Otx expression in the neural lineage at the 32-cell stage. However, it has not been determined whether differential inputs of all of these signaling pathways are really necessary. It is possible that differential activation of one of these signaling pathways is sufficient and the remaining signaling pathways are activated in all cells at similar levels. To address this question, we developed a parameter-free method for determining a Boolean function for Otx expression in the present study. We treated activities of signaling pathways as Boolean values, and we also took all possible patterns of signaling gradients into consideration. We successfully determined a Boolean function that explains Otx expression in the animal hemisphere of wild-type and morphant embryos at the 32-cell stage. This Boolean function was not inconsistent with three sensing patterns, which represented whether or not individual cells received sufficient amounts of the signaling molecules. These sensing patterns all indicated that differential expression of Otx in the neural lineage is primarily determined by Efna.d, but not by differential inputs of Fgf9/16/20, Admp, and Gdf1/3-r signaling. To confirm this hypothesis experimentally, we simultaneously knocked-down Admp, Gdf1/3-r, and Fgf9/16/20, and treated this triple morphant with recombinant bFGF and BMP4 proteins, which mimic Fgf9/16/20 and Admp/Gdf1/3-r activity, respectively. Although no differential inputs of Admp, Gdf1/3-r and Fgf9/16/20 signaling were expected under this experimental condition, Otx was expressed specifically in the neural lineage. Thus, direct cell–cell interactions through Efna.d play a critical role in patterning the ectoderm of the early ascidian embryo.A complex system of multiple signaling molecules often produce differential gene expression patterns in animal embryos. In the ascidian embryo, four signaling ligands, Ephrin-A.d (Efna.d), Fgf9/16/20, Admp, and Gdf1/3-r, coordinately induce  Otx expression in the neural lineage at the 32-cell stage. In addition to determining a Boolean function, our method determined sensing patterns, which represented whether or not individual cells received sufficient amounts of the signaling molecules. The sensing patterns predicted that differential expression of Otx in the neural lineage is primarily determined by Efna.d, but not by differential inputs of Fgf9/16/20, Admp, and Gdf1/3-r. We confirmed this prediction by an experiment. As a result, we found that only Efna.d signaling pathway is differentially activated between ectodermal cells and the remaining signaling pathways are activated in all ectodermal cells at similar levels.It is often difficult to understand a complex system of multiple signaling molecules in animal embryos only with experimental procedures. Although theoretical analysis might solve this problem, it is often difficult to precisely determine parameters for signaling gradients and kinetics of signaling molecules. In the present study, we developed a parameter-free method for determining a Boolean function for understanding a complex signaling system using gene expression patterns of signaling molecules and geometrical configurations of individual cells within the embryo. In the ascidian embryo, four signaling ligands, Ephrin-A.d (Efna.d), Fgf9/16/20, Admp, and Gdf1/3-r, coordinately induce  Ciona intestinalis, and all 16 have the potential to express Otx upon induction ], where Xo represents expression of gene o, and Xa and Xb represent the signaling states of a and b. Inputs and outputs are considered in binary space. If a signal sufficiently activates its intracellular pathway, it is represented as ‘1’, and otherwise as ‘0’ hereafter. Because there are 4 (= 22) possible combinations of input signaling states in each cell in this hypothetical system, there are 16 (= 4×4) possible states in the whole system , are called "sensing patterns" hereafter, because they represent how individual cells sense individual signaling inputs. In this hypothetical system, signaling molecule a comes from the upper side of b comes from the lower side. Obviously, eleven sensing patterns are incompatible with the following two simple principles, which we call Rule 1 and Rule 2.Here we introduce a method for determining a Boolean function of gene expression directed by extracellular signals within a population of equivalent cells. Before formalizing neural induction of the ascidian embryo, we first considered a Boolean function describing a simple hypothetical biological system illustrated in e system . These sa comes from the upper side of a should not be weaker in cell I than in cell II. Namely, whenever the signaling state of a in cell II is ‘1’, the signaling state of a in cell I is always ‘1’. For this reason, among the 16 sensing patterns shown in b should not be weaker in Cell II than in Cell I; therefore, sensing patterns 5, 7, 13 and 15 cannot stand.Because signal Rule 1, there are three possible combinations of the signaling states of a, , , and . Similarly, there are three possible combinations of the signaling states of b. These numbers are calculated as [(the number of cells) + 1] (formula 1); in this hypothetical system, 2 + 1 = 3. Because we consider two signaling molecules in the hypothetical system, there are 9 (= 32) combinations of the signaling states, as shown in In other words, according to o is not uniquely determined. Hence, these four patterns cannot stand , and loss-of-function of signaling molecule b induces ectopic expression of gene o in cell II (Experiment 2). If we assume sensing pattern 2, Experiment 1 does not alter the sensing pattern but the expression of o is lost in cell I. Hence, sensing pattern 2 is not consistent with Rule 2 under this experimental condition. In other words, the assumption of sensing pattern 2 necessitates F = 1 and F = 0 simultaneously, and therefore this assumption cannot stand. Similarly, sensing patterns 9 and 10 cannot stand, leaving only sensing patterns 12 and 14.Next, we introduce hypothetical loss-of-function of signaling molecules a and b , in whica activates o and b represses o, the logical considerations enumerated above indicate that these two hypothetical experiments cannot distinguish the two possibilities shown in Our method raises two important points. First, although the results of the above hypothetical experiments might lead to the intuitive interpretation that a and b, because outputs for F and F are predicted to be different between these two possibilities. Thus, this method can provide a practical direction of experiments.Second, b is tethered to the plasma-membrane of the signaling source, and therefore only cell II but not cell I can receive b, as shown in b. By considering this case in the same way as in We also consider a similar case, in which signaling molecule b is expressed additionally in cell I and cell II, as shown in b only from cell II and cell II receive b from both of cell I and the signaling source, the strength of signaling b should not be weaker in cell II than in cell I. Namely, whenever the signaling state of b in cell I is ‘1’, the signaling state of b in cell II is always ‘1’. This assumption is the same as the one we consider in the first hypothetical system shown in Finally, we consider another case, in which membrane-tethered signaling molecule Otx in the neural lineage (a6.5 and b6.5) of the 32-cell Ciona embryos (Otx expression in two pairs of cells (a6.5 and b6.5) within 16 equivalent ectodermal cells (eight pairs of cells) in the animal hemisphere  is represented by a Boolean function, Xotx =F.We next applied this logic to the signaling system that induces the neural marker gene  embryos . Previoumisphere  2–4]. T–4. TOtx 4)8]. As in the case of the hypothetical biological systems, we first screened individual sensing patterns with Rule 1 and Rule 2.In this biological system, there are four binary input variables and eight pairs of equivalent cells, and the number of possible sensing patterns is 4,294,967,296 [= , according to formula 1. In other words, the number of possible sensing patterns was reduced to 6,561 (= 94).Because the mid-to-late 32-cell period is crucial for induction of  pathway , we usedOtx is expressed only in a6.5 and b6.5, sensing patterns in a6.5 and b6.5 cells should not be the same as those of the remaining six pairs of cells. In addition, we used data from previous studies in which Admp, Efna.d, Fgf9/16/20 and Gdf1/3-r genes were knocked down and overexpressed [Rule 2, three sensing patterns remained , F, F, and F were not determined. To determine output values for these functions, we examined Otx expression in two double morphants: Admp and Fgf9/16/20, and Fgf9/16/20 and Gdf1/3-r. Otx expression in the animal hemisphere was lost in these morphants  and F gave the same result of Xo =1  and F gave different outputs of Xo =1 and Xo =0, respectively. Hence, Efna.d signaling was the only candidate that could pattern the ectoderm. Similarly, in the case of sensing pattern 3, although Admp signal was not sufficiently active in a6.5, this was not important for Otx expression; both of F and F gave the same result of Xo =1  and F gave the same result, Xo =1. Therefore, even if Otx expression in early 32-cell embryos is directed by a Boolean function different from the one in the mid-to-late 32-cell embryo, our analysis indicated that Efna.d is the limiting factor for patterning the ectoderm of the 32-cell embryo.Even under sensing pattern 4, the Boolean functions compatible with this sensing pattern indicated that Efna.d is a critical factor for patterning the ectoderm. Sensing pattern 4 showed that Efna.d signaling and Gdf1/3-r signaling are not sufficiently active in a6.5 and b6.5, implying that these two factors are candidates for a factor for patterning the ectoderm. However, all eight Boolean functions compatible with sensing pattern 4 indicated that Gdf1/3-r signaling cannot pattern the ectoderm under this sensing pattern, because Otx expression is expanded throughout epidermal cells of Efna.d morphants [Fgf9/16/20, Admp, and Gdf1/3-r. First we confirmed our previously result that Fgf9/16/20/Admp/Gdf1/3-r morphants do not express Otx in the animal hemisphere [Fgf9/16/20/Admp/Gdf1/3-r morphants in sea water containing recombinant bFGF and BMP4 proteins, which mimic Fgf9/16/20 and Admp/Gdf1/3-r activity [Otx is expressed on average in two cells of Fgf9/16/20 morphants incubated with 1 ng/mL of bFGF [Otx was expressed predominantly in the neural lineage, as in control embryos Otx expre embryos ; Table 1proteins ; Table 1pression ; Table 1Otx expression in the animal hemisphere of the mid-to-late 32-cell ascidian embryo, based on a theoretical analysis using data obtained in previous studies [Otx is specifically expressed in the neural lineage under either of these sensing patterns, the choice of sensing patterns by 32-cell embryos might not be strictly determined. In other words, these sensing patterns might represent fluctuations of signaling and robustness of this system.We determined a Boolean function for  studies , 7, 18 aOtx for the expression in the neural lineage contains multiple Ets-binding sites and Smad-binding elements (SBEs) [Otx in the neural lineage. The Boolean function in The cis-regulatory module of s (SBEs) , 4 , and the file names are shown in The contact areas of individual animal blastomeres of the 32-cell embryo with cells expressing Admp, Efna.d, Fgf9/16/20 and Gdf1/3-r were calculated using four different 3D-virtual embryos, which were reconstructed virtually from several series of confocal images  and the C. intestinalis (type A) adults were obtained from the National Bio-Resource Project for Ciona. The morpholino oligonucleotides for Fgf9/16/20, Admp, Gdf1/3-r, and Efna.d used in this study were those used in our previous study [us study . These mus study . Human rFgf9/16/20, CG.KH2012.C3.716 for Efna.d, CG.KH2012.C2.421 for Admp, CG.KH2012.C4.547 for Gdf1/3-r, and CG.KH2012.C4.84 for Otx.Identifiers for genes examined in the present study are as follows: CG.KH2012.C2.125 for S1 Figa and b. After a sufficient period of time, gene o is expressed only in cell I but not in cell II. Signaling molecule a is freely diffusible, while signaling molecule b is tethered to the cell membrane of its signaling source. (B) A Boolean function that describes expression of gene o. The 16 logically possible sensing patterns are shown in the second row. Because signaling molecule b is tethered to the cell membrane of its signaling source, signaling b is never transmitted to cell I. Hence, sensing patterns, 5–8 and 13–16, are incompatible and enclosed by boxes. Sensing patterns and Boolean functions incompatible with Rules 1 and 2 (see text) are indicated by ‘X’ in the third row. The fourth and fifth rows show sensing patterns appearing in Experiments 1 and 2, which are shown in (C). Sensing patterns incompatible with Rules 1 and 2 are shown in magenta in the second, fourth and fifth rows. Only sensing pattern 12 stands. The sixth row shows a Boolean function compatible with the sensing pattern among all of the logically possible Boolean functions shown in o in this hypothetical system.(A) A hypothetical biological system, consisting of two initially equivalent cells I and II, and two signaling molecules (PDF)Click here for additional data file.S2 Figa and b. After a sufficient period of time, gene o is expressed only in cell I but not in cell II. Signaling molecule a is freely diffusible, while signaling molecule b is tethered to the cell membrane. Signaling molecule b is expressed in the signaling b source cell (shown in the bottom), cell I and cell II. (B) A Boolean function that describes expression of gene o. The 16 logically possible sensing patterns are shown in the second row. Sensing patterns and Boolean functions incompatible with Rules 1 and 2 (see text) are indicated by ‘X’ in the third row. The fourth and fifth rows show sensing patterns appearing in Experiments 1 and 2, which are shown in (C). Sensing patterns incompatible with Rules 1 and 2 are shown in magenta in the second, fourth and fifth rows. Only sensing pattern 12 stands. The sixth row shows a Boolean function compatible with the sensing pattern among all of the logically possible Boolean functions shown in o in this hypothetical system.(A) A hypothetical biological system, consisting of two initially equivalent cells I and II, and two signaling molecules (PDF)Click here for additional data file.S3 FigCompatible sensing patterns shown in  are show(PDF)Click here for additional data file.S4 FigOtx expression in the normal and experimental conditions in the early 32-cell embryos. Sensing patterns 1, 2, and 3 are compatible with Boolean function A, whereas sensing pattern 4 is compatible with Boolean functions A to H, as shown in the bottom row of (D). Boolean functions A to H are shown in (E).(A–C) Estimated order of signaling strength of the Admp, Efna.d, Fgf9/16/20 and Gdf1/3-r pathways for the ectodermal cells of the animal hemisphere of three early 32-cell virtually reconstructed embryos. (A), (B), and (C) show the order in the first, second, and third embryos that appear in (PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


