Human T-lymphotropic virus type 4 (HTLV-4) is a new deltaretrovirus recently identified in a primate hunter in Cameroon. Limited sequence analysis previously showed that HTLV-4 may be distinct from HTLV-1, HTLV-2, and HTLV-3, and their simian counterparts, STLV-1, STLV-2, and STLV-3, respectively. Analysis of full-length genomes can provide basic information on the evolutionary history and replication and pathogenic potential of new viruses.Homo sapiens sapiens during the Middle Pleistocene suggesting that early humans may have been susceptible hosts for the ancestral HTLV-4.We report here the first complete HTLV-4 sequence obtained by PCR-based genome walking using uncultured peripheral blood lymphocyte DNA from an HTLV-4-infected person. The HTLV-4(1863LE) genome is 8791-bp long and is equidistant from HTLV-1, HTLV-2, and HTLV-3 sharing only 62–71% nucleotide identity. HTLV-4 has a prototypic genomic structure with all enzymatic, regulatory, and structural proteins preserved. Like STLV-2, STLV-3, and HTLV-3, HTLV-4 is missing a third 21-bp transcription element found in the long terminal repeats of HTLV-1 and HTLV-2 but instead contains unique c-Myb and pre B-cell leukemic transcription factor binding sites. Like HTLV-2, the PDZ motif important for cellular signal transduction and transformation in HTLV-1 and HTLV-3 is missing in the C-terminus of the HTLV-4 Tax protein. A basic leucine zipper (b-ZIP) region located in the antisense strand of HTLV-1 and believed to play a role in viral replication and oncogenesis, was also found in the complementary strand of HTLV-4. Detailed phylogenetic analysis shows that HTLV-4 is clearly a monophyletic viral group. Dating using a relaxed molecular clock inferred that the most recent common ancestor of HTLV-4 and HTLV-2/STLV-2 occurred 49,800 to 378,000 years ago making this the oldest known PTLV lineage. Interestingly, this period coincides with the emergence of Homo sapiens, the propensity of STLVs to cross-species into humans, the fact that HTLV-1 and -2 spread globally following migrations of ancient populations, all suggest that HTLV-4 may be prevalent. Expanded surveillance and clinical studies are needed to better define the epidemiology and public health importance of HTLV-4 infection.The inferred ancient origin of HTLV-4 coinciding with the appearance of  Deltaretroviruses are a diverse group of human and simian T-lymphotropic viruses  that until lately were composed of only two distinct human groups called HTLV types 1 and 2 . Two newSTLV-1 has a broad geographic distribution in nonhuman primates (NHPs) in both Asia and Africa thus providing humans with historical and contemporaneous opportunities for exposure to this virus ,4,5,9,10Pan paniscus), the zoonotic relationship of this divergent virus to HTLV-2 is less clear dCTP using Klenow enzyme (Invitrogen). The DNA-binding reaction was incubated for 1 h at room temperature using 5 ng of labeled probe and binding buffer  supplemented with 2 ug of sheared salmon sperm DNA, 1 ug poly-dI-dC , and 5 ug 293 cell extract in a final volume of 15 ul. The supershift was performed by adding 1 ug of anti-c-Myb monoclonal antibody  or non-specific PC10 monoclonal antibody  to the binding reaction for 1 h at room temperature. Unlabeled double-stranded  non-specific oligonucleotide contained mutations at three positions (underlined) within the predicted c-Myb binding site. Specific and non-specific competitors were added in a 100-fold excess over labeled probe. DNA-protein complexes were resolved on a 4% non-denaturing polyacrylamide gel in 0.5× Tris-borate-EDTA at 150 V for 2.5 h.The double-stranded oligonucleotide probe representing the c-Myb binding site within the HTLV-4 LTR  was end-labeled with [α-EF488483. GenBank accession numbers for the complete PTLV genomes used in this paper are [HTLV-1(ATK) = J02029], [HTLV-1(ATL-YS) = U19949], [HTLV-1(Mel5) = L02534], [HTLV-1 (Boi) = L36905], [STLV-1(TE4) = Z46900], [STLV-1(Tan90) = AF074966], [HTLV-2(MoT) = M10060], [HTLV-2(Kay96) = AF356584], [HTLV-2(Gab) = Y13051], [HTLV-2(SP-WV) = AF139382], [HTLV-2(G2) = L11456], [HTLV-2(G12) = L11456], [HTLV-2(Efe) = Y14365], [STLV-2(Pan-p) = U90557], [STLV-2(pp1664) = Y14570], [HTLV-3(2026ND) = DQ093792], [HTLV-3(Pyl43) = DQ462191], [STLV-3(CTO604) = NC_003323], [STLV-3(Ph969) = Y07616], [STLV-3(TGE2117) = AY217650], [STLV-3(NG409) = AY222339], [STLV-3(Ppaf3) = AF517775], [STLV-5(MarB43) = AY590142].The complete HTLV-4(1863LE) proviral sequence has been deposited in GenBank with accession number Some authors  have applied for a patent for the discovery of HTLV-4.WMS conceived, designed and coordinated the study, analyzed, acquired and interpreted the data, and wrote the manuscript. MS, RRG, and AK helped design the study, performed detailed phylogenetic analysis of the sequences, and helped write the manuscript. SHQ and HJ together obtained the full-length genome of HTLV-4, analyzed the sequences, and participated in writing the manuscript. SJM and KNP helped characterize the LTR regulatory elements and participated in writing the manuscript. NDW, DSB, TMF, and WH helped design the study, assisted in analysis of the data, and participated in writing the manuscript. All authors read and approved the final manuscript.versus divergence plots in different HTLV-4 (1863LE) genes using 1st + 2nd or 3rd codon positions (cdp). Genetic distances were calculated with the Tamura and Nei 1993 (TN93) model and plotted against the estimated number of transitions and transversions for each pair-wise comparison using the DAMBE program. Figure S2. Evolutionary relationship of major genes and the entire genome of HTLV-4(1863LE) to other PTLVs by using either Neighbor-Joining  or maximum likelihood  methods. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (100–1000 replicates) is shown at the branch nodes. Branch lengths are drawn to scale and only bootstrap values greater than 70% are shown. Branches leading to PTLV-1, HTLV-2, and PTLV-3 sequences are drawn in red, blue, and green, respectively. The branches leading to HTLV-4(1863LE), STLV-2, and to the divergent STLV-5(MarB43) strain are drawn in magenta, purple, and yellow, respectively.Supplementary figures. Figure S1. Pair-wise transition  and transversion  Click here for file


Molecular characterization of the FMS-like tyrosine kinase 3 receptor (FLT3) in cytogenetically normal acute myeloid leukemia (AML) has recently been incorporated into clinical guidelines based on correlations between FLT3 internal tandem duplications (FLT3-ITD) and decreased disease-free and overall survival. These mutations result in constitutive activation of FLT3, and FLT3 inhibitors are currently undergoing trials in AML patients selected on FLT3 molecular status. However, the transient and partial responses observed suggest that FLT3 mutational status alone does not provide complete information on FLT3 biological activity at the individual patient level. Examination of variation in cellular responsiveness to signaling modulation may be more informative.Using single cell network profiling (SCNP), cells were treated with extracellular modulators and their functional responses were quantified by multiparametric flow cytometry. Intracellular signaling responses were compared between healthy bone marrow myeloblasts (BMMb) and AML leukemic blasts characterized as FLT3 wild type (FLT3-WT) or FLT3-ITD. Compared to healthy BMMb, FLT3-WT leukemic blasts demonstrated a wide range of signaling responses to FLT3 ligand (FLT3L), including elevated and sustained PI3K and Ras/Raf/Erk signaling. Distinct signaling and apoptosis profiles were observed in FLT3-WT and FLT3-ITD AML samples, with more uniform signaling observed in FLT3-ITD AML samples. Specifically, increased basal p-Stat5 levels, decreased 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 were observed in FLT3-ITD AML samples. Preliminary analysis correlating these findings with clinical outcomes suggests that classification of patient samples based on signaling profiles may more accurately reflect FLT3 signaling deregulation and provide additional information for disease characterization and management.These studies show the feasibility of SCNP to assess modulated intracellular signaling pathways and characterize the biology of individual AML samples in the context of genetic alterations. AML is associated with a wide range of genetic alterations, including mutations in receptor tyrosine kinases (RTKs) that perturb intracellular signaling networks which play a role in leukemia pathogenesis and are manifested in the clinical heterogeneity of the disease. For example, internal tandem duplications in the juxtamembrane region or tyrosine kinase 1 domains of the FLT3 RTK are reported to result in autonomous, ligand independent signaling with consequent increases in survival and proliferation The length of the DNA insertion that constitutes the ITD and the mutational load or allelic ratio of mutated to wild type (WT) FLT3 receptor vary among patient leukemia samples. Although length of ITD has not been consistently reported to be associated with clinical outcomes, higher levels of ITD mutational load have been associated with worse outcomes in multiple studies Many studies have provided insight into intracellular signaling pathways regulated by the FLT3 receptor system in vitro exposure to modulators such as growth factors, cytokines or therapeutic agents known to be important for myeloid biology and clinical application (Single cell network profiling (SCNP) using multiparameter flow cytometry is a distinct proteomic platform for analyzing and interpreting protein expression and pathway activity under baseline and modulated conditions. Using