Histone deacetylases (HDACs) are potential therapeutic targets of polyglutamine (pQ) diseases including Huntington’s disease (HD) that may function to correct aberrant transcriptional deactivation caused by mutant pQ proteins. HDAC3 is a unique class 1 HDAC found in both the cytoplasm and in the nucleus. However, the precise functions of HDAC3 in the two cellular compartments are only vaguely known. HDAC3 directly binds to huntingtin (Htt) with short pQ and this interaction is important for suppressing neurotoxicity induced by HDAC3. With long pQ Htt, the interaction with HDAC3 is inhibited, and this supposedly promotes neuronal death, indicating that HDAC3 would be a good therapeutic target for HD. However, the knockout of one HDAC3 allele did not show any efficacy in reducing neurodegenerative symptoms in a mouse model of HD. Therefore, the role of HDAC3 in the pathogenesis of HD has yet to be fully elucidated. We attempted to resolve this issue by focusing on the different roles of HDAC3 on cytoplasmic and nuclear Htt aggregates. In addition to supporting the previous findings, we found that HDAC3 preferentially binds to nuclear Htt over cytoplasmic ones. Specific HDAC3 inhibitors increased the total amount of Htt aggregates by increasing the amount of nuclear aggregates. Both cytoplasmic and nuclear Htt aggregates were able to suppress endogenous HDAC3 activity, which led to decreased nuclear proteasome activity. Therefore, we concluded that Htt aggregates impair nuclear proteasome activity through the inhibition of HDAC3. Our findings provide new insights regarding cross-compartment proteasome regulation. In polyglutamine (pQ) diseases, the gene transcription machinery required for proper neuronal function is impaired, and this may result from the sequestration of essential proteins for transcription There are four classes of HDACs and among them, class I or IIa HDACs have been previously suggested as therapeutic targets for pQ diseases Another possible cause of this discrepancy is that unlike HDAC1 or 2, which only functions at the nucleus, HDAC3 can shuttle between the cytoplasm and the nucleus where it can have different roles. Therefore, the effect of HDAC3 inhibition on HD models can depend on the balance of nuclear vs. cytoplasmic aggregates. In the case of pQ diseases, nuclear aggregates exhibit a far higher toxicity than cytoplasmic aggregates To overcome these issues, we utilized highly specific HDAC3 inhibitors made by a click chemistry-based combinatorial fragment assembly technique  [24]. Th2 at 37°C. Cells were transfected with plasmids using Lipofectamine 2000  following the manufacturer’s protocol. The transfection efficiency was 60–75% for HeLa cells and >90% for 293T cells.HeLa and 293T cells were grown in 95% air and 5% COCells were incubated with CellTiter 96 Aqueous solution for an hour and absorbance at 490 nm was measured by the Spectra Max 384 Plus colorimetric plate reader .The filter trap assay was performed as previously described 5′- GATCCCCGATGCTGAACCATGCACCTTTCAAGAGAAGGTGCATGGTTCAGCATCTTTTTA-3′ and 5′- AGCTTAAAAAGATGCTGAACCATGCACCTTCTCTTGAAAGGTGCATGGTTCAGCATCGGG-3′ and was inserted into the pSuper vector . HDAC3 inactive mutants were PCR generated with oligonucleotide primers 5′ -TCGGGTGCTCTACATTGCCATTGCCATCCACCATGGTGA-3′ and 5′ -TCACCATGGTGGATGGCAATGGCAATGTAGAGCACCCGA-3′.HDAC3 cDNA was cloned from a cDNA library with oligonucleotide primers 5′ -CATGGCCAAGACCGTG- 3′ and 5′ -AAAGAAATTCCTTGGGACACA-3′. The HDAC3 knockdown construct was made with oligonucleotide primers, Details about HDAC3 inhibitors T130, T247, and T326 were previously published Pan-HDAC activity was assayed using the Flour-de-lys HDAC assay kit . The fluorometric assay was performed using the Spectramax Gemini XS (Molecular Devices) with an excitation wavelength of 360 nm and emission at 460 nm. HDAC3 activity was assayed using the HDAC3 activity assay kit (Sigma Aldrich) with excitation at 380 nm and emission at 500 nm.Western blot images were obtained using a LAS 3000 Mini . Digital images were analyzed by Multi Gauge software (Fujifilm).To prepare lysates for immunoprecipitation, cells were sonicated in 50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP40, 1 mM ethylenediaminetetraacetic acid (EDTA), and Complete protease inhibitor cocktail  and centrifuged at 20,000×g for 15 min. Lysates were incubated with 1 µg of anti-FLAG M2 antibody immobilized agarose beads (Sigma) for 4 h at 4°C and washed for four times with lysis buffer and subjected to SDS-PAGE and Western blot analysis. For the glutathione S-transferase (GST) pull-down assay, cells were lysed in 20 mM HEPES, pH 7.5, 100 mM NaCl, 0.1% Triton X-100, 10% glycerol, and Complete protease inhibitor cocktail (Roche). GST or GST-HDAC3 (500 ng) was mixed with glutathione sepharose beads  and incubated with the lysates for 2 h at 4°C. Beads were washed four times with the lysis buffer and subjected to SDS-PAGE and Western blot analysis.Cells were fixed with 4% paraformaldehyde and a standard immunocytochemistry procedure was performed. Visualization of the primary anti-HDAC3 antibody . was done with the Alexa 546 secondary antibodies (Life Technologies). Nucleus was visualized by Hoechst 33258 (Sigma Aldrich). Images were obtained using an Axioplan 2 fluorescent microscope and Axiocam HRc CCD camera system .Proteasome activity was measured using a 20S Proteasome Activity Assay Kit  following the manufacturer’s protocol. The fluorometric assay was performed using a Spectramax Gemini XS (Molecular Devices) with an excitation wavelength of 380 nm and emission at 460 nm.Proteasomes were purified using the Proteasome purification kit (Enzo Life Sciences) following the manufacturer’s instructions.Total RNA was extracted with TRIzol (Life Technologies) and cDNA was generated by ReverTra Ace qPCR RT Kit . Quantitative PCR (qPCR) was performed with the HT-7900 system . For qPCR, the probe set Mr04097229_mr was used to measure EGFP mRNA, and HuGAPDH and HuACTB (Applied Biosystems) were used as internal controls.Samples were incubated at 60°C in 4×LDS buffer (Life Technologies) for 15 min and subjected to SDS-PAGE with Mini-PROTEAN TGX gels  and transferred to PVDF membrane with the Trans-Blot Turbo Blotting System and Trans-Blot Turbo Transfer Pack (Bio-RAD). For the primary antibodies, Anti-GFP (Roche), anti-20S proteasome antibody , anti-actin antibody , anti-FLAG antibody (Sigma Aldrich), anti-HDAC3 antibody , anti-GST antibody (Millipore), anti-HSP90 antibody (Millipore), anti-SP1 antibody (Millipore), anti-acetylated lysine antibody , anti-HSP70 antibody  were used.nPPPPPPPPPPPQLPQPPPQAQPLLPQPQPPPPPPPPPPGPAVAEEPLHRP” which was followed by an EGFP sequence.Stable HeLa cell lines expressing green fluorescent protein (GFP) fused to huntingtin exon-1 (Htt-ex1) with a nuclear export signal (NES), or nuclear localization signal (NLS) was previously published Statistical analysis was performed using the GraphPad Prism 6 software . The significance was tested with t-tests or ANOVA with Dunnett’s multiple comparisons.Cytoplasmic and nuclear fractions were extracted using NE-PER Nuclear and Cytoplasmic Extraction Kit .Generation of aggregated over soluble species is essential for Htt toxicity. To understand the effect of HDAC3 inhibition on the amount of Htt aggregates, we used stable HeLa cell lines that express Htt exon-1 (Htt-ex1) with various pQ lengths  [23]. Thth and 168th aspartates; therefore, we mutated these amino acids to alanine, which successfully resulted in the loss of deacetylase activity Click here for additional data file.Figure S2The amount of 20S proteasome in each cellular compartment was not affected by HDAC3 inhibition HeLa cells were incubated with indicated amount of HDAC3 inhibitors for 48h, and nuclear and cytoplasmic fractions were extracted. HSP90 and Sp1 blots are shown for the purity of the fractions. There was a slight increase of cytoplasmic HDAC3 and a slight decrease of nuclear HDAC3 upon addition of inhibitors. Molecular weight markers are shown at the left side.(TIF)Click here for additional data file.Figure S3A, B: HDAC3 inhibitor does not alter the acetylation level of Htt-ex1s. E3 (NES) or N3 (NLS) cells were incubated with indicated amount of T326 for 48 h. Cells were lysed and immunoprecipitated by anti-GFP antibodies immobilized to protein G agarose beads. After a rigorous wash, they were run on SDS-PAGE and western blotted by anti-GFP (upper panel) or anti-acetylated lysine antibodies (lower panel). *are non-specific bands.(TIF)Click here for additional data file.Figure S4A: HDAC3 inhibitor does not affect the acetylation of the proteasome. The 293T and HeLa cells were incubated with indicated HDAC inhibitors at 10×IC50, and the proteasome was extracted from sonicated cell lysates. Purified proteasomes were analyzed by western blotting using anti-20S proteasome antibody (upper panel) or anti-acetylated lysine antibodies (lower panel). The “beads” lane indicates the negative control without any cell lysates. *are non-specific bands. B: HDAC3 inhibitors do not affect the amount of HSP70 chaperone. Indicated amounts of HDAC3 inhibitors were added to HeLa cells and the cell lysates were analyzed by western blotting by anti-HSP70 antibody. Anti-actin blot is shown for loading control. Molecular weight markers are shown at the left side.(TIF)Click here for additional data file.Figure S5HDAC3 does not bind to the proteasome. Proteasome was purified from extracts of HeLa cells sonicated in PBS and analyzed with anti-HDAC3 or 20S proteasome antibody. Molecular weight markers are shown at the left side.(TIF)Click here for additional data file.Table S1IC50 of HDAC3 inhibitors used in this study and previously reported studies.(DOCX)Click here for additional data file.Table S2Name of HeLa cell lines used in this study.(DOCX)Click here for additional data file.


