,136–138.tivation ,139–142.tivation , is exchtivation . Howevertivation ,146. A K[in vitro. It has th C-Raf . Thus, aDepending on its phosphorylation state, KSR1 is found to be localized at the plasma membrane or in the cytoplasm. As with Raf, KSR is held in the cytoplasm by the bound 14-3-3 protein, but it translocates from the cytoplasm to the plasma membrane upon treatment with growth factors in a process regulated by Cdc25C-associated kinase 1 (C-TAK1). C-TAK1 is constitutively associated with KSR1 and phosphorylates its Ser392 residue. Since Ser392 is the site that mediates the binding to 14-3-3 proteins, phosphorylation of this residue by C-TAK1 governs cytoplasmic sequestration of KSR1 in unstimulated cells . Upon grApart from 14-3-3, another KSR1 inhibitor has been identified. IMP  inhibits signal propagation by disrupting KSR1 homo-oligomerization, which is necessary to join Raf with its substrate, since it seems that different complexes of KSR1-MEK, KSR1-C-Raf and KSR1-B-Raf exist in the cells (reviewed in ). Thus, As already mentioned, the three tiers of the MAPK signaling cascade are initially activated at the plasma membrane. However, at later stages, the complexes are present in endosomes, which is required to achieve a proper signaling response. It seems that the endocytosis of the activated receptors and their associated signaling complexes is crucial for maximal MAPK activation, and activated Ras, C-Raf, MEK1 and ERK1 can be found on endosomes ,150–153.MEK partner 1 (MP1) is a widely expressed small protein that was originally identified in a yeast-two hybrid screen using MEK1 as a bait and was correspondingly named after its ability to bind to MEK1 . MP1 facDepletion of p14 and MP1 led to a pronounced inhibition of the MAPK signaling pathway. Interestingly, reduced ERK activation during the later stages of signaling was observed, while the early phase of ERK activation at the plasma membrane was unaffected. However, overexpression of p14 was not sufficient to inhibit MAPK signaling, pointing to the fact that p14 does not disrupt the formation of signaling complexes in the way a highly overexpressed MP1 or other scaffolds do .Consistent with its role in endosomal localization of MP1, p14 knockdown resulted in a mislocalization of MP1-MAPK scaffold complexes from late endosomes to the cytoplasm. However, p14 does not contain clearly identifiable membrane localization signals, yet it appears to be important for the localization of the complex in the membranes. Later findings have shown that the endosomal localization of MP1/p14 complexes is facilitated by a small adaptor protein, p18, which is associated with lipid rafts by means of myristoylation and palmitoylation . InteresThe multifunctional adaptor proteins, ß-arrestins, were originally discovered as proteins that desensitize or terminate G protein coupled receptor (GPCR) signaling. Upon ligand stimulation, GPCRs are phosphorylated by the family of GPCR kinases (GRKs), and this event promotes the recruitment of ß-arrestins to the GPCRs. Consequently, the interaction with ß-arrestins sterically prevents a further coupling of GPRCs with the trimeric G proteins and disrupts the normal activation of the second messenger signaling cascade ,160. LatFour members of the ß-arrestin family have been described so far, and they show different, but overlapping, expression patterns. Arrestin 2 (ß-arrestin 1) and arrestin 3 (ß-arrestin 2) are ubiquitously expressed, whereas arrestin 1  is localized to retinal rods and cones and arrestin 4 (X-arrestin) is found only in retinal cones. All four members are highly similar, sharing 70% sequence identity, and they are highly conserved across species (reviewed in ).In addition to their role as desensitizers of GPCR signaling, ß-arrestins have been shown to mediate the clathrin-dependent endocytosis of seven transmembrane spanning receptors and to act as adaptors for different E3 ubiquitin ligases –166. OneFollowing ß2 adrenergic receptor activation, the MAPK signaling cascade is activated in a biphasic way. The first phase of the activation is rapid and transient, with the signal peaking 2–5 min after stimulation. The second sustained phase is slower, lasting up to 30 min. The signal peaks 5–10 min after stimulation, and this phase of MAPK activation was found to be mediated by ß-arrestins ,169. ß-aHaving in mind all the aforementioned roles that ß-arrestins play in different signaling pathways, one would expect significant developmental defects after genetic ablation of the arrestins. However, ß-arrestin 1 knockout mice develop normally, but display increased cardiac contractility in response to adrenergic receptor agonists . SimilarFRS2 owes its name to the major and the first described function of this protein as a substrate for the fibroblast growth factor (FGF) receptor , althougWhereas FRS2 plays a role in the most upstream part of the MAPK pathway by recruiting signaling components to the RTKs, MORG1 is a scaffold protein in the downstream part of the ERK cascade . InteresMORG1 