In particular, the phenylalanine-glycine (FG) repeat containing cytoplasmic filament nucleoporin NUP214, and transport receptor transportin-1 (TNPO1) were consistently required for full MX2, and interferon-mediated, anti-viral function. Both proteins were shown to interact with the triple arginine motif, and confocal fluorescence microscopy revealed that their simultaneous depletion resulted in diminished MX2 accumulation at the nuclear envelope. We therefore propose a model whereby multiple components of the nuclear import machinery and nuclear pore complex help position MX2 at the nuclear envelope to promote MX2-mediated restriction of HIV-1.Human myxovirus resistance 2 (MX2/MXB) is an interferon-induced post-entry inhibitor of human immunodeficiency virus type-1 (HIV-1) infection. While the precise mechanism of viral inhibition remains unclear, MX2 is localized to the nuclear envelope, and blocks the nuclear import of viral cDNAs. The amino-terminus of MX2 (N-MX2) is essential for anti-viral function, and mutation of a triple arginine motif at residues 11 to 13 abrogates anti-HIV-1 activity. In this study, we sought to investigate the role of N-MX2 in anti-viral activity by identifying functionally relevant host-encoded interaction partners through yeast-two-hybrid screening. Remarkably, five out of seven primary candidate interactors were nucleoporins or nucleoporin-like proteins, though none of these candidates were identified when screening with a mutant RRR11-13A N-MX2 fragment. Interactions were confirmed by co-immunoprecipitation, and RNA silencing experiments in cell lines and primary CD4 The movement of large molecules into the cell nucleus is regulated at specific sites within the nuclear envelope termed nuclear pores. To infect cells productively, human immunodeficiency virus type-1 (HIV-1) must traverse the nuclear envelope to enable integration of the viral DNA into the genomic DNA of host cells. We, and others, have previously identified a cell-encoded protein, human myxovirus resistance 2 (MX2), which is expressed upon initiation of an innate immune response and prevents accumulation of HIV-1 DNA within the nucleus, thus imposing a block to infection. Here, we reveal that components of the nuclear pore complex, and nuclear import machinery, are required for MX2-dependent inhibition of HIV-1 infection. We show that MX2, which is localized at the cytoplasmic face of the nuclear envelope, interacts with multiple protein components of the nuclear pore complex, as well as transport receptor transportin-1, via a functionally required triple arginine motif at its amino-terminus. We speculate that these interactions facilitate MX2-mediated inhibition of HIV-1 nuclear import by situating the protein at the nuclear envelope. However, the relationship between CA binding and viral suppression is not straightforward, since mutations in CA that permit escape from viral inhibition do not prevent CANC nanotube interactions with MX2 [Human myxovirus resistance 2 (MX2/MXB) is an interferon-stimulated gene (ISG), and a key contributor to the type-1 interferon-induced post-entry inhibition of human immunodeficiency virus type-1 (HIV-1) infection . The MX2with MX2 –7.in vitro [et al, higher-order oligomerization of MX1 was found to be essential for anti-viral activity against IAV [Human MX2 is a large dynamin-like GTPase, and is most closely related to human MX1/MXA. MX1 has long been established as a potent interferon-induced restriction factor for a range of DNA and RNA viruses including influenza A virus (IAV), though it does not inhibit retroviruses such as HIV-1 , 9. Indein vitro . In a reinst IAV . Howeverinst IAV , 13, 16.MX2 possesses an extended N-terminal domain that distinguishes it from MX1, and exists as two isoforms owing to the presence of an internal initiation methionine codon at position 26. However, only the longer 78 kDa isoform displays anti-HIV-1 activity and is associated with the nuclear envelope (NE); the shorter 76 kDa isoform is cytoplasmic (as is human MX1), and is not anti-viral. Indeed we, and others, have previously shown that the N-terminal 91 amino acids of full-length MX2 are required for anti-viral activity , 18, andTo address the role of the N-terminal domain in MX2 function, we therefore sought to identify biologically relevant host-encoded binding partners using yeast two-hybrid (Y-2-H) screening. Strikingly, most of the candidate interactors with the wild-type N-terminal sequence were nucleoporins (NUPs) or nucleoporin-like proteins. We then determined the functional importance of these interactions using a combination of biochemical, genetic and virological experiments: specifically, we confirmed these interactions through co-immunoprecipitation assays, and used RNA silencing-mediated depletion to demonstrate that multiple components of the nuclear pore complex (NPC) and the nuclear import machinery promote MX2-mediated inhibition of HIV-1 infection.A yeast two-hybrid screen was performed to identify candidate interaction partners with the MX2 N-terminal domain (N-MX2). The N-terminal 91 amino acids of wild-type MX2, or mutant RRR11-13A MX2, were employed as bait fragments to screen against prey fragments from a human leukocyte cDNA library. Candidate interactors were assigned a Predicted Biological Score (PBS) . CandidTo validate these interactions, co-immunoprecipitation analyses were performed by co-expression of hemagglutinin (HA)-tagged wild-type MX2 or RRR11-13 mutant MX2 and FLAG-tagged candidate interactors in 293T cells, HA-specific immunoprecipitation, and immunoblot detection of associated FLAG-tagged proteins . These dNUP358, NUP214, NUP98 and NUPL2 have been described as cytoplasmic filament nucleoporins  and have+ CXCR4+ cells using the doxycycline-inducible EasiLV lentiviral vector system [We investigated whether these candidates, as well as selected other nuclear import factors and NPC components, are functionally linked with MX2 anti-viral activity. A focused siRNA “screen” incorporating other nucleoporins previously implicated in HIV-1 infectivity such as NUP153 , 33, NUPr system , 17, 19.Depletion of a number of nucleoporins  increaseDue to the observed lack of impact of nucleoporin knockdown on infection of U87-MG cells, we asked whether functional redundancy between nucleoporin-dependent HIV-1 nuclear import pathways in these cells may have occluded the detection of MX2-dependent infection phenotypes. We therefore investigated the nucleoporin requirements for MX2 inhibition in a second cell line, selecting HeLa cells since many previous studies relating to HIV-1 nuclear import have been conducted in this line. Human MX2 or CD8 (negative control) was overexpressed in HeLa cells using a puromycin selectable lentiviral vector system. Puromycin-selected transduced cells were treated with the same targeted siRNA pools used in the previous experiments prior to challenge with HIV-1/GFP, and monitoring of transduction efficiency was performed by flow cytometry .In notable contrast to the observations in U87-MG cells , depletiWe next wished to confirm whether endogenous FG-nucleoporins interact with IFN-induced endogenous MX2. U87-MG cells were first treated with either a non-targeting control siRNA, or anti-NUP214 siRNA followed by the addition of IFNα for 24 h to induce MX2 expression. Immunoprecipitation with mab414, a mouse monoclonal antibody which recognizes FG-repeat regions within multiple nucleoporins including NUP358, NUP214, NUP153 and NUP62 , was theSince NUP214 and TNPO1 were required for full MX2-mediated HIV-1 inhibition in both U87-MG cells and HeLa cells, potential interactions between these proteins and MX2 were explored further. A series of co-immunoprecipitation experiments were performed using 293T cells transfected with HA- and FLAG-tagged constructs, HA-specific immunoprecipitation, and immunoblot detection of associated FLAG-tagged proteins . TNPO1 i+ CXCR4+ derived cell line was engineered where the MX2 alleles were inactivated using CRISPR-Cas9 genome editing, and the effect of NUP214 and/or TNPO1 depletion by siRNA treatment was analyzed. As a control, we used siRNAs targeting MX2, as well as a CRISPR-Cas9 control cell line (CTRL CRISPR). As expected, IFNα treatment of CTRL CRISPR cells resulted in a severe reduction in the percentage of infected cells  was substantially reduced when compared to the CTRL CRISPR cells  of NUP214 and TNPO1 in MX2 in anti-viral activity using a more physiologically relevant model, we tested the effect of depleting these two proteins, as well as MX2, in primary CD44 donors . Upon ta4 donors . These dHuman MX2 localizes at the NE , 39; we In seeking to define host co-factors required for MX2 inhibition of HIV-1, we started by performing an unbiased Y-2-H screen using the functionally essential N-terminal domain as bait. Remarkably, most of the candidate interactors with wild-type N-MX2 were members of a specific subfamily of nucleoporins, the cytoplasmic filament FG-nucleoporins  or FG-repeat containing nucleoporin-like proteins also associated with the cytoplasmic face of the NE  ; Fig 7. + T cells  , 25, 42.in vitro evidence indicates that such mutant CA proteins retain the ability to interact with MX2 [+ MT4 cells where CA mutant viruses that have lost the ability to interact with CYPA remain MX2 resistant [The pathway(s) through which nuclear import of HIV-1 proceeds remains unclear, as do the molecular details of inhibition by MX2. There is evidence to suggest that HIV-1 CA is a key specificity determinant in both processes . Certainwith MX2 , 7. CYPAwith MX2  but not esistant . Many ofesistant , 33. Theesistant , with muesistant . However2-5-PY), no clear role for it in HIV-1 suppression is suggested since the inactive RRR11-13 mutant is still capable of binding to TNPO1  in some cell types. Nevertheless, in light of their known functions and sites of localization , we propOnce localized at the NPC, MX2 is presumably then positioned to inhibit the nuclear import of HIV-1 nucleoprotein replication complexes , 18, 20,et al., reported the use of RNA silencing to show that several nucleoporins impact HIV-1 infection as well as MX2 anti-viral function [During the review of this work, Kane function . Our res+ CXCR4+ cell line has been described [+ T cells were obtained through the Infectious Diseases BioBank at King’s College London (ethics reference MM2-220518) and were isolated from peripheral blood mononuclear cells (PBMCs) of anonymous healthy volunteer donors with written consent under overall permission from the Southampton and South West Hampshire Research Ethics Committee (B) . CD4+ T cells were purified by density gradient centrifugation using LymphoPrep (Axis-Shield) and isolated by negative selection using the CD4+ T Cell Isolation Kit (Miltenyl Biotec) following manufacturer’s instructions. Activation of the cells was achieved using Dynabeads Human T-Activator CD3/CD28 (ThermoFisher) and 50 U/ml recombinant IL-2 (rIL-2) (Roche) for 48 h in medium consisting of RPMI 1640-GlutaMAX containing 10% heat-inactivated autologous serum, 100 U/ml penicillin and 100 U/ml streptomycin. After activation, cells were maintained in medium containing 30 U/ml of rIL-2. IFNα-2b  was added to cultures for 24 h prior to infection, as indicated.Human 293T cells, HeLa cells and parental U87-MG cells were obtained from the American Tissue Culture Collection (ATCC). The generation of the U87-MG CD4escribed . All celantisense.WPRE) encoding MX2-FLAG or Photinus pyralis luciferase-FLAG; lentiviral vectors conferring puromycin selection (LV-puromycinR) (pRRL.sin.cPPT.CMV/CDS-IRES-puromycinR.WPRE) constitutively expressing CD8-FLAG or MX2-FLAG; and the NL4-3/nef-IRES-Renilla reporter virus  have been described [Construction of pCAGGS based expression constructs encoding FLAG- or HA-tagged GFP, MX1 or MX2 has been described previously , 17. pCAescribed .www.hybrigenics-services.com). Amino acids 1 to 91 of human MX2 (N-MX2) were used as bait to screen against a human leukocyte cDNA library. Predicted interactions with native N-MX2 were compared to those identified using mutant RRR11-13A N-MX2 as a bait domain. Interactions were awarded a Predicted Biological Score (PBS) to assess the reliability of each interaction, primarily based on the number of independent prey fragments identified for each interaction compared to the probability of random selection [A yeast two-hybrid screen to probe for interaction partners with the N-terminal domain of MX2 was performed by Hybrigenics Services ) by Dounce homogenization in experiments with PNRC1, KLHL6 and hRIP; or isotonic lysis buffer  and lysed by sonication (20 s) for all other experiments. Lysates were cleared by centrifugation at 1,500 × g for 10 min, and for hypotonic lysates, KCl and Triton X-100 were added at final concentrations of 100 mM and 0.3%, respectively. A sample from each whole cell lysate was saved in order to confirm protein expression levels. HA-tagged proteins were immunoprecipitated using anti-HA magnetic beads (Pierce) for 2 h at 4°C, or FLAG-tagged proteins were immunoprecipitated with anti-FLAG (M2) magnetic beads (Sigma) under the same conditions. Beads were washed a further 4 times in wash buffer for hypotonic lysates  or isotonic lysis buffer for isotonic lysates before the addition of sample buffer . HA-, FLAG- and T7 tagged proteins were resolved on 10% acrylamide gels by SDS-PAGE and detected by immunoblotting using HRP-conjugated anti-HA , anti-FLAG , or anti-T7 antibody . Images were visualized by chemiluminescence  on a LI-COR Odyssey Fc imaging system (LI-COR) and band intensities of input and precipitated proteins were quantified using ImageJ software.