S-glutathionylation, the reversible formation of mixed disulfides between glutathione(GSH) and cysteine residues in proteins, is a specific form of post-translational modification that plays important roles in various biological processes, including signal transduction, redox homeostasis, and metabolism inside cells. Experimentally identifying S-glutathionylation sites is labor-intensive and time consuming, whereas bioinformatics methods provide an alternative way to this problem by predicting S-glutathionylation sites in silico. The bioinformatics approaches give not only candidate sites for further experimental verification but also bio-chemical insights into the mechanism of S-glutathionylation. In this paper, we firstly collect experimentally determined S-glutathionylated proteins and their corresponding modification sites from the literature, and then propose a new method for predicting S-glutathionylation sites by employing machine learning methods based on protein sequence data. Promising results are obtained by our method with an AUC (area under ROC curve) score of 0.879 in 5-fold cross-validation, which demonstrates the predictive power of our proposed method. The datasets used in this work are available at http://csb.shu.edu.cn/SGDB. S-Glutathionylation, the reversible formation of protein mixed disulfides between cysteine residues and glutathione(GSH), is an important form of posttranslational modification that occurs during oxidative stress as well as under normal physiological conditions S-glutathionylation is induced along with other forms of thiol oxidation . Since S-glutathionylation is a reversible modification, likely catalyzed by glutaredoxins (Grx), it can serve to protect critical protein thiols from irreversible oxidation and inactivation. Operationalized proteins also serve as a storage form of GSH under oxidative stress, where the oxidized glutathione (GSSG) is otherwise exported from the cell. It has been recently hypothesized that glutathionylation is a general mechanism for protein function and cell signaling, similar to the protein phosphorylation system mediated by kinases and phosphatases S-glutathionylation has been shown to cause either inhibition or activation of a number of proteins. Generally, these proteins fall into the following functional categories: metabolism and energy, cell signaling (particularly kinases and phosphatases), calcium homeostasis, redox homeostasis, protein folding and degradation, and the cytoskeleton  S-glutathionylation with pathogenesis of many human diseases including metabolic disorders, cardiovascular diseases, lung diseases, cancer, and neurodegenerative diseases S-glutathionylation proteins, including spectrophotometric assays, high performance liquid chromatography assays, liquid chromatography-mass spectrometry, etc S-glutathionylation proteins have been identified, but only a minority of them have their S-glutathionylation sites identified. Experimentally identifying S-glutathionylation sites is a labor-intensive and time consuming procedure. Despite some bioinformatics approaches have been proposed to predict catalytic redox-active cysteine S-glutathionylation sites due to the scarcity of S-glutathionylation sites and protein structure data.Recently, various experimental methods were used to identify S-glutathionylation sites by employing machine learning methods based on protein sequences. Despite the incompleteness of protein structures, it is found that the flanking residues of a functional site in sequence can give the context information of this residue in protein structure and are widely used to predict post-translational sites S-glutathionylation sites based on protein sequences. Firstly, all the S-glutathionylation proteins and their corresponding modification sites are manually collected from the literature. Secondly, a series of classifiers are built to predict S-glutathionylation sites based on support vector machines (SVMs). Especially, different features are extracted from protein sequences for prediction of S-glutathionylation sites. Results obtained in 5-fold cross-validation demonstrate the effectiveness of our method with an AUC score of 0.879. To our best knowledge, this is the first work that use bioinformatics methods to predict S-glutathionylation sites, which can provide biologists putative S-glutathionylation sites for future experimental verification.In this paper, we present a bioinformatics framework to predict S-glutathionylation sites. To validate our proposed method, 5-fold cross-validation was utilized for our collected S-glutathionylation site dataset with following indexes.In this work, each cysteine residue and its flanking sequence was represented as a feature vector (see Methods). In this way, the supervised classifier can be constructed to predict S-glutathionylation. In particular, the results obtained by reduced amino acid triplet prove that the amino acid triplet provides spatial context information of cysteine without considering the specific direction of amino acid triplet. The good performance of binary amino acid profile may attribute to its position specific information on amino acids, while other descriptors fail to describe the neighborhood information of cysteine and therefore lead to poor results. Furthermore, S-glutathionylation sites.With the feature vectors available, for each feature extraction method, a classifier was constructed. In literature, different window sizes has been used to predict post-translational modification sites. For example, the upstream/downstream seven residues of phosphorylation site were considered (i.e. the window size is15) in GPS In constructing SVM classifier, there are generally three types of kernels that can be used, including linear kernel, polynomial kernel and radial basis function kernel (i.e. Gaussian kernel). In this work, we compared the performances of different kernels. S-glutathionylation motifs, which can be used to predict new S-glutathionylation proteins and provide insights into the functions related to S-glutathionylation sites. For this, we firstly employed PROSITE S-glutathionylation sites (positive samples) to search for conserved sequence motifs. Then, we obtained the function annotations of these motifs from InterPro database S-glutathionylation due to the close functional connections between phosphorylation and S-glutathionylation S-glutathionylation because of the key role of S-glutathionylation in cellular oxidative stress regulation. These results indicate that PS00112 and PS00071 are probably catalytic motifs of GSTs, which may help to understand the mechanism of S-glutathionylation. With more S-glutathionylation proteins coming in the future, we believe more such conserved S-glutathionylation motifs will be found.In literature, it is found that some post-translational modification sites are conserved and contained in motifs that are found in different proteins. Therefore, in this work, we investigated whether there exist any characteristic S-glutathionylation is a reversible post-translational modification that is important to many biological processes. Identification of protein S-glutathionylation sites is essential to the understanding of the functions of proteins. In this work, a framework by employing machine learning approach was developed to predict S-glutathionylation sites. With only protein sequence information, our method can obtain promising results with an AUC score of 0.879. Our method can provide biologists candidate S-glutathionylation sites for future experimental verification.S-glutathionylation. For example, low thiol pKa and big surface exposure of cysteine residue were considered to contribute to S-glutathionylation. The correlation between accessible surface area (ASA) of cysteine residue and susceptibility of S-glutathionylation was derived from the hypothesis that electrostatic interactions were involved in S-glutathionylation S-glutathionylation susceptibility of four Cys residues in cyclophilin A  and showed that Cys62 which exposes a smaller surface of its side chains than Cys161 is the target of S-glutathionylation while Cys161 is not. Cysteines with thiolate anions at a lowering of pKa value are inclined to have electrostatic interactions with neighboring positively charged amino acid residues. Cytoplasmic proteins that contain this type of cysteine are redox-sensitive and can form a mixed disulfide with GSH. Therefore, a cationic environment renders the protein thiol group highly reactive and particularly susceptible to S-glutathionylation In the literature, structural information is also found important to S-glutathionylation cysteine and non-S-glutathionylation cysteine. We collected the structure information for 39 S-glutathionylation proteins by querying the PDB database with both their names and primary sequences. As a result, 36 proteins were retrieved with their 3D structures from PDB database and were kept for further analysis. Subsequently, PROPKA 2.0 In this work, we also investigated the ASA and pKa value of S-glutathionylation cysteine and non-S-glutathionylation cysteine, where the ASA of S-glutathionylation cysteine tends to be larger than that of negative samples. In It is found that cysteine thiols with a bigger ASA are more likely to form a mixed disulfide with GSH. In contrast, if cysteine thiols are buried in protein, the formation of disulfide bond will be difficult. S-glutathionylation cysteines and non-S-glutathionylation cysteines. In our dataset, 15 of 47 (31.9%) positive samples have a pKa value S-glutathionylation cysteine tend to have lower pKa value compared to non-S-glutathionylation cysteine with a significant S-glutathionylation sites. With more structural data available in the future, our method can be easily extended to include the structural information to improve prediction accuracy.The above results imply that structural information can indeed help to predict S-glutathionylation proteins were collected by employing text mining techniques on published papers retrieved from PubMed. Furthermore, S-glutathionylation sites were manually identified and used as positive samples. Cysteines that were not validated to undergo S-glutathionylation in experiment were excluded from the database. To construct a supervised classifier for prediction of new S-glutathionylation sites, we randomly selected some cysteines except for known S-glutathionylation sites from the S-glutathionylation protein as negative samples, and the number of negative samples in one S-glutathionylation protein is at most three times of that of positive samples in the same protein so that the balance between positive and negative samples is ensured.S-glutathionylation proteins, where 75 cysteines undergo reversible S-glutathionylation and 158 cysteines that are not susceptible to S-glutathionylation. Since the S-glutathionylation of cysteine is determined by its position in the protein spatial structure, the flanking sequences of cysteine were taken into account and regarded as the spatial neighborhood of cysteine due to the incompleteness of protein structure data. Finally, the data set consists of 75 positive samples and 158 negative samples, where some cysteines in the negative samples were discarded due to their terminal positions in the protein sequence.In total, the data set contains 39 To be used as input for a classifier, each cysteine should be represented as a feature vector. In this work, different feature extraction methods were employed to describe a peptide sequence. S-glutathionylation. For each sample, we summed up the values of all amino acids within the sample and got a 10-dimensional vector. The first type of sequence descriptors considered in feature extraction is the physicochemical features of amino acids, which were taken from the AAIndex database We next consider two sequence profiles Bi-profile Bayes and binary profile. The Bi-profile Bayes method was firstly applied to predict methylation sites and provided significant improvement compared to other methods Binary profile is a simple encoding method that utilizes the position-specific information of each amino acid, and has been successfully used to predict the caspase cleavage sites Since all structural and functional information are encoded by amino acids in protein sequences, three different feature extraction approaches were used to describe each sample based on their amino acid compositions, including single amino acid composition, pairwise amino acid composition and triplet-wise amino acid composition.For the single amino acid composition, the occurrence frequency of each amino acid in the sample was used to encode a sample. For example, a sample will be represented as a 20-dimensional vector if we consider the 20 amino acids in nature. In this work, except for the natural amino acids, the 20 amino acids can be grouped into different clusters based on distinct properties, thereby leading to reduced amino acids. Here, the 3 reduced amino acids and 7 reduced amino acids were used For pairwise amino acid composition, all possible combinations of two amino acids were considered and the occurrence frequency of each amino acid pair within the sample was used to describe the samples. As a result, each sample can be represented as a 400-dimensional feature vector. This encoding approach has been widely used in literature For triplet-wise amino acid composition (ThrAA), all possible combinations of three amino acids were considered and the occurrence frequency of each amino acid triplet within the sample was used to describe the sample. The amino acid triplets was proved to be an effective descriptor of proteins for predicting protein-protein interactions As the values of 10 physicochemical properties are described in different range, each feature value is normalized as following:Table S1The effects of different window sizes on SVM performance.(DOC)Click here for additional data file.Table S2The values of the ten physicochemical properties for each amino acid. NA: Number of atoms; NE: Number of electrostatic charge; NP: Number of potential hydrogen bonds; HB: Hydrophobicity; HL: Hydrophilicity; PP: Propensity; IP: Isoelectric points; MA: Mass; EN: Expected number of contacts within 14 Å sphere; EI: Electron-ion interaction potential.(DOC)Click here for additional data file.


