An ability to develop sequence-defined synthetic polymers that both mimic lipid amphiphilicity for self-assembly of highly stable membrane-mimetic 2D nanomaterials and exhibit protein-like functionality would revolutionize the development of biomimetic membranes. Here we report the assembly of lipid-like peptoids into highly stable, crystalline, free-standing and self-repairing membrane-mimetic 2D nanomaterials through a facile crystallization process. Both experimental and molecular dynamics simulation results show that peptoids assemble into membranes through an anisotropic formation process. We further demonstrated the use of peptoid membranes as a robust platform to incorporate and pattern functional objects through large side-chain diversity and/or co-crystallization approaches. Similar to lipid membranes, peptoid membranes exhibit changes in thickness upon exposure to external stimuli; they can coat surfaces in single layers and self-repair. We anticipate that this new class of membrane-mimetic 2D nanomaterials will provide a robust matrix for development of biomimetic membranes tailored to specific applications. Biomimetic membranes can be used for various applications such as sensors and separations. Here, Chen et al. report the assembly of lipid-like peptoids into stable and self-repairing 2D membrane nanomaterials that change in thickness when under external stimuli. Molecular self-assembly is the key to building up well-defined functional structures in biology1234345Peptoids (or poly-N-substituted glycines)711121314N-(2-carboxyethyl)glycine (Nce) and six nonpolar residues, N-[2-(4-chlorophenyl)ethyl]glycines(N4-Clpe)  group on the nonpolar residues. Pep-6, which has six N-[2-(3-chlorophenyl)ethyl]glycines (N3-Clpe) residues, formed membranes with slightly different packing (-7 with six N-[2-(2-chlorophenyl)ethyl]glycines (N2-Clpe) did not self-assemble into crystalline membranes  or changing the polar domains from Nce6 to Nce3 (Pep-9), non-carboxyl-containing Nte3 [Nte=N-2-(2-(2-methoxyethoxy)ethoxy)ethylglycine] (Pep-10) or (NceNae)3 [Nae=N-(2-aminoethyl)glycine]  or a mixture of Nce and Nae residues (Pep-11) self-assembled into biomimetic membranes glycine] residues  images  images . These T process . We infe4-Clpe domains and the flexible hydrophilic Nce domains. To minimize the exposure of hydrophobic groups to water and to bring carboxyl groups on adjacent peptoid chains next to each other, the flexible hydrophilic domains need to be floppy, but they could align laterally to form strips along the x direction  with half the peptoids oriented up along the z axis and half oriented down. The side chains were oriented along the y axis, and the peptoids were stacked along the x axis. The system was solvated with 2 nm of explicit SPC water in the z direction. Peptoids were modelled with the AMBER03 forcefield20π-stacking in the hydrophobic N4-ClPe region and disorder in the carboxyl tails. In addition, simulated X-ray diffraction data for Pep-1 membranes is in good agreement with the experimental data (x direction (15.9 contacts per nm2 of contact area) than the y direction (0.86 contacts per nm2 of contact area); this indicates that peptoid packing in the membrane is anisotropic, which is consistent with our experimental results showing the anisotropic membrane formation process ethyl]glycine (Ntrp) that mimics tryptophan—a redox-active residue that facilitates electron transferN-[2-(4-hydroxylphenyl)ethyl]glycines (Ntyr) that mimics tyrosine—a redox-active residue significant for electron transfer during photosynthesis in nature2 (ref. N-(2-thiolethyl)glycine (Nse) that mimics cysteineN-[4-(2-phenyldiazenyl)phenyl]glycines (Nazo); and N-[benzo-15-crown-5-ether]glycines (Nbce) that could build ion channels28N-[(1-pyrenemethyl)]glycines (Npyr) and Nhex were also used to test the tolerance of peptoid membrane assembly in incorporating highly hydrophilic and hydrophobic objects.While peptoids Pep-re2 ref. ; N-, formed 2D membranes. X-ray diffraction results confirmed that they have highly crystalline structures similar to Pep-2  and X-ray diffraction results showed that all these peptoids formed 2D nanomembranes with similar structures to Pep-2 . As showto Pep-2 . The conPep-2 containing Ncd  for HPLC purification. Lyophilized and HPLC-grade peptoids were dissolved in the mixture of water and acetonitrile (v/v=1:1) to make a 5-mM clear solution, which was then transferred to a 4°C refrigerator for slow evaporation. Suspensions or gel-like materials containing a large amount of crystalline membranes were formed after a few days.Detailed information on materials and methods is available in the 8All relevant data are available from the authors.How to cite this article: Jin, H. et al. Highly stable and self-repairing membrane-mimetic 2D nanomaterials assembled from lipid-like peptoids. Nat. Commun. 7:12252 doi: 10.1038/ncomms12252 (2016).Supplementary Figures 1-57, Supplementary Methods and Supplementary References


PED/PEA-15 mice hydrogen peroxide-induced DNA fragmentation was lower compared to WT islets. TUNEL analysis showed that PED/PEA-15 overexpression increases the viability of Ins-1E beta-cells and enhances their resistance to apoptosis induced by hydrogen peroxide exposure. The activity of caspase-3 and the cleavage of PARP-1 were markedly reduced in Ins-1E cells overexpressing PED/PEA-15 (Ins-1EPED/PEA-15). In parallel, we observed a decrease of the mRNA levels of pro-apoptotic genes Bcl-xS and Bad. In contrast, the expression of the anti-apoptotic gene Bcl-xL was enhanced. Accordingly, DNA fragmentation was higher in control cells compared to Ins-1EPED/PEA-15 cells. Interestingly, the preincubation with propranolol, an inhibitor of the pathway of PLD-1, a known interactor of PED/PEA-15, responsible for its deleterious effects on glucose tolerance, abolishes the antiapoptotic effects of PED/PEA-15 overexpression in Ins-1E beta-cells. The same results have been obtained by inhibiting PED/PEA-15 interaction with PLD-1 in Ins-1EPED/PEA-15. These results show that PED/PEA-15 overexpression is sufficient to block hydrogen peroxide-induced apoptosis in Ins-1E cells through a PLD-1 mediated mechanism.The small scaffold protein PED/PEA-15 is involved in several different physiologic and pathologic processes, such as cell proliferation and survival, diabetes and cancer. PED/PEA-15 exerts an anti-apoptotic function due to its ability to interfere with both extrinsic and intrinsic apoptotic pathways in different cell types. Recent evidence shows that mice overexpressing PED/PEA-15 present larger pancreatic islets and increased beta-cells mass. In the present work we investigated PED/PEA-15 role in hydrogen peroxide-induced apoptosis in Ins-1E beta-cells. In pancreatic islets isolated from Tg PED/PEA-15 is a small cytosolic protein highly conserved among mammals, ubiquitously expressed The immunohistochemistry and morphometric analysis of islets from these animals evidenced that islets were larger and showed a more elongated and irregular shape despite a normal distribution of alpha- and beta-cells, compared with islets from control animals. Quantitative morphometry also showed a two fold increase of islets and beta-cells mass per unit of total pancreatic area in beta-tg mice without significant difference in their pancreas weight Rat Ins-1E beta-cell line was kindly donated by P. Maechler (University of Geneva). Ins-1E cells cultured in regular RPMI 1640 medium with 11 mM glucose, supplemented with HEPES, 5% fetal calf serum, 50 µM 2-mercaptoethanol, 1 mM pyruvate, and antibiotics, as described in CTRL and Ins-1EPED/PEA-15 cells were treated as indicated with hydrogen peroxide (70 µM) and propranolol (150 µM). Time of incubation and concentration of propranolol were set performing preliminary dose/response sulforhodamine experiments to avoid toxicity (data not shown). The D4 cDNA was amplified by PCR from HeLa cells and cloned into pcDNA3-HA vector as previously described CTRL and Ins-1EPED/PEA-15 cells were evaluated by qualitative PCR using the following human primers:The cDNA of PED/PEA-15 cloned in pcDNA3 expression vector as previously described 5′-CGATCAAGCTTCGCTGGAACTTCACAAAAAT-3′5′-CGATCTCTAGAGGATACGAGAATGCGTCAGG-3′PED/PEA-15 mice PED/PEA-15 mice and their non-transgenic littermates  were used for all studies. All procedures and euthanasia were conducted in according to the guidelines and approved by the Institutional Animal Care and Utilization Committee TgPED/PEA-15 mice by collagenase digestion, handpicked under a stereomicroscope as described in Islets were isolated from 4-month-old C57Bl/6J mice (WT) and Tg4 cells in 100 µl of medium/well were plated in 96-well flat-bottomed microtiter plates. 24 h after plating  culture medium was replaced with fresh medium either containing or not containing the hydrogen peroxide. At the end of hydrogen peroxide exposure, cells were fixed with 50% TCA for 2 h at 4°C and stained with 0.4% SRB (Sigma Aldrich) dissolved in 1% acetic acid for 30 minutes. The plates were then washed four times with 1% acetic acid to remove unbound stain, air-dried and solubilized in 100 µl of 10 mM unbuffered Tris base (tris(hydroxymethyl)aminomethane) solution. The optical density of treated cells was detected at 490 nm. Each sample was run in octuplet, and each experiment was repeated three times.The sulforhodamine B (SRB) assay was used as described previously PLUS kit  in vitro.The mechanism of cell death was quantitatively determined using the Cell Death Detection ELISAPED/PEA-15 and control cells were processed as described by the manufacturer's instructions. Necrotic cells were removed by centrifugation and the pellet (apoptotic fraction) was analyzed by ELISA. Measurements were made at 405 nm against an ABTS solution as a blank (reference wavelength ∼490 nm) using an Infinite F200 Tecan multimode reader.After treatments, Ins-1EPED/PEA-15 and control cells were fixed with 4% paraformaldehyde for 1 h and permeabilized in a 0.1% Triton X-100 - 0.1% sodium citrate solution for 2 min on ice. Subsequently, 50 µl of TUNEL reaction mixture was added to each sample and cells were incubated for 1 h at 37°C. Cells were counterstained with DAPI before mounting For TUNEL staining the In Situ Cell Death fluorescent kit  was used, following the manufacturer's instructions. Briefly, Ins-1EPED/PEA-15 and control cells plated on coverslips were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 in PBS for 20 min. Cells were exposed to blocking buffer  for 1 h and incubated overnight with primary antibody anti-cleaved Caspase-3  diluted 1∶400 in blocking buffer. Antigen-bound primary Caspase-3 antibody was detected by 1 h incubation with a 1∶200 dilution of Rhodamin RedX conjugated donkey anti-rabbit secondary antibody . All incubation steps were separated by washing in 0.1% Triton X-100 in PBS for 3×5 min Ins-1EPED/PEA-15 and control cells were solubilized in lysis buffer  for 2 h at 4°C. Cell lysates were clarified by centrifugation at 5,000 g