Among the multiple aberrant Tax-induced effects on cellular processes, persistent activation of transcription factor NF-κB, which is activated only transiently upon physiological stimulation, is essential for leukemogenesis. We and others have shown that Tax induces activation of the IκB kinase (IKK) complex, which is a critical step in NF-κB activation, by generating Lys63-linked polyubiquitin chains. However, the molecular mechanism underlying Tax-induced IKK activation is controversial and not fully understood. Here, we demonstrate that Tax recruits linear (Met1-linked) ubiquitin chain assembly complex (LUBAC) to the IKK complex and that Tax fails to induce IKK activation in cells that lack LUBAC activity. Mass spectrometric analyses revealed that both Lys63-linked and Met1-linked polyubiquitin chains are associated with the IKK complex. Furthermore, treatment of the IKK-associated polyubiquitin chains with Met1-linked-chain-specific deubiquitinase (OTULIN) resulted in the reduction of high molecular weight polyubiquitin chains and the generation of short Lys63-linked ubiquitin chains, indicating that Tax can induce the generation of Lys63- and Met1-linked hybrid polyubiquitin chains. We also demonstrate that Tax induces formation of the active macromolecular IKK complex and that the blocking of Tax-induced polyubiquitin chain synthesis inhibited formation of the macromolecular complex. Taken together, these results lead us to propose a novel model in which the hybrid-chain-dependent oligomerization of the IKK complex triggered by Tax leads to trans-autophosphorylation-mediated IKK activation.The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is crucial for the development of adult T-cell leukemia (ATL), a highly malignant CD4 trans-autophosphorylation-mediated IKK activation.NF-κB is a key transcription factor that regulates many physiologically important cellular processes. However, persistent activation of NF-κB leads to chronic inflammation, autoimmunity and malignancy. Infection with the human retrovirus HTLV-1 causes adult T-cell leukemia, and HTLV-1 Tax-mediated persistent NF-κB activation is crucial for leukemogenesis. Therefore, a better understanding of the precise mechanism underlying aberrant NF-κB activation is essential to develop new therapeutic approaches. Ubiquitination is one of the major post-translational modifications that regulate various intracellular signaling pathways. We and others have shown that Tax activates NF-κB through activation of the IκB kinase (IKK) complex by generating Lys63-linked polyubiquitin chains. However, the molecular mechanism underlying Tax-induced IKK activation remains less well understood. Here, we demonstrate precisely how HTLV-1 Tax utilizes the ubiquitin system to activate the IKK complex. The IKK complex-associated Lys63/Met1-linked hybrid polyubiquitin chains are generated through the Tax-mediated recruitment of linear ubiquitin chain assembly complex (LUBAC) to the IKK complex. Furthermore, the hybrid chains are required for the Tax-induced formation of the active macromolecular IKK complex. Accordingly, we propose a novel model in which Tax triggers Lys63/Met1-linked hybrid-chain-dependent oligomerization of the IKK complex, leading to  The HTLHAM/TSP) –5. Tax aHAM/TSP) . Among tHAM/TSP) –9.NF-κB plays critical roles in immune responses, inflammation, bone metabolism, cell proliferation and survival . NF-κB iThe importance of ubiquitination in the regulation of NF-κB activity is well established . UbiquitIn this study, we show that Tax induces generation of hybrids of K63 and M1 chains by recruiting LUBAC to the IKK complex, leading to the formation of the active macromolecular IKK complex. Thus, we propose a previously unidentified mechanism by which K63 and M1 chains cooperate in Tax-induced IKK activation.E. coli into S-100 cytosolic extracts prepared from the Jurkat human T cell line, HEK293T cell line or mouse embryonic fibroblast (MEF) cells results in IKK activation [Ubc13−/− MEFs further confirmed that Ubc13 is required for Tax-induced IKK activation , TRAF2/TRAF5-deficient (Traf2−/−/Traf5−/−), TRIM25-deficient (Trim25−/−) and Riplet-deficient (Rnf135−/−) MEFs [E. coli or Sf9 cells can efficiently activate IKK  MEFs  and were/−) MEFs . We then/−) MEFs  and the /−) MEFs . Some zivate IKK , neithervate IKK . These rRbck1−/−) MEFs, Sharpin-deficient (cpdm) MEFs or MEFs in which the RING-IBR-RING region  of HOIP was ablated (HOIPΔlinear) were used  were tested. Tax failed to induce IKK activation when cytosolic extracts from HOIL-1L-deficient  antibody that can recognize monoubiquitin and any type of polyubiquitin linkages or an anti-M1 chain-specific antibody. Both antibodies clearly detected smeared bands only when cytosolic extracts were incubated with Tax , lane 2,3), tetramer (Ub4), pentamer (Ub5) and hexamer (Ub6) of recombinant K63 chains , which remained at the top of separating gels  MEFs, and cell-free assays were performed. Tax induced phosphorylation of IKK and IκBα equally well in NEMO-WT- and NEMO (K285R/K309R)-expressing cytosolic extracts  Genetic evidence revealed that both Ubc13 (an E2 enzyme for K63 chain synthesis) and each component of LUBAC (the only E3 enzyme for M1 chain) are crucial for Tax-induced IKK activation. 3) Mass spectrometric analyses revealed that both K63 and M1 chains are associated with the IKK complex only in the presence of Tax. 4) Cell-free experiments with chain type-specific DUBs revealed that K63/M1-linked hybrid chains are associated with the active IKK complex. 5) Tax-induced IKK activation requires the ability of NEMO to bind to both K63 and M1 chains. 6) The formation of the active macromolecular IKK complex (active Taxisome) requires the synthesis of the K27, K63 and M1 chains. Based on 4), 5) and 6), a single hybrid chain may bind to multiple NEMO molecules, and a single NEMO may act as a bridge between the hybrid chains, which may explain why generation of the hybrid chain is required for the macromolecular IKK complex. Regarding how the formation of the macromolecular complex leads to IKK activation, there are two potential mechanisms. The first is that the formation of the macromolecular complex could induce an efficient physical interaction between IKK complexes, so that trans-autophosphorylation between IKK complexes results in full activation of IKK. The second possible mechanism is that the formation of the macromolecular complex could somehow recruit an IKK kinase (IKKK) such as TAK1, which phosphorylates and activates IKK in response to cytokine stimulation [trans-autophosphorylation model.Extensive studies on the role of Tax in ATL development have demonstrated that Tax is involved in leukemogenesis largely through its ability to constitutively activate NF-κB –9. It hamulation . We prevmulation . In addimulation , leadingIn contrast to our model, conflicting results have been reported on the following three points. The first point is the involvement of IKKK. Yin et al.  demonstrInconsistent with our model, the regular complex dots in , which wInterestingly, cell-free experiments using IsoT and viral OTU revealed that both unanchored and anchored polyubiquitin chains are required for Tax-induced IKK activation. Although the critical roles of unanchored and anchored polyubiquitin chains in Tax-induced IKK activation are still controversial, our experiments using Tax KR mutants support the idea that the polyubiquitin chains conjugated to Tax belong to the class of substrate-anchored polyubiquitin chains required for Tax-induced IKK activation. By taking advantage of specific DUBs that degrade only one type, we showed for the first time that unanchored and anchored polyubiquitin chains cooperate in Tax-induced IKK activation.Although the addition of the DN ubiquitin mutant K27R inhibited IKK activation by Tax, we did not detect K27 chains associated with the active Taxisome via the ubiquitin-AQUA method. This may be because an undetectable amount of K27 chains is involved in the generation of the hybrid chains as their component or because K27 chains act as initial triggers of the hybrid chains synthesis but are dissociated from the Taxisome afterwards. Further studies are needed to identify the precise role of K27 chains in Tax-induced IKK activation.Tax binds to LUBAC through the UBL domain of HOIL-1L and RBR domain of HOIP. The UBL domain of HOIL-1L is involved in the association with HOIP, while the RBR domain of HOIP is the catalytic active site . TherefoHuman cDNAs encoding dominant-negative mutants of E2 enzymes were generated via PCR and inserted into the pRK5 vector. pMRX-Cre was obtained from S. Akira (Osaka University). Expression vectors for HOIL-1L-HA, Myc-HOIP and their deletion mutants were generated as previously described . Human aThe following antibodies were used: anti-p-IκBα (9246), anti-IκBα (9242), anti-p-IKKα/β (2697), anti-NEMO (2695) and anti-ubiquitin (3936) ; anti-GST (sc-459), anti-Myc (sc-789) and anti-HA probe (sc-805) (Santa Cruz Biotechnology); anti-Flag M2 (F3165) (Sigma); anti-tubulin (CP06) ; anti-HOIL-1L (NBP-1-88301) ; anti-HOIP (ARP43241) (Aviva Systems Biology); anti-Sharpin (14626-1-AP) (Proteintech); anti-linear polyubiquitin-specific (AB130) (LifeSensors); and anti-Ubc13 (37–1100) (ThermoFisher Scientific). The anti-Tax antibody was generated as previously described .HEK293T cells (purchased from ATCC), Plat-E cells (provided by T. Kitamura) and mouse embryonic fibroblasts  were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS). Jurkat cells (purchased from ATCC), the Jurkat-derived cell line JPX-9 cells , and HTLV-1-infected cell line HUT102, MT-2, and MT-4 cells (provided by J. Fujisawa) were maintained in RPMI1640 supplemented with 10% heat-inactivated FBS. Sf9 cells were maintained in Sf900IIISFM (Thermo Fisher Scientific) supplemented with 10% FBS. The transfection of HEK293T cells was performed by the calcium phosphate method. siRNAs were transfected using NEPA21 Super Electroporator (NEPAGENE). Control stealth siRNA and the following stealth siRNAs (Thermo Fisher Scientific) were used: HOIP-1 sense/anti-sense, 5′-GGUACUGGCGUGGUGUCAAGUUUAA-3′/5′-UUAAACUUGACACCACGCCAGUACC-3′; HOIP-2 sense/anti-sense, 5′-CACCACCCUCGAGACUGCCUCUUCU-3′/5′-AGAAGAGGCAGUCUCGAGGGUGGUG-3′.For lentivirus production, HEK293T cells were transfected with the self-inactivating lentiviral vector construct, the packaging construct and the VSV-G- and Rev-expressing construct. After 48 h of incubation, culture supernatants were collected and centrifuged at 50,000 x g for 1 h at 20°C to concentrate lentivirus. MT-2 or MT-4 cells were infected with the lentivirus at 400 x g for 2 h at 20°C. After 48 h, puromycin (Wako) was added to the medium, and puromycin-resistant cell pools were used for further experiments. The following target sequences were used: Tax-1, 5′-GGCCTTCCTCACCAATGTTCC-3′; Tax-2, 5′-GGCAGATGACAATGACCATGA-3′; Tax-3, 5′-GCCTACATCGTCACGCCCTAC-3′; HOIP-1, 5′-GCTGCAGCTTTCAGAATTTGA-3′; HOIP-2, 5′-GCACTGCCCATCCTGTAAACA-3′; HOIP-3, 5′-GCTCCTTTGGCTTCATATATG-3′; Control, 5′-GATTTCGAGTCGTCTTAATGT-3′.Ubc13+/+ or Ubc13fl/fl MEFs were incubated with the retrovirus containing polybrene  for 8 h. After 48 h, puromycin was added to the medium, and puromycin-resistant cell pools were used for further experiments.For retrovirus production, Plat-E cells were transfected with pMRX-Cre vector. After 24 h, culture supernatants were collected, and 6-Tax, His6-M22, GST-viral OTU (WT) and its catalytic inactive mutant 1A were expressed in E. coli and purified. His6-TRAF6 and His6-Tax were expressed in Sf9 cells using the Bac-to-Bac Baculovirus Expression System (Thermo Fisher Scientific) and purified. GST, GST-HOIL-1L, GST-HOIP and GST-Sharpin were generated using the wheat germ cell-free protein synthesis system and purified [6-UBE1 (E-304), UbcH5c (E2-627), UbcH7 (E2-640) and His6-Ubc13/Uev1A (E2-664) were purchased from BostonBiochem. Recombinant