HTLV-I is associated to Adult T-cell leukemia (ATL) characterized by genetic disorders and an altered mRNA expression. Individuals who have a coinfection between HTLV-I and Strongyolides stercoralis (SS) might have an increased risk to develop ATL. The mechanism for the synergism between these two infectious agents is unclear. In this study we evaluate the mRNA expression in Peripheral Blood Mononuclear Cells (PBMC) from three different groups, to determine mRNA profiles similarities or divergences among patients with a)ATL, b)HTLV-I-SS and c)Asymptomatic carriers (AC).mRNA expression of eighty genes involved in the DNA damage pathway (Sabiosciences-Qiagen) were evaluated among HTLV-1 positive patients: a)ATL (n=6), b)HTLV-I-SS (n=8) and c)Asymptomatic carriers(n=6). RNA was extracted from PBMCs and analyzed by using PCR superarray plates from Sabiosciences-Qiagen, one plate was used to analyze 80 genes for every patient. Sabiosciences sample sheet was used for calculations.The number of genes differentially expressed among these groups was: a) nine genes between ATL and AC, P values range 0.045 to 0.0073, b) nine genes between HTLV-I—SS and AC, P values range 0.0497 to 0.0098 and c) two genes between ATL and SS, P values 0.027 and 0.046.Two genes, the G-2 and S-phase expressed 1 gene and the Fanconi anemia, complementation group G gene were commonly increased in ATL and HTLV-I—SS compared to AC. Similar profiles of mRNA expression regarding of DNA damage pathway were observed among ATL and HTLV-I—SS groups, suggesting some shared regulatory mechanisms during infection in vivo.


Cyanide-resistant non-phosphorylating respiration is known in mitochondria from plants, fungi, and microorganisms but is absent in mammals. It results from the activity of an alternative oxidase (AOX) that conveys electrons directly from the respiratory chain (RC) ubiquinol pool to oxygen. AOX thus provides a bypath that releases constraints on the cytochrome pathway and prevents the over-reduction of the ubiquinone pool, a major source of superoxide. RC dysfunctions and deleterious superoxide overproduction are recurrent themes in human pathologies, ranging from neurodegenerative diseases to cancer, and may be instrumental in ageing. Thus, preventing RC blockade and excess superoxide production by means of AOX should be of considerable interest. However, because of its energy-dissipating properties, AOX might produce deleterious effects of its own in mammals. Here we show that AOX can be safely expressed in the mouse (MitAOX), with major physiological parameters being unaffected. It neither disrupted the activity of other RC components nor decreased oxidative phosphorylation in isolated mitochondria. It conferred cyanide-resistance to mitochondrial substrate oxidation and decreased reactive oxygen species (ROS) production upon RC blockade. Accordingly, AOX expression was able to support cyanide-resistant respiration by intact organs and to afford prolonged protection against a lethal concentration of gaseous cyanide in whole animals. Taken together, these results indicate that AOX expression in the mouse is innocuous and permits to overcome a RC blockade, while reducing associated oxidative insult. Therefore, the MitAOX mice represent a valuable tool in order to investigate the ability of AOX to counteract the panoply of mitochondrial-inherited diseases originating from oxidative phosphorylation defects. c oxidase (COX). In plants, fungi, microorganisms, and some lower animals , in addition to the normal pathway, a cyanide-resistant alternative oxidase (AOX) exists. It maintains electron transfer to oxygen even when the normal pathway is blocked. This provides a bypath that releases constraints on the energy producing machinery and prevents the production of deleterious superoxide molecules. Thus, preventing the energy producing machinery blockade and excess superoxide production by means of AOX should be of considerable interest. However, because of its energy-dissipating properties, AOX might produce deleterious effects of its own in mammals. Here we show that the AOX can be safely expressed in a mammal with major physiological and molecular parameters being unaffected. We also show that the AOX is active in vivo where it counteracts the energy producing machinery blockade and reduces in vitro the associated oxidative insult. Up to now, efficient therapies against mitochondrial-associated diseases are lacking dramatically. Therefore, in view of our results, the MitAOX mice represent a precious tool to assess the AOX therapeutic capacity against the panoply of inherited mitochondrial diseases.In mammalian mitochondria, the energy-producing machinery is powered by the electron transfer to molecular oxygen, a mechanism whose terminal step is mediated by the cyanide-sensitive cytochrome  We show also that the presence of the AOX conferred cyanide-resistance to mitochondrial substrate oxidation and decreased ROS production under conditions of RC inhibition. Importantly, the AOX also conferred cyanide-resistance to intact organ respiration and significantly prolonged the survival of the whole organism in the presence of this deadly poison. Taken together, these results indicate that the AOX is active Ciona intestinalis AOX gene was recoded to maximize its expression in the mouse and introduced into early mouse embryos by germ-line lentiviral transduction on a mixed genetic background (CD-1/B6). We used the ubiquitously active, chimeric CAG promoter, together with the Woodchuck hepatitis virus Post-transcriptional Regulatory Element (WPRE) to further enhance AOX gene expression  and COX I , being much higher in exocrine than in endocrine tissue , showed the highest cyanide resistance, which was less in heart and nearly inexistent in liver  might affect the activity of the AOX, we next tested cyanide-resistance of substrate oxidation as a function of O2 concentration  we determined the ADP/O values in WT and MitAOX mice brain mitochondria . Any sigsumption , therebysumption  were fousumption . Becausentration . We obseo-optode . AOX thuo-optode . This wao-optode . NoticeaIn order to evaluate a potential detrimental effect of AOX expression, we next investigated a number of physiological and behavioral variables in pups and mature MitAOX animals . Cardiorin vivo, anesthetized MitAOX mice were exposed to gaseous cyanide. The deadly effect of cyanide on mammals has been previously shown to result mostly from the inhibition of the mitochondrial cytochrome oxidase rather than to its binding to other metalloenzymes Finally, in order to assess the capacity of the AOX to compensate for a cytochrome pathway blockade C. intestinalis AOX expressed in the MitAOX does not interfere/compete significantly with the cytochrome pathway, being functional only upon blockade of this latter when the pool of ubiquinone becomes highly reduced. Accordingly, the expression of the C. intestinalis AOX in the mouse did not result in any deleterious consequence, whilst spectacularly increasing the survival of the mouse in the presence of a lethal concentration of gaseous cyanide. The protective mechanism provided by the AOX to organisms naturally harboring the enzyme was therefore fully preserved when the oxidase was expressed in the mouse.Our data show for the first time that a functional AOX can be expressed in a mammal and transmitted between generations, conferring significant cyanide-resistance to mitochondrial substrate oxidation and tissue respiration as well as the whole organism. As previously observed in cultured human cells DQH2 from 0.53 to 0.38 mM) DQH2 less than 20 µM) The AOX protein typically has such a high Km for reduced quinones  and water. Animal management was in accordance with Good Laboratory Practice Guidelines Ciona intestinalis AOX cDNA sequence in vitro one-step recombination to the attR1/R2-containing plasmid pTrip The Lentiviral vectors containing the CAG-AOXopt-WPRE construct were generated as previously described GGATGAGCCCAATATCGAAG) and AOXopt-R (CTGAAACGAAAATGCCTTGG) primers. For Southern blot analysis the DIG System from Roche Applied Science was used.Mouse genomic DNA was extracted from frozen tail samples using the MasterPure DNA Purification Kit according to the manufacturer's instructions . The transmission of the AOX transgene was verified by PCR on tail genomic DNA using AOXopt-F  and 1 mg.ml−1 bovine serum albumin, plus substrates or inhibitors as described 2; Bionef, Paris, France). Superoxide plus hydrogen peroxide production by isolated brain mitochondria was quantified using Amplex Red fluorescence in 1.5 ml of medium consisting of 125 mM KCl, 14 mM NaCl, 1 mM MgCl2, 20 µM EGTA, 4 mM KH2PO4 and 20 mM HEPES (pH 7.2) to which were added 2 IU of purified superoxide dismutase Respiratory chain enzyme activities were spectrophotometrically measured using a Cary 50 UV–visible spectrophotometer  i.p.) and perfused through the ascending aorta with 20 ml of saline followed by 100 ml of 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (PB). Brains were post-fixed overnight in 4% paraformaldehyde at 4°C, cryoprotected in 30% sucrose in PB  and frozen in liquid isopentane at −45°C. Rabbit polyclonal anti-AOX antibody (1∶300), mouse monoclonal anti-Core1 , and mouse anti-complex IV subunit I antibody  were used for double-labeling fluorescent immunohistochemistry and proceeded as in Three-month-old mice were deeply anesthetized with chloral hydrate  were collected and processed for immunohistochemistry as described previously TOT), tidal volume (VT), and ventilation (VE) calculated as VT/TTOT) were measured noninvasively in unanaesthetized, unrestrained 6-day old pups using whole-body flow barometric plethysmography as described previously t-test (Statview 5). Values of p<0.05 were considered as significant. Motor coordination and fatigue resistance of older animals (2 months) were assessed by Rotarod test as previously described Breathing variables . Once anesthetized, one WT and one MitAOX mouse were placed in a 5.2 L airtight acrylic glass chamber maintained at 28°C . Cyanide gas (451 ppm) was produced in the chamber by injecting 100 mM KCN into a Figure S1Cox1 distribution profile in the MitAOX and WT mice. Immunohistochemical study of MitAOX and WT brain stained with COX I antibody showing mitochondrial distribution.(TIF)Click here for additional data file.Figure S2Respiratory chain activities and oxygen consumption by WT and MitAOX mitochondria. A, Respiratory chain enzyme activities from MitAOX and WT mice brain homogenates were spectrophotometrically measured as described in materials and methods. B, Oxygen consumption and cyanide resistance of succinate oxidation by brain mitochondria from WT and MitAOX mice measured using a standard Clark electrode (a) or using a fluorescence-based micro-optode (b) as described under material and methods. C, Effect of cyanide as a function of oxygen tension under similar conditions measured with the micro-optode. For the sake of comparison, slopes were adjusted by modulating mitochondrial protein used to record oxygen consumption, allowing an easier comparison of the effect of low oxygen tension.(TIF)Click here for additional data file.Table S1Polarographic determination of substrate oxidation rates and cyanide resistance by brain or pancreas mitochondria. Experimental conditions as described in (DOCX)Click here for additional data file.