viable cells, measurements of endogenous proteins in relevant signaling pathways are made before and after lication . SCNP inAlterations in kinases, phosphatases and transcription factors can lead to aberrant changes in intracellular protein networks resulting in increased proliferation and survival, and blocks in differentiation, and contributing to the pathogenesis of AML. In this report, using diagnostic non-M3 AML patients, we show that SCNP characterizes AML signaling pathways  and provThis study utilized two separate cohorts of patients. The first sample set (study one) consisted of 34 cryopreserved diagnostic peripheral blood mononuclear cell (PBMC) samples collected between September 1998 and September 2007 from AML (non-M3) patients treated at hospitals affiliated with the University Health Network (PMH/UHN), University of Toronto. In comparison to the general United States (US) AML population, patients in this group tended to be younger (74%<60 years), female (62%), of Asian ancestry (29%), and had primary refractory (NR) leukemia (74%) . Most leThe second sample set (study two) consisted of 83 cryopreserved diagnostic bone marrow mononuclear cell (BMMC) samples collected from AML (non-M3) patients treated at MD Anderson Cancer Center (MDACC) between September 1999 and September 2006 . This saTen healthy BMMC taken from male and female volunteers aged between 24–80 years (mean = 66 years) were analyzed as controls. Two BMMC samples were used as controls in study one and study two. Additionally, eight control BMMC samples were analyzed separately in order to characterize FLT3L induced signaling in healthy myeloblasts.To analyze modulator-antibody combinations, hereafter defined as signaling nodes, several metrics were developed to describe pathway activity: Basal , Fold Change (modulator-induced), and Total  . “Node |SCNP was utilized to compare WT FLT3 receptor signaling in healthy BMMb versus FLT3-WT leukemic blasts see , activatTo assess signaling differences between FLT3-WT and FLT3-ITD AML samples, we used SCNP to measure the activities of pathways that regulate hematopoietic cell proliferation and differentiation, drug transport, apoptosis, and DNA damage response/apoptosis .p≤0.05  (In order to determine the integrity of the DNA damage response (DDR) and apoptotic machinery, the ability of etoposide to induce DNA damage (increased p-Chk2 levels) and apoptosis (increased cleaved PARP levels), was measured in leukemic blasts. FLT3-ITD samples were more sensitive to ctively) , S4. Simctively) , S4Although the signaling nodes were analyzed independently in the primary analysis, several of the top-ranking nodes stratifying FLT3-ITD from FLT3-WT AML samples were correlated with each other. Pearson correlation coefficients computed for all signaling nodes with a p≤0.05 from study one  showed .To determine if different signaling nodes in combination could provide superior stratification of FLT3-ITD and FLT3-WT AML samples, all possible pairs of the 80 signaling nodes with AUC of the ROC ≧0.7 or lowest p≤0.05 from study 1  were evaTo understand how FLT3 receptor and NPM1 mutational status relate to intracellular biological pathways in diagnostic AML samples, we assessed IL-27 induced Jak/Stat signaling and FLT3L induced PI3K and Raf/Ras/MAPK signaling responses in FLT3 receptor and NPM1 molecular defined subgroups. NPM1 mutational status was available for all patients in study one but only for a subset in study two; hence the interpretation of the results of these analysis are limited by the small number of samples in each subset. For all nodes analyzed, the FLT3-WT/NPM-WT subgroup demonstrated the most variable signaling responses and often contained samples with the most elevated signaling . In contTo assess if functional intracellular pathways characterization using SCNP may stratify (beyond molecular characterization) AML for clinical outcomes, the signaling profiles of samples with clinical outcomes not predicted by molecular findings  were analyzed.Signaling profiles were assessed in samples from two groups of Cytogenetically Normal (CN) AML patients, each representing a clinically extreme example of an “outlier” (based on the molecular characterization): 1) FLT3-WT AML who experienced disease relapse within three months after initial remission and 2) FLT3-ITD AML in complete continuous disease remission for two or more years. In the first study there were no patient samples that met these two criteria. In the second study there were two FLT3-WT and two FLT3-ITD samples respectively . ClinicaThe wide range of signaling responses observed in FLT3-WT AML samples made identification of signaling outliers in this subgroup challenging. Examination of the signaling profiles in the two FLT3-WT samples with relapse <3 months (MD3–19 and MD3–37) showed low p-S6 and p-Erk in response to FLT3L, similar to induced signaling observed in FLT3-ITD samples . In addiROC: 0.92)  were associated with CR duration greater than two years (AUCC: 0.92) .While genetic and molecular heterogeneity may ultimately underlie the different responses to therapy observed among patients with AML, there appears to be a finite number of intracellular signaling pathways through which these differences are expressed. These pathways  play important roles in proliferation, survival, cell cycle progression, and differentiation in both normal and leukemic hematopoietic cells The present studies illustrate the value of using SCNP to characterize the biologic complexity of AML at the signaling pathway level. Although this approach has been applied previously to AML using a limited panel of nodes While much has been learned from experiments in leukemic cell lines there is little published data on WT FLT3 receptor signaling in healthy bone marrow myeloblasts (BMMb) and primary leukemic blasts Canonical Stat pathways, activated by cytokines, regulate an array of cellular functions ranging from survival and apoptosis to proliferation and differentiation This study is also the first to show different signaling responses between FLT3-WT and FLT3-ITD in AML samples for IL-27 induced Jak/Stat pathway activity. Most studies characterizing the biology of IL-27 have been performed on lymphocytes where this cytokine plays a major role in immune regulation. However, the IL-27 receptor is present on other cell types, including those of the myeloid lineage, where its activation has been shown to enhance proliferation and differentiation of mouse and human hematopoietic stem/progenitor cells Finally, analysis of the apoptosis pathway showed that FLT3-ITD samples were more sensitive in vitro to apoptosis inducing agents than FLT3-WT samples. Aside from differences in cell source (primary leukemia blasts versus cell lines) the continuous exposure of cells to DNA damaging drugs may account for the difference between our data and published literature, where the introduction of FLT3-ITD into cell lines has been associated with resistance to DNA damage induced apoptosis Taken together these data show that SCNP uncovers important heterogeneity in AML and has potential as a platform for understanding leukemia pathway dependency in the individual patient, information that could be valuable for the selection of therapeutic strategies in the era of personalized medicine. This method promises to reduce the complicated genetic picture that is emerging for AML to the functional status of a few distinct signaling pathways.However, whether the SCNP methodology will provide clinically useful and actionable information beyond existing molecular markers remains to be proven .FLT3 gene has shown not to be very informative in predicting the activity of any of these FLT3 receptor inhibitors and their effects on signaling transduction remains unknown. In this regard, SCNP could be tested as a tool to identify AML patients who could benefit from administration of such inhibitors alone or in combinations with other standard agents and/or targeted inhibitors. Further studies in the context of clinical trials are warranted.Lastly, multiple therapeutics that target FLT3 receptor  are in development for the treatment of AML. To date, the characterization of AML based on the mutational status of the In accordance with the Declaration of Helsinki, all patients provided written informed consent for the collection and use of their samples for research purposes. All studies were approved by Institutional Review Boards (IRB): University Health Network Research Ethics Board (study one), M.D. Anderson Cancer Center Institutional Review Board (study two), and Independent Review Consulting, Inc. . Clinical data were de-identified in compliance with Health Insurance Portability and Accountability Act regulations.Sample inclusion criteria included diagnosis of AML, French-American-British classification as M0 through M7 AML (excluding M3), collection prior to the initiation of induction chemotherapy, molecular determination of the presence or absence of FLT3-ITD, post-thaw cell viability of >50% and >500 cells in the leukemic cell population (defined below). All samples underwent fractionation over Ficoll-Hypaque prior to cryopreservation with fetal calf serum and 10% dimethyl sulfoxide and storage in liquid nitrogen.2 × 3 days, cytarabine 100–200 mg/m2 continuous infusion × 7 days). The second sample set consisted of 83 cryopreserved BMMC samples collected from AML (non-M3) patients treated at MDACC between September 1999 and September 2006. Induction chemotherapy consisted of one or two cycles of cytarabine (200 mg/m2 to 3 g/m2) in combination with an anthracycline (daunorubicin or idarubicin) or an additional anti-metabolite , and sometimes an experimental