TM2D3 was enriched in Icelanders (~0.5% versus <0.05% in other European populations). In 433 LOAD cases and 3903 controls from the Icelandic AGES sub-study, P155L was associated with increased risk and earlier onset of LOAD . Mutation in the Drosophila TM2D3 homolog, almondex, causes a phenotype similar to loss of Notch/Presenilin signaling. Human TM2D3 is capable of rescuing these phenotypes, but this activity is abolished by P155L, establishing it as a functionally damaging allele. Our results establish a rare TM2D3 variant in association with LOAD susceptibility, and together with prior work suggests possible links to the β-amyloid cascade.We performed an exome-wide association analysis in 1393 late-onset Alzheimer’s disease (LOAD) cases and 8141 controls from the CHARGE consortium. We found that a rare variant (P155L) in  TM2D3 gene. This variant was discovered to be more common in the Icelandic population, where it was significantly associated with both increased risk and earlier age of onset of AD. Lastly, in order to examine the potential functional impact of the implicated variant, we performed additional studies in the fruit fly. Our results suggest that P155L causes a loss-of-function in TM2D3, in the context of Notch-Presenilin signal transduction. In sum, we identify a novel, rare TM2D3 variant in association with AD risk and highlight functional connections with AD-relevant biology.Alzheimer’s disease (AD) is the most common cause of dementia in the older adult population. There is substantial evidence for an important genetic contribution to AD risk. While prior work has comprehensively evaluated the contribution of common genetic variants in large population-based cohorts, the role of rare variants remains to be defined. Here, we have used a newer genotyping array to characterize less common variants, including those likely to impact the function of encoded proteins, in a combined cohort of 1393 AD cases and 8141 control subjects without AD. Our results implicate a novel, amino acid-changing variant, P155L, in the  APP, [MIM: 104760]) and the presenilin-1 and -2 (PSEN1 [MIM: 104311] and PSEN2 [MIM: 600759]) genes cause familial early-onset (<65 years) AD  [PLD3 [MIM: 615698] [UNC5C [MIM: 603610] [AKAP9 [MIM:604001]  ,18, PLD3 615698] , UNC5C [ 603610] , and AKA:604001] . These f in LOAD ,23. Impoulations . The twomericans , and PLDulations .To date, the identification of rare susceptibility variants in LOAD has been hampered by poor representation on genotyping arrays used for large GWAS ; moreoveTable 1), using logistic regression-based scores  but not TM2D3[P155L] can suppress the neurogenic phenotype in the developing fly embryonic peripheral nervous system (PNS). (A-B) Maternal effect neurogenic defects in amx mutants can also be seen in the PNS. Compared to control embryos (A), maternal amx mutant embryos (B) show increased number of Sensory Organ Precursor cells (SOPs), labeled by Senseless , due to defective lateral inhibition. Embryos are counterstained with DAPI (blue). (C-F) PNS phenotypes in embryos laid by amx mutant females with or without amx[+], TM2D3[+], and TM2D3[P155L] genomic rescue constructs. Thoracic segments  of stage 11 embryos are shown. TM2D3[+] can rescue the lateral inhibition defects in SOPs in some  but not all embryos (E’). In contrast, all embryos from amx mutant females with TM2D3[P155L] exhibit neurogenic PNS phenotypes . Scale bars = 100μm for A-B, = 50μm for C-F.(TIFF)Click here for additional data file.S1 TableA) and SKAT (B) discovery analysis. Complete results areavailable in dbGAP (accession phs000930). A: Direction = AGES-CHS-FHS-RS, Alleles = non-coding/coding, AF displayed as percentage (%). B: cumAF = cumulative allele frequency displayed as percentage (%), N_SNPs = number of SNPs in test.Top results of exome-wide single variant ((PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Phosphorylation of the C-terminal tail of the heavy neurofilament subunit (NF-H) impacts neurofilament (NF) axonal transport and residence within axons by fostering NF-NF associations that compete with transport. We tested the role of phosphorylation of a GSK-3β consensus site (S493) located in the proximal portion of the NF-H tail in NF dynamics by transfection of NB2a/d1 cells with NF-H, where S493 was mutated to aspartic acid (S493D) or to alanine (S493A) to mimic constitutive phosphorylation and non-phosphorylation. S493D underwent increased transport into axonal neurites, while S493A displayed increased perikaryal NF aggregates that were decorated by anti-kinesin. Increased levels of S493A co-precipitated with anti-kinesin indicating that reduced transport of S493A was not due to reduced kinesin association but due to premature NF-NF interactions within perikarya. S493D displayed increased phospho-immunoreactivity within axonal neurites at downstream C-terminal sites attributable to mitogen-activated protein kinase and cyclin-dependent kinase 5. However, S493D was more prone to proteolysis following kinase inhibition, suggesting that S493 phosphorylation is an early event that alters sidearm configuration in a manner that promotes appropriate NF distribution. We propose a novel model for sidearm configuration. Summary: We demonstrate that phosphorylation of a critical site regulates neurofilament transport, proteolysis and interaction with other axonal cytoskeletal elements, and present evidence that it does so by altering protein conformation. This article has an associated First Person interview with the first author of the paper as part of the supplementary information. The cytoskeleton provides structural support to axons allowing for axonal outgrowth and for maintenance of synaptic connections formed by outgrowing axons . MammaliThe C-terminal tail domains of NF-H and NF-M undergo extensive post-translational modification by phosphorylation . PhosphoA GSK3β consensus sequence is located at the beginning of the NF-H tail domain; however, the consequences of phosphorylation of this site remain unclear , 2009. TFollowing knockdown of endogenous NF-H and expression of NF constructs, a significant increase in S493D was observed within axonal neurites versus that of wtH or S493A, while reduced levels of S493A were observed within axonal neurites verus wtH A. Cells To examine the potential effect of S493 phosphorylation on downstream NF-H tail domain phosphorylation, immunoblots were probed with monoclonal antibody RT97, which reacts with a conformationally dependent phospho-epitope located within the NF-H tail downstream of S493 . CytoskeAssociation of NF-H, S493A, and S493D with kinesin , we manipulated the activity of a number of known NF kinases  in cells expressing wtH, S493A and S493D A. UpreguSince NF steady-state levels are regulated by proteolysis, and proteolysis is suppressed by MAPk and cdk5 phosphorylation events  and 5. GWe next examined the consequence of inhibition of the above NF kinases by treatment of cells with pharmacological agents active against each kinase  on totalLevels of RT97-immunoreactivity were also examined . InhibitWe next examined the impact of kinase inhibition on subunits