A variety of mutations are accumulated in the genome of HTLV-1 infected T-cells during ATL development. To elucidate the mechanism of ATL development a mouse model of ATL was established by infecting HTLV-1 to humanized NOG mice and the infected mice recapitulate the ATL-like symptoms and die of leukemia within several months of infection. Analysis of gene expressions in the humanized mouse model of ATL demonstrated the induction of APOBEC3B (A3B) gene in the HTLV-1 infected human T-cells. A3B is a member of the APOBEC family of cellular cytidine deaminase and was recently identified as the mutational source in multiple human cancers. We have previously shown that HTLV-1 infected CD25 (-) CD4 T-cells but not CD25 (+) CD4 T-cells in ATL model mouse express a small amount of Tax mRNA even though both cell populations consist of identical infected-cell clones. As the A3B expression in HTLV-1 infected CD25 (+) T-cells was similar to, or rather higher than that in CD25 (-)T-cells, Tax appears not to be involved in the induction A3B. In contrast, ex vivo culture of infected T-cells, in which Tax expression was highly enhanced in both CD25 (+) and CD25 (-)T-cells, resulted in the suppression of A3B expression. However, ectopic expression of HTLV-1 in Jurkat cell by the transfection of HTLV-1 molecular clone plasmid enhanced the expression of A3B gene. It is thus suggested that HTLV-1 infection but not Tax induces the A3B expression. Interplay between HTLV-1 infection and A3B and the possible involvement of A3B in the mutagenesis of host cell genome are currently investigated.


Intracellular signaling networks transmit signals from the cell membrane to the nucleus, via biochemical interactions. The goal is to regulate some target molecules, to properly control the cell function. Regulation of the target molecules occurs through the communication of several intermediate molecules that convey specific signals originated from the cell membrane to the specific target outputs.In this study we propose to model intracellular signaling network as communication channels. We define the fundamental concepts of transmission error and signaling capacity for intracellular signaling networks, and devise proper methods for computing these parameters. The developed systematic methodology quantitatively shows how the signals that ligands provide upon binding can be lost in a pathological signaling network, due to the presence of some dysfunctional molecules. We show the lost signals result in message transmission error, i.e., incorrect regulation of target proteins at the network output. Furthermore, we show how dysfunctional molecules affect the signaling capacity of signaling networks and how the contributions of signaling molecules to the signaling capacity and signaling errors can be computed. The proposed approach can quantify the role of dysfunctional signaling molecules in the development of the pathology. We present experimental data on caspese3 and T cell signaling networks to demonstrate the biological relevance of the developed method and its predictions.This study demonstrates how signal transmission and distortion in pathological signaling networks can be modeled and studied using the proposed methodology. The new methodology determines how much the functionality of molecules in a network can affect the signal transmission and regulation of the end molecules such as transcription factors. This can lead to the identification of novel critical molecules in signal transduction networks. Dysfunction of these critical molecules is likely to be associated with some complex human disorders. Such critical molecules have the potential to serve as proper targets for drug discovery. Each cell in the human body includes many biomolecules, which continuously communicate with each other . IntraceSignal transmission in communication systems is accomplished via digital techniques, where different types of data and signals such as voice, music, image, video, text, etc., if not already in the digital format, are first digitized and converted to sequences of 0’s and 1’s ,7]. Dig. Dig7]. In the area of systems biology, digital models and methods have been used for various purposes, and have also been verified using experimental data. The interested reader can refer to some review articles on this subject –13]. Th. Th13]. Inspired by communication engineering and signal transmission concepts, in this paper we develop a systematic framework to quantitatively model how the signals that ligands provide upon binding can be lost in a pathological signaling network, due to the presence of some dysfunctional molecules. We also show how the lost signals result in message transmission error, i.e., incorrect regulation of target proteins at the channel output. Furthermore, we show how dysfunctional molecules affect the capacity of signaling networks and how the contribution of each signaling molecule to the signaling capacity and signaling errors can be computed. The proposed approach can quantify the role of dysfunctional signaling molecules in the development of the pathology.et al. ,7],,[7],Quan,,[7],Qua Networks–24],[34,[34Quant Networks.Click here for file