As the major sensor of UPR in eukaryotes, IRE1, in response to endoplasmic reticulum (ER) stress, catalyzes the unconventional splicing of HAC1 in yeast, bZIP60 in plants and XBP1 in metazoans. Recent studies suggest that IRE1p and HAC1 mRNA, the only UPR pathway found in yeast, evolves as a cognate system responsible for the robust UPR induction. However, the functional connectivity of IRE1 and its splicing target in multicellular eukaryotes as well as the degree of conservation of IRE1 downstream signaling effectors across eukaryotes remains to be established. Here, we report that IRE1 and its substrate bZIP60 function as a strictly cognate enzyme-substrate pair to control viral pathogenesis in plants. Moreover, we show that the S1P/S2P-bZIP17/bZIP28 pathway, the other known branch of UPR in plants, does not play a detectable role in virus infection, demonstrating the distinct function of the IRE1-bZIP60 pathway in plants. Furthermore, we provide evidence that bZIP60 and HAC1, products of the enzyme-substrate duet, rather than IRE1, are functionally replaceable to cope with ER stress in yeast. Taken together, we conclude that the downstream signaling of the IRE1-mediated splicing is evolutionarily conserved in yeast and plants, and that the IRE1-bZIP60 UPR pathway not only confers overlapping functions with the other UPR branch in fundamental biology but also may exert a unique role in certain biological processes such as virus-plant interactions.The unfolded protein response (UPR) signaling network encompasses two pathways in plants, one mediated by inositol-requiring protein-1 (IRE1)- bZIP60 and S1P/S2P-bZIP17/bZIP28, but only one, IRE1p-HAC1, in yeast. Despite recent significant progress in understanding UPR, a functional connectivity of IRE1 and its splicing target has not been established in multicellular eukaryotes. It is unknown if a single UPR branch in plants has any unique biological functions. Given that all eukaryotes are equipped with the IRE1-mediated pathway, are IRE1 downstream signaling effectors conserved among kingdoms and at what degree? Here, we show that IRE1 and its substrate bZIP60 function as a matched enzyme-substrate pair to mediate virus-host interactions in plants. We further provide evidence that a single UPR branch, IRE1-bZIP60, rather than S1P/S2P-bZIP17/bZIP28, determines viral pathogenesis, indicating that the two UPR arms may have distinct functions in plants. Finally, we demonstrate that the spliced form bZIP60 and HAC1p, rather than IRE1, are functionally replaceable to cope with abiotic stress in yeast, suggesting that the downstream signaling of the IRE1-mediated splicing is evolutionary conserved in plants and yeast. These data shed new lights into UPR in multicellular eukaryotes.The unfolded protein response (UPR) is crucial to life as it regulates gene expression in response to stress in the endoplasmic reticulum (ER). There are two functionally overlapping UPR branches in plants, e.g., IRE1- Inaa 41–80  22,49].1–238 in .1–238 inaa 41–80  and S8. onal NLS , NLS3, ional NLS  Figs. cDreatment . Intrigureatment  and S14Breatment . Previoureatment  and S14Areatment  and S14Bbzip60-2 mutant displayed an anti-biotic stress phenotype, i.e., inhibition of viral infection, whereas the other independent mutant bzip60-1 exhibited the same susceptible phenotype as wild type plants  and a C-terminal truncated mRNA (bZIP60ΔC), respectively  accumulation ) of NtbZIP60 mRNA folded by M-Fold. Open red boxed area is magnified in detail in NtbZIP60 with different free energy, 17 forms (89.5%) could fold into twin hairpin loop.Lowest free energy form ((TIF)Click here for additional data file.S5 FigTwo conserved regions of these mRNA were found, which correspond to the NLS/ZIP and TMD domains in protein level see . The ide(TIF)Click here for additional data file.S6 Figbzip60-1 (A) and bzip60-2 (B) mutants using the primers indicated by arrows was aligned with the wild type bZIP60 DNA. Note that the gray-shaded regions represent the consistent sequences, without showing the indicated nt. The sequences marked in red represent the part of T-DNA sequences inserted into bZIP60 genome in the two mutants. The 23 nt shaded in yellow represent the target of unconventional splicing. A red triangle indicates that the positions of T-DNA insertion in the bZIP60 genomic DNA are at 41 and 1116 in the bzip60-1 and bzip60-2 mutants relative to the first ATG, respectively. (A) The red and blue arrows indicate the start codons of two in-frame ORFs (bZIP60 and bZIP60ΔN), respectively. The nucleotides in an open box represent the changeable site used for T-DNA insertion. Colonies—(8/8) and Colonies_ (3/3) indicated that the sequencing carried out on 8 (A) and 3 (B) selected colonies generated the same result.The sequence of the genomic DNA amplification products from (TIF)Click here for additional data file.S7 Figbzip60-1 mutant (A) treated by DMSO, Tm, and infected with TuMV as well as from the bzip60-2 mutant (B) was aligned with the wild type bZIP60 cDNA. The gray-shaded regions represent the sequences that are consistent with wild type bZIP60 cDNA. The red triangles indicated the corresponding position of T-DNA insertion in cDNAs. (A) The two start codons are shaded in green. bZIP60-1218 R is a bZIP60 specific primer, and AAP is a universal primer provided by 5′ RACE kit  bZIP60 S7 and LB2 were used for amplifying 3′ end of bZIP60 in the bzip60-2 mutant. An in-frame stop codon (Stop ΔC) was introduced due to the T-DNA insertion, generating bZIP60ΔC2, indicated by a blue hand-arrow. Note that bZIP60ΔC1 without T-DNA tail was also analyzed in The sequence of the cDNA amplification products from the  kit see . The nt (TIF)Click here for additional data file.S8 Fig(A) Amino acid sequence alignment of bZIP60, bZIP60ΔN and bZIP60ΔC was shown. Identical sequences are shaded in gray. Due to the frame-shift mediated by the removal of 23 nt from bZIP60 U mRNA, the C-terminus of bZIP60 S is different from that of bZIP60 U, and thus lose the TMD (in purple) of bZIP60 U. The amino acids 138–197 indicated by a line above sequence are defined as bZIP domain according to the prediction by SMART and the previous report [(B) Amino acid sequence alignment of HAC1p U and HAC1p S was shown. Identical sequences are shaded in gray. Due to the removal of 252 nt from HAC1 mRNA via unconventional splicing, HAC1p S gains an AD  [s report ,22. The s report  are thers report . Note thin blue) . The amiin blue) . The seqin blue) .(TIF)Click here for additional data file.S9 Fig(A) and (B) qRT-PCR analysis of total bZIP60 transcripts (A) and bZIP60 S (B) level in unopened and opened flowers from the wild type and the two bzip60 mutants. The abundance of bZIP60 and bZIP60 S was normalized to that of Actin II transcripts. Data represent means with SD of three biological replicates. ** P<0.01, *** P<0.001, unpaired two-tailed Student’s test. ns, non-significant. (C) Detection of bZIP60 U and bZIP60 S in 3-week-old seedlings from the wild type, bzip60-1 and bzip60-2 mutants. Seedlings were treated with 2 mM DTT or 5 μg/mL Tm for 2 h in liquid MS medium  were not detectable in the bzip60-2 mutant. bZIP60 U (bZIP60ΔN U for bzip60-1 mutant) could be detected in the wild type and bzip60-1 mutant regardless of stress treatment or not. Lower level of bZIP60 S (bZIP60ΔN S) was found in the bzip60-1 mutant under DTT or Tm treatment, compared to the wild type.dium see . 0.1% DM(TIF)Click here for additional data file.S10 Fig(A) Both bZIP60 and bZIP60ΔN folds into kissing hairpin loop containing two splicing sites indicated by scissors. Each loop contains three conserved amino acids (red). (B) The percentage of predicted different free energy forms of bZIP60 mRNAs with the twin kissing hairpin is compared between full length bZIP60, bZIP60ΔN without the first 44 nt and with a T-DNA sequence (TGTTATT) (-44 nt), and bZIP60ΔN without the first 150 nt (-150 nt). n indicates the total number of predicted bZIP60 mRNAs structure.(TIF)Click here for additional data file.S11 Fig(A), one is NLS/ZIP and the other TMD that is absent in spliced proteins (B) (see Two conserved regions of these proteins ware found on unspliced proteins  (B) see . A schem(TIF)Click here for additional data file.S12 Fig(A) The strength of homo-interactions of bZIP60 S and bZIP60ΔN S was tested by a liquid selective QDO medium containing AbA. The transformed cells were cultured in DDO medium overnight, pelleted, washed and diluted to an OD600 = 0.5 by a selective QDO medium with different concentrations of AbA. Data represent means with SD of three biological replicates. Note that the homo-interaction of bZIP60 S and bZIP60ΔN S could no longer support yeast growth in the presence of 1000 ng/mL AbA, compared to controls  Homo-interactions of bZIP60 S and bZIP60ΔN S in living cells by sensitized emission FRET assay. Note that only cells co-expressing bZIP60 S-CFP and bZIP60 S-YFP or bZIP60ΔN S-CFP and bZIP60ΔN S-YFP exhibited FRET signal. Experiments were repeated three times with similar results. Bars = 50 μm.also see . (B) Hom(TIF)Click here for additional data file.S13 Fig(A) Phenotypes of the wild type and ire1b-4 mutant at 12 dpi after inoculation with buffer or TuMV. Note that although ire1b-4 developed slower TuMV symptoms during a little early stage of virus infection (12 dpi), it eventually produced same viral symptoms at 21 dpi, observed in white light and ultraviolet (UV) lamp (B), compared to the wild type. (D), (E) and (F) Phenotypes of the wild type, two IRE1A mutants (ire1a-3 and ire1a-2), bzip17, bzip28 and bip2-2 at 18 dpi after inoculation with buffer or TuMV. Note that the mutant mutants all developed typical TuMV symptoms, compared to the wild type. (C) and (G) qRT-PCR analysis of TuMV CP in the wild type and the single mutants. At 12 dpi (for ire1b-4) (C) or 18 dpi (G) after inoculation with buffer or TuMV, RNA was extracted from the systemic leaves, and qRT-PCR was carried out. Actin II was used as an internal control for quantitative RT-PCR. Data represent means with SD of three biological replicates. * P<0.05, unpaired two-tailed Student’s test. ns, non-significant.(TIF)Click here for additional data file.S14 FigbZIP60 S with mutated NLS3 and NLS1  and to rescue the ER-stress sensitive phenotype of CRY1 Δhac1::TRP strains (B), compared to the untransformed cells and the transformed cells with HAC1p S. The assays were conducted according to the procedures presented in detail in Figs (A) Data represent means with SD of three experiments. *** P<0.001, unpaired two-tailed Student’s test. ns, non-significant. (B) Experiments were repeated three times with similar results.see Figs  and 9, b0ΔC2 see  failed t(TIF)Click here for additional data file.S15 FigΔire1::KanMX6 strains with an integrated pRS304 4 x UPRE-GFP reporter. The cells were transformed with CEN-ARS plasmids expressing yeast IRE1, Arabidopsis IRE1A or IRE1B under the control of a GPD promoter. The transformed yeasts were selected and grown in the 2 x SD medium deficient in TRP and URA with 250 μg/mL G418  and 2% raffinose, using monosodium glutamate  as nitrogen source. ER stress was induced by 2 mM DTT in the presence of 2% glucose. Experiments were repeated three times with similar results. Bars = 20 μm.Functional complementation was tested by confocal imaging of CRY1 (TIF)Click here for additional data file.S16 FigN. benthamiana seedlings were pre-treated with 0.1% DMSO, 4-PBA (1 mM), TUDCA (1 mM) or 4-PBA (1 mM) plus TUDCA (1 mM) for 6 h (see N. benthamiana seedlings were taken under UV light (A), and RNA was extracted from the indicated leaves. (B) TuMV accumulation indicated by CP mRNA level was analyzed by qRT-PCR. Data represent means with SD of three experiments. *P<0.05, **P<0.01, ***P<0.001, unpaired two-tailed Student’s test, ns, non-significant. 6 h see . The pre(TIF)Click here for additional data file.S17 FigThe relevant entry vectors were cloned into the Gateway version of bimolecular fluorescence complementation (BiFC) vectors to fuse the split YN and YC at the C-termini of 6K2, IRE1A and IRE1B. The sets of constructs were subjected to the transient expression system, and YFP signal was captured under the confocal at excitation 514 nm and emission 525–600 nm. BiFC assay demonstrated the homo-interactions of TuMV 6K2, IRE1A and IRE1B, but no interaction of TuMV 6K2 with IRE1A or IRE1B. Experiments were repeated three times with similar results. Bars = 50 μm.(TIF)Click here for additional data file.S18 Fig(A) The wild type, bzip60-1 and bzip60-2 mutants were grown on half-strength MS medium for 9 d after germination. (B) Root lengths were measured in 9-d-old seedlings. Box plots represent the value range and the variability of root lengths. The boundaries of each box represent the lower 25th and upper 75th percentiles, and the horizontal line within the box represents the median value. The spacing within the box indicates the degree of dispersal in the data. The lines at the top and bottom of the box (whiskers) represent the minimum and maximum. Outliers are indicated by solid circles. Statistical analysis was conducted and showed no difference in root length among wild type and mutants.(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S1 Text(DOC)Click here for additional data file.


