Entamoeba histolytica. Previous studies have reported unconventional myosins to play significant role in membrane based motility as well as endocytic processes. EhMyosin IB is the only unconventional myosin present in E. histolytica, is thought to be involved in both of these processes. Here, we report an interaction between the SH3 domain of EhMyosin IB and c-terminal domain of EhFP10, a Rho guanine nucleotide exchange factor. EhFP10 was found to be confined to Entamoeba species only, and to contain a c-terminal domain that binds and bundles actin filaments. EhFP10 was observed to localize in the membrane ruffles, phagocytic and macropinocytic cups of E. histolytica trophozoites. It was also found in early pinosomes but not early phagosomes. A crystal structure of the c-terminal SH3 domain of EhMyosin IB (EhMySH3) in complex with an EhFP10 peptide and co-localization studies established the interaction of EhMySH3 with EhFP10. This interaction was shown to lead to inhibition of actin bundling activity and to thereby regulate actin dynamics during endocytosis. We hypothesize that unique domain architecture of EhFP10 might be compensating the absence of Wasp and related proteins in Entamoeba, which are known partners of myosin SH3 domains in other eukaryotes. Our findings also highlights the role of actin bundling during endocytosis.Motility and phagocytosis are key processes that are involved in invasive amoebiasis disease caused by intestinal parasite  Entamoeba histolyica is a highly motile human pathogen which eats the blood cells and immune cells by phagocytosis during progression of Amoebiasis disease. E. histolytica infections are a major concern in the developing countries. Myosins are motor proteins that move over actin cytoskeleton to drive the cellular processes. Unconventional myosins are a type of myosin which are different from myosin present in muscles, and are involved in regulation of membrane dependent processes crucial for cellular movement and endocytosis. In contrast to other eukaryotes, Entamoeba has only one unconventional myosin, Myosin IB which shows more similarity with metazoan myosins rather than amoeboid myosins. Myosin IB has been shown to be involved in phagocytosis. The exact role played by Myosin IB in the phagocytic process is still not fully understood.Entamoeba as well as in other organisms. Actin binding studies indicated that the c-terminal domain of EhFP10 binds to actin filaments and leads to formation of thicker actin bundles. Myosin IB interaction with EhFP10 inhibits the formation of actin bundles. Through our results, we could hypothesize that the presence of a unique GEF like EhFP10 could compensate for the absence of WASP proteins in E. histolytica which have been found to interact with the myosin I SH3 domain in other organisms and regulate actin dynamics during endocytosis. Our study reveals a rare interaction of a myosin with a GEF, which interact to regulate actin bundling. EhMyosin IB is different from other amoeboid myosins and lies between the metazoan and amoeboid myosins like human myosin IE. Hence, the findings have broader implication to completely understand the closure stage of a phagocytic and pinocytic cup.SH3 domain is present at the c-terminal tail of Myosin IB which has been found to interact with proteins that regulate the actin cytoskeleton in other organisms. In this work, we have identified EhFP10 as one of the interacting proteins of EhMyosin IB SH3 domain through a co-crystal structure and biophysical experiments. Our localisation studies demonstrated the involvement of EhFP10 in phagocytosis and pinocytosis. This is the first report of the involvement of a FYVE domain containing GEF in pinocytosis. We have also analysed that EhFP10 has a unique c-terminal domain not present in any other FYVE family GEFs in  Entamoeba histolytica is the causative agent of amoebiasis disease in humans, a major public health problem in developing countries. Amoebiasis is the third-leading cause of deaths resulting from parasitic infections  and class II [xPxxPx(K/R)] types , 32. To utilized . The datutilized . The scrutilized  as descrP motifs . Since nEhMySH3 was predicted to preferentially bind pseudo-symmetrical and class II ligands . P2 residues A3 and P4 formed the first XP motif, which fit in the first consensus hydrophobic pocket made by residues Y9 and Y52 of EhMySH3; and I5 and P6 formed the second XP motif, which occupied the second hydrophobic pocket made by residues P49 and W36 of EhMySH3. The c-terminal residue R9 of P2 was observed to interact with residue E34 of the n-Src loop present in the specificity pocket of EhMySH3. In other available ligand-bound SH3 structures, terminal Arg or Lys residues interact with a residue of the RT loop, which forms the specificity pocket. In our case, though the electron density for the terminal R9 residue was not complete, after refinement we obtained better electron density near the n-Src loop, though there could be an alternate conformation near E18 in the RT loop, likely to be one of the probable orientations for residue R9. Overall, we concluded that the EhFP10 peptide, P2 binds to PxxP motif of EhMySH3 in a class II peptide binding orientation and, due to its pseudo-symmetrical sequence, did so in a way resulting in the clustering of the two EhMySH3 domains , followed by a FYVE domain (439–511) and a c-terminal domain (512–876) . Based oE. histolytica, EhFP10 was found to be the only GEF to have this unique c-terminal domain. The sequences of all Dbl-homology GEFs had highly conserved RhoGEF domain but different c-terminal domain. In a BLAST search across all databases, we could not find any GEF homologous to EhFP10. EhFP10 gene is present in all Entamoeba species. Homologues of EhFP10 of non- pathogenic strain E. dispar and E. invadens (pathogenic in reptiles), showed 95% and 35% identity with that of E. histolytica molecule respectively. Maximum differences were observed in the c-terminal domain  of 200 nm for EhMySH3-cterEhFP10 . In immunostained cells, GFP-EhFP10 was found mainly in the cytoplasm in proliferating cells. It was also seen in some membrane invaginations and cup-like projections  was added to the media, and endowed the media with red fluorescence. The presence of red fluorescence within internalized vesicles enriched with GFP-omplete. .EhFP10-overexpressing cells were utilized for this purpose. RBC membranes were stained with lipophilic, red fluorescent dye (Thermo Fisher). EhFP10 was seen to be enriched at the site of pseudopod formation near the cell membrane as soon as RBC came in contact with E. histolytica cells, and it later became enriched in the extending pseudopods during the cup progression and was present until phagosome scission  of progressing pseudopods while actin was present both at the tip and the base of the cup. As the cups progressed towards closure, all three proteins were found to colocalize at the tip of the closing pinocytic and phagocytic vesicles  was observed, there was an enrichment of vesicles . As the vesicles . Images yosin IB .EhFP10 and EhMyosin IB in both of the processes.These observations clearly pointed out the difference in molecular mechanisms governing macropinocytosis and phagocytosis and the involvement of EhFP10 has a c-terminal domain that shows similarity with APC basic and tau domains, a direct involvement of EhFP10 in actin dynamics was investigated. Purified recombinant cterEhFP10 domain was observed to bind and bundle purified rabbit muscle F-actin in centrifugation-based actin binding  was observed in the supernatant  and to help in the formation of the Arp2/3-mediated endocytic complex. These observations led us to further investigate the potential for EhFP10 to serve as a binding partner of the EhMyosin IB SH3 domain. SPR studies and the co-crystal structure of peptide P2 from cterEhFP10 with the EhMySH3 domain, as well as a pull-down assay validated the proposed interaction.The SH3 domain of myosin I isoforms in yeast interact with proline-rich regions of Vrp1 and recruits the Arp2/3 complex . In amoedynamics , 42. Thu EhMySH3  and predEhFP10 has a unique domain organisation, not present in any homologous GEFs. It has sequence similarity only with the APC basic domain from adenomatous polyposis coli protein and the tau protein domain, though it is very low. Both the APC basic and tau domains have been shown to bind actin filaments as well as microtubules and to each act as a molecular linker mediating actin-microtubule crosstalk  and class II type [xPxxPx(K/R)]. To identify proteins that interact with the c-terminal SH3 domain of utilized . All of utilized . From alEhMySH3 protein samples, each at a concentration of 30 mg/ml, were mixed with the respective peptides in different molar ratios and incubated overnight at 4°C. Crystallization trials were done with several commercial screens from Hampton Research and Molecular Dimensions. After four days at 4°C, small needle-like crystals appeared for the EhMySH3-P2 complex (mixed with in a molar ratio of 1:2) in crystal screen II (Hampton Research) conditions 0.2 M ammonium sulphate, 30% PEG 8000 and 0.2 M ammonium sulphate, 30% PEG 4000. Better crystals were obtained by macroseeding in 0.2 M ammonium sulphate and 30% PEG 8000 condition . The data sets were indexed and scaled using HKL2000 .For binding studies of EhFP10 domain, 1.175 ng/mm3 of EhMySH3 was immobilized on the activated chip surface and samples of the cterEhFP10 domain having different concentrations were passed over it. Association kinetics was studied for 400 s while the dissociation was monitored for 150 s. 1M NaCl was used for regeneration of the chip surface. SPR studies with F-actin was carried out by immobilizing 0.67 ng/mm3 of filamentous actin on the chip surface using the buffer 50 mM sodium acetate pH 4.1, 0.05 mM CaCl2, 0.05 mM ATP and 0.125 mM DTT. (A solution of 0.5 mg/ml F-actin was obtained by dissolving actin powder (Sigma) in 50 mM sodium acetate pH 4.0, 12.5 mM KCl, 0.5 mM MgCl2 and 0.25 mM ATP).For interaction studies with the purified cterAll of the data were recorded at 25°C. For the evaluation of the kinetics, differential sensorgrams were analyzed using Autolab SPR Kinetic Evaluation software.EhFP10 for two hours at 4°C. Following 3–4 washes with wash buffer , the proteins were eluted with 20 mM reduced glutathione, 50 mM Tris pH 7.4, and 150 mM NaCl. The fractions were loaded onto a 12% SDS-PAGE gel and analyzed.Purified GSTSH3 and GST were incubated for one hour at 4°C with glutathione Sepharose  pre-equilibrated in equilibration buffer . Then, it was washed thrice with the equilibration buffer and incubated with the same amount of purified cter2and 0.1 mM NaN3) and was allowed to polymerize for 1½ hours at RT by adding to its solution an Imix solution . cterEhFP10 and EhMySH3 proteins of various concentrations were each added to separate samples of the mixture containing polymerized F-actin in a total volume of 200 μl and incubated for 30 minutes at RT. Each of the resulting mixtures was centrifuged at 40,000 rpm for two hours in a Beckman ultracentrifuge. Pellet and supernatant