already incorporated into the Triton-insoluble cytoskeleton . GFP-S49Inhibition of CK1α selectively depleted Triton-soluble and -insoluble levels of S493D, but not those of wtH or S493A  and 5. FOur findings suggest that phosphorylation of S493 induced one or more C-terminal conformational changes since (1) S493D underwent significantly larger increases than did S493A or wt-NF-H following co-expression of NF kinases, and (2) S493D underwent significantly more calpain-mediated proteolysis than S493A or wt-H. Breakdown products for all three constructs were observed at 130-140 kDa and at 100 kDa. S493D displayed an additional fragment migrating at 110-120 kDa. However, only the largest (130-140 kDa) species retained the RT97 epitope. GFP-tagged NF-H in which the terminal 187 amino acids have been deleted migrates at approximately 150 kDa and is reactive with RT97, while the GFP-tagged NF-H rod lacking the entire sidearm migrates at 115-120 kDa and does not react with RT97 . RetentiPhosphorylation of S493 likely represents an early event in overall NF tail phosphorylation, since (1) S493 is phosphorylated prior to the appearance of immunoreactivity towards monoclonal antibody SMI-31  alters the configuration of the NF-H tail to allow extensive phosphorylation. Proline residues can exist as ormation . PIN1 dis isomer . Notablyresidues . However kinases , 2009. I kinases A. While  kinases . Phospho kinases A. Phosphcis-cis conformations, fully ‘open/extended’ trans-trans conformations, and ‘medial’ cis-trans and trans-cis conformations. All of these, like the conformation of individual prolines, are strongly influenced by the adjacent amino acid residues  . We did not scrutinize the entire tail sequence of all mammals, but of the 92 mammals for which the NF-H sequence is available, 74% displayed a double-proline motif between the proximal E segment (containing GSK3b and/or CK1a consensus sequences) and before the initial KSP-consensus sequence, and all but three of these were immediately preceded by an S or T as part of a CK1a consensus sequence (Table S1). The model proposed herein is also consistent with the proposed behavior of the NF-H tail domain prior to and following phosphorylation as determined by atomic force microscopy and computer analyses  . The firanalyses , 2009, win situ (Mouse neuroblastoma cells (NB2a/d1) were utilized due to their ease of culture and ability to replicate rapidly while still modeling the dynamics of an axon. NB2a/d1 cells express and phosphorylate all three NF subunits , and exhin situ .Culture reagents were purchased from Sigma-Aldrich  unless otherwise indicated. Cells were propagated on either plastic tissue culture dishes or glass coverslips coated with laminin and poly-D-lysine and were maintained in high-glucose DMEM containing 10% fetal bovine serum  with antibiotics. Axonal neurite outgrowth was induced by addition of 1 mM dibutyryl cyclic AMP (dbcAMP) for 3 days . Cells wRattus norvegicus NF-H tagged on the N-terminus with GFP , each construct was co-transfected with a plasmid that expressed a shRNA targeting an untranslated region (UTR) at the 5′ end of the endogenous ous NF-H . Prior s+ which are active against mitogen-activated protein kinase kinase 1 (MKK1), cdk5, CK1, and GSK3β respectively  , murine 2 and 0.5 mM CaCl2] was added, lysates were incubated (30 min on ice), homogenized on ice with a loose-fitting Teflon pestle in a borosilicate glass homogenizer  and centrifuged . The resultant (Triton-soluble) supernatants were decanted and (Triton-insoluble) sedimented material was resuspended in the same buffer containing 8 M Urea. Samples were normalized according to protein concentration as determined by Pierce BCA Assay  and diluted in 187.5 mM Tris-HCl (pH 6.8) containing 6% (w/v) SDS, 30% glycerol, 0.03% (w/v) bromophenol blue, and 2% β-mercaptoethanol for SDS-PAGE.Cells were harvested after 3 days of differentiation for SDS-PAGE analysis using established methods with minor modifications . CultureTriton-soluble fractions were made 1% SDS and diluted 1:5 (vol/vol) in 60 mM Tris-HCl (pH 7.6) containing 190 mM NaCl, 6 mM EDTA, 1.25% Triton X-100, 1 mM PMSF, protease inhibitor cocktail and PhosStop . FractioSamples were subjected to SDS-PAGE with 4-15% polyacrylamide Tris-Glycine gels , and transferred for 1 h to nitrocellulose membranes . The resImmunoreactive species were detected with a 1:10,000 dilution of the appropriate alkaline phosphatase-conjugated secondary antibody (Sigma Aldrich) and BCIP/NBT substrate (BioRad). All samples intended for analytical comparison were either run on the same gel, or, in the case of large sample sets, on multiple gels that were electrophoresed, transferred to nitrocellulose, and visualized simultaneously, under identical conditions. Quantification of immunoreactive species was carried out using ImageJ (NIH) software of scanned images of processed nitrocellulose replicas. An average background signal of equivalent area was quantified in two adjacent regions within the same lane and subtracted from quantification of each immunoreactive species . GFP-tagP<0.05 were considered to be statistically significant; comparison with *P<0.1 but >0.05 were considered to represent a trend towards significance. For presentation, lines within a single treatment condition indicate which columns are being compared; in instances where no connecting lines are present, asterisk(s) indicate comparison of that condition versus the corresponding untreated control condition.Statistical analyses were performed using one-way ANOVA with Fisher's post hoc analyses (StatPlus Software). Comparisons with **Cells were cultured on laminin and poly-D-lysine-coated coverslips and co-transfected with shRNA directed against NF-H and constructs expressing wt-H, S493A, or S493D as described above . CultureFluorescent intensity was quantified within axonal neurites and soma. RT97 immunoreactivity within perikarya versus axonal neurites were compared as an index of NF phosphorylation level and relative distribution of phospho-H. The percentage of cells containing perikaryal GFP and phospho-H aggregates was also quantified. Cells containing non-diffuse tanged/aggregated immunoreactivity within ≥50% of their perikarya were scored as ‘possessing aggregates’ or ‘aggregated’. Kinesin immunoreactivity was compared as an index of distribution of this motor protein within perikarya and neurites by calculating a ratio of somal/neurite densitometric levels in cells displaying GFP (as confirmation of expression of respective constructs). In efforts to approximate the consequence of expression of NF construct on differential distribution of kinesin immunoreactivity, we also quantified this ratio for cells within cultures transfected with S493D but expressing no detectable GFP signal. These cells were considered to represent the distribution of kinesin in the absence of detectable expression of NF constructs, and in additional analyses this ratio was subtracted from the ratio of all values of transfected cells. Statistical analyses were performed using one-way ANOVA with Fisher's post hoc analysis (StatPlus Software).