INPP5F gene in gliomas is significantly correlated with lower overall patient survivals. These findings suggest that INPP5F is a potential tumor suppressor in gliomas via inhibition of STAT3 pathway, and that deregulation of INPP5F may lead to contribution to gliomagenesis.Glioblastoma (GBM), the most common type of primary malignant brain tumors harboring a subpopulation of stem-like cells (GSCs), is a fast-growing and often fatal tumor. Signal Transducer and Activator of Transcription 3 (STAT3) is one of the major signaling pathways in GSCs maintenance but the molecular mechanisms underlying STAT3 deregulation in GSCs are poorly defined. Here, we demonstrate that Inositol Polyphosphate-5-Phosphatase F (INPP5F), one of the polyphosphoinositide phosphatases, is differentially expressed in GSCs from glioma patients, and is identified as an inhibitor of STAT3 signaling via interaction with STAT3 and inhibition of its phosphorylation. Constitutively expressed INPP5F showed to suppress self-renewal and proliferation potentials of glioblastoma cells and reduced tumorigenicity of glioblastoma. In addition, loss of  STAT3 belongs to a family of transcription factor (TFs) and plays central important role in cell growth8INPP5F is frequently inactivated via chromosome deletion and mRNAs suppression in GBM. Based on this, we hypothesized that INPP5F might act as a tumor suppressor through regulation of oncogenic proteins by phosphorylation inhibition. Here, we showed that INPP5F regulates the STAT3 pathway via interaction and inhibition of STAT3 phosphorylation, and suppress gliomagenesis. Our data provide insight into the molecular underpinning of this potential tumor suppressor gene in glioblastomas.To identify glioblastoma related genes and improve novel targeting therapeutics, many studies have been done to characterize gene alterations on glioma surgery specimens by gene expression and single nucleotide polymorphism (SNP) genotyping arrays and the profiles are stored and could be analyzed in databases such as TCGA (The Cancer Genome Atlas)INPP5F is one of these highly altered genes, by looking for naturally occurring alterations within the INPP5F locus that could affect INPP5F expression and/or activity in gliomas. We analyzed gene expression data from TCGA and REMBRANDT (INPP5F and adjacent genes' locus including BAG3 (BCL2-associated athanogene 3) and MCMBP (Minichromosome Maintenance Complex Binding Protein). In 447 glioblastoma samples examined, 385 (86%) exhibited Ch10q25.2-10q26.11 deletion (log2 ratio less than -0.5) and 7 have shown INPP5F gene deletion only  have a short survival, significantly different from that with unaltered INPP5F (34 cases), p = 0.0144. Similar results observed in patients with all types of gliomas; Cases with INPP5F deletion (n = 103) showed a significant lower survival rate than that with INPP5F unaltered (n = 69), (−6). Although the retrospective data cannot determine whether INPP5F could be an independent predictor of survival, these data that INPP5F loss in glioma patients is inversely correlated with survival do suggest that the expression of INPP5F might be a positive prognostic factor for glioma patients.To further investigate the tumor suppressor effect of INPP5F in gliomas, we analyzed the INPP5F gene expression and glioma patient survival data contained in the REMBRANDT dataset. As shown in Finally, to evaluate the effect of INPP5F on glioma pathogenesis, INPP5F overexpressing U87 cells were injected into the periventricular cortex of adult female severe combined immunodeficiency (SCID) mice. Consistent with patients survival, mice inoculated with INPP5F overexpressing U87 cells lived significantly longer than control U87 inoculated animals . The datINPP5F located in chromosome 10q26.11 is one of the highly altered glioma-related genes. The expression of INPP5F is significantly suppressed in glioblastomas and all types of gliomas, which correlated with decreased survival rates in glioma patients, suggesting that INPP5F might be a tumor suppressor in glioma pathogenesis.In this study, we analyzed TCGA and REMBRANDT databases and identified that Glioblastoma represents one of the most lethal human cancers with frequent biological relevant alterations. Among these, the RTK/PI3K/AKT pathway is one of the three critical signaling pathways that drive glioma growth19In the present study, we demonstrate that INPP5F strongly inhibits STAT3 activation by interaction with its coiled-coil domain and inhibits its phosphorylation, by which plays essential role in maintaining glioblastoma cells' phenotype and tumorigenic potential. STAT3, as a member of the STAT transcription factors family, is important in central nervous system development, reprogramming to ground state pluripotency, and essential for maintenance of embryonic stem (ES) cell biology, as well as glioblastoma tumorigenesis22242530in vivo experiments  should be inactive. Using the STAT3-YF mutant we identified that INPP5F interacts with STAT3 directly and such an interaction is STAT3 phosphorylation site independent , the fin32eriments .INPP5F deletion is correlated with a decreased survival rate for glioma patients, and overexpressed INPP5F suppresses glioblastoma cell proliferation, GSCs' self-renewal, and gliomagenesis through inhibition of STAT3 pathway. In addition, as a number of STAT3 pathway related inhibitors are now in clinical trials, and loss of INPP5F expression may provide a marker for selecting patients who will respond to these drugs.Taken together, for the first time, our study identified a novel role of INPP5F as a tumor suppressor in glioma tumorgenicity. Antibodies against to INPP5F (H00022876-B01P) were purchased from Novus Biologicals, Littleton, CO; Stat3 (sc-7179), p-Stat3 (sc-8059), GFP (sc-8334) and Actin (sc-8432) from Santa Cruz Biotechnology, Dallas, TX; V5 (A01724) from GenScript, Piscataway, NJ; Lamin B1 (33-2000) from Invitrogen . Recombinant IL-6 was purchased from R&D System. The plasmids pLenti6/UbC/V5-DEST, pDEST47-DEST and ViraPower mix were purchased from Life Technologies; and INPP5F ShRNAs were from Open Biosystems .Glioblastoma stem cells (GSCs) were cultured in NBE media consisting of neurobasal medium, with N2 and B27 supplements (Life Technologies), and human recombinant bFGF and EGF  as previous describedINPP5F (F: GCCACCATGGAGCTCTTCCAAGCCAAG GAC and R:GCCACCAATCTGAATTATCCGTGTCTGGCATTGTATAAAC) and STAT3  genes were inserted into Gateway vector pCR8/GW/TOPO and subsequently into pLenti6/UbC/V5-DEST. Site-directed mutagenesis was performed to generate STAT3 Y705F, using a Quick Change Site-Directed Mutagenesis kit  according to the manufacturer's instructions. To change a base, STAT3 entry clone was used as a template. Primers (F: CCAGGTAGCGCTGCCCCATTC CTGAAGACCAAGTTTATC and R: GATAAACTTGGTCTTCAGGAATGGGGCAGC GCTACCTGG) were used. The nucleotide sequences of the mutated plasmids were confirmed by DNA sequencing. Lentivirus was produced in 293T cells with packaging mix . Plasmids were transfected with Lipofectamine and Plus reagent (Life Technologies). After 3 hrs, media were changed to that with 5% FBS. Virus-laden supernatants were collected at 24, 48 and 72 hrs. The supernatant was filtered and concentrated by ultracentrifugation, and viral titer was determined by serial dilution.5 cells per well and incubated at 37°C for 2 and 5 days. At the time of quantification, cells in each well were counted using Vi-CELL XR (BCM). For the limiting dilution assay, GSC cells were plated into 96-well plates at various seeding densities  and incubated at 37°C for up to 2 weeks. Each well was then examined for spheres formation. Soft agar colony formation assay was performed using standard protocol with a minor modification. Briefly, low-melting-point agar (Difco) was melted and mixed with DMEM containing serum medium at a 1:1 ratio to make a supporting bottom layer (1%) in a 2 ml/well (6-well plate). The bottom agar layer was allowed to solidify at room temperature for 20 min. The top layer containing 0.4% agar (2 ml/well) was prepared by mixing stock agar solutions with 100,000 cells in DMEM containing serum medium, and then laid on top of the supporting agar layer. Cell colonies were allowed to form at 37°C for 2–3 weeks. Experiments were performed at least in triplicates for each condition, and colonies in each well were counted.Cultured glioblastoma cells were dissociated into single-cell suspensions. For proliferation assay, GSCs  and U87 cells were plated into 6-well plates with 2 × 10Cells were collected and lysed in M2 buffer . Immunoprecipitations were performed with cell lysates using Immunoprecipitation Kit –Dynabeads Protein G , according to the manufacturer's instruction. Cell lysates were separated by SDS-PAGE and analyzed by immunoblot. The proteins were visualized by enhanced chemiluminescence (ECL) according to the manufacturer's instruction (Pierce).Nuclear fractions were prepared using Nuclear Extract Kit  and the STAT3 activity analysis assay was performed using TransAM STAT3 kit  according to the manufacturer's instructions. Absorbance (450 nm) with a reference wavelength of 655 nm was detected by using a plate leader .Cells cultured in chamber slides  were fixed with 4% paraformaldehyde fixative (PFA) for 30 min at 4°C, permeabilized with 0.5% Triton X-100 in PBS, and blocked with 1% BSA. The cells were then stained with primary antibodies followed by Alexa Fluor-conjugated secondary antibodies (Life Technologies), and examined under confocal microscope .https://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp). The .CEL files were normalized and detection calls were generated using MAS5 algorithm in R ver. 2.12. The INPP5F expression data across all the patients were extracted and plot was created using Prism 6. INPP5F expression was also analyzed with REMBRANDT dataset of 577 brain tumors. For copy number analysis, 447 glioblastoma gene centered copy number data were downloaded from TCGA data portal. The copy number data of INPP5F and its neighboring genes BAG3 and MCMBP on chromosome 10 were extracted and the heatmap of these genes were created in MATLAB R2009b.For expression analysis, gene expression array data of 389 glioblastomas and non-brain tumors generated using Affymetrix HG_U133A platform was downloaded from TCGA data portal , purified using RNeasy Mini Kit (Qiagen), and then profiled to HG_U133 plus2 GeneChip Arrays according to the manufactory's instruction (Affymetrix). All .CEL files were normalized using MAS5 method and probe sets with only absent calls across all the samples were removed prior to data analysis. The fold changes of 1.2 were used for deriving INPP5F signatures. The hierarchical cluster analysis was performed using Partek software 6.6 (Partek Inc.) for Euclidean distance with complete linkage method. Gene set enrichment analysis was done using GSEA application . The enrichment scores were calculated by walking down the ordered list and the statistical significance of nominal p values of the enrichment scores were estimated using Kolmogorov-Smirnov statistics by constructing a cumulative null distribution with 1,000 permutations.The mRNAs of four GSC lines, 211, 827, 923, 1228 and one established glioblastoma cell line U87, with either An intracranial orthotopic model was utilized for evaluation of glioblastoma cells tumorigenicity according to an approved animal study proposal by NCI-ACUC. Cells were resuspended in 5 μl of HBSS and injected stereotactically into the striatum of female SCID mice (6–8 week age) using a stereotactic device . Following injection, animals were monitored daily for the development of neurological deficits. For histology examination, mice were euthanized by perfusion with 4% PFA under anesthesia, and brains were dissected, followed by overnight post-fixation in 4% PFA at 4°C. The 5 µm paraffin serial sections were prepared and stained with hematoxylin and eosin . Images were captured using a Leica DM1400B microsystem and Leica FW4000 version 1.2.1.p value ≤ 0.05 was considered significant. All values are shown as mean ± standard deviation (SD). Kaplan-Meier survival analysis was performed in Prism 4.0 software.Student's t test was used for data analysis and a H.S.K. conceived and designed the experiments; H.S.K., S.A. and H.S. carried out the experiments; and H.S.K., A.L. and W.Z. analysed the data. H.S.K., A.L. and W.Z. wrote the manuscript, and H.S.K., A.L., S.A., H.S. and W.Z. discussed the results and commented on the manuscript. All authors read and approved the final manuscript.Inositol Polyphosphate-5-Phosphatase F (INPP5F) inhibits STAT3 activity and suppresses gliomas tumorigenicity