+ CXCR4+ cells were transfected twice, 24 h apart, with siRNAs targeting NUP214 (Dharmacon siGENOME smartpool) or a non-targeting control siRNA (Dharmacon). After 72 h, cells were treated with IFNα (500 U/ml) for 24 h to induce MX2 expression, prior to harvesting. Cells without IFN were included as a negative control for MX2 induction. Harvested cells were lysed in isotonic lysis buffer, sonicated, and the lysate cleared as described previously. FG-nucleoporins were immunoprecipitated with mouse monoclonal antibody mab414 [For co-immunoprecipitation of endogenous FG-nucleoporins and IFN-induced MX2, U87-MG CD4y mab414  conjugat+CXCR4+ cells, ectopic expression of MX2 was achieved by transduction of cells with doxycycline-inducible EasiLV lentiviral vectors encoding FLAG-tagged human MX2 (or a FLAG-tagged luciferase control construct) for 6 h. After a rest period of 48 h, transduced cells were transfected twice, 24 h apart, with a panel of siRNAs  at 20 nM concentration using Lipofectamine RNAiMAX reagent (Invitrogen). After 16 h, transgene expression was induced by the addition of 0.5 μg/ml doxycycline for 72 h, prior to viral challenge. EasiLV transduction efficiency was typically above 90% and was assessed by measuring the percentage of cells expressing E2-crimson (co-expressed via an IRES) by flow cytometry . To assess HIV-1 infectivity, cells were challenged with HIV-1/GFP, a vesicular stomatitis virus G protein (VSV G)-pseudotyped 8.91 HIV-1 Gag-Pol based cytomegalovirus (CMV) immediate early-enhanced green fluorescent protein lentiviral vector as described previously [For experiments in U87-MG CD4eviously . Producteviously . In expeantibodies-online.com). Bound primary antibodies were detected with HRP-conjugated anti-mouse, anti-rat, anti-rabbit or anti-goat immunoglobulin secondary antibodies and visualized by chemiluminescence.For experiments in HeLa cells, constitutive overexpression of MX2 was achieved by transduction of cells with lentiviral vector LV-PuromycinR expressing FLAG-tagged human MX2 (or a FLAG-tagged CD8 control construct) . After 7U87-MG cells were transduced with VSV-G-pseudotyped HIV-1 lentiviral vector (LV) bearing plentiCRISPRv2. Specific guide RNAs targeting MX2 or red fluorescent protein (RFP) (as the control) were cloned into BsmBI-linearized lentiviral vector pLentiCRISPRv2 using the oligonucleotides (forward/reverse) caccgAATTGACTTCTCCTCCGGTA / aaacTACCGGAGGAGAAGTCAATTc for MX2 and caccgCTCAGTTCCAGTACGGCTCCA / aaacTGGAGCCGTACTGGAACTGAGc for RFP. Transduced cells were selected with 1 μg/ml puromycin. Single-cell clones were obtained by limiting dilution and grown in a 96-well plate in the absence of puromycin and MX2 depletion was validated by immunoblotting.+ T cells. The shRNA targeting sequences for MX2, NUP214 and TNPO1 were AAGATGTTCTTTCTAATTG, GGTGAGAATCTTTGACTCC and GCAAAGATGTACTCGTAAG, respectively. Lentiviral vectors were obtained by cotransfection of 293T cells with VSV-G, p8.91 and the modified pHSIREN-S vector at a ratio of 0.5:1:1.5, respectively. Supernatants containing lentiviral particles were concentrated by ultracentrifugation and human primary CD4+ T cells were transduced by spin-infection at 2000 x g for 2 h at room temperature. 48 h after transduction, 2.5 x 104 cells/well were seeded in a 96 well plate and treated or not with IFNα. 24 h later, cells were challenged with 30 ng p24Gag of NL4.3/Nef-IRES-Renilla at 2000 x g for 2 h at room temperature. After 48 h, infection was assessed by measuring Renilla luciferase activity on a luminometer.Modified versions of the HIV-1 based lentiviral vector pHRSIREN-S-SBP-∆LNGFR-W  where th4Cl, 2% goat serum, 2% bovine serum albumin) for 1 h. MX2-FLAG was detected using a mouse anti-FLAG monoclonal M2 (Sigma) and secondary donkey anti-mouse antibody conjugated to Alexa 594 (Invitrogen). Endogenous NUP214 was detected using rabbit anti-NUP214 polyclonal antibody (Abcam ab70497) and secondary donkey anti-rabbit antibody conjugated to Alexa 488 (Invitrogen). Endogenous TNPO1 was detected using rat anti-TNPO1 monoclonal L5G3 (antibodies-online.com) and secondary goat anti-rat antibody conjugated to Alexa 647 (Invitrogen). DAPI  staining was used to demarcate the nucleus (0.1 mg/ml for 5 min) Cells were visualized using a Nikon A1 point-scanning laser confocal microscope (Nikon Instruments). Blinded quantification of MX2 cellular localization was determined visually for 100 randomly selected cells.HeLa cells stably expressing MX2 bearing a C-terminal FLAG-tag were obtained by transduction with LV-PuromycinR  encodingS1 Fig+ CXCR4+ cells were transduced with EasiLV vectors expressing FLAG-tagged MX2 or Luciferase (control). After 48 h, transduced cells were transfected twice, 24 h apart, with a panel of siRNAs at a concentration of 20 nM. For the first siRNA transfection, cells were treated with specific siRNAs, most of these targeting candidate interactors from the Y-2-H screen (U87-MG CD4H screen  includin(TIF)Click here for additional data file.S2 Fig+ CXCR4+ cells were transfected twice, 24 h apart, with 20 nM siRNA targeting NUP358, NUP214, NUP153, NUP62, NUP98, hRIP, KLHL6, NUPL2 and PNRC1. After 72 h, protein levels were analyzed by immunoblotting, with α-tubulin or HSP90 included as loading controls. No reduction in target protein abundance was observed after treatment with siRNA targeting NUPL2, and PNRC1 expression was not detectable by immunoblot.Efficiency of siRNA-mediated depletion of endogenous proteins in U87-MG cells. U87-MG CD4(TIF)Click here for additional data file.S3 FigEfficiency of siRNA-mediated depletion of endogenous proteins in HeLa cells. HeLa cells were transfected twice, 24 h apart, with 20 nM siRNA targeting NUP358, NUP214, NUP153, NUP62, NUP98, hRIP, KLHL6, PNRC1, NUP88, NUP188, TNPO1, TNPO3 and NUP214 together with TNPO1 (and CTRL siRNA). After 72 h, protein levels were analyzed by immunoblotting, with α-tubulin included as loading control.(TIF)Click here for additional data file.S1 TableComplete list and PBS of known genes identified in the yeast-two-hybrid screens.(DOCX)Click here for additional data file.S2 TableQuantification of co-immunoprecipitation of FLAG-tagged NUP214-CTD, NUP98, NUPL2, RUNX3, PNRC1, KLHL6, hRIP or TNPO1 with HA-tagged GFP, MX1, MX2 or RRR11-13A MX2. Values represent the ratio between protein input (IN) and the protein detected in the IP, normalized against wild-type MX2 interactions.(DOCX)Click here for additional data file.


The complete genetic loss of Klf5 reduced oxidative stress, increased regeneration of reduced glutathione and decreased apoptosis of leukemic precursors. Klf5 regulation of glutathione levels was mediated by its regulation of glutathione-S-transferase Mu 1 (Gstm1), an important regulator of glutathione-mediated detoxification and protein glutathionylation. Expression of Klf5 or the direct Klf5 target gene Gstm1 inhibited clonogenic activity of Klf5∆/∆ leukemic B-cell precursors and unveiled a Klf5-dependent regulatory loop in glutamine-dependent glutathione metabolism. In summary, we describe a novel mechanism of Klf5 B-ALL suppressor activity through its direct role on the metabolism of antioxidant glutathione levels, a crucial positive regulator of leukemic precursor survival.High-risk B-cell acute lymphoblastic leukemia (B-ALL) remains a therapeutic challenge despite advances in the use of tyrosine kinase inhibitors and chimeric-antigen-receptor engineered T cells. Lymphoblastic-leukemia precursors are highly sensitive to oxidative stress. KLF5 is a member of the Krüppel-like family of transcription factors. KLF5 expression is repressed in B-ALL, including BCR-ABL1+ B-ALL. Here, we demonstrate that forced expression of KLF5 in B-ALL cells bypasses the imatinib resistance which is not associated with mutations of BCR-ABL. Expression of Klf5 impaired leukemogenic activity of BCR-ABL1+ B-cell precursors  Philadelphia chromosome-positive (Ph+) hematological malignancies arise from the reciprocal translocation, t9,22) q34;q11.2), which encodes the protein BCR-ABL1. BCR-ABL1 is the transformation driver of ∼5–30% of B-ALL in patients . In. Inhttp:s Figure . The exps Figure .KLF5 in BCR-ABL1 negative and positive B-ALL cell lines , pre-pro B, pro-B, pre-B, and immature B, and mature B-cell populations in the BM of Vav1-Cre; Klf5flox/flox mice  cells (LDBM) from Vav1-Cre; Klf5flox/flox or WT mice were transduced with p190-BCR-ABL expressing bicistronic EGFP-expressing vectors. EGFP+ B-cell precursors (B220+dim/CD43+/IgM-) were sorted and cultured in the absence of stromal cell with rm-IL-7 and rm-SCF. At 8-days of culture, the ex-vivo expansion of p190-BCR-ABL+, Klf5∆/∆ B-cell precursors was 2.5- to 3.5-fold increased  accession number GSE 115919). Using an unsupervised approach, we found that the expression of 140 genes was increased, and was repressed in 489 genes in leukemic Klf5Δ/Δ B-cell precursors  [Fas [Tnfsf10 [Irf7 [Casp4 [Ikzf1, Cbx5, Hmgb3, Ebf1 and Pax5 genes, all of which have been implicated in B-cell differentiation and associated with the progression of BCR-ABL1 B-ALL, were not affected by the loss of Klf5 in leukemic B-cell precursors  , Fas [37c5) [Fas , Tnfsf10[Tnfsf10 , Irf7 [47 [Casp4   to validate the expression of the genes seen in the RNA-Seq data  followed by Q-PCR demonstrated the binding of Klf5 to the Gstm1 promoter  and pathway analysis of the RNA-seq data both pointed to dysregulation of glutathione–mediated detoxification and glutathione metabolism, which was greatest in the differential transcriptional regulation of the gene a Figure . The Gstr Figure  in leuke1 Figure . Remarka1 Figure  restored1 Figure , indicat13C515N2 glutamine into the cell culture media and assaying the steady-state levels of the heavy glutathione isotopologue M+5+1  pathway. KLF5 involvement in lymphoid leukemogenesis is however unclear. Zhu et al. proposed that KLF5 acts as a lymphoblastic leukemia oncogene by interacting with p53 to regulate survivin expression [KLF5 promoter in patient-derived BCR-ABL1+ leukemic cells [Both Klf5 and Klf4 are highly expressed in different lymphoid compartments and may act as lymphoproliferative suppressors . While spression . Herein,ic cells , suggestKlf5∆/∆ leukemic precursor cells in vitro and in vivo. Klf5 deficiency acted as an antioxidant, inhibited apoptotic transcriptional programs, including pro-apoptosis gene Fas, Tnfsf10, Irf7 and Casp4, and induced the anti-apoptosis gene Birc5, a finding opposite that to reported for Klf5 in other types of B-ALL [Our data indicate that KLF5-expression increased apoptosis of leukemic B-cell precursors, particularly in imatinib-resistant Ph+ B-ALL cells without affecting the differentiation of either leukemic or normal B-cell lymphopoiesis, suggesting that KLF5 may be a possible