agent. However, due to differences in cell number post-thaw in this study, the number of patients for which data was available for a given node/metric varied between 83 and 9. Standard clinical and laboratory criteria were used for defining complete response (CR) in both studies The first sample set consisted of 34 cryopreserved peripheral blood mononuclear cell (PBMC) samples collected from AML (non-M3) patients treated at hospitals affiliated with the PMH/UHN, University of Toronto, between September 1998 and September 2007. Induction chemotherapy consisted of one cycle of standard cytarabine-based induction therapy  as well as 2 cell lines were used in studies one and two as assay controls. Additionally, eight healthy BMMC controls were obtained from Williamson Medical Center and assayed in a separate study.FLT3 receptor gene mutational status was assessed at MDACC.In study one, an aliquot of cells were taken from each sample and DNA and RNA were isolated using TRIzol Reagent (Invitrogen) as described by the manufacturer. In study two, For the analysis of internal tandem duplication of the FLT3 receptor gene, PCR was performed on 500 ng genomic DNA using published primers 11F and 12R as described to identify ITD insertions in the JM and TK1 domains 5′-TTGCCCCTGACAACATAGTTGGA-3′, designed for a cDNA template.For the screening of FLT3 receptor D835 mutation by RFLP analysis PCR was performed on 500 ng genomic DNA using published primers 17F and 17R as described 5′-[6-FAM]-GATGTCTATGAAGTGTTGTGGTTC-3′ and reverse primer 5′-GTTTCTAAGGACAGCCAGATATC.In study one, Real-time quantitative PCR assays for NPM1 mutations A and B: RQ-PCR were performed on 100 ng cDNA using published primers and Taqman probe cNPM-F, c. Probe, cNPM mut.A-R, cNPM mut.B-R as described in vitro exposure of AML samples to etoposide, Ara-C/daunorubicin or staurosporine.The analysis included response to chemokines, cytokines, growth factors such as: SCF and FLT3L- mediated PI3K/Akt and MAPK pathway activation (important for maintaining the hematopoietic stem cell pool 3), pelleted, and stained with cocktails of fluorochrome –conjugated antibodies  . All flow cytometry data were analyzed with FlowJo  or WinList . Dead cells and debris were excluded by forward scatter, side scatter, and Amine Aqua Viability Dye measurement. Leukemic cells were identified as cells that fit the CD45 and CD33 versus right-angle light-scatter characteristics consistent with myeloid leukemia blasts and that lacked the characteristics of mature lymphocytes   [65] HeaIn SCNP 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-Stat5 as a readout. That signaling node is designated “FLT3L → p-Stat5”. Several metrics  were ≥0.40 for 33/34 samples in study one and 82/83 samples in study two suggesting that the majority of samples were capable of induced signaling responses.ROC) All signaling nodes  were indPearson correlation coefficients were computed between all pairs of signaling nodes.1 is the intercept for the model. N1 and N2 are the first and the second node/metrics respectively. The coefficients a1 and a2 are tested for their significance for being different from zero. Models in which both the nodes/metrics have a significant p-value (slope  =  0) are considered to be the nodes/metrics combinations with improved predictive value. All pairs for which the p-values were significant and the AUC for the model was greater than 0.90  were examined further.All possible pairs of the 80 node/metrics that were considered to be stratifying via univariate analysis (AUC ≥0.7 or lowest p≤0.05) were evaluated for their ability to complement each other in stratifying the FLT3 receptor mutational status. A logistic regression model of the formFor each node /metric that stratified FLT3-ITD from FLT3-WT the list of outlier donors that are outside of 1.5 times the inter-quartile range was created. In particular, the outliers from one class (e.g. FLT-3-ITD) that are more similar to the donors in the second class (e.g. FLT-3- WT) were identified and summarized in a table together with their clinical characteristics.Figure S1Survival curves. Survival curves in weeks for FLT3-ITD samples (dotted line) and FLT3-WT samples (solid line) are shown for A) Study 1 and B) Study 2.(0.59 MB TIF)Click here for additional data file.Figure S2Assessment of false discovery rate. Expected versus actual numbers of stratifying node/metrics at specific P-value thresholds (for FLT3-WT and FLT3-ITD stratification) demonstrate significantly higher numbers of actual stratifying node/metrics observed in A) Study 1 and B) Study 2 than would be found by chance alone.(0.77 MB TIF)Click here for additional data file.Figure S3Basal PI3K and Jak/Stat activity in FLT3-WT and FLT3-ITD samples. A) Box and whisker plots for basal levels of p-S6, p-Erk, p-Akt in Study 1 (left panels) and Study 2 (right panels). Similar basal levels of p-S6, p-Erk, p-Akt were observed in FLT3-WT and FLT3-ITD samples. B) Box and whisker plots for basal levels of p-Stat1, p-Stat3 and p-Stat5 in Study 1 (left panels) and Study 2 (right panels). Higher levels of basal p-Stat1, p-Stat3, p-Stat5 were observed in FLT3-ITD samples in Study 1 but not Study 2. Note: Absolute values are not comparable between studies due to different experimental configurations.(3.30 MB TIF)Click here for additional data file.Figure S4Additional FLT3L induced signaling readouts. Box and whisker plots for FLT3L induced p-Akt, p-Erk, and p-CREB in Study 1 (left panels) and p-Akt, and p-Erk in Study 2 (right panels). FLT3-ITD samples demonstrated generally lower FLT3L induced p-Akt, p-Erk, and p-CREB compared to FLT3-WT samples. Note: Absolute values are not comparable between studies due to different experimental configurations.(1.73 MB TIF)Click here for additional data file.Figure S5IL-27 and FLT3L signaling in FLT3-ITD and NPM1 molecular subgroups. A) Shown are box and whisker plots of FLT3L induced p-S6, p-Erk and p-Akt (Panel A) and for IL-27 induced p-Stat responses (Panel B) for Study 1 (upper rows) and Study 2 (lower rows) in molecular subgroups. FLT3 NPM1 molecular subgroups are coded by shape: FLT3-ITD, NPM1 mutated (circles); FLT3-ITD, NPM1-WT (squares); FLT3 WT, NPM1 mutated (+ signs); FLT3 WT, NPM1 WT (X signs). For both IL-27 induced Jak/Stat signaling and FLT3L induced PI3K and Raf signaling, the most variability and largest range of response were observed within FLT3-WT NPM1-WT AML samples. In contrast, more homogenous signaling responses were observed in samples with defined molecular alterations .(0.26 MB TIF)Click here for additional data file.Figure S6ROC statistics, mean value of apoptosis responses (Mean) and number (N) of patients per group are shown. B) Examples of apoptosis responses from FLT3-WT samples with CR Duration <2 years (left) and CR duration >2 years (right) are shown by overlaid histograms of cleaved PARP from unmodulated/basal (gray) and Ara/Dauno treated (black) conditions. Within FLT3-WT AML samples, apoptosis nodes Ara-C/Daunorubicin → c-PARP and Etoposide → c-PARP demonstrate higher induced apoptotic response in Complete Continuous Remission (CCR) patients with CR duration >2 years than in CR patients whose remission was <2 years.Apoptosis nodes predict CR duration within FLT3-WT samples. A) Descriptive statistics for the ability of apoptosis nodes  to stratify patients with CR duration of <2 years and patients with CR duration >2 years. P values, AUC(0.11 MB TIF)Click here for additional data file.Figure S7Example of gating analysis to define leukemic blast population. Flow cytometry plots and sequential gating scheme. A) Flow cytometry dot plot indicating how non-cellular debris were excluded using a FSC and SSC gate. B) Flow cytometry dot plot indicating how non-viable cells were excluded with a SSC and Aqua viability dye gate. This gating scheme ensured that only from live cells were analyzed for signaling responses. C) Flow cytometry dot plot indicating how lymphocytes were excluded and myeloid leukemic blasts included using additional surface markers . Note: In the subset of AML samples with phenotypically mature blasts  a small number of mature monocytes may also be included in this analysis, however, due to the abnormally high percentage of myeloid cells in these patients, the population analyzed likely contains predominately leukemic cells. Unfortunately, the side scatter properties of mature monocytes which clearly distinguish these cells from leukemic blasts are compromised by the fixation and permeabilization techniques used.(0.92 MB TIF)Click here for additional data file.Table S1List of nodes tested.(0.07 MB PDF)Click here for additional data file.Table S2Variance in signaling between healthy BMMb and FLT3 NPM1 molecular subgroups.(0.03 MB PDF)Click here for additional data file.Table S3Top-ranking nodes stratifying ITD from WT in Study 1 .(0.09 MB PDF)Click here for additional data file.Table S4Top ranking nodes stratifying ITD from WT in Study 2 .(0.08 MB PDF)Click here for additional data file.Table S5Pearson correlations between stratifying nodes for FLT3-ITD vs. FLT3-WT in Study 1.(0.21 MB PDF)Click here for additional data file.Table S6Combinations/pairs of nodes from linear regression analysis improve stratification of FLT3-ITD and WT samples in Study 1.(0.08 MB PDF)Click here for additional data file.Table S7Clinical characteristics of cytogenetically normal (CN) AML patient samples.(0.07 MB PDF)Click here for additional data file.Table S8Clinical characteristics of FLT3-WT vs. FLT3-ITD signaling outliers (Study 2).(0.06 MB PDF)Click here for additional data file.Table S9List of modulators, reagents and technical conditions.(0.08 MB PDF)Click here for additional data file.