Ethylene is a simple two carbon atom molecule with profound effects on plants. There are quite a few review papers covering all aspects of ethylene biology in plants, including its biosynthesis, signaling and physiology. This is merely a logical consequence of the fascinating and pleiotropic nature of this gaseous plant hormone. Its biochemical precursor, 1-aminocyclopropane-1-carboxylic acid (ACC) is also a fairly simple molecule, but perhaps its role in plant biology is seriously underestimated. This triangularly shaped amino acid has many more features than just being the precursor of the lead-role player ethylene. For example, ACC can be conjugated to three different derivatives, but their biological role remains vague. ACC can also be metabolized by bacteria using ACC-deaminase, favoring plant growth and lowering stress susceptibility. ACC is also subjected to a sophisticated transport mechanism to ensure local and long-distance ethylene responses. Last but not least, there are now a few exciting studies where ACC has been reported to function as a signal itself, independently from ethylene. This review puts ACC in the spotlight, not to give it the lead-role, but to create a picture of the stunning co-production of the hormone and its precursor. Figure 1). Figure 1). 13 years later, 6) dependent enzyme inhibitor, which was later known to inhibit the enzymatic conversion of SAM toward ACC. The identification of ACC as the precursor of ethylene was a major breakthrough in the understanding of the ethylene biosynthesis pathway in plants, and was part of the foundation for many new discoveries in the field of ethylene biology.The discovery of ethylene as a plant growth regulator can be attributed to the work of the Russian scientist Arabidopsis, eight of which encode functional ACC synthases . Expression of different members of the tomato ACO family in Escherichia coli showed that each isoform had a specific in vitro enzyme activity (The second ethylene biosynthesis protein is ACC-oxidase (ACO), which converts ACC to ethylene in the presence of oxygen. It took a long time before ACO activity could be demonstrated ong time , it was ong time . This ca and CN- . The reaoalanine . ACO belctivator . The subctivator , while octivator . Althougactivity .Arabidopsis  by ATP-dependent tonoplast carriers , was discovered by carriers . Nonethecarriers . The abif tomato .in vitro studies, stated that GACC was the most abundant ACC derivative in tomato (in vivo in tomato fruit during climacteric ripening (Arabidopsis genome contains four genes (GGT1-4), of which only GGT1 and GGT2 are catalytically active , which was discovered in crude tomato extracts of ACC-N-malonyltransferase . These cn tomato , anotherripening . The Aray active . Both GGlar GACC . A knocke number . The effArabidopsis tissue  showed that the JA-ACC-induced root inhibition was independent of JA signaling. Furthermore, the ethylene signaling mutant etr1-1 and the double mutant etr1-1 jar1-1 were insensitive to the JA-ACC treatment and displayed no inhibition of root growth, indicating that JA-ACC acts via the ethylene signaling pathway (A third derivative of ACC is jasmonyl-ACC (JA-ACC). This molecule was discovered by screening for amino acid conjugates of JA, using GC-MS . Four ams tissue . Interess leaves . Recombi pathway . Most liThe three above-mentioned derivatives of ACC  are perhaps not the only ones. Future metabolic studies might reveal additional conjugates of ACC. Nonetheless, these three derivatives can potentially play an important role in the regulation of the pool of available ACC, which in turn can affect eventual ethylene production levels, with physiological and developmental consequences. Genetic perturbations of the formation of ACC derivatives could be a useful tool to unravel their exact roles. In addition, a more detailed structural and biochemical characterization of the enzymes involved in the formation of ACC derivatives is essential.Pseudomonas sp. strain ACP , but so do other amino acids like L-alanine, DL-alanine, and DL-valine  promotor of some Rhizobia, linking ACC deaminase with nodule formation . It should be noted that the foliar applied ACC was also rapidly converted into MACC, which was not found to be transported via the phloem (ACS isoforms are expressed (but not exclusively) in the vascular tissue  secured the ability of ACO to produce ethylene in cells with a high ACO expression  during different developmental processes. Tissue specific gene expression profiling of maize root cells showed that there are differences between patterns . ACO wasobserved . Differepothesis . A similpothesis , 1999, wOf course one should take into account that gene expression levels not always reflect actual protein levels, and that post-translational modifications can play an important role in protein stability and activity. A targeted metabolomics and proteomics study by Besides long-distance, short-distance intracellular transport of ACC was also observed in barley and wheat mesophyll cells  and maizClearly, more research is needed to further unravel the exact molecular and biochemical mechanisms which assure intracellular, inter- and intra-tissue and long-distance ACC transport in plants, and their corresponding physiological effects.1-Aminocyclopropane-1-carboxylic acid holds a key position in many physiological processes as it is the direct precursor in the biosynthesis of ethylene. A balanced supply and consumption of ACC is essential to achieve the necessary production level of ethylene within a given spatial and temporal context. As shown above, the pool of ACC is regulated by a complex interaction of production, consumption, modification, and transport. Interestingly, recent findings have suggested a perhaps even more important role for ACC, as a signaling molecule independent from ethylene .fei1 fei2 mutant displays a severe defect in anisotropic root growth due to a reduced cellulose microfiber content in the cell wall at the root tip . The fei1 fei2 phenotype can be reversed by the application of ethylene biosynthesis inhibitors, but not by ethylene signaling inhibitors. The application of both aminooxy-acetic acid (AOA) or α-aminoisobutyric acid (AIB) specifically inhibits ethylene biosynthesis and can reverse the phenotype of the fei1 fei2 mutant . AOA is an inhibitor of PLP-dependent enzymes, and will affect the activity of ACS resulting in a reduced ethylene production. AIB on the other hand is a structural analog of ACC and acts as a competitive inhibitor of ACC preventing ethylene production at the level of ACO. Ethylene signaling inhibitors such as 1-methylcyclopropane (1-MCP) and silver ions, did not affect the fei1 fei2 phenotype . Similarly, genetics showed that the fei1 fei2 mutant crossed with etr1-3 (a mutation in the ethylene receptor causing severe ethylene insensitivity), nor ein2-50  could reverse the phenotype. All together this study showed that the typical fei1 fei2 phenotype was not affected by ethylene signaling, but could be reversed by ethylene biosynthesis inhibitors. This suggests that the signal reversing the fei1 fei2 phenotype originated independent from ethylene signaling, involved ACS and is possibly ACC itself  into the hexuple mutant acs2,4,5,6,7,9 creating an octuple mutant line with complete or severe inhibition of ACS function. The lines that showed a very strong silencing of ACS8 and ACS11, displayed embryo lethality. This suggests that ethylene biosynthesis (or ACC biosynthesis) is essential for Arabidopsis viability, while this is not the case for the single (ctr1 and ein2) and double (ctr1 ein2) ethylene signaling mutants , could shed light on the role of ACC as a signaling molecule. It also still needs to be elucidated whether this unique title of “signaling molecule” is to be awarded to ACC, or rather to one if its (unknown) downstream derivatives.All together these reports suggest a role for ACC as a signaling molecule to regulate plant development and growth, independent from ethylene. The exact molecular mechanism by which ACC signaling operates, and whether or not there is an ACC receptor and downstream signaling components, remains to be investigated. Future biochemical studies with specific ethylene biosynthesis and signaling inhibitors, in combination with genetics to create higher order ethylene biosynthesis/signaling mutants  ACC-deaminase, adding another layer of metabolic complexity to the regulation of ACC levels. It is also well established that ACC can be easily transported over short (intracellular and intra-tissue) and long-distances (via the xylem and phloem), providing the plant with an elaborate system to control local and remote ethylene responses. Last but not least, ACC has been identified as a potential signaling molecule, independent of ethylene. This property of ACC is perhaps the most exciting, opening new avenues in ACC research, with potentially profound effects on plant physiology. The molecular mechanism by which ACC is signaling and the identity of other putative signaling components in such an ‘ACC pathway’ remain to be discovered.Bram Van de Poel and Dominique Van Der Straeten conceived the topic and wrote the manuscriptThe 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.


Most common diseases, e.g., cancer are driven by not one, but multiple cell surface receptors that trigger and sustain a pathologic signaling network. The largest fraction of therapeutic agents that target individual receptors/pathways eventually fail due to the emergence of compensatory mechanisms that reestablish the pathologic network. Recently, a rapidly emerging paradigm has revealed GIV/Girdin as a central platform for receptor cross-talk which integrates signals downstream of a myriad of cell surface receptors, and modulates several key pathways within downstream signaling network, all via non-canonical activation of trimeric G proteins. Unlike canonical signal transduction via G proteins, which is spatially and temporally restricted, the temporal and spatial features of non-canonical activation of G protein via GIV is unusually unrestricted. Consequently, the GIV●G protein interface serves as a central hub allowing for control over several pathways within the pathologic signaling network, all at once. The relevance of this new paradigm in cancer and other disease states and the pros and cons of targeting the GIV●G protein interface are discussed. Heterotrimeric G proteins are one such major signaling hub that are essential components of the signal gating machinery in healthy eukaryotic cells. They serve as molecular switches for signal transmission via 7 transmembrane domain G protein coupled receptors (GPCRs) to intracellular effectors . Activatiseconds .hyperactivation of G proteins” is a bona-fide basis for oncogenic signaling via trimeric G proteins.The importance of maintaining the critical balance between G protein activation and deactivation and the loss of such balance in cancer has been highlighted by studies on several cancer-associated mutants of trimeric G protein α-subunits and GPCRs . ThDespite the insights gained, the rare oncogenic driver mutations in G proteins in a handful of cancers do not explain the basis for deregulated G protein signaling in the vast majority of cancers that do not harbor mutant G or GPCR proteins. A growing body of work by us and others , 45, 46 Most of these diseases, if not all, are characterized by cellular processes  that are driven by more than one receptor or one class of receptors, and most often require synergistic signaling of diverse classes of receptors. GIV appears to serve as a platform on which crosstalk between diverse classes of receptors either directly (in the case of RTKs ) or indiversus GEF-OFF states has also been described in the context of some other cellular processes and diseases, e.g., fibrosis, wound healing, diabetes [Given the broad landscape of signaling pathways that GIV modulates, and its ubiquitous nature of expression, it is not surprising that deregulation of GIV-GEF drives several pathophysiologic conditions . In the diabetes , most, iFinally, the ability to manipulate a broad signaling network and reset it from an unstable pathologic to a stable physiologic state downstream of multiple receptors by fine-tuning GIV's GEF function is an attractive concept because of many reasons: 1) Eliminates the need to block physiologic signaling via cell surface receptors; 2) Overcomes the limitations of unknown upstream and downstream components; 3) Preserves the utility of biomarkers/therapeutic targets despite re-wiring of signaling pathways during the course of disease progression. Recently, in a proof-of-concept study  using ceIn conclusion, through the studies on GIV, we have obtained a sneak preview of just how large the footprint of oncogenic signaling via trimeric G proteins could be. Because GIV is just one of the members of the rheostat family, we are likely seeing only the proverbial tip of the iceberg, and a lot more must be known before any of these findings can be translated to transformative and impactful therapies and/or biomarkers for personalized care.