The study of tumor viruses paves the way for understanding the mechanisms of virus pathogenesis, including those involved in establishing infection and dissemination in the host tumor affecting immune-compromised patients. The processes ranging from viral infection to progressing malignancy are slow and usually insufficient for establishment of transformed cells that develop cancer in only a minority of infected subjects. Therefore, viral infection is usually not the only cause of cancer, and further environmental and host factors, may be implicated.HTLV-I, in particular, is considered as an oncovirus cause of lymphoproliferative disease such as adult T cell leukemia/lymphoma (ATL) and disturbs the immune responses which results in HTLV-I associated meylopathy/tropical spastic parapresis (HAM/TSP). HTLV-I infection causes ATL in a small proportion of infected subjects (2-5%) following a prolonged incubation period (15-30 years) despite a strong adaptive immune response against the virus. Overall, these conditions offer a prospect to study the molecular basis of tumorgenicity in mammalian cells. In this review, the oncogencity of HTLV-I is being considered as an oncovirus in context of ATL. Human tumor virusesOncogenecity refers to viruses that may cause cancers. Generally, the viruses that associated with malignancies are known as tumorviruses. Some researchers prefer to consider human tumor viruses as a distinct group of viruses, however, the known tumorviruses such as Hepatitis B and C viruses, Epstein-Barr virus (EBV), human herpesvirus 8 (HHV-8), Human papillomavirus (HPV), and Human T lymphotropic virus type I (HTLV-I) are various in case of families, genomes and life cycles. The aforementioned viruses are associated with malignancies such as hepatocellular carcinoma (HCC), Burkitt’s lymphoma, nasopharyngeal carcinoma (NPC), Kaposi’s sarcoma (KS), Multicentric Castleman’s Disease (MCD) and Adult T cell leukemia/lymphoma (ATL) .HBV is a double-stranded DNA virus of the Hepadnaviridae family which infects 350 million people worldwide. Hepatitis B can lead to liver diseases ranging from the acute hepatitis to chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC) -2. HCV iKaposi’s sarcoma-associated herpesvirus (KSHV) is the most recently discovered human herpesvirus. It is the aetiologic agent of Kaposi’s sarcoma (KS), a tumor which affects more frequently on patients diagnosed with AIDS that does not receive any treatment. KSHV is also a probable cause of two lymphoproliferative diseases: multicentric Castleman’s disease and primary effusion lymphoma .Human T-cell lymphotropic virus type I (HTLV-I) was the first discovered human retrovirus  and it hHTLV-I biology and pathogenesisGenome structure and organizationThe HTLV-I is an enveloped dimeric positive- sense single-stranded RNA virus, and like other retroviruses, the linear genome encodes the structural and enzymes such as gag, env, and pol,  18). Mo. Mo18). The full length of HTLV-I mRNA encodes the gag protein (p55) which is then cleaved by the viral protease making the matrix p19), capsid (p24), and nucleocapsid (p15) proteins. A sequence, from the 3 end of gag to the 5 end of pol, encodes the protease which results from ribosomal frameshifting. The HTLV-I long-terminal repeat (LTR) at both 5’ and 3’ ends of the genome contains the viral promoter and regulatory elements. In addition, the viral mRNA encodes the pol protein, single spliced mRNA encodes the env protein and a double spliced mRNA encodes the Tax and Rex regulatory proteins , capsid .Tax proteinThe HTLV-I trans-activator (Tax) is a 40-kDa protein containing 353 amino acid residues located within the U3 region of the LTR -23. An aTax interacts with CREB and the p300/CBP co-activator family on three 21 bp-repeats in the HTLV-I LTR and is reported to stabilize the formation of CREB/ATF-dimers bound to the DNA -29. The RexRex is a 27-kDa phosphoprotein encoded by ORF III that localizes the nucleolus of infected cells -42. Rex,Rex is a specific RNA binding protein that binds to a cis-acting Rex responsive element (RxRE), a highly stable structure located within the R region of the viral LTR -45. Rex-p12IORF I encodes a 12-kDa protein (p12I). Although, p12I expression is difficult to be displayed n HTLV-I- infected cells, some evidences have suggested its importance . While pThis viral protein is found to have weak oncogenic activity, shares amino acid similarities with other viral oncoproteins , and binTwo variants of the p12I protein have been demonstrated: one has a Lysine at position 88 which is found in HTLV-I strains from TSP-HAM patients; the second has an Arginine at position 88 which is found in HTLV-I strains from all ATL patients and healthy carriers .It has been suggested that p12I has significant roles during the early stage of HTLV-I infection establishment. Furthermore, the ability of p12I for binding to the MHC I heavy chain and making it susceptible for degradation may decrease the viral peptide MHC I- complexes on the infected cell surface and protect them from lysis by cytolytic T lymphocytes (CTLs) .Transmission of HTLV-IHTLV-I can infect some cell types including T cells, B cells, and synovial cells. Many of the studies demonstrated that the transmission of HTLV-I should occur by healthy infected cells and is very inefficient by free virion (REF). This is because HTLV-I transmits naturally by the cell-to-cell manner. Whilst an HTLV-I-infected cell attaches to uninfected cells, the HTLV-I-infected cells form “virological synapses” with uninfected cells . The recHTLV-I-infected cells in the human body are transmitted via three major routes: (1) mother-to-infant transmission (mainly breast-feeding), (2) sexual transmission, (3) parenteral transmission. It is worth noting that fresh frozen plasma from seropositive donors does not transmit HTLV-I . MoreoveClonal proliferation of HTLV-I-infected cellsAfter transmission, HTLV-I is disseminated by de novo infection and clonal proliferation of infected cells. Tax plays a central role for increasing the number of HTLV-I-infected cells by promoting proliferation and suppressing apoptosis; proliferation of infected cells seems to be oligoclonal  60). A . A 60). HTLV-I clonal cells are more heterogeneous and less stable during sero-positivity than in long-term carriers . SeveralCell-to-cell spreading is the main rout of HTLV-I transmission. Tax mainly promotes T cell proliferation of HTLV-I infected cells. However, other HTLV-I proteins can enhance this process. Cytolytic T cells (CTLs) can prevent the proliferation by killing of Tax expressing targets cells. The expression of Tax is inactivated by several mechanisms, suggesting that Tax is not necessary in ATL stage and HBZ may be more effective. Idea from  with modOncogenic function of HTLV-I proteinsImmortalization / transformation of T cellsMany studies have demonstrated that HTLV-I genes (e.g. tax and HBZ), can induce various cellular dysfunctions, genetic and epigenetic alterations, and the host immune system may be involved in the leukemogenesis of ATL . HoweverImmortalization of lymphocytes infected by HTLV-I requires Tax, however, Tax-immortalized cells, but nontranformed cells are still IL-2 dependent. Therefore, infected cells could not pass the G1 check point without exogenous growth factors such as IL-2. In addition the inactivation of tumor suppressor gene p53 contributes to the accumulation of genetic mutations . AlternaCD4+-CD8+ cells, and immature CD4--CD8- cells from bone marrow can be transformed by HTLV-I. Transformed cells not only display an activated phenotype but also in some cases retain T-cell functional properties. Among the functional activities of HTLV-I, transformed cells have reported to be able to induce suppressor-cell activity  and retaHTLV-I and apoptosisThe programmed cell death is one of the major effectors mechanisms in NK cell and CTLs for killing the virus infected and malignant cells. The effect of HTLV-I infection on programmed cell death is less clear. Tax has been shown to repress Bax gene-expression as is shown in et al, 2006, to fit for HTLV-I. Apoptosis is one of the main mechanisms of immune system for killing virus infected or malignant cells. The extrinsic pathway of apoptosis (left side) is triggered by the binding of ligands to death-inducing membrane proteins. This signaling event leads to the recruitment of Fas-associated death receptor (FADD) which in turn activates caspase 8. Consequently, caspase 8 activates ‘effector’ caspases  and releasing caspase-activated DNase (CAD) which in turn lead to cell death. During conditions of cellular stress, such as DNA damage and growth-factor deprivation, the intrinsic apoptosis pathway (the pathway that is activated most frequently by the tumor suppressor protein p53) is activated (right side). The intrinsic apoptotic pathway converges on the disruption of mitochondrial membranes and consequently, after some molecular activation events, results in releasing CAD. The balance of the pro- and anti-apoptotic bcl-2 family proteins determines whether apoptosis proceeds. There is overlap between the intrinsic and extrinsic apoptotic pathways, as caspase 8 can cleave and activate the bcl-2 family protein.With modification from Rezaee Therefore, manipulation of both the intrinsic and extrinsic pathways is very important for dissemination of virus to evade from NK cell and CTLs antiviral activities. The precise mechanism that HTLV-I inhibits apoptosis is unclear, but it is assumed that Tax may have the central role by suppressing p53 and BAX and stimulation of survival signals via PI3K and NF-κB pathways.Activation of NF-κB by immune stimuli extrinsic and intrinsic pathways, which are based on degradation of IkB or processing of p100, respectively. The canonical pathway is stimulated by diverse cellular stimuli, such as antigens and cytokines, and is dependent on the trimeric IKK complex as well as certain upstream kinases, such as MEKK3 and PKCψ. The noncanonical pathway responds to a subset of TNF family members, including BAFF