was also shown to be important for the regulation of the stability of hypoxia-inducible factor-1 (HIF-1) . As a pa+/− mice are normal [+/− mice are partially protected from renal and cerebral ischemia-reperfusion injury [MORG1 is ubiquitously expressed, with the highest expression levels in heart, brain, liver, kidney and testis, while lower amounts were detected in lung, spleen and skeletal muscle . MORG1 ke normal –198. Hown injury ,198. Furn injury . DecreasMembrane microdomains, also known as lipid rafts, participate in cellular signaling events by providing a specialized surrounding where membrane receptors, their respective signaling partners and adaptor proteins meet to initiate downstream signaling. Due to their enrichment in sphingolipids and cholesterol, these microdomains exhibit a so-called liquid ordered state in the membranes and are able to float in low density fractions of detergent resistant membrane (DRM) preparations ,200. ProN-terminal globular SPFH (stomatin/prohibitin/flotillin/HflK/C), also called PHB (prohibitin homology), domain contains acylated residues and hydrophobic stretches that, together with the C-terminal flotillin-domain, enable flotillins to firmly associate with membrane microdomains. In addition, the predicted coiled-coil structures in the C-terminus of flotillins are essential for proper homo- and hetero-oligomerization [Structurally, flotillins are organized in two major domains. The rization –210. Subrization –215. Howet al., showing that a knockdown of flotillin-1 in breast cancer cells yields a reduced expression of cyclin D1 and results in an arrest of the cell cycle in the G1/S phase [Interestingly, flotillins are downstream transcriptional targets of ERK signaling. Their transcription is regulated not only by ERK, but also by the retinoic X receptor complexes, which is detectable as an enhanced activity of the flotillin promoter . On the /S phase .Flotillins have been functionally implicated to participate in several cellular processes, such as clathrin-independent endocytosis ,219, orgq signaling [Recently, our group and others found implications of flotillins in MAPK signaling pathways, for example, in EGF receptor , insulinignaling . Furtherignaling . Upon grignaling ,228,229.ignaling .Recently, we described a dual role for flotillin-1 during EGFR activation and MAPK downstream signaling . Early, etc. However, recent research efforts have revealed that rather than utilizing linear cascades, signaling proceeds through intertwining communication networks. The plethora of scaffolding proteins that have been identified in recent years are required for many aspects of signaling. Scaffolders not only increase the efficiency and specificity of signaling, but they also facilitate the crosstalk between different signaling pathways. For example, the MP1/p14 complex plays an important role in the crosstalk between Rac/p21-activated kinase 1 (PAK1) and ERK signaling pathways. It regulates the PAK1-dependent activation of MEK1 in response to adhesion, thus representing a point of convergence for integrating growth factor signaling and cell adhesion to extracellular matrix (ECM) [etc.) of a signaling response is extremely important in regulating the final cellular outcome of the signaling, as, e.g., MAPK activation can result in either proliferation or differentiation of the target cells.Originally, a simplified view of signaling pathways with a linear flow and amplification was presented, which can still be found in many text books, ix (ECM) ,231. ImpBut why do we need so many different scaffolding proteins for a single signaling pathway, such as the MAPK cascade? These scaffolds are usually part of and actually even vital for the formation of multiprotein signaling complexes, and they frequently even bind to the same cascade components. How can a specific function still be accomplished? Upon a closer look, the different scaffolds show many features that distinguish them from the others. Some scaffolds show a very specific cellular localization and may thus regulate or restrict the signaling in specific subcompartments, as, for example, the MP1/p14 scaffold in endosomes, Sef1 in the Golgi or GIT1 (G protein coupled receptor kinase interacting 1), RACK1 (receptor for activated protein C kinase 1) and paxillin in focal adhesions . FurtherSome scaffolders, such as MORG1 and flotillin-1, appear even to be “superscaffolds” that are not only capable of binding to the MAPK components, but also to other scaffolds. For example, MORG1 binds to Raf, MEK and ERK, but also associates with the MP1 scaffold in the endosomes , whereas