High frequency of neurologic diseases including urinary symptoms of overactive bladder (OAB) have been documented in HTLV-1 infected subjects. Moreover OAB may be the initial manifestation of HTLV-1 associated myelopathy (HAM). The aim of this study was to evaluate the immune response, proviral load and plasma levels of soluble Interleukin-2 receptor (sIL-2R) and beta 2 microglobulin (β2M) in HTLV-1 carriers, patients with HTLV-1 OAB and in patients with HAM to determine if these parameters are markers of neurologic disease. Cells and serum from carriers, HTLV-1 OAB and HAM/TSP patients were used. HAM/TSP had higher levels of β2M and sIL-2R than HTLV-1 OAB and carriers. Cells from HTLV-1 OAB produced spontaneously more proinflammatory cytokines than carriers. TNF-α and IL-17 expression as well as proviral load were similar in HAM/TSP and HTLV-1 OAB and were higher than in carriers. In contrast to HAM/TSP, levels of Th1 chemokines were similar in HTLV-1 OAB and carriers. Different from HAM/TSP addition of regulatory cytokines in cell cultures from HTLV-1 OAB decreased spontaneous IFN-g. This study showed that while sIL-2R determination helps in identifying HAM/TSP, both sIL-2R and β2M did not help to monitor neurologic disease progression. Additionally we showed that HTLV-1 OAB and HAM/TSP have in common many immunological features as well as proviral load. However, as HTLV-1 OAB were still able to down regulate their inflammatory response and express levels of chemokines similar to carriers this may explain why they have not yet developed the spinal cord damage observed in HAM/TSP.NIH , Brazilian National Research Council (CNPq).


Articular cartilage is a unique load-bearing connective tissue with a low intrinsic capacity for repair and regeneration. Its avascularity makes it relatively hypoxic and its unique extracellular matrix is enriched with cations, which increases the interstitial fluid osmolarity. Several physicochemical and biomechanical stimuli are reported to influence chondrocyte metabolism and may be utilized for regenerative medical approaches. In this review article, we summarize the most relevant stimuli and describe how ion channels may contribute to cartilage homeostasis, with special emphasis on intracellular signaling pathways. We specifically focus on the role of calcium signaling as an essential mechanotransduction component and highlight the role of phosphatase signaling in this context. The unique biomechanical properties of articular cartilage are attributed to the structure, composition, and organization of its extracellular matrix (ECM) macromolecules. The cartilage ECM is mainly composed of a collagen fiber network (type II collagen with type IX and XI) and large aggregating proteoglycans (PGs) entrapped within. The fixed negative charges on the glycosaminoglycan (GAG) side chains of PGs attract counteracting ions, which drive the movement of large amounts of osmotically obliged water into the matrix . The hig+, K+, Ca2+) passing the relatively “fixed” FCD  distributed along the PGs o independent protein kinase C (PKC)α-mediated hyperpolarization upon hyperosmotic stimulation [Pritchard et al.  found thmulation .Osmotic loading is known to modulate chondrocyte height, width, and volume in situ, and OP may modulate cell shape in accordance with the primary collagen fibril direction , as wellChondrocyte shrinkage by raised hyperosmotic pressure (≥480 mOsm) may protect cells. While most cell-based chondral repair strategies aim at re-differentiation of routinely expanded, dedifferentiated chondrocytes, van der Windt et al. showed that dedifferentiation can be delayed by harvesting and expanding cells under elevated  osmolarity . Interesi is also osmolarity-dependent and its contribution to sulfated GAG production remains speculative.The effects of OP, to a certain extent, also depend on the culture model: in alginate, higher proliferation rates, with diminished sulfated GAG production, were found at 280 mOsm . Of noteFinite element modeling showed that charged tissues (or synthetic matrices) always support larger loads than uncharged tissues. This load support derives from three sources: intrinsic matrix stiffness, HP, and OP .A precisely set balance between the activities of protein kinases and phosphoprotein phosphatases is crucial to regulating chondrogenesis and maintaining the chondrocyte phenotype. All of the major protein kinase families, including protein kinase A (PKA), PKC, mitogen-activated protein kinase (MAPK), and CaMK, as well as all major protein phosphatases  play fundamental roles in molecular regulation in chondrocytes . These s2+-dependent serine/threonine phosphoprotein phosphatase calcineurin  has been identified as a potential target to improve the chondrocyte phenotype. The Cn inhibitor FK506  increases the expression of chondrogenic markers during in vitro expansion in hypoosmotic culture medium [The Cae medium . Isolatie medium . Interese medium . Generale medium  and in te medium , where FThe exact mechanism underlying the effects of Cn inhibition under different osmolarities is not yet understood, but Cn is known to induce FGF18, which can suppress noggin and facilitate BMP-related chondrogenesis-like effects . This pa2+/calmodulin-dependent manner [Cn regulates the activity of NFAT family members in a specific and Cat manner . For a dt manner . NFATs ht manner .After the original study by Glimcher’s group had shown that all four NFATc1–4 proteins are expressed in the cartilage , Greenbl2+-dependent NFAT proteins (NFATc1–4). In contrast, osmotic stress, rather than Ca2+ signaling primarily regulates the more distantly related fifth family member NFAT5, as discussed above.Greenblatt’s cartilage-specific NFATc1 and NFATc2 double mutant mice showed accelerated cartilage degeneration and expression of OA markers, such as increased expression of genes encoding proteases involved in ECM degradation such as MMP13, ADAMTS-5, and hypertrophic chondrocyte markers, including COL10A1, and reduced expression of Sox9 and PRG4, encoding lubricin. Intriguingly, NFATc1 protein expression is restricted to the superficial zone of articular cartilage, and its mRNA expression is reduced around cartilage lesions in human osteoarthritic patients . A numbeV3.2 T-type Ca2+ channels may be involved in Cn–NFAT-dependent modulation of Sox9 expression [2+ channels [A recent study showed that lentiviral shRNA-mediated Nfatc2 knockdown in articular chondrocytes in vitro largely matches the in vivo phenotype and also upregulates pro-inflammatory cytokines . In tracpression ••. A prechannels .2+ signaling and Cn–NFAT signaling, which may be integrated through NFAT5. NFAT activity and expression in chondrocytes in vitro seems also to be dependent on both Notch and Wnt5a signaling, at least in growth plate chondrocytes [Pharmacological inhibition of Cn by FK506 promotes chondrogenic marker expression in dedifferentiated human adult chondrocytes, probably through upregulation of TGFβ1 . NFAT acdrocytes , 135, anOverall, in vivo and in vitro data from mice and humans strongly suggest a dynamic control of NFATc2 expression in articular cartilage and a crucial role of NFAT family members in cartilage homeostasis and joint health. NFATs might be involved in distinguishing articular from growth plate chondrocytes, the origins of which are still not understood completely . Novel sA main function of articular cartilage is to absorb shock during joint movements. Chondrocytes are sensitive to mechanical load, one of the most physiological stimuli that trigger the activation of key signaling molecules. Although appropriate mechanical stimuli are essential for limb development , differeAlthough mechanosensitive ion channels, primary cilia, and the actin cytoskeleton have all been implicated as mechanosensors in chondrocytes, downstream pathways are even less well characterized. Of the major signaling pathways, integrins and focal adhesion kinases (FAKs), the ERK, and the PI 3-kinase/Akt pathways have been reported . It is oSerially passaged articular chondrocytes, deprived of their ECM, rapidly lose their characteristic phenotype. Signaling events that control the re-differentiation of dedifferentiated chondrocytes have only partially been analyzed. Chondrocyte re-differentiation in micromass cultures may be mediated by PKC-dependent ERK1/2 regulation, whereas chondrocyte dedifferentiation is under a separate control by PKCα and ERK1/2 . In a di2+ signaling and changes in cytosolic Ca2+ concentration are closely related to cell proliferation and differentiation in chondroprogenitor cells, and Ca2+ release from intracellular stores and influx through plasma membrane ion channels are key factors controlling chondrogenesis [2+-activated K+ channel (BK-like channel) [2+ influx and subsequent alterations in Ca2+ signaling have been documented in chondrocytes upon both compressive loading and HP [2+ transients via activation of P2X and P2Y receptors that cause a combination of extracellular Ca2+ influx and intracellular Ca2+ release in agarose-embedded chondrocytes [2+ signaling is coupled to mechanosensation in chondrocytes remains an open question.Intracellular Caogenesis . Variouschannel)  or the tchannel) ••. Mechag and HP . Furtherdrocytes . How exa2+ signaling via P2X and P2Y purinergic receptors, inducing aggrecan mRNA expression and sulfated GAG secretion in a 3D chondrocyte culture system [A promising candidate for a mechanosensory organelle on chondrocytes is the primary (non-motile) cilium, first identified on articular chondrocytes almost 40 years ago . Tissue e system . These fThe effects of electrical, magnetic, and mechanical stimulation on articular cartilage are summarized in Fig.