target in overcoming the resistance to ABL TKI seen in a fraction of Ph+ B-ALL patients. We found that exogenous expression of Klf5 resulted in reduced B-cell precursor colony formation and reduced cell survival of of B-ALL .Klf5∆/∆ B-cell precursors. We hypothesized that in BCR-ABL1+ B-ALL, KLF5 may function as a negative regulator of oxidative stress through direct regulation of GSH metabolism. We found that forced Gstm1 expression repressed and restored B-cell precursor colony formation; Klf5 deficiency increased glutathione biosynthesis, which was restored to normal levels by forced expression of Gstm1; and finally, the expression of Klf5 or Gstm1 downregulated glutathione biosynthesis to the basal levels seen BCR-ABL expressing leukemic precursors.In our data set, we found that GSTM1, a critical enzyme involved in the detoxification process of oxidative species and associated with poor-prognosis pediatric B-ALL –54, is aOxidative stress results from an imbalance between oxidant and anti-oxidant levels within the cell. BCR-ABL is a major inducer of oxidative stress through multiple pathways, which ultimate result in increased genomic instability, especially in imatinib-resistant leukemia –57. WhenGlutamine is the most abundant amino acid in plasma and in intracellular pools, being consumed at significantly higher rates than other amino acids by tumor cells, and it is essential for GSH biosynthesis . GlutamaIn conclusion, Klf5 is efficiently downregulated in imatinib-resistant p190-BCR-ABL B-ALL leukemic cells and it acts as a negative regulator of the anti-oxidant glutathione. Klf5 is a negative regulator of leukemic B-cell precursor survival. Expression of Klf5 reduces leukemic B-cell precursor survival, and its role in controlling anti-oxidant glutathione levels depends on its transcriptional activating role on the expression of glutathione transferase genes. The leukemic suppressor effect of Klf5 on precursor survival can be restored by downstream overexpression of Gstm1.Our group has reported the generation and validation of Mx1-Cre and Vav1-Cre transgenic mice driving the deletion of Klf5 , 32. C57Normal BM CD34+ cells, and B-ALL mononuclear cells (BCR-ABL1+) specimens were obtained under Institutional Review Board-approved protocols and donor informed consent from Cincinnati Children’s Hospital Medical Center. B-ALL cell lines REH, RS4;11, SEM, KOPN8, RCH-ACV, NALM-1, SD-1, BV-173, SUP-B15, and NALM-20 were purchased from American Tissue Culture Collection  or the Deutsche Sammlung von Mikroorganismen und Zellkulturen . Z-181 and Z-119 were kindly provided by Dr. Zeev Estrov . REH, KOPN8, RCH-ACV, SD-1, Z-181, BV-173, Z-119, NALM-1, and NALM-20 were cultured in RPMI-1640 medium  containing 20% fetal bovine serum (FBS) , 2 mM L-glutamine (Thermo Fisher Scientific), 100 U/ml penicillin and 100 μg/mL streptomycin (Thermo Fisher Scientific). RS4;11 was cultured in alpha-MEM  containing 20% FBS, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. SEM was cultured in Iscove’s Modified Dulbecco’s Medium  containing 20% FBS, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. SUP-B15 was cultured in McCoy’s 5A medium (Thermo Fisher Scientific) containing 20% FBS, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. In some experiments, imatinib mesylate  was added to the culture medium.Retroviral and lentiviral transduction have been described previously . In brieflox/flox littermates were isolated and transduced with a retroviral bicistronic vector encoding p190-BCR-ABL with enhanced green fluorescent protein (EGFP) as a reporter. For the Klf5 in vivo rescue experiments, lentiviral particles encoding either full-length mKlf5 cDNA cloned in a pCDH-EF1-MCS-IRES-RFP vector (System Biosciences), or the empty vector, were used for stable expression in p190-BCR-ABL+ B-cell lymphoid precursors. Transduced LDBM cells (1 × 106) were intravenously transplanted into lethally (7 + 4.75 Gy) irradiated congenic CD45.1+ mice. The multiplicity of infection of p190-BCR-ABL was kept low to allow only one copy of BCR-ABL per transduced cell, and the multiplicity of infection of lentiviral mKlf5 was kept as 10. Peripheral blood was collected through retro-orbital puncture on Day 18 to test the engraftment of donor cells. Mice were then administered poly I:C intraperitoneally (5 mg/kg) every other day for a total of five times to delete the Klf5 gene. Mice were sacrificed when they showed signs of extreme hunched posture with labored breathing and/or symptoms of moribund or rear leg paralysis were noted. BM and spleens were dissociated and processed for flow cytometric analysis.We have previously reported our protocol of transduction/transplantation of leukemic BM cells . In briein vitro analysis. Early B-cell precursors were defined as EGFP+/B220+dim/CD19+/CD43+/membrane IgM-, and isolated by FACS , San Jose, CA) under sterile conditions. LDBM cells were stained with biotinylated lineage markers , Gr-1 , CD4 , CD8a , CD3e (clone 145-2C11), CD11b (clone M1/70), and Ter119 (clone Ly-76), streptavidin APC-Cy™7, allophycocyanin (APC)-conjugated c-kit (clone 2B8) and phycoerythrin (PE)-Cy7-conjugated Sca-1 (clone D7) antibodies for LSK cell population, and sorted (FACS Aria II). Commercially available fluorochrome-conjugated anti-mouse or anti-human antibodies were used , including B220 , CD11b (clone M1/70), CD3e (clone 145-2C11), CD43 (clone S7), CD93 (clone AA4.1), CD19 (Clone 1D3), IgM (clone R6-60.2), mouse anti-human CD34 (clone 581), and mouse anti-human CD19 (clone SJ25C1). Analysis was performed using a flow cytometer . KLF5 expression of CD34+CD19+ cells from normal donor and BCR-ABL1+ patients was carried out by flow cytometry analysis. Samples were prepared according to manufacturer’s protocol using BD Pharmingen Transcription Factor Buffer Set. Anti-KLF5 antibody (1:2000)  and FITC Goat Anti-Rabbit IgG  were used.LDBM cells were obtained after centrifugation in Histopaque-1083 . Transduced LDBM cells or harvested leukemic BM cells from B-ALL mice were purified for early B-cell precursors for further B-cell lineage colony-forming units (CFU-preB) were quantified after 9 days of culture of whole BM cells or after 7 days culture of sorted p190 BCR-ABL expressing B-cell precursors in M3134 methylcellulose (StemCell Technologies) supplemented with 30% FBS , 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/mL streptomycin, 100 μM β-mercaptoethanol (Fisher-Scientific), 1% BSA (Roche Diagnotics), 20 ng/mL of recombinant mouse IL-7 (PeproTech), and 100 ng/mL of recombinant mouse SCF (PeproTech) .ex-vivo generated B-cell precursors. For live cell gating analysis, we used 7-aminoactinomycin D .Cell cycle was analyzed by 5-bromo-2’-deoxyuridine (BrdU) incorporation on gated red fluorescent (mCherry +) ALL cell line cells or on gated EGFP+ B-cell precursors following the manufacturer’s protocol . Apoptosis was evaluated by flow cytometric determination of annexin V (BD Pharmingen) binding on gated mCherry+ B-ALL cell lines or on gated EGFP+ primary or ® Reverse Transcription Reagents , followed by amplification of cDNA . Cycle threshold (Ct) values of individual genes were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and values calculated using the 2−ΔCt method.Total mRNA was isolated from ALL cell line cells or p190-BCR-ABL expressing mouse B-cell precursors according to the manufacturer’s protocol . mRNA was reverse-transcribed using TaqManFACS sorted purified B-cell precursors or ALL cell line cells were lysed in radioimmunoprecipitation assay (RIPA) buffer and extracts were electrophoresed on SDS-PAGE. For immunoblotting, separated proteins were transferred to a PVDF membrane. The membrane was then blocked with 5% non-fat milk in tris-buffered saline (TBS) for one hour at room temperature (RT). A guinea pig anti-Klf5 antibody  (1:4000)Klf5Δ/Δ + vector and Klf5Δ/Δ + Gstm1 groups were labeled with 4 mM L-glutamine . At time points 0 and 24 hours, the cells were collected and centrifuged, and cell pellets and supernatant were collected and frozen in liquid nitrogen. 1 × 106 sorted purified p190-BCR-ABL+ B-cell precursors from leukemic WT (n = 2) or Klf5Δ/Δ (n = 3) mice, or from cultured WT + vector, WT + Gstm1, Klf5Δ/Δ + vector and Klf5Δ/Δ + Gstm1 groups were extracted in 1 ml of ice-cold lysis/extraction buffer (methanol: acetonitrile: water 5:3:2) as previously described [T-test, ANOVA), principal component analysis  and hierarchical clustering analysis (HCA) using GraphPad Prism  and GENE-E (Broad Institute) respectively.Targeted metabolomics analysis was performed as previously reported . Cultureescribed . Proteinn = 3 samples) mice or Klf5Δ/Δ (n = 3 samples) mice was isolated . RNA-seq was determined  in the CCHMC DNA Sequencing and Genotyping Core following standard procedures. Data analysis was performed using the Strand NGS v3.0 software (Agilent Technologies). The differentially expressed genes in the Klf5Δ/Δ versus WT samples were identified by applying the moderated t-test with the absolute fold change cut-off ≥2.0 and the p-value cut-off ≤ 0.05. Gene set over-representation analysis for the differentially expressed genes was performed by employing the Ingenuity Pathway Analysis v01–07 platform  for the collections of GO gene sets and signaling pathways. The significance of gene set over-representation was estimated based on the hypergeometric test with the p-value cut-off ≤ 0.05. Differential expression was confirmed by real time (RT)-PCR for selected genes.Total RNA from sorted purified p190-BCR-ABL+B-cell precursors of leukemic WT (The screen was performed from the Achilles v2.20.2 dataset (Broad Institute)  derived p-value. The genome-wide gene level dependency z-scores were illustrated through boxplots associated to individual lineages and through a hockey plot.Dependency z-scores for each gene were identified based on the DEMETER computational method that uses the depletion values induced by each shRNA construct to infer the effect of suppressing its intended target (on-target) gene along with the effect of activating an off-target seed. DEMETER models each depletion value as a sum of two unobserved quantities: gene knockdown and seed-based effects and then estimates these quantities by fitting the model to the full dataset. The KFL5 dependency z-scores across the 501 lines were computed based on the three KLF5 hairpins with reliable scores for on-target gene suppression . The differences in KLF5 dependency z-scores in B-ALL versus solid tumor cell lines in Achilles data were evaluated based on the Mann-Whitney nonparametric test with the cut-off 0.05 applied to the Chromatin immunoprecipitation (ChIP) assay was performed  according to the manufacturer’s protocol. Sorted purified WT p190-BCR-ABL+ B-cell precursors were prepared. Following the immunoprecipitation using anti-Klf5 antibody , quantitative PCR (Q-PCR) was performed using the primers designed to encompass the putative Klf5 binding site. Immunoprecipitation using normal rabbit IgG and amplification of GAPDH gene serve as negative control.t test or Anova test, and differences in survival were examined using the Log-Rank test. Level of significance was established at p < 0.05.Data are presented as mean ± SD or mean ± SEM when several independent experiments have been analyzed. Statistical significance was determined using the unpaired Student-