and CD40L, and requires NIK and its downstream kinase IKKα but not IKKβ or IKKγ. Tax activates both NF-κB pathways by physically targeting two different IKK complexes, both requiring the adaptor protein IKKγ. Formation of the noncanonical Tax/ IKK complex requires the interaction of Tax with both IKKγand p100.Therefore, manipulation of both the intrinsic and extrinsic pathways is very important for dissemination of virus to evade from NK cell and CTLs antiviral activities. The precise mechanism that HTLV-I inhibits apoptosis is unclear, but it is assumed that Tax may have the central role by suppressing p53 and BAX and stimulation of survival signals via PI3K and NF-κB pathways.Proviral load Recently, HTLV-I proviral load levels have been evaluated as important predictors of development of ATL and HAM/TSP. Many studies show that HTLV-I proviral load are significantly higher in ATL and HAM/TSP compared to HTLV-I healthy carriers -75, 76--76-77. Proviruses of HTLV-I are clonally integrated in ATL patients, but randomly integrated in HAM/TSP patients. The efficiency of HTLV-I replication is shown by the proportion of PBMCs that carry HTLV-I provirus . Moreoveet al, unpublished data). Counting HTLV-I infected cells in healthy carriers and ATL patients , forming a nucleoprotein complex on the three viral cyclic AMP-responsive elements (CREs) in the HTLV-Ipromoter. Each three 21 bp repeats contains a cAMP response element (CRE) core. In the presence of Tax, gene expression driven by multiple copies of the 21- bp repeat element can increase up to 100-fold or higher. Cellular basic domain-leucine zipper (bZip), transcription factors-CREB, ATF-1, the 21-bp repeats, and Tax form stable ternary complexes ,101. In Activation of NF-κB pathway by taxNF-κB/Rel family of transcription factors are suppressed by I-κB proteins in cytoplasm, . During The non-canonical pathway of NF-κB activation is important for B-cell proliferation and lymphoid organogenesis and is activated in response to some inducers such as lymphotoxin β and B cell activating factor . Differeet al.  and IKKα, and is independent of IKKγ . Phosphoet al.  have proet al. . Finallyet al. , 123-124HBZThe complementary strand of HTLV-I proviral genome generates distinct 2.6 kb and 2.9 kb transcripts, driven by promoter in the 3’ LTR . SubsequWhen expressed exogenously HBZ interferes with Tax mediated transactivation by hetrodimerizing with CREB-2 and preventing recruitment of CREB-2 to the TRE and cyclic AMP response element sites in a dose dependent manner , providiOther cellular transcriptional factors appear to be the target of HBZ functions. HBZ interacts with Jun-D and stimulates its transcriptional activity  while suATLATL is the most common outcome of infection with HTLV-I, and in areas of endemicity, it occurs in 2-5% of HTLV-I-infected individuals . It was ATL can be presented in one of several forms . Although there are significant geographic variations in the contribution of these forms in the initial presentation, the most common presentation is acute leukemia.The most benign detectable form of ATL is an asymptomatic pre-leukemic phase which is usually diagnosed incidentally when the examination of a peripheral blood smear reveals abnormal lymphocytes with characteristics of lobulated nuclei .Tax-mediated immortalization of HTLV-I-infected T cells toward leukemogenesis by genetic and epigenetic changes in provirus and host genes. However, HTLV-I gene expression also can be recognized by T cells result in activation of host immune system which eliminates Tax-expressing target cells. Therefore, the host immune system pressure in such circumstances is in favor of proliferation of Tax negative HTLV-I infected cells.Activation of the PI3 kinase pathway and ATLSome studies have reported constitutive activation of the phosphoinositide 3-kinase (PI3K) pathway as a common characteristic of HTLV-I- and Tax-transformed cells . TreatmeSomatic changes in ATL cellsMutation of p53, and deletion of p16 have been reported in ATL with a poor prognosis .ThereforEpigenetic changes are recognized as mechanisms implicated in oncogenesis as well as genetic changes. Since genetic changes in specific genes in ATL cells have not been identified except for p53 and p16, and there is no consistent chromosomal change, it is possible that epigenetic changes such as DNA methylation  play an Tax protein and cell cycle deregulationSeveral lines of evidence indicate that p40tax is the oncogene responsible for viral lymphocyte-transforming and leukemogenic properties . MechaniIn mammalian cells, G1-progression is controlled by the sequential activation of several cyclins and cyclin-dependent kinases (CDKs), starting with CDK4, CDK6 and CDK2. Tax activates CDK4, CDK6 and CDK2 leading to phosphorylation of retinoblastoma (Rb) tumor suppressor proteins and liberation of the transcription factor E2F , 164. MoIn general, Tax shortens the length of G1 and accelerates entry into S phase , 168. In Cyclin D2 expression is also overregulated by interleukin-2 receptor (IL2-R) signals - 171. TaThe 40 N-terminal amino acids of Tax are sufficient to bind cyclin D2 and CDK4. Within CDK4 a N- and a C-terminal domain are relevant for Tax binding. Taken together, these findings suggest that Tax stimulates G1- to S-phase transition by supporting the association of CDK4 and cyclin D2. Furthermore, these studies support the conclusion that CDK4 activity is stimulated through conformational changes of the enzyme directly mediated by Tax . Cell cycles checkpoints are activate in DNA damage. Tax disrupts the DNA damage-induced G1/S checkpoint (controlled by cyclin E/cdk 2), in part by inactivation of p53. Normally when DNA damage is detected during G1, p53 activates the cdk inhibitor p21waf1, which in turn, binds and inactivates cdk2. Although p53 is inactivated, Tax-expressing cells have higher levels of p21waf1 because Tax transactivates the p21waf1 promoter -178; 16. Loss ofHowever, more studies should be conducted to reach more reasonable mechanisms to understand how HTLV-I can pass the cell cycle checkpoints and push the cell toward mitosis and proliferation leading to cell transformation. T-lymphocyte activation is a way for clonal expansion in the immune system for encountering pathogens and is highly regulated. HTLV-I Tax has developed numerous mechanisms to subvert this control and induce cell cycle promotion in the absence of physiological signals. For instance, high levels of IL-2 are required to promote the progression of resting T cells through the cell cycle. After binding to its receptor (IL-2R), IL-2 triggers its production of as well as IL-2Ra. Cells expressing the high-affinity IL-2R are more sensitive to IL-2 and continue to proliferate . In firsATL cells secrete an increased amount of TGF-β because of Tax transactivation of the TGF-β promoter -190; howAlthough Tax enhances TGF-β production, infected cells are resistant to TGF- β growth inhibition because Tax disrupts the TGF- β signaling pathway and interferes with the function of growth inhibiting proteins induced by TGF-β.An additional disruptive effect of Tax emanates from its impact on the ability of the cell-cycle machinery to regulate DNA replication and cell division ,192-194.Despite the fact that nearly 70% of all cancers demonstrate aneuploidy (including both ATL and HTLV-I-infected cells), only rarely have genetic defects which are identified in mitotic checkpoint genes, implying that other events must alter function of the mitotic spindle checkpoint (MSC) -196. ResActivation of NF-κB by HTLV-I NF-κB activation by immune stimuliThe NF-κB proteins are normally sequestered in the cytoplasm by physical interaction with a family of inhibitory proteins, including IkBa, IkBb, and related proteins . The NF-The latent NF-κB complexes can be activated by diverse immune stimuli, such as antigens, cytokines, and microbial components, which target two alternative NFkB signaling pathways: the canonical and noncanonical pathways , 199.Aberrant activation of NF-κB by HTLV-IUnder normal conditions, the signals mediating NF-κB activation in T cells as well as most other cell types are transient and stimulate predominantly the canonical NF-κB pathway. Such a signaling mechanism ensures the rapid, but short-lived, nuclear expression of NF-κB members that are required for temporal proliferation and survival of antigen-stimulated T cells. This mechanism is achieved through different levels of regulation. First, following stimulation by an antigen, both the T cell receptor (TCR) and its proximal signaling molecules are downregulated, thus preventing persistent signaling through the cell surface receptor . Second,A main point of Tax-stimulated NF-κB activation is the marked induction of nfkb2 gene product, p52, as well as the canonical NF-κB members -209 Fig. In normOver the past decades, significant progress has been achieved through understanding the mechanism of NF-κB activation by Tax. Indeed, Tax binds to several NF-κB members, including RelA, p50, and p52 , 211. TaSome studies revealed that Tax induces the degradation of both IkBa and another IkB member, IkBb -214. SinIn addition to mediating IkB degradation and nuclear translocation of NF-κB, IKK also regulates phosphorylation of the RelA subunit of NF-κB, a modification that is required for the transactivation function of NFkB. Although IKKβ is essential for Tax-induced nuclear translocation of the canonical NF-κB  or defect .Conducting molecular research for better understanding of molecular behavior of HTLV-I can assist the researcher to find selective therapeutic agents for malignancy and autoimmunity. With regard to the oncogenesis of Tax and its impact on survival signaling pathways such as NFkB and PI3K-Akt pathways, new therapeutic opportunities for ATL has been presented in two collaborative studies , 226. MoBased on the molecular interactions between HTLV-I and host, there are more hopes that these activities have evolved under selective pressure to become highly specific for malignancy (ATL) and Autoimmunity (HAM/TSP).