In search of novel biomarkers of HTLV-1- transformed cells with relevance to oncogenesis, we identified the tumour marker and actin-bundling protein Fascin (FSCN1) to be specifically and strongly upregulated in both HTLV-1-transformed and ATLL-patient-derived CD4+ T cells. Fascin is important for migration and metastasis in various types of cancer. Here we report that a direct link can exist between a single viral oncoprotein and Fascin expression, as the viral oncoprotein Tax of HTLV-1 was sufficient to induce high levels of Fascin. In contrast, Tax-2 of HTLV-2 could not induce Fascin. Expression of Fascin was also detectable in primary CD4+ T cells of ATLL-patients after they expressed Tax spontaneously. Immunofluorescence analysis revealed that Fascin could colocalize with Actin in the cytoplasm and at the membrane of HTLV-1-transformed cells. We found a novel mode of transcriptional regulation of Fascin by showing the importance of NF-κB signals for Tax-mediated induction of Fascin in T cells. Chemical and dominant-negative inhibition of the NF-κB pathway as well as a NF-κB-deficient Tax-mutant led to a strong decrease of Fascin mRNA and protein in Tax-transfected Jurkat cells. Additionally, in HTLV-1-/Tax-transformed cells Fascin transcripts were strongly reduced after chemical inhibition of IκB-kinase. Knockdown of Fascin by lentiviral transduction of shRNA decreased the invasive capacity of ATLL-derived cells into extracellular matrix. Thus, we have clearly shown that the tumour marker Fascin can be induced by the viral Tax oncoprotein through NF-κB signals. Our data suggest that Fascin up-regulation by Tax contributes to the development of HTLV-1-associated pathogenesis.


Uncovering novel components of signal transduction pathways and their interactions within species is a central task in current biological research. Orthology alignment and functional genomics approaches allow the effective identification of signaling proteins by cross-species data integration. Recently, functional annotation of orthologs was transferred across organisms to predict novel roles for proteins. Despite the wide use of these methods, annotation of complete signaling pathways has not yet been transferred systematically between species.Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and humans. Using orthology data from InParanoid and signaling pathway information from the SignaLink database, we predict 88 worm, 92 fly, and 73 human novel signaling components. Furthermore, we developed an on-line tool and an interactive orthology network viewer to allow users to predict and visualize components of orthologous pathways. We verified the novelty of the predicted signalogs by literature search and comparison to known pathway annotations. In C. elegans, 6 out of the predicted novel Notch pathway members were validated experimentally. Our approach predicts signaling roles for 19 human orthodisease proteins and 5 known drug targets, and suggests 14 novel drug target candidates.Here we introduce the concept of ‘signalog’ to describe potential novel signaling function of a protein on the basis of the known signaling role(s) of its ortholog(s). To identify signalogs on genomic scale, we systematically transferred signaling pathway annotations among three animal species, the nematode C. elegans and D. melanogaster. In humans, signalogs may identify novel drug targets and new signaling mechanisms for approved drugs.Orthology-based pathway membership prediction between species enables the identification of novel signaling pathway components that we referred to as signalogs. Signalogs can be used to build a comprehensive signaling network in a given species. Such networks may increase the biomedical utilization of  D. melanogasterSignal transduction pathways are involved in the control of various cellular processes, including cell growth, proliferation, differentiation and stress response in divergent animal phyla Most of these methods predict new gene or protein properties (annotations) on the basis of sequence homology and similarities between known functions. Similar annotation transfer approaches have been applied to predict structural properties , expression profiles, and physical interactions for thousands of proteins C. elegans, D. melanogaster and humans In large-scale analyses, protein-protein interaction data are usually obtained from HTP experiments, such as yeast two-hybrid screens. However, the low abundance of extracellular, membrane-bound and nuclear signaling components  makes these experimental techniques only moderately efficient for identifying signaling interactions. Accordingly, several signaling pathway databases have been created manually by collecting relevant data from the literature Interestingly, in two different organisms the same signaling pathway is often known at different levels of detail. This may be due to evolutionary divergence or to differences between the current coverage of the two organisms' interaction maps. Therefore, large-scale pathway annotation transfer between these 3 organisms can extend our current knowledge of their signaling pathways. Note that in cases of rapid evolution, orthology-based predictions are less reliable as even the orthologs exist, they no longer participate in the same signaling pathway The topology of signaling pathways is important for selecting possible novel drug target candidates C. elegans, D. melanogaster and humans, and verify their novelty and predictive power, using