CCND1), Ajuba LIM Protein (AJUBA), and heart development protein with EGF like domains 1 (HEG1) were identified as common target genes of miR-193b-3p and -5p. A reporter assay and an Ago2-RIP experiment showed that both miRNAs directly bind to the 3′ untranslated region (3′UTR) of the target mRNA. Knockdown of target gene reduced the proliferative and metastatic potential of primary and metastatic lung cancer cells. Our results demonstrate miR-193b is a dual-strand tumor suppressor and a novel therapeutic target for lung cancer.Emerging studies suggest that microRNAs (miRNAs) play multiple roles in cancer malignancy, including proliferation and acquisition of metastatic potential. Differentially expressed miRNAs responsible for the malignancy of lung cancer were searched by miRNA microarray using a previously established brain metastatic lung cancer model. Twenty-five miRNAs were down-regulated in brain metastatic lung cancer cells. Among those, miR-193b-3p and -5p were chosen for further studies. Their function in metastatic potential and proliferation was examined using Transwell invasion, wound healing, and colony forming assays. The underlying mechanism of tumor-suppressor miR-193b-3p and -5p was explored using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), Western blot, Argonaute 2-RNA immunoprecipitation (Ago2-RIP), and reporter assays. Both strands of miR-193b were down-regulated in brain metastatic lung cancer cells and in tissues from lung cancer patients. Overexpression of miR-193b-3p and -5p inhibited invasive and migratory activities and diminished clonogenic ability. Conversely, inhibition of miR-193b-3p or -5p increased the metastatic potential and colony forming ability. Cyclin D1 ( Despite the remarkable development of lung cancer therapies, lung cancer has been classified as a major cause of death . Its unfMicroRNAs (miRNAs) are highly conserved small non-coding RNAs ranging from 19 to 25 nucleotides in length . They arWe have investigated the molecular mechanism whereby lung cancer cells acquire malignant characteristics such as metastatic and proliferative potential. Particularly, we examined the effects of miR-193b-3p and -5p, which are down-regulated in metastatic lung cancer cells on malignant phenotypes of lung cancer cells. Both strands of miR-193b suppress the expression of common target genes. Based on our findings, we identified miR-193b as a dual-strand tumor suppressor.2. For knockdown of miR-193b targets, cells were plated at a density of 3.5 × 105 cells/dish. After 24 h, transfected with the indicated small interfering RNAs (siRNAs) or control (CTRL) siRNA using Lipofectamine 2000 . The Opti-MEM™  is used for transfection medium. The 6 h later, the transfection medium was changed to Dulbecco’s modified Eagle’s medium  supplemented with 10% (v/v) FBS. Control siRNA (UUCUCCGAACGUGUCACGU), Ajuba LIM Protein (AJUBA) siRNA (GUGUCAAUGGCUCUGUGUA), Cyclin D1 (CCND1) siRNA (GCACAUCUUGGCUAUGUAA), heart development protein with EGF like domains 1 (HEG1) siRNA (UCGAGAAGAAACAGUAGAGUAAC) and matrix metalloproteinase 16 (MMP16) siRNA (CGUGAUGUGGAUAUAACCA) were synthesized by Bioneer (Korea). The overexpression plasmid pcDNA3.2/V5 hsa-mir-193b (Plasmid #26318) was purchased from Addgene. Synthetic miRNA mimics  and miRNA antisense inhibitors (Anti-193b-3p and Anti-193b-5p) were purchased from Ambion (U.S.A.). To verify miRNA overexpression, the levels of each miRNA in three independent sets were assessed by RT-qPCR using specific TaqMan primers (Supplementary Table S1).Brain metastatic lung cancer cells PC14PE6/LvBr4) were previously established by repeated left-ventricle injection of lung adenocarcinoma PC14PE6 cells ,14. ParePE6/LvBr4® Green PCR Master Mix . The following primers were used for amplification: Primary-miR-193b #1 , Primary-miR-193b #2 , AJUBA , CCND1 , HEG1 , MMP16  and GAPDH . The expression levels of miR-193b-3p (MIMAT0002819) and miR-193b-5p (MIMAT0004767) were assessed using specific TaqMan primers .Cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed using radioimmunoprecipitation (RIPA) buffer containing protease and phosphatase inhibitor cocktail . Cell lysates were clarified by centrifugation, and the protein concentration was quantified by using Bradford solution . Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and transferred to polyvinylidene fluoride membranes . After blocking with 5% bovine serum albumin (BSA) (5 g BSA in 100 ml TBST) or 5% skimmed milk (5 g skimmed milk in 100 ml TBST), membranes were incubated with the indicated primary antibodies overnight. Ajuba , cyclin D1 , and glyceraldehyde 3-phosphate dehydrogenase  antibodies were used. After 48-h post-transfection, Total RNA was isolated using TRIzol reagent . Chloroform  was added, and the homogenate was allowed to separate into a clear upper aqueous layer, RNA was precipitated from the aqueous layer with isopropanol . Isolated total RNA was converted into cDNA using the SuperScript III First-Strand Synthesis System . The mRNA was quantified using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) (ABI PRISM™ 7900HT) and Power SYBRAJUBA, CCND1, and HEG1 mRNA in Ago2-RIP was assessed by RT-qPCR. Direct binding of miRNAs to the target mRNA was determined by calculating the relative enrichment of target mRNA.For reporter assay, we constructed dual luciferase vectors  containing wild-type (WT) or mutated (MT) miR-193b-3p and miR-193b-5p binding sites  in the 3′UTR of the target genes. Mutated reporter vectors were constructed by substituting four or five nucleotides in the seed region, abolishing the interaction between miRNA and target mRNAs. Briefly, cells transfected with Con-miR or Pre-miR (Pre-193b-3p or Pre-193b-5p) were seeded into 24-well plates. After 24 h, cells were transfected with either WT or MT luciferase vector. After incubation for 20 h, luciferase expression was measured using a Dual-GLO™ Luciferase Assay System (Promega E2940). In addition to the reporter assay, we assessed the direct interaction between miRNA and target gene mRNA by performing Argonaute 2 (Ago2)-RNA immunoprecipitation (Ago2-RIP). Ago2 antibody   and Dyna2 cells/well and cultured in complete medium. After incubation for 2 weeks, cells were fixed with 4% (v/v) paraformaldehyde and stained with 0.2% crystal violet . Clonogenicity was assessed by counting the number of colonies. Metastatic potential was determined by measuring the invasive and migratory abilities of cancer cells. The invasiveness was assessed using BioCoat™ Matrigel® Invasion Chambers . 2 h before start cell trypsinization, lower chamber and upper chamber were filled with serum-free medium. Equal numbers of cells in serum-free medium were added to the upper chamber. Cellular invasion was triggered by the addition of complete medium containing FBS to the lower chamber. After 24 h, cells were fixed with methyl alcohol  and stained with hematoxylin solution  and 0.5% eosin Y   (H&E). Quantification of invasiveness was performed by counting the number of invaded cells from at least ten fields. The migratory activity was assessed by wound closure assay. Transfected cells were seeded into 12-well plates with high density and then a straight line was scratched by white pipette tips. After 24 h, the migrated distance was measured by AxioVision microscope software  and Image J.To determine clonogenicity, a colony-forming assay was performed. Briefly, transfected cells were plated into six-well plates at a density of 3 × 10in vitro model of brain metastatic lung cancer through repeated left ventricle injection and found that brain metastatic lung cancer cells (PC14PE6/LvBr4) had higher metastatic potential, including migratory and invasive abilities, than parental lung cancer cells (PC14PE6) did [Previously, we established an PE6) did ,16. To sPE6) did . For exaPE6) did ,18. To cSince miR-193b-3p and 5p were down-regulated in brain metastatic PC14PE6/LvBr4 cells, we checked if overexpression of miR-193b-3p or -5p could inhibit the metastatic potential including migratory and invasive abilities. As shown in To confirm the effect of both miR-193b strands on the proliferative potential, we performed a colony forming assay. The number of colonies was significantly decreased in PC14PE6/LvBr4 cells transfected with miR-193b-3p or -5p precursors F. SimilaTo gain further insight in the underlying molecular mechanism of miR-193b-3p and -5p in the malignant processes of lung cancer, we searched for common targets of miR-193b-3p and -5p using the TargetScan prediction algorithm and found that four genes  are putative common targets of miR-193b-3p and -5p A. List oHEG1 mRNA level by RT-qPCR. Similarly, the mRNA level of common target genes was higher in PC14PE6/LvBr4 cells than in parental PC14PE6 cells  regulated by both strands of miR-193b. To test whether miR-193b-3p and -5p directly bind to the 3′UTR of target mRNAs, Ago2-RIP and luciferase reporter assay were performed. miR-193b-3p and -5p was abolished by the disruption of the direct binding between miR-193b and the 3′UTR of the target mRNAs by point mutations in the seed sequence. These results demonstrate that miR-193b-3p and -5p inhibit the expression of common target genes  through direct interaction with the 3′UTR of their mRNA.In addition to the Ago2-RIP experiment, the direct interaction between miR-193b and target mRNAs was investigated using the luciferase reporter assay. We constructed dual luciferase vectors containing MREs for each miR-193b-3p and -5p in the 3′UTR from target mRNAs including CCND1, AJUBA, and HEG1  and reduced PC14PE6/LvBr4 and A549 invaded cells A,B. Addi49 cells E,F. CollThe action mechanism of miR-193b-3p and -5p consistent with our findings is summarized in In general, miRNAs repress the expression of target genes by degrading the mRNA or inhibiting translation in a sequence-dependent manner . AccumulAs a tumor suppressor, miR-193b-3p has been reported to be down-regulated and to inhibit cancer malignancy in various types of cancer. Stathmin 1 (STMN1) was identified as a direct functional target of miR-193b in colorectal cancer . Ectopicin silico predictions and transcriptome analyses, KRAS and MAX are identified as direct targets of miR-193b and miR-365. Since miR-365 was not down-regulated in metastatic lung cancer, our study excluded the effect of miR-365 on malignant phenotypes of lung cancer cells.Several pathways have been identified to regulate the expression of miR-193b. In prostate cancer, miR-193b is an epigenetically regulated tumor suppressor . It was It was revealed that miR-193b-3p and -5p regulate malignant phenotypes of lung cancer through the suppression of their common target genes . Cyclin D1 encoded by the CCND1 gene is one of the frequently altered and overexpressed cell cycle regulators in cancers . As an o


Down-regulating mitochondrial protease LonP1 increased mitochondrial TDP-43 levels and exacerbated TDP-43-induced mitochondrial damage as well as neurodegeneration. Together, our results demonstrate that TDP-43 induced mitochondrial impairment is a critical aspect in TDP-43 proteinopathy. Our work has not only uncovered a previously unknown role of LonP1 in regulating mitochondrial TDP-43 levels, but also advanced our understanding of the pathogenic mechanisms for TDP-43 proteinopathy. Our study suggests that blocking or reversing mitochondrial damage may provide a potential therapeutic approach to these devastating diseases.Mutations in or dys-regulation of the TDP-43 gene have been associated with TDP-43 proteinopathy, a spectrum of neurodegenerative diseases including Frontotemporal Lobar Degeneration (FTLD) and Amyotrophic Lateral Sclerosis (ALS). The underlying molecular and cellular defects, however, remain unclear. Here, we report a systematic study combining analyses of patient brain samples with cellular and animal models for TDP-43 proteinopathy. Electron microscopy (EM) analyses of patient samples revealed prominent mitochondrial impairment, including abnormal cristae and a loss of cristae; these ultrastructural changes were consistently observed in both cellular and animal models of TDP-43 proteinopathy. In these models, increased TDP-43 expression induced mitochondrial dysfunction, including decreased mitochondrial membrane potential and elevated production of reactive oxygen species (ROS). TDP-43 expression suppressed mitochondrial complex I activity and reduced mitochondrial ATP synthesis. Importantly, TDP-43 activated the mitochondrial unfolded protein response (UPR mt in both cellular and animal models of TDP-43 proteinopathy. LonP1, one of the key mitochondrial proteases in UPRmt, protects against TDP-43 induced cytotoxicity and neurodegeneration. Our study uncovers LonP1 as a modifier gene for TDP-43 proteinopathy and suggests protecting against or reversing mitochondrial damage as a potential therapeutic approach to these neurodegenerative disorders.TDP-43 proteinopathy is a group of fatal neurological diseases. Here, we report a systematic examination of the role of mitochondrial damage in TDP-43 proteinopathy using patient brain tissues, as