Recently we have demonstrated the importance of RBPjk-dependent Notch signaling in the regulation of mesenchymal stem cell (MSC) differentiation during skeletogenesis both in vivo and in vitro. Here we further performed RBPJK loss-of-function experiments to demonstrate for the first time that RBPJK deficient MSC shows enhanced differentiation and osteogenesis acts via up-regulation of the BMP signaling. In the present study, we first compared the spontaneous and osteogenic differentiation in normal and recombination signal binding protein for immunoglobulin kappa J region (RBPJK) deficient human bone marrow-derived mesenchymal stem cells (MSCs). It was found that RBPJK highly expressed in fresh isolated MSCs and its expression was progressing down-regulated during spontaneous differentiation and even greater in osteogenic media inducted differentiation. Deletion of RBPJK in MSCs not only enhances cell spontaneous differentiation, but also significantly accelerates condition media inducted osteogenic differentiation by showing enhanced alkaline phosphatase (ALP) activity, Alizarin red staining, gene expression of Runx2, Osteopontin (OPN), Type I collagen (COL1a1) in culture. Additionally, BMP signaling responsive reporter activity and phosphor-smad1/5/8 expression were also significantly increased upon removal of RBPJK in MSCs. These data proved that inhibition of Notch signaling in MSCs promotes cell osteogenic differentiation by up-regulation of BMP signaling, and RBPJK deficient MSC maybe a better cell population for cell-based bone tissue engineering. Mesenchymal stem cells (MSCs) hold promise for healing of injured tissue because of their capacity to differentiate into multiple cell lines and their availability from a wide variety of sources. AlthougHuman bone marrow derived MSCs are well known not only by their anti-inflammatory and immuno-regulatory properties, but alsVarious signaling factors have been implicated in the regulation of MSC osteoblastic differentiation, including Wnt, BMP14]14], FGF and PTH. RecentlHuman bone marrow aspirate was harvested from the iliac crest of donors (n = 6) for MSC isolation. The mean age of the donors was 55 years (range 47–65) comprised four males and two females with no history or evidence of genetic disease or malignancy. The ethics committee of Anhui Provincial Hospital approved the use of the tissue material for this study . Written informed consent was obtained from all donors and kept in locked file box since these patients were in spine fusion surgery using their own bone marrow aspirate.6 cells/cm2 in alpha-minimum essential medium (α-MEM) supplemented with 15% FBS, 2 mM L-glutamine, 0.5% antibiotic/antimycotic solution  for 3 days. Cells were then washed 3–5 times with 1XPBS and incubated at 37°C in a humidified 95% air and 5% CO2 atmosphere, cultured up to 80% confluence, and then trypsinized , centrifuged, and re-plated at a density of 2000 cells/cm2 for subsequent expansion. All experiments were performed with cells after the second or the third passage around 2 weeks.Method for isolation of human MSC based on red blood cell (RBC) lysis with ammonium chloride. After iMSC phenotype was evaluated after 2 weeks’ expansion by the expression of CD34-APC , CD45-FITC , CD90–PE , CD73-PE (BD Biosciences Pharmingen) and CD105-FITC . Cells were incubated for 30 min at room temperature with antibodies and flow cytometry was performed on a LSR-II flow cytometer (Beckton Dickson). Isotype matched controls were included for each antibody and used to set the electronic gates on the flow cytometer. The data were analyzed using FlowJo software (Tree Star).http://www.genechem.com.cn). For lentiviral infection, 2,000 cell/mm2 MSCs were seeded in 6-well plates and incubated for 24 h at 37°C prior to be added with shRNA lentivirus condition medium in the presence of 8 μg/ml polybrene (Sigma-Aldrich). The infected MSC cultures were harvested at various time points . RBPJK expression in cells was detected by Real time RT-PCR and Western blot analysis.RBPJK-specific (shRBPJK) and control (shCo) short hairpin RNA (shRNA) lentiviral particles were prepared by GeneChem  at 450 nm.Cell proliferation assays were performed using a BrdU ELISA Kit (Roche). Briefly, shRNA lentiviral infected MSCs were cultured at 2000 cells/cm2 to confluence on standard culture dishes, growth media was changed every 3 days till harvesting at day 3 and 14. RNA isolation and ALP staining were performed as previously described . Ad. Ad30]. Previous study demonstrates a RBPJK-dependent regulation of bone formation in vivo, in that deletion of RBPJK in limb bud progenitor cells leads transit increases bone mass . We thenGiven the crucial roles of BMP signaling in bone tissue developmental processes and Runx2 is one of the target gene induced by BMP signaling, it will be important to investigate the interaction between RBPJK and BMP signaling components during differentiation of MSCs. Here RBPJK-deficient MSCs exhibited an increased BMP signaling reporter activity and enhanced expression of phosphor-smad1/5/8 when compared with the controls, suggest that RBPJK deletion may promote MSC differentiation towards osteoblast by inducing Runx2 through upregulation of BMP signaling. In addition to the canonical Smad-dependent signaling pathway, mitogen-activated protein kinases (MAPKs) such as p38 or ERK1/2 can also be activated by BMPs via Smad-independent mechanisms. To eliminate the possible involvement of MAPK signaling in RBPJK deletion induced cell differentiation, phosphorylated active ERK1/2 and p38 were further detected by Western Blot. Our results clearly showed deletion of RBPJK did not induce activation of the MAPK signaling and demonstrated that RBPJK activity in MSCs can be attributed only to its BMP-activated Smad signaling pathway.In conclusion, our study provides novel and important insights into the role of RBPJK during early MSC maintenance and differentiation. We demonstrate for the first time that deletion of RBPJK in MSCs effectively enhances the cell osteogenic differentiation in vitro likely in part by co-activating Smad-dependent BMP signaling. Since RBPJK deficient MSC is a better MSC population for bone tissue engineering, identification of factors could be used to transient suppression of RBPJK expression to enhance the MSC osteoblastic differentiation during bone tissue regeneration become one of our future research focuses.


Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia. The HTLV-1 transactivator protein Tax has been identified as a critical component in the proliferation and transformation of human primary T-cells. This 40 kDa phosphoprotein not only manipulates chromatin remodeling within the host, but also subverts host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Here we utilized a combination of filtration and ultracentrifugation methods to enrich for exosomes from culture supernatants of HTLV-1 infected cells. We then employed western blots, mass spectrometry, and cytokine arrays to proteomically characterize the host and viral components in these exosomes. Additionally, RT-PCR was used to determine the presence of viral transcripts in these exosomes. Our results demonstrate that exosomes derived from HTLV-1 infected cells contain traditional exosome proteins. Furthermore, our proteomics studies revealed that these exosomes contain viral components such as gp46 and Tax, as well as inflammatory mediators including IL-6 and IL-10. We also investigated the presence of HTLV-1 mRNA transcripts of Env, Tax, HBZ, and 5’LTR contained within exosomes. Moreover, we evaluated the functional impacts of treating naïve recipient cells with exosomes secreted from HTLV-1 infected cells, and determined that the exosomes were able to induce a response in reactive oxygen species production. Our observation that the viral protein Tax is contained within exosomes and may be transmitted in an extracellular capacity raises important implications to pathogenesis associated with HTLV-1 infections.


Vibrio coralliilyticus. In Vibrio cholerae, infection by a bacterial virus (bacteriophage) results in the conversion of non-pathogenic strains to pathogenic strains and this can lead to cholera pandemics. Pathogenicity islands encoded in the V. cholerae genome play an important role in pathogenesis. Here we analyse five whole genome sequences of V. coralliilyticus to examine whether virulence is similarly driven by horizontally acquired elements. We demonstrate that bacteriophage genomes encoding toxin genes with homology to those found in pathogenic V. cholerae are integrated in V. coralliilyticus genomes. Virulence factors located on chromosomal pathogenicity islands also exist in some strains of V. coralliilyticus. The presence of these genetic signatures indicates virulence in V. coralliilyticus is driven by prophages and other horizontally acquired elements. Screening for pathogens of coral disease should target conserved regions in these elements.Disease is an increasing threat to reef-building corals. One of the few identified pathogens of coral disease is the bacterium  Vibrio are well-known for their roles as pathogens in both terrestrial and aquatic environmentsVibrio are pathogenic2Vibrio cholerae, a bacterium that causes the acute diarrhoeal disease cholera45V. cholerae, TCP (toxin co-regulated pilus) and an exotoxin called cholera toxin (CTX), are both introduced via horizontal gene transfer (HGT) and are encoded by a pathogenicity islandV. cholerae, CTXφ prophage transmits toxin genes among the bacteria that express the TCP prophage receptor. This process of inducing virulence in an otherwise harmless bacterium through prophage infection and integration of the prophage genome into the bacterial host genome is called lysogenic conversionSpecies of the gammaproteobacterium Vibrio species, V. coralliilyticus in particular, as the pathogen responsible for some instances of white syndrome (WS) disease1416Symbiodinium, and/or its photosynthetic pigments from the coral tissue resulting in paling of the coral tissues. If these coral algal endosymbionts are not regained following bleaching, coral mortality is inevitable2 day−1)17The health of coral reefs is declining on a global scale91112V. coralliilyticus in coral disease and bleaching has been attributed to increases in sea surface water temperatures14V. cholerae, has not yet been considered. Inconsistent findings on the role of Vibrio in coral disease/bleaching events have been reported in the literature1622V. coralliilyticus, identified as the causative pathogen of WS in the coral Pocillopora damicornis14V. coralliilyticus encodes for a zinc-metalloprotease that causes cellular damage in coral tissue17V. coralliilyticus strain (P1) between a wild-type and zinc-metalloprotease-deficient mutant in infection experimentsV. coralliilyticus strains may also be at play and contribute to virulence. Moreover, the possible role of a prophage as an alternative driver of virulence in V. coralliilyticus in these studies has not been addressed.Pathogenicity of the coral-associated V. coralliilyticus by examining in silico five publically available V. coralliilyticus whole genome sequences20252627V. coralliilyticus genomes that share similarities to prophages integrated in pathogenic V. cholerae strain genomes and supports our hypothesis.The aim of this study was to assess whether a prophage could potentially drive virulence of V. coralliilyticus associated with corals found in different locations worldwide , ace (accessory cholera enterotoxin) and cep (core-encoded cepin) are requiredzot and ace genes are identified as minor coat proteins of the CTXφ prophage but also exhibit toxic activity by encoding for toxins that play a direct role in disrupting the intestines of humans with cholera diseaserstB gene , a minor capsid protein gene similar to ace  and a gene encoding a Zot-like protein   .V. parahaemolyticus, a pathogen commonly associated with shellfish (Pseudomonas phage CTXφ-like genome found in P1 (V. cholerae prophage K139V. cholerae strains but it is still unclear whether or how this prophage may increase pathogenic host fitnessPseudomonas CTXφ-like prophage in P1 (An intact prophage was detected in the OCN014 genome that shares similarity to a plasmid-like prophage VP882hellfish b and 2. nd in P1 . PHAST33ge in P1 b and 2. ge in P1  online.V. choleraeVibrios to secrete the exotoxin cholera toxin (CTX) and is encoded on a pathogenicity island within the Vibrio chromosomedif-like region that accommodates an insertion site for the prophages CTXφ and f237 of V. choleraeV. parahaemolyticusV. cholerae with the presence of a second pathogenicity island (CPI-II)V. choleraeV. choleraeV. cholerae is physically located close to the CTXφ genomeThe synergistic effect of a number of genes, including toxicity genes introduced by CTXφ, and a series of genes clustered in pathogenicity islands on the bacterial genome result in the evolution of Vibrio choleraeStaphylococcus aureus, Clostridium botulinum, Corynebacterium diphtheriae, Streptococcus pyrogenes and Escherichia coli8Vibrio cholerae from a non-pathogenic to a virulent strain, resulting in often devastating cholera outbreaks among human populations45Vibrio coralliilyticus, and future studies should rigorously address this previously overlooked possibility.Prophage infections can transform ordinary bacteria to virulent pathogens, shown in a wide range of species including Pocillopora damicornis14V. cholerae strains, suggests that similar prophage-driven virulence mechanisms could operate in both the human and coral system. Extracellular zinc-metalloproteases expressed by V. coralliilyticus strain P1 were previously reported to be the driver of tissue damage in WS infections17V. coralliilyticus strains.BAA450 was reported over a decade ago as causing bleaching and tissue necrosis in the coral species Montipora capitataVibrio prophage K139 that is also present in the V. cholerae O1 O395 genome  in white plague diseaseVibrio shiloi in bleaching of the coral Oculina patagonica16V. coralliilyticus in coral bleaching and white syndrome disease141541V. coralliilyticus has generated much interest, but very little is understood about the dynamics of infection. The parallels between the Vibrio cholerae paradigm and an equivalent scenario in corals is highly relevant and comparable, as disease in both human and coral populations are influenced by environmental factors that are being altered through anthropogenic influences and changes in climatic conditions.There have been greater than 30 coral diseases described41Vibrio infections in corals. Previous studies have described the presence of viruses associated with coral diseases5253V. coralliilyticus genomes sequenced to date have been isolated from corals with disease/bleaching symptoms, at present we cannot make comparisons and form conclusions on the presence of these prophages in genomes of V. coralliilyticus isolated from disease-free samples. We anticipate future sequencing efforts to include genomes of V. coralliilyticus from healthy corals, or other such sources, to enable an in-depth comparison. In this present study, we indicate possible signatures to target in V. coralliilyticus to screen for prophages. We envisage our in silico analysis to be a foundation and starting point for future experimental research on the role of lysogenic conversion in pathogenicity of Vibrio and the environmental conditions that potentially trigger virulence. Coral disease is not caused by a single factor but is the result of a complex interplay among biotic and abiotic factors and prophages are a potential central driver of virulence.We encourage further experimental studies into the role of viruses in V. coralliilyticus genomes were downloaded from the NCBI database, March 2015. The V. coralliilyticus strain names and their accession numbers are as follows and summarised in V. coralliilyticus ATCC-BAA-45014V. coralliilyticus P115V. coralliilyticus OCN008V. coralliilyticus OCN014V. coralliilyticus RE98V. coralliilyticus genome sequences were curated and analysed using Ortho MCL v2.0.9V. cholerae representative reference genomes were downloaded from NCBI: V. cholerae O1 2010EL-1786V. cholerae O1 O395V. cholerae El Tor N16961V. coralliilyticus genomes.For interrogative and comparative analysis, whole genome sequences of five published How to cite this article: Weynberg, K. D. et al. From cholera to corals: Viruses as drivers of virulence in a major coral bacterial pathogen. Sci. Rep.5, 17889; doi: 10.1038/srep17889 (2015).