both bioinformatics and experimental methods. We also show the utility of the signalog concept in drug target discovery.Here we introduce the concept of ‘signalog’ to predict a protein as a novel component of a signaling pathway based on the signaling pathway membership of its ortholog in another organism. We identify signalogs on genomic scale in 8 signaling pathways, including the MAPK, TGF-β, and WNT pathways  C. elegans, D. melanogaster, and H. sapiens were compiled by applying uniform curation rules to keep the level of detail identical for all pathways examined; proteins were assigned to pathways based on literature data; and interactions were listed manually from original publications presenting biochemical evidence To predict a role for a protein (i.e. provide a successful annotation), the quality of the original sources from which the annotation transfer (prediction) can be performed is crucial. The original pathway data, including the lists of proteins of 8 distinct signaling pathways and their interactions were obtained from the SignaLink database  were applied to find known signaling proteins by orthology searches C. elegans, D. melanogaster, and H. sapiens), we listed those proteins that have no known signaling interactions but have at least one signaling pathway member ortholog in the other two species. Similarly to the concept of functional orthology signalog proteins . Note also that in SignaLink In each of the three species examined  searched the literature with semi-automated methods for already known annotations, (ii) compared the list of signalogs and their predicted pathway memberships to known pathway annotations in pathway databases, and also (iii) compared the ortholog predictions to previously published interolog predictions.To grade the novelty of signalogs  and quantify the confidence level of each prediction, we performed semi-automated searches using PubMed, UniProt, GO, Wormbase, FlyBase, iHOP, and Chilibot web services Next, we applied interologs to verify the novelty of our ortholog predictions.  that have no paralogs, i.e., homologs in the same organism. These genes encode diverse proteins: their orthologs in the other two species include receptors, co-factors, and transcription factors  of 6 signalogs. Out of the 21 Notch signalogs in  factors . Neithernum-1(ok433)V, FX02407 aqp-6(tm2407)V, RB1267 D1009.3(ok1349)X, RB1011 crb-1(ok931)X, and MT2343 dpy-19(e1259)III lin-12(n137)III/unc-32(e189)III lin-12(n137n720)III. The allele n137 is a gain-of-function mutation in lin-12, which confers ectopic vulval induction in mutant hermaphrodites. These lin-12(n137) mutant animals exhibit a Multivulva (Muv) phenotype . Strains were cultured as described previously lin-12, we applied RNA interference- (RNAi) based gene downregulation. For RNAi, 0.9 kbp (nsh-1) - 1.0 kbp long (F10D7.5) cDNA fragments were amplified by RT-PCR, and were cloned into the vector pPD129.36. The constructs were then transformed into E. coli HT115. RNAi experiments were performed as described nsh-1: 5′-GGC TTT AAT GGC TTC ACG AG-3′ and 5′-AAG GAA GAA CTG TCG CTT GC-3′, for F10D7.5: 5′-AAG CGT TGA TCC GTA GAC ATC-3′ and 5′-TCG AGA TTG ACG AGA ACG TG-3′, and for lin-12: 5′-CGC TTC ATA TTG GCT CAT GTC-3′ and 5′-CCA GCT TCG CAT TTA TTA TTC AC-3′.Wild-type strain was used as Bristol N2 num-1(ok433), aqp-6(tm2407) D1009.3(ok1349), and crb-1(ok931) single mutant animals looked superficially wild-type, we treated these mutant animals with lin-12 double-stranded RNA (dsRNA), and the average number of vulval protrusions were determined at their young adulthood. lin-12(gf) mutant animals were also treated with nsh-1 or F10D7.5 dsRNA, and tested for the penetrance and expressivity of the Muv phenotype. The significance of results was tested with Chi square tests.As To examine the drug target relevance of the predicted signaling pathway member proteins, we downloaded additional information from DAVID C. elegans, D. melanogaster and H. sapiens, respectively, which had previously not been assigned to a signaling system, but have at least one ortholog in the other two species that is clearly associated with a signaling pathway. We hypothesized that these 253 proteins function in the same signaling pathways as their orthologs. Thus, we named the predicted signaling components signalog proteins, or briefly signalogs. Note that in contrast to an interolog, a signalog is a single protein (not an interacting pair) that has an ortholog annotated to a signaling pathway. In the three species examined our in silico approach predicted 301 novel signaling pathway annotations in total . For the complete list of the predicted signalog proteins and their pathway annotations, see We identified novel signaling pathway components based on the signaling pathway memberships of orthologs in another organism. We found 88, 92, and 73 proteins in C. elegans a similarly high number of signalogs was predicted as a potential Hedgehog component . Note that a canonical Hh pathway has not been identified in C. elegans due to