fractions were collected and analyzed using 10% SDS-PAGE followed by Coomassie blue staining.G-actin was purified from rabbit muscle powder (sigma) . For F-aEhFP10 and EhMySH3 proteins of various concentrations were each added to separate samples of the mixture containing polymerized F-actin in a total volume of 50 μl and incubated for 30 minutes at RT. Each mixture was centrifuged at 12,000 rpm for 10 minutes. Pellet and supernatant fractions were collected and analyzed using 10% SDS-PAGE followed by Coomassie blue staining.For bundling assays, cterPurified alpha-actinin from rabbit skeletal muscle (cytoskeleton Inc.) was used as positive control and Bovine serum albumin (SRL) was used as negative control for both binding and bundling studies.EhMy1TD for mEhMyosin1B and EhGEFD for rEhFP10 Ab) were dialyzed against PBS. Six mice were immunized subcutaneously with 100 μg of protein per mice per injection with an interval of two weeks between each injection. The first dose of the protein was emulsified with complete Freund’s adjuvant while the following doses were emulsified with incomplete Freund’s adjuvant. Following the immunization series, the serum of each mouse was stored in aliquots at -80°C. To check the titer of the polyclonal antibody, western analysis of the E. histolytica lysate was performed.The purified antigenic proteins , 120 mM KCl, 0.15 mM CaCl2, 25 mM HEPES (pH 7.4), 2 mM EGTA, 5 mM MgCl2]. The washed cells were then resuspended in 0.8 ml of complete cytomix buffer  containing 200 μg of plasmid DNA and subjected to two consecutive pulses of 3000 V/cm (1.2 kV) at 25 μF . The transfectants were initially allowed to grow without any selection. Drug selection was initiated after two days of transfection in the presence of 10 μg/ml G-418 for constructs with luciferase reporter gene.3 medium . The cel3 medium . BrieflyEhFP10, washed cells were fixed with 3.7% pre-warmed paraformaldehyde for 60 min at room temperature. After the cells were fixed, they were again harvested at 280 xg for 7 min. In each of the two cases, the pellet was heated at 80°C for one minute, and then resuspended in 2x SDS dye without β-ME. The lysate was heated at 100°C for 7 minutes and centrifuged at 13000 rpm for 15 minutes to pellet down the debris. The supernatant was aliquoted and stored, and quantification was achieved by performing a BCA.One million trophozoites growing in log phase were harvested by centrifugation at 280 g for 7 min at 4°C. The pellet was then washed with chilled PBS pH 7.2 and pelleted. For probing E. histolytica trophozoites and transfectants were resuspended in incomplete TY1-33 medium and transferred onto acetone-cleaned coverslips placed in a Petri dish. The cells were allowed to adhere for 5 min at 37°C. The culture medium was discarded and the cells were fixed with 3.7% pre-warmed paraformaldehyde for 30 min. After fixation, the cells were permeabilized with 0.1% Triton X-100/PBS for 5 min, washed with PBS and then quenched for 30 min in PBS containing 50 mM NH4Cl. The coverslips were blocked with 1% BSA/PBS for 2 h, followed by incubation with primary antibody at 37°C for 1.5 h. The coverslips were washed three times with 1% BSA/PBS and then incubated with secondary antibody for 45 min at 37°C. Antibody dilutions used were mEhMyo1B at 1:100, anti-GFP (Sigma) at 1:100, rEhFP10 Ab at 1:150, anti-rabbit/mice Alexa 488, Alexa 556 and Pacific blue-410 (Molecular Probes) at 1:250, TRITC-Phalloidin at 1:250. Cells were further washed with 1% BSA/PBS twice and then PBS once and mounted on a glass slide using 1, 4-diazbicyclo  octane (DABCO) (55) 2.5% in 80% glycerol. The edges of the coverslip were sealed with nail-paint to avoid their becoming dried out. Confocal images were made using an Olympus Fluoview FV1000 laser scanning microscope.EhFP10 tagged at its N-terminus with GFP, (NGFP-EhFP10) were plated onto a 35 mm glass bottom dish and allowed to adhere to the dish. RBCs were labelled using CFDA SE dye . RBCs were collected by pricking a finger with a needle and collecting the blood in PBS. The cells were washed with PBS twice followed by incubation in PBS containing 10 μM CFDA for 10 min at 37°C with intermittent tapping. The reaction was stopped by washing the RBCs with PBS and then the RBCs were kept in ice. Labeled RBC or TRICT-Dextran (Sigma) containing media was added and time-lapse imaging was done using a spinning disk confocal microscope . The temperature was maintained at 37°C with the help of a chamber provided along with the microscope. The images were captured at 500 ms intervals. The raw images were processed using NIS element 3.20 analysis software.Amoebic cells expressing EhFP10, myosin IB and actin proteins were used for quantitation. Data with P < 0.05 were considered to be significant.All of the statistical analyses were done using GraphPad PRISMA 7 software using Student’s two-tailed t-test. Images of cells which were immunostained for all three proteins S1 VideoE. histolytica cells.Live cell movie showing localisation of GFP tagged EhFP10 protein (green) within (AVI)Click here for additional data file.S2 VideoE. histolytica cells activated for phagocytosis. RBC are red in colour.Live cell movie showing localisation of GFP tagged EhFP10 protein (green) within (AVI)Click here for additional data file.S3 VideoE. histolytica cells performing macropinocytosis. Media is red in colour. Engulfed media can be seen as red circular regions within the cell.Live cell movie showing localisation of GFP tagged EhFP10 protein (green) within (AVI)Click here for additional data file.S1 Fig(A)EhMy1TD, (B) GSTSH3, (C)EhGEFD, and (D) cterEhFP10. (E) Intact mass analysis of cterEhFP10: MS spectrum depicting a major peak equivalent to 28 kDa i.e., the molecular weight of cterEhFP10 although its runs on 12% SDS-PAGE gel at around 35 kDa.(TIF)Click here for additional data file.S2 FigEhMySH3 were obtained in 30% PEG 8000, 0.2 M ammonium sulphate condition of Crystal Screen II (Hampton Research) at 4°C.(A) Initial hits of EhMySH3 crystals were obtained after macroseeding with a few crystals from initial hits in the same condition.(B) Diffractable (TIF)Click here for additional data file.S3 FigEntamoeba of E. histolytica. . Inspection of the alignment showed that the maximum differences could be seen in the c-terminal domain region involved in actin reorganisation. E. dispar is 95 percent identical to E. histolytica while E. invadens is only 35 percent.A multiple-sequence alignment of EhFP10 sequences from various species of (TIF)Click here for additional data file.S4 Fig(A) Images from immunofluorescence studies with anti-GFP antibody in GFP-EhFP10-overexpressed cells, and EhFP10-specific antibodies showing both to colocalize well.(B) Bar graph depicting colocalization of GFP-tagged EhFP10 and untagged EhFP10. (C) Western blot depicting a band at 100 kDa equivalent to EhFP10 protein in wild type HM1 total lysate. Prebleed was used as a negative control. Ehcoactosin was used as a loading control. (D) Western blot depicting a band at about 130 kDa in lysate of GFP-EhFP10 cells while the GFP vector control showed only a band corresponding to GFP. Images from immunofluorescence studies in wild-type E. histolytica cells showed EhFP10 localized in membrane ruffles and cup-like projections and within pseudopod extensions and closing vesicles, during both pinocytosis and phagocytosis.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


FSA) is a phytotoxin produced by several Fusarium species and has been associated with plant disease development, although its role is still not well understood.Fusaric acid (FSA biosynthetic gene (FUB) cluster in Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) reduced the FSA production, and resulted in decreased disease symptoms and reduced fungal biomass in the host banana plants.Mutation of key genes in the FSA, both banana leaves and pseudostems exhibited increased sensitivity to Foc TR4 invasion. Banana embryogenic cell suspensions (ECSs) treated with FSA exhibited a lower rate of O2 uptake, loss of mitochondrial membrane potential, increased reactive oxygen species (ROS) accumulation, and greater nuclear condensation and cell death. Consistently, transcriptomic analysis of FSA‐treated ECSs showed that FSA may induce plant cell death through regulating the expression of genes involved in mitochondrial functions.When pretreated with FSA from Foc TR4 functions as a positive virulence factor and acts at the early stage of the disease development before the appearance of the fungal hyphae in the infected tissues.The results herein demonstrated that the  Musa spp.) is one of the most important economic crops for many Asian and African countries. Fusarium oxysporum f. sp. cubense (Foc) causes Fusarium wilt (Panama disease), one of the most destructive diseases of banana trees  and ‘Silk’  appeared and became the dominant cultivar for the industry. Foc race 2 affects the hybrid triploid cultivar ‘Bluggoe’ (ABB) and other cultivars closely related to ‘Bluggoe’  and the Foc ‘tropical’ race 4 (TR4) strains that affect the Cavendish bananas in the subtropics and tropics, respectively. Between these two strains, Foc TR4 is the more virulent one as it can infect Cavendish bananas under both stressed and nonstressed conditions   genes   ‘tropical’ race 4 (TR4) strain II5 (VCG01213) was used as the wild‐type (WT) for fungal transformation, targeted gene disruption and complementation experiments. All Foc strains were cultivated on potato dextrose agar (PDA) at 28°C.Fusarium species were downloaded from the given link: http://www.broadinstitute.org/annotation/genome/fusarium_group/MultiDownloads.html. PhyML 3.1  were inoculated with Foc TR4 isolates at a concentration of 1000 conidia g−1 of soil.The TR4‐susceptible ‘Cavendish’ banana (AAA) cv ‘Brazilian’ plantlets with six to seven leaves  equipped with filter blocks with spectral properties matching those of green fluorescent protein (GFP) (488 nm) and root autofluorescence (543 and 595 nm) .et al., EF1α) from Foc TR4 was used to qualify fungal colonization and banana actin gene (MusaActin) was used as an endogenous plant control.Fungal biomass was determined using the method described previously  analysis as described previously (Li FocEF1α (Foc) and MusaActin (banana) were used as internal controls to normalize the data. The relative expressions of genes were calculated using the 2−ΔΔCT method. The gene‐specific primers used in the qRT‐PCR analysis are listed in Supporting Information Table Total RNA samples were extracted using an RNA Out Kit  following the manufacturer's instructions. For each sample, 1 μg of RNA was reverse‐transcribed into cDNA using a PrimeScript RT reagent kit with gDNA Eraser (Takara). The qRT‐PCR mixtures  were prepared using the SYBR Premix Ex Taq kit (Takara). The qRT‐PCR was performed with three technical replicates in a CFX96 real‐time PCR system (Bio‐Rad). fastqc was used to remove poor‐quality bases and low‐quality reads as well as sequencing adaptors used in the process. The trimmed reads were mapped to banana genome (Musa acuminate DH Pahang v2) using SOAP aligner