Entamoeba histolytica infects millions of people worldwide and is capable of causing amebic dysentery and amebic liver abscess. The closely related species Entamoeba dispar colonizes many more individuals, but this organism does not induce disease. To identify molecular differences between these two organisms that may account for their differential ability to cause disease in humans, we used two-dimensional gel-based (DIGE) proteomic analysis to compare whole cell lysates of E. histolytica and E. dispar. We observed 141 spots expressed at a substantially (>5-fold) higher level in E. histolytica HM-1∶IMSS than E. dispar and 189 spots showing the opposite pattern. Strikingly, 3 of 4 proteins consistently identified as different at a greater than 5-fold level between E. histolytica HM-1∶IMSS and E. dispar were identical to proteins recently identified as differentially expressed between E. histolytica HM-1∶IMSS and the reduced virulence strain E. histolytica Rahman. One of these was E. histolytica alcohol dehydrogenase 3 (EhADH3). We found that E. histolytica possesses a higher level of NADP-dependent alcohol dehydrogenase activity than E. dispar and that some EhADH3 can be localized to the surface of E. histolytica. Episomal overexpression of EhADH3 in E. histolytica trophozoites resulted in only subtle phenotypic differences in E. histolytica virulence in animal models of amebic colitis and amebic liver abscess, making it difficult to directly link EhADH3 levels to virulence differences between E. histolytica and less-pathogenic Entamoeba.The protozoan intestinal parasite  Entamoeba histolytica can result in disabling diarrhea or even death, while the morphologically identical and genetically similar Entamoeba dispar harmlessly colonizes the human intestine. Understanding the molecular differences between these two organisms by comparing their protein repertoire may help us to understand why E. histolytica invades into colonic tissue, while E. dispar remains a benign passenger. Here, we identify four proteins that appear to be differentially expressed between the two species and show that a metabolic enzyme, which would appear to be an unlikely candidate for a role in disease, is expressed at much higher levels in the pathogenic organism.Infection with  Entamoeba histolytica, a protozoan intestinal parasite, is the causative agent of amebic dysentery and amebic liver abscess Entamoeba dispar, is morphologically indistinguishable from E. histolyticaE. dispar is a commensal and does not cause disease in humans, even in immunocompromised individuals. Previous studies have identified a number of Entamoeba molecules that appear to be linked to virulence, including cysteine proteinases, amoebapores, the Gal/GalNAc lectin and peroxiredoxin, but the virulence phenotype is unlikely to be secondary to only one, or even a few proteins E. histolytica, and the related but nonpathogenic E. dispar, provides a powerful platform for more widespread screening for additional virulence factors of E. histolytica. Here we report the use of comparative proteomics of E. histolytica HM-1∶IMSS and E. dispar SAW760 to identify proteins that are differentially expressed between the two species, and the characterization of one of the differentially expressed proteins, EhADH3, identified by this screen.E. histolytica HM-1∶IMSS and E. dispar SAW760 were grown axenically in LYI-S-2 with 15% adult bovine serum medium at London School of Hygiene and Tropical Medicine 6E. histolytica or E. dispar trophozoites were harvested and washed 3 times in ice-cold PBS to remove serum and medium proteins, then lysed in a buffer formulated to minimize post-lysis proteolysis . Lysates were frozen at −80°C before analysis Trophozoite lysates were analyzed as previously described in situ with trypsin as previously described m/z = 842.51, 1045.56, and 2211.10) E. histolytica.Gel features were selected in the DeCyder software, then excised and transferred to a 96-well source plate. The gel pieces were digested Peptide pools from the gel features that were not identified using MALDI-MS/MS were analyzed using capillary reversed-phase HPLC-MS/MS using an electrospray-quadrupole time-of-flight mass spectrometer . Peptide pools from gel features that remained unidentified by either MALDI-MS/MS or quadrupole-TOF-LC-MS/MS were analyzed using nano-LC-linear-quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometry as previously described AAGGATCCATGACAATGCTTAATTTCACATA and reverse - TTCTCGAGTTAATAAATGCTATTAAGAATTTGGAGAT were used to amplify a EhADH3 transcript from HM-1∶IMSS genomic DNA. The fragment was inserted into pCR 2.1 TOPO vector , cut by BamHI and XhoI and cloned into pGEX-6p-1. The plasmid was expressed under 0.05 mM IPTG induction in BL21- Codon Plus RIL from Stratagene  at 18°C under shaking at 250 rpm for 48 h.Primers derived from the sequence of EhADH3 (Z48752.1) Escherichia coli cells were harvested by centrifugation at 1,500×g for 30 minutes 4°C and resuspended in 35 ml PBS with a Protease Inhibitor Cocktail Tablet from Roche . The cell suspension was passed through a French press twice at 12,000 PSI, and then centrifuged at 20,000×g for 30 minutes at 4°C. The supernatants were frozen at −80°C for later use. For purification on the GST column, 500 ml supernatant was thawed overnight at 4°C, filtered with a 0.22 um filters from MilliPore , and loaded on a B-PER GST Fusion Protein Purification Kit from Pierce  according the manufacturer's instructions. Following column washings with 200 ml PBS, 10 ml Wash Buffer 1, 10 ml Wash Buffer2 and 10 ml PBS, 1.5 ml PBS containing 120 ul PreScission Protease from GE Healthcare was added to the column and incubated overnight at 4°C. The column was eluted with 1 ml PBS, and the total volume (2.5 ml) containing partially purified EhADH3 was dialyzed against 25 mM MES pH 6.0 (Buffer A) overnight at 4°C. This material was then loaded onto a Resource Q ion exchange column from GE Healthcare. After column washes with Buffer A, EhADH3 was eluted by a salt gradient created by the addition of increasing amounts (0% to 100%) of Buffer B (Buffer A with 1 M NaCl) to Buffer A run over the column over 30 minutes at flow rate 2 ml/min. Every 2 ml fraction was analyzed at UV 260 nm for protein concentration and those with the highest OD were pooled, dialyzed against 10 mM Tris HCl pH 7.3 and then concentrated by Amicon Ultra-4 Centrifugal Filter Units from MilliPore. Purity of the recombinant EhADH3 was assessed by SDS-PAGE.To purify recombinant EhADH3, 1 L of the transfected BL-21 E. histolytica or E. dispar lysates used for analysis of alcohol dehydrogenase activity were prepared as previously described 4, 60 mM DTT, 0.2 mM NADP and 0.1 M of the substrate alcohol to a final volume of 1 ml. The rate of increase in absorbance was observed at 340 nm (1 OD = 6.22 mM cm−1 NADPH). A unit of enzyme activity was defined as one micromole of product formed per minute of incubation at room temperature.E. histolytica HM-1∶IMSS and E. histolytica Rahman, plasmid pNeoCass was used to construct plasmid pNeoADH3 E. histolytica HM-1∶IMSS cDNA: Forward- 5′ -AATTGGTACCATGACAATGCTTAATTTCACATATTAC - 3′, Reverse: 5′ - ATCCGGATCCTTAATAAATGTCATTAAGAATTTGGAG - 3′. The insert was then cut and ligated into pNeoCass yielding pNeoADH3. Transfection was performed as previously described 6 amebae were washed 2× with cold PBS, and once with cold, fresh cytomyx. The amebae were resuspended with 800 ul cytomyx and 60 ug DNA into a chilled 0.4 cm electroporation cuvette. A BioRad GenePulser XCell was set to 25 uF and 3000 V/cm. Two successive pulses were completed 30 s apart, and then amebae were placed in medium and grown for 72 h before G418 drug selection. These strains were maintained in culture medium BI-S-33 containing G418 as previously described In order to overexpress EhADH3 in strains 5 amebae were grown to log phase, chilled, pelleted at 4°C and 400×g for 1 min, and resuspended in 2 ml resuspension/blocking buffer, which consists of 50% v/v TYI-S-33 Approximately 2×106E. histolytica HM-1∶IMSS or E. histolytica Rahman trophozoites overexpressing EhADH3 or an equivalent number of the parental control cells were directly inoculated into the liver of male, 6 to 8 weeks old BALB/c or SCID mice, as previously described All our research on mice was approved by the Washington University Animal Studies Committee (ASC), and was conducted under AAALAC and USDA guidelines. For studies of amebic liver abscess, 1×106 trophozoites  and infection was assessed 24 h later. To measure the integrity of the intestinal permeability barrier, 20 h after infection human intestinal xenografts were intraluminally inoculated with fluoresceinated dextran, and serum levels of fluoresceinated dextran were measured using a fluorescent plate reader 4 h later Severe combined immunodeficient mice were engrafted in the subscapular region with human colonic xenografts as previously described E. histolytica and E. disparE. histolytica and E. dispar has significantly reduced, but not completely eliminated these issues for this comparison.Two-dimensional difference gel electrophoresis (2D-DIGE) allows protein lysates to be fluorescently labeled in such a way as to allow visualization of multiple channels representing up to three biological samples in a single physical gel while maintaining the ability to analyze chosen protein spots using modern mass spectrometric techniques E. histolytica HM-1∶IMSS lysates, varying only the concentration of protease inhibitor added to the cell lysate . No differences in spots were observed when a cutoff of 3-fold difference in fluorescent intensity was used, indicating that inter-sample full-length protein level differences secondary to endogenous proteases were not important at these protease inhibitor concentrations. To measure potential differences in spots based on sample preparation, lysates from the same E. histolytica HM-1∶IMSS isolate were captured one week apart, both in the logarithmic phase of growth. Again, with a 3-fold cutoff for differences, only 1 protein spot differed from more than 2800 computationally indexed spots (a five-fold cutoff reduces the number of observed differences to zero). Finally, we compared lysates from E. histolytica HM-1∶IMSS separately maintained in two distant laboratories (“Saint Louis” and “London”) using separate growth media  in three 2-D gels each containing distinct biological replicates of lysates from each species. The number of differentially expressed spots was a function of the cut-off used for differential expression, but requiring a minimum of 5-fold differential expression yielded an average of 141 (+/−16) spots expressed at a higher level in E. histolytica HM-1∶IMSS than E. dispar, with an average of 189 (+/−42) spots showing the opposite pattern. E. histolytica HM-1∶IMSS and E. dispar SAW760 . We postulate that increased grainin levels may contribute to a reduced virulence phenotype, since grainin levels were decreased in trophozoites recently obtained from amebic liver abscesses, and were higher in the proteome of the reduced virulence strain E. histolytica Rahman compared to E. histolytica HM-1∶IMSS E. dispar compared to E. histolytica is consistent with this hypothesis.Three proteins were present at higher levels in . dispar . Strikina Rahman  [3]. TheE. histolytica compared to E. dispar was a protein containing a LIM domain (gi67483283) (E. dispar sequence database yielded a truncated gene (77% complete) which is 99% identical to the E. histolytica gene (EDI_092410). In addition, the full length E. histolytica gene is nearly identical