Subsequently, different recombinant spidroins were spun into fibers through wet-spinning via a custom-made continuous post-drawing device. Mechanical tests of the synthetic fibers with four parameters  demonstrated that the extensibility of the fibers showed a positive correlation with spidroin size, consequently resulting in the extensibility of N-Sc4-C fiber ranked the highest (58.76%) among four fibers. Raman data revealed the relationship between secondary structure content and mechanical properties. The data here provide a deeper insight into the relationship between the function and structure of Flag silk for future design of artificial fibers.Spider silk is an attractive biopolymer with numerous potential applications due to its remarkable characteristics. Among the six categories of spider silks, flagelliform (Flag) spider silk possesses longer and more repetitive core domains than others, therefore performing the highest extensibility. To investigate the correlation between the recombinant spidroin size and the synthetic fiber properties, four recombinant proteins with different sizes [N-Scn-C ( Summary: A study of the relationship between the structure and property of synthetic spider silk-like fibers, which aims to aid with the designing of functional artificial fibers. Orb-weaving spiders can produce up to six kinds of silk and one sticky aggregate for different tasks . Spider n repeat motifs  that form β-turns, juxtaposed to (GGX)n motifs and spacer motifs that form helices and β-sheets. These motifs combine to form spring-like spirals that provide the excellent extensibility of Flag (Flagelliform spidroin (Flag) contains (GPGGX) of Flag . Flag ha of Flag . Therefon=1-4), were constructed, expressed and spun to the silk-like fiber by wet spinning. The spun fiber was subjected to continuous post-drawing by a custom-made device. This study shows that the mechanical properties (such as extensibility and toughness) are improved by the increase of functional repetitive domains. The best elongation is obtained from N-Sc4-C (58.76%), while the highest strength belongs to N-Sc3-C (107.54 MPa). The Raman data illustrates the mechanical test results from the structural respect. This study may indicate the correlation between the size of recombinant spidroin and the mechanical properties of the synthetic Flag fibers, which might be applied for various medical or industrial settings.Here, the aim of this study is to illustrate the correlation between the length of the functional repetitive domain and mechanical properties of the synthetic Flag fibers. Four recombinant Flag spidroin with different sizes, like N-Scn-C (Araneus ventricosus (A.v.) genomic library and aligned with the only accessible Flag repetitive domain sequence (A.v.Flag-CT) in the database (GenBank: EF025541) . The Sc amino acid sequence showed GPGGX and GGX  motifs were contained, which were also conserved within Flag spidroin from Nephila clavipes (N.c.)  in one repeat domain, our Flag silk gene contained less pentapeptide repetitive motifs , Nephila madagascariensis (N.m.), or Nephila clavipes (N.c.) (A Flag repetitive domain gene (Sc) encoding 231 amino acids  was screF025541) . The seqs (N.c.) . When coe motifs . Two vale motifs , were foe motifs . The Flas (N.c.) .Fig. 1.n=1-4) was constructed to produce the silk-like fibers. The N/C domains of the recombinant Flag spidroin were chosen from the MaSp2 as their structures and functional roles have already been reported and analyzed  were constructed: N-Sc1-C, N-Sc2-C, N-Sc3-C and N-Sc4-C shown in C. The coanalyzed .Fig. 2.in vivo (E. coli.The intein-mediated purification with chitin binding tag (IMPACT) system was chosen to purify the recombinant spidroin A Evans . N-termiin vivo . Althougin vivo . After oin vivo B. In accin vivo  the yielin vivo C are difThe lyophilized spidroin was re-solubilized in HFIP . The spiThe inner morphology of the fibers was determined by scanning electronic microscopy (SEM). Fiber samples were immersed in liquid nitrogen and subjected to brittle fracture . In thisFig. S2 shows the morphology of one fiber of each type under the light microscope, and the diameter of fibers was calculated. The stress-strain curves and mechanical testing data  are shown in Table S1)  . The spiable S1) . Interesable S1)  comparede S1) 3; B. We spe1) . From the divided peaks, the secondary structural fractions in fibers could be calculated via accounting the area of the peak  were constructed with the N/C non-repetitive domains  and the repetitive domain Sc . Meanwhile, another construct Sc1 was constructed without the terminal domain N/C as a control. The CD spectra of the Sc1 and N-Sc1-C were done to detect their secondary structure in PB buffer and HFIP . From the CD spectra we speculated that the N/C domain plays a functional role for the structure of the Sc1 in PB buffer or HFIP, and the HFIP may affect the protein structure. Though HFIP may change the protein structure, some reports proved that the conserved C-terminal domain of spider tubuliform spidroin 1 contributed to extensibility in synthetic fibers even in HFIP  and breaking stress (41.5 MPa). With the increase of repetitive domain Sc, which contains many GPGGX and GGX motifs contributing to extensibility , the proInterestingly, the N-Sc3-C fibers had the smaller diameter, though they showed the best strength. We speculated that the moderate molecule weight of N-Sc3-C spidroin supplies better overall organization. This may cause its diameter to be smaller than the others and contain the most β-sheet content (38.34%), providing a higher strength. Besides, some unknown mechanisms might exist resulting in these unusual changes. Further studies on these mechanisms may help to produce fibers that meet the specific requirements in various settings. Some reports also claim that synthetic fibers with small diameters have better properties than others with a large diameter . Apart fThe mechanical tests and Raman data analysis indicated there might be a molecular weight threshold of the functional repetitive domain relating to extensibility of the silk. In this study, the largest protein (N-Sc4-C: 99 kDa) possessed the highest extensibility. When compared to the native Flag silk with >200% extensibility, our constructed fibers were lower since the native Flag silk proteins were usually larger than 360 kDa . This fuIn this study, we investigated the correlation between the recombinant spidroin size and the synthetic fiber properties. Besides, the relationship between the structure and function of repetitive domain in synthetic fibers was also studied. Four different sizes of recombinant spidroin were constructed and generated into fibers by wet-spinning using a custom-made post-drawing device. The fiber morphology, mechanical properties, and secondary structure were comprehensively evaluated. The results indicated that the extensibility of the synthetic fibers was in positive correlation with the recombinant Flag spidroin size, and may imply a potential threshold of the protein size for excellent extensibility of the fibers . Furthermore, our study showed the relationship between the function and structure of the synthetic fibers, which was useful for functional protein design with various extensibility. In addition, these synthetic fibers from Flag spidroin with acceptable mechanical properties may provide a promising potential as the biomaterial for specific applications.Araneus ventricosus (A.v.) genomic library  and two enzyme recognition sites, BsaІ and BfuAІ, were added to amino-terminal (N) and carboxy-terminal (C) of the silk gene in pET32, respectively, to make clone pETSc1 with NdeІ and XhoІ. The N and C terminal non-repetitive domains were obtained according to the major ampullate spidroin 2 (MaSp2) of Argiope trifasciata and Argiope aurantia, by using E. coli gene codon .Chemical reagents and kits were purchased from Sangon . All restriction enzymes were purchased from New England BioLabs . The gene of the Flag repetitive domain was screened from an  library , and theBsaI/PstI and BfuAI/PstI followed by Sc1 insertion. The same procedures were used to generate pETSc3 and pETSc4. The N terminal non-repetitive domain was amplified by primers N-1 (5′-GGGAATTCATATGGGTGAACAAGGTGGTCTGTC-3′) and N-2 (5′-ACCGGTGGTCTCGCCAGCGTACGGGCCACGACCGCCTG-3′); while C terminal non-repetitive domain was amplified by primers C-1 (5′-GGCACCTGCTTCTGGCGGTGGTCAACGTGGTCCTCGCTC-3′) and C-2 (5′-GGCCTCGAGGCTCTTCCGCAACCCAGAGCCTGAGTCAG-3′). Underlining shows the site of restriction enzymes, NdeI, BsaI, BfuAI, XhoI, respectively. The N and C non-repetitive domains were cloned into the pETSc by NdeІ/BsaІ and BfuAІ/XhoІ, resulting in pET-N-Sc1-C, pET-N-Sc2-C, pET-N-Sc3-C and pET-N-Sc4-C, respectively.The cloning strategy was performed as previously described . Brieflyn=1-4) were digested by NdeІ and SapІ and ligated into pTWIN1 resulting in pT-N-Sc1-C, pT-N-Sc2-C, pT-N-Sc3-C and pT-N-Sc4-C, respectively. All the recombinant plasmids were sequenced.To construct the expression vector, the recombinant plasmids pET-N-Scn-C  to a final concentration of 0.8 mM. Upon induction, the temperature was decreased to 25°C for 16 h. The collected overnight cells were re-suspended in a lysis buffer  by 20% (w/v), lysed by a pressure homogenizer JN-3000 plus  at 1300 bar, and was further incubated for an additional 1 h at room temperature. Cell lysates were clarified by centrifugation at 21,000×g for 20 min and the supernatants were dialyzed in a column buffer .All expression clones (pT-N-Scn-C) in BL21 (DE3) cells were cultured overnight in 10 ml of Luria broth (LB) containing 100 μg/ml ampicillin in a shaking incubator at 37°C. The overnight culture was inoculated into 1 liter of LB containing 100 μg/ml ampicillin. When the ODThe purification of the silk proteins was performed according to the N-terminal cleavage protocol of the intein mediated purification with an affinity chitin-binding tag (IMPACT) system (New England Biolabs). All the purification buffers contained 4 M urea and the elution buffer added 100 mM DTT additionally. The IMPACT system carries an intein-chitin binding domain (intein-CBD) tag for one-step purification using a chitin resin. The target protein can be specifically released from the tag through induction of intein cleavage by the addition of a thiol-containing buffer (we used DTT) and elute from the column. Thus, the target protein can be collected without any extra tag. The purified recombinant spidroin was dialyzed in pure water to remove salts and centrifugation and freeze-drying.The lyophilized proteins were re-solubilized in HFIP (hexafluoroisopropanol) to make a 15∼20% (wt/v) spinning drop. The spinning drop was moved into a syringe and injected by a pump  into a coagulation bath. The spinning rate was 20 μl/min and the coagulation solvent was 90% methanol (v/v). The silk-like fiber was spun and drawn from the bath by a custom made device . There wBefore the mechanical tests, all fibers were fixed on testing cards and checked by the light microscope to ensure the fibers were of uneven thickness. The diameters of fibers were measured by Image Tool software. The mechanical tests of the synthetic fibers were conducted on a UTM T150  from Agilent. The gage length was 6 mm and the starting load strength was 750 μN. A minimum ten samples were tested for each type of silk.The secondary structures of the fibers were determined using Micro-Raman Spectroscopy System . For each sample, at least seven fibers were tested to assess sample uniformity, and the spectral decomposition of the amide I region was analyzed by the peak-fit software to calculate the secondary structure composition .