for 20 min, separated by SDS-PAGE, and transferred onto 0.45-µm Immobilon-P membranes  For Western blot analysis, after treatments Ins-1EPED/PEA-15 and control cells using TRIzol reagent (Life Technologies) according to manufacturer's instructions. After quantification, 1 µg of total RNA was reverse transcribed using Superscript III Reverse Transcriptase (Invitrogen) using random hexamers. Real-time RT-PCR was performed on an iCycler IQ multicolor Real Time PCR Detection System  using the comparative CT method (2-ΔΔCT) method with a Platinum SYBR Green qPCR Super-UDG mix  Total RNA was isolated from Ins-1EAGAGGCTGGCGATGAGTTTBcl- x- Fw 5′-3′ AGAAGAAGGCCACAATGCGABcl- xL Rv 5′-3′ GCTGCATTGTTCCCGTAGBcl- xS Rv 5′-3′ CCAGAGTTTGAGCCGAGTGAGCABAD – Fw 5′-3′ CTGTTATTGGCTGCCTGTCCCGBAD – Rv 5′-3′ GTAACCCGTTGAACCCCATT18s rRNA Fw 5′-3′ CCATCCAATCGGTAGTAGCG18s rRNA Rv 5′-3′ p<0.05. Data are expressed as means ± standard errors of the mean (SEM).For statistical analyses, one-way ANOVA followed by Tukey-Kramer multiple comparison test was performed using GraphPad Prism 5.00 . The statistical significance was set at PED/PEA-15 mice and their non-transgenic littermates . Islets were incubated 16 h in presence or absence of 10 µM hydrogen peroxide PED/PEA-15 mice only a 2-fold increase in apoptosis was observed upon hydrogen peroxide incubation.To investigate the role of PED/PEA-15 overexpression in beta-cells apoptosis, we isolated pancreatic islets from TgPED/PEA-15) or an empty vector (Ins-1ECTRL) PED/PEA-15 cells exposed to hydrogen peroxide and about 30% of apoptotic cells in Ins-1ECTRL cells  for shorter times (3–8 h) did not significantly modified anti- and pro-apoptotic genes expression  reduction of viability compared to untreated cells. In contrast, the preincubation of hydrogen peroxide treated Ins-1EPED/PEA-15 cells with propranolol reduced cell viability by ∼60%, and almost abolished the apoptosis protective effects conferred by PED/PEA-15 overexpression. Indeed, the viability of Ins-1EPED/PEA-15 cells treated with hydrogen peroxide in the presence of propranolol was comparable with that measured in Ins-1ECTRL cells in the same conditions  for shorter times (3–8 h) did not significantly modified cell viability , spanning residues 929–1030 15 cells . In Ins-c action .PED/PEA-15 mice compared to Wt mice. Moreover, sulforhodamine B staining showed a marked increase in viability of beta-cells overexpressing PED/PEA-15 upon hydrogen peroxide incubation compared to control cells. Both ELISA and TUNEL analysis showed that overexpression of PED/PEA-15 in Ins-1E beta-cell model protects from hydrogen peroxide induced apoptosis. Furthermore, several steps of the apoptotic cascade are modified by the overexpression of PED/PEA-15. Caspase-3 activation, induced by hydrogen peroxide exposure, is lower in Ins-1EPED/PEA-15, similar to PARP-1 cleavage. Also the mRNA expression levels of the antiapoptotic gene Bcl-xL in hydrogen peroxide treated Ins-1EPED/PEA-15 cells is increased, while the expression of proapoptotic genes such as Bcl-xS and Bad PED/PEA-15 cells compared to control cells upon hydrogen peroxide incubation. This is not surprising since the balance between pro- and anti-apoptotic members of the Bcl-2 family has been described to modulate apoptosis induced by hydrogen peroxide in several cell type including HeLa cells, neonatal rat cardiac myocytes and PC12 PED/PEA-15 cells and in myocytes derived from PED/PEA-15-overexpressing transgenic mice, D4 domain abrogates the PED/PEA-15-PLD-1 interaction and reduces protein kinase C-alpha activity to levels similar to controls. Interestingly, the peptide restores insulin-stimulated glucose uptake by approximately 70% PED/PEA-15 cells, despite the presence of phosphatidic acid. DAG is a second messenger produced by PLD-1 and activates classical PKC isoforms such as PKC alpha and beta PED/PEA-15 is a small scaffold protein, which modulates signalling pathways relevant to several human disorders such as cancer and Type 2 diabetes. In particular, PED/PEA-15 regulates proliferation by binding ERK1/2 and blocks apoptosis by interfering with both the intrinsic and the extrinsic pathways S1 FigurePED/PEA-15 and in Ins-1ECTRL cells upon incubation with 30–50 µM hydrogen peroxide for 3–8 h. The mRNA amounts of Bcl-xL (A), Bcl-xS (B) and Bad (C) remain unchanged both in Ins-1EPED/PEA-15 and in Ins-1ECTRL cells in response to hydrogen peroxide.Hydrogen peroxide effect on anti- and pro-apoptotic genes expression (dose-response). The expression levels of anti- and pro-apoptotic genes were examined by RT-PCR in Ins-1E(TIF)Click here for additional data file.S2 FigureCTRL and Ins-1EPED/PEA-15 (dose-response). Ins-1ECTRL and Ins-1EPED/PEA-15 were treated for the indicated times (3–8 h) with 30–50 µM hydrogen peroxide and then we evaluated cell viability by sulforhodamine B staining (A) and apoptosis measuring the level of DNA fragmentation (B). We did not find significant differences both in Ins-1ECTRL and Ins-1EPED/PEA-15 upon hydrogen peroxide treatment.Hydrogen peroxide effect on Ins-1E(TIF)Click here for additional data file.S3 FigurePED/PEA-15 and in Ins-1ECTRL cells performing proliferation curves analysis. To this aim, Ins-1EPED/PEA-15 and Ins-1ECTRL cells were seeded at a density of 2×104 cells in 60 mm dishes. After 24, 48, 72 or 96 h non-adherent cells were removed by gentle washing with PBS whether adherent cells were detached by trypsin treatment and counted using BIO-RAD TC10 automated Cell Counter . As shown in PED/PEA-15 compared to Ins-1ECTRL cells.Proliferation curves analysis. We evaluated cell proliferation in Ins-1E(TIF)Click here for additional data file.


Arabidopsis VQ motif-containing proteins have recently been demonstrated to interact with several WRKY transcription factors; however, their specific biological functions and the molecular mechanisms underlying their involvement in defense responses remain largely unclear. Here, we showed that two VQ genes, VQ12 and VQ29, were highly responsive to the necrotrophic fungal pathogen Botrytis cinerea. To characterize their roles in plant defense, we generated amiR-vq12 transgenic plants by using an artificial miRNA approach to suppress the expression of VQ12, and isolated a loss-of-function mutant of VQ29. Phenotypic analysis showed that decreasing the expression of VQ12 and VQ29 simultaneously rendered the amiR-vq12 vq29 double mutant plants resistant against B. cinerea. Consistently, the B. cinerea-induced expression of defense-related PLANT DEFENSIN1.2 (PDF1.2) was increased in amiR-vq12 vq29. In contrast, constitutively-expressing VQ12 or VQ29 confered transgenic plants susceptible to B. cinerea. Further investigation revealed that VQ12 and VQ29 physically interacted with themselves and each other to form homodimers and heterodimer. Moreover, expression analysis of VQ12 and VQ29 in defense-signaling mutants suggested that they were partially involved in jasmonate (JA)-signaling pathway. Taken together, our study indicates that VQ12 and VQ29 negatively regulate plant basal resistance against B. cinerea. In nature, plants are constantly threatened by various microbial pathogens. To protect themselves against pathogen infection, resisant plants have evolved an effective innate immune system. Upon infection by virulent pathogens, plants detect microbes or pathogen-associated molecular patterns (PAMPs) via transmembrane pattern recognition receptors (PRRs), resulting in PAMP-triggered immunity (PTI)PATHOGENESIS-RELATED GENE (PR) genesArabidopsis mutants deficient in SA biosynthesis  or responsiveness  are compromised to establish both basal defense and systemic acquired resistance4689101213R proteins-mediated resistance is often associated with activation of the salicylic acid (SA)-signaling pathway that induces a subset of Arabidopsis WRKY transcription factor family comprises 74 members which are subdivided into three major structural groups151718Botrytis cinerea and Alternaria brassicicolaPseudomonas syringaeP. syringaeWRKY11 and WRKY17 resulted in increased defense-related gene expression and enhanced basal defense to P. syringaeThe Arabidopsis272431B. cinerea263025et al. showed that VQ29 acts as a negative transcriptional regulator of light-mediated inhibition of hypocotyl elongation by interacting with PHYTOCHROME-INTERACTING FACTOR1 (PIF1)Recently, several WRKY transcription factors have been found to physically interact with a class of novel proteins, defined as VQ proteins, to regulate various physiological processes2425262728et al. showed that a number of Arabidopsis VQ genes were responsive to plant defense signalsVQ genes and the exact mechanisms underlying their involvement in defense responses remain largely unknown. To further clarify the functions of Arabidopsis VQ genes in plant defense, we chose VQ12 and VQ29 for further investigation. VQ12 and VQ29 were strongly induced by JA treatment and B. cinerea infection; and the proteins encoding by VQ12 and VQ29 were exclusively localized in the nucleus. Phenotypic analysis indicated that the resistance of vq29 mutant plants to B. cinerea was enhanced compared with that of wild type. Moreover, decreasing the expression of VQ12 and VQ29 simultaneously conferred the amiR-vq12 vq29 double mutant plants even greater resistance against B. cinerea. In contrast, the transgenic plants overexpressing VQ12 or VQ29 were much more susceptible to B. cinerea. Further investigation revealed that VQ12 and VQ29 physically interacted with themselves and each other to form homodimers and heterodimer. Taken together, our results provide evidence that VQ12 and VQ29 negatively regulate plant basal resistance against B. cinerea.Cheng Arabidopsis VQ12 (AT2G22880) and VQ29 (AT4G37710) encode two VQ motif-containing proteins with 114 and 123 amino acids, respectivelypromoterVQ12:GUS and promoterVQ29:GUS transgenic plants. β-Glucuronidase (GUS) staining showed that VQ12 was mainly expressed in the root, leaf, hypocotyl, and silique base  and SA, but not by abscisic acid (ABA) and 1-aminocyclopropane-1-carboxylate (ACC). Similarly, the expression level of VQ29 was also upregulated by MeJA treatment  35S promoter and these constructs were transiently expressed in leaves of tobacco (Nicotiana benthamiana). As shown in To determine the properties of VQ12 and VQ29 in more detail, we next analyzed their subcellular localizations. The full-length VQ12 and VQ29 were fused to the green fluorescent protein (GFP) protein under the control of the B. cinerea, we generated amiR-vq12 transgenic plants by using an artificial miRNA approach to repress VQ12 expressionVQ12 in amiR-vq12 transgenic lines 5 and 7 (amiR-vq12–5 and amiR-vq12–7) were reduced compared with those in wild-type plants with or without MeJA treatment  mutant harbors a T-DNA insertion in the promoter region (−136 bp from the translation start site) of VQ29 . As shown in amiR-vq12 lines and wild type in disease symptom development was observed. However, following infection with B. cinerea, the vq29 