The activation of nuclear factor kappa B (NF-κB) signaling has a central role in the development of adult T-cell leukemia/lymphoma (ATL) and many other cancers. However, the activation mechanism of the NF-κB pathways remains poorly understood. Recently, we reported that N-myc downstream-regulated gene 2 (NDRG2) is a negative regulator of the phosphoinositide 3-kinase (PI3K)/AKT pathway by promoting the active dephosphorylated form of PTEN at its C-terminus via the recruitment of PP2A. Additionally, the down-regulation of NDRG2 expression promotes the inactive phosphorylated form of PTEN, which results in constitutively active PI3K/AKT signaling in various cancer cell types. Here, we investigated the involvement of NDRG2 in modulating NF-κB signaling. The forced expression of NDRG2 in ATL cells down-regulates not only the canonical pathway by inhibiting AKT signaling but also the non-canonical pathway by inducing NF-κB-inducing kinase (NIK) dephosphorylation via the recruitment of PP2A. Therefore, NDRG2 works as a PP2A recruiter to suppress not only PI3K/AKT signaling but also NF-κB signaling, which is particularly important in host defenses or immune responses to Human T-cell leukemia virus type 1 (HTLV-1) infection. Furthermore, the loss of NDRG2 expression might play an important role in the progression of tumor development after HTLV-1 infection. Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus associated with an aggressive form of CD4+ T-cell leukemia termed adult T-cell leukemia/lymphoma (ATL). Although the molecular mechanism of leukemogenesis has not yet been completely elucidated, aberrations in signal transduction in HTLV-1-infected T cells play an important role in the development of the disease. Among them, nuclear factor kappa B (NF-κB) is constitutively activated in HTLV-1-infected T and ATL cells, and activation of the NF-κB signaling pathway is dependent or independent of HTLV-1 Tax expression along with many molecular events. Moreover, several studies have suggested that NF-κB plays an essential role in the pathogenesis of ATL and is one of important molecular targets for the prevention and treatment of ATL1234NF-κB comprises a family of transcription factors that play critical roles in inflammation, immunity, cell proliferation, differentiation, survival and cancer development6891011Recently, we identified N-myc downstream-regulated gene 2 (NDRG2) as a novel PTEN-binding protein that recruits protein phosphatase 2A (PP2A) to regulate the dephosphorylation of PTEN at Serine380, Threonine382 and Threonine383 in the C-terminal domain of PTEN. NDRG2 expression was down-regulated in ATL via genetic deletion and DNA promoter methylation, resulting in the activation of the PI3K-AKT pathway through increased PTEN phosphorylation at its C-terminus, representing its inactive form161820Therefore, we investigated whether NDRG2 expression could modulate NF-κB signaling in ATL and other solid cancers. In this manuscript, the forced expression of NDRG2 remarkably inhibited the canonical NF-κB pathway by inhibiting AKT signaling in ATL and solid cancer cells. In the non-canonical pathway, NDRG2 expression dephosphorylates NIK, which is overexpressed and highly phosphorylated (active) in ATL, through the recruitment of PP2A. Therefore, NDRG2 works as a recruiter of PP2A to suppress the important signaling pathways for defending against infection or immune responses, and the loss of NDRG2 expression might play an important role in the progression of tumor development.We recently showed that NDRG2 regulates the phosphorylation status of PTEN at its C-terminus by recruiting PP2A and suppresses PI3K/AKT signaling by dephosphorylating PTEN, the active form of PIP3 phosphatase20Because NDRG2 suppressed PI3K/AKT signaling through PTEN phosphorylation8To confirm the results, we established mouse embryonic fibroblasts (MEFs) from Ndrg2-deficient and wild-type mice, and these MEF cell lines were analyzed for the activation of PI3K/AKT and NF-κB signaling stimulated by LPS or TNFα. As shown in in vivo. Therefore, to explore whether NDRG2 regulates the canonical and non-canonical NF-κB pathways in ATL cell lines, we assessed the activation status of the canonical and non-canonical NF-κB pathways in three HTLV-1+ cell lines with various levels of HTLV-1 Tax expression  and whether NDRG2-introduction to these cell lines might affect the activation status of NF-κB signaling, including the NF-κB transcriptional activity. The canonical pathway was activated in all three HTLV-1+ cell lines, with high phosphorylation levels of IKK-Ser176/180 and IκBα-Ser32/36 that were not dependent on the expression level of Tax; additionally, the suppression of IKK phosphorylation and the accumulation of IκBα protein were detected in all NDRG2+ cell lines 2324252627ll lines . Furtherll lines , suggestll lines .In the next experiment to investigate whether the activation of the non-canonical pathway is regulated by PI3K/AKT signaling, we measured the activity of the non-canonical pathway after the treatment of the three HTLV-1+ cell lines with the PI3K inhibitor LY294002 and Wortmannin. As shown in Because we recently demonstrated that NDRG2 recruits PP2A to facilitate PTEN dephosphorylation, resulting in the suppression of phosphorylated AKTIn HTLV-1+ cells, NIK was overexpressed to activate the non-canonical pathway by down-regulating the negative regulator miR3129In the next experiment, we assessed whether NDRG2 expression might affect the phosphorylation status of NIK along with activation of the non-canonical pathway in three HTLV-1 cell lines . The phosphorylation of NIK-Thr559 was significantly decreased in NDRG2-expressing cells, although the NIK protein level was maintained under the introduction of NDRG2 . To confTo confirm the direct binding of NIK to NDRG2, co-immunoprecipitation of endogenous NIK with transfected Flag-NDRG2 and western blotting were performed in HUT102 cells treated with MG132, using anti-Flag and anti-NIK antibodies, and vice versa. The same experiment was performed in 293T cells by co-transfecting FLAG-tagged NDRG2 and HA-tagged NIK. In each experiment, NIK and NDRG2 could be co-immunoprecipitated in both cell lines .In the next experiment, to identify the serine/threonine phosphatase for NIK-Thr559, we assessed whether treatment with the phosphatase inhibitor OA modulates the dephosphorylation reaction resulting from NDRG2 expression. In this experiment, OA treatment prevented the NIK-Thr559 dephosphorylation reaction through the overexpression of NDRG2, suggesting that PP2A or other members might regulate the phosphorylation status of NIK with NDRG2 . Becausein vitro NIK dephosphorylation assay using synthetic phospho-Thr559 peptide . Myc-tagged PP2Ac significantly dephosphorylated the phospho-Thr559 peptide; however, PP1c or PP5c could not dephosphorylate the phospho-Thr559 peptide  were obtained form Sigma-Aldrich , MG132 was obtained from LifeSensors  and Okadaic Acid (OA) and Tumor Necrosis Factor α (TNFα) were obtained from Wako . The transfection reagent HilyMax and the cell proliferation/cell toxicity kit Cell Counting Kit-8 were purchased from DOJINDO . Primary antibodies were purchased as follows: goat polyclonal antibodies against NDRG2 (E20) and phospho-NIK (Thr559), rabbit polyclonal antibodies against IκBα (C-21), NEMO (FL-419), and NIK (H-248), and a mouse monoclonal antibody against p100/p52 (C-5) from Santa Cruz ; rabbit polyclonal antibodies against AKT (9272), phospho-AKT (Ser473) (D9E), PTEN (138G6), and phospho-PTEN (Ser380/Thr382/Thr383) (9554), rabbit monoclonal antibodies against phospho-IKKα/β (Ser176/180) (16A6), GSK3β (27C10), phospho-GSK3β (Ser9) (D85E12) and PP2A C Subunit (PP2Ac) (52F8), and a mouse monoclonal antibody against phospho-IκBα (Ser32/36) (5A5) from Cell Signaling Technology ; and mouse monoclonal antibodies against β-actin (AC-15) and Flag M2 (F3165) from Sigma-Aldrich. The mouse monoclonal antibody against Tax (MI73) was a kind gift from Dr. M. Matsuoka .The Flag-tagged NDRG2 expression vector (NDRG2/pCMV26) has been described elsewhereBlood samples were obtained with informed consent with approval by the Institutional Review Board of the Faculty of Medicine, University of Miyazaki. The collection of ATL cells from the patients and CD4+ lymphocytes from volunteers was performed as described previouslyThe Ndrg2-deficient mice were previously described2 at 37 °C. The HTLV-1 negative cell lines, IL2-independent ATL cell lines, and cell lines transfected with HTLV-1 were maintained in the same medium without IL2. Other cells were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% FBS.Jurkat and MOLT4 are HTLV-1-negative human T-ALL cell lines. KOB and KK1 are IL2–dependent ATL cell lines. SU9T-01, ED and S1T are IL2-independent ATL cell lines. MT2 and HUT102 are human T-cell lines transformed by HTLV-1 infection. Jurkat and MOLT4 cells were obtained from the Fujisaki Cell Center, Hayashibara Biochemical Laboratories . MT2 and HUT102 cells were a kind gift from Dr. Kuan-Teh. Jeang . KOB and KK1 cells were kind gifts form Dr. Y. Yamada . SU9T-01 and S1T cells were kind gifts from Dr. N. Arima . ED was a kind gift from Dr. M. Maeda . Human embryonic kidney 293T cells were obtained from RIKEN Bioresource Center . IL2-dependent ATL cell lines were maintained in PRMI1640 medium supplemented with 10% fetal bovine serum (FBS) and 50 JRU per ml recombinant human IL2 (Takeda) in a humidified atmosphere of 5% COCells were harvested from 6-well plates. For the extraction of protein in tissues, excised organs were homogenized in NP-40 lysis buffer  supplemented with a proteinase inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor tablet . The lysate was centrifuged for 10 min at 15000 x g (maximum) at 4 °C, and the supernatant was then collected. The protein concentration was determined by BCA protein assay  with bovine serum albumin (BSA) standard. Equal amounts of protein samples were loaded, separated by SDS-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane . The membranes were blocked in PBS–Tween (0.1%) with 1% BSA or 5% nonfat dried milk and were then probed with the primary antibodies diluted PBST-BSA, 5% nonfat dried milk or Can Get Signal Buffer . The bands were detected using Lumi-light Plus kit (Roche) and LAS-3000. Band intensities were quantified with the NIH Image J software. All primary antibodies were used at a dilution of 1:1000.The lysates were incubated with 1 μg of the indicated antibodies or normal IgG with constant rotation at 4 °C overnight and were then incubated with Protein G Sepharose 4 Fast Flow  or Flag M2 Affinity Gel (Sigma-Aldrich) for 2 h. The immunoprecipitates were washed 3 times with PBS, and the bound proteins were denatured with SDS sample buffer. Each sample was subjected to western blotting.Cells were transfected with pNF-κB-Luc and pRL-TK vectors using Amaxa or HillyMax. The NF-κB transcriptional activity was measured using a dual luciferase assay kit  according to the manufacturer’s instructions with a luminometer .Total RNA was isolated from cells using TRIzol reagent (Invitrogen). The cDNA was synthesized from 1 μg RNA with the RNA PCR kit with oligo(dT) primers (TaKaRa) and was then used as a template for RT-PCR analysis. Quantitative real-time PCR (qPCR) was carried out using the Applied Biosystems StepOne Real Time PCR System (Applied Biosystems) and the GeneAce SYBR qPCR Mixα . The expression levels of target genes were normalized by β-actin mRNA.2, 8 mM MgCl2, 1 mM dithiothreitol and 100 μg/ml BSA) at 30 °C for 60 min. The reactions were terminated by the addition of 40 μl of TE buffer  and 100 μl of BIOMOL Green reagent, and the absorbance was read at 620 nm after incubation for 20 min at room temperature.The NIK-Thr559 phosphopeptide (TGDYIPGpTETHMAPE) was obtained from the TORAY Research Center . The release of inorganic phosphates from the phosphopeptide was assessed using a malachite green assay kit . Immunoprecipitated samples were incubated with 100 μM NIK-Thr559 phosphopeptide in 10 μl of phosphatase assay buffer .