specific gene loss After a detailed analysis of the predicted pathway annotations of signalogs, we found that in each of the three species the EGF/MAPK pathway contains the largest portion of signalogs , which is consistent with the fact that this pathway contains the highest number of signaling components Next, we tested the concept of orthology-based pathway annotation transfer by testing whether known signaling proteins have known signaling pathway member orthologs. We analyzed the three organisms separately. For each organism we listed all signaling pathway proteins from SignaLink and then listed, with InParanoid (version 6.1) C. elegans and D. melanogaster, most signalogs  have not yet been characterized biochemically, while in humans, only 26% of the signalogs remain uncharacterized. Note that this lower rate is partly due to the larger abundance of literature information on signaling in humans compared to worms and flies . Taken together, we conclude that signalog prediction can effectively contribute to the identification of novel signaling components.To examine the novelty of the predicted signaling roles and to quantify their confidence levels, we first classified these predicted proteins into 5 groups on the basis of their known properties. These groups range from genes for which only the ORF is known to genes whose protein products have known molecular function(s) , but only 1 in the same pathway, as in our prediction. We conclude that, depending on the organism and the database, 20 to 47% of all signalog proteins are already present in KEGG or Reactome, however, the large majority (86.7%) of pathway memberships we predicted for them are novel.Next, we verified that our signolog predictions are novel in the sense that the predicted signaling roles have not been known or predicted by other resources yet. We compared the list of signalogs and their predicted pathway memberships to known pathway membership annotations from KEGG and Reactome databases. Note that the KEGG database contains pathway data for all three species examined, while Reactome database contains only human data Finally, to reveal the presence of signalogs in current orthology-based prediction databases, we used interologs (orthology-based predicted interactions). We compared the interologs generated for this test from the SignaLink dataset with the interologs listed for worms, flies and humans by 3 species-specific databases , while here we predict 34 pathway annotations for them. 15 out of these pathway annotations are identical (for 14 of the 27 shared genes). Interestingly, a large-scale study predicted that p38b functions in 2 pathways (EGF/MAPK and TGF), while in our study 2 additional pathways (JAK/STAT and WNT) were predicted for this gene. In conclusion, the small overlap of the predicted novel signaling pathway genes and their predicted pathways verifies the novelty of the signalogs listed in the current paper and the predictive power of the method.While this study was in progress, Yan C. elegans  genetically interact with lin-12, which encodes a nematode Notch receptor  secondary vulval cell fate during pattering of this tissue lin-12 and a selected signalog (gene) would clearly indicate the participation of that gene in Notch signaling.The Notch pathway controls cell growth, differentiation and proliferation during normal animal development C. elegans vulval development, six originally equivalent ventral epidermal cells, called vulval precursor cells , are specified into one of three distinct – primary (1°), secondary (2°) or tertiary (3°) – vulval cell fates by the combined action of different signaling systems, including the Ras/MAPK, WNT, Notch and synMuv (for synthetic Multivulva) pathways synMuv genes antagonizes Ras signaling to repress vulval fates in each VPC During lin-12 causes a Protruded vulva (Pvl) phenotype that is due to the misspecification of 2° vulval cells; in lin-12 loss-of-function (lf) mutant hermaphrodites each induced VPC [P(5–7.p] adopts a 1° fate (lin-12 gain-of-function (gf) mutations confer VPCs [P(3–8).p] to adopt 2° fates, resulting in multiple vulval protrusions, i.e., a Multivulva phenotype (Muv)  .C. elegans Notch signalogs  animals. We found that lin-12 RNAi treatment of wild-type animals cause a Pvl phenotype with 17% penetrance. As compared, lin-12 RNAi in aqp-6, crb-1, num-1, and D1009.3 single loss-of-function mutant background significantly increased the penetrance of the Pvl phenotype  and monitored the average number of vulval protrusions, compared to those found in lin-12(gf) animals expressing the control RNAi alone. Of these lin-12(gf) mutants, 93% were Muv and the number of vulval inductions was 3.27+/−0.19. The penetrance and expressivity of the Muv phenotype in lin-12(gf) mutants was reduced by nsh-1 or F10D7.5 RNAi treatment. Depletion of NSH-1 decreased the penetrance of the Muv phenotype to 83%, whereas silencing of F10D7.5 reduced it to 81%. Both RNAi interventions significantly reduced the effect of lin-12 hyperactivity on vulval induction (lin-12(RNAi) animals and lin-12(gf) mutants , the user can enter a search term (arbitrary protein or gene name/ID). The on-line tool understands a variety of names and IDs, and also name fragments. Next, an integrated UniProt API synonym search helps the user to select the actual protein To facilitate the adaptability of the signalog prediction concept, we developed an on-line tool. This tool is available at http://signalink.org and http://signalink.org/signalog. This viewer can simultaneously visualize known and predicted pathway membership information, i.e., pathway annotation transfers  have been annotated to signaling pathways, respectively ptp-2, which has not been annotated to any signaling pathway prior to our current study. On the other hand, the role of SHP-2 in multiple kinase pathways – including MAPK, JAK/STAT, and IGF – is well established. Thus, identifying novel signaling components via their orthologs may help future experimentation in model organisms and the description of the underlying disease mechanism.In http://signalink.org and in Finally, we tested the drug target relevance of human signalogs by examining 4 key drug-related properties: disease-relatedness, localization in the plasma membrane, enzymatic function, and kinase domain content see  [60]–[62C. elegans and D. melanogaster – and humans. In addition, we developed and on-line tool allowing the users to predict signalogs and visualized known pathway data and predictions simultaneously with an ortholog network viewer. This viewer has distinctive features that can facilitate the interactive (user-defined) investigation of orthologous signaling networks, and it can visualize the predicted pathway components and their possible interactions, leading to the establishment of additional signalogs in later updates of the SignaLink database. The novelty of our predictions was verified by analyzing key properties, known pathway annotations, and already predicted interactions of signalogs. In C. elegans, we experimentally validated the signaling role of 6 predicted novel Notch pathway proteins. We anticipate that signalogs and, especially, orthodisease proteins of model organisms  gene to a signaling pathway with known phenotypes. Biological research often focuses on altering the function(s) of a single selected protein. However, this may cause undesired dysregulation of a signaling pathway, interfere with multiple cellular processes and lead to pleiotropic effects. Therefore, constructing more comprehensive signaling networks and identifying novel signaling components can certainly improve the design and evaluation of experiments.In the postgenomic era novel tools and methods are constantly needed to integrate genomic information with cellular processes. Current knowledge on signaling pathways is far from complete. ‘White spots’ can often be filled with the orthology-based transfer of ‘complete’ signaling system annotations between species, for example, by the method presented here. This method can be effectively used for the prediction of novel signaling proteins. The InParanoid database contains more than 100 eukaryotic genomes and its downloadable algorithm can be applied to other genomes as well, thus, orthology information is not a limiting factor for signalog predictions. Currently, signalog predictions for other genomes are limited mainly by the absence of proper signaling pathway data sources. Most importantly, the curation rules of databases should be uniform across all analysed organisms and signaling pathways. Such databases would be essential for signalog predictions both as seeds and as reference data sets. Unfortunately, for species not yet listed in the SignaLink database, current comprehensive signaling maps (databases) do not contain data curated with these guidelines in sufficient quantities that would allow the prediction of novel signaling components. The on-line tool was designed such that extensions can be added easily and we will include more species and pathways as soon as they become available. Based on the results, examples, and experimental work of this study, we believe that the predicted signaling pathway memberships  will be a good source of functional hypotheses to be experimentally verified in all three investigated organisms.Table S1Predicted signaling pathway member proteins . Protein names, species-specific identifiers, UniProt identifiers, and pathway annotations for all signalog proteins in the three investigated species.(XLS)Click here for additional data file.Table S2List of human signalog proteins and their drug target relevances. (1) Current drug target proteins and their signaling roles predicted in the current paper. (2) Predicted pathway annotations and further properties of proteins that are possible novel drug targets.(XLS)Click here for additional data file.Text S1Experimental verification of signalog proteins. Evaluation tables containing numerical experimental results and p-values quantifying the significance of experimental results.(PDF)Click here for additional data file.