mutant plants showed reduced disease symptom with restricted disease lesions in comparison with wild-type plants . Since vq29 and amiR-vq12 vq29 plants acquired more resistance against B. cinerea, the pathogen-induced expression of PDF1.2, the antifungal gene THIONIN2.1 (Thi2.1), and the pathogenesis response gene PR4, were also analyzed3439PDF1.2, Thi2.1 and PR4 transcripts were increased in vq29 and amiR-vq12 vq29 plants after B. cinerea infection  were fused to the Gal4 DNA-binding domain of the bait vector (BD-VQ12 and BD-VQ29). After screening, more than 30 independent colonies were isolated. Among these positive clones, clones encoding VQ proteins were frequently represented. To confirm their physical interactions, the full-length coding sequences of all 34 Arabidopsis VQ proteins were cloned and introduced into the prey vector (AD-VQ). The BD-VQ12 or BD-VQ29 and AD-VQ plasmids were co-transformed into yeast. As shown in To understand how VQ12 and VQ29 modulate plant resistance against To characterize which domain of VQ12 or VQ29 is responsible for the interactions with themselves or each other, VQ12 and VQ29 were divided into N-terminal parts containing the VQ motif and C-terminal portions . MoreoveWe further re-examined the properties of different domains of VQ12 and VQ29 in more detail by analyzing their sub-cellular localization. Different domains of VQ12 and VQ29 were fused to the GFP protein and these fused proteins were transiently expressed in leaves of tobacco. As shown in Arabidopsis, several signaling pathways are involved in defense responses, such as the SA-, ET- and JA-signaling pathways. To analyze which pathway VQ12 and VQ29 are involved in, we monitored their expression in various defense-related mutants, including sid2 (SA-biosynthesis mutant), npr1 , coi1  and ein2 481214B. cinerea, the basic expression levels of VQ12 and VQ29 were not affected in those mutants  mutant and all transgenic plants used in this study were derived from the Arabidopsis Columbia (Col-0) ecotype. Arabidopsis plants were grown in growth chambers at 22°C with a 10-h light/14-h dark photoperiod. The mutant lines npr1, sid2, coi1 and ein2 were kindly gifted by Dr. Zhixiang Chen . The plant hormones MeJA, SA, ABA and ACC were purchased from Sigma-Aldrich. Taq DNA polymerase was purchased from TaKaRa Biotechnology Co. Ltd. Other common chemicals were obtained from Shanghai Sangon Biotechnology Co. Ltd.The PstDC3000 infection was performed as described inB. cinerea spores and plant inoculation was performed as described previously5 spores/ml in Sabouraud maltose broth (SMB) buffer was placed on each leaf. Induction treatments with the plant hormones MeJA, SA and ACC were performed as described in Chen et al.Agrobacterium tumefaciens strain EHA105. For transient expression, N. benthamiana leaves were infiltrated with the bacterial cell suspensions . After infiltration, the plants were incubated at 24 °C for 4 h, and then the infected leaves were sectioned for observation. GFP and DAPI fluorescence were observed under a confocal laser scanning microscope .Full-length VQ12 and VQ29 fused to GFP were cloned into the pOCA30 binary vector, downstream of the CaMV 35S promoter. The constructs were then transformed into vq29 mutant contains a T-DNA insertion in the promoter of VQ29 gene. We confirmed the T-DNA insertion by PCR using a combination of a T-DNA border primer (5′-AAACGTCCGCAATGTGTTAT-3′) and a gene-specific primer . The insertion mutant was further confirmed by qRT-PCR. To generate the VQ12 and VQ29 overexpression constructs, the full-length VQ12 and VQ29 gene coding sequences were PCR-amplified from Arabidopsis gDNA and cloned into the pOCA30 vector in the sense orientation behind the CaMV 35S promoterVQ12 expression we used an artificial miRNA approach as described in Liang et al.amiR-vq12 sequence was designed using WMD3 (http://wmd3.weigelworld.org), with an AthmiR319a backbone to drive its expression38amiR-vq12 precursor was cloned into the pOCA30 vector. All constructs were transformed into Arabidopsis plants using the Agrobacterium-mediated flower dip method. Homozygous transgenic amiR-vq12–5 line were crossed with vq29 to generate amiR-vq12 vq29 homozygous plants. VQ12OX4/VQ29OX3 plants were generated through genetic crosses of VQ12OX4 and VQ29OX3 homozygous transgenic lines.The ACTIN2 (AT3G18780) was used as an internal control. The gene-specific primers for qRT-PCR are listed in Total RNA was extracted using the Trizol reagent (Invitrogen). One microgram of DNase-treated RNA was reverse-transcribed in a 20 μl reaction mixture using Superscript II (Invitrogen). After the reaction, 1 μl cDNA was subjected to qRT-PCR using a SYBR Premix Ex Taq kit (Takara) on a Roche LightCycler 480 real-time PCR machine. At least three independent biological samples were used for qRT-PCR analysis for each reported result. VQ12 and VQ29 were amplified from genomic DNA using gene-specific primers . The cDNA library was obtained from Clontech . Yeast two-hybrid screening was performed as described in Hu VQ12 and VQ29 were cloned into pFGC-nYFP to generate N-terminal in-frame fusions with N-YFP, while VQ12 and VQ29 CDSs were inserted into pFGC-C-YFP to form C-terminal in-frame fusions with C-YFP. The plasmids were transformed into A. tumefaciens strain EHA105, and infiltration of N. benthamiana was performed as described previouslyThe cDNA sequences for the N-terminal 173-amino acid eYFP (N-YFP) and C-terminal 64-amino acid (C-YFP) fragments were cloned into pFGC5941 to generate pFGC-nYFP and pFGC-cYFP, respectivelyAccession codes:Arabidopsis Genome Initiative numbers for the genes discussed in this article are as follows: VQ12 (AT2G22880), VQ29 (AT4G37710), PDF1.2 (AT5G44420), VQ1 (AT1G17147), VQ2 (AT1G21320), VQ3 (AT1G21326), VQ4 (AT1G28280), VQ5 (AT1G32585), VQ6 (At1G32610), VQ7 (AT1G35830), VQ8 (AT1G68450), VQ9 (AT1G78310), VQ10 (AT1G78410), VQ11 (AT1G80450), VQ13 (AT2G33780), VQ14 (AT2G35230), VQ15 (AT2G41010), VQ16 (AT2G41180), VQ17 (AT2G42140), VQ18 (AT2G44340), VQ19 (AT3G15300), VQ20 (AT3G18360), VQ21 (AT3G18690), VQ22 (AT3G22160), VQ23 (AT3G56710), VQ24 (AT3G56880), VQ25 (AT3G58000), VQ26 (AT3G60090), VQ27 (AT4G15120), VQ28 (AT4G20000), VQ30 (AT4G39720), VQ31 (AT5G08480), VQ32 (AT5G46780), VQ33 (AT5G53830), VQ34 (AT5G65170).How to cite this article: Wang, H. et al.Arabidopsis VQ motif-containing proteins VQ12 and VQ29 negatively modulate basal defense against Botrytis cinerea. Sci. Rep.5, 14185; doi: 10.1038/srep14185 (2015).


T-VEC, an intralesionally-delivered oncolytic immunotherapy, is a herpes simplex virus-1 engineered to selectively replicate in tumors and stimulate an anti-tumor immune response through expression of GM-CSF. T-VEC has the ability to lyse various cancer cell types in vitro. To furt7 or 108 PFU/mL) and volumes (up to 4 or 8 mL). MTD for HCC is determined separately from non-HCC tumor types; HCC cohorts will not proceed until safety at respective dose levels are determined in non-HCC. Six T-VEC doses injected under ultrasound or computed tomography guidance q21 (±3) days are planned, with an investigator option to continue for up to 6 additional doses. The first dose of T-VEC in all dose cohorts is given at 106 PFU/mL. Part 2: 7 expansion cohorts for each cancer type with 10 patients each administered the MTD of T-VEC determined from Part 1.Approximately 100 patients will be enrolled. Primary objective: evaluate maximum tolerated dose (MTD) of intrahepatic injection of T-VEC by patient incidence of dose-limiting toxicities (DLTs). Key secondary objectives: overall safety, efficacy, and biodistribution of T-VEC. Key eligibility criteria: breast, colorectal, gastroesophageal, kidney, lung cancer or melanoma with liver metastases (non-HCC) or hepatocellular carcinoma (HCC); measurable liver tumors suitable for injection; ECOG performance status 0-1; life expectancy ≥5 months; ≥1 prior standard systemic anticancer therapy (non-HCC); Child-Pugh A-B7; no detectable hepatitis B/C viral load; not a candidate for surgery or locoregional therapy of liver tumors with curative intent or planned systemic anticancer therapy; tumor in < 1/3 of the liver; no macroscopic intravascular invasion. The study consists of two parts. Part 1: 3+3 dose escalation of 3 sequential dose cohorts each administering T-VEC in increasing concentrations (10


SUMO-binding proteins interact with SUMO modified proteins to mediate a wide range of functional consequences. Here, we report the identification of a new SUMO-binding protein, ZNF261. Four human proteins including ZNF261, ZNF198, ZNF262, and ZNF258 contain a stretch of tandem zinc fingers called myeloproliferative and mental retardation (MYM)-type zinc fingers. We demonstrated that MYM-type zinc fingers from ZNF261 and ZNF198 are necessary and sufficient for SUMO-binding and that individual MYM-type zinc fingers function as SUMO-interacting motifs (SIMs). Our binding studies revealed that the MYM-type zinc fingers from ZNF261 and ZNF198 interact with the same surface on SUMO-2 recognized by the archetypal consensus SIM. We also present evidence that MYM-type zinc fingers in ZNF261 contain zinc, but that zinc is not required for SUMO-binding. Immunofluorescence microscopy studies using truncated fragments of ZNF198 revealed that MYM-type zinc fingers of ZNF198 are necessary for localization to PML-nuclear bodies (PML-NBs). In summary, our studies have identified and characterized the SUMO-binding activity of the MYM-type zinc fingers in ZNF261 and ZNF198. Small Ubiquitin-related Modifiers (SUMOs) are reversible post-translational protein modifications that are conjugated to lysine residues on substrate proteins by an enzymatic cascade. Interactions between SUMO-modified proteins and SUMO-binding proteins containing SUMO-interacting motifs (SIMs) regulate a wide range of functional consequences including formation of multi-protein complexes and changes in protein localization, activity, solubility, and stability The archetypal consensus SIM consists of four hydrophobic amino acids adjacent to a cluster of negatively charged residues To address SUMO paralog-selectivity, we initiated high-throughput studies using protein microarrays to identify novel SUMO-1, SUMO-2, and SUMO-2 polymeric chain binding proteins. Among the identified proteins was ZNF261, which contains a stretch of unique tandem zinc fingers called MYM-type zinc fingers . Tandem MYM-type zinc fingers are found in three other human proteins including ZNF198, ZNF262, and ZNF258 ZNF198 is the best studied member of the MYM-type zinc finger family of proteins. Translocation of the ZNF198 and Fibroblast Growth Factor Receptor 1 (FGFR1) genes has been linked to atypical myeloproliferative disease In this report, we characterized ZNF261 as a newly identified SUMO-binding protein. Our analysis revealed that ZNF261 and ZNF198 interact with SUMO through the MYM-type zinc fingers rather than a consensus SIM. We also provide evidence that the MYM-type zinc fingers in ZNF198 are important