well as cellular and animal models. Our data show that TDP-43 induces severe mitochondrial damage, accompanied by activation of UPR TDP-43 proteinopathy is characterized by the presence of TDP-43 immunoreactive inclusion bodies in the affected tissues. Clinically, TDP-43 proteinopathy manifests as a spectrum of different neurodegenerative diseases, ranging from dementia  and motor neuron disease (MND) to traumatic brain injuries –4. FTLD Mitochondrial damage is associated with a range of neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD) and MNDs –19. Mitomt), which includes induction of mitochondrial chaperones assisting in proper protein folding, and of proteases promoting clearance of misfolded proteins  with different concentrations of purified LonP1 protein for 90 min at 37°C. The reaction products were analyzed by Western blotting using the corresponding specific antibodies to detect TDP-43 and LonP1 proteins.A cDNA encoding the human LonP1 protein (amino acid residues 115–959) was cloned into vector pET32M3C [a modified version of the pET32a vector ], expressed as an N-terminal thioredoxin and 6XHis-tagged protein and purified from E. coli  following the published protocol . PurifieTotal RNA was isolated from HEK293 cells or fly heads using TRizol reagent (Invitrogen) as described previously . cDNA syThe adult fly locomotor assay was carried out as described previously with minor modifications . BrieflyThe protein solubility was examined as described previously with minor modifications . Brieflyt-test. Multiple group comparisons were performed using a one-way or two-way analysis of variance (ANOVA) followed by post-hoc tests. The bar graphs with error bars represent mean ± standard error of the mean (SEM). Significance is indicated by asterisks: *, P < 0.05; **, P< 0.01; ***, P< 0.001.Data were collected in Excel (Microsoft) and analyzed using GraphPad Prism 6 unless specified otherwise. Differences between two groups were analyzed using a Student’s S1 TableAll samples used were sequenced and confirmed that there were no mutations in known genes associated with ALS or FTLD, including TDP-43, FUS, C9orf72, GRN, SOD1 and MAPT, as reported previously . Age, ge(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 FigA) Western blotting experiments revealed that the Myc-tagged TDP-43 (Myc) was detected in the purified mitochondria 24 hr following induction of TDP-43 expression, and that the endogenous (Endo) TDP-43 was detected in the purified mitochondria from both control and TDP-43 expressing cells. Western Blotting experiments were performed as described for B, C). Quantification of mitochondrial size (B) or percentage of damaged mitochondria in EM analyses of the corresponding HEK293 cells, vector control (Ctr) or cells expressing Wt or A315T-mutant TDP-43. Approximately 200 mitochondria  were quantified for each group. Data represents 3 independent experiments [one-way ANOVA with Bonferroni post hoc test (***: P<0.001)].((TIF)Click here for additional data file.S2 Fig(A) Induction of either Wt or A315T-mutant TDP-43 expression does not affect the total cellular ATP level. The total cellular ATP level was measured in cells expressing the control, or Wt or A315T-mutant TDP-43 at different time points after induction of TDP-43 expression using tetracycline (1ug/ml Tet). (B) Induction of either Wt or A315T-mutant TDP-43 expression does not lead to a general reduction in the mRNA levels of respiratory complex I genes. Quantitative RT-PCR experiment was performed at 24 hr post-induction using specific primers to examine the expression of a number of components of the complex I, including ND3, ND6, NDUFAB1, NDUFS8, NDUFAF4 and NDUFA13. Data represent 3 independent experiments and are analyzed using a one-way ANOVA with Bonferroni post hoc test .(TIF)Click here for additional data file.S3 Fig(A) A diagram illustrating the heat-shock induction strategy. Adult flies were collected after eclosion and subjected to heat shock daily at 28°C for 4 hours followed by culturing at 25°C for 20 hours every day.  TEM analyses revealed that the expression of A315T-mutant TDP-43 under the GMR-Gal4/Tubulin-Gal80ts driver leads to age-dependent progressive retinal degeneration and mitochondrial damage, whereas the retinae and mitochondria of control flies retained their normal morphology even at day 30 post-induction (Day30 post-Ind).TDP-43 expression was induced by heat shock in the retinae (GMR) or all neurons (Elav) under the Gal4 driver containing a Tubulin-Gal80ts regulatory element. (TIF)Click here for additional data file.S4 Fig(A) Confocal ratiometric imaging of VNC motor neurons of larvae expressing control RFP (Ctr), Wt or A315T-mutant TDP-43 revealed increased mitochondrial ROS production (excitation for reduced/oxidized mito-roGFP2-Grx1: 405-nm/488-nm). Scale bars: 25μm. (B) Quantification of the mitochondrial redox index (405-nm/488-nm) in MNs of fly larvae expressing Ctr, Wt or A315T-mutant TDP-43. Thirty-two to forty images were taken from more than 15 flies in each group . Data represent 3 independent experiments, analyzed using a one-way ANOVA with Bonferroni post hoc test (***:P<0.001). Fly genotypes: Ctr: OK371-Gal4/UAS-mito-roGFP2-Grx1/UAS-RFP; Wt: OK371-Gal4/UAS-mito-roGFP2-Grx1/UAS-Wt-TDP-43-RFP;A315T: OK371-Gal4/UAS-mito-roGFP2-Grx1/UAS-A315T-TDP-43-RFP.(TIF)Click here for additional data file.S5 Fig TDP-43-induced cytotoxicity is enhanced by the LonP1 inhibitor, but not by proteasome or autophagy inhibitors. Control (Ctr) cells or cells expressing Wt or A315T-mutant TDP-43 were induced with Tet (1μg/mL) for 24 hours. Following PBS washes to remove Tet, cells were cultured for an additional 24hours in media containing LonP1 inhibitor CDDO , proteasome inhibitor MG132  or autophagy inhibitor 3MA . Cell viability or cytotoxicity was determined using a CytoTox-ONE Homogeneous Membrane Integrity kit (Promega) in cells treated with CD, MG or MA. Data represent 4 independent experiments and are analyzed using StatPlus (one-way ANOVA with Bonferroni post hoc test). (C) Increased LonP1 expression suppresses TDP-43 cytotoxicty see  without  Western blotting analyses show increased LonP1 expression following LonP1 transfection (+) in cells expressing control vector (Ctr), Wt or A315T-TDP-43. The total TDP-43 levels in cell lysates were not changed by LonP1 overexpression (OE). (D) TDP-43 degradation by LonP1 is ATP-dependent. The in vitro degradation assay was carried out as described for (TIF)Click here for additional data file.S6 Fig(A) There was no detectable interaction between ClpP and TDP-43 in the co-Immunoprecipitation assay. (B) Down-regulating ClpP did not affect mitochondrial TDP-43 protein level. ClpP was down-regulated in HEK293 stable inducible cells expressing Wt or A315T-mutant TDP-43. ClpP down-regulation did not alter mitochondrial TDP-43 levels. Data represent 3 independent experiments.(TIF)Click here for additional data file.S7 FigA) qPCR experiment was carried out to determine mRNA levels of Drosophila Lon (dLon) in the corresponding fly lines expressing vector control (Ctr) or siLon (#1 and #2) or overexpressing Lon (dLonOE). Because siLon#1 line showed consistently more robust down-regulation (reducing dLon expression to ~30% of the control level), this line was used in subsequent experiments. Data represent 3 independent experiments. Overexpression of dLon in control flies led to retinal degeneration, preventing us from testing effect of dLonOE in TDP-43 flies. (B) Western blotting experiments using anti-TDP-43 antibody showed that the total TDP-43 was expressed in eyes at equivalent levels in TDP-43 and TDP-43/siLon flies at day 20 following heat shock. (C) Western blotting experiments using anti-TDP-43 antibody showed that the total TDP-43 was expressed at equivalent levels in heads of TDP-43 and TDP-43/siLon flies at day 4 following heat shock. The pan-neuronal marker Elav was used as a loading control in panels B and C.((TIF)Click here for additional data file.S8 Fig(A). Mitochondria were purified from heads of flies expressing control or Wt or A315T-mutant TDP-43 under the GMR-Gal4 driver. Purified mitochondria were sequentially extracted in RIPA buffers containing 0.5% NP-40 or 2%SDS and finally 8M Urea (see B). Quantification of WB band intensity shown in panel A. Data from 3 experiments were analyzed by a Student’s t-test. Data represent 3 independent experiments.Urea see . Corresp(TIF)Click here for additional data file.S9 Fig(A) Down-regulation of Lon in control (Ctr) flies did not affect retinal morphology or mitochondrial ultra-structure. (B) Down-regulating Lon in control flies did not affect the number of rhabdomeres per ommatidium, with 81 and 89 ommatidia analyzed in Ctr and siLon groups respectively. (C) Quantification of EM data shows that down-regulating Lon in control flies did not alter the percentage of damaged mitochondria, with 431 mitochondria in Ctr group and 521 mitochondria in siLon group examined. Data represent 3 independent experiments, analyzed using a one-way ANOVA with Bonferroni post hoc test .(TIF)Click here for additional data file.S10 FigElectron-dense aggregates (marked by black arrows) were detected inside mitochondria in flies expressing A315T-mutant TDP-43 when Lon was down-regulated by the specific siRNA. These structures were not detected in other groups, including the control flies or flies expressing Wt (with or without Lon knockdown) or A315T-mutant TDP-43 alone.(TIF)Click here for additional data file.


Scientific Report 10.1038/s41598-018-33638-x, published online 15 October 2018Correction to: The Acknowledgements section in this Article was omitted. The Acknowledgements section should read:“This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2016R1C1B1015761).”


BRAF alterations, primarily the V600E mutation and rarely V600D and FXR1-BRAF fusion [Desmoplastic infantile ganglioglioma (DIG) and desmoplastic infantile astrocytoma (DIA) are rare, low-grade neuroepithelial neoplasms . BRAF alF fusion , 10, 12,F fusion , underscBRAF alteration in a DIG in a 3-month-old female patient who had seizures, apnea, and a right postcontrast enhancing temporal solid multicystic mass  at low variant allelic frequency (approximately 15%) was the only clinically relevant alteration identified Additional file 2:DNA NGS results. (TIF 699 kb)Additional file 3:Sanger sequencing results. (TIF 522 kb)Additional file 4:RNA sequencing results. (TIF 443 kb)Additional file 5:In silico protein modelling. (TIF 690 kb)


Overweight older adults with osteoarthritis (OA) face increased risk for disability; however, no evidence-based programs target weight and OA simultaneously. Fit & Strong! (F&S!) is an 8-week evidence-based exercise program for persons with OA that improves lower extremity (LE) strength and mobility out to 18 months. F&S! Plus, a weight loss version of F&S! was tested against standard F&S! in a comparative effectiveness trial. Two and six-month trial outcomes were previously presented, this session will present maintenance outcomes at 12 and 18 months. This trial randomized 413 participants, 210 to F&S! and 203 to F&S! Plus. The mean sample age was 67.9, 86% female, and 92% African American. At 12 months, significant and marginally significant between-group differences favoring F&S! Plus were seen in several outcomes, including weight (p=.049), BMI (p=.04), waist circumference (p=.004), LE physical function (p=.09), 6-minute distance walk (mobility) (p=.08), 30-second chair stands (LE strength) (p=.46), and anxiety & depression (p=.08). At 18 months, only LE strength remained significantly improved for the F&S! Plus group (p=.045), however several within-group improvements remained statistically significant for both groups out to 18 months, including weight, BMI, waist circumference, LE pain & physical function, 30-second chair stands (LE strength), anxiety & depression, and self-efficacy for weight-management. F&S! Plus showed significant improvements over standard F&S! for several outcomes at the conclusion of the intervention (2 months) and many were maintained out to 12 months, with LE strength continuing to 18 months. Both groups showed significant within-group improvements out to 18 months.