FLT3) internal tandem duplication (ITD) mutations result in constitutive activation of this receptor and have been shown to increase the risk of relapse in patients with acute myeloid leukemia (AML); however, substantial heterogeneity in clinical outcomes still exists within both the ITD mutated and unmutated AML subgroups, suggesting alternative mechanisms of disease relapse not accounted by FLT3 mutational status. Single cell network profiling (SCNP) is a multiparametric flow cytometry based assay that simultaneously measures, in a quantitative fashion and at the single cell level, both extracellular surface marker levels and changes in intracellular signaling proteins in response to extracellular modulators. We previously reported an initial characterization of FLT3 ITD-mediated signaling using SCNP. Herein SCNP was applied sequentially to two separate cohorts of samples collected from elderly AML patients at diagnosis. In the first (training) study, AML samples carrying unmutated, wild-type FLT3 (FLT3 WT) displayed a wide range of induced signaling, with a fraction having signaling profiles comparable to FLT3 ITD AML samples. Conversely, the FLT3 ITD AML samples displayed more homogeneous induced signaling, with the exception of patients with low (<40%) mutational load, which had profiles comparable to FLT3 WT AML samples. This observation was then confirmed in an independent (verification) cohort. Data from the second cohort were also used to assess the association between SCNP data and disease-free survival (DFS) in the context of FLT3 and nucleophosmin (NPM1) mutational status among patients who achieved complete remission (CR) to induction chemotherapy. The combination of SCNP read outs together with FLT3 and NPM1 molecular status improved the DFS prediction accuracy of the latter. Taken together, these results emphasize the value of comprehensive functional assessment of biologically relevant signaling pathways in AML as a basis for the development of highly predictive tests for guidance of post-remission therapy.FMS-like tyrosine kinase 3 receptor ( AML with NPM1 WT/FLT3 WT or carrying both NPM1 mutation and FLT3 ITD are considered to have intermediate-risk disease Nucleophosmin /AKT and the RAS/RAF/ERK kinase signal transduction pathways which ultimately affect cell survival and proliferation FLT3 ITD mutation demonstrated decreased FLT3 ligand (FLT3L)-induced activation of the PI3K and RAS/RAF/ERK pathways, decreased IL-27-induced activation of the JAK/STAT pathway, and heightened apoptotic responses to agents inducing DNA damage FLT3 ITD samples compared to FLT3 WT AMLs suggesting that the latter represented a more heterogeneous and biologically distinct group of leukemias. On the basis of these observations we hypothesize that assessing patient samples for the presence of FLT3 signaling deregulation may provide clinically relevant prognostic information that is independent of AML mutational status.Previously, we reported the use of SCNP to functionally characterize FLT3 signaling in healthy bone marrow (BM) and samples obtained from adult patients (<60 years) with AML In the current study this hypothesis was tested and confirmed in two sequential cohorts  using diIn accordance with the Declaration of Helsinki, all patients provided written informed consent for the collection and use of their samples for research purposes. Institutional Review Board approval was obtained from Independent Review Consulting, Inc.  on August 31, 2009. Clinical data were de-identified in compliance with Health Insurance Portability and Accountability Act regulations.FLT3 WT and 8 FLT3 ITD AML. The second (verification) study consisted of 104 cryopreserved BMMC samples collected from patients ≥60 years old enrolled in Phase 3 trials E3999 or E3993 for whom ITD mutational status , response and DFS were available . Both trials excluded PML/RARα positive acute promyelocytic leukemia.The first (training) study consisted of 46 cryopreserved bone marrow mononuclear cell (BMMC) samples collected at the time of diagnosis from patients ≥60 years of age with morphologically and immunophenotypically confirmed AML, enrolled in Phase 3 trial E3999. Samples included 38 vailable . These iSamples were collected prior to the initiation of anthracycline/cytarabine (Ara-C)-based induction chemotherapy and BMMC were cryopreserved in the ECOG Leukemia Tissue Bank until release for this study. The sample sets used for the present study represent a subset of the subjects enrolled on the ECOG studies which were selected based on the availability of stored aliquots of BMMC samples. In order to be included in the experimental analysis, BMMC specimens had to contain a minimum of 2 million viable cells post-thaw.FLT3 ITD samples, was also available.Based on data obtained in the training study, prespecified sample inclusion criteria for the verification study were modified to include sample quality elements and require that the percentage of leukemic (identified using CD45 and side scatter) cells that were cleaved PARP (cPARP) negative (non-apoptotic leukemic cells) was greater than 25% at the 15 minute time point  and that molecular assay data, including percentage of mutational load for the Healthy BMMC (n = 6) were obtained from patients at the time of hip replacement surgery from Williamson Medical Center (TN). These samples  were from predominately Caucasian donors who ranged in age from 60–87 years, (median = 71 years).FLT3 ITD donors depending on the condition tested.SCNP assays were performed as described previously FLT3 ITD mutational status were examined in vitro exposure  Modulated cell signaling pathways previously shown to be associated with FLT3 WT and any of the FLT3 ITD groups both in variance (using Levene Test) or magnitude (using student's t-test) (data not shown). Healthy bone marrow myeloblasts (BMMb) were identified in BMMC samples using right-angle light scatter and surface markers such as CD45 and CD34.Flow cytometry data were acquired on an LSR II and/or CANTO II flow cytometer using the FACS DIVA software  and analyzed with WinList . Dead cells and debris were excluded by forward scatter (cell size), side scatter (granularity), and Amine Aqua Viability Dye measurement. Leukemic cells and normal myeloblasts were identified as cells that fit the CD45 versus right-angle light-scatter characteristics of myeloblasts In SCNP assay terminology, a “signaling node” is used to refer to a proteomic readout in the presence or absence of a specific modulator. For example, the response to FLT3L treatment can be measured using p-AKT as a readout. That signaling node is designated “FLT3L→p-AKT”. The normalized assay readouts for surface and intracellular markers are evaluated using metrics that are applied to interpret the functionality and biology of each signaling node. They are referenced following the node e.g. “FLT3L→p-AKT | Fold”, “G-CSF→p-STAT5 | Total” and are defined in detail below.Median fluorescence intensity (MFI) was computed for each node from cell fluorescence intensity levels. Equivalent Number of Reference Fluorophores (ERF) 2(ERFUnmodulated/ERFAutofluorescence) to measure basal levels of signaling in the resting, unmodulated cells; B) Fold Change ERF (“Fold”): defined as log2(ERFModulated/ERFUnmodulated) to quantify the responsiveness of a protein or pathway to a specific modulator; C) Total Phospho ERF : defined as log2(ERFModulated/ERFAutofluorescence) to assess the magnitude of total activated protein after modulation; D) Uu metric, which is the Mann-Whitney U statistic comparing the fluorescence intensity values of the modulated and unmodulated cells scaled to the unit interval , was used to assess the proportion of cells which showed modulated signal; E) Ua metric, (comparing ranking of the antibody stained cells in the modulated state vs. autofluorescence) was used to quantify the percentage of cells showing signal induction post-modulation. Finally, for surface markers, the Percent Positive (“PercentPos”) was used to quantify the frequency of cells positive for a surface marker relative to a control antibody. For measures of induced signaling, one metric per node was generally used as Fold and Uu metrics gave highly concordant results . For short term signaling, the the Fold metric wastipically used based on the potentially higher dynamic range of this metric with the exception of p-AKT intracellular readout (measured in the Alexa Fluor AF647 channel) which displayed low (near zero in unmodulated wells) absolute median intensity values which contribute to less reproducible Fold metrics. A rank based metric Uu which approximates the proportion of cells that were induced was found (based on reproducibility data) to be more robust in such situations. Similarly, apoptosis readouts were measured using rank based Uu or Ua metrics which approximate the proportion of cells undergoing apoptosis and were previously found to be more stable than metrics based on the absolute levels of cPARP staining or Fold metrics (data not shown).The ERF values were then used to compute a variety of metrics to quantify functional changes in signaling proteins as follows see also : A) BasaFLT3 ITD, PCR was performed on 500 ng genomic DNA using published primers 11F and 12R to identify ITD insertions in the JM and TK1 domains FLT3 ITD mutational load was determined using densitometry analysis software (Image J) quantifying the percentage of FLT3 ITD mutant PCR signal intensity from DNA electrophoresis gels.An aliquot of cells was taken from each sample to isolate DNA and RNA using TRIzol Reagent (Invitrogen) as described by the manufacturer. For the analysis of NPM1 mutations A and B were performed to analyze the NPM1 mutational status. RQ-PCR was performed on 100 ng cDNA using published Taqman primers for mutation A (cNPM-F and cNPMmutA-R) or mutation B (cNPM-F and cNPM-mutB-R) and a common probe (c.Probe), as described NPM1 mutation.Real-time quantitative PCR assays for u metric. The Uu metric gives each node an equivalent scale/range of signaling (from 0 to 1) which is preferable for PCA analysis. The first two principal components were then used to visually compare FLT3 ITD, FLT3 WT and Healthy BMMb sub-groups.Principle component analysis FLT3 ITD samples were pre-defined and specified prior to unblinding in the second study; those with any percentage of mutation  (FLT3 ITD+), those with mutational load greater or equal to 30% (FLT3 ITD+ 30), 40% (FLT3 ITD+ 40) or 50% (FLT3 ITD+ 50). The variance in the range of FLT3L-induced pS6 levels observed in each of these sub-groups and FLT3 WT samples was computed by using the median as the central measure. Levene's test for the equality of variance was applied to compare each of the FLT3 ITD sub-groups with the FLT3 WT group using the method ‘levene.test’ from the package ‘lawstat’ in the R software package (version 2.13.0). The p-value from the Levene's test is reported for each of the comparisons.Four sub-groups of Disease-free survival (DFS) was defined as the number of days from CR until relapse, death from any cause, or last contact and therefore is only applicable to patients who achieved CR.FLT3 ITD mutational status or molecular classification along with an interaction term. Molecular classification combined FLT3 and NPM1 molecular status into a single coding of 1 for those patients with good prognosis (FLT3 WT and NPM1 MUT), 2 for those with intermediate (FLT3 WT and NPM1 WT or FLT3 ITD and NPM1 MUT) and 3 for those with poor prognosis (FLT3 ITD and NPM1 WT). Additional modeling of DFS was also performed only among the patients with CR and FLT3 WT, where two SCNP nodes (each of which represented a different pathway) were combined to model DFS. The p-value for the model  and the p-value (slope = 0) for each of the components of the model are reported. In addition, within the CR and FLT3 WT group, Cox-proportional hazard models for DFS with two SCNP nodes were built with NPM1 mutation status or cytogenetics as