allowing up to two base mismatches per read. The RPKM (reads per kb per million reads) was calculated to reflect the expression level of banana transcripts. For gene expression comparison, differentially expressed genes were identified with the criterion of the absolute log2 (RPKM fold‐change) value ≥ 1 and false discovery rate ≤ 0.05.Total RNA was extracted from Cavendish banana embryogenic cell suspensions (ECSs) treated with FSA for 0, 6 and 24 h using an Illumina standard library preparation kit. Each treatment had three biological replicates. RNA quality and integrity were confirmed with a minimum RNA integrity number (RIN) value of 7. RNA‐Seq reads were generated via Illumina HiSeq 2000 platform at Beijing Novogene Biotech Co, Ltd . et al., −1 PMSF, 0.1% Triton X‐100 and protease/phosphatase inhibitor cocktails (Roche). For Western blotting, protein samples (20 μg each) were resolved via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. Protein detection via Western blotting used the anti‐HSP70 and anti‐AtpB (1 : 10 000) antibodies .The protein isolation and Western blot analysis of banana ECSs were performed as previously (Gao Ψm) was analyzed via JC‐1 staining  cellulase RS , 0.15% (w/v) pectolyase Y 23 , 204 mM KCl and 67 mM CaCl2 (pH 5.6). The enzyme–cell suspension mixture was incubated on a shaker at 80 rpm for 3 h at 28°C. The protoplasts were washed twice with 204 mM KCl and 67 mM CaCl2. The cells were collected by centrifugation at 600 g for 5 min each time.The ECSs of Cavendish banana (AAA) cv ‘Brazilian’ were used as donor materials to isolate protoplasts 7 d after the final subculture. The cells were centrifuged at 600 l‐glutamine and 2 mM sodium pyruvate, pH 7.4), and protoplasts were seeded onto poly‐d‐lysine‐coated XF 96‐well cell culture microplates (Seahorse Bioscience) at a density of 1 × 105 cells per well. The optimal concentrations of the pharmacological agents (oligomycin (8 μM), phenylhydrazone  and antimycin A (5 μM)) were determined by titrations before the FSA treatment assay. During the FSA treatment assay, four compounds were serially injected, shifting the bioenergetic profile of the cells. Basal respiration is primarily controlled by ATP synthase and proton leakage. Injection of the ATP coupler oligomycin blocks ATP synthase and residual respiration due to a proton leak, resulting in a significant decrease in OCR. FCCP uncouples oxygen consumption from ATP production, which induces high artificial proton conductance into the membrane. A candidate uncoupler or the respiration inhibitor FSA was added after FCCP treatment. Finally, the electron transport complex III inhibitor antimycin A was added to inhibit total mitochondrial respiration. The results were averages recorded from four replicate wells for each treatment group.The impact of FSA on cellular respiration was determined by measuring the oxygen consumption rate (OCR) of the protoplasts using an XF96e Flux Analyzer  in real time. Thirty minutes before the assay, the growth media were replaced with XF assay media  or the NADPH oxidase inhibitor (DPI), for 5 min. Samples were then double stained with CM‐H2DCFDA (to detect ROS) and CMXRos . Images were taken using a confocal laser scanning microscopy (LSM 710).Protoplasts from 7‐d‐old banana ECSs were treated with 0.1% MEOH (CK) or 300 μM FSA, or co‐incubated with the ROS scavenger An alkaline comet assay reagent kit  was used according to the manufacturer's instructions to evaluate DNA damage in the banana protoplasts, including single‐strand breaks (SSBs), double‐strand breaks (DSBs) and alkali labile sites (ALSs). FSA (300 μM) was added to the assays in 24 and 48 h reactions. Comet parameters, including the tail DNA (%), tail moment  and tail length (μm), were assessed with a comet assay image analysis system .et al., imagej software as described previously  with a CCD camera . The experiments were repeated at least three times.Trypan blue staining was performed to visualize cell death as described previously (Houterman −1) or xanthine + xanthine oxidase  in 10 mM PBS (pH 7.0) for 3 h to generate H2O2 or O2, respectively. Two hours after each treatment, the discs were inoculated with the Foc spore suspension (100 spores ml−1 concentration). In the antioxidant assays, the leaf discs were treated for 1 h with 10 μM diphenyleneiodonium chloride (DPI) to inhibit the NADPH oxidase activities, similar to the procedure used for the FSA treatment. The leaf inoculation was carried out using a spore suspension of the pathogen as described previously . The experiments were repeated at least three times.In order to alter the level of oxidative burst, a ROS‐generating system or a ROS‐scavenger was used as described previously  cluster in F. verticillioides, F. fujikuroi and strains of F. oxysporum  at the Broad Institute ( http://www.broadinstitute.org/annotation/genome/fusarium_group/MultiDownloads.html), together with the genomes of F. verticillioides str. 7600 and F. fujikuroi str. IMI 58289 as comparison controls . A specific replacement was observed on the FUB6/FUB7 intergenic region in F. oxysporum f. sp. cubense II5. This region is 2865 bp and showed no sequence similarity to the same intergenic region that was homologous in other F. oxysporum isolates (1281–1320 bp in length). From a blast search against other Fusarium genomes, no homologous sequence of the II5 FUB6/FUB7 intergenic region could be identified in any other F. oxysporum isolate. However, a 148‐bp region of the sequence showed 93% similarity to a sequence of similar length that presents in multiple locations in the genome of Fusarium poae, suggesting that the region in II5 could have been introgressed from a distant Fusarium species.It was reported that there are 12 genes in the Fusaric acid biosynthetic , their corresponding complementation mutants also were obtained   Fig. . Their erse Fig. . The othy 6 Fig. .FUB mutants of the Foc isolate and their corresponding complementation strains were tested for the productions of FSA both in cultures and within banana plants. According to the LC‐MS/MS analysis of the liquid media cultured with the mutants, FSA production was severely impaired in fub1 (3%), fub3 (5%), fub4 (1%) and fub10 (4%). Mutants fub2 and fub5 still showed 30% and 50% of FSA relative to the WT, respectively  of the LC‐MS/MS detection , the fub mutants and their complementation mutants, were able to infect the Cavendish banana plantlets when inoculated with 1000 conidia g−1 of soil, resulting in wilting symptoms on the banana plantlets. However, it took much longer for disease symptoms to develop for the mutants (c. 51 dpi) compared to the WT and the complementation strains (c. 37 dpi)  at P < 0.01 when compared with that of WT‐inoculated plants  was added onto fub mutant‐inoculated banana plantlets. Notably, the ability of each fub mutant to invade banana was restored by FSA at 40 dpi and the addition of FSA even enhanced banana infection with the WT Foc TR4  were inoculated with GFP‐tagged Foc TR4 (GFP‐TR4) isolates at a concentration of 1000 conidia g−1 soil. In the root system, no fungal spores or hyphae were found on the root surface at 2 dpi  plantlets with six to seven leaves  developed only a natural browning over time . Based on global RPKM‐expressing values, principal component and hierarchical cluster analysis showed that the three biological replicates of each sample were clustered into one group Fig. a,b. In tMitochondria produce the energy currency ATP for cells through respiration and to regulate cellular metabolisms. To investigate the effects of FSA on mitochondrial respiration, a mitochondrial stress test was performed to determine the OCR in banana ECS protoplasts in microtiter plates when FSA and other metabolism‐altering chemicals were added. Two different concentrations of FSA (50 or 300 μM) were used in two independent assays. Oligomycin (8 μM), FCCP 6 μM) and antimycin A (5 μM) were injected sequentially, and the OCR was measured after each injection. As expected, before the addition of the metabolism‐altering compounds, the protoplasts were in a quiescent state until the injection of oligomycin, which decreased the OCR due to the inhibition of ATP synthase  was significantly decreased after the FSA treatment by c. 61.3% over 8 h , which reacts with H2O2 to produce electron‐dense precipitates of cerium perhydroxide that can be visualized using electron microscopy  was used to observe the impact of FSA on potential nuclear condensation. Although the control cells treated with 0.1% MEOH showed normal nuclei (smooth nuclear), the FSA‐treated banana protoplasts showed apoptotic nuclei with condensed chromatins Fig. a. ComparFusarium strains  cluster and the creation of their deletion and complementation strains have facilitated our understanding of the impact of FSA on phytotoxicity. Brown and colleagues deleted nine of the 12 genes to inhibit FSA production ‘tropical’ race 4 (TR4) reduced the production of FSA both in vitro and in vivo, when tested on banana plantlets  is produced at high concentrations in many phytopathogenic and nonpathogenic Foc TR4 to discriminate among the resistant, tolerant and sensitive banana varieties or germplasm (data not shown). At high concentrations, it was difficult to find a difference in pathogenicity between the fub mutants and the wild‐type (WT) because they were both able to induce disease rapidly in the banana plantlets. However, by decreasing the inoculation concentration of the fub mutants in the soil, delayed wilting symptoms and a decreased disease index were noticed . Similar to FSA and SECRETED INTO XYLEM (SIX)1 , no fungal colonization was observed in the banana pseudostems, but FSA was already present in the pseudostems as early as 6 dpi  is the predominant phytotoxin in cereal grains infected with Fusarium species , neither a chemotactic response in Foc TR4 nor growth of the fungus toward FSA were found. Because of these observations, the present authors believe that FSA produced in Foc TR4 is not an allelochemical or chemotaxin. Instead, it is more likely a virulent molecule adventuring into the uninfected tissues first and preparing for the upward progression of the pathogen invasion.The FSA secreted by et al., et al., Although there were several studies investigating the mode of action of FSA  are toxic to plants and can inhibit both O2 uptake and host cell growth by increasing the membrane permeability and inhibiting mitochondrial activity , O2 uptake was stimulated . Mitochondria play key roles in apoptosis, especially by mediating ROS eruption  should be directed to the Fig. S1 Generation of fub mutants.Fig. S2 Relative expression of FUB genes.Fig. S3 Global transcriptome analysis based on RNA‐seq.Fig. S4 Enriched pathways among all genes that are significantly up‐/downregulated by FSA treatments.Fig. S5 Enriched genes associated with mitochondrial function and cell death in 6 h or 24 h FSA‐treated samples.Fig. S6 Verification of RNA‐seq data by qRT‐PCR and Western blotting.Table S1 The primer used in this study.Table S2 Abbreviation list in this study.Click here for additional data file.