Publisher's Note added on 10 September 2009: There is a pagination error in Molecules2009, 14(3) with pages 1134-1144 missing. Thus, pages 1134-1144 are taken as a blank pages.


However, the molecular mechanisms responsible for their reprogramming are not fully understood. Here we show identification of transcription factors that mediate PGC reprogramming. We selected genes encoding transcription factors or epigenetic regulatory factors whose expression was significantly different between PGCs and PSCs with in silico analysis and RT-qPCR. Among the candidate genes, over-expression (OE) of Bcl3 or Klf9 significantly enhanced PGC reprogramming. Notably, EGC formation was stimulated by Klf9-OE even without bFGF. G-protein-coupled receptor signaling-related pathways, which are involved in PGC reprogramming, were enriched among genes down-regulated by Klf9-OE, and forskolin which activate adenylate cyclase, rescued repressed EGC formation by knock-down of Klf9, suggesting a molecular linkage between KLF9 and such signaling.Primordial germ cells (PGCs) are precursors of eggs and sperm. Although PGCs are unipotent cells  Oct4, Klf4, Sox2, and cMyc, which are known as the Yamanaka factors [Dead-end 1 (Dnd1) [Dnd1 functions has revealed the molecular mechanisms of conversion of germ cells into pluripotent early teratoma cells in embryos, including control of genes involved in enhancement of the cell cycle in germ cells [Cellular reprogramming to establish pluripotent stem cells (PSCs) is a topic in basic research and may have medical applications. Various types of differentiated somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forced expression of pluripotency-associated genes such as  factors ,2. For s1 (Dnd1) ,4. More rm cells ,6. In adrm cells –9, indicIn initial studies, reprogramming of mouse PGCs to pluripotent embryonic germ cells (EGCs) was induced by addition of leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) with serum on feeder cells expressing membrane-bound Steel factor also known as stem cell factor and kit ligand. Subsequent studies revealed that bFGF can be replaced by retinoic acid (RA) or forskolin (FK: an activator of adenylate cyclase) , TrichosBlimp1 is down-regulated shortly after PGC reprogramming begins [Blimp1 enhances PGC reprogramming, and BLIMP1 represses the pluripotency network in embryonic stem cells (ESCs), together suggesting that BLIMP1 functions as a pluripotency gatekeeper in PGCs [In addition to intra-cellular signaling, the importance of transcriptional regulation including repression of the transcription factor, BLIMP1 playing a role on PGC specification , for PGCg begins . In addi in PGCs . HoweverMCH and C57BL/6 mice were purchased from CLEA Japan and Japan SLC, respectively. The Oct4-deltaPE-GFP  transgenE12.5 or E13.5 Oct4-deltaPE-GFP transgenic embryos were collected and dissected in Dulbecco’s modified Eagle medium  containing 10% fetal bovine serum (FBS). The genital ridges of male and female embryos were dissected. Tissue samples containing PGCs, were incubated with 1.2 mg/ml collagenase (SIGMA) in PBS containing 10% FBS for 1h at 37°C. To prepare single-cell suspensions for flow cytometry, tissues were dissociated by pipetting, and were filtered through a nylon mesh . A Bio-Rad S3e cell sorter was used to sort and collect PGCs with intense GFP expression.Vasa-RFP (VR15) ESCs  were culTotal RNA samples were purified using RNeasy Micro Kit (QIAGEN) according to the manufacturer’s instruction. RNAs were reverse-transcribed using SuperScript III (Invitrogen) and random primers (Promega). Expression levels of genes were quantified using the SYBR Green Master Mix (Applied Biosystems) with the primers shown in Klf9-OE E13.5 PGCs and control PGCs cultured for 1 days, with TruSeq RNA sample prep kit v2 (Illumina). The libraries were clonally amplified on a flow cell and sequenced on HiSeq2500  with 51-mer single-end sequence. Image analysis and base calling were performed using Real-Time Analysis Software . For gene expression analysis, reads were mapped to the mouse genome (UCSC mm10 genome assembly and NCBI RefSeq database) using TopHat2 and Bowtie. Cufflinks was used to estimate gene expression levels based on reads per kilobase of exon per million mapped reads (RPKM) normalization. Differentially expressed genes (DEGs) were extracted from the Cuffdiff results. The PANTHER  Classification System ver13.0 (http://pantherdb.org/) was used for pathway analysis, and Venny 2.1 (http://bioinfogp.cnb.csic.es/tools/venny/) was used for Ven diagrams. The microarray data of PGCs, ESCs and 1-day cultured PGCs in previous studies; GEO:assession: GSE30056 (E9.5 PGC and ESC) [RNA-seq libraries were prepared from 500 ng of total RNA purified from and ESC) , GSE6761and ESC) , were anBcl3, Klf9, Nupr1, Psrc1, Tbx3, and Tead4 were amplified from VR15 ESC cDNA using the primer sets shown in EcoR I/Not I site of CSII-EF-MCS lentivirus vector by using In-Fusion HD Cloning Kit (Takara Bio) according to the manufacturer’s instructions. For Bnc1-, Isl2-, p53-, and Klf9-knock-down (KD) vectors, pairs of oligonucleotides shown in Age I/ EcoR I site of pLKO.1 lentivirus vector. Lentivirus particles were produced as described previously [For over-expression (OE) vectors, coding regions of eviously . Brieflyeviously  and storPGC culture for reprogramming was carried out as described previously ,25 with Statistical analysis was performed using the Student’s t-test. P values < 0.05 were considered to be statistically significant.To identify candidate genes that control PGC reprogramming into EGCs, we re-analyzed published microarray data ,22,23 anNext, we confirmed whether the expression of the 39 candidate genes was significantly different between ESCs and PGCs with RT-qPCR. We selected six and two genes whose expression was more than 10 times higher or lower, respectively, in PGCs from E12.5 embryos than in two different ESCs  as possibly important genes for PGC reprogramming .p53-KD, which enhances PGC reprogramming by suppressing apoptosis [Bcl3-OE and Klf9-OE significantly enhanced EGC formation  or knock-down (KD) of the candidate genes whose expression was up- or down-regulated, respectively, in ESCs compared with PGCs. For OE or KD, we infected E12.5 PGCs with lentivirus vectors and cultured the cells in conditions required for EGC formation. poptosis , was incormation . We conformation . The remormation .Bcl3 and Klf9 in ESCs were 30~150 folds higher than those in PGCs . We identified 781 and 959 differentially expressed genes with two times higher or lower expression, respectively, in Klf9-OE PGCs compared with control PGCs  pathway were enriched among genes commonly down-regulated by nditions . These dnditions . PreviouS1 FigThe expression of candidate genes was determined with RT-qPCR, and relative expression levels in VR15-ESCs and BVSC-ESCs compared with those in E12.5 PGCs are shown. Genes whose expression is less than 10 times higher (A) or lower (B) in ESCs compared to PGCs are shown. Error bars show the SE of three independent experiments. *p<0.05, **p<0.01, ***p<0.001.(TIF)Click here for additional data file.S2 FigBcl3 or Klf9 expression in Bcl3-OE or Klf9-OE PGCs by infecting the lenti-virus vectors at MOI 5 (A) and MOI 0.2 (B) after culturing for 2 days. The expression was determined by RT-qPCR.  Relative efficiency of EGC formation by Bcl3-OE or Klf9-OE (MOI 0.2) (C) or by Bcl3-OE and/or Klf9-OE (MOI 5) (D) in PGCs compared with that of control. EGC colonies are identified by alkaline phosphatase staining. Error bars show the SE of four , two (B) independent experiment. *p < 0.05, **p < 0.01, ***p < 0.001. Induction of (TIF)Click here for additional data file.S3 FigBnc1-KD, Isl2-KD, Nupr1-OE, Psrc1-OE, Tead4-OE, and Tbx3-OE PGCs compared with control is shown. Error bars show the SE of three independent experiment. *p < 0.05. **p < 0.01.Relative efficiency of EGC formation by (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 TableGenes highlighted in gray are G alpha (i)-related genes.(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


Triterpenoids are the most abundant secondary metabolites present in marine organisms, such as marine sponges, sea cucumbers, marine algae and marine-derived fungi. A large number of triterpenoids are known to exhibit cytotoxicity against a variety of tumor cells, as well as anticancer efficacy in preclinical animal models. In this review efforts have been taken to review the structural features and the potential use of triterpenoids of marine origin to be used in the pharmaceutical industry as potential anti-cancer drug leads. Geodititoxicity .35–38), and erylosides F (39) . Four compounds induced the early apoptosis of Ehrlich carcinoma cells, where erylosides F3 have shown the highest activity at a concentration of 100 µg/mL [Five cytotoxic triterpene glycosides, erylosides F1-F4 (s F (39)  were iso00 µg/mL .40–48) and sodwanones K-W (49–61), have been isolated from the Indo-Pacific sponge Axinella wltneri [46), H (47), and I (48) have been found to have cytotoxic activity. The compounds have shown cytotoxicity activity against cell cultures of P-388 murine leukemia, A-549 human lung carcinoma, HT-29 human colon carcinoma, and MEL-28 human melanoma. Sodwanones G (46), H (47), I (48) showed high specificity towards human lung carcinoma cell line A-549, where the specificity of sodwanone G was prominent (46) [49), L (50), and M (51) were found to be cytotoxic to P-388 murine leukemia cells [57) was evaluated against 13 human tumor cell lines [60) inhibited both hypoxia-induced and iron chelator -induced HIF-1 activation in T47D breast tumor cells (IC50 15 µM), and sodwanone V (60) was the only sodwanone that inhibited HIF-1 activation in PC-3 prostate tumor cells (IC50 15 µM).The special group of triterpenoids named sodwanones: sodwanones A-I ( wltneri . Sodwanoent (46) . The cytia cells . The bioll lines . Sodwano40) and sodwanone T (58) inhibited hypoxia-induced HIF-1 activation in T47D cells , and sodwanone V (60) showed cytotoxicity to MDA-MB-231 breast tumor cells (IC50 23 µM). Sodwanone derived compounds, 3-epi-sodwanone K (62), 3-epi-sodwanone K 3-acetate (63), 10,11-dihydrosodwanone B (64) have been isolated from Axinella sp., and 62 and 64 also inhibited hypoxia-induced HIF-1 activation in T47D cells  and 63 was cytotoxic to T47D cells (IC50 22 µM) [Sodwanone A (0 22 µM)  Figure 0 and sod65–83 (65), raspacionins A (66), raspacionins B (67), 21-deacetylraspacionin (68), 10-acetoxy-21-deacetyl-28-hydroraspacionin (69), 10-acetoxy-21-deacetyl-4-oxo-28-hydroraspacionin (70), 10-acetoxy-15,21-dideacetyl-4-oxo-28-hydroraspacionin (71), 10-acetoxy-15-deacetyl-4-oxo-28-hydroraspacionin (72), 10-acetoxy-4-acetyl-15-deacetyl-28-hydroraspacionin (73), 10-acetoxy-15-deacetyl-4-21-dioxo-28-hydroraspacionin (74), 10-hydroxy-4,21-dioxo-28-hydroraspacionin (75), 21-oxoraspacionin (76), 15-deacetyl-21-dioxo-raspacionin (77), 4,21-dioxo-raspacionin (78), 10-acetoxy-4,21-dioxo-28-hydroraspacionin (79), 10-acetoxy-4-acetyl-21-oxo-28-hydroraspacionin (80), 10-acetoxy-4-acetyl-28-hydroraspacionin (81), 10-acetoxy-28-hydroraspacionin (82) and 10-acetoxy-21-deacetyl-4-acetyl-28-hydroraspacionin (83), have been isolated from red sponge, Raspaciona aculeuta Johnston (family Raspailiidae), and from the Mediterranean sponge Raspaciona aculeata. All the compounds have showed cytotoxicity against MCF-7 tumor cell line with IC50 values between 4 and 8 µM [Raspacionin triterpinoids 65–83 , namely and 8 µM ,35,36.Siphonochalina siphonella is a rich source of sipholane triterpenoids including sipholenols  , sipholenones  , and siphonellinols  . Sipholenol A (84) and sipholenone A (sipholenol B) are the major sipholane triterpenoids [84), sipholenol I (91), sipholenol L (94), sipholenone A (95), sipholenone E (96), siphonellinol C (97), and siphonellinol D (98) have found to show potent reversal of multidrug resistance in cancer cells that over expressed P-glycoprotein. These compounds enhanced the cytotoxicity of several P-glycoprotein substrate anticancer drugs, and significantly reversed the multidrug resistance phenotype in P-glycoprotein-overexpressing multidrug resistant cancer cells KB-C2 and KB-V1 in a dose-dependent manner [The Red Sea sponge rpenoids . Sipholerpenoids . Sipholet manner ,40 , B (101), and C (102), pervicoside C (103) and holothurin A (104) isolated from Holothuria fuscocinerea Jaeger on human leukemia HL-60 and human hepatoma BEL-7402 cells was analyzed and all compounds have shown a potent cytotoxicity towards both cell lines. However, fuscocineroside C was found to be the most potent  in HL-60 and BEL-7402 cell lines respectively [Triterpenoid glycosides (saponins) are the major and most abundant type of compounds isolated from sea cucumbers. Saponins are generally perceived as highly active natural product and the sea cucumber saponins have been well characterized for their anti-cancer activities. The cytotoxicity of five triterpene glycosides, fuscocineroside A  and A4 (106) found to be strongly cytotoxic to cancer cell lines; human epidermoid carcinoma (KB) and human hepatocellular carcinoma (Hep-G2), with IC50 values of 0.87 and 0.32 µg/mL (for compound 7) and of 1.12 and 0.57 µg/mL (for compound 8), respectively [The triterpene glycosides from the sea cucumber ectively   also exhibited significant cytotoxicity against different human tumor cell lines while showing the highest activity towards human colorectal carcinoma (HCT-116) cells (IC50 = 0.14 µM) with more potency than the employed positive control, 10-hydroxycamptothecin (IC50 = 0.84 µM) [108) and C (109) have also shown potent cytotoxicity against human tumor cell lines, adenocarcinomic human alveolar basal epithelial cells (A549), HCT-116, HepG2, and human breast adenocarcinoma (MCF-7) cell lines. The cytotoxicity of the compounds on A549  and HCT-116  cells were more potent than the positive control V-16 (110) and E (111) have also been tested for their anticancer activities in above human cancer cell lines and revealed a significant activity with IC50 values in the range of 3.36–7.77 µg/mL [108 and 109 are potent cytotoxic agents compared to compounds 110 and 111. It has been reported that the length and type of sugar moieties of glycosides play an important role in terms of cytotoxic activity against tumor cells and this observation clearly indicates that.Arguside A (0.84 µM) . Argusidrol V-16 . However77 µg/mL  Figure 7 also exin vitro cytotoxicity of impatienside A (112) and bivittoside D (113) were evaluated extensively by employing seven human cancer cell lines and the results showed that both glycosides exhibited in vitro cytotoxicities similar to or better than that of the potent anticancer drug etoposide (V-16) in four human tumor cells, A549 , HCT-116 , DU-145 , KB . The structural differences between glycosides 112 and 113 limited to their holostane skeleton, and no significant difference in the cytotoxicity of the two glycosides was found. However, pervicoside C (103), an analogue of 113 having the same aglycone but a different sugar chain, isolated from Holothuria fuscocinerea Jaeger, exhibited weak activities against HCT-116 and A549 cancer cells, with IC50 values of 18.7 and 28.6 µg/mL, respectively [Moreover, the ectively . Accordi114) and griseaside A (115) are identified as promising anticancer agents due to their significantly higher cytotoxicity against four human tumor cell lines, A549  , HL-60 , BEL-7402 , and human acute lymphoblastic leukemia cell line (Molt-4)  compared to the positive control HCP  [17-Dehydroxyholothurinoside A ( 2.2 µM)  Figure 4 and gri116) has also been tested for its anticancer potential against eight human tumor cell lines  and has exhibited cytotoxicity with IC50 values in the range of 0.15–3.20 µg/mL [116 has shown more potent cytotoxicity towards CAKI-1 (IC50 = 0.15 µg/mL) and KB-VIN (IC50 = 2.81 µg/mL) cell lines. Three new triterpene glycosides, intercedensides A (117), B (118), and C (119) from Mensamaria intercedens Lampert, were widely studied for their anticancer activity employing 10 human tumor cell lines . Interestingly all compounds showed a significant cytotoxicity against all tumor cell lines within the IC50 value range of 0.7–4 µg/mL, and the compounds 117 and 119 showed similar potencies, while compound 118 was generally more potent in all cell lines. Furthermore, compound 117 also exhibited significant in vivo antineoplastic activity against mouse Lewis lung cancer and mouse S180 sarcoma, with 48.39% and 57.48% tumor reduction levels [Hillaside C  showed a significant cytotoxicity (IC50 = 0.75–3.50 µg/mL) against ten tumor cell lines  [121) from Pseudocolochirus violaceus exhibited significant cytotoxicity against cancer cell lines MKN-45 (human gastric adenocarcinoma) and HCT-116 with IC50 values in the range of 0.052–0.442 µM and both compounds showed significantly higher activity against HCT-116 compared to the positive control HCP [A new sulfated triterpene glycoside from ls-A431) . Further122) and B (123) showed significant cytotoxicity (IC50 = 0.75–3.50 µg/mL) against ten tumor cell lines  [Moreover, the sulfated triterpene glycosides, philinopsides A (and PC3)  , cucumarioside A2-2 (125), echinoside A (126) and ds-echinoside A (127) have been reported against several cancer types in vitro and in vivo (in vivo studies have confirmed that frondoside A (100 µg/kg/day) effectively decreased the growth of breast cancer xenografts in athymic mice without exerting any side-effects [−1) to the mice bearing H22 hepatocarcinoma tumors has reduced the tumor weight by 49.8% and 55% respectively [Even though a number of saponin compounds have been isolated and identified as potent cytotoxic agents only few of them have been studied to unravel the mode of their cytotoxicity. Among them detailed cytotoxic mechanisms of frondoside A ( in vivo . All fou-effects . Moreoveectively . These s128) and (21a)-21-hydroxythyrsiferol (129) were isolated and identified from the marine red alga Laurencia mariannensis, which was collected off the coast of Hainan and Weizhou Islands of China. Both compounds have displayed significant cytotoxic activity against P-388 tumor cells with IC50 values of 0.6 and 6.6 mg/mL, respectively [Two cytotoxic squalenoid-derived triterpenoids, laurenmariannol , isodehydrothyrsiferol (131) and 10-epidehydrothyrisiferol (132), isolated from Laurencia viridis, have shown potent cytotoxic activities against a number of cancer cell lines [The red seaweed ll lines ,57.133), 22-hydroxy-15(28)-dehydrovenustatriol (134), 1,2-dehydropseudo-dehydrothyrsiferol (135), and secodehydrothyrsiferol (136) exhibited significant cytotoxic activity against a panel of cancer cell lines [137), thyresenol A (138) and thyrsenol B (139) were also isolated from Laurencia viridis, and these compounds have exhibited significant inhibitory action on protein phosphatase at a concentration of 10 mM. Moreover, they have shown potent cytotoxic activity against P388 cell line [Polyethers, iubol , l5-anhy-drothyrsiferyl diacetate diacetate (141), magireol-A (142), magireol B (143) and magireol C (144) were isolated from Japanese red alga Laurencia obtuse [Five cytotoxic triterpenoids 28-anhy-drothyrsiferyl diacetate , and cyclosadol (146) were isolated from brown algae Kjellmaniella crassifolia, two compounds were obtained from this species for the first time. 3-epicyclomusalenol (145), and cyclosadol (146) have been reported to have moderate chemo preventive effects [Two cycloartane-type triterpenoids, 3-epicyclomusalenol  were isolated from the fermented extract of Xylarialean sp. A45, an endophytic fungus of Annona squamosa L., and their structures were determined by NMR spectroscopy. These compounds have shown modest cytotoxic activities against human tumor cell line HepG2 [Triterpenoids are frequently found in marine source, but have rarely been reported from marine-derived fungi. Rainer Ebel have reviewed 7 triterpenoids from marine-derived fungi . In 2011ne HepG2  (Figure et al. [Even though not much research in this area has been carried out, the anti-cancer activity of triterpenes is believed to be directly correlated to their structural features. As suggested by many authors the bioactivity of the triterpenes is a result of its strong membranolytic activity, and this membranolytic activity is a function of the structural features of the glycosides . In tritet al.  have repet al. .Up to date the vast chemodiversity in the oceans has paved the way for natural product chemists to mine for new bioactive compounds. Among them triterpenoids are one of the most studied classes of compounds. Due to the extreme environments in the oceans, survival demands have resulted in the evolution of these sophisticated toxic compounds and this fact is confirmed by the proven toxicity of these compounds in biochemical studies. Triterpenoids derived from sea cucumbers, sponges and algae have been used as ingredients in Traditional Chinese Medicine for years. Even though there are many lead compounds with promising potential to be used as drugs for cancer therapy, the cytotoxicity itself would be a constraint for this purpose, because most of the compounds could be cytotoxic towards normal cells in addition to the cancerous cells. In identification of therapeutics from natural products the preference is given to the compounds having high specificity towards the cancer cells in their cytotoxic action, while minimizing the damage to normal cells . TherefoMoreover, the possibility of continuous supply of the product and the ecological importance of the triterpene sources such as sponges, sea cucumbers and algae are factors of importance before entering to the drug development phase. Sustainable production of compound through chemical synthesis or culturing of these marine organisms should be ensured. The structural complexities have challenged the chemical synthesis and thus it would limit the entering of these compounds to drug development phase. However, with the advances in synthetic chemistry and understanding of triterpene biosynthetic processes, new opportunities for exploitation of these compounds as drug leads are opening up.