K48- and K63-linked Ub2-Ub7 chains  were purchased from BostonBiochem. Recombinant M1-linked Ub2-Ub7 chains (BML-UW1010-0100) were purchased from Enzo Life Sciences.Hispurified . Ubiquit2, 10 mM KCl, 0.5 mM dithiothreitol (DTT) and protease inhibitor cocktail (Roche)) and then lysed with a Dounce homogenizer. Cell debris was removed via ultracentrifugation at 100,000 x g for 1 h at 4°C to prepare the S-100 cytosolic extract. Cytosolic extracts (10 mg/ml) were incubated with recombinant His6-Tax in ATP buffer (50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 2 mM ATP, 5 mM NaF, 20 mM β-glycerophosphate, 1 mM Na3VO4, and protease inhibitor cocktail) in the presence or absence of various recombinant DUBs. After incubation at 30°C for 1 h, the reaction mixtures were subjected to immunoblotting or immunoprecipitation.Jurkat cells and MEFs were suspended in hypotonic buffer (10 mM Tris-HCl (pH 7.5), 1.5 mM MgCl2, 10 mM NaF, 1% NP-40, 10 mM β-glycerophosphate, 1 mM Na3VO4, 1 mM DTT, 5 mM N-ethylmaleimide (NEM) and protease inhibitor cocktail), followed by centrifugation at 22,000 x g for 15 min at 4°C to remove the insoluble fraction. For detection of polyubiquitination of Tax, the reaction mixtures were boiled for 10 min in the presence of 1% SDS to remove noncovalently attached proteins. The mixtures were then diluted 10-fold in IP buffer to reduce the SDS concentration to 0.1%. The cell lysates or the cell-free reaction mixtures were subsequently incubated with the antibodies plus protein G-sepharose. The immunoprecipitates were washed five times and subjected to immunoblotting. For immunoblotting, immunoprecipitates or cell lysates were separated via SDS-PAGE and transferred to PVDF membranes . The membranes were then incubated with the primary antibodies. Immunoreactive proteins were visualized with anti-rabbit or anti-mouse IgG conjugated to horseradish peroxidase, followed by processing with an ECL detection system.Cells were lysed in IP buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1 mM MgCl6-Tax at 4°C for 1 h. After incubation, the beads were washed and subjected to immunoblotting.Glutathione sepharose was incubated with 300 ng of GST, GST-HOIL-1L, GST-HOIP or GST-Sharpin at 4°C for 1 h in binding buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM DTT, 2.5 mg/ml BSA and protease inhibitor cocktail). The beads were then incubated with 500 ng of His6-Tax in ATP buffer. After incubation at 30°C for 1 h, the reaction mixtures were subjected to immunoprecipitation with an anti-NEMO antibody in IP buffer. The immunoprecipitates were washed three times with IP buffer without NEM and incubated with Otubain-1 (5 μM), AMSH (5 μM), OTULIN (5 μM) or USP2 (5 μM) at 37°C for 1 h in DUB buffer (50 mM HEPES-KOH (pH 7.5), 100 mM NaCl, 1 mM MnCl2, 0.01% Brij-35 and 2 mM DTT). After incubation, the reaction mixtures were subjected to immunoblotting.Jurkat cytosolic extracts (10 mg/ml) were incubated with recombinant His6-Tax in ATP buffer. After incubation at 30°C for 1 h, the reaction mixtures were subjected to immunoprecipitation with an anti-NEMO antibody in IP buffer. The immunoprecipitates were analyzed via the ubiquitin-AQUA method as described previously [Jurkat cytosolic extracts (10 mg/ml) were incubated with recombinant Hiseviously . The immAfter the cell-free reaction, the reaction mixtures were mixed with NativePAGE Sample Buffer (Thermo Fisher Scientific). Electrophoresis was performed using NativePAGE Running Buffer (Thermo Fisher Scientific) containing 0.002% G-250. The gels were soaked in denaturation buffer (10 mM Tris-HCl (pH 6.8), 1% SDS and 0.006% 2-mercaptoethanol) for 30 min at 60°C, followed by transfer to PVDF membranes and immunoblotting.The total RNA was isolated from MT-4 cells transfected with control or HOIP siRNA with Trizol reagent (Thermo Fisher Scientific). cDNA was synthesized from 2.0 μg of total RNA with Prime ScriptII (Takara). Quantitative real-time PCR analysis was performed on CFX Connect (Bio-Rad). The level of GAPDH expression was used to normalize the data. The following primers were used: IL-6 sense/anti-sense, 5′-CCTGAACCTTCCAAAGATGGC-3′/5′-TTCACCAGGCAAGTCTCCTCA-3′; IL-1β sense/anti-sense, 5′-TTCGACACATGGGATAACGAGG-3′/5′-TTTTTGCTGTGAGTCCCGGAG-3′; MMP-9 sense/anti-sense, 5′-ATGTACCGCTTCACTGAGGG-3′/5′-TCAGGGCGAGGACCATAGAG-3′; GAPDH sense/anti-sense, 5′-TGCACCACCAACTGCTTAGC-3′/5′-GGCATGGACTGTGGTCATGAG-3′.HEK293T cells were transfected with the plasmids encoding wild type or various Tax mutants together with 20 ng of luciferase reporter (3xκB-luc) and 30 ng of β-actin-β-galactosidase plasmid. After 48 h, the luciferase activity was measured using the Luciferase Assay System (Toyo Ink). β-galactosidase activity was used to normalize the transfection efficiency.6-UBE1 (0.1 μM), the indicated E2 (0.2 μM), ubiquitin (25 μM) and His6-TRAF6 or His6-Tax in the presence of ATP (2 mM). After incubation, the reaction mixtures were analyzed by immunoblotting.The reactions were performed at 37°C for 1 h in the reaction buffer containing His2. After electrophoresis, the gels were washed with transfer buffer containing 10 mM EDTA for 15 min. The gels were further washed with transfer buffer without EDTA for 10 min, and the samples were transferred to PVDF membranes, followed by immunoblotting.After cell-free reactions, the reaction mixtures were separated using 6% polyacrylamide gels containing 20 μM Phos-tag acrylamide (Wako) and 40 μM MnClt-test . Data are presented as the means ± SD.Statistically significant differences between mean values were determined using Student’s S1 Fig6-Tax and ATP (2 mM) in the presence of MG132 (10 μM). The reaction mixtures were analyzed by immunoblotting with the indicated antibodies.Jurkat cytosolic extracts were incubated with recombinant His(TIF)Click here for additional data file.S2 Fig(A-D) Cytosolic extracts were prepared from cIAP1/cIAP2-deficient (A), TRAF2/TRAF5-deficient (B), TRIM25-deficient (C), Riplet-deficient MEFs (D) and corresponding WT MEFs and subjected to cell-free analyses. The depicted results are representative of three independent experiments.(TIF)Click here for additional data file.S3 Fig(A) A schematic representation of the various Tax mutants used in (B) and (C). (B) HEK293T cells were transfected with expression plasmids encoding Tax or various Tax mutants. After 60 h, the cells were treated with MG132 (20 μM) for 2 h, and the cell lysates were subjected to immunoblotting with the indicated antibodies. (C) HEK293T cells were transfected with plasmids encoding Tax or various Tax mutants together with a 3xκB-luc reporter. After 48 h, luciferase activity was measured. The results are given as the mean ±S.D. (n = 3). (D) Jurkat cytosolic extracts were incubated with recombinant His6-Tax purified from Sf9 cells or E. coli in the presence of ATP (2 mM). The reaction mixtures were analyzed by immunoblotting with the indicated antibodies. His6-Tax from Sf9 is larger than that from E. coli due to the difference in the length of linker sequence between His-tag and Tax protein. (E) Recombinant His6-Tax purified from Sf9 cells or E. coli (left) or His6-TRAF6 (right) was incubated with UBE1 , the indicated E2 (0.2 μM) and ubiquitin (25 μM) in the presence of ATP (2mM). The reaction mixtures were analyzed by immunoblotting with an anti-Ub antibody. The depicted results are representative of three independent experiments.(TIF)Click here for additional data file.S4 Fig(A) Jurkat cytosolic extracts were incubated with recombinant His6-Tax and ATP (2 mM) in the presence of DN ubiquitin mutants or HA-ubiquitin (50 μM). The reaction mixtures were separated via Phos-tag SDS-PAGE, followed by immunoblotting with an anti-HOIP antibody. (B) Jurkat cytosolic extracts were incubated with recombinant His6-Tax and ATP (2 mM) in the absence or presence of increasing amounts of the IKKβ inhibitor TPCA-1 . The reaction mixtures were separated via regular SDS-PAGE. Dots denote the phosphorylated form of HOIP. The depicted results are representative of three independent experiments.(TIF)Click here for additional data file.


The type III receptor tyrosine kinase FLT3 is one of the most commonly mutated oncogenes in acute myeloid leukemia (AML). Inhibition of mutated FLT3 in combination with chemotherapy has displayed promising results in clinical trials. However, one of the major obstacles in targeting FLT3 is the development of resistant disease due to secondary mutations in FLT3 that lead to relapse. FLT3 and its oncogenic mutants signal through associating proteins that activate downstream signaling. Thus, targeting proteins that interact with FLT3 and their downstream signaling cascades can be an alternative approach to treat FLT3-dependent AML. We used an SH2 domain array screen to identify novel FLT3 interacting proteins and identified ABL2 as a potent interacting partner of FLT3. To understand the role of ABL2 in FLT3-mediated biological and cellular events, we used the murine pro-B cell line Ba/F3 as a model system. Overexpression of ABL2 in Ba/F3 cells expressing an oncogenic mutant of FLT3 (FLT3-ITD) resulted in partial inhibition of FLT3-ITD-dependent cell proliferation and colony formation. ABL2 expression did not alter the kinase activity of FLT3, its ubiquitination or its stability. However, it partially blocked FLT3-induced AKT phosphorylation without affecting ERK1/2 and p38 activation. Taken together our data suggest that ABL2 acts as negative regulator of signaling downstream of FLT3. The mammalian genome encodes more than 500 protein kinases that contribute to the regulation of almost all cellular events. Around 60 protein kinases are characterized as receptor tyrosine kinases which are regulated by extracellular stimuli including growth factors , 2. The The mammalian Abelson (ABL) family of non-receptor tyrosine kinase includes the two members ABL and ABL2 . ABL and ABL2 transduce signals from upstream receptors and regulate numerous biological processes such as cell survival, apoptosis, response to genotoxic stress, morphogenesis and cell motility . ABL famReceptor tyrosine kinases such as FLT3 signal through proteins that associate with the activated receptor. In order to identify novel FLT3-interacting proteins we used an SH2 domain array . SeventyTo explore the role of ABL2 in oncogenic FLT3-ITD-mediated biological responses, we generated Ba/F3 cells overexpressing both FLT-ITD and ABL2 or empty control vector (EV). FLT3-ITD surface expression was analyzed by flow cytometry and the total expression was determined by western blotting. The results demonstrate an equal surface Figure  as well Wild-type FLT3 activates the PI3K/AKT, RAS/ERK and p38 pathways in response to FL-stimulation while FLT3-ITD constitutively activates those pathways –25. SincA majority of the proteins that associate with receptor tyrosine kinases affect either receptor activation, ubiquitination or stability , 27. We ns, not significant.Signaling downstream of receptor tyrosine kinases is tightly controlled by various associating proteins such as kinases, phosphatases, ubiquitin E3 ligases, adaptors and scaffolding proteins. In order to maintain a proper balance in signaling, many interacting proteins enhance receptor signaling, but some of them turn off the signal by dephosphorylating the receptor or directing the receptor for ubiquitin-mediated degradation. Thus, the level of signaling is maintained at a level that is appropriate for the cell. Too much RTK signaling can be deleterious and lead to induction of apoptosis . Here weThe recombinant SH2 domain of ABL2 displayed a high affinity for multiple phosphopeptides corresponding to known FLT3 tyrosine phosphorylation sites. In addition to the ABL2 SH2 domain, the SH2 domains of CRK, CRKL, FYN, ABL, LCK, SOCS6, SYK etc. also displayed a considerable affinity for those phosphopeptides. Many of those proteins have already been verified by biochemical methods and identified as critical regulators of FLT3 signaling , 8, 29. FLT3 and its oncogenic counterpart, FLT3-ITD, exert survival, proliferative and transforming signals by activating mainly three different signaling cascades, i. e. the PI3K/AKT, RAS/ERK and p38 pathways , 25. WheLipofectamine 2000 and horseradish peroxidase (HRP)-coupled secondary anti-mouse and anti-rabbit antibodies were from ThermoFisher Scientific . Cycloheximide and mouse anti-FLAG (M2) antibody were from Sigma-Aldrich . FLT3 ligand (FL) was from ORF Genetics . Rabbit anti-phospho-AKT (pSer473), mouse anti-phosphotyrosine (4G10), mouse anti-mono-ubiquitin antibodies were from Abcam , Merck Millipore  and Covance Research Products , respectively. Rabbit anti-phospho-ERK1/2 (pThr202/pThr204), goat anti-AKT, rabbit anti-ERK2 and anti-goat secondary antibodies were from Santa Cruz Biotechnology . The phycoerythrin-labeled anti-FLT3, mouse anti-phospho-p38 and anti-p38 antibodies were from BD Transduction Laboratories . Rabbit anti-FLT3 antibody was described previously .Expression plasmids pcDNA3-FLT3-WT, pcDNA3-FLT3-ITD, pMSCVpuro-FLT3-WT and pMSCVpuro-FLT3-ITD plasmids were previously described . Human fCOS-1 and Ba/F3 cells were purchased from DSMZ . EcoPack cells were from Clontech . Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100μg/ml streptomycin was used for maintaining COS-1 and EcoPack cells. RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 10 ng/ml recombinant murine interleukin-3 (IL-3), 100 units/ml penicillin and 100μg/ml streptomycin was used for Ba/F3 cells.Lipofectamine 2000 was used for transient transfection. COS-1 cells were transfected with pCMV-Myc-FLAG and pcDNA3 plasmids according to the manufacturer's protocol.Retroviral vector pMSCV was used to produce virus particles in EcoPack packaging cells. EcoPack cells were transfected with 10μg pMSCVpuro-FLT3-WT, pMSCVpuro-FLT3-ITD, pMSCVneo-ABL2-Myc-FLAG or pMSCVneo constructs using Lipofectamine 2000 followed by collection of virus-containing supernatants after 72 hours of transfection. Then Ba/F3 cells were infected with FLT3-WT or FLT3-ITD virus particles. Cells were selected using 1.2 μg/ml puromycin for two weeks and FLT3 expression was checked by flow cytometry and Western blotting. Ba/F3-FLT3-WT and Ba/F3-FLT3-ITD cells were further infected with ABL2 or empty vector virus particles and then further selected against 0.8 mg/ml G-418 for 2 weeks. ABL2 expression was verified using Western blotting. All stably transfected Ba/F3 cells were maintained in Ba/F3 medium as described above and recommended previously .Ba/F3 cells were washed three times and then starved of IL3 and serum for 4 hours in RPMI-1640 before stimulation with 100 ng/ml of FL. Cells were then washed with ice-cold PBS and lysed in 1% Triton X-100 lysis buffer. For immunoprecipitation of 1 ml cell lysates one μg primary antibody was used. The Western blotting and immunodetection procedures were described elsewhere , 33.Screening of SH2 domain specificity of the various phosphopeptides was performed by Dr. Shawn Li's Laboratory, University of Ontario, as a service. Peptide synthesis, array constructions and assay conditions were essentially as described elsewhere .Ba/F3-FLT3-ITD cells expressing ABL2 or empty vector were washed three times and resuspended in RPMI 1640 supplemented with 10% FBS, 100 units/ml penicillin and 100μg/ml streptomycin. Cells were then seeded in a 96-well plate . Two days after seeding, cell viability was counted by PrestoBlue  assay.Ba/F3-FLT3-ITD cells expressing ABL2 or empty vector were washed three times and resuspended in RPMI 1640 supplemented with 10% FBS, 100 units/ml penicillin and 100μg/ml streptomycin. Cells were then seeded in a 12-well plate . Two days after seeding, apoptosis was measured by flow cytometry using Annexin V and 7-aminoactinomycin D (7-AAD) apoptosis kit .Ba/F3-FLT3-ITD cells expressing ABL2 or empty vector were washed three times and 1,000 cells were mixed with 500 μl of 80% methylcellulose medium and seeded in a 24-well plate. Cells were incubated for 7 days and colonies were counted by two individual researchers.Ba/F3-FLT3-WT cells expressing ABL2 or empty vector were washed three times with PBS and treated with 100 μg/ml cycloheximide for 30 minutes. Cells were then stimulated with FL for 30 minutes in presence of cycloheximide followed by lysis in Triton X-100 lysis buffer. Cell lysates were subjected to the Western blotting analysis.Blots were quantified using ImageJ software and statistical analysis was performed using GraphPad Prism 5.0. Data were presented as the mean ± SE. One-way ANOVA with Bonferroni's post-tests and student's t-test were used to check statistical significance.


Nrf2 andangiotensinogen (Agt) gene expression; and upregulated heterogeneousnuclear ribonucleoprotein F and K (hnRNP F and hnRNPK) expression in Akita mice with T1D. In immortalized rat renal proximaltubular cells, insulin suppressed Nrf2 and Agt butstimulated hnRNP F and hnRNP K gene transcriptionin high glucose via p44/42 mitogen-activated protein kinase signaling. Transfectionwith small interfering RNAs of p44/42 MAPK, hnRNP F, orhnRNP K blocked insulin inhibition of Nrf2 genetranscription. Insulin curbed Nrf2 promoter activity via a specificDNA-responsive element that binds hnRNP F/K, and hnRNP F/K overexpression curtailedNrf2 promoter activity. In hyperinsulinemic-euglycemic mice,renal Nrf2 and Agt expression was downregulated,whereas hnRNP F/K expression was upregulated. Thus, the beneficialactions of insulin in diabetic nephropathy appear to be mediated, in part, bysuppressing renal Nrf2 and Agt gene transcriptionand preventing Nrf2 stimulation of Agt expression via hnRNP F/K.These findings identify hnRNP F/K and Nrf2 as potential therapeutic targets indiabetes.Oxidative stress induces endogenous antioxidants via nuclear factor erythroid2–related factor 2 (Nrf2), potentially preventing tissue injury. Weinvestigated whether insulin affects renal Nrf2 expression in type 1 diabetes (T1D)and studied its underlying mechanism. Insulin normalized hyperglycemia, hypertension,oxidative stress, and renal injury; inhibited renal  Nrf2 gene transcription via a putative IRE inthe Nrf2 gene promoter that binds hnRNP F/K.Our present study identifies an inhibitory action of insulin on renal Preclinl lines) , howeverl lines) . In contl lines) . Bardoxol lines) . The reaAgt) and Nrf2 gene expression, and blocks Nrf2stimulation of Agt gene transcription, in type 1 diabetes (T1D) AkitaCat-transgenic mice  overexpression in renal proximal tubular cells(RPTCs) prevents hypertension and nephropathy, attenuates renal angiotensinogen, which bind to a putativeinsulin-responsive element (IRE) in the rat Agt genepromoter  inhibitor], wortmannin and Ly-294,002(specific inhibitors of phosphatidylinositol 3-kinase), and oltipraz (an Nrf2activator) were purchased from Sigma-Aldrich Canada Ltd. .U0126 (a p44/42 MAPK inhibitor) was obtained from Cell Signaling Technology . Dulbecco’s modified Eagle medium and penicillin/streptomycinand fetal bovine serum were procured from Invitrogen, Inc. .Insulin implants  were sourced from Linshin . pGL4.20 [Luc/Puro] vector containing luciferase reporter came from PromegaCorporation . The pGL4.20 construct, containing the ratAgt gene promoter N-1495 to N+18 or the ratNrf2 gene promoter N-1960 to N+111, has beendescribed previously  withspecific primers  were provided by Ambion,Inc. . Restriction and modifying enzymes were supplied by Invitrogen,Inc., and New England Biolabs. Oligonucleotides were synthesized by Integrated DNATechnologies . QuickChange II Site-Directed Mutagenesis Kit andLightShift Chemiluminescent electrophoretic mobility shift assay (EMSA) Kit wereprocured from Agilent Technologies  and Thermo Scientific , respectively. Primer biotin-labeling kitwas purchased from Integrated DNA Technologies.eviously . ThehnR primers , confirm primers . The othinsulin2 gene (C57BL/6-Ins2Akita/J) were purchasedfrom Jackson Laboratories .Adult male wild-type (WT) and heterozygous Akita mice with mutatedad libitum. Animal care and procedures were approvedby the Centre de recherche du Centre hospitalier de l’Université deMontréal Animal Care Committee and followed the Principles of LaboratoryAnimal Care .Male Akita mice (age 10 weeks) were divided into 2 groups with and without insulinimplants at week 12 until week 16 .Non-AkiBlood glucose levels and systolic blood pressure (SBP) were measured with anAccu-Chek Performa System  and BP-2000tail-cuff pressure monitor , respectively .The micGlomerular filtration rate (GFR) was estimated with fluorescein isothiocyanate inulin. KidneysSeparate hyperinsulinemic-euglycemic clamp experiments were performed on consciousmale C57Bl/6 mice (age 12 to 14 weeks) after a 4-hour food restriction .Serum and urinary Agt and angiotensin II (Ang II) levels were quantified byenzyme-linked immunosorbent assay  .http://rsb.info.nih.gov/ij/).Kidney sections  were stained with standard periodic acid Schiff or Masson’strichrome or processed for immunohistochemistry  . TubularROS generation as an index of oxidative stress was assessed by dihydroethidium  staining of frozen kidney sections  and by lAgt, pGL4.20-Nrf2, pGL4.20-hnRNPF, and pGL4.20-hnRNP K, respectively, containingAgt, Nrf2, hnRNP F, and hnRNP K gene promoters,were transfected into IRPTCs. Stable transformants were selected in the presence of0.6 mg/L of puromycin    or HG (25 mmol/L d-glucose) DMEM containing 1%depleted fetal bovine serum and insulin (10−7 mol/L) for up to 24hours ± p44/42 MAPK inhibitors (PD98059 or U0126), phosphatidylinositol3-kinase inhibitors , or the Nrf2 activator oltipraz. Thecells were then harvested, and promoter activity was measured by luciferase assay weresynchronized overnight in serum-free DMEM containing 5 mmol/L se assay. IRPTCs p44/42 MAPK, Nrf2, hnRNP F, or hnRNP KsiRNAs , Kelch-like ECH-associated protein 1 (Keap1),Nox1, Nox2, Nox4, and β-actinmRNA levels in RPTs and IRPTCs were quantified by real-time quantitative PCR(RT-qPCR) with specific primers .Western blotting (WB) was undertaken .The relP < 0.05 values wereconsidered statistically significant.Values were expressed as mean ± standard error of the mean (SEM). Data wereanalyzed using 1- or 2-way analysis of variance, as appropriate, followed by aBonferroni multiple comparison testing. Supplemental Fig. 1(a)], and increasedextracellular matrix proteins in the glomeruli and tubules[Supplemental Fig. 1(b)]. Glomerular tufts, RPTCvolume, and renal tubule lumen areas were augmented significantly in Akita micecompared with WT controls. Insulin treatment normalized these changes  and remained significantly elevated for thestudy’s duration .Agt, HO-1, and Nrf2 immunostaining increased in RPTCs of Akita mice compared with WTcontrols. Treatment with insulin normalized these changes . Keap1 ipression , i–iv frita mice . ConsistNrf2 and Agt gene promoteractivity in NG and prevented HG stimulation of Nrf2 andAgt gene promoter activity in IRPTCs in a time-dependent manner.In contrast, insulin stimulated hnRNP F and hnRNP Kgene promoter activity in NG and HG in IRPTCs in a time-dependent manner.PD98059 and U0126, but not wortmannin or Ly-294, 002, prevented insulin inhibition ofNrf2 gene promoter activity . Transienttransfection of p44 MAPK and p42 MAPK siRNAsattenuated the expression of respective p44 MAPK and p42 MAPK in IRPTCs, whereasscrambled siRNA had no effect [Supplemental Fig. 3(b)]. Transfection withp44 MAPK or p42 MAPK siRNAs or both reversedinsulin inhibition of Nrf2 and Agt gene promoteractivity . DHE staining,ROS generation, Cat, Nox1, Nox2, and Nox4 mRNAexpression [Supplemental Fig. 3(d–i)] did not differfrom RPTs of saline-infused and hyperinsulinemic mice. In contrast, hyperinsulinemiadecreased Agt and increased hnRNP F and hnRNP K immunostaining , leading us tospeculate that the lower Cat expression and activity observed in