for localization to PML-nuclear bodies (PML-NBs). Taken together, our findings establish that the MYM-type zinc fingers are legitimate SUMO-binding domains.Anti-GST , anti-myc , anti-PML  were used.2.The U2OS cell line used in this study is an established cell line that is commercially available from American Type Culture Collection . U2OS cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 10 mM Hepes. Cells were cultured at 37°C in a humidified incubator with 5% COCD measurements of ZNF261 samples (75 µM) were performed on a Jasco J-810 spectropolarimeter using a 0.2 mm pathlength cuvette (Jasco). Protein absorbance was measured in the UV range between 260 and 190 nm. Samples were scanned at 100 nm/min and 10 scans were collected per sample. High-tension voltage was kept below 500 V during all scans. Treated samples were dialyzed overnight against 50 mM EDTA, 100 mM sodium acetate pH 5.5 or 6 M GuHCl.2 at 10°C in an An60-Ti rotor on a Beckman Optima XL-I. Recombinant MYM SIMs from ZNF261 (amino acids 236–490) were run in the analytical ultracentrifuge at three different concentrations  at rotor speeds of 15,000, 20,000, and 25,000 rpm. Three different concentrations of SUMO-2(x2)  were added to 20 µM MYM SIMs from ZNF261 and run in the analytical ultracentrifuge using the same protocol. Approach to equilibrium was monitored using SEDFIT Equilibrium analytical ultracentrifugation experiments were performed in 20 mM HEPES, pH 7.6, 350 mM NaCl, 5 mM DTT, and 10 µM ZnClCells grown on coverslips were washed with phosphate-buffered saline (PBS) and then fixed and permeabilized at room temperature with PBS containing 2% formaldehyde/0.2% Triton X-100. After 10 min, cells were washed with PBS and fixed with 2% formaldehyde in PBS for 20 min at room temperature. Cells were washed with PBS and incubated with the indicated primary antibodies for 1 hour. Cells were washed and incubated with Alexa Fluor–conjugated goat anti-rabbit immunoglobulinG (IgG) or Alexa Fluor–conjugated goat anti-mouse IgG (Invitrogen) for 1 hour. Coverslips were washed with PBS and mounted with mounting solution containing 100 mM tris (pH 8.8), 50% glycerol, 2.5% DABCO (Sigma), and 4′,6′-diamidino-2-phenylindole (0.2 mg/ml). Images were obtained on a Zeiss Observer.Z1 microscope.U2OS cells were transfected, incubated for 24 hours, and lysed in buffer A  containing 1 mM PMSF, 0.3 µM aprotinin, 10 µM leupeptin, 1 µM pepstatin A, 0.025 U/µl benzonase, and 10 mM N-ethyl-maleimide (Sigma). Cell lysates were sonicated on ice and centrifuged at 14,000 rpm and 4°C for 10 min. Soluble cell lysates and anti-Flag-agarose beads  or anti-myc-agarose beads  were incubated for 3 hours and washed five times with buffer A. Bound proteins were eluted with SDS-PAGE loading buffer.in vitro transcription and translation in rabbit reticulocyte lysate in the presence of [35S]methionine according to manufacturer's instructions (Promega). In vitro translated proteins (10 µl) were diluted into 100 µl of assay buffer and incubated with the immobilized proteins for 1 h at room temperature. Where indicated binding reactions were performed in the presence of 50 mM EDTA, 100 mM sodium acetate pH 5.5, or 10 mM 1,10-phenanthroline, or 10 mM 1,7-phenanthroline. Unbound proteins were removed by washing and bound proteins were eluted with SDS sample buffer and resolved by SDS-PAGE and autoradiography. Equal loading of immobilized proteins was verified by anti-GST immunoblot after binding. Relative binding was determined by loading equal cpm of radiolabeled protein per well followed by liquid scintillation detection of eluted proteins. All binding assays were repeated in triplicate.Recombinant GST, GST-tagged SUMO-1, SUMO-2, SUMO-2(QFI), or SUMO-2(x3) (8 µg protein) were diluted into 100 µl of 1X PBS, 0.05% Tween 20 and incubated in glutathione-coated 96-well plates (Pierce Biotechnology). Following overnight incubation at 4°C, wells were blocked for 1 h at room temperature with 2% bovine serum albumin in assay buffer . ZNF261 and ZNF198 proteins were produced by Binding assays using recombinant His-tagged ZNF261 (amino acids 1-495) proteins were performed following overnight dialysis against assay buffer, or 50 mM EDTA, 100 mM sodium acetate pH 5.5, or 1,10-phenanthroline, or 1,7-phenanthroline. Binding assays were performed as described above and bound proteins were eluted with SDS sample buffer and detected by anti-His immunoblot.ZNF261 (amino acids 1-495) entry clone was obtained from the Ultimate Human ORF collection (Invitrogen). To generate a clone for protein expression in bacteria or rabbit reticulocyte lysate, recombination reactions were performed between the entry clone and Gateway pDEST17 Vector according to manufacturer's instructions (Invitrogen). Amino acid substitutions in ZNF261 were generated by PCR-based QuikChange site-directed mutagenesis (Stratagene) using the wild-type plasmid. ZNF261 and ZNF198 fragments were generated by PCR, digested with appropriate restriction enzymes, and ligated into pcDNA6/myc-His A (Invitrogen). All mutations and constructs were verified by DNA sequence analysis. Full-length ZNF198 cDNA was a gift from Hongtao Yu . The construct for expression of FLAG-PML (isoform III) in mammalian cells was provided by Pier Paolo Pandolfi .2 in E. coli and purified using Ni-NTA agarose according to Manufacturer's instructions (Qiagen). For analytical ultracentrifugation experiments, MYM domains (amino acids 236–490) were expressed as His-tagged thioredoxin fusion proteins. Cultures were grown in the presence of 10 µM ZnCl2 at 37°C. When the OD reached ∼0.7, expression was induced with 50 µM IPTG. The temperature was adjusted to 16°C and expression was carried out overnight. His-tagged MYM domains were purified using HisTrap columns according to Manufacturer's instructions . Purified His-tagged MYM domains were incubated with His-tagged TEV protease overnight at 4°C. The protein sample was applied to a HisTrap column and untagged MYM domains were collected in the flow-through. Untagged MYM domains were separated from impurities using a MonoS column  followed by gel filtration over a Superdex 200 column . GST, GST-tagged SUMO-1, SUMO-2, SUMO-2(x3), and SUMO-2(QFI) were purified by affinity chromatography on glutathione-Sepharose 4B beads  according to manufacturer's instructions.Recombinant His-tagged ZNF261 (amino acids 1-495) was expressed in the presence of 10 µM ZnClTransfections were performed in 35×10–mm tissue culture dishes with 1.0 µg of DNA and 2.5 µl of Lipofectamine 2000 (Invitrogen) per transfection or 60×10-mm tissue culture dishes with 2.5 µg of DNA and 6.25 µl of Lipofectamine 2000 (Invitrogen) per transfection. Immunofluorescence or immunoprecipitation experiments were performed 24 hours later.3 and a final volume of 1.5 ml. A standard calibration curve was generated from samples containing 0, 1, 5, 10, 50, 100, 500 µg/L Zn2+. Prior to analysis ZNF261 (amino acids 1-495) samples were dialyzed overnight against 25 mM HEPES, pH 7.3, 350 mM NaCl, 5 mM β-mercaptoethanol, 10 µM ZnCl2 (Untreated), or 50 mM EDTA, 100 mM sodium acetate pH 5.5, or 6 M GuHCl, or 10 mM 1,10-phenanthroline, or 10 mM 1,7-phenanthroline.Zinc content of ZNF261 (1-495) samples (3 µM and 0.5 µM) were determined by ICP-MS (Inductively Coupled Plasma Mass Spectrometry) using an Agilent 7500ce ICP-MS (Agilent). Samples were diluted to a final concentration of 1% HNO2-Cys-X19-22-Cys-X3-Cys-X13-19-Cys-X2-Cys-X19-25-Cys-X2-Cys where X is any residue 2-Cys-X19-24[F/Y]-Cys-X3-Cys-X3[F/Y] giving rise to 9 MYM-type zinc fingers in ZNF198 and ZNF261 We performed SUMO-binding assays on protein microarrays containing ∼4,000 unique human proteins that are known or predicted to function as transcription factors To validate the protein microarray binding results, full-length ZNF261 was expressed in rabbit reticulocyte lysate and incubated with GST, GST-tagged SUMO-1, SUMO-2, SUMO-2(x3) immobilized on glutathione-coated plates . Consistin vitro SUMO-binding assays  . When py protein . Adding  protein . Similar protein . Taken tTo further characterize interactions between the MYM-type zinc fingers of ZNF261 and SUMO-2, we determined the dissociation constant and binding stoichiometry of a ZNF261:SUMO-2 complex using equilibrium analytical ultracentrifugation. Experiments were performed with a recombinant fragment of ZNF261 containing the first two MYM-type zinc fingers (amino acids 237-490) and SUMO-2(x2), a linear fusion of two consecutive SUMO-2 proteins. Analytical equilibrium ultracentrifugation of ZNF261(1-495) alone revealed that this fragment behaves as a monomer in solution . The samThe archetypal consensus SIM forms a β-strand that inserts between the α-helix and second β-strand in SUMO 2-Cys-X19-22-Cys-X3-Cys-X13-19-Cys-X2-Cys-X19-25-Cys-X3-Cys” where X is any amino acid The MYM-type zinc finger consensus sequence is “Cys-X2+ content after denaturation with 6 M guanidine hydrochloride, a condition in which 10% of the Zn2+ ion remained bound relative to untreated protein. After treatment with EDTA or 1,10-phenanthroline, the percent of Zn2+ ion remaining bound to ZNF261(1-495) was 7% and 28% compared to untreated protein, respectively. As expected, >90% of the Zn2+ ion remained bound to ZNF261 after treatment with 1,7-phenanthroline, the non-chelating analog of 1,10-phenanthroline. Thus, MYM-type zinc fingers are zinc-binding motifs and zinc can be removed using standard chelating agents.To determine whether various chelating agents effectively remove zinc from the MYM-type zinc finger, we subjected recombinant ZNF261(1-495) to several chelating treatments and measured the zinc content by ICP-MS . As a nein vitro binding assays in the absence and presence of metal chelators. Full-length ZNF261 was expressed in reticulocyte lysate and incubated with GST and GST-tagged SUMO-2(x3) in the absence or presence of metal chelators. Surprisingly, similar amounts of ZNF261 were pulled down by SUMO-2(x3) in standard assay conditions and in the presence of either EDTA, 1,10-phenanthroline, or 1,7-phenanthroline  . GST andTo further demonstrate that MYM-type zinc fingers can bind SUMO independently of zinc we performed binding assays using ZNF261(1-495) containing cysteine residues within the MYM-type zinc fingers mutated to alanine. ZNF261(1-495) contains two MYM-type zinc fingers each with 8 cysteine residues for a total of 16 cysteine residues. Wild-type and cysteine mutant ZNF261 proteins were transcribed and translated in rabbit reticulocyte lysate and binding assays were performed under standard assay conditions. Minimal differences in SUMO-binding activity were observed between wild-type and cysteine mutant ZNF261 proteins containing 1, 2, or 4 cysteine residues mutated to alanine . Taken tWe hypothesized that MYM-type zinc fingers bind to SUMO in the presence of metal chelators because the secondary structure of the MYM-type zinc finger is not significantly perturbed by zinc removal. To test this hypothesis we measured the secondary