an additional covariate to examine if the SCNP nodes remained significantly associated with DFS. This data set contained several patients with missing cytogenetics data; in particular among the CR group 5 patients were coded by ECOG as Unknown cytogenetics. For modeling purposes, patients with missing cytogenetics were considered intermediate-risk, as is standard in clinical practiceModel+ and those with less than 1.0 were plotted on the curve labeled Model−.All DFS modeling was performed as a continuous variable. Cox-proportional hazards regression was used to develop models for DFS using the package ‘survival’ in the R Software package (version 2.13.0). Due to the small size of the data set, the complexity of these models was limited to combining only one SCNP node at a time with either the NPM1 mutational status were associated with FLT3 ITD mutational status . Incubation of cells with FLT3L resulted in the phosphorylation (p) of S6, ERK, AKT, and STAT5 at 5, 10 and 15 minute time-points in healthy BMMb and leukemic blasts from AML with or without FLT3 ITD mutation. Induced signaling in the FLT3 ITD AML samples (N = 8) was muted except in patients with low (<40%) mutational load (FLT3 WT AML samples (N = 38) showed a high level of heterogeneity, with some samples showing patterns of signaling similar to that of healthy BM and FLT3 ITD AML  mutational load. Similar results were seen in response to etoposide although an additional sample in the FLT3 ITD group  displayed a low apoptosis response that was not explained by low mutational load .Apoptosis was evaluated as the proportion of cells that accumulated above the background of cPARP using the Ua metric . FLT3 WTnal load . While tFLT3 ITD AML, FLT3 WT AML and healthy BMMbs (FLT3 ITD samples (with the exception of samples with mutation load <40%) formed tight but distinct clusters, suggesting tightly regulated, though discrete, signaling across these pathways. The FLT3 WT AML samples, on the other hand, spanned a wide range of signaling and did not display distinct clusters, overlapping both the signaling patterns of the healthy BMMb and FLT3 ITD samples.PCA was used to combine information across multiple nodes, i.e. FLT3L-mediated induction of p-AKT, p-S6, p-ERK, and p-STAT5, IL-27-mediated induction of p-STAT1, p-STAT3 and p-STAT5, and etoposide-induced p-Chk2 and cPARP. Inspection of the first two principal components showed distinct signaling patterns among the three groups of samples: hy BMMbs . Both heFLT3 ITD patients in the training study, association with clinical outcomes could not be performed in this set.Due to the small number of FLT3 ITD than FLT3 WT AML, we chose to analyze the variance of FLT3L-induced p-S6 signaling as the primary study objective, a common and relevant downstream readout in the FLT3 receptor signaling pathway. Sub-groups were defined based on the FLT3 ITD mutational load: those with 0% ITD , those with measureable FLT3 ITD levels , and inside the latter group, those with ≥30% , ≥40%  and ≥50%  mutational load. Variance of signaling was pre-specified to be largest in the FLT3 WT group followed in order by FLT3 ITD+, FLT3 ITD+ 30, FLT3 ITD+ 40, and least in FLT3 ITD+ 50. After unblinding clinical and mutational data, the results confirmed that in the verification sample set the FLT3L-induced p-S6 responses were muted and more homogeneous in FLT3 ITD AML samples with high mutational load compared to FLT3 WT AML  different from the FLT3 ITD+ 50 group and marginally (Levene Test p = 0.097) different from the FLT3 ITD+ 40 group , while 39 patients experienced CR after induction chemotherapy; 37% of these CRs remained disease-free for at least one year . The SCNwith DFS , althougl curves .FLT3 mutational status improved DFS modeling. Given the small size of the data set, the complexity of the models was limited. Each regression model included a single node-metric term, FLT3 ITD status, and an interaction term between the node-metric and mutational status. Inclusion of the interaction term was motivated by the difference in range of signaling for a majority of the nodes between FLT3 ITD and FLT3 WT samples. Nine of 11 nodes examined were identified to have a significant association (pModel≤0.05) with DFS when both node and FLT3 mutational status were considered  when tested with FLT3 and NPM1 mutational status together (i.e. molecular classification) as shown in We first examined whether combining signaling nodes with nsidered . Specifimodeling , Fig. 7AFLT3 mutational status. To assess whether SCNP nodes provided independent information in improving the accuracy of modeling DFS, additional analyses were performed. Specifically, focusing on the FLT3 WT patients (u metric) was combined with the signaling node (FLT3L→pS6 using log2(Fold) metric) using a Cox-proportional hazard regression model for DFS. This model revealed a significant association of both nodes with DFS (Model<0.05) when accounting for the presence of NPM1 mutation in the FLT3 WT group (Model<0.05)   after coFLT3 ITD AML samples  was verified and shown to be correlated with FLT3 ITD mutational load FLT3 and NPM1 mutational status improved the accuracy of modeling DFS over that provided by mutation status alone. While the relatively small number of samples analyzed in this hypothesis generating study will necesitate further study in larger sample sets, these findings demonstrate that SCNP provides insights into critical signaling pathways of individual AML cases, information which is potentially clinically relevant. Functional data such as this could ultimately be used to inform post-remission therapy selection and aid in the selection of patients for treatment with inhibitors of signaling pathways.Herein, SCNP was used to functionally characterize FLT3 signaling in patients age 60 years or older with AML. In two sequential studies, the homogeneity of FLT3 pathway signaling abnormalities observed in FLT3 ITD samples displayed a blunted response to FLT3L modulation  compared to their FLT3 WT counterparts. These data confirm prior findings FLT3 ITD AML.Ligand binding to the FLT3 receptor initiates a signaling cascade that results in the differential activation of RAS/RAF/MAPK, PI3 kinase, and/or STAT pathways in AML FLT3 ITD samples were more sensitive to etoposide-induced in vitro apoptosis than FLT3 WT samples. These data indicate the presence of an intact apoptotic machinery in FLT3 ITD samples and suggest their increased in vitro susceptibility to continuous exposure of apoptosis-inducing drugs. While these data may seem counter-intuitive based on the poor survival prognosis associated with FLT3 ITD AML, FLT3 ITD has been reported to be associated with increased reactive oxygen species and with susceptibility to double-stranded DNA breaks with inaccurate but permissive repair FLT3 ITD myeloblasts which eventually cause disease relapse.FLT3 WT group followed by FLT3 ITD AML in increasing order of mutational load. The impact of FLT3 mutational load has been evaluated in several studies but has not yet been standardized for application in clinical practice. A high ratio of the ITD allele compared to the WT allele or a high percentage of FLT3 ITD mutation confers unfavorable prognosis, and multivariate analysis has shown allelic burden to be an independent prognostic factor for worse overall survival (OS) and DFS FLT3 ITD/FLT3 WT ratios of 0.5 or higher in the context of mutated NPM1 have an adverse impact on both event-free survival and OS FLT3 ITD , suggesting that these cell types have tightly regulated distinct signaling pathways. Conversely, FLT3 WT AML samples demonstrated diffusely scattered heterogeneous signaling patterns. These data suggest that prognostication based on similarities in FLT3 receptor signaling may be equally, and if used in modeling, potentially even more informative than molecular characterization and mutational load alone.PCA analysis that combined information across multiple SCNP nodes showed distinct clusters in healthy BMMb and FLT3 receptor and NPM1 mutations (a subset of patients in need of markers associated with disease relapse) is of note and warrants further investigation in a larger patient cohort. In summary, this work demonstrates the potential utility of combining SCNP measurements of disease-relevant signal transduction pathways with molecular characterization to yield improved accuracy of DFS predictions in AML samples compared to predictions from molecular characterization alone. These results emphasize the value of comprehensive functional assessment of biologically relevant signaling pathways in AML as a basis for the development of highly predictive tests for guidance of post-remission therapy, including treatment with agents interfering with the FLT3 signaling pathway.Finally, the observation that SNCP nodes from multiple pathways were found to be independent and yet significantly associated with DFS in the subgroup of patients lacking Figure S1Metrics Overview. (A) Overview of SCNP metrics based on ERF values. (B) The Uu metric compares the ERF values of the modulated and unmodulated wells that has been scaled to the unit interval . Ua is the same as the Uu metric except for the autofluoresence control is used as the reference instead of the unmodulated well.(TIFF)Click here for additional data file.Table S1List of experimental conditions and reagents used. (A) List of modulators and technical conditions used. (B).List of antibodies and reagents used.(TIFF)Click here for additional data file.Table S2NPM1 mutational status does not account for association between SCNP nodes and DFS among FLT3 WT donors.(TIFF)Click here for additional data file.


Objective(s): Systemic lupus erythematosus (SLE) is an autoimmune disease with unknown etiology. Some environmental factors can induce SLE in genetically susceptible individuals; for example, sun exposure and some viral infections may emerge the disease manifestations. Human T lymphotropic virus type 1 (HTLV-I) can dysregulate the human immune system, and the role of this virus in the pathogenesis of autoimmune diseases is under investigation. There are conflicting data about the role of HTLV-I in the pathogenesis of several autoimmune diseases such as SLE. In this study, we have focused on the correlation between HTLV-I infection and SLE in the northeast of Iran, an endemic area for the virus. Materials and Methods: One hundred and thirty women with SLE and 915 healthy controls were screened for HTLV-I by enzyme linked immunosorbent assay (ELISA). Western blot method was used for confirmation of the positive results done by ELISA in the patients and the control group.Results: Two (1.5%) of the patients and 23 (2.5%) of the healthy controls were HTLV-I seropositive. There was not a statistical difference between patients and controls in the number of HTLV-I seropositive samples (P=0.49). Conclusion: This cross-sectional case-control study did not find any association between HTLV-I and SLE. With regard to the previous studies, these controversies may stem from differences in ethnic background. Geographical and environmental factors should also be taken into account. Human T lymphotropic virus type I (HTLV-I) is a C-type retrovirus which is associated with two main types of disease; HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T cell leukemia (ATL) -2.The virus is endemic in southwestern Japan, parts of Africa, and Central and South America -4. MashhThis virus with several routes of transmission not only induces HAM/TSP in a small proportion of HTLV-I carriers, but also is associated with other autoimmune diseases -7. Although pathogenesis of HAM/TSP remains unknown; autoimmune imbalance in the efficiency of HTLV-I-specific cytotoxic T lymphocytes (CTL), HLA associations, and HTLV-I antibody titer  bear in Taken together, in addition to escape from self-tolerance, molecular mimicry, and inflammatory cytokines , 15-18, + T cells into different organs such as central nervous system damages surrounding