to the LIM domain protein found elevated in E. histolytica strain HM-1∶IMSS when compared to strain E. histolytica Rahman One of the proteins expressed at higher levels (average of 14.1-fold) in 7483283) . The LIME. histolytica HM-1∶IMSS than E. dispar were alcohol dehydrogenase 2 and alcohol dehydrogenase 3   E. histolytica fermentation, growth and survival E. histolytica lysates, but there is no direct experimental evidence that it serves this function in vivoThe two other proteins found at higher levels in ctively) . This fiE. histolytica alcohol dehydrogenase 3 is an NADPH- dependent alcohol dehydrogenase whose physiologic role in E. histolytica metabolism remains unknown E. histolytica resequencing data show that EhADH3 has no closely related inparalog , differing from initial reports suggesting multiple copies of this gene. Our DIGE analysis  which are 90% identical and 95% similar to EhADH3 in derived amino acid sequence. This difference in expression was confirmed by immunoblotting using polyclonal sera that showed EhADH3 was present at 5.4-fold higher levels in E. histolytica compared to E. dispar by densitometry , being present at higher levels in E. histolytica than its close homologues in E. dispar, but we cannot exclude the possibility than other more distant alleles of EhADH3 and EdADH3 could show different expression patterns.analysis  indicateitometry . AdditioE. histolytica than E. dispar, we first analyzed purified recombinant EhADH3 to determine the optimal substrate specificity for the ADH3 enzyme. As shown in E. histolytica and compared it to that found in protein-matched lysates from E. dispar. As shown in E. histolytica HM-1∶IMSS than in lysates of E. dispar. These data are consistent with higher levels of EhADH3 in E. histolytica than E. dispar, but we cannot be absolutely certain that all of the NADP-dependent ADH activity in these lysates (with ethanol as substrate) is secondary to the Entamoeba ADH3 enzyme family.To determine whether there is physiologic evidence that ADH3 levels are higher in E. histolytica virulence we performed immunolocalization studies using polyclonal antiserum to recombinant EhADH3 with live trophozoites. As shown in E. histolytica HM-1∶IMSS trophozoites, and pre-incubation of the anti-EhADH3 antibodies with recombinant EhADH3 prior to staining significantly inhibited their ability to bind to the trophozoites' surface. These data indicate that at least some of the EhADH3 is on the surface of E. histolytica trophozoites, increasing the likelihood of interactions with host molecules. We used the same antibodies to determine whether ADH3 could be detected on the surface of E. dispar, and saw a similar staining pattern, but with much decreased overall intensity (data not shown).As a first step towards determining whether EhADH3 could play a role in S. pneumoniae 6-phosphogluconate-dehydrogenase, which mediates binding to buccal epithelial cells E. histolytica HM-1∶IMSS trophozoites from binding to Chinese Hamster Ovary cells (data not shown) We also explored whether EhADH3 might play some role in trophozoite adherence to host cells or host macromolecules due to its presence on the amebic plasma membrane. Some bacterial dehyrogenases have also been surface-localized and linked to adherence, including the E. histolytica virulence, E. histolytica HM-1∶IMSS trophozoites and E. histolytica trophozoites of the reduced virulence E. histolytica Rahman strain were transfected with a plasmid designed to overexpress EhADH3. Immunoblotting of lysates from wild type and transfected trophozoites with antibodies to EhADH3 confirmed successful overexpression of EhADH3 in the transfectants  compared with the parental wild type E. histolytica HM-1∶IMSS strain , this difference was not statistically significant (P = 0.25). We then looked at whether overexpression of EhADH3 in E. histolytica Rahman would alter the ability of this strain to cause amebic liver abscesses in mice. We were unable to detect any increase in the size or presence of amebic liver abscesses in mice undergoing liver challenge with E. histolytica Rahman transfected with EhADH3 compared to challenge with either wildtype E. histolytica Rahman trophozoites or E. histolytica Rahman trophozoites expressing the pNEO control plasmid (data not shown).We then examined whether E. histolytica Rahman trophozoites expressing EhADH3 , compared to either human intestinal xenografts infected with either E. histolytica Rahman trophozoites transfected with the pNEO control plasmid  or wildtype E. histolytica Rahman trophozoites . Using our assay for damage to the intestinal permeability barrier E. histolytica Rahman trophozoites expressing EhADH3 and human intestinal xenografts infected with either control, nor any obvious histological differences in sections from infected intestinal xenografts (data not shown). Interpretation of all of these results is complicated by the fact that we were only able to obtain an approximately 2-fold increase in EhADH3 production, and this may have been insufficient to detect a phenotypic difference between tranfectants and controls. Expression of antisense constructs did not reduce EhADH3 levels in E. histolytica HM-1∶IMSS (data not shown).In order to study phenotypic effects in colonic disease, we employed the SCID-hu human intestinal xenograft mouse model of amebic colitis E. histolytica HM-1∶IMSS and the commensal E. dispar. However, when we limited our analysis to those spots that showed the greatest magnitude difference and were consistently reproduced, we identified 4 proteins that were differentially expressed between the two species. Remarkably, 3 of the 4 were identical to proteins identified in a comparison of E. histolytica HM-1∶IMSS and the reduced virulence E. histolytica Rahman strain In summary, our proteomic comparisons identified a number of differences between the virulent E. histolytica HM-1∶IMSS, EhADH3. We found that EhADH3 does possess alcohol dehydrogenase activity but, unexpectedly, can be localized to the trophozoite surface. However, we were not able to detect an adherence function for EhADH3, and an approximate two-fold overexpression of EhADH3 in E. histolytica HM-1∶IMSS failed to result in increased virulence. A three-fold overexpression of the EhADH3 gene in the reduced virulence E. histolytica strain E. histolytica Rahman also did not lead to trophozoites capable of causing increased tissue damage in animal models of amebic liver abscess or amebic colitis; however, it did lead to an increased inflammatory response. Thus, while EhADH3 is more abundant in E. histolytica HM-1∶IMSS than either E. dispar or E. histolytica Rahman, we cannot directly link EhADH3 to the increased virulence of E. histolytica HM-1∶IMSS.We focused our efforts on characterizing one of the proteins expressed at higher levels in


BMC Biology, demonstrates that Hh signals through the cilium in zebrafish, clarifying the evolution of Hh signal transduction.The Hedgehog (Hh) signaling pathway differentially utilizes the primary cilium in mammals and fruit flies. Recent work, including a study in http://www.biomedcentral.com/1741-7007/8/65               See research article:  Drosophila melanogaster [et al.  in BMC Biology [iguana gene product, originally implicated in the regulation of Hh signaling in zebrafish, has in fact a conserved role in ciliogenesis.Hedgehog (Hh) signaling is an evolutionarily conserved signal transduction pathway that is essential for a range of developmental patterning events, including specifying growth and polarity of the vertebrate limb and neural tube, and is misregulated in a number of cancers, for example basal cell carcinoma and medulloblastoma . It has nogaster . Subsequ Biology , show thDrosophila, Gli in vertebrates). In the absence of Hh signaling, Ci/Gli transcriptional regulators undergo proteasome-dependent limited proteolysis to a truncated form in which they act as transcriptional repressors, a process that also requires the scaffolding protein Costal-2/Kinesin family member 7 (Cos-2/Kif7). Hh signaling causes Ci/Gli proteins instead to be converted, by an unknown mechanism, to transcriptional activators that translocate to the nucleus and activate target genes. This modification occurs as a consequence of the activity of the G-protein coupled-like receptor Smoothened (Smo), which in the absence of Hh is repressed by the Hh receptor Patched (Ptc). When secreted Hh binds Patched (Ptc), Ptc-mediated repression of Smo is disrupted and Ci/Gli's are activated, possibly through modulation of Cos-2/Kif7 conformation and localization.Most of the important components of the Hh signal transduction pathway have been known for some time to be conserved from invertebrates to vertebrates . The funA requirement for the cilium in this pathway was first suggested on the basis of ENU mutagenesis screens in mice, which showed that genes required for ciliogenesis are also required for Hh signal transduction (first reported by Kathryn Anderson's group and reviewed in ). DisrupSubsequent investigations with overexpressed and endogenous Hh pathway components showed that many are localized to the primary cilium (reviewed in ), and thift57, ift88 and ift172 mutant embryos, in which intraflagellar transport is disrupted, do not exhibit overt loss-or gain-of-function Hh phenotypes. It now seems likely that this is due to the maternal contribution of transcripts and/or proteins for these genes, as zebrafish embryos lacking both maternal and zygotic ift88 function completely lack cilia and exhibit disrupted Hh signaling [you-too/gli2a mutant, and localizes predominantly to the distal tip of the primary cilium [ptc1 and ptc2 genes, which creates a constitutively activated Hh signaling state, results in increased intensity of the Gli2a-GFP signal at the tip of the cilium compared with wild-type siblings. In a smo mutant background, where Hh signaling is abrogated, Gli2a-GFP signal intensity is reduced, although it is unclear whether this is due to a reduction in Gli2a-GFP protein levels or solely reflects dispersal of the signal along the length of the axoneme and the basal body. These data, with a prior report of Smo localization to primary cilia in zebrafish [Surprisingly, initial analysis in zebrafish suggested that cilia might not be involved in Hh signal transduction in this important model species, as ignaling . Kim andignaling . They wey cilium . Gli2a tiguana gene product in Hh signaling. The iguana (igu) locus was first identified as a modulator of Hh signaling in zebrafish, and found to encode a homolog of the human DAZ-Interacting Protein 1 (DZIP1), prominently expressed in embryonic stem cells and the germline [iguana mutants and morphants (morpholino injected embryos) [igu mutants have severely truncated primary ciliary axonemes, indicating effects on cilium formation [et al propose that Igu may be an essential component of a multi-protein complex required for cilium formation [A second issue addressed by Kim and colleagues is the role of the germline ,7. Igu, embryos) ,7 are exembryos) , which aembryos)  now showembryos) , and iguormation . On the ormation .Schmidtea mediterranea, which is a focus of considerable interest because investigation of its regenerative capacity may lead to a better understanding of how regeneration is controlled. Knockdown studies of Igu and other ciliary proteins in planaria show that igu is required for ciliogenesis, though not for Hh signaling [This proposal is consistent with recent RNA interference studies in the planarian ignaling ,10. It iignaling . Thus, Iet al.  observed a reduction in the number of motile cilia in Kupffer's vesicle, but not in the pronephros. Others by contrast have found a reduction in cilia in both regions of the embryo, and varying effects on left-right asymmetry and kidney cyst formation [igu deficiency on motile cilia may be secondary to its effects on Hh signaling, which is required for the expression of foxj1, a master regulator of the motile cilium formation program [igu/dzip1 and dzip1-like [igu and dzip1-like morphants and mutants will be necessary to resolve this issue.Although there is a growing consensus on the conservation of the Hh signaling pathway, the role of the primary cilium, and how Igu connects the two, there is some disagreement on the part played by Igu in the construction of motile cilia. These specialized cilia contain an additional central pair of microtubules, as well as accessory dynein motors and macromolecular machines, and are not implicated in Hh signaling but have a critical role in the specification of left-right asymmetry , and in fluid flow in the zebrafish kidney and brain. Motile cilia are found in Kupffer's vesicle in fish (where they specify left-right asymmetry), the ventral canal of the spinal cord and ependymal cells of the brain, and the pronephros. Kim ormation ,9. Effec program . Furtherip1-like ,9, and/oThe function of Igu in ciliogenesis appears to be conserved in most metazoa, but its role in Hh signaling has thus far been shown only in vertebrates. It is not yet known whether Hh signaling utilizes cilia in most organisms (and fruit flies have thus 'lost' the cilium-Hh connection), or if Hh signal transduction co-opted cilia later in evolution ,10. The