Undaria pinnatifida, which is called Mi-Yoek in Korea, has been traditionally consumed as a health food in East Asian countries. Recent studies have reported that U. pinnatifida has beneficial effects on arteriosclerosis, inflammation, fat metabolism, and tumors. In this study, we examined the anti-senescence effects of ethanol extracts of U. pinnatifida (UP-Ex) in human bone marrow mesenchymal stem cells (hBM-MSCs). UP-Ex protected hBM-MSCs against oxidative injury, as determined by MTT assays. This effect was confirmed by immunoblot analysis of the oxidation-sensitive protein p53 and the apoptotic protein cleaved caspase-3. Excessive intracellular reactive oxygen species (ROS) accumulation induced by oxidative stress was moderated in UP-Ex-treated hBM-MSCs (UP-Ex-MSCs). Similarly, expression of the ROS-scavenging enzymes superoxide dismutase 1 (SOD1), SOD2, and catalase was recovered in UP-Ex-MSCs. Excessive ROS induced by long-term cell expansion (passage 17) was significantly decreased along with restoration of the senescence proteins p53, p21, and p16 in UP-Ex-MSCs. UP-Ex treatment also improved the ability of these replicative, senescent hBM-MSCs (passage 17) to differentiate into osteocytes or adipocytes, suggesting that UP-Ex ameliorates the functional decline of senescent stem cells and may provide better therapeutic efficacy in stem cell therapy.The brown alga Abbreviations: hBM-MSCs: human bone marrow mesenchymal stem cells; DCF: 2′,7′-dichlorodihydrofluorescein; DCFH-DA: 2′,7′-dichlorofluorescein diacetate; MTT: 3-2,5-diphenyltetrazolium bromide; PBS: phosphate-buffered saline; PFA: paraformaldehyde; RIPA: radioimmunoprecipitation assay; ROS: reactive oxygen species; SOD1: superoxide dismutase 1; SOD2: superoxide dismutase 2. Antioxidants protect cells from these impairments and attenuate cellular senescence. In the present study, we examined the anti-senescence and anti-oxidative effects of Human BM-MSCs were purchased from CEFO . Cells were cultured as described in our previous study , washed two or three times with tap water, drained after elimination of residual salts, and dried at room temperature. The dried seaweed powder was extracted with 80% ethanol (20 times volume) for 1 week and filtered through 150-mm Qualitative Filter Paper . The ethanol was evaporated using a Rotary Evaporator , and the dried extract stored at −70°C for one day. After freezing, the final, dried extract was obtained by lyophilizing for 3 days in a freeze dryer . The stock solutions for experimental assays were prepared by solubilizing 20 mg of UP-Ex in 1 mL of DMSO/ethanol (ratio = 1:1).4 cells/well) and 96-well plates (8 × 103 cells/well) and incubated at 37°C for 24 h. Cells (80% confluence) were then treated with UP-Ex for 24 h at 37°C and incubated with 20 μM DCFH-DA at 37°C for 1 h. After washing with phosphate-buffered saline twice, the cells were incubated with hydrogen peroxide  for 1 h at 37°C. Levels of iROS were measured as described in our previous study , which converts to the detectable fluorescent product 2′,7′-dichlorodihydrofluorescein (DCF) inside cells. Cells were seeded in both 24-well plates  according to the manufacturer’s instructions. Briefly, 8 × 10Total proteins were extracted with 400 μL RIPA buffer containing 2 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and protease inhibitor cocktail . Immunoblot analysis was performed with specific antibodies for SOD1, catalase, p53, p21, or p16 , SOD2 , cleaved caspase 3 , or β-actin  for 16 h at 4°C. The appropriate horseradish peroxidase-conjugated secondary antibodies  were used for enhanced chemiluminescence detection . The bands were quantified using the Image J software .The ability to differentiate into osteocytes and adipocytes was evaluated in P-17 UP-Ex-treated hBM-MSCs (UP-Ex-MSCs) according to previously described procedures . Statistical comparisons between groups were analyzed using a paired p < .05, n = 4). To examine the anti-oxidative effect of UP-Ex, cells were exposed to 1 mM H2O2 for 1 h following pre-incubation with UP-Ex (0–10 μg/mL) for 24 h. The viability was significantly increased in UP-Ex-MSCs compared to untreated hBM-MSCs . Similarly, 5 μg/mL UP-Ex protected hBM-MSCs exposed to 1 or 2 mM H2O2 . To further examine these protective effects at the molecular level, apoptosis-related proteins (p53 and cleaved caspase-3) were measured by immunoblot analysis. The expression of p53 and cleaved caspase-3 was decreased in UP-Ex-MSCs compared to untreated hBM-MSCs  for 24 h, and cell viability was measured by an MTT assay. Toxicity was not detected at ≤10 μg/mL UP-Ex treatment , whereas the hBM-MSCs with/without UP-Ex treatment showed no change in the steady state . Immunoblot analysis of antioxidant enzymes showed that the expression of SOD1, SOD2, and catalase was decreased in P-17 cells and restored in P-17 UP-Ex-MSCs . In addition, the senescence proteins p53, p21, and p16 were increased in P-17 cells and this increase was reversed in P-17 UP-Ex-MSCs . These data suggest that UP-Ex reverses cellular senescence through recovery of antioxidant enzymes.Our previous study showed that stem cells made senescent by long-term expansion accumulate iROS and exhibit reduced antioxidant enzyme expression  cultured hBM-MSCs were treated with 5 μg/mL UP-Ex for 24 h and differentiated into osteocytes and adipocytes. P-17 hBM-MSCs exhibited reduced differentiation potential compared to P-7 cells, and UP-Ex treatment of P-17 cells restored this potential compared to untreated P-17 hBM-MSCs . These rU. pinnatifida , it may also improve the differentiation potential of stem cells. In support of this hypothesis, long-term cultured hBM-MSCs (P-17) pretreated with UP-Ex could be differentiated into osteocytes or adipocytes, confirming the differentiation potential of UP-Ex treated P-17 MSCs . This reU. pinnatifida moderates excessive iROS and reverses replicative senescence with an increase in antioxidant enzymes in hBM-MSCs. This study has also shown that UP-Ex treatment restored the differentiation potential of replicative senescent (P-17) hBM-MSCs, suggesting that UP-Ex ameliorates the functional decline of senescent stem cells. Combined treatment with UP-Ex may provide better therapeutic efficacy in stem cell therapy.In conclusion, we have shown that an ethanol extract of


For preparation of the anode, the glucose oxidase is sequentially layer-by-layer-assembled with the same linkers onto the metallic cotton fibers. The resulting biofuel cells exhibit a remarkable power density of 3.7 mW cm−2, significantly outperforming conventional biofuel cells. Our strategy to promote charge transfer through electrodes can provide an important tool to improve the performance of biofuel cells.Electrical communication between an enzyme and an electrode is one of the most important factors in determining the performance of biofuel cells. Here, we introduce a glucose oxidase-coated metallic cotton fiber-based hybrid biofuel cell with efficient electrical communication between the anodic enzyme and the conductive support. Gold nanoparticles are layer-by-layer assembled with small organic linkers onto cotton fibers to form metallic cotton fibers with extremely high conductivity (>2.1×10 Biofuel cells offer biocompatibility and operation at mild conditions, but application is limited by relatively low output power. Here the authors use layer-by-layer assembly for glucose oxidase-coated metallic cotton fibers for use as electrodes in a hybrid biofuel cell to achieve high output power. Despite these advantages, the relatively low power output of BFCs compared to other power sources, resulting from slow electron transfer, poor utilization efficiency of the enzyme, and limited mass transport, has restricted their practical applications. In particular, poor electrical communication between the enzyme and the conductive support has a critical effect on the power output of BFCs6. To overcome this problem, redox mediators between the enzyme and the conductive support have been introduced to improve the electrical communication between the two, thereby enhancing the power performance of the cell. To the best of our knowledge, the best power performance reported to date for these mediated electron-transfer-based BFCs (MET-BFCs) is 2.18 mW cm−27. However, the incorporation of a redox mediator in BFCs can lead to several issues, such as toxicity, instability, the complex synthesis of redox mediators, and a reduced open-circuit voltage. Thus, recent research efforts have focused on the development of direct electron transfer-based BFCs (DET-BFCs) with improved performance8.Biofuel cells (BFCs)11, graphene12, and porous carbon13) with large surface areas, 3D porous structures for facile mass transport, and high electrical conductivities (<300 S cm−1) have been proposed as supports having favorable electrical communication with the enzyme layer. For example, Zebda et al. demonstrate that DET-BFCs based on CNT supports exhibited a significantly higher power output of 1.25 mW cm−2 compared to previously reported DET-BFCs fabricated by solution drop casting and high mechanical compression5. Despite all the merits of the carbon substrates for the BFCs, their biosafety for long-term operation in the in vivo systems is still controversial due to the cellular toxicity nature15. Furthermore, in most cases, the enzymes have been directly deposited onto these carbon supports through conventional physical adsorption, which has limitations in terms of the precise control of the interfacial distance, conformation, and stability between the enzyme and the conductive support, resulting in poor utilization of the enzymes for catalyzing electrochemical reactions.A variety of conductive carbon materials 16. However, the catalytic activity and stability are strongly dependent on the physiological conditions of the mammal’s body fluid such as pH, temperature, and chlorine concentration20. As an alternative, catalytic metal nanoparticles (NPs) such as Pt, Pd, and Au with high ORR activity have been considered as the competent candidates to the abiotic ORR catalysts22. Despite the improved performance, conventional methods of depositing NPs onto the substrates, such as the mixing of NPs with carbon-substrates23 or chemical reduction of the metal precursor, etc.24 have poor controls over the structures of NPs on the substrates. In addition, chemical reduction methods often cause particle agglomeration between adjacent NPs with the reduced electrochemical surface area, which hinders high mass loading of NPs on the substrates25. Therefore, it is ideal to incorporate the catalytic NPs onto the substrates in a controlled, 3D spatial arrangement without agglomeration, which can facilitate electron and ion transport for the efficient electrocatalytic reaction.With the advances of the anode for the glucose oxidation reaction through glucose oxidase (GOx), the proper design of the cathode to improve the oxygen reduction reaction (ORR) activity is also crucial to obtain the high-power density and efficiency. In this regard, the cathodic enzyme, such as laccase or bilirubin oxidase, have been intensively studied due to their excellent ORR activity38. However, as bulky insulating polyelectrolytes are generally LbL-assembled with the desired functional components  to build up multilayers, the mutual electron transfer is significantly suppressed due to the increased contact resistance40. Therefore, the ideal LbL assembly for BFC applications should replace bulky polymer electrolytes with small-molecule linkers that can facilitate the electrical communication between the active materials as well as enable the efficient assembly of the catalysts with a controlled structure and loading amount.Layer-by-layer (LbL) assembly methods offer effective opportunities to prepare enzymatic or conductive multilayer films with tailored thickness, functionalities, and