Human adenovirus type 55 (HAdV55) is a newly identified re-emergent acute respiratory disease (ARD) pathogen with a proposed recombination of hexon gene between HAdV11 and HAdV14 strains. The identification of the neutralizing epitopes is important for the surveillance and vaccine development against HAdV55 infection. In this study, four type-specific epitope peptides of HAdV55 hexon protein, A55R1 (residues 138 to 152), A55R2 (residues 179 to 187), A55R4 (residues 247 to 259) and A55R7 (residues 429 to 443), were predicted by multiple sequence alignment and homology modeling methods, and then confirmed with synthetic peptides by enzyme-linked immunosorbent assay (ELISA) and neutralization tests (NT). Finally, the A55R2 was incorporated into human adenoviruses 3 (HAdV3) and a chimeric adenovirus rAd3A55R2 was successfully obtained. The chimeric rAd3A55R2 could induce neutralizing antibodies against both HAdV3 and HAdV55. This current study will contribute to the development of novel adenovirus vaccine candidate and adenovirus structural analysis. Human adenovirus (HAdV) has been recognized as a common cause of acute respiratory disease (ARD) ,2,3. To The adenovirus capsid icosahedron is composed of three major structural proteins: hexon, penton base, and fiber. The hexon protein is the major antigenic determinant recognized by neutralizing antibodies (NAbs) ,24,25,26The current study sought to map the neutralizing epitopes of HAdV55. Furthermore, we tried to incorporate the epitopes on the surface of HAdV-3 hexon to prepare a novel bivalent vaccine candidate against both HAdV3 and HAdV55. This study expands current knowledge about HAdV and will be useful for HAdV vaccine development.Previous studies suggest that human adenovirus serotype-specific NAb epitopes may be exposed on the virion surface and reside within any of the seven hypervariable regions (HVRs) ,30,31,33To test the antigenicity of the predicted neutralizing epitopes, ELISAs were performed by coating synthetic peptides and reacting with anti-HAdV55 sera. The synthetic peptide A14R1 (ETTEERQNEDGENDEKA) and A11R1 (EGVKNTTGEERV), and the purified HAdV55 virions were used as the controls. in vitro neutralization tests were performed with serially diluted anti-peptides sera , anti-PBS and anti-HAdV55 sera, neutralizing HAdV55 cultured in AD293 cells. After continuous observation for 72 h, the anti-A55R1, anti-A55R2, anti-A55R4, anti-A55R7, anti-A11R1, and anti-HAdV55 sera could neutralize HAdV55 infection; whereas, the anti-A14R1, anti-PBS and all preimmune sera could not neutralize HAdV55 infection, even at the lowest dilution, 1:8  and an enhanced green fluorescent protein (eGFP) with an E3 region deletion were obtained as previously described . HAdV-14t 260 nm .The HVRs of HAdV55 hexon protein were determined by multiple sequence alignment (MSA) with the available hexon protein sequences of species B human adenoviruses using the protein BLAST program   or ClustThe four predicted epitope peptides were synthesized by GL Biochem Ltd. , and then purified and analyzed by highperformance liquid chromatography (purity ≥90%). The Cys was added on the N-terminal of synthetic peptides for conjugation. Then, each peptide was chemically linked to the carrier protein keyhole limpet hemocyanin (KLH). Meanwhile, two control peptides (A14R1 from HAdV14 and A11R1 from HAdV11) were synthesized by the same method.The plasmid pBRAdΔE3GFP encoding a HAdV-3 GZ-01 genome and eGFP with an E3 region deletion and the shuttle vector pBRLR was previously constructed by us ,33. In tEscherichia coli (E. coli) strain BJ5183. The successful creation of the constructs was confirmed by restriction digestion and sequencing analyses.Briefly, the mutated fragment H3A55R2 was produced by overlapping PCR extension mutagenesis, and then cloned into pBRLR to generate shuttle vectors pBRLR-H3A55R2. Finally, the LR-H3A55R2 fragment was cloned into the pBRAdΔE3GFP vector to generate the HAdV-3 hexon HVR2 mutagenesis vector pBRAd3-A55R2 using homologous recombinant technology in 2 for 6–10 days and were examined daily for evidence of cytopathic effect. The virus was harvested and designated rAd3A55R2. Finally, the mutant virus was cultured with AD293 cells in a total of twenty 100 mM dishes, then harvested and purified by standard CsCl gradient centrifugation as described above. The full-length modified hexon genes of the viruses were identified by sequencing.To rescue viruses, these modified plasmids were digested with AsisI to linearize genomic DNA, then transfected into AD293 cells grown in 30-mm dishes using Lipofectamine LTX with Plus reagents  according to the manufacturer’s instructions. The transfected cells were cultured at 37 °C with 5% CO9 VPs per mouse  rAd3A55R2, rAd3EGFP and inactivated HADV55 (inactivated with 20 mmol/L beta-propiolactone at 4 °C for 24 h) viron, or 50 μg KLH-conjugated peptides in QuickAntibody-Mouse5W adjuvant , followed by one additional boost 3 weeks later with the same doses and method. Phosphate buffered saline (PBS) in the same adjuvant was injected to mouse as the control. Blood samples were collected from the mice at two weeks after the final immunization and sera were isolated, heat-inactivated and kept frozen for serology tests. The animal procedures used in this work were evaluated and approved by the Ethic Committee of the First Affiliated Hospital of Guangzhou Medical University . They complied with all relevant guidelines and the National law for Laboratory Animal Experimentation. The animal experiments were conducted in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animals were housed in individually and received humane care. During injection and sample collection, the mice were anesthetized with 1.5% isoflurane or 1mL/kg weight 3% pentobarbital sodium to minimize suffering. Female Balb/c mice aged 4–6 weeks were used in the immunization experiments. Mice in groups of 5 were injected intramuscularly (i.m.) with 5 × 1010 VPs/mL) in carbonate–bicarbonate buffer (pH 9.6), and were washed once with 0.05% Tween-20 in phosphate-buffered saline (PBST) and blocked for 2 h with 5% skim milk in PBST. Then antisera of adenoviruses or peptides serially diluted in PBS were added and incubated for 1 h at 37 °C. After being washed three times with PBST, the plates were added with a 1:8000 dilution of HRP-Conjugated goat anti-mouse IgG (H + L) secondary antibody  and incubated for 1 h. After washing four times with PBST, the plates were developed with tetramethylbenzidine (TMB) substrate, stopped with 2 M H2SO4, and analyzed at 450 nm using an ELISA plate reader . Endpoint titer was defined as the highest dilution at which the OD450 was at least 0.1 above wells receiving no sera. Wells receiving no sera always had an OD450 of <0.1. Each assay was performed independently at least three times and with at least two parallel reactions for each well.For ELISAs, the ELISA plates  were coated overnight at 4 °C with synthetic peptides (about 2 µg/mL) or purified virus particles  of purified adenoviruses were detected by the typic method with AD293 cells. All of the antipeptides sera and anti-rAd3A55R2, anti-PBS sera as a negative control, and anti-HAdV55, anti-rAd3EGFP sera as positive controls, were serially 2-fold diluted  in DMEM, and 50 µL aliquots of each dilution were mixed with 50 µL of virus with 100 TCID50. The antibody-virus mixtures were incubated at 37 °C for 1 h and transferred to 96-well plates containing 70%–90% confluent monolayers of AD293 cells. The monolayers were cultured for 96 h, after which the infection was observed by microscopy and the neutralization titers were determined as the reciprocal of the highest serum dilution that completely inhibited visually observable cytopathic effect (CPE).t tests. Comparisons among groups were performed by ANOVA with Bonferroni’s test to account for multiple comparisons and p values of less than 0.05 were considered statistically significant.Statistical significance was determined using Prism 5.0 software . Comparisons between two groups were made with Student’s


Ingenuity pathway analysis revealed a more significant upregulation of death receptor signalling, driven by H5N1 than with H5N8 infection at 3 and 7 dpi. Early induction of immune response-related genes may elicit protection in H5N8-infected mice, which correlates with moderate pathogenicity in vivo. Collectively, our data provide new insight into the underlying mechanisms of the differential pathogenicity of avian influenza viruses.Emerging outbreaks of newly found, highly pathogenic avian influenza (HPAI) A(H5N8) viruses have been reported globally. Previous studies have indicated that H5N8 pathogenicity in mice is relatively moderate compared with H5N1 pathogenicity. However, detailed mechanisms underlying avian influenza pathogenicity are still undetermined. We used a high-throughput RNA-seq method to analyse host and pathogen transcriptomes in the lungs of mice infected with A/MD/Korea/W452/2014 (H5N8) and A/EM/Korea/W149/2006 (H5N1) viruses. Sequenced numbers of viral transcripts and expression levels of host immune-related genes at 1 day post infection (dpi) were higher in H5N8-infected than H5N1-infected mice. Dual sequencing of viral transcripts revealed that in contrast to the observations at 1 dpi, higher number of H5N1 genes than H5N8 genes was sequenced at 3 and 7 dpi, which is consistent with higher viral titres and virulence observed in infected lungs  The continuing spread of highly pathogenic avian influenza virus (HPAI) among wild birds and poultry is alarming and requires urgent active surveillance; it attracts public concern owing to its potential to become pandemic. HPAI AH5N1) viruses have been discretely involved in reassortment events that have either generated H5 strains with novel viral neuraminidase (NA) subtypes or donated gene components to other co-circulating strains viruses 235Our previous work demonstrated that the MDk/W452(H5N8) strain is less pathogenic than is the EM/W149(H5N1) strain in BALB/c miceA better understanding of the global gene changes underlying the multi-step progression of pathogenicity