Dendritic cells are antigen-presenting cells that play an essential role in linking the innate and adaptive immune systems. Much research has focused on the signaling pathways triggered upon infection of dendritic cells by various pathogens. The high level of activity in the field makes it desirable to have a pathway-based resource to access the information in the literature. Current pathway diagrams lack either comprehensiveness, or an open-access editorial interface. Hence, there is a need for a dependable, expertly curated knowledgebase that integrates this information into a map of signaling networks.We have built a detailed diagram of the dendritic cell signaling network, with the goal of providing researchers with a valuable resource and a facile method for community input. Network construction has relied on comprehensive review of the literature and regular updates. The diagram includes detailed depictions of pathways activated downstream of different pathogen recognition receptors such as Toll-like receptors, retinoic acid-inducible gene-I-like receptors, C-type lectin receptors and nucleotide-binding oligomerization domain-like receptors. Initially assembled using CellDesigner software, it provides an annotated graphical representation of interactions stored in Systems Biology Mark-up Language. The network, which comprises 249 nodes and 213 edges, has been web-published through the Biological Pathway Publisher software suite. Nodes are annotated with PubMed references and gene-related information, and linked to a public wiki, providing a discussion forum for updates and corrections. To gain more insight into regulatory patterns of dendritic cell signaling, we analyzed the network using graph-theory methods: bifan, feedforward and multi-input convergence motifs were enriched. This emphasis on activating control mechanisms is consonant with a network that subserves persistent and coordinated responses to pathogen detection.http://tsb.mssm.edu/pathwayPublisher/DC_pathway/DC_pathway_index.html.This map represents a navigable aid for presenting a consensus view of the current knowledge on dendritic cell signaling that can be continuously improved through contributions of research community experts. Because the map is available in a machine readable format, it can be edited and may assist researchers in data analysis. Furthermore, the availability of a comprehensive knowledgebase might help further research in this area such as vaccine development. The dendritic cell signaling knowledgebase is accessible at  The innate immune system represents the first line of defense against attack by viral, bacterial, and parasitic infections. Dendritic cells (DCs), which are found in abundance in peripheral tissues such as skin, lung, and mucosal surfaces, act as a bridge between the innate and adaptive immune systems: recognition of a 'danger' signal initiates the maturation of DCs, which ultimately activate cells of the adaptive arm of the immune system, B and T cells -3. DCs ein vitro and rendering them responsive to tumor antigens may lead to tumor regression [http://sbml.org/), and be accessible to the community for updates and corrections.DCs detect pathogens via pattern recognition receptors (PRRs), which recognize various molecular structures referred to as pathogen-associated molecular patterns (PAMPs), e.g. lipopolysaccharides, lipoteichoic acids, flagellin and nucleic acids. Membrane-associated PRRs, like the Toll-like receptors (TLRs) and C-type lectin receptors (CLRs) respond to extracellular pathogens, while cytosolic PRRs, including RIG-I-like receptors (RLRs) and NOD-like receptors (NLRs) sense intracellular pathogens -7. Pathogression . Traditigression . Therefohttp://www.genome.jp/kegg/pathway.html[http://www.reactome.org/cgi-bin/frontpage?DB=gk_current[http://stke.sciencemag.org/cm/) biological pathways database, and Ingenuity Systems, a commercial subscription-based knowledgebase. The KEGG pathways do not integrate all PRRs, but rather depict each type of PRR-derived pathway separately and with little detail. Although Reactome is abundantly annotated and organism-specific, it does not provide cell-type specific information. STKE features 19 immunity-related pathways, the majority of which, however, have not been updated in recent years. Similar to Reactome, Ingenuity Systems offers a great diversity of annotations, including literature references from various biological models and many other database resources, yet it presents a fairly basic version of each signaling network and is not cell-type specific. In contrast, the group of Kitano [Current databases of signaling networks of the innate immune response include free online resources such as the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway database hway.html, Reactomk_current, Sciencef Kitano  construcf Kitano , is compBased on a manual curation of the published literature, we have assembled an extensive and detailed map of the signaling pathways involved in DC response to pathogens, as described in human DCs and mouse models. The DC pathway map, which is web-accessible, includes the following annotations: a list of interactions, GeneIDs, PubMed IDs (PMIDs), along with summary notes. In order to provide a discussion forum for the community and an opportunity for direct feedback and contribution, we have linked the DC map to a public wiki. Thus, it should represent a valuable resource for the research community, and conceivably initiate a community-wide interactive process. Additionally, using computational methods we delineated the regulatory motifs that are present in the DC signaling network.http://celldesigner.org/; it was then web-published using BioPP, a software suite which converts CellDesigner-SBML formatted pathways into a web-viewable format [To summarize the complexity of the pathogen recognition response in DCs, we have developed a map of signaling events occurring in those cells upon viral and bacterial infection. This map was assembled in CellDesigner, a free process diagram editor for gene-regulatory and biochemical networks e format . Hence, vice versa has been omitted. The network consists of 249 entities or nodes and 213 reactions or edges. Among the 118 protein species, 20 were classified as receptors and 2 as truncated proteins, the remainder being comprised of intracellular proteins as well as transcription factors. The reactions can be classified into 122 state transitions , 9 heterodimer associations, 4 dissociations, 36 transcriptional activations, 8 unknown transitions, 8 transport reactions, 21 inhibitions, and 4 translations.At present, the DC signaling network consolidates manually curated information from 167 peer-reviewed journal articles. Target publications are mainly reports on human DCs and mouse models, as well as studies in heterologous expression systems. In particular, as the main interest of our research group is monocyte-derived DCs obtained from human blood donors, namely conventional DCs, we have not yet included plasmacytoid DCs in the knowledgebase. To avoid adding more visual complexity to the diagram, we have chosen not to depict response genes, but rather represent their respective RNAs. For simplicity, transcription factors and other regulatory complexes are directly connected to the RNAs of the response genes they activate. Additionally, the molecular machinery required for the transportation of transcription factors from the cytoplasm to the nucleus and The DC signaling network Figure  can be dThere are 13 different members in the TLR family of proteins identified across mammalian species (TLR1-13). We omitted TLR10, which is not functional in mice, and TLR 12 and TLR13, which are lost in humans. TLRs recognize a wide variety of PAMPs ranging from lipids, lipoproteins, glucans to nucleic acids . They arThe RLRs RIG-I and MDA5 activate NFκB and IRF3 independently of TLRs. RIG-I interacts with either ssRNA or dsRNA through an RNA helicase domain, and recruits adaptor MAVS via a CARD-CARD association ,30. MAVSCLRs recognize carbohydrate structures present on bacterial or fungal pathogens . They aThe NLR family of intracellular PRRs includes NOD1, NOD2, and NALP3. NOD1 and NOD2 recognize distinct motifs derived from the peptidoglycan bacterial cell wall, while NALP3 responds to multiple stimuli . NOD1 aMultiple PRRs can recognize a specific pathogen, thereby activating separate or shared pathways. Generally, the collaboration between PRRs typically results in enhanced transcriptional and cellular responses, i.e. synergistic inflammatory responses, as compared to each PRR. The fact that several receptors may be involved in pathogen recognition renders the system more robust against immune evasion by the pathogenic microbe. Not only can it scale up the inflammatory response, but it may also tailor it to the type of microbe encountered. Importantly, a better understanding of the effective interplay between DC signaling pathways may eventually be exploited in the development of vaccines. Hence, TLRs, RLRs, NLRs, and CLRs can cross talk and synergize to orchestrate immune responses effectively. Cross talks observed between PRRs that belong to the same family, or between different types of PRRs, are detailed below and on the DC map Figure . Additioet al. speculated that synergistic TLR stimulation may result from sustained signaling, or possibly imply complementary signaling pathways that have yet to be identified [TLR3/4 synergize with endosomal TLR7-9 to increase the production of Th1- polarizing cytokine IL-12p70 by more than 20-fold compared to either receptor alone; the mechanisms underlying this synergy are not fully understood -39. It iTLR3 and RLRs have been shown to be important in recognizing RNA viruses in different cellular compartments. TLR3 is present in endosomal membranes and binds to the dsRNA phagocytosed from viral infected apoptotic cells -42. In cActivation of NFκB and of the