structure of untreated and EDTA-treated recombinant ZNF261(1-495) by CD spectropolarimetry. As a negative control, we completely denatured ZNF261(1-495) with 6 M GuHCl and observed that the CD spectrum showed a significant loss of ZNF261 secondary structure . SimilarA previous study reported that ZNF198 localizes to PML-NBs and that SUMO modification of ZNF198 on residue K963 is required for this localization To characterize interactions with SUMO-modified proteins in vivo, we immunopurified myc-tagged full-length ZNF198 and truncation mutants from transiently transfected cells. We detected high molecular mass SUMO-2 conjugates in immunopurifications of full-length ZNF198, as well as N- and C-terminal deletion mutants . NotablyTo investigate whether ZNF198 interacts directly with PML in mammalian cells, we co-expressed FLAG-tagged PML and myc-tagged ZNF198 and performed reciprocal immunopurification experiments. Using anti-myc and anti-Flag agarose beads, we were able to co-purify ZNF198 and PML from mammalian cell extracts . UnfortuHere we report the identification and characterization of the SUMO-binding activity of the MYM-type zinc fingers in ZNF261 and ZNF198. Our characterization of the SUMO-binding activity of the MYM-type zinc finger in ZNF261 and ZNF198 includes the following findings: (1) MYM-type zinc fingers preferentially bind to SUMO-2, (2) the MYM-type zinc finger:SUMO-2 complex binding stoichiometry is 1∶1, (3) MYM-type zinc fingers and classical SIMs interact with a common surface on SUMO-2, (4) each MYM-type zinc finger coordinates two zinc atoms, (5) MYM-type zinc fingers bind to SUMO in the absence of zinc, (6) removal of zinc does not significantly perturb the secondary structure of the MYM-type zinc finger, and (7) MYM-type zinc fingers and the N-terminus of ZNF198 mediate localization to PML-NBs.ZNF198 contains three predicted classic SIMs (two in the N-terminus and one in the zinc-finger domain), however, SUMO-binding assays using truncated ZNF198 fragments revealed that these predicted SIMs are not involved in binding to SUMO-2 modified HDAC1 Although SUMO-binding activity has been formally mapped to the MYM-type zinc finger domains in only two proteins, and to individual MYM-type zinc fingers in only ZNF261, we propose that these zinc fingers represent a signature SIM. However, whether all MYM-type zinc fingers function individually as SIMs, and whether they all share similar affinities for different SUMO paralogs, remains to be formally tested. The four MYM-type zinc finger proteins expressed in mammals each contain 5 tandem MYM-type zinc fingers, suggesting a potential to interact preferentially with poly-SUMO chains, proteins mono-sumoylated at multiple tandem sites, or with multiple sumoylated proteins. Consistent with individual MYM-type zinc fingers functioning as SIMs to enhance binding to poly-SUMO, we detected more robust interactions between a ZNF261 fragment with two zinc-finger motifs and poly-SUMO-2 compared to a fragment with a single zinc-finger motif and monomeric SUMO-2. We measured the binding affinity of the two N-terminal MYM-type zinc fingers of ZNF261 for SUMO-2(x2) as ∼50 µM. In comparison, recent studies demonstrated affinities between ∼15–70 µM for interactions between individual tandem classic SIMs present in RNF4 and SUMO-2(x2) The mechanisms used by SUMO-binding proteins to differentiate between SUMO-1 and SUMO-2 have not been completely defined. For several SUMO-binding proteins the charged residues adjacent to the consensus SIM confer preferential SUMO-1 binding Our ICP-MS analysis revealed 2 zinc atoms bound per MYM-type zinc finger, however, SUMO-binding activity and secondary structure were not significantly perturbed by zinc chelation. This effect may be related to the formation of stabilizing disulfide bridges between cysteine residues, as has been reported for two zinc finger proteins T4 gp32 metalloprotein and DnaJ An important consequence of SUMO modification is formation of multi-protein complexes assembled by interactions between modified proteins and proteins containing SIMs. For example, PML-NBs are formed by association of SUMO modified PML with proteins containing SIMs including PML In this study we revealed that the MYM-type zinc-finger domain mediates SUMO-binding. Moreover, a previous study demonstrated that the ZZ Zinc finger motif in HERC2 is a SUMO-binding motif In summary, our studies have demonstrated that the MYM-type zinc fingers in ZNF261 and ZNF198 function as SIMs. MYM-type zinc finger containing proteins function in transcriptional regulation Figure S1The N-terminus of ZNF261 does not bind SUMO.In vitro expressed ZNF261(1-495) truncation fragments were incubated with GST or GST-tagged SUMO-2(x3). Bound proteins were eluted with SDS-sample buffer and analyzed by SDS-PAGE and autoradiography.(TIF)Click here for additional data file.Figure S2Zinc chelation does not significantly perturb SUMO-binding activity or secondary structure of ZNF261. A: Schematic Diagram of his-tagged ZNF261(1-495) used for SUMO-binding assays and CD spectropolarimetry. B: Immobilized GST or GST-tagged SUMO-2(x3) was incubated with recombinant His-tagged ZNF261(1-495) and bound proteins were visualized by SDS-PAGE followed by immunoblot analysis with anti-His antibody. Prior to incubation, ZNF261(1-495) was dialyzed overnight against assay buffer or assay buffer containing EDTA or GuHCl. C: CD spectra for 75 µM ZNF261(1-495) untreated (diamonds), EDTA treated (triangles), and GuHCl treated (squares) were obtained.(TIF)Click here for additional data file.


DMPK gene. The pathomechanism of DM1 is driven by accumulation of toxic transcripts containing expanded CUG repeats (CUGexp) in nuclear foci which sequester several factors regulating RNA metabolism, such as Muscleblind-like proteins (MBNLs). In this work, we utilized very short chemically modified antisense oligonucleotides composed exclusively of locked nucleic acids  complementary to CUG repeats, as potential therapeutic agents against DM1. Our in vitro data demonstrated that very short, 8- or 10-unit all-LNAs effectively bound the CUG repeat RNA and prevented the formation of CUGexp/MBNL complexes. In proliferating DM1 cells as well as in skeletal muscles of DM1 mouse model the all-LNAs induced the reduction of the number and size of CUGexp foci and corrected MBNL-sensitive alternative splicing defects with high efficacy and specificity. The all-LNAs had low impact on the cellular level of CUGexp-containing transcripts and did not affect the expression of other transcripts with short CUG repeats. Our data strongly indicate that short all-LNAs complementary to CUG repeats are a promising therapeutic tool against DM1.Myotonic dystrophy type 1 (DM1) is an autosomal dominant multisystemic disorder caused by expansion of CTG triplet repeats in 3′-untranslated region of  According to several reports, an unknown mechanism may lead to CUGBP1 phosphorylation resulting in its increased cellular stability (Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy in adults, affecting 1 in 6–10 000 live birth. Clinical features of DM1 mainly present myotonia, weakness and atrophy of skeletal muscles (PK) gene . The DM1eet foci . Expanderocesses ,5. Anothtability .exp-containing RNA (exp RNA with nuclear proteins (such as MBNLs) without causing significant degradation of targeted transcript, by either antisense oligomers (ASOs) (exp transcripts could be induced by either RNA interference (RNAi) tools  (O-methoxyethyl (MOE)/DNA gapmers  antisense oligomer-induced degradation of toxic CUGning RNA –16 and (s (ASOs)  or smalls (ASOs) . In celli) tools ,9, ribozi) tools , endogeni) tools  or shorti) tools ,14 or inechanism ,16. For -2′O-Me) , locked  gapmers . For an ent tool .exp RNA with nuclear proteins in vitro and in vivo. The LNA is a ribonucleotide homolog with a characteristic 2′-O,4′-C-methylene bridge  when bound to either DNA or RNA  and control fibroblasts derived from non-DM1 patients (cell line GM07492) were purchased from the Coriell Cell Repositories. Cells were grown in Eagle's minimal essential medium (EMEM) (Lonza) supplemented with 10% fetal bovine serum (FBS) (Sigma), 1% antibiotic antimycotic (Sigma) and 1% non-essential amino acids solution (Sigma), in 5% CO2 at 37°C. HeLa and HEK293 cells were grown in Dulbecco's modified Eagle's medium medium (DMEM) supplemented with 10% FBS (Sigma), 1% antibiotic antimycotic (Sigma) and, in 5% CO2 at 37°C. Control (non-DM1) myoblast line (9936) and DM1 patient-derived myoblasts  were obtained from Telethon Biobank Network and were grown in F10 HAM medium (Sigma) supplemented with 20% FBS (Sigma), 0.39-μg/ml dexamethazon (Sigma), 10-ng/ml epidermal growth factor (Sigma), 25-μg/ml insulin (Sigma), 1% antibiotic antimycotic (Sigma), in 5% CO2 atmosphere at 37°C. Cell transfection experiments were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions with thermally denatured oligomers (in appropriate concentration) or DT960 plasmid (1 μg). For transient overexpression of DMPK ex11–15 (DT960CUG) HEK293 or HeLa cells were transfected with PO-LNA-CAG-10 at 125- and 250-nM final concentration, 24 h after delivery of DT960 plasmid. The total RNA was isolated 48 h after treatment or after the time specified in figure legends.Fibroblasts derived from DM1 patients  cDNA. RT-PCR was performed in using GoTaq Flexi DNA Polymerase (Promega). All PCR experiments involved at least three independent biological replicates and three technical repeats. Primer sequences and PCR conditions are specified in Supplementary Table S1. The RT-PCR products were stained with Ultra Safe Blue (Syngene) according to manufacturer's instructions and separated in 2% agarose gel. The DNA bands were visualized on G:BOX system (Syngene) and the intensity of each band was measured using GeneTools software (Syngene). EC50 was determined in Graph Pad Prism using log(inhibitor) versus response model: Y = Bottom + (Top-Bottom)/(1+10∧[=((X-LogEC50))). Real-time quantitative RT-PCR analysis was performed using the 7900HT Fast Real-Time PCR System (Applied Biosystems). The Power SYBR Green PCR Master Mix (Applied Biosystems) and specific primer set (0.2 μM of each) were used for amplification (Supplementary Table S1). The experiments were carried out in triplicate technical repeats for three biological replicates. The relative quantification in gene expression was determined using the ΔΔCt method. The GAPDH-specific signal was used as a reference.The total RNA was isolated from cells or temporal arteritis (TA) muscles using TRI Reagent (Sigma) according to manufacturer's protocol. First strand of cDNA was synthesized using SuperScript II/III Reverse Transcriptase (Invitrogen) with Random Primers (Promega) according to manufacturer's instruction. The quality of the reverse transcription products was assessed by polymerase chain reaction (PCR) amplification of the glyceraldehyde-3-phosphate dehydrogenase (Bpm1 polymorphism in exon 10 of DMPK gene (The DM1-derived myoblast