during infection could help develop potential therapeutic strategies for emerging infectious diseases910111213In the present study, we used the RNA-seq technology to comprehensively annotate dual host/pathogen transcriptomes following infection of mice with H5N8 and H5N1. Our work provides a global view of virus type-specific and post-infection time-specific mRNA profiles, which illustrate the role and mechanisms of viral replication efficiency and host response during avian influenza infection of mice.To gain global and dynamic gene expression profiles of A/MD/Korea/W452/2014- - and A/EM/Korea/W149/2006- -infected BALB/c mice, RNA-seq was performed to explore the transcriptomes from mouse lungs derived at 1, 3, and 7 day post infection (dpi). H5N1 was used to compare the pathogenicity with recently isolated H5N8 virus. More than 37 million 100-bp paired-end reads were generated. The base quality of RNA-seq reads was checked and analysed using an Agilent RNA 6000 Nano Kit . Each saq-value < 0.05 were identified. Using the 41,388 annotated genes in the mouse genome database, gene expression was quantified and compared between the control and the virus-infected groups, and differentially expressed genes (DEGs) with a p-values . Additionally, further analysis of DEGs were visualized by hierarchical clustering analysis, scatter plot and MA plot . At 7 dpi, the transcript counts for HA, M, NP, PA, and PB1 were found to be significantly different between the H5N1- and H5N8-infected mice. These results suggest that viral gene expression levels also vary for these avian influenza viruses in a temporal dependent manner.RNA-seq enabled dual analysis of both host and viral transcripts within the same sample. Paired-end reads from influenza-infected samples were mapped to the influenza virus genome. Interestingly, viral transcript counts of hemagglutinin (HA), neuraminidase (NA), and nonstructural protein 1 (NS1) of H5N8 were found to be significantly higher than those of H5N1 at 1 dpi. Given that host transcriptome data also showed more upregulated DEGs for H5N8 in comparison with H5N1 at 1 dpi, these data imply a strong correlation between sequenced numbers of viral transcripts and expression levels of host immune-related genes. Overall, the pattern of influenza virus gene expression was different between H5N8- and H5N1-infected mice in a time-dependent manner. The average number of reads was approximately 10 to 100 times higher in H5N1-infected mice than in H5N8-infected mice at 3 and 7 dpi . Signifivs 8.76 log2-fold change (fc) values). We also determined DEGs that were upregulated in virus-infected groups only but not in mock-infected groups. Among those genes, IL-24, which is known to be an important regulator of tumorigenesis2 fc values; 2 fc values; To further analyse the characteristics of DEGs, we focused on the 10 DEGs showing the most marked up or down-regulation from H5N8- and H5N1-infected mice  and 2. ATo examine the biological roles of the DEGs, a gene ontology (GO) enrichment analysis was applied to the up-regulated genes. GO term statistical analysis of genes in this cluster confirmed that defence response to virus were significantly over-represented  and 4. ATo investigate possible biological interactions of DEGs and identify important functional networks, datasets representing genes with altered expression profiles derived from RNA-seq analyses were imported into the Ingenuity pathway analysis (IPA) tool. The highest activated networks (high z-score) were identified using IPA. in vivoNext, the expression of different pathways was analysed with IPA, and several gene interaction networks were created. DEGs due to viral infection were indicated as either red (upregulated) or green (downregulated)  and 6. Win vivo. Consequently, these data collectively suggest that higher upregulation of DEGs from both viral and host transcriptomes induced by H5N1 infection at 3 and 7 dpi may have led to more robust immune activation, elevating persistent activation and recruitment of immune cells  and resulting in cytokine storm-like severe pathogenicity in lethal HPAI A(H5N1) viral infection in mice.We further generated a network map of death receptor signalling for H5N8- and H5N1-infected mice at 3 dpi . FasL, TSince 2013, cases of HPAI H5N8 infection in wild birds and poultry have emerged and caused economic and social losses globally, including in South Korea, China, Europe, and the United States461718196 log10 EID50/mL (EID50 is 50 percent embryo infectious dose) of MDk/W452(H5N8) induced only 6% reduction in body weight and 40% lethality within the 14-day observation period, whereas EM/W149(H5N1) infection was highly lethal and replicated systemically in various tissuesin vivo (Our data highlight distinct and dynamic changes in expression patterns of host genes following MDk/W452(H5N8) virus infection in mouse lungs compared with those observed following H5N1 virus infection. Previously, we have demonstrated that intranasal inoculation of mice with 10sin vivo . NotablyPreviously, our data have shown that virus titres from H5N8-infected mouse lungs were lower than those from H5N1-infected mouse lungs, suggesting a moderate viral replication pattern of H5N8 in comparison to that of H5N1RNA-seq analysis of influenza virus infection has been performed in mouse, crow, and chicken samples1021232425et al., H5N1-infected mice also induced an increased secretion of IFN-γin vivo results suggest that the cytokine storm-like phenotype is the hallmark of severely lethal HPAI A(H5N1) virus infection in miceIt was also interesting to note that IFN-γ was robustly expressed in the top 10 upregulated genes of H5N8-infected lungs. Previous studies demonstrate that IFN-γ treatment significantly reduced the number of T and NKT cells in the lungs at the inflammatory phase following infection, thus contributing to protection against viral pathogenesisin vivo. The increase in the replication of H5N1virus in lungs may have resulted in the induction of immune cell recruitment with robust production of chemokines (such as CXCL10 and CXCL11); in contrast, H5N8 replication was relatively moderate and thus caused a relatively moderate induction of these chemokines at both 3 and 7 dpi. This observation may reflect a positive correlation with host responses related to virus sensing and virus-mediated inflammation, such as TREM1 signalling, acute phase response signalling, and virus receptor signalling, as shown by the IPA heat map analysis . They were approved by the Laboratory Animal Research Center , which is a member of the Institutional Animal Care and Use Committee of Chungbuk National University, and were performed in a level 3 biosafety laboratory (BSL3) in Chungbuk National University (permit No. CBNU-BSL-14-001). The methods were carried out in accordance with the approved guidelines.50 and 50% tissue culture infectious dose (TCID50) end-point titrations. All experiments with HPAI H5 viruses were conducted in an enhanced biosafety level 3 (BSL3-1) containment facility approved by the KCDC.HPAI H5 viruses were isolated from wild bird faecal samples in the winters of 2006–2007 (A/environment/Korea/W149/2006 [EM/W149(H5N1)]), and 2013–2014 ]) and grown in specific pathogen-free 10-day-old embryonated chicken eggs. Supernatants  were harvested at 48 hours post inoculation (hpi) and aliquoted into cryovials (1 mL each). Stock viral titres were determined by EID2–107 EID50/50 mL of MDk/W452(H5N8) and EM/W149(H5N1). Body weights and survival patterns were recorded daily for 14 days. For virological and pathological examinations, groups of mice were inoculated intranasally with 105 EID50/50 mL of virus, and six mice per group were euthanised at 1, 3, 5, 7 and 9 dpi to examine the growth kinetics of the virus in mouse lungs.Six-week-old female BALB/c mice  were used for all infection experiments. Ten mice per group were inoculated intranasally with 10PRJNA290088(SUB1025618) (http://www.ncbi.nlm.nih.gov/sra/).RNA quality was assessed by analysis of rRNA band integrity on an Agilent RNA 6000 Nano Kit . Prior to cDNA library construction, poly (A) mRNA was enriched using 2 μg of total RNA and magnetic beads with oligo(dT) primer. The purified mRNAs were then disrupted into short fragments, and double-stranded cDNAs were immediately synthesised. The cDNAs were subjected to end-repair, poly (A) addition, and connection with sequencing adapters using the TruSeq RNA Sample Prep Kit . Suitable fragments, automatically purified using a BluePippin 2% agarose gel cassette , were selected as templates for polymerase chain reaction (PCR) amplification. The final library sizes and qualities were evaluated electrophoretically using an Agilent High Sensitivity DNA Kit , and the fragment was found to be between 350 and 450 bp. Subsequently, the library was sequenced using an Illumina HiSeq 2500 sequencer . Three biological replicates from RNA-seq run were performed per time point. RNA-seq data from this study have been submitted to the NCBI Sequence Read Archive (SRA) under the accession number of q-value was calculated based on the p-value using the p adjust function of the R package with default parameter settings. DEGs were identified based on a false discovery rate (FDR) q-value threshold of less than 0.05. Genes were considered differentially expressed with displaying a change of more than two-fold with a q-value of less than or equal to 0.05 for at least one time point.Low-quality reads were filtered according to the following criteria: (i) reads containing more than 10% of skipped bases (marked as ‘N’s); (ii) reads containing more than 40% of bases with quality scores less than 20; and (iii) reads with average quality scores of each read of less than 20. The whole filtering process was performed using in-house scripts. Filtered reads were mapped to the mouse reference genome (Ensembl release 77)p-values < 0.001 were considered statistically significant.The GO database classifies genes according to the three categories of biological process, cellular component, and molecular function, and predicts the function of the selected genes. To characterise the identified genes from DEG analysis, a GO-based trend test was performed using Fisher’s exact test; p-values (Fisher’s exact test) and activation z-scores were calculated by IPA. mRNAs that were upregulated or downregulated at least 2-fold at each time point were included for IPA analysis.Data were analysed using QIAGEN’s IPA . The canonical pathways and functional processes of biological importance that were the most significant were identified using the list of DEGs identified with RNA-seq and the Ingenuity Pathways Knowledge Base. The Ingenuity Knowledge Base contains the largest database of manually curated and experimentally validated physical, transcriptional, and enzymatic molecular interactions. Furthermore, each interaction in the Ingenuity Knowledge Base is supported by previously published information. The list of DEGs was overlaid onto a global molecular network developed from information contained in the Ingenuity Pathways Knowledge Base, and networks were then algorithmically generated on the basis of connectivity. Pathway enrichment How to cite this article: Park, S.-J. et al. Dynamic changes in host gene expression associated with H5N8 avian influenza virus infection in mice. Sci. Rep.5, 16512; doi: 10.1038/srep16512 (2015).