Vaccinia virus (VACV) oncolytic therapy has been successful in a number of tumor models. In this study our goal was to generate a double recombinant vaccinia virus (VV-GMCSF-Lact) with enhanced antitumor activity that expresses exogenous proteins: the antitumor protein lactaptin and human granulocyte-macrophage colony-stimulating factor (GM-CSF). Lactaptin has previously been demonstrated to act as a tumor suppressor in mouse hepatoma as well as MDA-MB-231 human adenocarcinoma cells grafted into SCID mice. VV-GMCSF-Lact was engineered from Lister strain (L-IVP) vaccinia virus and has deletions of the viral thymidine kinase and vaccinia growth factor genes. Cell culture experiments revealed that engineered VV-GMCSF-Lact induced the death of cultured cancer cells more efficiently than recombinant VACV coding only GM-CSF (VV-GMCSF-dGF). Normal human MCF-10A cells were resistant to both recombinants up to 10 PFU/cell. The selectivity index for breast cancer cells measured in pair cultures MCF-7/MCF-10A was 200 for recombinant VV-GMCSF-Lact coding lactaptin and 100 for VV-GMCSF-dGF. Using flow cytometry we demonstrated that both recombinants induced apoptosis in treated cells but that the rate in the cells with active caspase −3 and −7 was higher after treatment with VV-GMCSF-Lact than with VV-GMCSF-dGF. Tumor growth inhibition and survival outcomes after VV-GMCSF-Lact treatment were estimated using immunodeficient and immunocompetent mice models. We observed that VV-GMCSF-Lact efficiently delays the growth of sensitive and chemoresistant tumors. These results demonstrate that recombinant VACVs coding an apoptosis-inducing protein have good therapeutic potential against chemoresistant tumors. Our data will also stimulate further investigation of coding lactaptin double recombinant VACV in clinical settings. A number of recent studies have demonstrated the successful application of oncolytic viruses (OVs) as effective therapeutics to tumors that have become resistant to conventional chemotherapy –4. The aLister, Wyeth and Western Reserve and all of them exhibit broad tumor tropism. The most explored oncolytic VACV is JX-594  and this has shown promising results in clinical trials [Wyeth strain for inactivation of the viral thymidine kinase gene by insertion of two transgenes of the human GM-CSF and β-galactosidase E.coli under the control of synthetic early-late and natural virus P7.5k promoters, respectively [9) treatment was well-tolerated. Intravenous Pexa-Vec administration allows the virus to spread through the blood to distant metastases and to replicate there with the accompanying dose-dependent expression of GM-CSF while the slow-dividing normal tissues are tolerant to TK− virus strain replication [Wyeth VACV strain.The genome organization, lysis capacity and wide tumor tropism of vaccinia virus make it an ideal agent for cancer treatment and a model for the construction of recombinant viruses with enhanced antitumor activity. Vaccinia includes many strains such as l trials . It was ectively . The regectively , 9–11. Pectively . Overalllication –14. Howelication . The postk gene and virus growth factor gene (vgf) in the WR (Western Reserve) VACV strain leads to lack of virus replication in non-dividing cells with efficient destruction of cancer cells [The alternative approach for oncolytic VACV construction is based on using highly virulent VACV strains in which more genes of virulent factors are inactivated. It was demonstrated that synchronous suppression of the er cells , 17. Comer cells . A phaseer cells .gm-csf gene into the site of deletion of the tk gene [ClinicalTrials.gov Identifier: NCT00574977).The vvDD was used to construct the JX-963 strain with the insertion of the human  tk gene . JX-963  tk gene . Cytosintk and vgf genes that would selectively target tumor cells without decreasing its oncolytic capacity. Two transgenes could be simultaneously inserted into the VACV genome to enhance the therapeutic efficacy of recombinant VACV - the gm-csf gene and the gene of cytotoxic protein.GL-ONC1 (GLV-1h68) is another VACV based on the Lister strain and has being studied in phase I and II clinical trials in patients with therapy-resistant peritoneal carcinomatosis . Thus thvgf gene could attenuate the virulence of recombinant VACV against non-transformed cells as well as enhance its cytotoxic activity against cancer cells.In our laboratory we have developed a very promising selective inducer of cell death, the protein lactaptin. Lactaptin is a fragment of human milk kappa-casein (residues 57–134) that induces the death of various cultured cancer cells, primary endometrial cells and has no effect on the viability of non-malignant mesenchymal stem cells (MSCs) –26. Furttk gene is inactivated by insertion of the human gm-csf gene was engineered and characterized by the expression of the secreted form of GM-CSF in the infected human and animal cells at the level of 1 - 40 μg/ml in the culture medium [vgf gene region.Here we exploited the medium virulence L-IVP strain  that was used for anti-smallpox vaccination in Russia up to 1980 , 29. Thue medium , 32. Thiin vitro and in vivo. We investigated the biological effect of VV-GMCSF-Lact on the growth properties and apoptosis of human cancer cells and thus its antitumor activity against drug resistant mouse tumors. Together, our results demonstrate that recombinant VACV armed with lactaptin enhances its therapeutic efficacy in tumor growth delay and prolongs the survival of chemoresistant tumor-bearing mice when administered either intravenously or intratumorally.The objectives of this study were to generate a new double recombinant vaccinia virus VV-GMCSF-Lact (L-IVP strain) and to analyze its antitumor potential Pat) gene as a selective marker. This method has been shown to be highly efficient for the construction of recombinant VACVs coding the apoptosis-inducing protein apoptin [vgf gene region of the VV-GMCSF-S1/3 recipient strain genome through the generation of a low stable genetic construction after a single crossing-over. This low stable genetic construction contains the lactaptin and Pat expression cassettes, which include VACV promoters and transgene sequences. Puromycin selection enriched the virus population with puromycin-resistant recombinants. Under the conditions of puromycin cancellation the selected recombinants have lost the marker Pat gene by intramolecular