Akita mice at 16 weeksof age might be due to exhaustion of the scavenging system.Cat expression and activity, but not Nrf2, and AgtmRNA expression and stimulated p44/42 MAPK phosphorylation in RPTs of Akita mice at 24weeks [Supplemental Table 1 andSupplemental Fig. 5(a–c)]. These findings areconsistent with those of Lizotte et al. , normalized e et al. , who repin vitro (10−7 M or 573 ng/mL)was at least 200-fold higher than the mean circulating insulin level in Akita micebearing insulin implants (2.3 ± 1.1 ng/mL), similar to those reported (3.4± 0.4 ng/mL) by others ].The insulin level used y others . HoweverNrf2 and Agt as well as stimulation ofhnRNP F/K gene transcription. At present, we do not understand theexact mechanism by which insulin decreases nuclear Nrf2 accumulation in Akita mice.Studies of Zheng et al. (p-Nrf2 (s-40) without apparent effect on cytoplasmic Nrf2 andp-Nrf2 (s-40) in Akita mice and increases p44/42 MAPKphosphorylation .During oxidative stress, PKC-δ phosphorylates Nrf2 at serine 40to enhance its nuclear translocation  in the respective promoters. Hu etal.  in Nrf2 gene promoter,subsequently suppressing Nrf2 gene transcription. This possibility issupported by our finding that hnRNP F/K overexpression considerably decreasesNrf2 gene promoter activity, and hnRNP F/K siRNAreverse insulin downregulation of Nrf2 gene transcription. DNA sequenceanalysis discerned 2 GC-rich regions, nucleotides N-463 toN-444 (5′-cgcgccccgcccccgcggga-3′) andN-607 to N-592(5′-ggggcccgggctccc-3′), in the Nrf2 gene promoter.Nucleotides N-463 to N-444 contain the core sequence5′-ccccgcccc-3′, which is homologous to the core sequenceof IRE  of the ratAgt gene promoter  specifically binds to RPTC nuclearproteins, and the addition of antihnRNP F or antihnRNP K antibody yields a supershift ofbiotinylated-labeled IRE binding with nuclear proteins on EMSA. Thesedata demonstrate that hnRNP F/K bind to a putative IRE(N-463 to N-444) and inhibit Nrf2gene transcription. It is noteworthy that hnRNP F/K are not restricted toNrf2 gene expression but also affect the expression ofAgt  compared with insulin implants in Akita mice (after 4 weeks of insulinimplantation). Such rapid transcription is consistent with other studies of upregulatedand downregulated genes in muscles and liver within 2 to 4 hours undereuglycemic-hyperinsulinemic conditions  may exaggerate renal dysfunction via activation of the intrarenal RAS,thereby enhancing renal fluid and salt reabsorption.Finally,  pathway . It has pathway , however pathway . Our stuNrf2 genetranscription and prevents Nrf2 stimulation of intrarenal Agt geneexpression via hnRNP F/K, indicating that Nrf2 activation may amplify renal dysfunctionvia intrarenal RAS activation in diabetes. Our study identifies renal hnRNP F/K and Nrf2as potential targets for the treatment of hypertension and kidney injury indiabetes.In summary, our data demonstrate that insulin inhibits


Although potent androgen receptor pathway inhibitors (ARPI) improve overall survival of metastatic prostate cancer patients, treatment-induced neuroendocrine prostate cancer (t-NEPC) as a consequence of the selection pressures of ARPI is becoming a more common clinical issue. Improved understanding of the molecular biology of t-NEPC is essential for the development of new effective management approaches for t-NEPC. In this study, we identify a splice variant of the MYST/Esa1-associated factor 6 (MEAF6) gene, MEAF6-1, that is highly expressed in both t-NEPC tumor biopsies and neuroendocrine cell lines of prostate and lung cancers. We show that MEAF6-1 splicing is stimulated by neuronal RNA splicing factor SRRM4. Rather than inducing neuroendocrine trans-differentiation of cells in prostate adenocarcinoma, MEAF6-1 upregulation stimulates cell proliferation, anchorage-independent cell growth, invasion and xenograft tumor growth. Gene microarray identifies that these MEAF6-1 actions are in part mediated by the ID1 and ID3 genes. These findings suggest that the MEAF6-1 variant does not induce neuroendocrine differentiation of prostate cancer cells, but rather facilitates t-NEPC progression by increasing the proliferation rate of cells that have acquired neuroendocrine phenotypes. Next generation androgen receptor (AR) pathway inhibitors (ARPIs) prolong survival of patients with metastatic castrate resistant prostate cancer (CRPC) , 2. HoweThere is accumulating evidence indicating that t-NEPC is clonally derived from adenocarcinoma (AdPC) precursors , 9. WholThe transition of AdPC to t-NEPC is a complicated process that may involve both cell differentiation and proliferation, two distinguishable and coordinated processes controlled by multiple genes. Luminal epithelial AR signaling is critical in the maintenance of epithelial cell differentiation in the adult prostate . As a reUsing whole-transcriptome sequencing of AdPC and NEPC patient biopsies from the Beltran  and Vancp = 0.0034 in the VPC cohort and p = 0.0002 in the Beltran cohort), while total MEAF6 mRNAs remained unchanged , while MEAF6-2 mRNA levels in NEPC were not statistically different between NEPC and AdPC (p = 0.338) . In contrast, MEAF6-2 mRNA levels were not statistically different in AdPC lines from NCI-H660, -H69, and -H82 cell lines  and Beltran (32 AdPC and 6 NEPC samples) cohorts were analyzed by a computational tool, COMPAS , which id Figure . These r) Figure . Increas) Figure  and clin) Figure . Additio) Figure . These rs Figure . FurtherTo determine whether SRRM4 regulates MEAF6 splicing, we transiently transfected SRRM4 expression vector in LNCaP cells. SRRM4 did not alter the levels of total MEAF6 transcripts Figure . InsteadTo study the biological functions of MEAF6-1 in prostate cancer cells, we constructed LNCaP and PC-3 cell lines that stably express exogenous MEAF6-1 or MEAF6-2 via lentiviral transduction . NeitherUnder 2D culture conditions, BrdU incorporation assays demonstrated that enhanced expression of MEAF6-1 but not MEAF6-2 in both LNCaP and PC-3 cells induced a 40–50% increase of cell proliferation rates Figure . Since NTo profile the MEAF6-1 transcriptome, we compared gene microarrays using LNCaP(CTL), LNCaP(MEAF6-1), and LNCaP(MEAF6-2) stable lines. There were 2044 genes differentially regulated by MEAF6-1 and 2702 genes by MEAF6-2. DAVID  for gene ontology (GO) analyses indicated that the functions of both gene groups regulated by MEAF6-1 or MEAF6-2 are very similar. GO terms indicated that MEAF6-1 and MEAF6-2-regulated genes are associated with cell cycle, cellular response to stress, cell division, regulation of cell cycle, and DNA replication. Since MEAF6-1, but not MEAF6-2, promotes cell proliferation, anchorage-independent growth and invasion, we further stratified a group of 159 genes that were specifically regulated by MEAF6-1 Figure . InteresTo study the impacts of MEAF6-1 on xenograft growth, we transplanted PC-3(CTL) or PC-3(MEAF6-1) cells subcutaneously into nude mice. Tumor uptake at ~3 weeks was similar in both groups. However, tumor volume of PC-3(MEAF6-1) xenografts increased significantly faster than control tumors Figure . Mice weThis study reports that a functionally reprogrammed MEAF6 gene alternatively spliced by neuronal splicing factor SRRM4 facilitates NEPC progression. Spliced by SRRM4, the MEAF6-1 variant does not mediate neuroendocrine differentiation of prostate cancer cells, but rather promotes cell proliferation and invasion to accelerate tumor growth. MEAF6-1 exerts these actions mainly via the ID1 and ID3 genes, which are well characterized inhibitors of cell differentiation as well as enhancers of cell proliferation and multipotency preservation . These fTo our knowledge, this is the first report that characterizes the biological functions of the MEAF6 gene with a particular focus on the actions of MEAF6 gene splicing and its role in NEPC progression. It is known that MEAF6 is a component of 4 different MYST family of HAT complexes in cells that modulate gene transcription through posttranslational modifications of histones . HoweverWe report that MEAF6-1 facilitates NEPC progression mainly by activating the ID1 and ID3 gene networks. The ID family of proteins are inhibitors of bHLH, ETS, and PAX transcription factors and RB family members which regulate cell differentiation . ID protIn summary, our studies demonstrate that SRRM4-mediated MEAF6-1 splicing is a facilitator for NEPC progression.VPC and Beltran cohort RNA-seq data was previously analyzed and published , 19. RNA2 at 37C.LNCaP, C4-2, PC-3, 22Rv1, DU145, NCI-H660, and VCaP cell lines were purchased from American Type Culture Collection . LNCaP95 (aka LN95) cells were a generous gift from Dr. Alan Meeker of Johns Hopkins University. Small cell lung cancer (SCLC) cells, NCI-H69 and -H82, were generously provided by Dr YZ Wang from the Vancouver Prostate Centre. 293T cells were generously provided by Dr. Paul Rennie and Dr. Ralph Buttyan from the Vancouver Prostate Centre. VCaP, PC-3, DU145 and 293T cells were cultured in DMEM with 10% fetal bovine serum (FBS), whereas LNCaP and 22Rv1 cells were cultured in RPMI-1640 medium with 10% FBS. LNCaP95 was maintained in phenol-free RPMI-1640 medium with 10% charcoal-stripped serum (CSS) (Hyclone). NCI-H660 was cultured in HITES medium  with 10% FBS. NCI-H69 and NCI-H82 suspension cells were maintained with RPMI-1640 medium supplemented with 10% FBS, 100 ug/ml streptomycin, and 100units/ml penicillin. Cells were all incubated in 5% COReal-time qPCR assays were performed as previously described , 35–37. Cells were transfected with Silencer Select (Ambion) siRNA targeting ID1 (siRNA ID: s7106) or ID3 (siRNA ID: s7110), or with Silencer Select Negative Control No. 1 siRNA (Ambion) using Lipofectamine 3000 (Invitrogen) according to manufacturer's protocol. Lipofectamine 3000 was also used for transient DNA plasmid transfections. Flag-SRRM4 expression vector was previously described ). Colony numbers were counted according to their diameters (100–300 um or > 300 um). Three independent biological replicates were performed for all the assays.Cell proliferation, migration, and invasion assays were described previously , 36. Brivs LNCaP(MEAF6-1), LNCaP(CTL) vs LNCaP(MEAF6-2), and LNCaP(MEAF6-1) vs LNCaP(MEAF6-2). Unpaired t-test with P value cut-off of 0.05, a fold change cut-off of 1.5, and Benjamini-Hochberg multiple testing correction were applied. LNCaP(CTL) vs LNCaP(MEAF6-1) transcriptomes were then uploaded and analyzed in IPA  for Pathway prediction algorithms.Gene microarray methods were described previously in detail . In summ6 cells per line) was implanted subcutaneously in bilateral flanks of 6–8-week old male nude mice (Nu/Nu). Tumor volume (V = length * width * high * 0.5236) was measured three times a week. All animal procedures were under the guidelines of the Canadian Council on Animal Care.To construct PC-3 xenografts, each PC-3 cell line (1 ×10t-test was carried out using GraphPad Prism (version 6) to determine differences between groups with the level of significance set at p < 0.05 as *p < 0.01 as **, and p < 0.001 as ***.All results are expressed as the mean ± SEM. One-way ANOVA or Student