Current human papillomavirus (HPV) major capsid protein L1 virus-like particles (VLPs)-based vaccines in clinic induce strong HPV type-specific neutralizing antibody responses. To develop pan-HPV vaccines, here, we show that the fusion protein E3R4 consisting of three repeats of HPV16 L2 aa 17–36 epitope (E3) and a modified human IgG1 Fc scaffold (R4) induces cross-neutralizing antibodies and protective immunity against divergent HPV types. E3R4 was expressed as a secreted protein in baculovirus expression system and could be simply purified by one step Protein A affinity chromatography with the purity above 90%. Vaccination of E3R4 formulated with Freunds adjuvant not only induced cross-neutralizing antibodies against HPV pseudovirus types 16, 18, 45, 52, 58, 6, 11 and 5 in mice, but also protected mice against vaginal challenges with HPV pseudovirus types 16, 45, 52, 58, 11 and 5 for at least eleven months after the first immunization. Moreover, vaccination of E3R4 formulated with FDA approved adjuvant alum plus monophosphoryl lipid A also induced cross-neutralizing antibodies against HPV types 16, 18 and 6 in rabbits. Thus, our results demonstrate that delivery of L2 antigen as a modified Fc-fusion protein may facilitate pan-HPV vaccine development. More than 150 HPV genotypes have been identified with different epithelial tropisms Currently there are two licensed HPV major capsid protein L1 virus-like particle (VLP)-based vaccines, Cervarix, a bivalent HPV16/18 vaccine, and Gardasil, a quadrivalent HPV16/18/6/11 vaccine. Although some cross-reactivity has been observed between closely related HPV genotypes, the protection provided upon vaccination with HPV L1 VLP vaccine is largely HPV type-specific, indicating that vaccination provides very little cross-protection to the HPV types not covered by the vaccines Vaccination with recombinant minor capsid protein, L2, or peptides derived from L2 results in the production of cross-neutralizing antibodies that are protective in animal models The neutralizing titers produced by L2 vaccination are considerably lower than that induced by L1 VLP vaccination, particularly against heterologous HPV types The Fc receptors for IgG (FcγRs), expressed on dendritic cells (DCs) and APCs, can bind and internalize antigen-IgG immune complexes via the interaction with the IgG, resultsing in enrichment of exogenous antigens in DCs, which facilitates DC maturation and antigen-specific T cell responses and humoral responses. Recombinant antigen-immunoglobin Fc-fusion proteins were shown to increase the immunogenicity of the fused antigens and elicit neutralizing antibody responses to HIV A recombinant ligand for FcγRs, R4, is an Fc-like fusion protein that contains a tandem repeat composed of 4 copies of the hinge and CH2 regions (HCH2) of human IgG1 for each chain Four- to six-week-old female BALB/c mice and New Zealand white rabbits were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, and kept in the animal facility of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. All animal work was done in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, and all experimental protocols were approved by the Institutional Animal Care and Use Committee.Groups of 5 mice were vaccinated on weeks 0, 2, 4 and 6 with 1.7 nmol of E3R4, E12R4, R4, E3 peptide  or phosphate-buffered saline (PBS) formulated with complete Freunds adjuvant (CFA) for priming dose and with incomplete Freunds adjuvant (IFA) for booster immunizations. Groups of 2 rabbits were vaccinated subcutaneously on weeks 0, 2, 4 and 6 with 4 nmol of E3R4 alone or with Alum (Sigma-Aldrich) and monophosphoryl lipid A (Sigma-Aldrich) (Alum-MPL) adjuvant. Sera were collected at weeks 6 and 8 and stored at -20°C.6 293TT cells as suggested by Prof. Reinhard Kirnbauer. The titer of PsV  was determined by GFP expression in 293TT cells as described in http://home.ccr.cancer.gov/lco/pseudovirusproduction.htm. L1/L2 expression plasmids, pLucf plasmid, were generously provided by John Schiller, Susana Pang, Chris Buck, Martin Müller and Tadahito Kanda.Pseudoviruses of HPV16, 18, 45, 58, 6, 11 and 5  with encapsidated reporter plasmid pLucf which encoding both luciferase and green fluorescence protein (GFP), or plasmid pEGFP-N1 (Clonetech) which encoding GFP were produced in 293TT cells as previously described 2SO4. Plates were read at 490 nm, and antibody titer was determined as the reciprocal of the highest serum dilution with an OD 490 greater than 0.2 and 2-fold higher than control sera at the same dilution.To assess the binding of L2R4 proteins to RG-1, ELISA plates were coated with 100 ng of each protein , or 5 ng  of E3 peptide at 4°C overnight. ELISA was performed as previously described 4 to 2.0×104 IU of HPV PsVs (depending on the PsV stock) encoding GFP. Sera collected at week 8 were assessed by end-point dilution ELISA as described above. Binding titer was determined as the reciprocal of the highest serum dilution with an OD 490 greater than 0.2 and 2-fold higher than control sera at the same dilution. Statistical significance was determined by one-way ANOVA and Bonferroni multiple comparison test (GraphPad Prism 5). P value <0.05 was considered to be statistically significant.To measure cross-reactive antibody responses to HPV capsids in antisera, ELISA plates were coated with 1.5×10HPV PsV-based neutralization assays were performed as previous reports 5 to 1.0×107 IU of PsVs (encapsidated reporter plasmid pLucf) and 1% carboxymethyl cellulose  was intravaginally instilled using a positive-displacement pipette. Forty-eight hours post-PsV challenge, mice were vaginally instilled with 0.4 mg of 5′-F-Luciferin (CellCyto Life Sciences). Three minutes later, luciferase signals were acquired for 5 min with a biofluorescence imaging (BFI) system of the LB 983 NightOWL II (Berthold Technologies), and analyzed with IndiGO 2 software (Berthold Technologies). Statistical significance was determined by one-tailed unpaired t-test.Mice were treated with 3 mg of progesterone subcutaneously four days before PsV challenge. The immunized mice were intravaginally pretreated with 50 µl of 4% nonoxynol-9  at six hours prior to PsV challenge, R4 and PBS injected female mice were used as controls. 20 µl of PsV preparation containing 2.5×10in vitro.We first examined the expression of L2R4 proteins in the culture medium by SDS-PAGE since they are expressed as secreted proteins. We observed E3R4 (120 kDa) or E12R4 (130 kDa) band by Coomassie blue staining , which wP<0.001) (P<0.001), which may be due to the instability of E12R4  , suggestof E12R4 . Thus, tin vitro neutralization assay. As proof-of-concept, a panel of PsVs encoding GFP from divergent HPVs, including HPV16, 52, 58 , HPV6, 11 , HPV18, 45  and HPV5 (beta-1), were used in neutralization assays. Although the cross-neutralizing antibody levels in the antisera after the third immunization of E3R4 or E12R4 were very low , we did observed broad cross-neutralizing antibody responses induced by E3R4 after the fourth immunization (P<0.05). E12R4 only induced weak anti-HPV16 neutralizing antibody response  and the neutralizing antibody titers against heterologous PsVs in E12R4 antisera were all below 50. Although E12R4 elicited significantly higher cross-reactive antibody responses than E3 measured by HPV capsid-based ELISA assay (P>0.05), suggesting that the majority of serum antibodies induced by E12R4 were not neutralizing antibodies. We didn't detect any neutralizing antibodies in R4 scaffold control antisera (not shown). Collectively, we show that Freunds adjuvanted E3R4 vaccine induces broad cross-neutralizing antibody responses in mice.HPV capsid-based ELISA assay detects both non-neutralizing and neutralizing antibodies, and the neutralizing antibodies are thought to be the primary immune mechanism of protection by HPV vaccination. Thus, we further examined the cross-neutralizing antibody responses induced by L2R4 proteins using PsV-based nization . The meaSA assay , there win vivo protection against multiple HPV types using the vaginal challenge assay. Mice were immunized with E3R4 or R4 formulated with Freunds adjuvant for four times with 2-week intervals. Eleven months after the first immunization, mice were vaginally challenged with divergent PsVs  encoding luciferase. Consistent to relatively high-levels of cross-neutralizing antibodies in antisera  which were shown to induce enduring protective antibody against HPV58 for 12 month after vaccination Neutralizing antibodies binding to linear epitopes in HPV16 L2 aa 17–36, 65–81 and 108–120 have been described In the current study, we used R4, a modified IgG1 Fc, as a scaffold to display L2 epitopes and observed strong effects of R4 scaffold on enhancing the immune responses induced by L2 epitopes. Fc of IgG is considered as an important fusion tag for co-expressing several viral proteins to promote correct folding of the fusion proteins, facilitate purification, enhance the binding to APCs expressing FcγR and improve the immunogenicity of fused proteins Previous studies have shown that the immunogenicity of HPV16 L2 aa17–36 (RG-1 epitope) is relatively low It is worthy to note that unlike Freunds adjuvanted E3R4 immunization in mice, Alum-MPL adjuvanted E3R4 immunization in rabbits only induced low-titers of neutralizing antibodies against HPV16, 18 and 6. Adjuvant Taken together, our data demonstrate that a modified human IgG1 Fc can be used as a scaffold to display L2 antigen to induce cross-neutralizing antibodies and protective immunity against divergent human papillomavirus types. This type of fusion protein can be expressed with high yield and easily purified with high purity. Therefore, delivery of L2 antigen by a modified Fc scaffold opens a new avenue for pan-HPV vaccine development.


Organ/space (O/S) infection in colorectal surgery remains a major health problem. In Catalonia, the VINCat Program has monitored 24,832 procedures during 2007-2014, showing a steady rate of O/S infection over the years, 8.2% (95% CI 7.9 - 8.6). Improving awareness of stakeholders could be an easy strategy for assembling quality programs within health systems.Evaluating excess costs of organ/space infections associated with elective colorectal surgery in the Catalan Health System.We selected a sample of 10 different sized hospitals that provided data to VINCat from January 2012-June 2014. To estimate the excess of cost we based on differences between lengths of stay in patients with and without O/S infection and extra-cost related to readmission/need of ICU and re-operation.A total of 2276 patients underwent elective colorectal surgery. O/S infection occurred in 193 (8.5%). Patients with O/S were more frequently men ; underwent a rectal procedure  and had NNIS index ≥ 1 . Median length of stay was 3 fold higher  when O/S occurred which accounted for an extra cost of €3,052 per patient. Within the group of O/S infection, 45/193(23%) patients were re-admitted with a median length of stay of 13 days [IQR 8 - 17)]; 117/193 (60%) required re-operation and 56/193 (29%) required intensive care unit stay with a median length of stay of 5 days [IQR 3 - 12]. This added an additional cost of €2,235 per patient. Accordingly, 193 O/S infections accounted for an overall excess cost of €1,020,391.O/S infection represents an important excess of cost for the Catalan Health System. The reinforcement of quality strategies can lead to a strong reduction in the use of resources and relieve its current financial constraints.None declared.