recombination, which resulted in a hybrid population with the recombinant (VV-GMCSF-Lact) and original (VV-GMCSF-S1/3) genotypes.Recombinant VACVs were obtained via the transient dominant selection technique with the use of the puromycin resistance .By the same method the control recombinant VV-GMCSF-dGF containing the GM-CSF transgene in the d Figure . This cotk and vgf loci. Specific primer positions are depicted in Figure tk gene region that corresponded to the gm-csf gene sequence whereas DNA of the parental VACV L-IVP strain produced a 241 b.p. fragment  parameter. MDA-MB-231 cells were treated with recombinant viruses with different multiplicity (0.1 - 10 PFU/cell) and real time monitoring was performed Figure . VV-GMCSMDA-MB-231 cancer cells were treated with recombinant VACVs (0.05 PFU/cell and 0.5 PFU/cells) for 8 h and 48 h and then were analyzed for apoptosis by flow cytometry as described in the Methods. We found that the two recombinant VACVs were unable to induce a significant level of cell death after 8 h of viral infection Figure . The ratNext, the activation of caspase −3 and −7 in MDA-MB-231 cells treated with recombinant VACVs was analyzed  and medium tumor growth suppression by VV-GMCSF-dGF (36%)  Figure . Cycloph) Figure . The rat7 PFU per mouse on day 8 and day 14 after tumor cell implantation. We found that tumor inhibition was 70% for VV-GMCSF-Lact-treated mice there was no significant difference in tumor growth between mice treated with cyclophosphamide and the control group  we analyzed the pooled annexin V+ population which was larger in VV-GMCSF-Lact-treated cells [The mechanism by which vaccinia virus induces tumor cell death remains partly unclear. In general, caspase-dependent apoptosis is a universal cellular defense mechanism against viruses and other intracellular pathogens, including vaccinia virus, which interferes with virus production and transmission to neighboring cells . Howevered cells . It is lin vitro with activation of effector caspase-3 and −7, so here we hypothesized that the expression of the lactaptin transgene could also lead to effector caspase activation. Indeed we found that treatment of the cells with VV-GMCSF-Lact increased the size of the cell population with active caspase−3 and −7 in comparison with control VV-GMCSF-dGF. Thus, VACV-dependent expression of apoptosis-inducing proteins may promote the alteration of the route of death of infected cancer cells to apoptosis. This could be a helpful strategy to reinforce the oncolytic potential of recombinant VACVs.Of interest, vaccinia virus attenuates caspase activity by encoding various inhibitors of apoptosis, including F1L and B13R, which promote to immune escape by the virus as well as by the infected cancer cell , 47. As in vivo. For oncolytic VACV therapy a wide dose range of recombinant viruses has been successfully used in experimental trials, ranging from 105 to 108 PFU per mouse. These doses of recombinant attenuated vaccinia viruses were reported to be well tolerated and effective against various types of cancer in mice [In the current study the main task was to analyze the therapeutic potential of VV-GMCSF-Lact  in mice , 52. It  in mice . In thisThe use of OVs is a promising approach for the treatment of various types of cancer but it is particularly important that oncolytic viruses seem to be ideal candidates to target drug resistant tumors. In contrast to cytotoxic chemotherapeutics, the typical mechanisms of drug resistance such as drug efflux pumps and defective apoptotic signaling do not work in virotherapy . We usedIn summary, this is the first study to demonstrate the antitumor potential of a new recombinant virus coding human GMCSF and lactaptin. While further work will be necessary to clarify the molecular events taking place during VACV-induced cancer cell death, our data strongly support the therapeutic efficiency of VV-GMCSF-Lact in immunodeficient and immunocompetent tumor-bearing mice.African green monkey kidney fibroblasts (CV-1) and non-tumorigenic human breast epithelial cells (MCF 10A) were obtained from the American Type Culture Collection . RLS cells were generously provided by Dr. V. I. Kaledin (Institute of Cytology and Genetics SB RAS). Cancer cell lines MDA-MB-231, MCF-7, A549, U87MG, BT549 and BT20 were obtained from the Russian cell culture collection . MDA-MB-231 cells were grown in Leibovitz media  supplemented with 10% fetal bovine serum , 2mM L-glutamine, 250 mg/ml amphotericin B and 100 U/ml penicillin/streptomycin. Other cancer cells were cultivated in Iscove's modified Dulbecco's media (Sigma) with 10% FBS , 2mM L-glutamine (Sigma-Aldrich), 250 mg/mL amphotericin B and 100 U/ml penicillin/streptomycin . MCF 10A were cultured in MEGM BulletKit . CV-1 cells were grown in Dulbecco's modified Eagle's mediun  supplemented with 10% of FBS and antibiotics (100 U/ml penicillin/streptomycin).2 in air at 37°C and were passaged with 0.05% trypsin-EDTA every 2–4 days.Cells were grown in a humidified atmosphere of 5% COtk gene between nucleotides 81277 and 81308 (GenBank Acc. KP233807.1).The Lister strain of vaccinia virus  was obtained from the State Collection of Viral and Rickettsial Disease Agents of the State Research Center of Virology and Biotechnology “Vector”. Parental and recombinant viruses were grown in a monolayer of CV-1 cells and purified as described previously . The VV-tk gene in the pXJP5.2 were substituted with the sequences of the left (L-flank) and right flank (R-flank) of the VACV vgf gene, respectively. In the first step, L-IVP DNA (GenBank Acc. No. KP233807.1) was used as a template to amplify the flank's regions with the following primers: R-flank up 5′ – ctcccgaattcTCAGAAAACCCAAACACTACAACGT (EcoRI) and R-flank low 5′ – cttctcagctgGGAAACACCGATATGTGGAGGC (PvuII) and L-flank up 5′ – cccccatgcatCACCATCATATCAACGCTGGTAACTAT (Nsil) and L-flank low 5′ – tttccgtcgacTCAGTGTGTGTTTATGACAAGATTGGG .Plasmid pXJP5.2  was usedEndonuclease restriction sites were included in the primers' sequence. In the second step, the native VACV P7.5K promoter was substituted with the synthetic P7.5 synth promoter using oligonucleotides with the indicated duplex structure under annealing conditions:SalI P7.5synth5′ TCGACGGCCAAAAATTGAAAAACTAGATCTATTTATT GCACGCTAGCAAGCTTGGATCGC 3′3′ GCCGGTT TTTAAC TTT TTGATCTAGATAAATAACGTGCGATCGTTCGAACCTAGGCTTAA 5′Nhe I HindIII BamH EcoRIThis substitution allowed us to avoid the