Spodoptera frugiperda Sf21 and Trichoplusia ni Tn368 cells, whereas AcMNPV defective in hcf-1 (host cell-factor 1) gene productively infects only Sf21 cells, indicating that HCF-1 is indispensable for the AcMNPV productive infection of Tn368 cells. Here, we demonstrated that HCF-1 protein transiently expressed in Tn368 cells promotes the DNA synthesis of Hyphantria cunea MNPV (HycuMNPV), Orygia pseudotsugata MNPV and Bombyx mori NPV, which are normally unable to replicate in Tn368 cells. We also demonstrated that a recombinant HycuMNPV harboring the hcf-1 gene successfully replicates in Tn368 cells, generating substantial yields of progeny viruses and polyhedra. These results indicate that HCF-1 encoded by AcMNPV is an essential viral factor for productive NPV infection of Tn368 cells. Taken together with the previous findings on HRF-1 (host range factor 1), the present results provide strong evidence that viral genes acquired through horizontal gene transfer play an important role in baculovirus evolution, serving to expand the host range of baculoviruses.Baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) replicates in both  Baculoviridae, are large, enveloped, insect-pathogenic viruses that contain a double-stranded circular DNA genome of 80 to 180 kbp1. NPVs generally exhibit a narrow host range and are capable of productively infecting only one or a few closely related insect species. Notably, however, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), and Anagrapha falcifera and Rachiplusia ou MNPVs, which are close relatives of AcMNPV, are capable of infecting over 30 lepidopteran insect species within 12 families5.Nucleopolyhedroviruses (NPVs), which belong to members of the family p143, ie-2, lef-7 (late expression factor-7), p35, iaps (inhibitors of apoptosis), host range factor 1 (hrf-1), and host cell-specific factor 1 (hcf-1)8. The p143, ie-2, lef-7, and hcf-1 play a role in viral DNA replication or late gene expression, whereas the p35 and iaps are involved in an antiviral defense system of insects, serving as anti-apoptotic genes. These genes are differentially required for optimal viral replication in a cell-line specific manner. For example, recombinant AcMNPV defective in functional p35 gene normally replicates in Tn368 cells derived from the cabbage looper Trichoplusia ni, but induces massive apoptosis in Sf21 cells derived from the fall armyworm Spodoptera frugiperda, yielding little or no progeny viruses10. Similarly, AcMNPV harboring p143 gene derived from Bombyx mori NPV in place of its native p143 replicates successfully in non-permissive B. mori BmN-4 or Bm5 cells12. Notably, the HRF-1 and HCF-1 have been shown to contribute to AcMNPV replication exclusively in Lymantria dispar Ld652Y cells and Tn368 cells, respectively16. Despite the identification and characterization of these genes, the molecular mechanisms underlying the host range determination of NPVs remain largely elusive.Several viral genes that determine the host range of NPVs have been identified and include hcf-1 gene, however, productive infection only occurs in Sf21 cells, indicating that AcMNPV requires HCF-1 for the productive infection of Tn368 cells15. The HCF-1 protein contains a putative RING-finger domain, which is involved in the formation of functional HCF-1 dimers or higher-order structures in the nucleus of infected Tn368 cells18. It was also demonstrated that transiently expressed HCF-1 protein represses expression from the hcf-1 promoter and this repression activity of HCF-1 is required for the efficient expression of viral late genes and production of polyhedra in Tn368 cells18. However, the functional role of HCF-1 protein in AcMNPV-infected Tn368 cells has not been conclusively determined.AcMNPV replicates to high titers in both Sf21 and Tn368 cells. In recombinant AcMNPV defective in the hcf-1 gene also successfully replicated in Tn368 cells, indicating that hcf-1 gene embedded in the HycuMNPV genome functions effectively for HycuMNPV productive infection of Tn368 cells. In contrast, Orgyia pseudotsugata MNPV (OpMNPV) and Bombyx mori NPV (BmNPV) were capable of viral DNA replication in HCF-1-expressing Tn368 cells, but not the synthesis of viral structural or polyhedral proteins. Taken together, these results indicate that HCF-1 protein is an essential viral factor for the productive NPV infection of Tn368 cells, but is not sufficient to promote viral protein synthesis of certain NPVs in infected Tn368 cells.In the present study, we demonstrated that transiently expressed HCF-1 protein promotes the productive infection of non-permissive Tn368 cells by Hyphantria cunea MNPV (HycuMNPV). Recombinant HycuMNPV harboring the T. ni cells. Tn368 cells were first transfected with plasmids pFBD/hcf-1 and pFBD/luc, which express HCF-1 and luciferase proteins, respectively, under transcriptional control of the Drosophila melanogaster heat shock protein 70 (HSP70) gene promoter  or the luciferase gene (HycuBac/luc)  Fig.  to deteruc) Fig. . The lucuc) Fig. .Figure 3gp64 early promoter, which is occasionally observed upon infection with group I alphabaculoviruses even when inoculated viruses are unable to replicate productively. Immunoblot analysis showed that the luciferase and HCF-1 proteins were expressed at high levels from 24 to 72 h post-infection  genes, which is essential for viral DNA replication and expression of late genes in transient assays in Tn368, but not Sf21, cells14. In AcMNPV-infected Tn368 cells, the HCF-1 protein is synthesized early during infection and localizes in punctuate nuclear structures18. Recombinant AcMNPV defective in functional hcf-1 gene replicates normally in Sf21 cells, but exhibits various mutant phenotypes in Tn368 cells, including defective viral DNA replication and late gene transcription, and complete cessation of cellular and viral protein syntheses15. These results collectively imply that the HCF-1 protein is involved in viral DNA replication and late gene expression, as well as cellular protein synthesis, in AcMNPV-infected Tn368 cells. However, the present results also showed that although OpMNPV and BmNPV DNAs were synthesized successfully in HCF-1-expressing Tn368 cells, major capsid protein VP39 and polyhedrin were not detected, and neither BVs nor polyhedra were produced. Quantitative qRT-PCR analysis revealed that transcripts from the vp39 genes of HycuMNPV and OpMNPV, but not those of BmNPV or LdMNPV, increased markedly at 48 h post-infection in HCF-1-expressing Tn368 cells , and Clanis bilineata NPV, which is more distantly related to AcMNPV27, suggesting that the hcf-1 gene of AcMNPV was also acquired by a gene acquisition event. Notably, despite marked differences in structure and mode of action in virus-infected cells, HCF-1 and HRF-1 proteins have convergent functional roles in the host range expansion of NPVs to non-permissive cell lines derived from T. ni and L. dispar, respectively. Taken together with previous findings for the hrf-1 gene20, the present study indicates that several NPVs contain genes that are acquired by gene acquisition events and contribute to evolution of baculoviruses in terms of the host range expansion.The Trichoplusia ni28 were maintained at 28 °C in TC100 medium (AppliChem) supplemented with 0.26% tryptose broth (Sigma) and 10% fetal bovine serum (FBS). FRI-SpIm (SpIm) cells derived from Spilosoma imparilis29 were grown at 28 °C in MM medium30 supplemented with 3% FBS. The following clonal isolates of four NPVs were used in these experiments: BmNPV N9 (BmNPV) from the silkworm B. mori31, HycuMNPV N9 (HycuMNPV) from the fall webworm H. cunea32, LdMNPV A21-MPV (LdMNPV) from the gypsy moth L. dispar33, and OpMNPV from the Douglass-fir tussock moth, O. pseudotsugata34.Tn368 cells derived from 35. Infected Tn368 and SpIm cells were washed three times with TC100 and MM media, respectively, and used for analyses at the indicated time points. The time zero of infection was defined as the time when the viral inoculum was removed from the cell culture.Tn368 and SpIm cells were infected with viruses at varying multiplicity of infection (MOI) during a 60-min incubation at room temperature, as described previously36 was obtained by inverse PCR using primers P1 and P2 (see Supplementary Table hcf-1 open reading frame (ORF) was PCR-amplified from pHSHAHishcf-1VI+12 using primers P3 and P4, which contained 15-nt extension sequences complementary to the ends of linearized pFBD/pIE1-2/Egfp. The amplified hcf-1 ORF region was cloned into linearized pFBD/pIE1-2/Egfp using an In-Fusion HD Cloning Kit, yielding pFBD/pIE1-2/Egfp/pHS/HAHisHcf-1 (pFBD/hcf-1). Control plasmid pFBD/pIE1-2/Egfp/pHS/HAHisLuc (pFBD/luc) was constructed by replacing the hcf-1 gene of pFBD/hcf-1 with the luciferase gene that was amplified by PCR from pGL (Promega) using primers P5 and P6. hcf-1-defective linearized pFBD/hcf-1 was obtained by inverse PCR using primers P7 and P2. pFBD/hcf-1 and pFBD/luc were used as expression plasmids and for recombinant HycuMNPV bacmid construction. The construction of pIE1-2/Egfp, which expresses the EGFP protein, was described previously37. The plasmid sequences were confirmed by nucleotide sequencing using a BigDye Terminal v3.1 Cycle Sequencing Ready Reaction Kit and ABI 3130 Genetic Analyzer (Applied Biosystems), as described previously38.Plasmids pFBD/hcf-1 and pFBD/luc, which express HCF-1 and luciferase, respectively, under control of the HSP70 promoter  , yielding pHycuIE1-IE0. For construction of pHycuIE1-miniFKan-IE0, pHycuIE1-IE0 was linearized by inverse PCR using primers P10 and P11. The miniF-Kan region was PCR-amplified from AcMNPV bacmid DNA (Invitrogen) using primers P12 and P13, which contained 15-nt extension sequences complementary to the ends of linearized pHycuIE1-IE0. The obtained amplicon was cloned into linearized pHycuIE1-IE0 using an In-Fusion HD Cloning Kit, yielding pHycuIE1-miniFKan-IE0.HycuBac containing a miniF replicon, 40. Viral DNA was extracted from BVs and electroporated into DH10B Escherichia coli cells using a MicroPulser (BioRad). Positive transformants were selected on LB agar containing kanamycin (50 μg/ml), IPTG (24 μg/ml) and X-Gal (60 μg/ml). HycuBac DNA was isolated from LacZ-positive and kanamycin-resistant colonies using a Qiagen Plasmid Midi Kit (Qiagen). HycuBac was then transfected into SpIm cells and culture medium containing vHycuBac BVs was collected at 168 h post-transfection. Working stocks of vHycuBac were amplified in SpIm cells.pHycuIE1-miniFKan-IE0 was co-transfected with HycuMNPV genomic DNA into SpIm cells using Lipofectin (Invitrogen), as described previouslyhcf-1 (HycuBac/hcf-1) or luciferase gene (HycuBac/luc)  and HycuBac/luc (vHycuBac/luc) were prepared as described above for vHycuBac.Recombinant HycuBac harboring 41. Briefly, polypeptides from virus- and mock-infected cells were resolved on 12% SDS-polyacrylamide gels and blotted onto Immobilon-P (Millipore) or nitrocellulose membranes (Advantec Toyo) using a Trans-Blot Turbo system (Bio-Rad). GP64 and HA-tagged proteins (HCF-1 and luciferase) were probed with monoclonal antibodies against AcMNPV GP64 (Clontech) and HA , respectively, and were then reacted with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (Zymed Laboratories). VP39 and polyhedrin proteins were probed with polyclonal antibodies against BmNPV/AcMNPV VP3942 and BmNPV polyhedrin proteins35, respectively, and were then reacted with HRP-conjugated goat anti-rabbit IgG antibody (Zymed Laboratories). Positive signals were visualized using ECL Western Blotting Detection Reagent , ECL Select Western Blotting Detection Reagent  or an HRP Conjugate Substrate Kit (Bio-Rad).Immunoblot analyses of viral structural proteins, polyhedrin, and hemagglutinin (HA)-tagged HCF-1 and luciferase proteins were performed as described previouslyie1 gene copies of HycuMNPV (hycu-ie1), OpMNPV (op-ie1), BmNPV (bm-ie1) and LdMNPV (ld-ie1) was performed by qPCR analysis, as described previously36. Briefly, total DNAs were extracted from virus-infected Tn368 cells using a QIAamp DNA Mini Kit (Qiagen) and subjected to qPCR analysis using a StepOnePlus Real-Time PCR system (Applied Biosystems) with the gene-specific probes Pb-1, Pb-2, Pb-3 and Pb-4 . The plasmids were constructed by cloning each partial sequence, which was amplified using the primers P22 and P23 for hycu-ie1, P24 and P25 for op-ie1, P26 and P27 for bm-ie1, and P28 and P29 for ld-ie1, into EcoRV-digested pBlueScript KS+ using DNA Ligation Kit Mighty Mix (Takara) or into pCR4-TOPO using a TOPO TA Cloning Kit (Invitrogen).Quantification of vp39 genes of HycuMNPV (hycu-vp39), OpMNPV (op-vp39), BmNPV (bm-vp39) and LdMNPV (ld-vp39) was performed by qRT-PCR, as described previously40. Briefly, total RNAs were extracted from virus-infected Tn368 cells using a SYBR Green Cells-to-CT Kit (Ambion), treated with DNase I using an RNAqueous-4PCR Kit (Ambion) and reverse-transcribed to cDNAs using a SYBR Green Cells-to-CT Kit. The resultant cDNAs were subjected to qRT-PCR analysis using a StepOnePlus Real-Time PCR system with the gene-specific primers P30 and P31, P32 and P33, P34 and P35, and P36 and P37 for hycu-vp39, op-vp39, bm-vp39 and ld-vp39, respectively. The number of each vp39 mRNA was calculated using a standard curve generated from 10-fold dilutions of plasmids containing entire or partial sequences of each vp39 ORF . The plasmids were constructed by cloning each sequence using the primers P38 and P39 for hycu-vp39, P40 and P41 for op-vp39, P42 and P43 for bm-vp39, and P37 and P44 for ld-vp39 into pCR4-TOPO using a TOPO TA Cloning Kit.Quantification of mRNAs expressed from the 35. Briefly, monolayer cultures of these cells (0.5 × 106 cells) were prepared in 35 mm culture dishes  and inoculated with 200 μl of the virus suspensions to be titrated. After a 60 min adsorption period, the viral inoculum was replaced with 3 ml of 0.75% SeaPlaque agarose (FMC) in the medium appropriate for each cell line. The cultures were incubated at 28 °C for 5–6 days and formed plaques were counted under a microscope.Plaque assays were performed using SpIm cells for HycuMNPV, BM-N cells for BmNPV and Ld652Y cells for OpMNPV and LdMNPV, as described previously43.The transfection of SpIm and Tn368 cells with expression plasmids and bacmids was performed using Lipofectin (Invitrogen) and FuGENE (Promega), respectively, as described previouslyDataset 1