Nonetheless, no anti-tumor protection was observed in mice deficient in CD8+ T cells. Also, the vaccine promoted high activation of E6/E7-specific T cells and in a therapeutic-approach, E6E7 protein conferred full anti-tumor protection in mice. These results show a potential use of this E6E7 multi-epitope antigen as a new and promising antigen for the development of a therapeutic vaccine against tumors induced by HPV.Cervical cancer is a common type of cancer among women worldwide and infection with high-risk human papillomavirus (HPVs) types represents the major risk factor for the etiopathogenesis of the disease. HPV-16 is the most frequently identified HPV type in cervical lesions and expression of E6 and E7 oncoproteins is required for the uncontrolled cellular proliferation. In the present study we report the design and experimental testing of a recombinant multi-epitope protein containing immunogenic epitopes of HPV-16 E6 and E7. Tumor preventive assays, based on the engraftment of TC-1 cells in mice, showed that the E6E7 multi-epitope protein induced a full preventive anti-tumor protection in wild-type mice, as well as in mice deficient in expression of CD4 Cervical cancer is the fourth most common type of cancer among women worldwide . Persist+ helper T cells and CD8+ cytotoxic T lymphocytes (CTLs)  of HPV16 were synthesized at the Center for Applied Toxinology–CAT, Butantan Institute as described by Carpino and Han [E6 and E7 synthetic peptides representing H2-DBamHI/PvuII and cloned into pAE vector [EcoRI enzyme designed to flank the ubiquitin cDNA. The construction was designed to express the recombinant protein with a 6 x His tag at the N-terminus. The nucleotides sequence was confirmed by DNA sequencing using Big Dye Terminator Reaction Kit version 3.1 and ABI PRISM 3100 Genetic Analyzer (Applied Biosystems).For construction of a vector expressing the E6E7 multi-epitope protein, the synthetic coding sequence of E6E7Ub protein was excised from pUC57 (GenScript Inc.) using E. coli strain. Isolated colonies grown on LB agar were transferred into 0.5 L of LB with ampicillin and incubated in a shaker at 37°C until the optical density at 600 nm (OD600) reached 0.6. Protein expression was induced by adding 1 mM isopropyl-β-thiogalactopyranoside and the cultures were incubated at 37°C for 4 h. Cells were harvested by centrifugation and lysed using a French press. Bacterial inclusion bodies were washed in washing buffer  and solubilized with binding buffer .The E6E7 recombinant protein was expressed in the BL21(DE3) Star pLysS (Novagen) ™ Fast Flow . The columns with adsorbed protein were washed three times with binding buffer containing 60 mM, 80 mM and 100 mM imidazole. The E6E7 protein was eluted with elution buffer . Stepwise dialysis was performed until urea reached a concentration of 2 M. The protein was quantified by spectrophotometry  according to its molar extinction coefficient.The recombinant protein was purified on Chelating-SepharoseThe recombinant protein was characterized by SDS-PAGE and mass spectrometry .4 TC-1 cells into the right flank. Tumor growth was monitored daily. The mice were euthanized by CO2 inhalation once the tumors reached 1.5–2.0 cm in size. TLR4 (5–6 mice/group), CD4+ T-cells (8 mice/group) or CD8+ T-cells knockout (5 mice/group) animals were immunized with E6E7 or PBS/2 M urea, following the above protocol. A group of five wild-type C57BL/6 mice immunized with PBS/2 M urea was used as a control of tumor growth in all these cases.Groups of five C57BL/6 mice were immunized subcutaneously (s.c) in the right flank with three doses of E6E7 (18 μg/dose) or 100 μl PBS/2 M urea at 14 days intervals. After two weeks from the last dose, mice were challenged (s.c) with 7.5 x 104 TC-1 cells. After three days, the immunization protocol was initiated, being injected (s.c) three doses of E6E7 (18 μg/dose) or 100 μL PBS/2 M urea at 7 days intervals. Tumor growth was monitored daily using a caliper, measuring two opposing diameters. The results are presented in measurement as mm2 (length x width). Following the above protocol of therapeutic immunization, TLR4 knockout animals were immunized with E6E7 (4 mice) or PBS/2 M urea (4 mice). In parallel, a group of five wild-type C57BL/6 mice immunized with PBS/2 M urea was used as a control of tumor growth.Groups of five C57BL/6 animals were first injected (s.c) into the right flank with 7.5 x 10Immune response generated by E6E7 vaccines was evaluated using enzyme-linked immunosorbent spot (ELISPOT) assays to detect cells secreting IFN-γ.4Cl, 10 mM KHCO3 and 0.1 mM EDTA, pH 7.2), centrifuged and suspended in serum free RPMI media (Gibco). 105 cells were plated in a 96-well plate (Corning Life Science) previously coated with anti-mouse IFN-γ (BD Bioscience) and incubated for 48 hours at 37°C in 200 μl of RPMI supplemented with 2% fetal bovine serum, 10 ng/mL IL-2 and 10−6 M β-mercaptoethanol. E6-specific  and/or E7-specific  peptides were then used as stimuli at a concentration of 2μg/mL for 48 hours. After incubation, the spots were revealed according to the manufacturer instructions (mouse IFN-γ ELISPOT kits—BD Bioscience). Plates were dried and the number of spots counted.The ELISPOT assay was performed using peripheral blood mononuclear cells (PBMCs) from animals immunized according to prophylactic protocol and challenged with TC-1 cells 20 days before the bleeding. Blood was treated with the ACK lysing buffer  or Mantel-Cox curves using log-rank tests (Percentage of tumor-free mice). P < 0.05 was considered significant. All analysis and graphics were done using GraphPad Prism version 5.+ responses, known to play an important role in tumor clearance. Also, we added to the E6E7 antigen sequence epitopes that are presented to CD4+ T-cells by MHC class II known to induce long lasting cytotoxic responses [The E6E7 antigen was designed to contain HPV E6 and E7 immunogenic epitopes based on published data reporting antigen specific cellular immune responses detected in patients and/or C57BL/6 mice. MHC class I-restricted epitopes were included as an attempt to induce T CD8esponses  Fig 1A)+ respons29–38, E649-67, E6127-141 [7-20, E743-77 and E779-93 [Considering the high polymorphism of HLA genes in the human population, we elected E6 and E7 regions that presented the higher number of established epitopes covering several human HLA types . Thereby6127-141 ,16–21, E E779-93 ,15,19,22 E779-93 .6 tag was expressed as inclusion bodies in E.coli and purified under denaturing protocol. E6E7 protein showed apparent molecular masses of 17.4 kDa in reducing gel , an agonist of TLR4, are expected to be present in the vaccine preparations. To evaluate the possible impact of LPS contamination on the induced anti-tumor immune responses, we performed prophylactic immunization experiments with TLR4 knockout mice (C57BL10/ScCr). Vaccination with three doses of E6E7 multi-epitope antigen conferred 100% of antitumor protection in TLR4 knockout mice. All mice immunized with PBS/2 M urea developed tumor within 10 days after challenge  mice, we investigated the contribution of CD4+ and CD8+ T-cells to mediate an effective antitumor response in vaccinated animals. Similar to results observed with the wild-type mice  and/or E7 (RAHYNIVTF49-57) specific peptides. We observed high IFN-γ secretion in PBMC cells from mice immunized with the E6E7 vaccine stimulated with E6 peptide or with E7 peptide  containing both CLT and Th cells epitopes of E6 and/or E7 resulted in the induction of robust E7-specific CD8al model ,26. In cal model . TA-CIN al model . Howeveral model –31.The peptide based vaccines have emerged as promising approaches for the development of therapeutic HPV vaccine as they are considered to be safe, easy to produce and specific . NonetheOur approach to overcome the limited immunogenicity of the HPV antigens consisted of a recombinant protein containing overlapping MHC I and II epitopes of E6 and E7. In this strategy, epitopes are linked together by a short amino acid motif (AAY) which are a proper substrate for proteasome mediated cleavage or binding to the transporter associated with antigen processing (TAP) . After t49-57) along to the HLA- restricted epitopes of E6 and E7 seems to have collaborated substantially for the efficient protection of the E6E7 vaccine against the TC 1 cells in mice. Indeed, this E7 immunodominant epitope (RAHYNIVTF) has been previously used in several therapeutic vaccines that were tested in C57BL/6 mice, resulting in excellent immune protection against tumor which expresses HPV-16 E7 protein [In fact, the multi-epitope E6E7 protein has proven to be highly immunogenic in mice without the enhancement of any adjuvant, as three doses of vaccination was sufficient to promote full prophylactic and therapeutic anti-tumor protection in mice. The inclusion of epitope restricted to the H-2Db allele , being a indicative of the significance of the present selected E6 and E7 epitopes as a whole to induce the activation of tumor-specific T cells.Although the immunodominant epitopes of E7 are likely to be critical for the immune response conferred by the E6E7 vaccine in mice, the selected epitopes of E6 have also contributed to the vaccine efficacy, as we can observe high levels of IFN-γ production by PBMCs, derived from E6E7 immunized animals, stimulated with E6+ T-cells knockout mice did not reduce the anti-tumor effects. Despite CD4+ T-cells of being crucial for the maintenance of the immune response through secretion of cytokines, such as IL-2 and IFN-γ, and for playing an important role in anti-tumor effect against genital warts and cervical cancer [+ T-cells than CD4+ T-cells response [+ T-cell responses in the absence of CD4+ T-cells [+ T-cells, but not CD4+ T-cells.As observed in the prophylactic assay, the vaccination of CD4l cancer , studiesresponse . Also, a T-cells . Similarin vitro and resulted in a similar antitumor effect when compared to E6E7 (data not shown).Despite these encouraging results in mice, the multi-epitope E6E7 vaccine will probably require the reinforcement of adjuvants to be effective in clinical trials. However, due to the high immunogenicity of the E6E7 antigen, the effectiveness of an adjuvant or an immune stimulating fusion molecule could not be evident in this current study. Actually, as an attempt to enhance the Class I MHC antigen presentation, we have produced a fusion protein composed of E6E7 and ubiquitin. In preliminary studies, this strategy seemed to increase the activation of E6/E7-specific CD8+ T-cells In an effort to circumvent the poor immunogenicity of peptide or protein-based vaccines, much of the research in this area has explored strategies employing adjuvants or fusion protein . The comIn this current study, we demonstrated that immunization of mice with the E6E7 protein containing CTL and T helper epitopes of E6 and E7 can promote high activation of specific T-cells and can efficiently inhibit the tumor growth in a prophylactic and therapeutic context. Hereby, the immunogenicity of this E6E7 antigen can be intensely explored to reach the idealized vaccine against cervical tumor cells.S1 FigThe aminoacid sequences of the E6E7 protein. E6 (blue) and E7 (green) peptides are distinguished by color. Peptides identified by mass spectrometry are underlined.(TIF)Click here for additional data file.S1 Material and Methods(DOCX)Click here for additional data file.S1 Table(TIF)Click here for additional data file.


Schizosaccharomyces pombe deletion mutant library and found that deletion of the dbl2 gene led to missegregation of chromosomes during meiosis. Analyses of both live and fixed cells showed that dbl2Δ mutant cells frequently failed to segregate homologous chromosomes to opposite poles during meiosis I. Removing Rec12 (Spo11 homolog) to eliminate meiotic DNA double-strand breaks (DSBs) suppressed the segregation defect in dbl2Δ cells, indicating that Dbl2 acts after the initiation of meiotic recombination. Analyses of DSBs and Holliday junctions revealed no significant defect in their formation or processing in dbl2Δ mutant cells, although some Rec12-dependent DNA joint molecules persisted late in meiosis. Failure to segregate chromosomes in the absence of Dbl2 correlated with persistent Rad51 foci, and deletion of rad51 or genes encoding Rad51 mediators also suppressed the segregation defect of dbl2Δ. Formation of foci of Fbh1, an F-box helicase that efficiently dismantles Rad51-DNA filaments, was impaired in dbl2Δ cells. Our results suggest that Dbl2 is a novel regulator of Fbh1 and thereby Rad51-dependent DSB repair required for proper meiotic chromosome segregation and viable sex cell formation. The wide conservation of these proteins suggests that our results apply to many species.To identify new proteins required for faithful meiotic chromosome segregation, we screened a  dbl2 gene led to missegregation of chromosomes during meiosis. Analysis of live dbl2Δ cells by fluorescence microscopy showed that chromosomes frequently failed to segregate during the first meiotic division. Further cytological and biochemical analyses revealed that this segregation defect is due to persistent intermediates of DNA double-strand break repair, also called DNA joint molecules. Our results indicate that Dbl2 is required for formation of Fbh1 DNA helicase foci at the sites of DNA double-strand break repair in order to process DNA joint molecules and allow segregation of chromosomes during meiotic divisions. Our bioinformatics searches revealed that Dbl2 is highly conserved in fungi, animals and plants, suggesting that Dbl2 plays a similar role in other organisms–the formation of viable sex cells and healthy progeny.Meiosis produces haploid gametes from diploid precursor cells. This reduction of chromosome number is achieved by two successive divisions after only a single round of DNA replication. To identify novel regulators of meiosis, we screened a library of fission yeast deletion mutants