composition on substrates of various sizes and shapes utilizing complementary interactions41, such as the superior biocompatibility42, more facile surface modification43, and higher electrical conductivity. In this case, the MCFs act as not only the conductive host for depositing the anodic enzyme materials, but also the electrocatalytic cathode for ORR. More importantly, it is worth noting that the loading amount of the metal NPs in the MCF can be easily increased forming the 3D porous structure according to the increase of the bilayer number, and thereby achieving both the higher electrical conductivity and the improved ORR activity without cathodic enzymes. As a result, our hybrid MCF-BFC provides a remarkable power density of 3.7 mW cm−2 using a fixed external resistance without the aid of redox mediators, significantly exceeding the power density of the previously reported conventional DET- and MET-BFCs.Here, we introduce a highly porous metallic cotton fiber-based BFC (MCF-BFC) fabricated using small-molecule linker-induced LbL assembly of the active catalysts, which significantly boosts the DET rate between the enzymes and the conductive supports Fig. . In part4 S cm−1) and yet still maintain the intrinsic 3D porous structure of cotton fibers with conformal coatings of the active layers. With its extremely high electrical conductivity and highly porous 3D structure, MCF can facilitate charge transfer to the conformally coated enzyme layers as well as the mass transport of glucose (for oxidation by GOx) and oxygen (for reduction by Au NP) through the electrodes, thus enabling effective electrocatalytic reactions. Furthermore, the robust covalent bonding between the Au NPs and TREN and the electrostatic bonding between GOx and TREN increase the operational stability of the MCF-BFCs. Given that these unique characteristics of MCFs play a decisive role in enhancing the power output of hybrid BFCs, this approach can contribute to the design of other high-performance electrodes for various electrochemical applications, including other energy storage devices and sensors.For the fabrication of the MCF, instead of bulky polymers such as polyelectrolytes, we employ an amine-functionalized small molecule, tris-(2-aminoethyl)amine (TREN), that can electrostatically interact with GOx as well as covalently interact with Au NPs. First, LbL assembly of TREN and tetraoctylammonium bromide-stabilized Au NPs (TOA-Au NPs) was carried out on cotton fibers. In this case, the amine groups of TREN can covalently adsorb on the surface of Au NPs due to its stronger affinity than the ammonium group of TOA. Then, negatively charged GOx was electrostatically LbL-assembled onto the TOA-Au NP/TREN multilayer-coated cotton fibers using positively charged TREN in water. Based on this unique LbL assembly of conductive NPs and enzyme materials through the same small-molecule linker allowing different interactions, we fabricate entirely different MCF electrodes that have extremely high electrical conductivity (>2.1×1043 were LbL-assembled with TREN onto a cotton fiber composed of multiple hydrophilic microfibrils (cellulose fibers containing hydroxy groups). The TOA-Au NPs were uniformly and densely coated onto all the microfibrils that make up the cotton fiber (~200 μm diameter) without disrupting the inherently porous structure  was more than ten times higher than that loaded onto nonporous nylon fiber, which has the same dimension (~200-μm-diameter) as a cotton fiber (0.8 mg cm−2 for n = 20) 20 multilayers . It should be noted that the conductivity of bare CNT fibers does not exceed 600 S cm−1 without additional post-treatment45. Furthermore, the formed MCF retains its intrinsic flexibility even though Au NPs are densely loaded onto the bare cotton fiber  groups during the LbL assembly. As confirmed by Fourier transform infrared (FTIR) spectroscopy -coated Si wafer  generated the characteristic peaks of TOA ligands (C−H stretching vibrations at 2928 and 2856 cm−1) and the amine groups of PEI (N−H bending at 1654 and 1554 cm−1). The subsequent adsorption of TREN onto the outermost TOA-Au NP layer  eliminated the C−H stretching peaks corresponding to the TOA ligands with long alkyl chains, while the N−H bending peaks were intensified by TREN adsorption. As a result, this sequential ligand exchange reaction allows TREN to act as a new molecular linker between adjacent Au NPs instead of the bulky TOA ligands, which minimizes the distance and contact resistance between the Au NPs.The high electrical conductivity of MCFs is caused by the unique adsorption mechanism, in which the bulky and hydrophobic TOA ligands of the TOA-Au NPs are sequentially replaced by small TREN ligands containing amine  as a function of the number of bilayers (n) at a scan rate of 5 mV s−1 using a three-electrode cell configuration in 20 mmol L−1 phosphate-buffered saline (PBS) solution under ambient conditions. Cyclic voltammetry (CV) scans showed a gradual increase in cathodic current densities when the number of bilayers (n) was increased from 5 to 20 due to the increased active surface area from the introduced Au NPs46  of 4 Ω relative to those of the 5-MCF electrode  compared to 5-MCF (10.9 Hz) indicates better mass transport for the highly porous structure with sufficient Au NP coverage .We also investigated the electrochemical properties of the (TOA-Au NP/TREN)s46 Fig.  (please 2), ambient, and oxygen-rich PBS solutions  under ambient condition, and −20.7 mA cm−2 (with an ESR of 29 Ω) under O2 condition  for the 20-MCF electrode outperforms the current density level (−3.0 mA cm−2) of nonporous bulk Au wire with the same diameter in ambient conditions . The LbL-assembled GOx can catalyze the oxidation of glucose to gluconolactone, and the electrons generated can be directly transferred from the enzyme to the MCF electrode  multilayers on (TOA-Au NP/TREN)-coated quartz glass was investigated by ultraviolet-visible (UV−Vis) spectroscopy  or the decrease in mass changes (ΔM) after deposition of each TREN layer can be attributed to the partial desorption of weakly adsorbed GOx during the adsorption of TREN 20 and (GOx/TREN)30 are adsorbed on the substrates like a single GOx layer and double GOx layers, respectively. As a result, the adsorption conformation of thin and uniform (GOx/TREN)m multilayers can facilitate the electron transfer through the enzyme layers.We prepared MCF-based anodes through the additional deposition of electrostatically LbL-assembled (anionic GOx/cationic TREN) multilayers onto 20-MCF in PBS solution. In this process, GOx, with an isoelectric point of pH 4.3, has a net negative charge in pH 7.4 PBS solution, which can induce the electrostatic adsorption of positively charged TREN molecules  at a scan rate of 5 mV s−1  showed that the overall current density of the electrode steadily increased with increasing number of bilayers (m) from 0 to 30. This bilayer (m)-dependent anodic performance can be explained by different adsorption behavior of the GOx layer on the inner and outer surface of the MCFs. In other words, in the case of the GOx/TREN multilayers onto the interior of highly porous MCFs, it is considered that their thicknesses will be much thinner with going from the outer surface to the center region of MCFs because the repetitive deposition of bulky GOx layers can block up the porous structure of MCFs. Therefore, the overall anodic current density of MCF-based anode electrode can be gradually increased up to the thickness of GOx/TREN multilayers allowing efficient electron tunneling. The anodic current density level measured at +0.6 V approached a maximum value of ~50 mA cm−2 , indicating the excellent electrode stability caused by the precisely controlled deposition of GOx using TREN 30 multilayers onto nonporous electrode , which indicate the effective electrode conformation of the MCF-based anode for facile electrochemical kinetics.In this case, the 30-GOx/20-MCF anode maintained ~97.5% of its initial current density after continuous operation for 1 h  groups of GOx). In line with this high affinity, glucose could be easily diffused into the outermost TREN-coated multilayer films, and therefore the outermost TREN-coated electrode exhibited much lower electron transfer resistance than the outermost GOx-coated electrodes in spite of the increase of layer number. These similar phenomena were also demonstrated by Willner group49. Considering that all the BFCs shown in our study are based on the outermost TREN-coated electrodes as well as the highly porous electrodes, the electron transfer resistances of MCF-based electrodes can be significantly lowered compared to those of other nonporous electrodes.We also observed that the electron transfer kinetics at the interface of the electrode are significantly influenced by a kind of outermost layer of GOx/TREN multilayers m–coated electrodes can be also used as in vivo glucose sensors m multilayers were deposited onto (TOA-Au NP/TREN)20 multilayer-coated Si wafers, the (GOx/TREN)10-coated flat substrate anode in a 300 mmol L−1 glucose solution exhibited a maximum normalized current density level of ~0.01 mA cm−2 at +0.6 V 20-coated cotton fiber (i.e. PEI-MCF) was prepared as a comparison target of the (TOA-Au NP/TREN)20-coated cotton fiber (i.e. TREN-based MCFs). In this case, the PEI-based MCF showed an extremely low electrical conductivity of ~0.02 S cm−1 compared to that of the TREN-based MCF (~3.5×103 S cm−1) due to the use of bulky insulating PEI  despite their similar physical characteristics such as total thickness and surface morphology  of GOx was immobilized onto each MCF (i.e. PEI- and TREN-based MCFs), the electrooxidation response of the PEI-based MCF anode was significantly low compared to that of the TREN-based MCF Ks) was estimated using the Laviron method53 for a surface-controlled electrochemical system  vs. scan rate (log v), which was recorded from the CV data. As a result, the 30-GOx/20-MCF anode exhibited a relatively large Ks value of 6.0 ± 0.1 s−1 and a small redox peak potential separation (∆Ep) of 60 mV at a scan rate of 100 mV s−1. Here, small ∆Ep (60 mV at a scan rate of 100 mV s−1) indicates fast heterogeneous electron transfer process, and the reaction is not a diffusion-controlled process but a surface-controlled one, as expected for immobilized system . Additionally, a large ks of 6.0 ± 0.1 s−1 obtained from 30-GOx/20-MCF suggests that the electrode is significantly effective in facilitating the direct electron transfer of GOx57. As a result, the formed 30-GOx/20-MCF electrode provides efficient catalytic kinetics with smaller ∆Ep and larger ks -modified electrode, 0.3 s−1 for single-walled carbon nanotubes (SWNT)-modified electrode, 3.96 s−1 for the porous TiO2 electrode, and 6.28 s−1 for GOx-carbon nanodots on the glassy carbon electrode)58.To further investigate the catalytic kinetics of 30-GOx/20-MCF  anode, the apparent heterogeneous electron transfer rate , a bulky polymer linker, onto 20-MCF for the preparation of (GOx/PEI)30/20-MCF electrodes  than the (GOx/TREN)30/20-MCF  electrode  of bilayer number of (TOA-Au NP/TREN)n multilayers onto cotton fibers).We also highlight that the use of a small-molecule linker between neighboring enzymes can allow facile charge and/or mass transport through the enzyme layer. For comparison between small-molecule and polymer linkers, GOx was electrostatically LbL-assembled with PEI  of its initial power density and voltage retention of ~83% after continuous operations for 20 days   of (TOA-Au NP/TREN)n from 20 to 120 in the cathode and investigated its effect on the power performance of MCF-BFCs  at n = 120  reported to date (Supplementary Table −2) obtained from our membrane-free and mediator-free MCF-BFC is in stark contrast with those measured from other mediator-free enzymatic fuel cells . For example, the power output shown in our system is nearly three