line (10009) is heterozygous for MPK gene ,30. Thisin situ hybridization (FISH) was performed with the use of DNA/LNA probe (CAG)6-CA, labeled at the 5′-end with Cy-3 as previously described (The RNA fluorescence escribed . Microsc100 transcript was transcribed and 5′-labeled as previously described (100 (1 nM) with PO-LNA-CAG-8, PO-LNA-CAG-10, PO-LNA-CAG-12, PS-LNA-CAG-8, PS-LNA-CAG-10 or 2′OMe-CAG-21 of indicated concentrations (ranging from 0 to 1.25 μM). Reactions were performed in a volume of 10 μl, in 1x filter binding buffer (FBB)  and incubated at 37°C for 30 min. The samples were run on a native 8% polyacrylamide (PA) gel in 0.5 x TBE at 100 V for 1 h. The gel was subsequently dried and the signal was detected O/N, visualized on IP through FLA-1500 (FujiFilm) and quantified using Multi Gauge software (FujiFilm). Kd measurement of the CUGexp/LNA–CAG interaction was based on the percentage of free CUGexp using one phase decay curve in Graph Pad software.The (CUG)escribed . ElectroMBNL1 gene (NP_066368) containing glutathione-S-transferase, GST-tag, at N-terminus and His6-tag at C-terminus was prepared as previously described (100 (0.1 nM), PO-LNA-CAG-8, PO-LNA-CAG-10 (0.5 nM) and PO-2′OMe-CAG-21 (0.2 nM) were incubated with the indicated concentration of the protein (ranging from 0 to 500 nM) in 1 x FBBa (FBB with 250-mM NaCl and 15-mM KCl) at 37°C for 30 min. To analyze the inhibition of MBNL1binding to (CUG)100 by PO-LNA-CAG and PS-LNA-CAG, 0.1 nM of labeled (CUG)100 was first incubated with the indicated concentrations of oligomers (ranging from 0 to 10 μM) at 37°C for 30 min, and then 15 nM of MBNL1 was added to each sample and incubated as before. Twenty-five microliter of samples was loaded onto filter binding apparatus with nitrocellulose  and nylon  membranes pre-wetted in 1x FBBa. The signal from membranes was visualized on IP through FLA-1500 and quantified using Multi Gauge software (FujiFilm). Ki of the prevention of (CUG)100/MBNL1 complexes by LNA-CAG was measured by Graph Pad using two-phase decay curve.The MBNL1 isoform carrying sequence encoded by exons 1–4 of escribed . Filter HSALR transgenic mice  were previously described .Group data are expressed as mean ± standard deviation. Statistical significance of RT-PCR results was determined by a two-tailed Student's exp RNA with several nuclear proteins  of designed oligomers differ significantly and amount to 19, 35, 49 and 56oC, respectively 100  for 8-mer and 10-mer oligomers were very low and accounted to ∼4 and ∼7 nM, respectively . As control, we used long 21-mer CAG repeat oligomer, with 2′-O-methyl modification in each position (PO-2′OMe-CAG-21) with predicted Tm of 75°C. The Kd value for control oligomer was within the same range as for all-LNAs (5–10 nM).In the initial 0 Figure . The shoy Figure  and b. Pin vitro. For this purpose we first used FBA and measured the affinity of MBNL1 to the transcript containing 35 CUG repeats (CUG)35, derived from normal DMPK allele, and a three times longer mutant transcript, (CUG)100. MBNL1 bound both types of transcripts with similar affinity . Then, using the same method, we tested the interaction between radiolabeled (CUG)100 and MBNL1 in the absence or presence of each oligomer. PO-LNA-CAG-8 showed the lowest inhibition constant (Ki) which was about 100-fold lower than Ki for a 10-mer and 300-fold lower than Ki for a 12-mer. The inhibitory potential of the control 2′OMe-CAG-21 oligomers resembled the one observed for PO-LNA-CAG-12 500, (CUG)1000 and (CUG)2000, respectively. These cells display several DM1-specific molecular phenotypes, such as the formation of CUGexp nuclear foci, sequestration of MBNL1 and aberrant alternative splicing of several MBNL-sensitive exons. In an initial attempt to find alternative exons that could serve as useful markers for splicing correction analyses in DM1 cells, we examined roughly 20 alternatively spliced transcripts known to be sensitive to MBNL level, and ∼20 transcripts regulated by other splicing factors either affected in DM1 (CUGBP1) or not affected in DM1 . The reverse transcription PCR (RT-PCR) assays showed that most of the tested alternative splicing events were not informative in our cellular models due to either low expression level or presence of only one alternative splicing isoform (100% of either exon inclusion or exon exclusion isoform) . For further studies we chose only six MBNL-sensitive splicing events, namely MBNL1 exon 7  (APLP2 exon 14 (NOVA1-sensitive) (ECT2 exon 4 (FOX2-sensitive)  , APLP2 ensitive) , ECT2 exnsitive)  . These data suggest that short all-LNAs could potentially be able to interact with CUGMBNL1 and MBNL2, we observed a shift from DM1-specific splicing isoform  toward normal, non-DM1 isoform  in cells transfected with 125 nM of PO-LNA-CAG-8, 125 nM of PO-LNA-CAG-12 and all tested concentrations of PO-LNA-CAG-10 1000 and (CUG)2000, with three different concentrations of each PO-LNA-CAG oligomer . After 48 h from single transfection, we analyzed two MBNL1-regulated splicing events. For both assayed transcripts, 0 Figure .OMe-CAG-21 oligomer which showed high affinity toward CUG repeat RNA hairpin in our in vitro tests. Similar oligomer, but with additional phosphorothioate modification (PS-2′OMe), was previously shown in DM1 models as an effective tool inducing degradation of expanded CUG repeat RNA , and we hypothesize that this could be caused by lower stability of phosphodiester containing oligomer compared to its PS-modified version. As the PO-LNA-CAG-10 showed the highest potency, we have decided to use it for all further analyses.As a control we used PO-2′peat RNA . We did 50 values ranged from ∼5 nM  to ∼40 nM (for exon 19 of PHKA1). At 125 nM of PO-LNA-CAG-10, most tested transcripts showed splicing pattern characteristic for non-DM1 cells  in DM1 fibroblasts. We monitored alternative splicing correction of six studied MBNL-sensitive splicing events 48 h after transfection. All tested exons were sensitive to low nanomolar concentrations of PO-LNA-CAG-10. LogEC–250 nM iIn order to determine specificity of PO-LNA-CAG-10, we designed three control oligomers containing 10 LNA residues: scrambled sequence with the same but mixed nucleotide composition (PO-LNA-CTR3) and two other oligomers (PO-LNA-CTR1 and PO-LNA-CTR2) with random sequences . These results suggest that PO-LNA-CAG-10 very specifically corrects the alternative splicing of MBNL-sensitive exons in DM1 cells, most probably due to its high binding specificity toward CUGFinally, to determine the duration of PO-LNA-CAG-10 activity in dividing cells, we performed a time-course experiment Figure . The DM1exp-containing transcript  reduction of mutant DMPK mRNA level in treated cells . Additionally, we tested the effect of PO-LNA-CAG-10 on artificial transcript containing CUGexp in HEK293 and HeLa cells transiently transfected with DT960 vector expressing DMPK exons 11–15, with 960 interrupted CTG repeats in exon 15 1000 cells and found that neither of the five tested transcripts, exp foci. Three DM1 cell lines differing in CUG-repeat expansion size, (CUG)500, (CUG)1000 and (CUG)2000, respectively, were treated for 48 h with PO-LNA-CAG-10, PO-LNA-CAG-8 or control oligomers. The results of RNA fluorescent in situ hybridization (FISH) revealed a significant reduction in the average number of nuclear CUGexp foci. In striking contrast to untreated or control LNA-treated cells which contained ∼60% of nuclei with three or more foci, all DM1 cells treated with PO-LNA-CAG-10 contained only ∼15% of such nuclei . Therefore, we wondered whether additional chemical modification of these oligomers could improve their bioavailability. For this purpose, two fully phosphorothioated all-LNAs consisting of either 8 (PS-LNA-CAG-8) or 10 (PS-LNA-CAG-10) LNA residues first were tested in vitro. The EMSA and FBA showed that both oligomers have very high affinity to long CUG repeat hairpin (Kd = 0.8 nM for PS-LNA-CAG-8 and 5 nM for PS-LNA-CAG-10) and strong inhibitory effect on MBNL1 binding (Ki = 3.2 nM for PS-LNA-CAG-8 and 262 nM for PS-LNA-CAG-10), which are comparable with PO-LNA-CAG-8 and PO-LNA-CAG-10 .Recently, Stein and collaborators used an intracellular delivery method called gymnosis  that doeMBNL2, NCOR2 and INSR, respectively) . Moreover, FISH analyses of CUGexp foci in DM1-fibroblasts revealed that gymnotically delivered PS-LNAs reduced the average number of CUGexp foci in DM1 cells after both, 72- and 216-h-long treatment. The reduction was minor after 72 h, however, upon extended incubation time the effect was much more prominent and similar to the one observed upon canonical transfection of 25-nM PS-LNA with lipofectamine . Taken together, our data confirm that PS-LNAs can be effectively delivered into cells by gymnosis, but to reach comparable effectiveness as with lipofectamine-delivered PS-LNAs, the concentration of the oligonucleotides must be two orders of magnitude higher.To analyze the potency of PS-LNA oligomers in cells, we first delivered them by lipofection. PS-LNAs were able to induce alternative splicing correction to a similar extent as unmodified PO-LNA-CAGs . Next, fully phosphorothioated LNA oligomers were delivered gymnotically into (CUG)1000 fibroblasts by incubating the cells for 72 or 216 h in a medium containing from 0.5 to as much as 10 μM of a specific oligomer. Neither experiment carried out for 72 h caused any significant alternative splicing changes of four tested MBNL-sensitive exons (data not shown). However, three times longer treatment with PS-LNA-CAG-8, at 10-μM concentration but not at 0.5-μM concentration, resulted with slight but statistically significant correction of three out of the five analyzed splicing events  mouse model in which skeletal muscles express the human alpha actin transcript containing ∼220 CUG repeats in the 3′ UTR. This mouse model shows a number of molecular features observed in DM1 patients, including formation of nuclear CUGexp foci that sequester Mbnl proteins, alternative splicing abnormalities of Mbnl-sensitive exons and myotonic discharges in skeletal muscles   . Needle charges) . Moreoveo Figure . AltogetO-Me, 2′-F, 2′-MOE groups or LNA modification often improve the oligomers potency, stability and their overall pharmacokinetic properties , the cellular concentration of the oligomer reached the level sufficient enough to reduce the efficacy of CUGdelivery ,46,47. Fo CAG-25 ,56.HSALR mice lasted minimum 14 days. This is at least twice as long as activity of siRNA directed against CUGexp in the same mouse model (Our experiments demonstrated that all-LNAs are stable molecules in cellular environment. In proliferating cells the duration of beneficial effect of PO-LNA-CAG-10 on splicing correction was relatively long and reached ∼20 days in spite of significant dilution of the oligomer after several cell divisions. Moreover, the therapeutic effects in differentiated muscle fibers of se model . Moreovese model  and LNA/Supplementary Data are available at NAR Online.