Furthermore, blockage of Shp2 by its specific inhibitor PHPS1 or by siRNA-mediated depletion was found to reduce the production of IL-25 in epithelial cells as well as the up-regulated LPS-triggered activation of JNK but not p38. To confirm the role of intra-bronchial epithelial Shp2 in OVA-induced allergic reaction, we generated CC10-rtTA/(tetO)7-Cre/Shp2f/f mice, where Shp2 was conditionally knocked out in bronchial epithelial cells. Surprisingly, specific deletion of Shp2 in bronchial epithelial cells showed a mild but insignificant effect on the expressions of epithelium-derived cytokines as well as TH2 and TH17 polarization following allergen-induced murine airway inflammation. Collectively, our data suggested that deletion of Shp2 impaired IL-25 production in bronchial epithelial cells in vitro, but might yet have minor influence on OVA-induced allergic reaction in vivo.Shp2 played an important role in cigarette-smoke-mediated inflammation, surfactant homeostasis and asthmatic airway remodeling. However, whether shp2 plays a key role in epithelium-associated allergic reaction is still unknown. In this study, LPS and OVA were observed to induce the production of IL-25 in bronchial epithelial cells  Asthma is a T-lymphocyte-controlled disease of the airway that is characterized with airway inflammation, overproduction of mucus and airway wall remodeling, leading to bronchial hyperreactivity and airflow obstruction. The rolTLR4 triggering on airway epithelial cells induces the production of innate pro-inflammatory cytokines, such as interleukin-25 (IL-25), interleukin-33 (IL-33), thymic stromal lymphopoietin (TSLP) and granulocyte-macrophage colony–stimulating factor (GM-CSF). These cShp2 (Src homology 2 domain-containing phosphatase 2) is a member of intracellular classical protein tyrosine phosphatases (PTPs). Recently, Shp2 had been shown to have extensive function in a wide variety of diseases. Of interest, Shp2 is known to be universally expressed in the lung. It was reported that Shp2 took an important role in cigarette-smoke-mediated IL-8 overproduction in bronchial epithelial cells and thatThis present study will determine the role of Shp2 in the production of epithelium-derived cytokines and the value of intra-bronchial epithelial Shp2 in allergic reaction.(Shp2f/f) mice (C57BL/6 background) were generous gifts from Dr. Gen-Sheng Feng ,,5], AlteActivation of TLR4 by LPS results in downstream cellular events that are mediated by MAPK p38 and JNK. To confirm that, serum-free Beas-2b cells were treated with different concentrations  of LPS for different periods of time . Phosphorylation levels of p38 and JNK were measured via immunoblotting. The results showed that LPS was powerful to activate MAPK p38 and JNK . Based oShp2 siRNA were used. Pretreated Beas-2b cells with PHPS-1 before LPS stimulation led to significant inhibition of the secretion of IL-25 in a concentration-dependent manner  stimulation. Results showed that Shp2 siRNA inhibited LPS induced phosphorylation of JNK but not that of p38 as well 7-Cre/Shp2f/f mice by crossing floxed Shp2 (Shp2f/f) with mice carrying the human CC10 promoter-rtTA and (tetO)7-CMV-Cre transgenes (CC10-rtTA/(tetO)7-Cre). We obtained four kinds of phenotypes: CC10-rtTA/(tetO)7-Cre/Shp2f/f, CC10-rtTA/Shp2f/f, (tetO)7-Cre/Shp2f/f and Shp2f/f. Triple transgenic mice CC10-rtTA/(tetO)7-Cre/Shp2f/f and littermate CC10-rtTA/Shp2f/f control mice were used for the experiments. When given doxycycline in drinking water, CC10-rtTA/(tetO)7-Cre/Shp2f/f mice expressed cre remonbinase in their bronchial epithelia cells to recognize the loxp/loxp sequence, leading to subsequent Shp2 inactivation 7-Cre/Shp2f/f mice. Then we obtained four genotypes as filial generation: Shp2f/f, CC10-rtTA/Shp2f/f, (tetO)7-Cre/Shp2f/f, and CC10-rtTA/(tetO)7-Cre/Shp2f/f (CC10-rtTA/(tetO)7-Cre/Shp2f/f mice showed that the Shp2 gene was disrupted when 2mg/ml DOX was administered to the mice for seven consecutive days (CC10-rtTA/(tetO)7-Cre/Shp2f/f mice after DOX exposure, and we found that Shp2Δ/Δ was not detectable in these organs , CC10-rtTA/(tetO)7-Cre/Shp2f/f:H2O (Shp2F/F) and CC10-rtTA/(tetO)7-Cre/Shp2f/f:DOX (Shp2△/△).Shp2 is universally highly expressed in lungs of healthy mice . Immunobtivation . For res/Shp2f/f . Genomicive days . Since we organs . Therefond Kopans, suggestF/F mice  and Genetic Control (Shp2F/F)   mice. ThF/F) mice  and Genetic Control (Shp2/F) mice . To furt/F) mice .in vivo. However, specific deletion of Shp2 in bronchial epithelial cells showed a mild but insignificant effect on the expressions of epithelium-derived cytokines as well as TH2 and helper TH17 polarization following allergen-induced murine airway inflammation. Collectively, our data suggest that Shp2 promote IL-25 production in bronchial epithelial cells via JNK activity in vitro but might yet have little influence in OVA-induced allergic airway inflammation in vivo.In the present study, our data indicated that LPS-contaminated OVA activated MAPK p38 and JNK, promoting IL-25 production in bronchial cells. We also found that blockage of Shp2 by its specific inhibitor PHPS1 or by siRNA-mediated depletion selectively down-regulated LPS-triggered activation of JNK rather than p38. We still determined that inhibition of Shp2 by its specific inhibitor PHPS-1 resulted in the reduction of IL-25 production in epithelial cells in vitro 7-Cre/Shp2f/f mice. Surprisingly, animal experiments showed specific knockout of Shp2 in bronchial epithelial cells have little effect on epithelium-related cytokine production in vivo. In other words, there are differences between data in vitro and in vivo. These differences are unlikely due to species differences in gene regulation between humans and mice, since we documented increased IL-25 in both the human bronchial cell line and primary mouse tracheal epithelial cells. Therefore, two points may aid understanding. Firstly, our previous work showed PHPS-1 alleviated eosinophilic airway inflammation and airway hyper-responsiveness as well as eosinophils in allergic wild-type mice, suggesting Shp2 is indeed involved in asthma pathogenesis. This present research aims to clarify whether intraepithelial Shp2 or non-epithelium-derived Shp2 play a role. Also, the findings suggest that not intraepithelial Shp2 but non-epithelium-derived Shp2 might be involved in airway inflammation[Along with our partners, we previously documented that Shp2 played an important role in acute cigarette-smoke-mediated IL-8 overproduction, loss oflammation. Secondllammation,24, alvelammation. Our datS1 FigSerum-free Beas-2bs were treated with different concentrations  of OVA for different periods of time . Phosphorylation levels of ERK, p38 and JNK were measured via immunoblotting.(TIF)Click here for additional data file.S2 Figp<0.001, n.s.p>0.05.(A) Serum-free Beas-2bs were treated with 20 ng/ml IL-13 for different periods of time . Phosphorylation levels of stat6 were measured via immunoblotting. (B) Beas-2bs were treated with 20 ng/ml IL-13 or 100 ng/ml LPS for 8 hours, supernatants were harvested, and IL-25 concentrations were measured through Elisa. (C) Shp2 siRNA were transfected into Beas-2bs, followed by LPS treatment 48 hours later. Cell total protein was extracted 30 min after LPS stimulation to detect the expression of P-stat6. ***(TIF)Click here for additional data file.S3 Fig(A) The cellular distribution of Shp2 (brown staining cells) in the lungs of healthy and allergic mice by immunohistology. (B) Total Shp2 protein expression in the lungs of healthy and allergic mice by immunoblotting. (C) Shp2 protein expression in MTECs that respectively isolated from healthy and allergic mice (immunoblotting).(TIF)Click here for additional data file.S4 FigCC10-rtTA/(tetO)7-Cre/Shp2f/f mice after 7-day treatment with DOX  and Shp2 allele was detected by PCR.Genomic DNA was isolated from the brain and liver of (TIF)Click here for additional data file.


Some of these major histocompatibility complex class I (MHC-I)-associated mutations result in reduction of in vitro viral replicative capacity. While these mutations can revert after viral transmission to MHC-I-disparate hosts, recent studies have suggested that these MHC-I-associated mutations accumulate in populations and make viruses less pathogenic in vitro. Here, we directly show an increase in the in vivo virulence of an MHC-I-adapted virus serially-passaged through MHC-I-mismatched hosts in a macaque AIDS model despite a reduction in in vitro viral fitness. The first passage simian immunodeficiency virus (1pSIV) obtained 1 year after SIVmac239 infection in a macaque possessing a protective MHC-I haplotype 90-120-Ia was transmitted into 90-120-Ia- macaques, whose plasma 1 year post-infection was transmitted into other 90-120-Ia- macaques to obtain the third passage SIV (3pSIV). Most of the 90-120-Ia-associated mutations selected in 1pSIV did not revert even in 3pSIV. 3pSIV showed lower in vitro viral fitness but induced persistent viremia in 90-120-Ia- macaques. Remarkably, 3pSIV infection in 90-120-Ia+ macaques resulted in significantly higher viral loads and reduced survival compared to wild-type SIVmac239. These results indicate that MHC-I-adapted SIVs serially-transmitted through MHC-I-mismatched hosts can have higher virulence in MHC-I-matched hosts despite their lower in vitro viral fitness. This study suggests that multiply-passaged HIVs could result in loss of HIV-specific CD8+ T cell responses in human populations and the in vivo pathogenic potential of these escaped viruses may be enhanced.CD8 + T-cell responses exert considerable control over replication of HIV and select for viral escape mutations. Recent studies have suggested that these major histocompatibility complex class I (MHC-I)-associated mutations accumulate in populations and make viruses less pathogenic in vitro. Other studies have shown that some of these escape mutations can revert after passage to MHC-I-disparate hosts. In an attempt to reconcile these apparently conflicting results, we serially passaged a virus isolate through MHC-I-mismatched hosts in the macaque AIDS model of simian immunodeficiency virus (SIV) infection. Here we show an increase in