intramolecular instability of the VV-GMCSF-Lact recombinant strain in which the lengthy 276 base P7.5k promoter is used for GM-CSF expression . The insClaI PE/L KpnI SpeI Nsil ClaI5′ CGATGGCCAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAAATAT 3′GGTACCACTAGTATGC3′ TACCGGTTTTTAACTTTAAAATAAAAAAAAAAAACCTTATATTTTAGC 5′CCATGGTGATCATACGTAPat) was inserted into the resulting plasmid using KpnI – Nsil sites. The DNA fragment coding the Pat gene was amplified by PCR using pGEM-Puro-UN-DS as a template and the primers:(ii) The puromycin resistance gene (GGTACCATGACCGAGTACAAGCCCACGG (KpnI) and Puro Low 5′- GCATCATGCATTCAGGCACCGGGCTTGCGGGTCA (Nsil) [Puro Up 5′- GCATCA (Nsil) . The PCRLact into the pVGF-PE/L-Pat under the control of the P7.5synth promoter we amplified lactaptin DNA using the lactaptin-coding plasmid pGSDI/RL2 [For insertion of the lactaptin gene GSDI/RL2  as a temAAGCTTACCATGAACCAGAAACAACCAGCA (HindIII) and FR2 Rev 5′ –CTATCGAATTCTTAGTGATGGTGATGGTGATGTG (EcoRI). The structure of the resulting plasmid pVGF-FR2-PE/L-Pat  into CV-1 cells (confluent monolayer) which were preinfected with the VV-GMCSF-S1/3 (0.05 PFU/cell). Viral particles from infected cells were released by a quick freeze-thaw cycle and sonicated to obtaining a homogenous viral suspension9 PFU/ml in sterile saline and aliquots were stored at −80°C.Three repetitive rounds of selection were carried out by passaging of recombinant viruses through the CV-1 cells with puromycin (10 μg/kg) to obtain Pat-resistant recombinant VACVs. Puromycin-resistant clones were isolated and purified. The VGF-gene deletion and targeted insertions in recombinant viruses were analyzed by PCR with primers Up35 5′- gtaagcaaagaatataagaatgaagcggtaatgat-3′ and Apa-L22 5′ –cgagcacaataccgggagatgg-3′. The size of the PCR-fragment obtained from individual virus DNA differed: the fragment from the parental VV-GMCSF-S1/3 was 584 bases, that from the VGF-deleted VV-GMCSF-dGF – was 423 bases and the lactaptin-coding recombinant VV-GMCSF-Lact – was 710 bases  were infected by recombinant VACVs and wild type L-IVP (1 PFU/cell). Twenty-four hours post virus infection the culture medium was harvested and a proteases inhibitor cocktail was added. Culture medium and cell lysates were centrifuged at 14000 rpm for 30 min at 4°C and supernatants were separated by 10% SDS-PAGE electrophoresis and transferred to a Trance-Blot nitrocellulose membrane  by a wet blotting procedure  using the ‘Mighty small transphor’ . To detect GM-CSF membrane with culture medium samples was incubated with rabbit polyclonal anti-GM-CSF  (0.2 μg/ml in TBS pH 7.4 with 0.1% Tween-20 and 5% skim milk) for 16h at 4°C and after that primary antibodies were detected using alkaline phosphatase-conjugated anti-rabbit IgG   and BCIP (5-bromo-4-chloro-3-indolyl phosphate) and NBT (NitroBluetetrazolium) as a phosphatase substrate. To detect lactaptin the membrane with cell lysates was incubated with mouse monoclonal anti-lactaptin as described in .in vitro was analyzed using 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilid (XTT) (Sigma-Aldrich) as described previously as well as by MTT assay -2,5-diphenyl-2H-tetrazolium bromide) (Sigma-Aldrich) [The cytotoxic activity of recombinant VACVs Aldrich) , 30.Cell proliferation and survival were monitored real-time using the iCELLigence RTCA  system (ASEA Biosciences) by measuring cell-to-electrode responses of the cells seeded in eight-well E-plates with integrated microelectronic sensor arrays as described previously . For the5 MDA-MB-231 cells per well were seeded into six-well plates in complete medium and treated 24 h later with recombinant VACVs. Next, cells were harvested with trypsin and stained with annexin V-FITC and propidium iodide (PI) according to the manufacturer's protocol.Plasma membrane phosphatidylserine exposure was investigated by flow cytometry using the BD Pharmigen Apoptosis Detection Kit (BD Biosciences). Here, 2×102. Single color analysis was made on a FACSCantoII flow cytometer (Becton Dickinson) with 488 nm excitation wavelength and green emission for FLICA-stained cells using FACSDiva Software (BD Biosciences). Cells were initially gated based on forward scatter vs. side scatter to exclude small debris, and ten thousand events from this population were collected.Caspase-3 and −7 activation in MDA-MB-231 cells that were treated with recombinant viruses at various time points was detected by using the Vibrant FAM Caspase-3 and −7 Asasy Kit  according to the manufacturer's protocol. In short, after incubation with viruses, the detached cells were collected and pelleted by centrifugation and adhesive cells were harvested with trypsin. For flow cytometry analysis, the identical specimens of all detached and adhesive cells were combined and incubated with FLICA (fluorescent inhibitor of caspases) working solution for 60 min at 37°C and 5% COAll animal experiments were carried out in compliance with the protocols and recommendations for the proper use and care of laboratory animals (ECC Directive 86/609/EEC). The protocol was approved by the Committee on the Ethics of Animal Experiments of the Administration of the Siberian Branch of the Russian Academy of Science. Mice were housed as described previously .6 cells in Matrigel per mouse) into the back of each mouse. All groups consisted of four mice. When tumors become palpable recombinant viruses were dissolved in saline and administered to mice i.v. via the tail vein (1×107 PFU). A second VACV injection was given 20 days after the first injection. On day 74 mice were sacrificed by CO2 asphyxiation, and the tumors were excised and weighed.Female SCID mice (line SHO-PRKDC SCID HR/HR1EW 43375) aged 6–8 weeks old from the SPF vivarium of the Institute of Cytology and Genetics SB RAS  were used for s.c. MDA-MB-231 cell transplantation  was calculated as V=(2 x b)π/6 x a, where a was the smaller of the two perpendicular tumor diameters.For virotherapy of chemoresistant tumor, female CBA mice aged 8–10 weeks old were intramuscularly transplanted by RLS lymphosarcoma (1500 cells per mouse). RLS-bearing mice were treated intratumorally (intramuscularly) or i.v. with 1×10Student's t-test was used to compare treatment effects in cell experiments. For mouse experiments, the data are expressed as mean ± SE. The Mann-Whitney U-test was used for comparison between the two groups. A p value of less than 0.05 was considered significant.