Thalassemia is an inherited genetic disorder requiring multiple transfusions to treat anemia caused by low hemoglobin levels. Thus, thalassemia patients are at risk for infection with blood-borne pathogens, including human T-cell lymphotropic viruses (HTLV) that are transmitted by transfusion of cellular blood products. Here, we examined the prevalence of HTLV among 234 US thalassemia patients using sera collected in 2008. Sera were tested for antibodies to HTLV-1/2 using EIA and a confirmatory Western blot (WB) that differentiates between HTLV-1 and HTLV-2. Demographic and clinical information were collected at study enrollment, including HIV and HCV status. Three patients (1.3%) were WB-positive, two were HTLV-1 and one could not be serotyped as HTLV-1/2. All three HTLV-positive persons were HIV-1 negative and only one was HCV seropositive. The HTLV seroprevalence was higher than that of HIV-1 (0.85%) and lower than HCV (18.8%) in this population. All three patients (ages 26-46 years) were diagnosed with beta-thalassemia shortly after birth and have since been receiving multiple transfusions annually. Two of the HTLV-positive patients confirmed receiving transfusions before HTLV blood screening was implemented in 1988. We identified a substantial HTLV-1 seroprevalence in US thalassemia patients that is much greater than that seen in blood donors. Our findings highlight the importance of HTLV testing of patients with thalassemia and other diseases requiring multiple transfusions, especially in recipients of unscreened transfusions. In addition, appropriate counseling and follow-up of HTLV-infected patients is warranted.