the in vivo virulence of an MHC-I-adapted virus despite a reduction in in vitro viral replication capacity. Only a few of the selected escape mutations reverted after transmission to MHC-I-disparate recipients. Results clearly showed that MHC-I-adapted SIVs that have been serially-transmitted through MHC-I-mismatched hosts can have higher in vivo virulence in MHC-I-matched hosts despite their lower in vitro viral fitness. This study suggests that HIVs may become less sensitive to CD8+ T cell responses and could have increased in vivo virulence by adaptation to MHC-I in human populations.CD8 Three A[+]E[–]P[–] macaques #411, #412, and #413 and three A[–]E[–]P[–] macaques #421, #422, and #423 were intravenously inoculated with 3pSIV1, whereas three A[+]E[–]P[–] macaques #414, #415, and #416 and three A[–]E[–]P[–] macaques #424, #425, and #426 were intravenously inoculated with 3pSIV2  consisting of 90-010-Ie+ (n = 6) and 90-120-Ib+ (n = 4) animals that were previously reported to show typical levels of viremia E[–]P[–]) macaques #421, #422, and #423, while 0.2 ml of 3pSIV2 was intravenously inoculated into three A[+]E[–]P[–] macaques #414, #415, and #416 and three A[–]E[–]P[–] macaques #424, #425, and #426 . Fragments of cDNAs encoding SIVmac239 (GenBank accession number M33262) Gag, Pol, Vif, Vpx, Vpr, Tat, Rev, and Nef were amplified from plasma RNAs by nested RT-PCR and subjected to direct sequencing by using dye terminator chemistry and an automated DNA sequencer (Applied Biosystems) as described before . Predomihttp://www.megasoftware.net/).For pyrosequencing, cDNA fragments corresponding to nucleotides (nt) 1760–2463 (containing entire Gag capsid [CA]-coding region), nt 5460–6340 (containing entire Vif-coding region), and nt 9257–10167 (containing entire Nef-coding region) were used for making fragmentation libraries using GS FLX Titanium Rapid Library Preparation Kit (Roche). The products were cleaned with Agencourt AMPure XP magnetic beads (Beckman Coulter) followed by quality control using Agilent 2100 Bioanalyzer (Agilent Technologies). Emulsion PCR was performed with GS junior Titanium emPCR Kit Lib-L (Roche). The emPCR products were deposited onto a GS Junior Titanium Pico Titer Plate and sequenced on the GS Junior System (Roche). Sequencing reads were analyzed by the GS Amplicon Variant Analyzer Software (Roche). After alignment of the FASTA files, populations of <1% were excluded. Molecular phylogenetic analyses were conducted by the Maximum Likelihood method using the MEGA6 software  (Roche) with stimulation by 2 μg/ml Phytohemagglutinin-L (Sigma) on the first 2 days. The culture supernatants on day 6 were added into HSC-F cells (a cynomolgus macaque T-cell line) [in vitro viral fitness with PBMC-derived and plasma-derived viruses. In addition, we constructed recombinant SIV clones by replacing the gag region (nt 1056–3408) in the wild-type pBRmac239 molecular clone with that amplified from plasma RNAs of macaques #31 and #32 at 1 year post-infection. We then obtained recombinant molecular clones whose gag had the predominant 3pSIV1 and 3pSIV2 sequences, respectively. COS-1 cells were transfected with these molecular clones to obtain recombinant SIV3p1gag and SIV3p2gag virus stocks. COS-1 cells were transfected with pBRmac239 to obtain the wild-type SIVmac239 stock used for comparison of in vitro viral fitness with these recombinant viruses, SIV3p1gag and SIV3p2gag. Titers of these virus stocks were measured by RT assay as described previously [in vitro replication capacity, HSC-F cells were infected with these viruses (5 x 104 HSC-F cells were infected with viruses having the same RT activity with the wild-type SIVmac239 corresponding to 0.2 ng of p27), and RT activity of the culture supernatants on day 4 post-infection was measured. In the competition assay for comparison of in vitro replication capacity of two kinds of viruses, HSC-F cells infected with individual virus stocks were cocultured to determine which viral genome sequences become dominant in the culture supernatants. HSC-F cells were infected with individual virus stocks  and their coculture started next day. Coculture was continued by transferring the culture supernatant into fresh HSC-F cells every 4 days. RNA was extracted from the coculture supernatant and the Gag CA (capsid)-coding region was sequenced. When only one viral sequence became dominant on day 2 after the coculture initiation, we confirmed that the virus became dominant even in the coculture of the virus-infected cells with larger numbers of the other virus-infected cells in which both viruses were equivalently detected on day 2.We recovered virus stocks from PBMCs and concentrated plasma samples obtained from macaques #11, #21, #22, #31, and #32 at 1 year post-infection. First, 1–5 x10ll line) , which wll line) . Then, tnt 1056–38 in the + T-cell responses by flow cytometric analysis detecting gamma interferon (IFN-γ) induction after specific stimulation as described previously [+ T-cell frequencies from those after antigen-specific stimulation. Specific CD8+ T-cell frequencies lower than 0.02% of CD8+ T cells were considered negative.We measured antigen-specific CD8eviously . Autologp values of < 0.05. Comparisons were performed by Mann-Whitney U-test or log-rank test.All statistical analyses were performed using Prism software  with significance set at S1 Figgag mutations shown in Frequencies of the (TIF)Click here for additional data file.S2 FigA, B) and Vif-coding  regions in macaques #11-#21-#31  and #11-#22-#32  are shown. The trees are drawn to scale, with branch lengths measured in the number of substitutions per site.NGS analyses were performed on SIV Gag CA- and Vif-coding cDNAs obtained from macaque #11 at 3 months (3M) and 1 year (1Y) post-infection and from macaques #21, #22, #31, and #32 at 1 week (1W), 3M, and 1Y post-infection. Phylogenetic analyses of NGS data on viral CA-coding ((TIF)Click here for additional data file.S3 FigA) and #11-#22-#32 (B) are shown. The trees are drawn to scale, with branch lengths measured in the number of substitutions per site.Phylogenetic analyses of NGS data on viral Nef-coding regions in macaques #11-#21-#31 ((TIF)Click here for additional data file.S4 Fig(A) or plasma-derived SIVs (B). PBMC-derived or plasma-derived virus-infected cells were cocultured with wild-type virus-infected cells to determine which viruses become dominant by detection of wild-type (Wt) or mutant (Mt) sequences in culture supernatant-derived viral gag cDNAs on day 16 (d16) after the coculture start. Representative results (on Gag CA residues at which compared viruses had different sequences) of two experiments on the coculture in which both kinds of viruses were equivalently detected on day 2 (d2) after coculture initiation are shown. In all competitions, the mutant sequences became undetectable on d16, indicating higher in vitro viral fitness of the wild-type SIV.Comparison between wild-type and PBMC-derived (TIF)Click here for additional data file.S5 Fig90-120-Ia+ (A) or 90-120-Ia- (B) macaques up to 3 months after WtSIV (black) or 3pSIV (red) infection are shown.To clearly see the acute phase portion of the data exhibited in (TIF)Click here for additional data file.S6 Fig90-120-Ia+ macaques other than #412 sharing 89-002-Ip-derived Mamu-B*007:02 with macaque #31 showed significantly higher setpoint viral loads (by Mann-Whitney U-test) (A) and shorter survival periods (by log-rank test) (B) than SIVmac239-infected.Even the five 3pSIV-infected (TIF)Click here for additional data file.


However in vitro, NVP-AST487 was superior to letrozole in inhibiting the GDNF-induced motility and tumor spheroid growth of MCF7-AROM1 cells and required in combination with letrozole to inhibit GDNF-induced motility in BT474-AROM3 aromatase expressing cells. These data indicate that inhibiting RET is as effective as the current therapeutic regimen of AI therapy but that a combination treatment may delay cancer cell dissemination and metastasis.The majority of breast cancers are estrogen receptor positive (ER+). Blockade of estrogen biosynthesis by aromatase inhibitors (AIs) is the first-line endocrine therapy for post-menopausal women with ER+ breast cancers. However, AI resistance remains a major challenge. We have demonstrated previously that increased GDNF/RET signaling in ER+ breast cancers promotes AI resistance. Here we investigated the efficacy of different small molecule RET kinase inhibitors, sunitinib, cabozantinib, NVP-BBT594 and NVP-AST487, and the potential of combining a RET inhibitor with the AI letrozole in ER+ breast cancers. The most effective inhibitor identified, NVP-AST487, suppressed GDNF-stimulated RET downstream signaling and 3D tumor spheroid growth. Ovariectomized mice were inoculated with ER+ aromatase-overexpressing MCF7-AROM1 cells and treated with letrozole, NVP-AST487 or the two drugs in combination. Surprisingly, the three treatment regimens showed similar efficacy in impairing MCF7-AROM1 tumor growth  In postmenopausal patients, aromatase inhibitors (AIs), which block the conversion of androgens to estrogens, are the first-line treatment choice -3-(trifluoromethyl)phenyl]-N'-[4-[[6-(methylamino)-4-pyrimidinyl]oxy]phenyl]-urea) and NVP-BBT594 (5-[[6-(acetylamino)-4-pyrimidinyl]oxy]-2,3-dihydro-N-[4-[(4- methyl-1-piperazinyl) methyl]-3-(trifluoromethyl)phenyl]-1H-indole-1-carboxamide) were provided by Novartis Pharmaceuticals, Basel (Switzerland). Cabozantinib and sunitinib were purchased from Tocris Bioscience.3 MCF7-AROM1 cells per well were seeded in 96-well ultra-low attachment plates in phenol red-free RPMI supplemented with 10% DCC FBS and 10 nM androstenedione [1 × 10enedione . After 24 cells were seeded per well and cultured overnight. The following day, cells were stimulated with GDNF (20 ng/mL) and treated with letrozole, NVP-AST487 or the combination as indicated. Medium was replaced every 3 days and colonies larger than 200 μm were counted at day 10.Growth factor reduced and phenol red-free Matrigel  was diluted 1:1 in PBS and 0.2 mL per well was plated into 24-well tissue culture dishes and left at 37°C for 2–3 h. Next, 1 × 10For motility assay, cells were serum starved overnight and seeded into the upper chamber of 8 μm-pore Transwells (Corning) in serum-free growth medium, with or without the indicated compounds. Cells were allowed to migrate towards complete growth medium for 48 hours. Non-moving cells were removed mechanically and the underside of the Transwells was stained with DiffQuick solution (BD Biosciences) as previously reported . Chemota7 MCF7-AROM1 cells were inoculated subcutaneously into ovariectomized female Ncr Foxhead nude 6- to 8-week-old mice (Harlan). Throughout the course of the experiment, mice were treated daily with androstenedione via intradermal injection (100 μg/day). Tumors were grown to ~8 mm diameter and then the mice were assigned to the following treatment groups. All treatments were given daily. Vehicle, 10% N-methyl-pyrollidone (NMP)/90% polyethylene glycol (PEG300); letrozole, 1 mg/kg in 150 μL of 10% NMP/90% PEG300; NVP-AST487, 50 mg/kg/d; combination, combination of letrozole and NVP-AST487. Tumor growth was assessed by caliper measurements of the two largest tumor diameters. Volumes were calculated according to the formula: a×b2×π/6, where a and b are orthogonal tumor diameters. All animal work was carried out with UK Home Office approval.MCF7-AROM1 xenografts were established as previously described . In brieFor RET immunohistochemistry (IHC), antigen retrieval was carried out by microwave irradiation for 5 minutes at full power (900 W) in citrate buffer pH 6.0. Anti-Ret (E1N8X) XP antibody  was optimized to give robust IHC staining of formalin-fixed paraffin-embedded material and was applied at 1/100 dilution see . The staStatistical analysis was conducted using GraphPad Prism Software as reported in the figure legends and Results.