Altogether, these studies, which provide important information regarding the pharmacology of the urotensinergic system and the conformational requirements for binding and activation, will ultimately lead to the development of potent and selective drugs.The urotensinergic system plays central roles in the physiological regulation of major mammalian organ systems, including the cardiovascular system. As a matter of fact, this system has been linked to numerous pathophysiological states including atherosclerosis, heart failure, hypertension, diabetes as well as psychological, and neurological disorders. The delineation of the (patho)physiological roles of the urotensinergic system has been hampered by the absence of potent and selective antagonists for the urotensin II-receptor (UT). Thus, a more precise definition of the molecular functioning of the urotensinergic system, in normal conditions as well as in a pathological state is still critically needed. The recent discovery of nuclear UT within cardiomyocytes has highlighted the cellular complexity of this system and suggested that UT-associated biological responses are not only initiated at the cell surface but may result from the integration of extracellular and intracellular signaling pathways. Thus, such nuclear-localized receptors, regulating distinct signaling pathways, may represent new therapeutic targets. With the recent observation that urotensin II (UII) and urotensin II-related peptide (URP) exert different biological effects and the postulate that they could also have distinct pathophysiological roles in hypertension, it appears crucial to reassess the recognition process involving UII and URP with UT, and to push forward the development of new analogs of the UT system aimed at discriminating UII- and URP-mediated biological activities. The recent development of such compounds,  Gillichthys mirabilis, urotensin II (UII), a somatostatin-like peptide, was first characterized as a spasmogenic agent  and endothelin-1 (ET-1), in driving a variety of cardiac and vascular disease processes  located on the inner nuclear membrane  with the C-terminal of the protein being localized either within or outside the nucleus. In fact, the topology of the nuclear membrane lumen is very similar to the extracellular space, which makes it a favorable environment for a binding site  of rat and mouse UT isoforms but is absent in the primate (human and monkey) receptor  , for the treatment of diabetic nephropathy. Similarly, GlaxoSmithKline started a phase I clinical trial for the use of an UT antagonist, i.e. SB1440115 , for the treatment of asthma. Finally, over the past few years, Boehringer-Ingelheim (European patent application EP2155748) as well as Janssen Pharmaceutical  filled several patents regarding UT antagonists but no phase I clinical trial was yet reported. With the recent discovery that UII and URP could exert common as well as different biological activities  physiological roles of the urotensinergic system i.e. to emanate from the initial activation of the receptor. However, multiple studies pointed out the ability of some ligands to selectively trigger specific signaling pathways, therefore having collateral and not linear efficacy  respectively, showed a specific behavior that has set them apart from known UT antagonists  played by the UII/URP/UT receptor system in physiology and pathology.These latest findings about the urotensinergic system will probably generate a considerable interest within the scientific community. First, the discovery of UT on the nuclear membrane and the presence of intracellular ligands open up new avenues in UT signaling physiology. In general, nuclear-localized receptors may regulate distinct signaling pathways, suggesting that biological responses mediated by GPCRs are not only initiated at the cell surface but might result from the integration of extracellular and intracellular signaling pathways. These receptors are therefore well-positioned to play major roles in the physiological and pathophysiological responses associated with their endogenous ligands. Finally, the discovery of allosteric modulators of the urotensinergic systems such as urocontrin, UCA, and rUII(1–7), will surely enable a better understanding of the urotensinergic system by allowing to discriminate 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.