tissue by releasing different cytokines and metalloproteinases  revised criteria  for SLE,All patients who were high risk for HTLV-I infection after SLE involvement, including patients with a history of suspicious sexual contact, intravenous drug use, and Medical Centre staff members, were excluded from the study. Consecutive patients without exclusion criteria were recruited from university hospitals, rheumatology clinics, and clinical lupus cohorts between January 2008 and March 2009. Disease activity was determined by the SLE Disease Activity Index (SLEDAI-2k) . DemograSerological assay and confirmation testsFive milliliters of blood were obtained from the patients and the healthy control group and stored at −20°C. Serum samples were screened for HTLV-I antibody by enzyme-linked immunosorbent assay  according to the manufacturer’s instructions. Western Blot (WB) was carried out for all positive ELISA samples for further confirmation of HTLV-I infection in patients and controls. All reactive samples on serologic screening were tested further by Western blot (WB) analysis with the MP Diagnostics HTLV Blot 2.4  according to the manufacturer’s instructions. Each anti-HTLV-I positive sample (provided by Abbott Diagnostics) was tested simultaneously with study samples to verify the test results.Statistic Analyses The statistical analysis was performed using the SPSS 11.5 program . Values were reported as mean±SD for normally distributed variables and median with interquartile range (IQR) for others. Kolomogrove-Smirnov test was applied for normal variable distribution. Two independent proportion test was used to evaluate statistical differences between the frequency of HTLV-I seropositive results, in patients and control group.P=0.9). Two (1.5%) of patients and 23 (2.5%) of controls had HTLV-I infection, which was confirmed by WB . The mean age of the patients was 29.78± 11.37 years. The range of age was 13 to69 years. The mean age of controls was 29.1± 18.5 years with range of 13-55 years. All participants were women. There was no significant difference between the age of the case under study and the control group (ed by WB .P=0.49).No statistical significant difference was observed between the patients and controls in the number of HTLV-I seropositive samples (The average of the disease duration in the patients was 3 (0.5-5.7) years. The frequency of important lupus manifestations in these patients (according to SLEDAI) are pointed here: renal involvement: 28.3%, seizure: 5.4%, psychosis: 3.3%, severe headache: 5.4%, myositis: 5.4%, arthritis: 33.9%, malar rash/discoid lupus erythematosus (DLE) /alopecia: 42%, serositis: 21.7%, retinal vasculits: 0.2%, fever: 3%, anemia: 43.4% (hemolytic anemia: 10%), lymphopenia: 40%, leukopenia: 11%, and thrombocytopenia: 14.9%. The mean values of immunologic laboratory parameters in these patients were anti ds-DNA (R): 1.3 (0.5-3)  and C3 (mg/dl): 87 ±46, C4 (mg/dl): 16.6±10 (mg/dl).9 /L) and seizures due to CNS involvement. The other one, was a 53-year-old patient from Neyshabur (the most endemic city in Khorasan province with regard to HTLV-I infection), with history of HAM/TSP since 4 years before the diagnosis of lupus, which was presented with arthritis, leukopenia (2.3×109/L), lymphopenia (0.6×109/L), positive ANA (1/360) and anti-dsDNA (800 U). The mean age of seropositive patients was 53.5±0.7 years.Clinical features of two HTLV-I seropositive lupus patients were as follows: one patient was 54 years old woman with antinuclear antibody (ANA) (1/180), anti double stranded DNA (ds-DNA) (300 U) and antiphospholipid positive tests in repeated measurements. This patient had lymphopenia .As mentioned above, leucopenia and profound lymphopenia were the persistent manifestations in these HTLV-I positive patients. HTLV-I seropositive patients were significantly older than other lupus patients (Some previous studies have reported the correlations between C-type retrovirus with human autoimmune diseases like SLE (40-41). HTLV-I as a subgroup of C-type retroviruses, plays a pivotal role in various autoimmune diseases . LymphocIn the present study, we investigated the association between HTLV-I infection and SLE in the northeast of Iran, Mashhad. This study revealed that HTLV-I infection was not a predisposing factor for SLE in this endemic area, because our SLE patients had the same rate of HTLV-I seroprevalence as the control group.et al. report. The age of HTLV-I-seropositive patients with SLE was significantly higher than that of seronegative patients. Furthermore, the age at onset of SLE in HTLV-I-seropositive cases was also significantly higher than that of seronegative cases, suggesting that HTLV-I may induce a unique autoimmune disease similar to SLE, many years after initial infection (et al evaluated the correlation between HTLV-I infection and different autoimmune diseases. They found no antibody against HTLV-I in seropositive patients. In addition, in seropositive HTLV-I group, nobody had positive antinuclear antibodies or antibodies to U1RNP(et al. suggested that type Cretroviruses played a role in immune activation in murine lupus (The results of our study confirm the results of the previous studies in endemic areas of HTLV-I , 30-31. nfection . The immnfection -16. Howe to U1RNP. Gourleyne lupus . Neverthne lupus , 30-31. The current study was not without limitations, for example, we used ELISA screening assay for all patients and the level of IgG directed against HTLV-I was detected. It has been reported that HTLV-I antibodies of a non-IgG class of immunoglobulin might not been detected during the screening of HTLV-I infection . The strTaken together, the overall outcome of our research is in line with the most of the studies that have shown no association between HTLV-I infection and SLE. Contradictions about the relationship between HTLV-I and SLE occurrence suggest that differences in ethnic background may contribute to this issue. Other conditions such as geographical and environmental factors should be taken into account.This cross-sectional case-control study did not find any association between HTLV-I and SLE. With regard to the previous studies, these controversies may stem from differences in ethnic background. Geographical and environmental factors should also be taken into account.


In this open-label, single arm study enrolling 24 HAM/TSP patients recently published in BMC Medicine, oral prosultiamine (300 mg/day for 12 weeks) was found to be effective by neurological, urological and virological evaluations. Notably, it increased detrusor pressure, bladder capacity and maximum flow rate, and improved detrusor overactivity and detrusor-sphincter dyssynergia. A significant decrease in HTLV-I copy numbers in peripheral blood following the treatment provided a rationale for using the drug. The trial has some limitations, such as the small numbers of participants, the open-label design, the lack of a placebo arm, and the short trial period. Nevertheless, the observation that such a safe, cheap drug may have excellent therapeutic effects on HAM/TSP, a chronic devastating illness occurring mainly in developing countries, provides support for future large-scale randomized controlled trials.Prosultiamine, a vitamin B1 derivative, has long been used for beriberi neuropathy and Wernicke’s encephalopathy. Based on the finding that prosultiamine induces apoptosis in human T lymphotropic virus type I (HTLV-I)-infected T cells, Nakamura http://www.biomedcentral.com/1741-7015/11/182.Please see related research:  Prosultiamine (Alinamin®), a well-known thiamine derivative, was first developed by Takeda Pharmaceutical Company in Japan in the 1950s. The drug is a homolog of allithiamine produced by thiol-type vitamin B1 and allicin. Prosultiamine is synthesized by substitution of allyl disulfide with propyl disulfide in the allithiamine structure, to increase its stability in blood and achieve efficient absorption from the gut. Prosultiamine is converted to vitamin B1 after absorption from the gut. The drug thus enables a long-lasting high blood concentration of vitamin B1, resulting in efficient access of vitamin B1 to nervous tissue. Prosultiamine has cured many patients with vitamin B1 deficiency resulting in beriberi neuropathy and Wernicke’s encephalopathy. Prosultiamine might also save patients suffering from another devastating neurologic illness associated with a human retrovirus, namely T lymphotropic virus type I (HTLV-I).+ T cells; such HTLV-I-infected CD4+ T cells are autoproliferative and tend to infiltrate central nervous system (CNS) tissue [+ T cells continuously kill HTLV-I-infected CD4+ T cells infiltrating the CNS [HTLV-I causes not only adult T cell leukemia but also chronic progressive myelopathy, known as HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) . HAM/TSP) tissue . Some au the CNS ,4.In HAM/TSP patients, HTLV-I proviral DNA loads in peripheral blood are markedly increased despite the presence of abundant cytotoxic T cells against HTLV-I ,6. Thereet al. [Recently, Nakamura et al.  discoveret al. .BMC Medicine by Nakamura et al. [The study recently published in a et al.  was an o2O on average) and bladder capacity (from 341.3 to 391.0 ml on average), and maximum flow rate (from 7.5 to 10.2 ml/s on average) together with improvements in detrusor overactivity (68.8% of the patients) and detrusor-sphincter dyssynergia (45.5% of the patients). Importantly, HTLV-I copy numbers in 104 peripheral blood mononuclear cells (PMBCs) decreased significantly after treatment compared with pretreatment levels , with some patients reaching a 30% to 50% decrease. Only three patients complained of mild epigastric discomfort during treatment; otherwise, no side effects were observed.Prosultiamine improved lower limb spasticity in 80% of the patients (by more than 1 grade for the degree of spasticity on MAS), which almost coincided with improvements in the times required for a 10 meter walk (4.4% to 36.8% decrease in 11 patients) and walking down a flight of stairs (2.3% to 53.2% decrease in 10 patients). Remarkably, both the N-QoL scores and urinary function tests as evaluated by urodynamic studies were significantly improved by the treatment compared with baseline levels; there were increases in detrusor pressure (from 16.8 to 27.5 cm/HThe number of participants in the present trial was small and the trial was open-label without a placebo arm; therefore, the current observations should be interpreted with caution and the findings should be confirmed by a large-scale, randomized, double-blind, placebo-controlled study in the future. Nonetheless, the improvement in urodynamic study findings is striking and could provide some proof of concept for the use of prosultiamine in HAM/TSP patients. Although placebo effects in the improved motor performance could not be fully eliminated, the urodynamic study findings support the notion that oral vitamin B1 derivatives have real therapeutic effects on HAM/TSP. The coincidence of the HTLV-I proviral DNA reduction in PBMCs by the drug with therapeutic efficacy may also support such a notion. Such a coincidence was also observed with IFNα . The samOral prosultiamine could safely alleviate HAM/TSP as evidenced by motor function evaluation and urodynamic studies, coinciding with a modest decrease in HTLV-I copy numbers in PBMCs. The drug appears to be promising, but the long-term benefits of the drug remain to be established by a large-scale, randomized, controlled study. The results of the present study encourage such investigations.JK is a consultant for Biogen Idec Japan, and has received honoraria from Bayer Healthcare and funding for a trip from Bayer Healthcare and Biogen Idec Japan. He is funded by a Research Grant for Nervous and Mental Disorders from the Ministry of Health, Labour and Welfare, Japan, and grants from the Japan Science and Technology Agency and the Ministry of Education, Culture, Sports, Science and Technology, Japan.