times higher than that of the previous DET-based compressed CNT electrode5 , and even higher than that of the reported osmium complex-based bi-scrolled carbon nanotube yarn MET-BFC7 (2.18 mW cm−2). Consequently, we demonstrated that our highly porous metallic cotton fiber-based BFC (MCF-BFC) fabricated by small-molecule linker-induced LbL assembly of the active catalysts can significantly realize the DET rate between the enzymes and the conductive supports. More significantly, the maximum power density of an MCF-BFC can be further increased by simply increasing the bilayers of Au NP layers in the cathode and/or in the anode. Moreover, these controlled electrode–electrode and/or BFC–BFC connections can further increase the overall power density of the device. For example, if two 120-MCF-BFCs were connected in series, the maximum power output measured was ~7.1 mW cm−2, which is an approximately twofold increase in the hybrid BFC performance  was purchased from Amano Enzyme Inc. (Japan). Tetraoctylammonium bromide (TOA), gold (III) chloride trihydrate (HAuCl4·3H2O), sodium borohydride (NaBH4), tris-(2-aminoethyl)amine (TREN) (Mw ~146), and polyethylenimine, branched  were purchased from Sigma-Aldrich.Cotton fibers with a diameter of 200 μm were purchased from Igonji (South Korea), and glucose oxidase (GOx) from 43. In brief, HAuCl4·3H2O in water  and TOA in toluene  were mixed with vigorous stirring, and then NaBH4 in water  was added to the two-phase mixture. After sufficient stirring (approximately 3 h), the aqueous solution was separated from the mixture, and then the remaining toluene solution was repeatedly washed using dilute aqueous H2SO4 (0.1 mol L−1), NaOH (0.1 mol L−1), and deionized water. After several washing steps and the removal of the aqueous solution, TOA-Au NPs dispersed in toluene were obtained.Tetraoctylammonium bromide-stabilized Au NPs (TOA-Au NPs) with a diameter of 8 nm were synthesized using a two-phase method in toluene2)-functionalized PEI  for 40 min. The resulting PEI-coated fibers, which were prepared through a hydrogen-bonding interaction between the NH2 of PEI and the OH groups of the cotton fibers, were then immersed in a TOA-Au NP solution (10 mg mL−1) for 40 min, and then washed with toluene to remove the weakly adsorbed TOA-Au NPs. In this case, the bulky TOA ligands bound to the surface of the Au NPs were almost completely replaced by NH2 groups of the PEI-coated fibers due to the high affinity between the Au surface and NH2 groups. Next, the formed TOA-Au NP-coated fibers were immersed in a solution of TREN in ethanol (1 mg mL−1) for 40 min. This was repeated, and the fibers were washed in ethanol to remove the excess TREN molecules, resulting in fibers coated with one bilayer (i.e. (TOA-Au NP/TREN)1-cotton fiber). The adsorption mechanism between the TOA-Au NPs and the NH2-functionalized TREN is the same as that between the TOA-Au NPs and PEI. These deposition and washing steps for the preparation of the MCFs were continuously repeated until the desired number of bilayers was obtained.Cotton fibers composed of hydroxy (OH) group-functionalized cellulose fibers were dipped in ethanolic solutions of amine (NH−1 in PBS) for 10 min, and then washed with PBS (pH 7.4) to remove weakly adsorbed GOx. During these deposition and washing steps, negatively charged GOx molecules in the pH 7.4 PBS aqueous solution were electrostatically adsorbed onto the positively charged MCF. It should be noted that the neutral NH2 groups of the outermost TREN layer of the MCF prepared in organic phases can be easily protonated to positively charged ammonium (NH3+) groups in pH 7.4 PBS aqueous solution because the pKa (pH with a degree of ionization of 50%) of ammonium groups is approximately 10. After the electrostatic deposition of GOx, a solution of positively charged TREN (1 mg mL−1) was deposited on the anionic GOx-coated MCFs for 10 min, which were then washed with PBS. These processes were repeated until an anode electrode (i.e. (GOx/TREN)m-coated MCF) with the desired number of bilayers (m) was obtained. To prepare the polymer linker-based fiber anode electrode, a solution of cationic PEI  was used instead of TREN. In this case, all the LbL assembly conditions, except the inclusion of polymer, were exactly the same as those used for the (GOx/TREN)m multilayers.The fabricated MCF electrode was immersed in a solution of GOx . The growth and loading amount of the LbL-assembled multilayers were examined using UV−Vis spectroscopy  on quartz glass, and a QCM , respectively. In the case of QCM measurement, the mass change (ΔM) per layer was calculated from the QCM frequency change (ΔF) using the Sauerbrey equation65,F0, A, ρq, and μq represent the resonant frequency (~5 MHz), the active area (cm2), the density (2.65 g cm−3), and the shear modulus (2.95×1011 g cm−1 s−2) of the QCM electrode, respectively, and this expression can be simplified to ΔF (Hz) = −56.6 × ΔMA (MA indicates mass per unit area).The surface and cross-sectional morphologies of the MCF-based BFC electrodes were characterized using FE-SEM  and AFM . The adsorption behavior and mechanism of formation of the (TOA-Au NP/TREN)2) were measured over the potential range −0.6 to +0.6 V using an electrochemical analyzer . All measurements were carried out in an electrochemical cell in batch mode, 50 mL PBS buffer  at 36.5 °C without stirring. Interelectrode spacing between anode and cathode electrode was ~1 cm, the membrane between two electrodes was not used in this study. In addition, one end of each BFC electrode was fixed by epoxy resin on the glass substrate for avoiding electrical shortage while measuring the power density with the continuous operation. The anodic and cathodic current densities were determined by CV at a constant voltage of +0.6 V for the anode and −0.6 V for the cathode, showing a maximum current density. The normalized current density was respectively obtained by subtracting the current density level in glucose-free PBS at +0.6 V for the anode, and subtracting the current density level in an oxygen-free PBS at −0.6 V for the cathode. The BFC power densities were determined by measuring the current output applying a fixed external resistance (in the range of 1 kΩ~10 MΩ) to control cell potential66. The experimental set-up for measuring the power density of the complete hybrid MCF-BFC was shown in Supplementary Figure n-MCF electrodes and the m-GOx/n-MCF electrodes were performed over the frequency range 0.2 Hz−100 kHz with a perturbation amplitude of 0.01 V. The impedance spectra (Nyquist plots) of the real (Z′) and imaginary parts (Z″) obtained from the electrochemical BFC cell were processed using the ZView software .In a three-electrode system composed of an MCF working electrode, Ag/AgCl reference electrode, and a Pt counter electrode, the top end of the working electrode was firmly connected to a copper wire using commercial silver paste . After the silver paste was dry, it was additionally insulated with an epoxy adhesive. Cyclic voltammograms of an MCF electrode (diameter 200 µm, length 5.0-mm, active external surface area 3.14 mmSupplementary InformationPeer Review File


AXIN2, LGR5). SFN inhibits Wnt signaling downstream of β-catenin degradation and induces the formation of nuclear β-catenin structures associated with closed chromatin. Co-expression of the transcription factors LEF1 or TCF4 prevented formation of these structures and rescued inhibition of Wnt/β-catenin signaling by SFN. Our findings provide a molecular basis explaining SFN effects in colorectal cancer cells and underline its potential for prevention and therapy of colorectal cancer.The naturally occurring isothiocyanate sulforaphane (SFN) from cruciferous vegetables is associated with growth inhibition of various cancer types, including colorectal cancer. Colorectal cancer is most frequently driven by hyperactive Wnt/β-catenin signaling. Here, we show that SFN treatment reduced growth of three unrelated colorectal cancer cell lines  via induction of cell death and inhibition of proliferation. Importantly, SFN inhibits Wnt/β-catenin signaling in colorectal cancer cells as shown by inhibition of β-catenin-dependent luciferase reporters and repression of β-catenin target genes ( Colorectal cancer is one of the leading causes for cancer-associated morbidity and mortality in industrialized countries therefore representing a major health issue . The vasLGR5, AXIN2, MYC and CCND1 . , 8. AXINd Figure . LGR5 exIn breast cancer cells, SFN was described to inhibit Wnt/β-catenin signaling by activating GSK3 . Since βInhibition of Wnt/β-catenin signaling in β-catenin S33Y expressing HEK293T cells is in line with inhibition of signaling in HCT116 cells which harbor a similar stabilizing β-catenin mutation. Moreover, SFN was also able to inhibit β-catenin S33Y-activated Wnt signaling in DLD1 cells indicating that SFN functions by a common mechanism in HEK293T cells and colorectal cancer cells Figure . FinallyTogether, these experiments suggest that SFN inhibits Wnt/β-catenin signaling downstream of β-catenin degradation at the level of nuclear import or activation of target genes in the nucleus. To investigate this further, the nuclear localization of β-catenin after SFN treatment was assessed. Transiently expressed β-catenin is localized diffusely throughout the cytoplasm and the nucleus, and sporadically forms nuclear puncta in a low percentage of cells. Importantly, SFN treatment significantly increased the percentage of cells showing these β-catenin puncta as well as the size of these puncta in different cell lines Figure . These pUpon co-expression of the transcription factor LEF1, which is known to associate with β-catenin to activate β-catenin-dependent transcription, β-catenin was completely recruited into the nucleus, as previously reported Figure  6]. Of . Of 6]. In this study, we characterize SFN as potent inhibitor of Wnt/β-catenin signaling in colorectal cancer cells. SFN inhibited β-catenin-dependent reporter activity and repressed β-catenin target gene expression in a dose-dependent manner at concentrations which can be achieved in the blood through oral SFN uptake . To our Mechanistically, SFN, most likely, prevents the formation of active β-catenin-based transcription complexes in the nucleus. Like ARID1B, a chromatin remodeling factor which inhibits Wnt/β-catenin signaling, SFN increased the formation of nuclear β-catenin puncta, which had previously been linked to closed chromatin structures and reduced β-catenin-dependent transcription . Increasin vitro. The observed growth inhibition of colorectal cancer cells by SFN is in line with previous studies  and Annexin V-FITC [BD Bioscience]). After 15 min incubation at RT in the dark, 15 μl of the stained cell suspension was pipetted on a microscope slide, covered with a coverslip and immediately analyzed with an Axioplan II microscope system (Carl Zeiss). Images were acquired using MetaMorph analysis software (Carl Zeiss).As previously described , cells wAXIN2 , LGR5  and GAPDH  was performed in a CFX96 Real-Time System (Bio-Rad) in triplicates. mRNA expression of the Wnt/β-catenin target genes AXIN2 and LGR5 was normalized to the housekeeping gene GAPDH.Cells were treated with indicated SFN concentrations for 48 h before the whole cellular RNA was isolated using the RNeasy mini kit (QIAGEN) according to manufacturer's instructions. cDNA was synthesized with the AffinityScript QPCR cDNA Synthesis Kit (Agilent Technologies). Quantitative PCR for For Western blotting, cells were lysed after 24 h treatment with indicated SFN concentrations in hypotonic extraction buffer  Figure  or lucifCells were treated with SFN concentrations indicated in Figure Expression plasmids for HA-Dvl2, RFP-caLrp6, YFP-β-catenin S33Y, GFP-β-catenin, HA-LEF1 and CFP-TCF4, and the siRNA against APC (5´-AAGACGUUGCGAGAAGUUGGA-3`) have been described previously –31.