However, the relationship between cytoplasmic aggregate formation and cell death remains unclear. In the present study, rat neural stem cell lines stably transfected with EGFP- or Sirius-expression vectors under the control of tubulin beta III, glial fibrillary acidic protein, or 2′,3′-cyclic nucleotide 3′-phosphodiesterase promoter were differentiated into neurons, astrocytes, and oligodendrocytes, respectively, in the presence of retinoic acid. The differentiated cells were then transduced with adenoviruses expressing DsRed-tagged human wild type and C-terminal fragment TDP-43 under the condition of proteasome inhibition. Time-lapse imaging analyses revealed growing cytoplasmic aggregates in the transduced neuronal and glial cells, followed by collapse of the cell. The aggregates remained insoluble in culture media, consisted of sarkosyl-insoluble granular materials, and contained phosphorylated TDP-43. Moreover, the released aggregates were incorporated into neighboring neuronal cells, suggesting cell-to-cell spreading. The present study provides a novel tool for analyzing the detailed molecular mechanisms of TDP-43 proteinopathy in vitro.TAR DNA-binding protein 43 (TDP-43) is a main constituent of cytoplasmic aggregates in neuronal and glial cells in cases of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We have previously demonstrated that adenovirus-transduced artificial TDP-43 cytoplasmic aggregates formation is enhanced by proteasome inhibition  TARDBP, a DNA/RNA-binding protein that localizes predominantly to the nucleus and shuttles between the cytoplasm and nucleus  and centrifuged at 100,000 × g for 30 min at 20°C. The supernatants were collected as urea soluble fraction (termed U). Protein concentration of each fraction was determined with BCA Protein Assay kit .For RIPA/urea fractionation, cells were lysed in RIPA buffer  containing phosphatase inhibitors as above, sonicated and incubated for 30 min on ice followed by centrifugation at 100,000 × Ten—fifteen micrograms of cell lysate were electrophoresed on 10% SDS/polyacrylamide gels under reduced conditions and transferred to PVDF membrane . The blotted membrane was blocked with 5% skim milk and incubated overnight with rabbit anti-TDP-43 C-terminus (405–414) or phospho-TDP-43 (pSer409/S410) antibodies  at dilutions of 1:3,000, followed by incubation with HRP-conjugated anti-rabbit IgG . Reactions were visualized by enhanced chemiluminescence detection using an ECL Western blotting detection kit  and the images were captured and modified with Ez-Capture (ATTO).Cells were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS), permeabilized with 100% methanol, washed with PBS, and immunostained overnight at 4°C with the following primary antibodies at 1:200 dilutions; mouse monoclonal TuJ1 , rabbit anti-phosphoTDP-43 (pSer409/410) (Cosmo Bio), rabbit anti-TDP-43 C-terminus , rabbit anti-GFP (Abcam), rabbit anti-GFAP , rabbit anti-ubiquitin (DAKO) and rabbit anti-CNP . The cells were then incubated with Alexa Fluor 488- or 633-conjugated goat anti-rabbit or anti-mouse antibodies (Thermo Fisher) at 1:400 dilutions for 45 min at room temperature, followed by incubation for 15 min with 2 μg/mL Hoechst 33342 (Thermo Fisher). After washings, coverslips were mounted on glass slides with Gelvatol . Immunostained cells were examined under an AX80TR microscope equipped with a DP70 CCD camera (Olympus) or a TCS SP8 confocal microscope .For electron microscopic analysis, cells on coverslips were fixed with 3% glutaraldehyde in 0.1 M PBS. After washings, cells were post-fixed with 1% osmium tetroxide in 0.1 M phosphate buffer, pH 7.4, dehydrated through graded ethanol steps, and embedded in Epon 812. The coverslips were peeled off and ultrathin sections were horizontally made, stained with uranyl acetate and lead citrate, and examined under a Hitachi H-7650 electron microscope.2 to start running the program. Prior to time-lapse imaging, cells were fed with new medium with or without 0.5 μM MG-132. In some experiments, cells were co-transduced with AxshNC/EGFP or AxshPSMC1/EGFP in place of MG-132 treatment. The dish was immediately set on a microscopic stage and the object areas were determined with the aid of EGFP (excitation at 475/28 nm and emission at 525/48 nm) and DsRed (excitation at 542/27 nm and emission at 597/45 nm) fluorescence observation. Sirius, one of the blue fluorescent proteins that have maximum excitation at 355 nm and maximum emission at 425 nm with improved photostability [Time-lapse live cell fluorescence imaging was performed using a DeltaVision microscopy system . The 1464RTBB3pEGFP or 1464RTBB3pSirius cells were seeded and differentiated on a PLL-coated 35-mm glass bottom dish  for 2 days and transduced with AxDsR-WT.TDP43, AxEGFP-WT.TDP43, AxDsR-CTF.TDP43, and/or AxEGFP-CTF.TDP43 for another 2 days. Two hours before imaging, incubation chambers were pre-warmed to 37°C and filled with 5% COtability , was cap5 cells/well), transduced with AxDsR-WT.TDP43 and AxDsR-CTF.TDP43, and subsequently treated with MG-132. The cells were rinsed twice with pre-chilled PBS and harvested by cell scraper. Collected cells were sonicated and the cell lysates corresponding to 2 × 104 transduced cells were inoculated onto 2 × 104 non-treated differentiated cells derived from 1464R cells on coverslips. Cells were fixed after 48 hrs and processed for immunocytochemistry as described above.To examine spreading of naked TDP-43 aggregates to naive cells, neuronal and glial cells differentiated from 1464R NSCs were cultured on PLL-coated 6-well plates (4 × 104) on coverslips. Cells were fixed after 48 hrs for immunocytochemistry.To examine cell-to-cell spreading of TDP-43 aggregates, differentiated 1464R cells that had been inoculated with sonicated cell lysates containing TDP-43 aggregates as described above were washed and seeded on naive differentiated 1464RTBB3pEGFP cells on coverslips. In some experiments, differentiated 1464R cells were treated with adenoviruses and MG-132, thoroughly washed in medium, and seeded on naive differentiated 1464RTBB3pEGFP cells (1 × 103) were treated with adenoviruses and MG-132, thoroughly washed in medium, dissociated, and overlayed on the differentiated 1464REGFPWT.TDP43 cells (3 × 104) maintained on coverslips. Cells were fixed after 3 or 5 days for immunocytochemistry. DsRed fluorescence and maximum fluorescence in TuJ1- or EGFP-positive area were quantified with ImageJ software .To examine the seeding activity of the aggregates, differentiated 1464R cells . Measured results were analyzed with two-tailed test and significance level was set at p<0.05. All results were analyzed and graphed with Prism 7 software .S1 Figa-d) Differentiated neurons were transduced with AxDsR-WT.TDP43 (DsR-WT)  or AxDsR-CTF.TDP43 (DsR-CTF)  (red) followed by the treatment with DMSO  or 0.5 μM MG-132 . Fixed cells were immunostained with phosho-TDP-43 (pS409/S410) (green) and TuJ1 (white), and counterstained with Hoechst 33342 (blue). Arrows indicate cytoplasmic aggregates. Scale bar = 20μm.((TIF)Click here for additional data file.S2 Figa-f) Differentiated neurons were transduced with AxEGFP-WT.TDP43 (EGFP-WT) , AxEGFP-CTF.TDP43 (EGFP-CTF)  or both  (green) followed by the treatment with DMSO (a-c) or 0.5 μM MG-132 (d-f). Fixed cells were immunostained with phosho-TDP-43 (pS409/S410) (green) and TuJ1 (white) and counterstained with Hoechst 33342 (blue). Arrows indicate cytoplasmic aggregates. Scale bar = 20μm.((TIF)Click here for additional data file.S3 Figa-c) Differentiated neurons were transduced with AxDsR-WT.TDP43 and AxDsR-CTF.TDP43 (DsR-(WT+CTF)) (red) followed by the treatment with DMSO (a), 0.5 μM MG-132 (b), or 1 μM lactacystin (c) for 24 hrs. Fixed cells were immunostained with phosho-TDP-43 (pS409/S410) (green) and TuJ1 (white) and counterstained with Hoechst 33342 (blue). Arrows indicate cytoplasmic aggregates. Scale bar = 20μm. (d-e) Differentiated neurons transduced with AxDsR-WT.TDP43 and AxDsR-CTF.TDP43 (DsR-(WT+CTF)) were treated with DMSO, 0.5 μM MG-132, 1 μM lactacystin, 0.1 μM epoxomicin, or 26 μM ALLN for 24 hrs. Sarkosyl soluble (S) and Sarkosyl insoluble (P) fractions were immunoblotted with antibodies for phosho (p)-TDP-43 (pS409/S410) (d) or TDP-43 (405–410) (e). The 72 and 50 kDa bands correspond to non-phosphorylated DsRed-tagged WT and CTF TDP-43, respectively. TDP-43 antibody also detects endogenous rat TDP-43 (arrowhead).((TIF)Click here for additional data file.S4 Figa) Time-lapse imaging of AxEGFP-WT.TDP43 and AxEGFP-CTF.TDP43 (EGFP-(WT+CTF); gray scale in top panel and green in bottom panels, respectively)-transduced 1464RTBB3pSirius neurons in the presence of MG-132. Cytoplasmic TDP-43 aggregates (arrow) are formed and remained in the insoluble material after cell collapse. See also b) Time-lapse imaging of AxDsR-WT.TDP43  and AxEGFP-CTF.TDP43 -transduced with 1464RTBB3pSirius neurons in the presence of MG-132. Cytoplasmic TDP-43 aggregates shows both EGFP and DsRed fluorescence (arrow). Scale bar = 20 μm. See also ((TIF)Click here for additional data file.S1 MovieImages were captured by every 15 min. The obtained serial images were converted into movie with 5 frames/second. Scale bar = 15 μm.(MP4)Click here for additional data file.S2 MovieImages were captured by every 15 min. The obtained serial images were converted into movie with 5 frames/second. Scale bar = 15 μm.(MP4)Click here for additional data file.S3 MovieImages were captured by every 20 min. The obtained serial images were converted into movie with 5 frames/second. Scale bar = 15 μm.(MP4)Click here for additional data file.S4 MovieImages were captured by every 15 min. The obtained serial images were converted into movie with 5 frames/second. Scale bar = 15 μm.(MP4)Click here for additional data file.S5 MovieImages were captured by every 15 min. The obtained serial images were converted into movie with 5 frames/second. Scale bar = 15 μm.(MP4)Click here for additional data file.S6 MovieImages were captured by every 15 min. The obtained serial images were converted into movie with 5 frames/second. Scale bar = 15 μm.(MP4)Click here for additional data file.


Bacillus velezensis strain S141 is a plant growth-promoting rhizobacterium isolated from soybean (Glycine max) rhizosphere that enhances soybean growth, nodulation, and N2 fixation efficiency by coinoculation with Bradyrhizobium diazoefficiens USDA110. The S141 genome was identified to comprise a 3,974,582-bp-long circular DNA sequence encoding at least 3,817 proteins. Bacillus species  are naturally occurring soil bacteria that aggressively colonize plant roots for better plant growth . Various species . Severalroducers , 3. BaciB. velezensis S141 genomic DNA was prepared using the Wizard Genomic DNA purification kit , and a genomic DNA library was constructed using the NEBNext ultra DNA library prep kit for Illumina  according to the manufacturer’s protocol. B. velezensis S141 genome sequencing was performed on an Illumina MiSeq platform. After quality filtering, 11,675,054 high-quality paired-end reads (2 × 300 bp) were assembled using CLC Genomics Workbench version 10.0.1 . Total read coverage was 300-fold. After trimmed (paired) assembly using the CLC Genomics Workbench, a draft genome with 19 scaffolds was obtained. Gaps between scaffolds were closed using PCR and Sanger sequencing to obtain the complete sequence of the circular chromosome. The genome sequence was automatically annotated using the Microbial Genome Annotation Pipeline (MiGAP)  genome sequence  (ipdC and dhaS encoding indole-3-pyruvate decarboxylase and indole-3-acetaldehyde dehydrogenase, respectively, to synthesize IAA from indole-3-pyruvic acid; putative iaaH encoding indole-3-acetamide hydrolase to synthesize IAA from indole-3-acetamide; and yhcX encoding nitrilase to synthesize IAA from indole-3-acetonitrile. Additionally, we found ysnE, which putatively encodes IAA transacetylase involved in the tryptophan-independent IAA biosynthesis pathway. Thus, we suggest that B. velezensis S141 possesses multiple genes that are functionally related to auxin production and play key roles in its ability to promote plant growth.The B. velezensis S141 genome sequence was deposited in DDBJ/EMBL/GenBank under accession number AP018402.


Several studies have documented changes in oxidative parameters and antioxidant enzymes in patients with schizophrenia . However, their relation to negative symptoms and the longitudinal clinical course is still unclear.The objectives of the present study are to: 1) analyze the association between oxidative stress biomarkers and negative dimension; 2) identify if these biomarkers could predict clinical outcomes in stable patients with schizophrenia at 1-year follow-up.A 1-year follow-up study of 57 stable outpatients with schizophrenia (≤10 years of illness) .PANSS, Clinical Assessment Interview of Negative Symptoms (CAINS) -Motivation/Pleasure (MAP) & Expression (EXP) domains-, Brief Negative Symptom Scale (BNSS). Oxidative stress biomarkers: homocysteine, hemolysis test (% hemolysis), lipid peroxidation subproducts (LPO), catalase activity in erythrocytes (CAT).Pearson correlations were performed to determine associations between biomarkers and clinical scores at baseline, and they were included in stepwise multiple linear regression analyses, considering potential confounding factors.The clinical course for each psychopathological domain was determined using the formula: [follow-up-baseline scores]. Positive values were interpreted as worsening, while negative improvement. Pearson correlation and multiple linear regression analyses were performed to determine if baseline levels of oxidative stress parameters were predictors of clinical changes at follow-up.1) Baseline associations: Final regression models identified that LPO level was a significant predictor of lower scores in PANSS-N, BNSS total, Avolition and Blunted Affect subscale of BNSS and CAINS-EXP .2) Longitudinal course: At 1-year follow-up, patients only improved significantly (p<0.05) in PANSS-Total [59.4 ± 16.4 - 54.5 ± 16.0 (t=3.362)], PANSS-General [29.7 ± 8.9 - 26.9 ± 7.9 (t=3.362)], Blunted Affect subscale [6.9 ± 5.0 - 5.9 ± 4.7 (t=2.489)], and almost significant (p<0.069) in CAINS-EXP and BNSS total score. No significant changes in BMI, waist circumference, smoking or antipsychotic equivalent doses were detected, but they were also considered in regression analyses. A higher percentage of hemolysis at baseline, with a decrease in equivalent doses of antipsychotics, both significantly predict an improvement in scores of PANSS-N , BNSS  and CAINS-EXP .Lower concentrations of LPO were related to greater severity of negative symptoms as avolition and blunted affect (inner world). Longitudinal analyses showed that higher % of hemolysis at baseline predict an improvement of negative dimension at 1-year follow-up. From our results, we hypothesize that there is an inverse relationship between oxidative stress and negative dimension in stable patients with schizophrenia during the first ten years of illness.