MAPK cascade occurs in the TLR and NLR signaling pathways  Figure. NOD2 agTLR2 and Dectin-1 are synergistic in mediating IL-12 and TNFα production  Figure . InteresAppropriate and accurate PRR responses to pathogen signals are essential for the host defense. Yet over-activated cytokine responses to infection can be detrimental to the host. In order to generate an effective but non-toxic response, control mechanisms that negatively regulate the degree and duration of PRR responses are needed. Such surveillance mechanisms operate at different nodes in the PRR-induced signaling cascades. Regulatory mechanisms, which include degradation, sequestration, or inhibition of signaling molecules, are represented schematically in Figure A20 functions as a feedback negative regulator of the TLR3 pathway by inhibiting TRIF-mediated induction of NFkB and IFNβ transcription Figure . It was SIKE was demonstrated to associate with IKKE and TBK1 by co-immunoprecipitation Figure . OverexpPIN1 is a peptidylprolyl cis-trans isomerase that promotes proteasomal degradation of phosphorylated IRF3 by augmenting its poly-ubiquitination  Figure . RNF125 SOCS proteins inhibit cytokine-mediated signal transduction by targeting JAK/STAT signaling Figure . Cytokinin vitro, and co-immunoprecipitation experiments indicated the association of PP2C with TAK1 [Additional negative regulators include IRAK-M and phosphatases. IRAK-M negatively regulates TLR signaling by preventing the dissociation of IRAK1 and IRAK4 from MyD88 and the formation of IRAK1-TRAF6 complexes, as revealed by co-transfection experiments into heterologous cells followed by co-immunoprecipitation . The autith TAK1 ,73. Earlith TAK1 ,75. Howeith TAK1 ,77.To identify motifs in our DC signaling network, we used a fully connected directed graph that was reconstructed from our DC map, as described in detail in Additional file Motifs have been shown to functionally implement signal processing within larger networks ,82. ObtaThe DC pathway map is a public knowledgebase that offers a platform for the curation, diffusion, and update of information about the signaling cascades activated upon viral and bacterial infection in DCs. Its manual curation is based on extensive literature searches, and its dissemination relies on the availability of a navigable map of nodes and edges through the internet. Pathway layout has been optimized manually in order to provide more clarity to the diagram. In particular, we have put nodes that interact with one another in close proximity. In the web-published diagram, each node is clickable and links to a complete list of interactions in which it is involved; interactions themselves are supported by hyperlinked PMIDs. Moreover, each node that belongs to either a Protein or RNA category has one or more hyperlinked GeneIDs, and is tied to a wiki page, which lists all interactions in which it is engaged. Wiki pages allow experts to contribute their remarks, suggestions or literature updates. We expect to update the map regularly with new data derived from the literature as well as experts' intellectual contributions on the wiki. Hence, the DC map represents both an online resource and an opportunity for community-wide collaboration. An xml version of the pathway is available for download on the website to allow biologists to modify and expand it in accordance with their own experimental observations. This xml file  kinase; IL: interleukin; IRAK: IL-1 receptor-associated kinase; IRF: IFN regulatory factor; IκB: inhibitor of κB; KEGG: Kyoto Encyclopedia of Genes and Genomes; LPS: lipopolysaccharide; LRR: leucine rich repeat; MAPK: mitogen-activated protein kinase; MAVS: mitochondrial associated viral stimulator; MDA5: melanoma differentiation associated gene 5; MDP: muramyl dipeptide; MGL: macrophage galactose-type lectin; MHC: major histocompatibility complex; MIC: multi-input convergence; MR: mannose receptor; MyD88: myeloid differentiation primary response gene 88; NALP3: NLR family, pyrin domain containing 3; NFκB: nuclear factor kappa B; NLR: nucleotide-binding oligomerization domain (NOD)-like receptor; NLRX1: NLR family member X1; NOD: nucleotide-binding oligomerization domain; PAMP: pathogen-associated molecular pattern; PIN1: peptidylprolyl cis/trans isomerase, NIMA (never in mitosis gene a)-interacting 1; PMID: PubMed ID; PRR: pattern recognition receptor; RICK: RIP (receptor-interacting protein)-like interacting caspase-like apoptosis regulatory protein kinase; RIG-I: retinoic acid-inducible gene-I; RIP1: receptor interacting protein 1; RLR: RIG-I-like receptor; RNF125: Ring finger protein 125; SBML: Systems Biology Mark-up Language; SIKE: Suppressor of IKKE; SOCS: suppressor of cytokine signaling; ssRNA: single-stranded RNA; STKE: Signal Transduction Knowledge Environment; SYK: spleen tyrosine kinase; TAK1: transforming growth factor β-activated kinase 1; TBK1: TANK (TNFR-associated factor family member-associated NFkB activator) binding kinase 1; TIR: Toll/IL-1R; TIRAP: TIR domain-containing adaptor protein; TLR: Toll-like receptor; TNFα: tumor necrosis factor-α; TRAM: TRIF-related adaptor molecule; TRIF: TIR-containing adaptor inducing IFNβ.SP was the initial curator and wrote the first manuscript draft. HP is the present curator and revised the manuscript. JS contributed to the curation, figure design and manuscript preparation. GN maintained and revised the custom software suite utilized for web-publishing. GN conceived and designed the network motif analysis. IN and GN performed the network motif analysis and participated in the manuscript writing. SCS organized and directed the project and revised the manuscript. All authors read and approved the final version.Supporting Methods & Figures S1-S2. This file describes the method employed for the computational motif detection analysis of the DC network. It also illustrates examples of motifs identified in the DC network in Figure S1, and instances of bifan motifs found in the entire graph in Figure S2.Click here for fileFANMOD size 4 output. This output file, obtained using the FANMOD program, contains all overrepresented motifs of size 4.Click here for fileDC signaling pathway. This file is an xml version of the DC signaling pathway map, which allows biologists to edit and/or expand it in accordance with their own experimental observations.Click here for file


Giardia duodenalis is prevalent in tropical settings where diverse opportunities exist for transmission between people and animals. We conducted a cross-sectional study of G. duodenalis in people, livestock, and wild primates near Kibale National Park, Uganda, where human-livestock-wildlife interaction is high due to habitat disturbance. Our goal was to infer the cross-species transmission potential of G. duodenalis using molecular methods and to investigate clinical consequences of infection.G. duodenalis in people from three villages of 44/108 (40.7%), with prevalence reaching 67.5% in one village. Prevalence rates in livestock and primates were 12.4% and 11.1%, respectively. Age was associated with G. duodenalis infection in people  and livestock , but other potential risk factors in people  were not. G. duodenalis infection was not associated with gastrointestinal symptoms in people, nor was clinical disease noted in livestock or primates. Sequence analysis of four G. duodenalis genes identified assemblage AII in humans, assemblage BIV in humans and endangered red colobus monkeys, and assemblage E in livestock and red colobus, representing the first documentation of assemblage E in a non-human primate. In addition, genetic relationships within the BIV assemblage revealed sub-clades of identical G. duodenalis sequences from humans and red colobus.Real-time PCR on DNA extracted from fecal samples revealed a combined prevalence of G. duodenalis in people and primates (assemblage BIV) and livestock and primates (assemblage E) underscores that cross-species transmission of multiple G. duodenalis assemblages may occur in locations such as western Uganda where people, livestock, and primates overlap in their use of habitat. Our data also demonstrate a high but locally variable prevalence of G. duodenalis in people from western Uganda, but little evidence of associated clinical disease. Reverse zoonotic G. duodenalis transmission may be particularly frequent in tropical settings where anthropogenic habitat disturbance forces people and livestock to interact at high rates with wildlife, and this could have negative consequences for wildlife conservation.Our finding of  Giardia duodenalis is a common protozoan parasite that infects multiple mammalian species, including humans. We analyzed G. duodenalis from people, livestock, and wild non-human primates in forest fragments near Kibale National Park, western Uganda, where habitat disturbance and human-animal interaction are high. Molecular analyses indicated that endangered red colobus monkeys were infected with G. duodenalis assemblages BIV and E, which characteristically infect humans and livestock, respectively. G. duodenalis infected people at rates of up to 67.5% in one village, and people age 15 years or younger were especially likely to be infected. G. duodenalis infection in people was not associated with other factors related to behavior and hygiene, and infected people were no more likely to have reported gastrointestinal symptoms than were uninfected people. These results demonstrate that G. duodenalis transmission from humans and domestic animals to wildlife may occur with ease in locations such as western Uganda, where habitat disturbance causes ecological overlap among people, livestock, and primates. This conclusion has conservation implications for wildlife such as red colobus, which are already endangered by habitat loss. Giardia is a genus of parasitic protozoan that infects the small and large intestines of a broad range of vertebrate hosts G. duodenalis ranges in clinical severity from asymptomatic to highly pathogenic G. duodenalis is also notable for cross-species transmission, including zoonotic transmission G. duodenalis ecology Canis familiaris), and cattle (Bos taurus) in Italy Gorilla beringei beringei) in Uganda G. duodenalis “assemblages” (A–G) are recognized, infecting a range of mammalian hosts and likely representing as many distinct species G. duodenalis in wild mammals have found that samples fall into assemblages A or B, which characteristically infect people, concluding that the animal hosts involved may represent reservoirs of infection for humans , and in surrounding forest fragments. This habitat consists primarily of moist semi-deciduous and evergreen forest, between approximately 1,100 and 1,600 m in elevation 2 and contain small populations of monkeys . Our previous investigations have shown that rates of human-primate-livestock bacterial transmission are elevated in these locations due to anthropogenic habitat disturbance related to timber harvesting, agriculture, and the collection of forest products (e.g. firewood) G. duodenalisFor the present study, we focused on three such forest fragments, Bugembe, Kiko 1, and Rurama, and the villages surrounding them. These sites, which are described in detail elsewhere Procolobus badius tephrosceles; n = 30) and the black-and-white colobus , and one omnivorous monkey species, the red-tailed guenon , from undisturbed areas within Kibale National Park, as well as from the highly disturbed Bugembe, Kiko 1, and Rurama forest fragments. We collected single fecal samples non-invasively from individual animals on 13 days, at the same time that we collected demographic information and behavioral observations, as previously described In May and June, 2007 (dry season), we collected fecal samples from two folivorous monkey species, the red colobus (n = 108) and their livestock  in villages surrounding each forest fragment. People and livestock in these villages use forest fragments intensively for such purposes as firewood collection and grazing, respectively, thus increasing ecological overlap with primates We sampled local human volunteers . 