BRIM7 is a phase 1B study evaluating the safety and efficacy of combined BRAF and MEK inhibition with vemurafenib + cobimetinib. The utility of FDG-PET as an early predictor of clinical benefit was also evaluated in this study.V600BRAF-mutated melanoma and ECOG PS 0-1 and were either naïve to BRAF inhibitor (BRAFi-naïve) or had disease progression on vemurafenib (vem-progressor). Pts in the dose-escalation portion received vemurafenib 720 or 960 mg BID continuously and cobimetinib 60, 80, or 100 mg QD 14 days (d) on/14 d off (14/14); 21 d on/7 d off (21/7); or continuously (28/0). Two dose levels were expanded: vemurafenib (720 mg and 960 mg BID) + cobimetinib 60 mg QD 21/7. FDG-PET scans were performed at baseline and on Day 15 of Cycles 1 and 2. Correlation between tumor glucose metabolism changes, baseline tumor burden and target lesion responses were evaluated.Eligible patients (pts) had advanced 128 pts were treated with vemurafenib + cobimetinib as of 21 June 2013; male 60%, median age 55 y (19-88), stage M1c 76% and BRAFi-naive 49%. Median duration of follow-up in vem-progressor and BRAFi-naïve pts was 3 and 10 months, respectively. Most adverse events (AE) were mild to moderate in severity and vem-progressor pts reported fewer AEs compared to BRAFi-naïve pts. Most common AEs in BRAFi-naïve pts (n=63) were non-acneiform rash (89%), diarrhea (81%), photosensitivity/sunburn (70%), fatigue (67%) and liver test abnormality (59%). Most frequent grade ≥3 AEs in BRAFi-naïve pts were liver test abnormality (19%), non-acneiform rash (13%), arthralgia (11%) and fatigue (10%).BRAFi-naive pts attained 85% confirmed response rate (RR), including 10% complete responses; median PFS was not reached at 10 months follow-up. Vem-progressor pts attained 15% confirmed RR, stable disease of 43%, and median PFS of 2.8 months. Preliminary FDG-PET analysis showed that the magnitude of reduction in tumor glucose uptake correlated with maximal tumor reduction, but the degree of correlation varied across time and in BRAFi-naïve and Vem-progressor pts.Vemurafenib + cobimetinib can be safely administered at the respective single-agent MTDs of vemurafenib (960 mg BID) and cobimetinib (60 mg 21/7). Preliminary efficacy of the combination is encouraging in BRAFi-naive patients. FDG-PET is a potentially useful marker of early biologic response to the combination.


In vitro antioxidant activities and neuron-like PC12 cell protective effects of solvent fractions from aged garlic extracts were investigated to evaluate their anti-amnesic functions. Ethyl acetate fractions of aged garlic had higher total phenolics than other fractions.in vivo anti-amnesic effects of aged garlic extracts by using Y-maze and passive avoidance tests.Antioxidant activities of ethyl acetate fractions from aged garlic were examined using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) and malondialdehyde (MDA) inhibitory effect using mouse whole brain homogenates. Levels of cellular oxidative stress as reactive oxygen species (ROS) accumulation were measured using 2',7'-dichlorofluorescein diacetate (DCF-DA). PC12 cell viability was investigated by 3--2,5-diphenyl tetrazolium bromide (MTT) and lactate dehydtrogenase (LDH) assay. The learning and memory impairment in institute of cancer research (ICR) mice was induced by neurotoxic amyloid beta protein (Aβ) to investigate in vivo tests.We discovered that ethyl acetate fractions showed the highest ABTS radical scavenging activity and MDA inhibitory effect. Intracellular ROS accumulation resulting from Aβ treatment in PC12 cells was significantly reduced when ethyl acetate fractions were presented in the medium compare to PC12 cells which was only treated with Aβ only. Ethyl acetate fractions from aged garlic extracts showed protection against Aβ-induced neurotoxicity. Pre-administration with aged garlic extracts attenuated Aβ-induced learning and memory deficits in both Our findings suggest that aged garlic extracts with antioxidant activities may improve cognitive impairment against Aβ-induced neuronal deficit, and possess a wide range of beneficial activities for neurodegenerative disorders, notably Alzheimer's disease (AD). All solvents used were of analytical grade. RPMI 1640 medium and fetal bovine serum was obtained from Gibco BRL .2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS), potassium persulfate, vitamin C, α-tocopherol, catechin, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), sodium bicarbonate, streptomycin, ferrous sulfate (FeSOAllium sativum) was supported by Namhae Garlic Research Institute , in December 2010 and was authenticated by Dr. JH Shin (Namhae Garlic Research Institute). Extraction of aged garlic was prepared by pouring 80% ethanol 3.5 L into the bottle containing 1 kg of aged garlic, and the mixture was steeped at room temperature (20°C) for 7 days-2,5-diphenyl tetrazolium bromide; LDH: Lactate dehydtrogenase; ICR: Institute of cancer rese; Aβ: Amyloid beta protein; AD: Alzheimer's disease; AGE: Aged garlic extract; FRG: Fresh raw garlic; DMSO: Dimethyl sulfoxide; NGF: Nerve growth factor; TCA: Trichloroacetic acid; TBA: Thiobarbituric acid; IACUC: Institutional animal care and use committee; HPLC: High performance liquid chromatography; RT: Retention time; ICV: Intracerebroventricular; SAC: S-allyl cysteine; DMDS: Dimethyl disulfide.The authors declare that they have no competing interests.HJH participated in the design of the study. JHJ, HRJ, YNJ and HJK conducted the experiments, analyzed the data and drafted the manuscript. JHS helped conduct the experiments. All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/13/268/prepub


It may be possible to overcome antigen escape by targeting two tumor antigens simultaneously, i.e. CD19 and CD20 using a tandem construct with two scFv-based CAR binding domains. Lentiviral vectors encoding chimeric antigen receptors comprised of anti-CD19 and anti-CD20 targeting domains expressed alone or in tandem were transduced into T cells from healthy donors to generate the corresponding CAR19, CAR20, CAR19_20  and CAR20_19 T cells . The transduced T cells were 50-70% CAR positive as determined by protein L flow cytometric analysis. Expression of CAR proteins of the expected molecular weight was confirmed by Western blot analysis of transduced T cells. When CAR-transduced T cells were combined with CD19+CD20+ Raji target cells, but not CD19-CD20- K562 cells, all four CAR T cell types demonstrated comparable efficient killing of leukemia targets (E:T ratio >2), and target-dependent induction of IFN-γ, as measured in co-culture supernatants by ELISA.Adoptive immunotherapy with chimeric antigen receptor (CAR) transduced T lymphocytes has shown promising results in both pediatric and adult B cell malignancies. Nevertheless, both CAR-based and antibody-based anti-CD19 therapies, in vitro co-culture experiments where we used very low E:T ratios to examine the potential for CAR-induced antigen loss on surviving leukemia cells. All CAR-T cells expressing an anti-CD19 scFv induced rapid loss of CD19 surface expression. In contrast, the CD20 surface marker was less prone to down-regulation by CAR-T cells expressing anti-CD20 scFv. Upon flow cytometric analysis of surviving leukemia cells on day 5, CD19 expression was reduced to 3%, 48%, 73%, 90% and 93% of control when co-cultured with CAR T cells expressing CAR19_20, CAR20_19, CAR19, CAR20, and control T cells, respectively. Similar results were seen when experiments were of longer, 7 days, or shorter, 1 day, duration. In conclusion, tandem CAR T cells are as effective as single CAR19 or CAR20 T cells in leukemia cell killing. Importantly, dual scFv-expressing CARs are more potent in preventing tumor antigen escape via target antigen down-regulation.We then began a series of


MK5 (MAPK-activated protein kinase 5) or PRAK (p38-regulated and -activated kinase) are alternative names for a serine/threonine protein kinase downstream to ERK3/4 and p38 MAPK. A previous gene targeting approach for MK5/PRAK (termed here MK5/PRAK-Δex8) revealed a seemingly tumor-suppressive role of MK5/PRAK in DMBA-induced one step skin carcinogenesis and Ras-induced transformation. Here we demonstrate that an alternative targeting strategy of MK5/PRAK (termed MK5/PRAK-Δex6) increased neither tumor incidence in the one step skin carcinogenesis model, nor Ras-induced transformation in primary cells. Interestingly, due to the targeting strategies and exon skipping both knockouts do not completely abolish the generation of MK5/PRAK protein, but express MK5/PRAK deletion mutants with different biochemical properties depending on the exon targeted: Targeting of exon 6 leads to expression of an unstable cytoplasmic catalytically inactive MK5/PRAK-Δex6 mutant while targeting of exon 8 results in a more stable nuclear MK5/PRAK-Δex8 mutant with residual catalytic activity. The different properties of the MK5/PRAK deletion mutants could be responsible for the observed discrepancy between the knockout strains and challenge the role of MK5/PRAK in p53-dependent tumor suppression. Further MK5/PRAK knockout and knock-in mouse strains will be necessary to assign a physiological function to MK5/PRAK in this model organism. The pathways regulating expression and activity of MK5/PRAK iewed in ,2), and iewed in . RemarkaThe physiological role of MK5/PRAK has mainly been analyzed using two independently generated mouse models: The “Δex6 mice”, in which exon 6 of MK5/PRAK is replaced by a neomycin cassette and which are kept in the 129xC57BL/6 background to increase viability and fertility . More inHere, we analyzed the apparent tumor-suppressive function of MK5/PRAK using the Δex6 mice generated by us previously . We repeIn previous experiments the one step DMBA treatment of mice resulted in superficial papillomas in 8% of wild type animals (1of 12) after about 300 days, whereas 54% 7 of 13) of Δex8/Δex8 mice and 40% (12 of 30) of Δex8/+ mice developed papillomas, indicating an important role of MK5/PRAK in tumor suppression ,13. To v of Δex8/WAF1 is a key marker molecule of tumor suppression and ras-induced senescence ATP from Hartmann Analytic, LMP Agarose from Bethesda Research Laboratories and SensiFAST SYBR No-Rox Kit and Vybrant DyeCycle Ruby Stain from Bioline.Antibodies against Ras, ERK3, p53, phospho-p53 (S15 (9284), S37 (9289) and S46 (2521)) and phospho-Hsp27 (Ser82) were obtained from Cell Signaling Technology, anti-PRAK antibody from Sigma and anti-GAPDH  antibody from Millipore. Anti-GFP (B2), anti-GST (B-14), anti-EF-2 (C-14), Hsp27 (C-20) and secondary antibodies were purchased from Santa Cruz Biotechnology. Control siRNA (Allstars negative control), HiPerFect transfection and TopTaq DNA polymerase reagent were obtained from Qiagen, mouse MK5/PRAK siRNA (sc-36311) from Santa Cruz Biotechnology, -ATP) were added to the reaction mix. Kinase reaction was carried out at 30°C for 1 h, was stopped by adding 8 μl of 4x Laemmli buffer and samples were heated to 96°C for 5 min. Protein samples were separated on a 7.5–22.5% polyacrylamide gradient gel. After separation gel was used for Western blotting as described earlier ATP on a set of peptides with the general sequence Y-A-X-X-X-X-X-S/T-X-X-X-X-A-G-K-K(biotin) having the indicated amino acid present fixed at the indicated position relative to the phosphorylation site. The substrate consensus motif is compared to the sequence surrounding S37 in p53 of human and mouse. The prominent basic residue (R) in minus-3 position is missing in both p53 sequences.Panel A) MCF-7 cells were stimulated by doxorubicin (1μM)- or UV (40J/mreporter . Co-tranreporter . The amoreporter . GST-MK5(TIFF)Click here for additional data file.S3 FigFour different primer combinations were used: the upper panel represents the results of PCR where the same primer combinations were used as described in  to discr(TIF)Click here for additional data file.S4 FigA) Representative fluorescence microscopic images (magnification 200x) of subcellular localization of GFP and recombinant GFP-MK5/PRAK fusion proteins transfected into HEK293 cells used for the quantification in B). A nuclear co-staining in the living cells was performed using Vybrant DyeCycle Ruby Stain (life technologies). B) Quantification of subcellular localization of GFP and the GFP-fusion proteins. From three independent transient transfections more than 100 cells of each transfection were counted. Cells were scored predominantly nuclear (N), predominantly cytoplasmic (C) or equally distributed between nucleus and cytoplasm (NC). The cells with very strong and apparently toxic overexpression resulting in NC were not excluded here. We normally avoid such cells in quantification of transient transfections with GFP fusion proteins, but here the fully unbiased counting is presented.(TIF)Click here for additional data file.S5 FigHis-ERK3 was detected in immunoprecipitates using GFP-nanobodies and in whole lysate (as control) by Western blot against ERK3. Western blot against GFP was used to detect GFP-MK5/PRAK and its mutant variants.(TIF)Click here for additional data file.S6 FigHEK293 cells were transfected with indicated constructs. 24 hr post-transfection cells were lysed in the plates and analyzed by Western blotting using the indicated antibodies. A slower migrating band of GST-ERK4, indicating its phosphorylation, appears when co-expressed with GFP-MK5/PRAK wild type, but not with GFP-MK5/PRAK-Δex6, GFP-MK5/PRAK-Δex8 or kinase-dead GFP-MK5/PRAK-T182A mutant.(TIF)Click here for additional data file.