HTLV-1 and HTLV-2 encode accessory proteins p30 and p28, respectively, which share partial amino acid homology and are required for viral persistence in vivo. The goal of this study was to identify host proteins interacting with p30 and p28 in order to understand their role in pathogenesis.Human T lymphotropic virus type-1 (HTLV-1) and type 2 (HTLV-2) are closely related human retroviruses, but have unique disease associations. HTLV-1 is the causative agent of an aggressive T-cell leukemia known as adult T-cell leukemia (ATL), HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), and other inflammatory diseases. HTLV-2 infection has not been clearly associated with any disease condition. Although both viruses can transform T cells Affinity-tag purification coupled with mass spectrometric (MS) analyses revealed 42 and 22 potential interacting cellular partners of p30 and p28, respectively. Of these, only three cellular proteins, protein arginine methyltransferase 5 (PRMT5), hnRNP K and 60 S ribosomal protein L8 were detected in both p30 and p28 fractions. To validate the proteomic results, four interacting proteins were selected for further analyses using immunoblot assays. In full agreement with the MS analysis two cellular proteins REGγ and NEAF-interacting protein 30 (NIP30) selectively interacted with p30 and not with p28; heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1) bound to p28 and not to p30; and PRMT5 interacted with both p30 and p28. Further studies demonstrated that reduced levels of PRMT5 resulted in decreased HTLV-2 viral gene expression whereas the viral gene expression of HTLV-1 was unchanged.The comparisons of p30 and p28 host protein interaction proteome showed striking differences with some degree of overlap. PRMT5, one of the host proteins that interacted with both p30 and p28 differentially affected HTLV-1 and HTLV-2 viral gene expression suggesting that PRMT5 is involved at different stages of HTLV-1 and HTLV-2 biology. These findings suggest that distinct host protein interaction profiles of p30 and p28 could, in part, be responsible for differences in HTLV-1 and HTLV-2 pathobiology. This study provides new avenues of investigation into mechanisms of viral infection, tropism and persistence. HTLV-1 d in vivo-13. Furtnditions ,15.The genome organization of the HTLV-1 and HTLV-2 provirus is very similar with 5’ and 3’ LTRs. Both viruses encode regulatory and accessory proteins apart from the typical structural and enzymatic proteins Gag, Pol and Env. Two key regulatory gene products are Tax-1/Tax-2 and Rex-1/Rex-2 corresponding to HTLV-1/HTLV-2, respectively.The Tax proteins encoded by both viruses are considered the primary oncoproteins ,17 requiin vitro viral infection and T cell transformation. Interestingly, both p30 and p28 are required for the establishment of viral persistence in the rabbit model, underlining the importance of these proteins in vivo[tax/rex mRNA in the nucleus [tax/rex mRNA [HTLV-1 p30 and HTLV-2 p28 are accessory proteins involved in the regulation of viral replication and persistence and could therefore affect the pathogenic outcome -27. Boths in vivo,27,28. Is in vivo. Both p3s in vivo,30. The s in vivo. Both p3 nucleus ,30. Howe nucleus . In addirex mRNA .Microarray studies and genome-wide screens have shown that p30 differentially modulates cellular gene expression ,34. ExprComparative studies of host protein interactions with HTLV-1 and HTLV-2 proteins have been largely focused on Tax-1 and Tax-2 ,40-42. SIn order to sample the p30 and p28 cellular proteome, we employed S-tag affinity purification  of ectopWe previously identified the interaction of p30 and REGγ using proteomic and molecular biology techniques . HoweverThe interaction of p30 with REGγ was further confirmed by immunoprecipitating REGγ from lysates of cells transfected with S-p30-HA and S-p28-HA and immunoblotted with anti-HA antibody. REGγ was able to co-immunoprecipitate p30 but not p28 Figure B. These To confirm the interaction of p28 with hnRNP H1, lysates from cells with transient expression of mock, S-GFP, S-p30-HA and S-28-HA were S-tag affinity purified and immunoblotted with anti-hnRNP H1 antibody. The presence of hnRNP H1 only in the p28 purification and not in GFP or p30 purifications . The analysis of all proteins that interact with p30 from Table Furthermore, we evaluated indirect involvement of p30 and p28 interacting host proteins in various cellular processes using Ingenuity Pathways Analysis . NEFA was initially thought to bind DNA; however, it has been shown to be a Golgi localized calcium-binding protein ,54. The tax/rex mRNA  and retain it in the nucleus and thus regulate viral gene expression at the post transcriptional level, our findings in Figure tax/rex mRNA in the nucleus as a part of the spliceosome to regulate viral gene expression. Further studies should provide insights into the mechanism of p30 and p28 function and avenues to develop novel therapeutic targets.Although both p30 and p28 specifically bind in vivo for SMN complex formation to join proteins and RNA [Both p30 and p28 interact with PRMT5, a type II PRMT enzyme that symmetrically dimethylates arginines . The int and RNA .To investigate the possible role of PRMT5 interaction with p30 in transcriptional regulation, we performed PRMT5 knockdowns. HTLV-1 gene expression did not change significantly under low levels of PRMT5 Figure A. The exIn summary, we identified 42 and 22 potential cellular proteins interacting with HTLV-1 p30 and HTLV-2 p28, respectively. Of these, only three cellular proteins overlapped indicating markedly different interacting profiles for these two viral proteins. PRMT5 interacted with both p30 and p28, but had a differential effect on the gene expression of HTLV-1 and HTLV-2 indicating that PRMT5 might have different roles in HTLV-1 and HTLV-2 viral biology. The differences in protein and cellular processes with which p30 and p28 engage could be one reason for the difference in viral infection outcomes. Elucidating the role of PRMT5 in HTLV-1 viral biology would provide insights into how HTLV-1 and HTLV-2 are utilizing a host protein differently to potentially result in different pathological outcomes. Further investigation to understand the functional relevance of these interactions is warranted to better understand the HTLV life cycle and its role in T-cell transformation in order to develop new therapeutic drug targets.6 cells were plated in 100 mm dishes and after 24 hr, cells were transfected with 10 μg of pTriEx4-Neo (Mock), 10 μg of pTriEX-Neo-S-GFP (S-GFP), 10 μg of pTriEx4-Neo-S-p30-HA (S-p30-HA), 10 μg of pTriEx4-Neo-S-p28-AU1 or 4 μg of pTriEx4-Neo-S-p28-HA(S-p28-HA) from Novagen, Madison, WI. For Flag-PRMT5 immunoprecipitation 5 μg of pBabe (empty vector) or 5 μg of Flag-PRMT5  was co-tranfected with 5 μg of S-p30-HA or S-p28-HA. The cells were harvested and lysed in 1x passive lysis buffer after 24 hr . 293T cells treated with siRNA or lentiviral particles expressing scrambled shRNA or PRMT5 shRNA were transfected with the HTLV-1 molecular clone, Ach (1.8 μg/well) and HTLV-2 molecular clone, Ph6neo (1.8 μg/well) of a six well plate using SuperFect . Oligofectamine  was used to transfect 293T cells with 100 nM of control siRNA or smart pool siRNA against REGγ and NIP30 . Stable knockdowns were produced by transducing 293T cells with lentiviral particles expresssing scramble or PRMT5 shRNAs  and selected for puromycin resistance.HEK 293T cells were cultured in DMEM containing 10% FBS, 2 mM L-glutamine, 100 mg/ml streptomycin, and 100 units/ml penicillin. Plasmids were transfected using SuperFect  according to the manufacturer’s instructions. Briefly 2x10Molecular proviral clones of HTLV-1 and HTLV-2 were transfected 24 hrs after siRNA treatment and incubated for 24 hrs. Supernatant was collected for measure of p19 Gag by ELISA  and cells were lysated and immunoblotted for p24 production.2O and subjected to shotgun proteomics. For subsequent immunoblotting the proteins were eluted by boiling the beads in SDS loading buffer. Immunoprecipitation was performed by rocking cell lysates overnight with specific antibody at 4°C and then with Sepharose protein A beads for 2 hrs (GE Sweden). Sepharose protein A beads were washed twice with high salt (1 M NaCl) containing radioimmunoprecipitation assay buffer and thrice with low salt (150 nM NaCl) radioimmunoprecipitation assay buffer and once with PBS. For western blotting proteins were eluted by boiling the beads in SDS loading buffer.Cell lysates were prepared with 1X passive lysis buffer  in the presence of protease inhibitor mixture . S-tag purification was performed by rocking cell lysates with S-beads  overnight at 4°C. The S-beads were washed once with high salt (1 M NaCl) containing radioimmunoprecipitation buffer ), three times with radioimmunoprecipitation buffer and once with PBS. The beads were suspended in ddHTM 3000 HPLC system from LC-Packings A Dionex Co. . The scan sequence of the mass spectrometer was based on the data dependant TopTenTM method. The resolution of a full scan was set at 30000 to achieve a high mass accuracy MS determination.Capillary-liquid chromatography-tandem mass spectrometry (Capillary-LC/MS/MS) for global protein identification was performed on a Thermo Finnigan LTQ Orbitrap mass spectrometer equipped with a microspray source  operated in positive ion mode. Samples were separated on a capillary column  using an UltiMatehttp://www.massmatrix.net/download). The resulting MGF files were searched using Mascot Daemon by Matrix Science version 2.2.2  and the database searched against the full SwissProt database version 57.5  or NCBI database version 20091013 . Considered modifications (variable) were methionine oxidation and the presence of carbamidomethyl cysteine. Three missed cleavages for the enzyme were permitted with a peptide tolerance of 1.2 Da, and the MS/MS ion tolerance was 0.8 Da. Mock transfected (empty pTriEx4-Neo) cell lysates treated similarly served as negative control. Search results were compiled and visualized using the Scaffold 3 sofware. Unweighted spectrum count and percent coverage provided semi-quantitative data analyses. Protein identifications were assigned using PeptideProphet. Proteins with 80% confidence were accepted with a minimum of one peptide displaying 95% threshold confidence level.The RAW data files collected on the mass spectrometer were converted to mzXML and MGF files by use of MassMatrix data conversion tools  ; rabbit anti-hnRNPH1 monoclonal (1:1000), mouse anti-hnRNPH1 (1:1000), mouse anti-HTLV p24 (1:1000) ; Rabbit anti-NIP30 monoclonal (1:1000), rabbit anti-REGγ (1:1000) ; mouse-anti β-actin (1:2000) ; rat anti-Flag (1:1000) horse anti-mouse (1:2000), and goat anti-rabbit antibodies (1:2000) anti-rat (1:2000) .NIP30, NEFA interacting protein 30; PRMT5, Protein Arginine methlytransferase 5; hnRNP H1, Heterogeneous nuclear ribonucleoprotein H1; HTLV-1 and -2, Human T-cell lymphotropic virus type 1 and 2; ATL, Adult T cell leukemia; HAM/TSP, HTLV-1 associated myelopathy/tropical spastic paraparesis.Rami Doueiri and Rajaneesh Anupam contributed equally to this work.Authors have no competing interests involved in data collection, materials and methods or conclusions.RD and RA contributed equally to designing and collecting the data presented in the paper. KBG headed the shot-proteomics data collection. MK helped with mass spectrometry-based proteomic data analysis and finalizing the manuscript. MDL and PLG conceived, coordinated and helped in writing the manuscript. All authors read and approved the final manuscript.Figure S1. Effects of REGγ and NIP30 knockdown on HTLV-1 gene expression. A) Two independent experiments (labeled 1 and 2) of control and REGγ siRNA treated 293T cells were transfected with Ach (HTLV-1 molecular clone). The knockdown of REGγ was confirmed by immunoblotting with anti-REGγ. The expression of HTLV p24 was monitored by immunoblotting. B) Negative siRNA and NIP30 siRNA treated 293 T cells were transfected with HTLV-1 molecular clone (Ach). The levels of NIP30 were tested by immunoblotting with anti-NIP30 antibodies. The expression of HTLV-1 p24 was monitored by anti-HTLV-1 p24 antibody. Equal loading of samples was confirmed by anti-actin antibodies. (JPEG 51 kb)Click here for file