1 ml of fecal material from each sample was homogenized with an equal volume of RNA-later nucleic acid stabilizing buffer (Ambion) and stored at −20°C in the field prior to transport to the United States.Concurrent with human fecal sample collection, a survey was administered to each participant. The survey focused on demography, gastrointestinal symptoms, patterns of land use, and interactions with animals during the four-week period prior to sample collection. The survey was administered in Rutooro  by trained field assistants who were also members of the local communities; response biases caused by the presence of foreigners were thereby avoided.G. duodenalis DNA using a real-time PCR targeting the small subunit RNA gene on an ABI Prism® 7000 Sequence Detection System , with primers, probe, and cycling conditions following published methods DNA was extracted from 500 µl of fecal+RNA-later suspension using the FastDNA Spin Kit for Soil , followed by an additional wash and ethanol precipitation in the presence of CTAB in order to remove potential PCR inhibitors G. duodenalis from samples that tested positive by real-time PCR, we conducted conventional PCR amplification and direct sequencing of four G. duodenalis genes. Conditions were optimized for each locus separately using the Failsafe PCR System  according to the manufacturer's recommendations and based on published primers and protocols for each gene: ef1-αgdhtpiTo infer genetic relationships among PCR products were electrophoresed on 2.0% agarose gels containing 0.5µg/ml ethidium bromide in 1× Tris-acetate-EDTA buffer at 100 volts for 30–45 minutes at room temperature . Each well contained 25 µl of PCR product and 4µl 6× loading dye or 6 µl of 0.5 mg/ml GeneRuler 100-bp DNA ladder . Amplicons were visualized under UV light, and bands of the predicted sizes were excised for DNA extraction using the Zymoclean Gel DNA Recovery Kit , according to the manufacturer's protocol.Following extraction, amplicons were sequenced in both directions on ABI 3730XL capillary sequencers located in the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana-Champaign. Sequences were edited using the computer program Sequencher, Version 4.2  and were queried against known sequences using BLAST G. duodenalis in 64 (23%) of 278 total fecal samples  or livestock . No similar differences were observed in the prevalence of G. duodenalis in livestock among these locations, however  than in individuals between 16 and 75 years , and this difference was statistically significant . Subadult livestock (n = 24) harbored G. duodenalis at a higher rate (25.0%) than did adult livestock , and this difference was also statistically significant , mirroring the trend in humans. We were unable to examine age as a risk factor for G. duodenalis infection in primates, due to the difficulty of determining the ages of individual wild primates.The prevalence of G. duodenalis infection related to land use, demography, and behavior  were used to identify risk factors for human behavior . No factbehavior . ResidenG. duodenalis and the reporting of gastrointestinal symptoms or the taking of medicines (P = 0.001). The lack of an association between infection status and the reporting of gastrointestinal symptoms held true even when individuals ≤15 years (n = 48) were analyzed separately .We found no association between infection with edicines . This waP = 0.002) and residence in Rurama . Residence in a household with another positive person or residence in a household with a positive animal, which were significant in our univariate analysis, fell out of the model as non-significant when all predictors were considered together.To account for potential confounding effects, we used multiple logistic regression with various strategies of model selection . Regardless of the method used, the same two variables were retained in the final model in all cases: age of ≤15 years . Our success in generating SSU-rDNA . Genetice lowest . Geneticour loci .G. duodenalis nucleotide sequences yielded trees that were topologically similar across loci, but that differed in their phylogenetic resolution  or the reporting of gastrointestinal symptoms . We were, however, limited by a small sample size of infected children ≤5 years of age for whom both clinical data and G. duodenalis assemblage were available (n = 6); this precluded meaningful analysis of an association between assemblage and clinical disease in this age category, which might have been expected based on published results Using our phylogenetic results, we were able to classify 36 human G. duodenalis transmission cycles in western Uganda. Our sequence analysis was able to resolve four G. duodenalis clades: one involving assemblage BIV in humans and primates, one involving assemblage E in livestock and primates, and two involving assemblages AII and BIII in humans. Our phylogeny based on gdh was additionally able to resolve sub-clades of G. duodenalis within assemblages, including from humans and red colobus within assemblage BIV and from livestock and red colobus within assemblage E. Although our small sample sizes precluded formal cladistic analyses of the directionality of cross-species transmission G. duodenalis in humans was higher than in either primates or livestock , who inferred assemblage E G. duodenalis in another primate - humans - in Egypt from tpi gene sequences, implying a possible cattle-human transmission link G. duodenalis in people, cattle, and mountain gorillas in Bwindi Impenetrable National Park, Uganda (approximately 200 km from our study site), where ecological overlap between species is also high, and inferred infection with assemblage A in all three species G. duodenalis in species other than humans may reflect real differences between sites; however, we also note that SSU-rDNA had the lowest phylogenetic resolution of any gene in our study . This observation again suggests that PCR-based methods are more sensitive than microscopy.Our results also demonstrate that the prevalence of ons e.g. . Our preG. duodenalis prevalence among human communities. People from Rurama community were infected with G. duodenalis at a rate approximately three times that of people in Bugembe or Kiko 1 communities, despite the roughly similar demographic composition of these communities and their close proximity to each other  found G. duodenalis at 28.1% prevalence in diarrheic Peruvian children as well as in 19.5% of nondiarrheic children, emphasizing the importance of asymptomatic patients in G. duodenalis transmission where hygiene and sanitation are poor G. duodenalis infection status and gastrointestinal symptoms.The lack of any measurable association between infection of people with G. duodenalis on human health, we cannot exclude the possibility that the parasite may be impacting the health of livestock or that of wild primates. Such effects could, in turn, have consequences for conservation. The Ugandan red colobus monkey, for example, is endangered and may be declining due to a combination of nutritional stress and parasitism Although we found little evidence for an effect of i.e. multilocus sequence tying), which might have improved phylogenetic resolution. Findings such as our discovery of assemblage E G. duodenalis in red colobus monkeys, which would be the first report of this assemblage in a non-human primate, should ideally be confirmed with additional sampling and sequencing. Also, we were unable to examine the relationship between infection status and Giardia-specific treatment in people, which could have altered prevalence and the clinical manifestations of infection. We therefore view the results of this study as indicating a strong need for future research into the epidemiology, cross-species transmission ecology, and clinical consequences of G. duodenalis infection in both humans and wildlife, especially where these species interact in anthropogenically disturbed habitats in the tropics.We emphasize that our results are based small sample sizes and short nucleic acid sequences. Moreover, variable amplification success among loci and samples precluded phylogenetic analysis of concatenated sequences (


According to the American Cancer Society approximately 242,000 new cases of prostate cancer will be reported in 2012, making it one of the most frequently diagnosed cancers in men. Moreover, with 1 in 9 men diagnosed with prostate cancer anticipated to die of the disease, prostate cancer is also the second-leading cause of cancer death in men. Although several new treatment options for men with advanced prostate cancer have recently become available, none are as yet curative.i.e., pharmacological depletion of androgens) has long been a mainstay for treatment of men with advanced prostate cancer. However, while androgen deprivation initially leads to tumor regression, ultimately it leads to a highly aggressive form of the disease, which has been called “castration-resistant prostate cancer” (CRPC) to indicate its continued dependence on androgen receptor signaling despite the absence of androgens [Because of the necessity of androgen receptor signaling for all aspects of normal prostate growth, as well as for prostate tumorigenesis, androgen deprivation therapy  model of CRPC and in vitro studies using human prostate cancer cell lines to investigate the consequences of dual targeting Akt/mTOR signaling, with an inhibitor of Akt, namely, MK-2206, and one that targets mTORC1, namely ridaforolimus (MK-8669) (Figure in vivo in the GEM models and in the human prostate cancer cell lines in vitro.Consequently, in our recent study we asked whether combined inhibition of upstream and downstream nodes in the Akt/mTOR signaling pathway is effective for suppressing CRPC . We usedWhy would dual targeting of two kinases in the same pathway yield improved results compared with targeting the pathways individually? In part, this reflects the fact that inhibition of mTORC1 alone has limited efficacy, in part due to feedback activation of PI3K/Akt signaling (Figure How might these findings be applied in the clinic? Currently, several studies have described a crosstalk between the PI3K/Akt/mTOR and AR signaling pathways. In particular, it has been suggested that castration-resistant tumors compensate for reduced AR signaling by activation of Akt/mTOR signaling, which highlights the therapeutic potential of combining inhibition of Akt/mTOR pathways with androgen receptor pathway inhibition. Indeed, Carver et al have suggested that inhibition of the PI3K pathway restores AR signaling in PTEN-deficient prostate cells, and that feedback regulation between AR and PI3K pathways may be reciprocal .Thus, based on recent findings by us and others, we propose that a therapeutic approach using dual inhibitors of Akt/mTOR signaling in combination with inhibitors of androgen receptor signaling, such as Abiraterone Acetate or Enzalutamide, may effectively block androgen signaling while simultaneously inhibiting a Akt/mTOR signaling and therefore an effective treatment course for CRPC. We await the results of the relevant clinical trials.