Prior to vaccination, we employed vector silencing to inhibit the CD8 T cell response against the immunodominant TMEV antigen, VP2121-130. We then monitored the resulting effect on the CD8 T cell response against the targeted tumor-specific antigen, ovalbumin. We demonstrate that employing vector silencing in the context of B16-OVA melanoma does not reduce tumor burden or improve survival, while TMEV-OVA vaccination without vector silencing controls tumor burden. Meanwhile, employing vector silencing during picornavirus vaccination against the GL261-quad cassette glioma resulted in a lower frequency of tumor antigen-specific CD8 T cells. The results of this study are relevant to antigen-specific immunotherapy, in that the virus vector-specific CD8 T cell response is not competing with tumor antigen-specific CD8 T cells. Furthermore, vector silencing may have the adverse consequence of reducing the tumor antigen-specific CD8 T cell response, as demonstrated by our findings in the GL261-quad cassette model.Virus vector-based vaccination against tumor-specific antigens remains a promising therapeutic approach to overcome the immune suppressive tumor microenvironment. However, the extent that the desired CD8 T cell response against the targeted tumor antigen is impacted by the CD8 T cell response against the virus vector is unclear. To address this question, we used picornavirus vaccination with Theiler’s murine encephalomyelitis virus (TMEV) as our vector against tumor-expressed ovalbumin (OVA Optimizing vaccines to effectively treat tumors remains a critical goal for immunotherapy. Virus vector-based immunotherapies are an attractive option, as viruses have been employed as oncolytics and can be engineered to express patient-specific tumor antigens . The utiWhen employing recombinant virus vaccination against tumor specific antigens, two CD8 T cell responses are generated. One response is directed against the targeted tumor antigen of interest. The other response is directed against epitopes derived from the virus vector ,7,8. Imp121-130 peptide antigen of the picornavirus, Theiler’s murine encephalomyelitis virus (TMEV), the vector utilized for our vaccine platform [b:VP2121-130 epitope can be silenced by administering VP2121-130 peptide intravenously (i.v.) one day prior to infecting with TMEV [121-130 peptide, the Db:VP2121-130 epitope-specific response can be inhibited indefinitely [in vivo. Using vector silencing, we sought to determine if the CD8 T cell response against the picornavirus vector alters the CD8 T cell response to the tumor-specific antigen during vaccination.In order to determine the effect of CD8 T cell responses directed against a picornavirus vector on the targeted tumor-specific antigen, we developed a method to inhibit CD8 T cell responses. Specifically, we silenced the response to the immunodominant VP2platform . Our gro257-264). We have demonstrated previously that vaccination with TMEV XhoI-OVA8 (TMEV-OVA) reduces tumor burden and improves survival in animals bearing B16-OVA melanoma and GL261-quad cassette glioma tumors, both of which express OVA257-264 as a model tumor antigen [In this study, we employ a picornavirus vaccination strategy using TMEV engineered to express ovalbumin antigen, SIINFEKL  for evaluating immunotherapy [257-264, into C57BL/6 mice [121-130 peptide. One group received mock E749-57 peptide. One day following vector silencing with VP2121-130 peptide, mice were intracranially vaccinated with TMEV-OVA or control TMEV. Mice were imaged weekly to assess tumor size. When animals became moribund, they were euthanized and brain-infiltrating lymphocytes were isolated. The frequency of Db:VP2121-130- and Kb:OVA257-264-specific CD8 T cells infiltrating the brain were quantified. We determined that TMEV-OVA vaccinated mice had low frequency of Db:VP2121-130-epitope specific CD8 T cells infiltrating the brain . The GL261-quad cassette line is engineered to express the model antigens OVA257-264, OVA323-339, human GP10025-33, and alloantigen I-Ea52-68. The GL261-quad cassette cell line expresses luciferase to assess tumor burden using bioluminescence imaging. 5.0x105 B16-OVA melanoma cells were implanted into the left hind leg of C57BL/6 mice  subcutaneously in a total volume of 50μL. 6.0x104 GL261-quad cassette cells were implanted by stereotactic injection into the right striatum of C57BL/6 mice  as previously described [The B16-OVA melanoma and GL261-quad cassette glioma cell lines were cultured as previously described ,14. The FEKL OVA27-264. Th49-57 (RAHYNIVTF) or VP2121-130, (FHAGSLLVFM) peptide  were administered i.v. one day prior to vaccination with TMEV or TMEV-OVA. B16-OVA melanoma-bearing mice were treated with peptide 6 days post tumor implantation, while GL261-quad cassette-bearing mice were treated with peptide 14 days following tumor implantation. Mice assessed during acute TMEV-OVA infection received i.v. peptide one day prior to viral infection.100.0μL of 1.0mg/mL E77 plaque forming units (PFU) wild-type TMEV or TMEV-OVA one day following peptide administration. Mice receiving i.c. vaccination were administered a single dose of 2.0x106 PFU TMEV or TMEV-OVA following anesthesia with 1–2% isoflurane. GL261-quad cassette-bearing mice were vaccinated in the opposite hemisphere of the brain from where GL261-quad cassette tumor cells were implanted.The recombinant TMEV XhoI-OVA8 (TMEV-OVA) picornavirus was generated as previously described . Briefly2 inhalation when tumors reached a diameter of 19mm, in accordance with Mayo Institutional Animal Care and Use Committee standards [B16-OVA tumor size was assessed via caliper measurement . Tumor diameter was measured daily as tumors became palpable. Mice were euthanized by COtandards .2 inhalation when they became moribund. Symptoms included weight loss, hunched posture, and difficulty moving. All animal work was completed following the Mayo Institutional Animal Care and Use Committee guidelines.GL261-quad cassette tumor burden was assessed using bioluminescence imaging. Prior to imaging, animals were administered 150mg/kg D-luciferin i.p. . Bioluminescence imaging was performed using an IVIS Spectrum system  running Living Image software as previously described . Animals6 splenocytes were isolated and stimulated with 10ng/mL IL-2 and 1μg/mL peptide  and Golgi Plug at a concentration of 1μL/1mL media . Cells were incubated for 5hr at 37°C and 5% CO2, then stained for surface markers, followed by permeablization and intracellular staining for IFNγ . For CNS-infiltrating immune cells, brains were homogenized and digested with collagenase type IV  or manual dounce homogenization  and centrifuged against a percoll density gradient at 7846g for 30 minutes to isolate immune cells. Peptide:MHC tetramers were constructed as previously described [Interferon-γ (IFNγ) stimulation was assessed by intracellular cytokine staining. 2x10escribed ,32. Antit test or Mann-Whitney Rank Sum Test if the data did not follow a normal distribution. Survival was assessed using a logrank (Mantel-Cox) test. GraphPad Prism 6.0  was used for statistical analysis.All data are presented as mean ± SEM. For comparisons of two groups, significance was determined using Student’s


We previously reported that EP3, a subtype of prostaglandin E2 receptors, negatively regulates eosinophilic infiltration in murine experimental allergic conjunctivitis induced by TLR3, which causes reduced eosinophilic conjunctival inflammation in TLR3/EP3 double knock-out (DKO) mice although in EP3-KO mice eosinophilic conjunctival inflammation is pronounced. We also documented that in human conjunctival epithelial cells, the EP3 agonist suppressed the production of cytokines such as CXCL10, CXCL11, IL6, CCL5, TSLP, and MCP-1 induced by polyI:C, a TLR3 ligand. EP3 was dominantly expressed in conjunctival epithelial cells, airway epithelial cells, and keratinocytes. To examine the effects of EP3 against polyI:C-inducible gene expression in conjunctival epithelium we performed gene expression analysis of the polyI:C-stimulated conjunctival epithelium in wild-type, EP3-KO-, and EP3/TLR3 DKO mice. Using GeneChip® we first examined the comprehensive effects of gene expression in polyI:C-stimulated conjunctival epithelium of wild-type mice. We found that after 6-hr stimulation, 31 transcripts were up-regulated more than 10-fold. Quantitative RT-PCR confirmed that 21 of the 31 transcripts were significantly (> 3-fold) up-regulated. Next, to identify the transcripts regulated by EP3 we compared the gene expression of these 21 transcripts in polyI:C stimulated conjunctival epithelium of wild-type and EP3-KO mice by quantitative RT-PCR. We found that all 21 transcripts were expressed significantly stronger in polyI:C stimulated conjunctival epithelium of EP3-KO mice. We also confirmed that the mRNA expression of these 21 transcripts was significantly reduced in polyI:C stimulated conjunctival epithelium of EP3/TLR3 DKO- compared to EP3- KO mice. GeneChip® analysis also showed that the number of 4 transcripts was more than 5 times greater in polyI:C stimulated conjunctival epithelium of EP3-KO- than wild-type mice although in wild-type mice these 4 transcripts were not significantly up-regulated after 6-hr polyI:C stimulation. Quantitative RT-PCR confirmed that the number of 2 of the 4 transcripts was more than 100-fold higher in polyI:C stimulated EP3 KO- than wild-type mice. In summary, we found that EP3 suppressed polyI:C, a TLR3 ligand, inducible genes in polyI:C stimulated murine conjunctival epithelium. Our findings suggest that EP3 and TLR3 in conjunctival epithelium play a critical role in regulating ocular surface inflammation.