Epichloë endophytes have been reported to produce epoxy-janthitrems, LpTG-3  and LpTG-4 . Epichloë epoxy-janthitrems are not well understood, the biosynthetic pathway and associated gene complement have not been described and while the literature suggests they are associated with superior protection against pasture insect pests and are tremorgenic in grazing mammals, these properties have not been confirmed using isolated and purified compounds. Whole genome sequence analysis was used to identify candidate genes for epoxy-janthitrem biosynthesis that are unique to epoxy-janthitrem producing strains of Epichloë. A gene, jtmD, was identified with homology to aromatic prenyl transferases involved in synthesis of indole diterpenes. The location of the epoxy-janthitrem biosynthesis gene cluster (JTM locus) was determined in the assembled nuclear genomes of NEA12 and E1. The JTM locus contains cluster 1 and cluster 2 of the lolitrem B biosynthesis gene cluster (LTM locus), as well as four genes jtmD, jtmO, jtm01, and jtm02 that are unique to Epichloë spp. that produce epoxy-janthitrems. Expression of each of the genes identified was confirmed using transcriptome analysis of perennial ryegrass-NEA12 and perennial ryegrass-E1 symbiota. Sequence analysis confirmed the genes are functionally similar to those involved in biosynthesis of related indole diterpene compounds. RNAi silencing of jtmD and in planta assessment in host-endophyte associations confirms the role of jtmD in epoxy-janthitrem production. Using LCMS/MS technologies, a biosynthetic pathway for the production of epoxy-janthitrems I–IV in Epichloë endophytes is proposed.Epoxy-janthitrems are a class of indole diterpenes with structural similarity to lolitrem B. Two taxa of asexual  Lolium perenne L.) is often infected with fungal endophytes that include sexual Epichloë species and their asexual derivatives. In the case of asexual Epichloë spp. endophytes, the host grass–endophyte interaction is considered mutualistic as both the plant and the fungus receive benefits [ symbiota was reduced when compared to non-transgenic E1 control plants, with significant differences observed for Alto-E1jtmD 129bp , Alto-E1jtmD 432bp-1  and Alto-E1jtmD 432bp-2  symbiota  reduction of epoxy-janthitrem was observed. When compared to Alto-E1 symbiota, abundance of paxilline was significantly reduced in Alto-E1jtmD129bp  and Alto-E1jtmD 432bp-2  symbiota. Interestingly, when compared to the control symbiota, Alto-E1jtmD432bp-1 symbiota produced the lowest levels of epoxy-janthitrem I–IV , yet no change in paxilline abundance was observed in these plants. Effects on upstream components of indole diterpene pathways as result of gene knockdown have not frequently been reported. One such study describes that the disruption of the terP cluster involved in terpendole E biosynthesis resulted in overproduction of upstream alkaloids [terP gene is involved in the metabolic conversion of terpendole E to its downstream counterparts; thus, terP knockout strains of Agrobacterium tumefaciens resulted in terpendole E accumulation [Production of the precursory metabolite paxilline was also measured. Paxilline abundance was perturbed in response to lkaloids . The termulation .Indole diterpene gene clusters identified to date are characterized by a core set of four genes for the synthesis of paspaline, and a suite of additional genes that encode multi-functional cytochrome P450 monooxygenases, FAD dependent monooxygenases and prenyl transferases that catalyse various regio- and stereo- specific oxidations on the molecular skeleton to generate a diversity of indole diterpene products .Epichloë is described based on analogy with other indole diterpene pathways, identification of biosynthesis intermediates using LC-MS, and the functional analysis of jtmD, which implicated the gene in epoxy-janthitrem biosynthesis . The extracted ion chromatograms of the compounds, observed in planta in Alto-E1 symbiota, are illustrated in . The obspaxG, paxM, paxB and paxC, are required for the biosynthesis of the first stable indole diterpene intermediate, paspaline. Genetic analysis of P. paxilli has established that paxilline biosynthesis then requires two cytochrome p450 monooxygenases, paxP and paxQ which utilise paspaline and 13-desoxypaxilline as their respective substrates [P. janczewskii and P. janthinellum, incorporation of labelled β-paxitriol, and not α-paxitriol, into penitrem A and E and janthitrem B and C, respectively, suggests that β-paxitriol is an immediate precursor for the complex indole diterpenes with B-stereochemistry [Saikia et al.  showed tbstrates . Ortholohemistry .Epichloë species, involving ltmG, ltmC, ltmM and ltmB in the synthesis of paspaline. Here, we propose that initial steps in the epoxy-janthitrem pathway require LtmP and LtmQ for the demethylation and hydroxylation of paspaline for the synthesis of β-paxitriol. JtmD and JtmO are required for the diprenylation and oxidative cyclisation to construct the distinct A/B ring in the janthitremane indole diterpenes. LtmF and LtmK are required for the prenylation and cyclisation of the epoxy-janthitrems II to IV consistent with the lolitrem biosynthesis (Genetic evidence suggests that the biosynthesis of epoxy-janthitrems proceeds along the same pathway as lolitrem B in other ynthesis . Jtm01 (Epichloë endophytes. Genes for epoxy-janthitrem biosynthesis are found clustered at a single locus, JTM, on chromosome III of the genome. Functional analysis of the jtmD gene using RNAi knockdown, revealed reduced abundance of epoxy-janthitrems I–IV, implicating JtmD in epoxy-janthitrem biosynthesis. While the role of JtmD and JtmO can be predicted based on comparison to previously well characterized indole diterpene pathways, further work is required to determine a role of Jtm01 and Jtm02 in epoxy-janthitrem biosynthesis. A biosynthetic pathway based on predicted protein function and comparison with other indole diterpene pathways is proposed. As the genes for epoxy-janthitrem production are now determined, it becomes possible to predict janthitrem production by screening strains for the presence of jtm genes and manipulate the pathway, using genome editing technology. A limitation to the study of epoxy-janthitrems in the past has been their unstable nature once isolated; it is now possible to synthesize sufficient quantities for isolation and use in accurate measurement as well as in insect/animal toxicity studies. There is also potential for creating novel indole diterpenes in Epichloë endophytes using the existing LTM gene clusters and adding new designer gene clusters using a targeted genome editing approach. Deeper understanding of epoxy-janthitrem production enables exploiting their potential use in pasture pest management, enhancing pasture persistence while minimizing the threat posed by their likely tremorgenicity.The work described here provides a genetic basis for epoxy-janthitrem biosynthesis in


MYC amplification or overexpression is common in Group 3 medulloblastoma and is associated with the worst prognosis. Recently, protein arginine methyl transferase (PRMT) 5 expression has been closely associated with aberrant MYC function in various cancers, including brain tumors such as glioblastoma. However, the role of PRMT5 and its association with MYC in medulloblastoma have not been explored. Here, we report the role of PRMT5 as a novel regulator of MYC and implicate PRMT5 as a potential therapeutic target in MYC-driven medulloblastoma.Expression and association between PRMT5 and MYC in primary medulloblastoma tumors were investigated using publicly available databases. Expression levels of PRMT5 protein were also examined using medulloblastoma cell lines and primary tumors by western blotting and immunohistochemistry, respectively. Using MYC-driven medulloblastoma cells, we examined the physical interaction between PRMT5 and MYC by co-immunoprecipitation and co-localization experiments. To determine the functional role of PRMT5 in MYC-driven medulloblastoma, PRMT5 was knocked-down in MYC-amplified cells using siRNA and the consequences of knockdown on cell growth and MYC expression/stability were investigated. In vitro therapeutic potential of PRMT5 in medulloblastoma was also evaluated using a small molecule inhibitor, EPZ015666.We observed overexpression of PRMT5 in MYC-driven primary medulloblastoma tumors and cell lines compared to non-MYC medulloblastoma tumors and adjacent normal tissues. We also found that high expression of PRMT5 is inversely correlated with patient survival. Knockdown of PRMT5 using siRNA in MYC-driven medulloblastoma cells significantly decreased cell growth and MYC expression. Mechanistically, we found that PRMT5 physically associated with MYC by direct protein-protein interaction. In addition, a cycloheximide chase experiment showed that PRMT5 post-translationally regulated MYC stability. In the context of therapeutics, we observed dose-dependent efficacy of PRMT5 inhibitor EPZ015666 in suppressing cell growth and inducing apoptosis in MYC-driven medulloblastoma cells. Further, the expression levels of PRMT5 and MYC protein were downregulated upon EPZ015666 treatment. We also observed a superior efficacy of this inhibitor against MYC-amplified medulloblastoma cells compared to non-MYC-amplified medulloblastoma cells, indicating specificity.Our results reveal the regulation of MYC oncoprotein by PRMT5 and suggest that targeting PRMT5 could be a potential therapeutic strategy for MYC-driven medulloblastoma. Medulloblastoma is the most common malignant pediatric brain tumor, accounting for nearly 20% of all childhood brain cancers . CurrentEpigenetic