A36R is deleted.Ectromelia virus (ECTV) is an orthopoxvirus and the causative agent of mousepox. Like other poxviruses such as variola virus , monkeypox virus and vaccinia virus , ECTV promotes actin-nucleation at the surface of infected cells during virus release. Homologs of the viral protein A36 mediate this function through phosphorylation of one or two tyrosine residues that ultimately recruit the cellular Arp2/3 actin-nucleating complex. A36 also functions in the intracellular trafficking of virus mediated by kinesin-1. Here, we describe the generation of a recombinant ECTV that is specifically disrupted in actin-based motility allowing us to examine the role of this transport step in vivo for the first time. We show that actin-based motility has a critical role in promoting the release of virus from infected cells in vitro but plays a minor role in virus spread in vivo. It is likely that loss of microtubule-dependent transport is a major factor for the attenuation observed when  Listeria monocytogenes; Autographa californica multiple nucleopolyhedrovirus (AcMNPV)) or across the external surface of the host plasma membrane (vaccinia virus (VACV); enteropathogenic Echerichia coli (EPEC)). The contribution of actin-based motility to disease progression is difficult to assess in these complex systems due to the pleiotropy of pathogen-encoded activators of actin polymerization and the lack of availability of in vivo experimental models that faithfully reproduce endogenous infections.Ectromelia virus (ECTV) is an orthopoxvirus endemic to mice that has been used extensively as a model for variola virus, the causative agent of smallpox . In commOur understanding of the actin-based motility of orthopoxviruses is primarily derived from VACV, the prototypal poxvirus used as the live vaccine to eradicate smallpox. However, actin-based motility is conserved in the orthopoxvirus genus including variola virus, monkeypox virus, ECTV  and furtA36R (the gene that encodes A36) or mutation of the critical tyrosine residues Y112 and Y132 (=VACV-A36YdF). As A36 is pleiotropic, only A36YdF specifically disrupts actin-based motility while leaving the kinesin-1 recruitment function intact  for 10 min at room temperature or ice-cold 100% methanol for 15 min. Cells were blocked in blocking buffer  and 2% FBS in CB) for 20 min and then incubated for 40 min with suitable primary antibodies diluted in blocking buffer. Primary antibodies used in this study were: anti-A36-Y112 . Fluoresw/v) bromophenol blue, 12.5% (v/v) β-mercaptoethanol) and boiled at 95 °C for 5 min. Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (resolving gel of 10% acrylamide-Bis solution (37.5:1), 0.375 M Tris-HCl, pH 8.8, 0.1% (w/v) SDS, 0.1% ammonium persulfate (APS), and 0.1% N,N,N=,N=-tetramethylethylenediamine (TEMED); stacking gel of 4% to 30% acrylamide-Bis solution (37.5:1), 0.375 M Tris-HCl, pH 6.8, 0.1% (w/v) SDS, 0.1% APS, and 0.1% TEMED). Resolved proteins from SDS-PAGE were transferred to nitrocellulose membranes  and probed with primary antibodies diluted in PBST-milk (5% (w/v) skim milk in PBS with 0.1% Tween 20). Primary antibodies used in this study were anti-A36-Y112 [BSC-1 cells were infected with virus at an MOI of 1 for 24 h. Infected cells were harvested in sodium dodecyl sulfate (SDS)-reducing sample buffer −/− (IFN-α/βR−/−) mice were challenged by injection subcutaneously in the right hind leg (hock) with 103 PFU virus and sacrificed at 5 days pi. Viral loads in livers, spleens, and lymph nodes were quantified by viral plaque assay on BSC-1 cell monolayers as described above and are expressed as log10 PFU per gram of tissue or per popliteal lymph node. C57BL/6 wild-type, C57BL/6 IFN-γThis study was performed in accordance with the recommendations in the Australian code of practice for the care and use of animals for scientific purposes and the Australian National Health and Medical Research Council Guidelines and Policies on Animal Ethics. The Australian National University Animal Ethics and Experimentation Committee approved all animal experiments (Protocol Numbers: J.IG.75.09 and A2012/041). Tribromoethanol  was used as the anesthetic (200–240 mg/kg body weight) given via intra-peritoneal injection prior to infection with virus. The respiration rate of the animals was monitored during anesthesia and recovery took place upon a warm table. Animals were euthanized by cervical dislocation. The animal ethics approval did not allow for mortality experiments and for all mice to be euthanized in the event that 20% of the mice succumbed to disease. For these reasons, all mice were euthanized at Day 5 post-infection.A36R gene was deleted in ECTV-Mos by replacing the gene with selectable (GPT resistance) and screenable (mCherry fluorescence) markers. This created the ΔA36 virus, which is deficient in both microtubule transport and actin-based motility [Res and ECTV-A36RY112F were made by rescue of the small plaque phenotype. ECTV-A36Res restored a wild type copy of A36R and was expected to fully restore function of virus. ECTV-A36Y112F carries a Y112F substitution, which was predicted to specifically disrupt the actin-based motility function of A36R while leaving other functions intact , with the exception of a large truncation at the C-terminal that removes three Asn-Pro-Phe (NPF) motifs that recruit intersectin-1 . Althougmotility . From ans intact B. Virusemutation C. A36R gene), cells were infected with ECTV-Mos, ECTV-ΔA36, ECTV-A36Y112F and ECTV-A36Res and the lysates collected at 48 h post infection (hpi). A36 was detected in ECTV-Mos, ECTV-A36Y112F and ECTV-A36Res at the expected size of 34 kDa  [Y112F-infected cells displayed a dramatic (15-fold) reduction in WV release (A36R fully restored actin-based motility and WV release (We then tested the ability of ECTV-A36 deleted A. Althou-A36YdF) . In line release B. Wild t release A,B.Y112F and ECTV-A36Res and plaques allowed to develop over six days. There were no significant differences in plaque size between ECTV-Mos, ECTV-A36Y112F and ECTV-A36Res although we were able to replicate the small plaque phenotype of ECTV-ΔA36  was quantified. To gain further insight into the effects of defective viral transport in vivo, three subsets of mice were used, either WT, IFN-γ−/− or IFN-α/βR−/−. Viral dissemination to the lymph nodes was reduced significantly by three orders of magnitude in ΔA36 in WT mice , conectively . YL126 hfamilies . The conY112F or VACV-A36YdF) does result in a reduction of cell-to-cell spread [Y112F plaques could be attributable to super-repulsion not playing a role in ECTV spread. In this case, the function of actin-based motility may lie solely in promoting release. We have previously suggested that actin-based motility in poxviruses may have evolved to promote WV release; a mechanism later coopted for super-repulsion. Although our data would appear to support this hypothesis, ECTV-A36R is still an early expressed gene. Alternatively, the small plaque size of ECTV may mask the contribution of actin-based motility rendering the assay less sensitive to small differences. Our results demonstrate that, while release of ECTV WV into the extracellular media is strongly defective in the absence of actin-based motility, loss of this motility has only minor consequences in vivo for the pathogenesis of ECTV. In contrast, the loss of the microtubule-based transport function of A36 results in a significant reduction of viral spread both in vitro and in vivo. We propose that the critical function of A36, in terms of virulence in vivo, lies in translocating WV to the cell surface. Ablation of actin-based motility did not significantly affect the cell-to-cell spread of ECTV in vitro. This result was somewhat unexpected given that in VACV a mutation in actin-based motility via deletion of one or both tyrosines (VACV-A36l spread . This sul spread . A possil spread ,32. Repul spread ,32. The Y112F, this was negligible in comparison to the effect of deleting A36. Our data also establish that intact IFN-γ and IFN-α/β signaling are critical for A36 function, particularly for virus dissemination to the lymph node, but less so for dissemination to the liver and spleen. While our ethics approval did not allow for mortality experiments, based on the viral load in liver and spleen of the IFN-γ−/− and IFN-α/βR−/− mice at Day 5, and based on our experience with the mousepox model, we predict that gene knock-out mice challenged with ECTV-ΔA36 would have survived, whereas those challenged