Benzothiazole (BTA) belongs to the heterocyclic class of bicyclic compounds. BTA derivatives possesses broad spectrum biological activities such as anticancer, antioxidant, anti-inflammatory, anti-tumour, antiviral, antibacterial, anti-proliferative, anti-diabetic, anti-convulsant, analgesic, anti-tubercular, antimalarial, anti-leishmanial, anti-histaminic and anti-fungal among others. The BTA scaffolds showed a crucial role in the inhibition of the metalloenzyme carbonic anhydrase (CA). In this review an extensive literature survey over the last decade discloses the role of BTA derivatives mainly as anticancer agents. Such compounds are effective against various types of cancer cell lines through a multitude of mechanisms, some of which are poorly studied or understood. The inhibition of tumour associated CAs by BTA derivatives is on the other hand better investigated and such compounds may serve as anticancer leads for the development of agents effective against hypoxic tumours. Five membered heterocyclic which incorporate oxygen, nitrogen and sulphur are found in broad spectrum therapeutic agents which have an enormous significance in drug discovery and drug development processesThe pharmacological profile of the drug used for the management of amyotrophic lateral sclerosis Riluzole  attracteThe BTA scaffold possesses a wide spectrum of biological activities such as anti-inflammatoryCancer is the most prominent, notably complex and lethal disease which became a serious concern of today’s medical science. It poses a great challenge to medical scientific community for development of drugs, medicines and procedures for safer treatment and cure of cancer disease2.2.1.1 (3–(5-fluorobenzo[d]thiazol-2-yl)phenol) and 2 (4–(5-fluorobenzo[d]thiazol-2-yl)phenol) having hydroxyl substituents on the third and fourth position of phenyl exhibited the best activity having GI50 values of 0.57 and 0.4 µM respectively against MCF-cell line as compared to BTA derivatives containing alkoxy, methyl sulphonyl and ethyl substituents on the benzothiazole (N-bis-benzothiazole and benzothiazolyl thiocarbamide derivatives and screened for cytotoxic activities against two human cell lines U-937 (human macrophage cell line), THP-1 (human leukaemia monocytic cell line) and B16-F10 (mouse melanoma cell line). The thiourea containing benzothiazole derivative 3 ), (4847.73 ± 2.39 µM)) and (34.58 ± 1.73 µM)) as compared to standard compound etoposide IC50 values (17.94 ± 0.89), (18.69 ± 0.94) and (2.16 ± 0.11 µM)) against U-937, B16-F10 and THP-1 cell lines respectivelyAiello et al. synthesised fluorinated 2-aryl benzothiazole derivatives and evaluate them for anti-tumour activities against cancer cell lines such as MDA-MB-468 (mammary gland/breast tissues derived from metastatic site) and MCF-7 cell line (human breast adenocarcinoma). The fluorinated BTA derivatives thiazole . Kumbharvative 3  demonstr4 , non-small cell lung cell lines , colon cancer cell lines , central nervous system cancer cell lines , melanoma cell lines , ovarian cancer cell lines , renal cancer cell lines , prostate cancer cell lines  and breast cancer cell lines  in comparison with reference drug 5-fluorouracil NSC 19893. The BTA scaffold 5 showed remarkable growth inhibitory activities against different human tumour cell lines such as leukaemia cell lines , non-small cell lung cell lines , colon cancer cell lines , central nervous system cancer cell lines , melanoma cell lines , ovarian cancer cell lines , renal cancer cell lines , prostate cancer cell lines  and breast cancer cell lines  as compared with reference drug 5-fluorouracil NSC 19893. The scaffolds 4 and 5 showed the best anticancer therapeutic potential due to presence of electron with drawing groups on para position of phenyl ringCaputo et al. afforded two types of five derivatives on the basis of an aryl amide and an aryl urea functionalities attached at C-2 of benzothiazole core and these scaffolds were screened against 60 human cancer cell lines. The urea moiety based fluorophenyl containing benzothiazole derivative 4  and cyan4  and A-498 (human kidney carcinoma cell line) with GI50 values of 0.542 µM and 1.02 µM respectively. This compound also possess efficacy against UO-31 and RXF 393 cell lines. The derivatives 7 and 9 because of 3,4-disubstitutedphenyl moiety, exhibited excellent anti-proliferative activities with low IG50 values of 1.85, 2.10 µM against RCC and ACHN cell lines respectively. The SAR study revealed the fact that sorafenib analogues possesses anti proliferative activity due to the presence of both urea spacer and phenyl disubstitution. Compound 8 demonstrated the highest CLogP value being the most lipophilic and potent derivative, in the low µM rangeEl-Damasy et al. synthesised the novel amide and urea based BTA series of 20 sorafenib analogues in which the pyridylamide privileged functionality was attached with an ether linkage at 6-position of the BTA ring. A selected group of 12 potent scaffolds were evaluated and appraised for anti-proliferative activities against sixty human cancer cell lines. These chlorotrifluoromethyl phenyl ureido picolinamide benzothiazoles 7 , bis-tri7 , MNK-45 (gastric cancer cell line), NCI-H226 (human lung cancer cell line), HT-29  and SK-N-SH (neuroblastoma cell line). The substituted 2-hydroxybenzylidene containing semicarbazide 10 -hydrazino moiety at the C-2 position which remarkably enhances the anti-tumour potential, whereas replacing the 4-hydroxy moiety with 4-methoxy decreased the activities against both cell linesGabr et al. obtained hydrazine derivatives by treating 2-amino-6-fluorobenzothiazole with hydrazine hydrate which was further treated with the suitable aldehydes to afford 27 different BTA Schiff base derivatives. These derivatives were screened for anti-tumour potential against Hela  and COS-7 (kidney fibroblast cancer) cell lines. The hydrazine based benzothiazole 11  exhibite12 , human lung cancer cell (H460), non-small cell lung cancer (A549) and human breast cancer (MDA-MB-231). Among these derivatives, the indole based hydrazine carboxamide scaffold 12  showed p2.2.13 (2–(1H-benzo[d]imidazol-2-ylthio)-N-(4-(benzo[d]thiazol-2-yl)-3-chlorophenyl) acetamide) (14 (N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(1-phenyl-1H-benzo[d]imidazol-2-yl-thio)-acetamide)  benzothiazole derivatives with reference to the heterocyclic substitution was benzimidazole Yurttas et al. obtained 2–(4-aminophenyl)BTA derivatives substituted with different heterocyclic rings and tested their antitumor potential against 60 human tumour cell lines. The BTA derivatives etamide)  and 14 (etamide)  showed r15 piperazin-1-yl)-2-oxoethyl)-4-chloro-benzenesulfonodithioamide) showed antiproliferative activity (CC50 = 8 ± 3 µM) against human derived DU-145 cell line (17 (N-(2–(4-(benzo[d]thiazol-2-yl) piperazin-1-yl)-2-oxoethyl)-2,5-dichloro benzenesulfonodithio-amide) demonstrated remarkable activities against several human derived cell lines such as HepG2 and DU-145  , DU-145 (human prostate cancer cell lines express androgen receptor), HepG-2 cell line (human liver cancer), WIL-2NS (Human splenic B-lymphoblastoid cells), MRC-5 (human lung fibroblast cell line), MCF-7 cell line (human breast adenocarcinoma), MT-4 (human T-cells containing an integrated HTLV-1 genome), SK-MES-1 cell line (human lung cancer) and SK-28 cell line (skin melanoma). Derivative ell line  whereas ctively) . Derivat50 values of these derivatives indicated that all compounds exhibited good potential against the aforementioned cell lines but the pyridine containing derivative 18 , MCF-7 (Breast) and HCT-116 . GIative 18  had a re2.4.in-vitro against different tumour cell lines. BTA derivatives 19 (N-(benzo[d]thiazol-2-yl)-2–(5-(1–(2-chlorophenoxy)propyl)-1,3,4-oxadiazol-2-ylthio)acetamide) and 20 (N-(benzo[d]thiazol-2-yl)-2–ethyl)-1,3,4-oxadiazol-2-ylthio) acetamide) exhibited remarkable activities against CCRF-CEM (leukaemia) cell lines. The CC50 values of compounds 19 and 20  indicated that the thiophene based acetamide benzothiazole derivatives 21 , morphol21 , HeLa, A549, HCT-116, HepG2 and SKRB-3 breast cancer cell line. The substituted bromopyridine acetamide benzothiazole derivative 29  showed p30 Kamal et al. reported the synthesis of novel phenyl pyridopyrimidinones based BTA derivatives which were examined against four different cancer cell lines such as ME-180, DU-145, MCF-7 and B-16. The pyridine containing pyrimidine benzothiazole 31  exhibite2.9.32  pathways, which regulate the equilibrium between apoptosis and cell proliferation. The SAR explained that the maximum cytotoxicity of derivative 34 against colon cancer cell line was due to the presence of methoxy group (–OCH3) in the phenylKambhare et al. synthesised the isoxazole pyrimidine based BTAs and evaluated them for anticancer activity by the MTT assay against different cell lines such as A549, Colo205, MCF-7 and U937 cell lines, in comparison with the standard drug etoposide. The pyridine containing pyrimidine derivative 34  demonstr35 , breast carcinoma (MCF-7), cervical carcinoma (HeLa), pancreatic carcinoma (MiaPaCa-2), colon carcinoma (SW 620), lung carcinoma (H 460), and diploid fibroblasts (WI 38). Among them, the pyrimidine based carbonitrile benzothiazole derivative 36  showed p2.11.37  cell line, human lung adenocarcinoma epithelial (A549) cell line, human breast adenocarcinoma (MCF-7) cell line, human colorectal adenocarcinoma (HT-29) cell line and mouse embryo fibroblast (NIH3T3) cell line. The piperidine based acetohydrazide derivative 37  showed m2.12.38  The sulphonamide scaffold based BTA 40  exhibite2.13.41 and the substituted chloromethylbenzamide benzothiazole 42 . The benzamide benzothiazole derivative azole 44  showed p45  phenyl]-2-aryloxyacetamide derivatives for cytotoxicity and anticancer activity against sixty human cancer cell lines derived from 9 neoplastic diseases, among which L (leukaemia), M (melanoma), RC  NSCLC  CC (colon cancer), OC (ovar ian cancer), BC (breast cancer), CNSC  and PC (prostate cancer). Derivatives 49 (N-(4-(benzo[d]thiazol-2-yl)-3-methoxyphenyl)-3–(4-chlorophenyl)propanamide) and 50 (N-(4-(benzo[d]thiazol-2-yl)-2-chlorophenyl)-2–(4-chlorophenylthio)acetamide)   exhibite51 . The SAR studies showed that the highest activity of compound 51 was due to the presence of three chlorine atoms in the compound as compared to other derivativesNoolvi et al. reported the synthesis of chloro substituted benzothiazole amines in order to produce isothiocyanates and thioureases. These BTA derivatives were tested for their anticancer activities. The dichlorophenyl containing chlorobenzothiazole 51  showed g52 (N-(4-methoxyphenyl)-acetamide substitutions on position 5 of the 4-thiazolidinones ring enhanced the anti-cancer potentialHavrylyuk et al. screened novel 4-thiazolidinone benzothiazole derivatives against ovarian, renal, prostate, leukaemia, melanoma, lung, colon, CNS and breast cancer cell lines. The thioxothiazolidine acetamide benzothiazole 52  showed t53  and liver cancer (HEPG2) cell lines. Among all these derivatives, nitrobenzylidene containing thiazolidine derivative 54  exhibite55  complexes containing benzothiazole derivatives and tested their anticancer activity against K-562 and KE-37 (human leukemic) cell lines. Among all these derivatives, the Ru(III) containing methylbenzothiazole 60  exhibite61 and derivative 62  cell line. Derivatives ative 62  exhibite63 and 64 benzothiazole-6-carbohydrazide 65  in Phase I/IIb clinical trials for the management of hypoxic metastatic tumours,Carbonic anhydrases  are zinc enzymes which catalyse the reversible interconversion between CO72, a CAI in clinical use for decades as lead molecule, a multitude of primary sulphonamides (e.g. compounds 73–75) as well as the secondary siulfonamide 76 were reported to act as highly efficient, frequently low nanomolar inhibitors against the tumour-associated isoforms CA IX and XIIex vivo or in vivo studies are available so far with these potent CA IX/XII inhibitors, but compounds belonging to other classes of sulphonamides were proved to possess significant antitumor effects in vivo when they act as potent inhibitors of these two CA isoforms,.Indeed, using the ethoxzolamide 4.Benzothiazole is a pharmacophore widely used in medicinal chemistry. This review points out to a growing interest in the development of lead or hybrid structures bearing the BTA moiety as antiproliferative and anticancer agents. The present work describes the potential of BTA scaffolds in the management of various types of cancers such as ovarian, prostate, central nervous system, renal, gastric, pancreatic, liver, breast and colon cancers. SAR studies reaveled that the anticancer activity of BTA scaffolds depends upon the nature of substituents present in these molecules, being multifactorial and not always easy to rationalise. The plethora of research on the anticancer profile of BTA derivatives mentioned in this review and their rationalisation based on the drug targets of these derivatives, when this was possible, may be useful for the development of novel such agents.