deregulation has emerged as a key driver in medulloblastoma tumorigenesis, particularly alterations in histone modifying enzymes such as histone methyl transferases , 10. FurPRMT5 represents a member of PRMT family proteins that methylate histone and non-histone proteins to regulate gene expression and cellular development . PRMT5 sRecently, PRMT5 was found to associate with aberrant MYC function in various cancers including brain tumors such as glioblastoma and neuroblastoma –23. HoweThe human medulloblastoma cell lines Daoy (HTB-186), D-283 (HTB-185) and D-341 (HTB-187) were purchased from American Type Culture Collection . HD-MB03 (ACC-740) human medulloblastoma cell line was purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen . ONS-76 (IFO50355) human medulloblastoma cell line was obtained from Sekisui-XenoTech . These Cell lines were authenticated by their respective companies using short tandem repeat profiling. All cell lines were tested for mycoplasma contamination using MycoSensor PCR Assay Kit . In this study, Daoy and ONS-76 were used as SHH medulloblastoma subgroup cell lines without MYC-amplification, whereas, D-341, HD-MB03 and D-283 were used as Group 3 medulloblastoma cell lines with MYC-amplified status. All these cell lines were cultured and maintained using Eagle’s minimal essential medium (EMEM) or RPMI-1640 media supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin  in a humidified incubator at 5% CO2 and 95% air atmosphere at 37 °C. The experiments were performed using no more than 10 passages for each cell line. The cell lysate of human normal brain cerebellum was purchased from BioChain Institute Inc. .www.r2.amc.nl) was used to investigate PRMT5 mRNA expression and its correlation with patient survival across medulloblastoma subgroups using publicly available datasets. The expression of PRMT5 mRNA in medulloblastoma was analyzed using a total 491 medulloblastoma tumors (5 independent cohorts) and 9 normal cerebellum samples. The survival analyses with respect to PRMT5 expression in medulloblastoma patients were performed using a separate cohort of 612 medulloblastoma samples from Cavalli (763 samples) dataset.The R2 Genomics Analysis and Visualization Platform  and PRMT5 siRNAs  were purchased from Santacruz Biotechnology . Each siRNA was dissolved in RNase-free water at 10 μM stock concentration and stored at -20 °C. The PRMT5 inhibitor EPZ015666 was purchased from Selleckchem Company . This inhibitor was dissolved in DMSO at 10 mM stock concentration and stored at -20 °C.Control (scrambled) and PRMT5 siRNA (a pool of 3 target-specific 19–25 nt siRNAs with 50 nM) were transiently transfected into medulloblastoma cells using Lipofectamine 2000  according to the manufacturer’s instructions. Following 72 h of transfections, cells were subjected to downstream analyses using western blotting and MTT assay.To examine the effects of PRMT5 inhibition on medulloblastoma cell growth, twenty thousand cells of each medulloblastoma cell line were plated in 96-well plates 24 h before the experiment. Then, these cells were transfected with PRMT5 siRNAs or treated with PRMT5 inhibitor for 72 h according to the experimental plan and the growth of these cells was determined using an MTT assay as described previously [The effect of PRMT5 inhibitor to induce apoptosis in medulloblastoma cells at 72 h, was determined using an Annexin-V:FITC flow cytometry assay kit  following the manufacturer’s instructions. For cell cycle analysis, the control and PRMT5 inhibitor-treated medulloblastoma cells for 24 and 48 h, were fixed with 75% ethanol and stained with propidium iodide using a propidium iodide flow cytometry kit .To determine protein stability, medulloblastoma cells were treated with 50 μg/ml cycloheximide  following siRNA transfection for 72 h. Following transfection, cell lysates from the indicated time points of cycloheximide treatments were subjected to western blotting.For co-immunoprecipitation, 500 μg protein lysate was precleared with 50 μl of protein A-Sepharose beads  for 1 h at 4 °C. Immunoprecipitation was performed in the presence of 8 μg of the indicated primary antibodies at 4 °C overnight. Immune complexes were captured by adding 50 μl of protein A-Sepharose beads and rotated at 4 °C for 2 h. After the supernatant was discarded, protein A-Sepharose beads were washed with PBS and lysed in 1x Laemmli buffer and then subjected to western blotting.The expression levels of indicated proteins in medulloblastoma cells were determined using western blot analyses as described previously . The priMethanol-fixed HD-MB03 cells on glass cover slips, and an antigen-retrieved medulloblastoma tumor section were washed with PBS and blocked in 1% BSA in PBS for 30 min. The tumor cells were then co-incubated with PRMT5  and MYC  antibodies overnight at 4 °C. Following three washes with PBS, the cells were further co-incubated with fluorochrome-conjugated anti-rabbit (Alexa-488) and anti-mouse (Alexa-647) secondary antibodies  for 1 h at room temperature. The cells were then washed three times with PBS and the cover slips were mounted on glass slides and visualized under confocal microscope. DAPI was co-incubated with the secondary antibodies to facilitate the visualization of the nuclei. Confocal images were taken using a Zeiss LSM 5 Pascal confocal microscope  using a 40x objective in the UNMC Confocal Microscopy facility.Frozen samples of normal cerebella and medulloblastoma tumor specimens were collected from the Children’s Hospital and Medical Center, Omaha and the University of Nebraska Medical Center after Institutional Review Board (IRB) approval. Normal cerebellum specimens were obtained from patients at autopsy. All normal and tumor samples were from the pediatric age group.Normal cerebellum and medulloblastoma tumor sections were deparaffinized with xylene and rehydrated with water. Antigen retrieval was performed using citrate buffer at 95 °C for 20 min. Sections were treated with 3% hydrogen-peroxide for 30 min to block peroxidase activity. Sections were blocked using 5% goat serum with 0.3% Triton-X-100 in PBS and incubated with PRMT5 (1:100) and MYC (1:100) rabbit-antibodies  overnight at 4 °C. Next day, primary antibodies were washed with PBS three times and incubated with appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. Following three washes with PBS, detection was performed using a DAB Peroxidase Substrate Kit  followed by counterstaining with hematoxylin. Sections were mounted in Paramount solution and visualized under an EVOS FL Auto Imaging System . Staining intensity was scored from 0 to 3, where signal detected at 10X was 3+, at 20X was 2+, at 40X was 1+, and no detection was 0. The percentage positive cells was scored from 1 to 4 scale, where < 25% scored 1, 25–50% scored 2, 50–75% scored 3; and > 75 scored 4. Composite score (0–12) was derived from the staining intensity and % positive cells.p-value) were calculated using independent Student t-tests or analysis of variance (ANOVA) and p-values < 0.05 were considered significant. The IC50 values of inhibitor EPZ015666 for each medulloblastoma cell line were determined using GraphPad Prism V6 software . Our analyses using these data showed a significant overexpression of PRMT5 in medulloblastoma compared to normal cerebellum tissues  of samples with all 4 molecular subgroups. We observed significantly higher expression of PRMT5 in Group 3 (MYC-driven) medulloblastoma compared to other 3 subgroups  dataset. Our survival analyses showed that high levels of PRMT5 expression correlated with poor survival of medulloblastoma patients, a pattern recapitulated in Group 3 medulloblastoma patients  cohort at the R2 genomic analysis platform. Results from this analysis showed that high expression of PRMT5 was strongly correlated  with high MYC in Group 3 medulloblastoma. Although results showed some degree of correlation  of these genes in the other three medulloblastoma subgroups, none of these medulloblastoma subgroups showed a significant correlation between PRMT5 and MYC expression  higher in MYC-driven medulloblastoma cell lines compared to non–MYC medulloblastoma cell lines and normal human cerebellum cells  compared to normal pediatric cerebellum (n = 4). We found that the protein levels of PRMT5 and MYC were significantly (p = 0.004) higher with more than 75% staining in Group 3 medulloblastoma samples than normal pediatric cerebellum tissues -mediated knockdown of PRMT5 on MYC expression and cell survival in MYC-amplified medulloblastoma cell lines. We used two cell lines D-341 and HD-MB03 in this study as both lines have previously been reported as well-established Group 3 medulloblastoma cell lines with high MYC expression. Consistently, our western blot results further confirmed strongly higher expression of MYC protein in these two cell lines compared to other medulloblastoma lines Fig. a. Using Previous studies , 23 in nTo determine the consequence of this physical interaction on post-translational MYC stability and influence on MYC expression, we performed cycloheximide (CHX) chase experiments in PRMT5 knocked-down HD-MB03 cells and measured the half-life of MYC protein. As shown in Fig. Given the potential for anti-neoplastic effects with PRMT5 knockdown, via reduction in cell viability and MYC expression, we investigated the therapeutic potential of PRMT5 inhibition using a recently developed potent PRMT5 inhibitor EPZ015666) . We firs5666 . WeWe next determined the ability of EPZ015666 to induce apoptosis in a representative MYC-amplified medulloblastoma cell line HD-MB03. The results of the apoptosis analyses  cohort at R2-Genomics platform.