and found that deletion of the  During meiosis, haploid gametes are produced from diploid precursor cells. The reduction of chromosome number is achieved by a single round of DNA replication followed by two rounds of chromosome segregation, termed meiosis I and meiosis II. While meiosis II is similar to mitosis in that sister centromeres segregate from each other, centromeres of homologous chromosomes (homologs) segregate to opposite poles in meiosis I ,2.Three meiosis-specific features are essential for proper segregation of chromosomes during meiosis I–formation of crossovers that connect homologs, mono-orientation of sister kinetochores, and a stepwise loss of sister chromatid cohesion. The formation of crossovers, as a result of meiotic recombination, and the attachment of sister kinetochores to microtubules emanating from the same spindle pole (mono-orientation) ensure that homologous centromeres are pulled in opposite directions on meiosis I spindles ,3.Drosophila) complexed with the protein phosphatase 2A (PP2A) . Th. Thdbl2 ions see . These psee mbs1  and ade6de6-3049 .dbl2Δ mutant cells. Consistent with the idea that Dbl2 has a function independent of the Mus81-Eme1 Holliday junction resolvase . spindle . These dS. pombe cells, Rad51 foci are present during meiotic prophase, when DSBs are formed and repaired, but are no longer present in metaphase I cells [dbl2Δ mutant cells Rad51 foci formed during meiotic prophase and, as in fbh1Δ mutant cells, remained visible during meiosis I and meiosis II and in mononucleate cells containing more than one spindle , the meiosis-specific Rad51 paralog Dmc1, or Rdh54 -tagged Fbh1 was expressed from a strong nmt promoter [dbl2+ cells but were strongly reduced in dbl2Δ mutant cells (dbl2Δ mutant cells because the stability of the Fbh1-YFP plasmid was similar in both dbl2+ cells (5.8% plasmid loss per generation) and dbl2Δ cells (6.7% plasmid loss per generation). We obtained similar results when we induced DNA lesions in vegetative cells using methyl methanesulfonate (MMS) [rad51Δ, rad52Δ, rad55Δ, rad57Δ, sfr1Δ and rad54Δ mutant strains, but the frequency of Fbh1-YFP foci was decreased by the dbl2Δ mutation in each of these strains, both in the absence and presence of CPT  ,54 , of the dbl2Δ chromosome segregation failure   , which iobserved . In conts81-Eme1 , is suppdbl2Δ and fbh1Δ are remarkably similar. In both mutants during meiosis, DSB formation and repair as well as recombination are close to wild-type levels, chromosomes frequently fail to segregate during meiosis I, Rad51 foci persist beyond meiotic prophase, and chromosome missegregation is suppressed by rad51Δ, rad52Δ or rad57Δ  with rad55Δ and rad57Δ [The phenotypes of 57Δ Figs ; Table 2tothecin  and showd rad57Δ .S. cerevisiae, the Mre11-Rad50-Nbs1 (MRN) complex is required for meiotic DSB formation even though its catalytic activity is apparently needed only after DSB formation, i.e., for the processing of DNA ends to allow Rad51-ssDNA filament formation [dbl2Δ mutants , Homo sapiens (ZGRF1) and Arabidopsis thaliana (AT4G10890)  and RAD54B (H. sapiens)  at the N-terminus. Our bioinformatic searches revealed that within the DUF2439 region Dbl2 is highly conserved in fungi, animals and plants. Orthologs can be detected in 4G10890) . The onlsapiens) . Intereswork and ). Similaactivity –74. Thusactivity ,76.rdh54 confers sensitivity to MMS but N-terminal truncations of Rdh54 that remove or truncate the DUF2439 domain do not affect MMS sensitivity [What is the role of the DUF2439 domain? Surprisingly, the N-terminal truncation that removes the DUF2439 domain had no effect on either Dbl2 focus formation at DSBs or on its ability to confer camptothecin-resistance . Similarsitivity . Interessitivity –79. The sitivity . SimilarS. cerevisiae. However, it has been proposed that S. cerevisiae Srs2 helicase is a functional counterpart of Fbh1 [Both Dbl2 and Fbh1 are evolutionarily conserved proteins present from yeast to humans, suggesting that our results may apply widely. A notable exception is the absence of an Fbh1 homolog in the budding yeast  of Fbh1 . The DblS. pombe strains [4Cl plates, and incubated at 25°C for 10–17 hr before examination [S. pombe was transformed using the lithium acetate method and genes deleted as described [S. pombe cells were performed as described [rad51Δ mutant cells, indicating that in our assays anti-Rhp51 antibody specifically detected Rad51  and fbh1∆ dbl2∆ (JG17777) cells harboring plasmid pMW651  were grown in selective EMM medium lacking leucine at 32°C to mid-log phase. The cells were washed with water and resuspended in nonselective YES liquid medium, incubated at 32°C for 18 hr (7 generations) and then plated on YES plates. Colonies were formed after 3 days at 32°C and replica-plated onto selective plates (EMM lacking leucine). The percentage of plasmid loss per generation was determined as described by Osman et al. [n et al. .SfiI and 3' SmaI or BamHI (Fbh1 only) restriction sites and then cloned into plasmid pGBKT7 (Clontech) for GAL4-DNA binding-domain bait constructs, or into plasmid pGADT7 (Clontech) for GAL4 activation domain prey constructs. Primers used were 5'-AAAAGGCCATGGAGGCCATGGATACAAGTTCCAATGTTTTTCATTATC-3' and 5'-AAAACCCGGGTCAAATAAAGTCACCATCTTCGTCCGAATC-3' for Dbl2, 5'-AAAAGGCCATGGAGGCCATGAGTGCTCAACATTTACATAGCTGCAAAT-3' and 5’-AAAAGGATCCCTACTGATCATGTACAGCAAACAATTGATTTTCAATAAATAGCATCGATCTTTTAAGCCG-3' for Fbh1, 5'-AAAAGGCCATGGAGGCCATGTCCGATGATTCTTTTAGTAGTGATGAAG-3' and 5'-AAAACCCGGGCTAAATCAGCATTCCTTTCATACGTTTCCTTTTC-3' for Fml1, and 5'-AAAAGGCCATGGAGGCCATGGCAGATACAGAGGTGGAAATGCAAGTTAG-3' and 5'-AAAACCCGGGTTAGACAGGTGCGATAATTTCCTTGGGATCACCAACACC-3' for Rad51. Bait and prey plasmids were introduced into S. cerevisiae strain PJ69-4a (Clontech) by standard lithium acetate-mediated transformation. Transformants were tested for bait-prey interaction by spotting onto SD minimal media lacking appropriate amino acids according to the manufacturer's instructions. We performed at least two independent transformations and growth tests.Full-length coding regions for Dbl2, Fbh1, Fml1 and Rad51 were amplified from meiotic cDNA with primers that added a 5' S. pombe Dbl2 as starting query and applying significant e-value thresholds (<0.001), we identified an N-terminally located region (Dbl2 amino acids 7 to 77) conserved in fungi, animals and plants, that is also described as the domain of unknown function DUF2439 in the Pfam database [S. cerevisiae YGR042W (= Mte1) and human ZGRF1, and reciprocal NCBI-Blast searches in the S. pombe proteome with the respective orthologous regions significantly yielded only Dbl2. The animal ZGRF1 protein family is extended, relative to yeast proteins, by one GRF zinc finger region and two AAA+ ATPase domains at the C-terminal half.In a series of NCBI-BLAST searches  within tdatabase . OrtholoS. pombe Dbl2 (residues 3–85) found Cryptococcus gattii Rad54b in iteration 3 , fission yeast Rdh54 in iteration 4 , and human RAD54b in iteration 6 . The relationship was confirmed in a reciprocal search with S. pombe Rdh54 (residues 49–128), which identified Dbl2 in iteration 6  and human ZGRF1 in iteration 4 . In an independent approach, using hmmsearch with the Pfam DUF2439 hidden Markov model [S. pombe Rdh54  and Dictyostelium fasciculatum RAD54b . Whereas Rad54 and RAD54b protein families share the same overall domain architecture, including their DEAD-like helicase domain, the N-terminal region is specific to each protein. In human RAD54b, the N-terminal region has been reported to bind to Rad51 and Dmc1 and branched DNA structures [To find distantly related DUF2439-containing proteins, we performed iterative NCBI-PSIBLAST searches. For 16 out of 17 queries, the NCBI-PSIBLAST search converged before it reached the specified threshold of 20 iterations. Other than the Dbl2 and ZGRF1 proteins, all significant hits were to an unidentified conserved domain in the Rdh54/RAD54b family. For example, ov model , Rdh54 aructures .S1 Figsod2 (telomeric) and the cen2 (centromeric) loci marked by LacI-GFP was scored in 100 h90dbl2Δ  anaphase I cells showing lagging chromatin and in 100 h90 wild-type anaphase I cells . The strains were fixed and immunostained for tubulin and GFP; DNA was visualized by Hoechst staining.The localization of the (TIF)Click here for additional data file.S2 Figdbl2Δ (JG17236) cells as well as in a mononucleate dbl2Δ zygote with two spindles.Strains were sporulated on SPA and at 10–17 hr fixed and immunostained for tubulin and GFP; DNA was visualized by Hoechst staining. Representative images show the Rec8-GFP signal during anaphase I in wild type (JG13990) and (TIF)Click here for additional data file.S3 Figdbl2Δ cells (JG17146) were fixed and analyzed by DIC microscopy.Sporulating wild-type (JG11355) and (TIF)Click here for additional data file.S4 Fig(A) As shown in dbl2+) and GP8664 (dbl2Δ). The results are from two independent experiments from which extracted DNA was digested with NotI and analyzed by pulsed-field gel electrophoresis and Southern blot hybridization using a probe at the left end of the 501 kb NotI fragment J (band at the top of the gel) [mbs1 and at mbs2 (indicated by arrows on the right) at the indicated time for each strain is shown below each blot. (B) Strains GP6656 (dbl2+), GP8664 (dbl2Δ), and GP8836 (rec12Δ dbl2Δ) were induced and their DNA analyzed as in ade6-3049 DSB hotspot on the 11.8 kb BsrGI fragment or near the mbs1 hotspot on the 10.5 kb BsrGI fragment [ade6-3049 hotspot persist in dbl2Δ but not in dbl2+ and are Rec12-dependent. (C) Analysis of DNA at both hotspots from independent inductions. (D) Quantification of data for ade6-3049 from blots in S4 Fig, panels A and B, and additional experiments. See the gel) . The fra(TIF)Click here for additional data file.S5 Fig(A)dbl2Δ (JG17148) and eme1Δ (JG17465) strains were crossed, and asci were subjected to tetrad analysis. From one such representative tetrad, growth of the four spore colonies with the indicated genotypes is shown. (B) 10-fold dilutions of wild-type strain (JG17894), dbl2Δ mutant strain (JG17895), eme1Δ mutant strain (JG17896) and dbl2Δ eme1Δ double mutant strain (JG17897) were spotted on YES plates and incubated at 32°C for 3 days. Three independent cultures of slow-growing strain JG17897 (dbl2Δ eme1Δ) were tested.(TIF)Click here for additional data file.S6 Fig(A and B)S. pombe strains expressing YFP-Fbh1 from plasmid pMW651 and carrying fbh1Δ (JG17775) or fbh1Δ dbl2Δ (JG17777) mutations growing in EMM2 medium without leucine at 32°C were treated with MMS (0.025%) for 4 hr and fixed; DNA was visualized with DAPI. The dbl2Δ mutant showed significantly fewer number of YFP-Fbh1 foci in G2 cells compared to those in dbl2+. The values reported are means of three independent experiments ± SEM. In each experiment 200 G2 cells were scored.(TIF)Click here for additional data file.S7 FigS. pombe wild-type strain (JG17460) and dbl2Δ mutant strain (JG17510) expressing Rad52-mCherry from the native promoter were grown to exponential phase in liquid YES medium, treated with either 5 μM CPT (A) or 0.025% MMS (B) for 4 hr, fixed, and examined by fluorescence microscopy; DNA was visualized with DAPI. Data are the means of three independent experiments ± SEM. Rad52-mCherry foci were scored in three sets of 200 G2 cells.(TIF)Click here for additional data file.S8 FigMultiple alignment of the indicated proteins from various species was performed with MAFFT   and visu(TIF)Click here for additional data file.S9 FigStrains expressing Dbl2 fused to the GAL4 transcription activation domain and Rad51, Fbh1 or Fml1 fused to the GAL4 DNA-binding domain were grown on SD plates lacking tryptophan and leucine  and then spotted at 5-fold serial dilutions on SD plates lacking tryptophan and leucine  or SD plates lacking tryptophan, leucine and histidine  or SD plates lacking tryptophan, leucine and adenine . The empty vectors pGADT7 and pGBKT7 containing GAL4 transcription activation domain and GAL4 DNA-binding domain, respectively were used as negative controls. Growth on plates without histidine or without adenine indicates interaction between the fusion proteins [(TIF)Click here for additional data file.S10 Figrad51Δ  prophase I cells. Cells were mated on SPA sporulation agar and at 10–17 hr fixed and immunostained for tubulin and Rad51, and examined by fluorescence microscopy; DNA was visualized by Hoechst staining.To test the specificity of anti-Rhp51 antibody, we analyzed subcellular localization of Rad51 using anti-Rhp51 polyclonal antibody (Cosmo Bio) diluted 1:500 in wild-type (JG11355) and (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.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S1 References(DOCX)Click here for additional data file.