The etiology of fibromyalgia and chronic fatigue syndrome (FM/CFS) is currently unknown. A recurrent viral infection is an attractive hypothesis repeatedly found in the literature since it would explain the persistent pain and tiredness these patients suffer from. The initial striking link of two distinct orphan retroviruses: the gamma retroviruses murine leukemia virus (MLV)-related virus and the delta retrovirus T-lymphotropic virus type 2 (HTLV-2) to chronic fatigue have not been confirmed to date.env gene was amplified in 20% of samples tested . Unexpectedly, no PCR amplifications from independent gDNA preparations of the same individuals were obtained. None of the positive samples showed presence of contaminating murine sequences previously reported by other investigators, neither contained additional regions of the virus making us conclude that the initial env amplification came from spurious air-driven amplicon contaminants. No specific HTLV-2 sequences were obtained at any time from any of the 154 quality-controlled gDNA preparations screened.Genomic DNA (gDNA) from 75 fibromyalgia patients suffering from chronic fatigue and 79 age-matched local healthy controls were screened for the presence of MLV-related and HTLV-2 related proviral sequences. The XMRV Previous associations between MLV-related or HTLV-2 retrovirus infection with chronic fatigue must be discarded. Thus, studies showing positive amplification of HTLV-2 sequences from chronic fatigue participants should be revised for possible undetected technical problems.To avoid false positives of viral infection, not only extreme precautions should be taken when nested-PCR reactions are prepared and exhaustive foreign DNA contamination controls performed, but also consistent amplification of diverse regions of the virus in independent preparations from the same individual must be demanded.The fact that our cohort of patients did not present evidence of any of the two types of retroviral infection formerly associated to chronic fatigue does not rule out the possibility that other viruses are involved in inciting or maintaining fibromyalgia and/or chronic fatigue conditions. Fibromyalgia and Chronic fatigue Syndrome (FM/CFS) are characterized by long-lasting debilitating fatigue, often accompanied by widespread muscle pain. Its incidence is rising and ranges between 0.5-6% with high prevalence in females . The higCurrently the FM/CFS diagnosis is made solely on clinically grounds, as no biological markers associated with the disease have been found. Alteration in cytokine profiling, decreased function of natural killer (NK) cells, presence of autoantibodies and reduced response of T cells to mitogens and other specific antigens have been reported in these patients -4. The oet al. reported the finding of a novel gammaretrovirus named xenotropic murine leukemia virus-related virus (XMRV) in about 67% (68/101) of CFS patient DNA samples prepared from peripheral blood mononuclear cell (PBMC) compared to only 3.6% (5/218) of the samples prepared from healthy controls ). The latter was amplified with the GAPDH specific primers designed Lombardi env set of primers under these same amplification conditions (data not shown). Although most bands had a smaller size, some of them showed a molecular weight close to the expected gag MLV or XMRV product size. However, sequencing of these PCR amplified fragments confirmed that all of them corresponded to unspecific amplification of human genomic sequences.In order to determine whether the samples analyzed could contain other retroviruses partially related to the MLV virus, the nested PCR amplifications were performed at lower annealing temperatures up to 5°C below primers melting temperature (Tm), which would allow annealing to, and thus amplification of non-perfect matches. Under these permissive amplification conditions, several bands were obtained with both the GAG-I-F and GAG-I-R set and more abundantly with the NP116/NP117 set Figure . No bandAn additional objective of the present study was to determine whether the participating fibromyalgia patients suffering of chronic fatigue were infected with the HTLV-2 retrovirus formerly related to chronic fatigue patients .Using a similar nested PCR based approach used to screen XMRV and MLV-related sequences the gDNA samples of patients and controls were rescreened; this time HTLV-2 sequence specific primers under formerly described conditions  were usegag sequences spiked into 1 μg of human gDNA showed that our nested-PCR assay detection threshold was 1–10 viral copies  or the healthy blood donor controls (n = 79) showed amplification of a HTLV2-specific sequence Table . In ordes Figure , panel CThis is the first report to our knowledge that evaluates the presence of the both families of retrovirus formerly associated with chronic fatigue: the gamma retroviruses MLV-related and the delta retrovirus HTLV-2 in a cohort of patients suffering from fibromyalgia and chronic fatigue.At present enough evidence exists to support the inexistence of a natural form of the XMRV virus in humans ,27,28. HOpposite to the actively studied relationship of the XMRV to chronic fatigue, the association or non-association of the orphan HTLV-2 retrovirus to the disease has not been clarified. The initial study which used PCR amplification of HTLV-2 proviral sequences followed by probe hybridization assays to find an association of HTLV-2 infection to CFS was followed by only a few negative studies -18. EvenThis, to our knowledge, constitutes the study with the largest number of patients n = 75) of fibromyalgia suffering from chronic fatigue examined for infection by HTLV-2. None of the samples assayed showed specific amplification of HTLV-2 or related sequences at any time making us conclude a non-association between HTLV-2 retroviral infection and the chronic fatigue condition of our patients. As we already proposed  HTLV-2 a5 of fibrgag band obtained by Gow et al.  with the Murine Leukemia Virus MCF1233 complete genome [Genbank: U13766]. For the first round PCR the primers selected were XMRV-env-outer-F 5′-TGTGAGACCACTGGACAGGC-3′and XMRV-env-outer-R 5′-GTAAGTCCTCCCAACAGCAG-3′ and for the nested PCR the primers XMRV-env-inner-F 5′- ACGCGGGTAAAAGGGCCAGC-3′and XMRV-env-inner-R 5′-AAGCCCAAATGGTCCCGGCG-3′ were used.Primers to amplify conserved gag XMRV sequences were the same used by Urisman et al. and Lombardi et al. to detect 413-bp XMRV gag sequences in prostate cancer and CFS patients, 419 F and 1154R (outer) and GAG-I-F and GAG-I-R respectively  (Click here for file


The proviral DNA of BM HTLV-1 CD4+ T cells revealed the presence of integrated provirus. The number of fibroblast progenitor cells, referred as colony-forming units-fibroblasts (CFU-F), were lower in HTLV-1 infected individuals (CFU-F per 5 x 105 cells in HAM/TSP (2.1±1.6), HAC (7.5±2.1) compared to healthy controls (10.4±1.1). HTLV-1 BM mesenchymal stem cells (MSC) isolated showed surface expression of CD105, CD73, and CD90, absence of hematopoietic markers such as CD45, CD34, and CD14; and in vitro differentiation to adipogenic and osteogenic cells. Proviral HTLV-1 DNA was detected in MSC from all HTLV-1 patients and the levels ranged from 2.5 to 25.7 copies per 105 cells. HTLV-1 MSC was also positive to HTLV-1 p19 protein as determined by confocal microscopy. The confirmation of active viral replication was performed in the concentrated supernatant obtained from MSC by real-time PCR of the pol and gag HTLV-1 gene. In conclusion, we suggest that HTLV-1 could infect and replicate in human MSC possibly by the contact with infected CD4+ T-cells.The characterization of cell types that are susceptible to HTLV-1 infection is essential to understand of the biology of virus infection and the pathophysiology of HTLV-related diseases. In the present study, we investigated bone marrow (BM) cells from asymptomatic carriers (HAC) and symptomatic HTLV-1 individuals. Initially, we observed an infiltrated of CD4FUNDHERP, CTC/FAPESP, INCTC, and CNPq.


MicroRNAs (miRNAs) are small non-coding RNAs that regulate protein expression. HTLV-1 is able to promote oncogenesis in T cells by altering the expression of miRNAs involved in the control of cell-cycle. It is not known whether HTLV-1 deregulates miRNAs expression in cells of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients. To asses if HTLV-1 infection might alter the expression of miRNAs involved in inflammatory response, we evaluated the expression of 84 miRNAs involved in inflammatory process in asymptomatic HTLV-1 carriers (AC) and HAM/TSP patients using the miScript miRNA PCR Array Human Inflammatory Response & Autoimmunity (SABioscience). For this purpose, fourteen HTLV-1-positive individuals were selected and classified into three groups: five asymptomatic carriers (AC), 4 HAM/TSP patients with EDSS score of 1-5 (=mild HAM/TSP), and 5 HAM/TSP patients with EDSS score of 5.5-9 (=severe HAM/TSP). Total RNA was isolated from PBMCs and pooled according to the groups. qBase software was used for normalization, ANOVA was used for comparisons and False Dicosvery Rate to correct for multiple comparisons. We found nine differentially expressed miRNAs between AC and HAM/TSP patients (mild and severe HAM/TSP). Twelve miRNAs were differentially expressed among mild HAM/TSP, severe HAM/TSP and AC groups. These findings support results previously reported in Adult T-cell leukemia/lymphoma (ATLL) cells, in which hsa-miR-145, miR-130a, miR181a and miR101a were found to be down-regulated and miR-30d was found to be up-regulated in comparison to those of healthy donors. Further analysis to confirm these findings are needed.


Adult T-cell Leukemia/Lymphoma is an aggressive malignancy caused by HTLV-1 infection. HTLV-2 is genetically related to HTLV-1, but does not cause a malignant disease. The HTLV-1 Tax (Tax-1) viral transactivator is required for HTLV-1 expression and modulates the classical and non-canonical NF-κB pathways. Interaction of Tax-1 with IKKγ/NEMO results in constitutive activation of NF-κB in HTLV-1 infected cells, and contributes to HTLV-1-driven leukemogenesis. Tax-1 transgenic mice develop leukemia, lymphomas or spontaneous osteolytic bone metastases demonstrating Tax-1 oncogenic properties in vivo. However, the cellular pathways and the partners involved in vivo have not been described. HTLV-2 Tax (Tax-2) has properties different from Tax-1, including different post-translational modifications and different intracellular localization. Thanks to the availability of collection of mutants and RNAi lines, Drosophila melanogaster allows simple and exhaustive genetic screens. We generated transgenic Drosophila models expressing either Tax-1 or Tax-2 in the compound eye and plasmatocytes (leukocyte-like cell). We demonstrate that Tax-1 but not Tax-2 induces a perturbation of the crystalline array of the ommatidia and increase in plasmatocyte proliferation indicating that Tax-1 but not Tax-2 has transforming potential in Drosophila. We further show that induction of the eye phenotype is primarily dependent on Kenny, the Drosophila homolog of IKKγ/NEMO, upstream of Relish (NF-κB) activation. Using this model we were able to identify a novel post-translational modification which Tax-1 undergoes in addition to the well-known ubiquitylation and SUMOylation. This novel Tax post-translational modification was confirmed in HTLV-I transformed cell lines. Altogether, these results show that the Drosophila system is useful for dissecting the molecular mechanisms of HTLV-1-induced cell transformation in vivo.


We found that some clones proliferated distinctively in monkeys with higher proviral load. T lymphocytes expressing Tax in the peripheral blood were largely CD4+. Notably, one of the monkeys surveyed in this study developed T-cell lymphoma in the brain, indicating that STLV-1 is oncogenic in Japanese macaques. We also assessed the molecular function of STLV-1 Tax and STLV-1 bZIP factor (SBZ). STLV-1 Tax activated NFAT, AP-1, canonical Wnt and canonical NF-kappa B pathways, whereas SBZ suppressed those signaling pathways. SBZ enhanced TGF-beta signaling, but STLV-1 Tax suppressed it. These findings suggest that STLV-1 Tax and SBZ have similar functions to their counterparts of HTLV-1. In addition, we found that administration of anti-CCR4 antibody, which is currently used in Japan for the treatment of ATL patients, efficiently reduced proviral load in STLV-1-infected Japanese macaques. Our study provides the evidence that Japanese macaques naturally infected with STLV-1 correspond to HTLV-1 carriers and are a suitable animal model to investigate the pathogenesis of HTLV-1 and novel therapeutic strategies.Various non-human primates are the natural hosts of simian T-cell leukemia virus type 1 (STLV-1). In the present study, we analyzed Japanese macaques naturally infected with STLV-1, and evaluated them as an animal model for HTLV-1 research. Approximately 60% of individuals in the colony are seropositive for STLV-1. Clonal proliferation of STLV-1