with ECTV-Mos, ECTV-A36Y112F or ECTV-A36Res would have all succumbed to mousepox. We speculate that if orders of magnitude lower doses of virus were used, we may have seen more significant differences between ECTV-Mos and ECTV-A36Y112F or between ECTV-ΔA36 and ECTV-A36Y112F. We conclude that viral release mediated by actin-based motility is not a major determinant to the virulence of ECTV. There is little consensus in the literature as to how WV release correlates to poxvirus virulence in vivo. While poxvirus WV release is complex and not fully characterized, several mutations that increase release have been identified. However, these are typically, but not exclusively, associated with a reduction in cell-to-cell spread as measured by plaque assay and these viruses are attenuated in vivo (for example, VACV-B5P189S; [Our results demonstrate only a minor role for actin-based motility for virus spread in vivo. While we observed a detectable reduction in viral titers in distal organs associated with ECTV-A36B5P189S; ,35,36,37B5P189S; . This stB5P189S; . ConversB5P189S; . While iB5P189S; ,39,40. FListeria, Rickettsia, Shigella, Mycobacteria, Burkholderia, and EPEC [Listeria monocytogenes motility was key to identifying the Arp2/3 complex as an inducer of actin nucleation [L. monocytogenes, such as the internalin–E-cadherin engagement during entry [L. monocytogenes, is also highly pleiotropic with a role in the aggregation of bacteria [Actin-based motility is utilized not only by poxviruses but also by a wide range of bacterial pathogens including species of and EPEC ,42. Undecleation . In manyng entry . ActA, tbacteria . In ECTV


However, the effect of miR-19a in regulating myocardial ischemic injury has not been previously addressed. The present study was to investigate the effect of miR-19a on myocardial ischemic injury and identified the potential molecular mechanisms involved. Using the H/R model of rat cardiomyocytes H9C2 in vitro, we found that miR-19a was in low expression in H9C2 cells after H/R treatment and H/R dramatically decreased cardiomyocyte viability, and increased lactate dehydrogenase (LDH) release and cardiomyocyte apoptosis, which were attenuated by co-transfection with miR-19a mimic. Dual-luciferase reporter assay and Western blotting assay revealed that PTEN was a direct target gene of miR-19a, and miR-19a suppressed the expression of PTEN via binding to its 3′-UTR. We further identified that overexpression of miR-19a inhibited the expression of PTEN at the mRNA and protein levels. Moreover, PTEN was highly expressed in H/R H9C2 cells and the apoptosis induced by H/R was associated with the increase in PTEN expression. Importantly, miR-19a mimic significantly increased p-Akt levels under H/R. In conclusion, our findings indicate that miR-19a could protect against H/R-induced cardiomyocyte apoptosis by inhibiting PTEN /PI3K/p-Akt signaling pathway.miRNAs have been implicated in processing of cardiac hypoxia/reoxygenation (H/R)-induced injury. Recent studies demonstrated that  Ischemic heart disease causes myocardial infarction, increasing the loss of cardiomyocytes, and has long been a leading cause of morbidity and mortality worldwide. Hypoxia affects mitochondrial oxidative metabolism, leading to a heart remodeling process. Reperfusion strategies are the current standard therapy for myocardial ischemia ,2. HowevRecent evidence has indicated that myocardial apoptosis contributes to myocardial I/R injury . I/R indmiRNAs are small (19–25 nts), noncoding, regulatory RNAs that can negatively regulate gene expression by complementary base pairing with the 3′-UTR of target mRNAs, leading to their degradation or repressing mRNA translation ,14. ClosmiR-138 inhibits the activity of PI3K/Akt signaling pathways, a major cardiac gap junction protein in mice, resulting in alleviating human coronary artery endothelial cell (HCAEC) injury and inflammatory response [miR-21 in aldosterone-mediated cardiac injury and dysfunction in male rats. The researchers found that miR-21 down-regulation aggravated aldosterone/salt-mediated cardiac hypertrophy, fibrosis markers gene expression, interstitial and perivascular fibrosis, and cardiac dysfunction.Recent studies have shown that the roles of miRNAs in the pathogenesis of heart diseases have attracted more attention. It has been shown that overexpression of response . Moreoveresponse  reportedmiR-19a is previously confirmed to be an important regulator in heart development. Chen and Li [miR-19a were significantly higher in pulmonary arterial hypertension (PAH) patients than in controls; circulating miR-19a was found to be a potential, independent biomarker for diagnosis of PAH. However, little information is available regarding the effects of miR-19a on hypoxic adaptation in cardiomyocytes.n and Li  found thmiR-19a expression was dramatically down-regulated in H9C2 cells during hypoxia/reoxygenation (H/R) injury. Therefore, our aim was to unravel the effects of miR-19a on apoptosis, and to investigate the underlying mechanisms in H9C2 cells.In the present study, we confirmed that miR-19a, the negative control miRNA, and the transfection kit (Lipofectamine 2000) were purchased from Invitrogen .Dulbecco’s modified Eagle’s medium (DMEM), FBS, and penicillin/streptomycin  were purchased from the Gibco Company . The precursor of 2 incubator . To establish the H/R model, the culture medium was then changed to serum-free DMEM and placed into an anaerobic chamber that was purged with 94% N2, 5% CO2, and 1% O2 for 24 h. To simulate reperfusion, the cells were again treated with culture medium with a gas mixture of 95% O2, 5% CO2 at 37°C, for 3 h. Cells under normoxia throughout the experiments were included as a control [The H9C2 was maintained in DMEM supplemented with 10% FBS and a 1% pen/strep solution at 37°C in a 5% CO control .5 cells/plate and cultured overnight. Then, the cells were transfected with the miR-19a mimic (50 nM) or a negative control RNA (50 nM) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocols. The transfected cells were harvested for RNA isolation at 24 h after transfection and protein extraction/apoptosis analysis at 48 h after transfection.Cells in exponential phase of growth were plated in six-well plates at 2 × 103 cells per well. After transfection and H/R treatment, the culture medium was replaced with 100 μl of CCK-8 solution (containing 90 μl of serum-free DMEM and 10 μl of CCK-8 reagent) and incubated for 2 h at 37°C, and then the absorbance at 450 nm was measured.Cell viability was assessed with the cell count kit-8 , according to the manufacturer’s instructions. H9C2 cells were plated in 96-well plates at 5 × 10g for 10 min, and supernatants were collected for analysis using an LDH assay kit . The results were divided by the value of maximal LDH release in each group, which were expressed as cell death rate.Cell death was assessed by the release of lactate dehydrogenase (LDH), a cytoplasmic enzyme, and a marker of membrane integrity. After treatment, samples were centrifuged at 10000 miR-19a-specific RT primers. The real-time quantitative RT-PCR (qRT-PCR) analysis for miRNAs was performed with the TaqMan Universal PCR Master Mix II by using TaqMan Small RNA Assay according to the manufacturer’s instructions (Life Technology). U6 was used as an endogenous control. Primers were used as indicated in The transfected cells were harvested for RNA isolation at 24 h post transfection. Total RNA was extracted with TRIzol Reagent (Invitrogen). For miRNA quantitative analysis, cDNA was synthesized using TaqMan MicroRNA Reverse Transcription Kit (Life Technology) using For mRNA quantitative analysis, cDNA was synthesized using a PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa). SYBR Premix Ex Taq II (TaKaRa) was used for relative quantitative real-time PCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was the internal control. Real-time qRT-PCR analysis was performed on an MX3000P real-time PCR system (Agilent). Primers were used as in miR-19a were calculated by ΔCT values normalizing to the U6. PTEN expression levels were normalized to the control gene(GAPDH) and were calculated by ΔCT method.The relative quantitation (RQ) values were plotted .The expression levels of For the ectopic expression of miRNAs, the protein was extracted at 48 h after transfection. Parental and transfected cells were washed with prechilled PBS and lysed in 1× RIPA buffer (Sigma). Then extraction and solubilization were performed. Equal amounts of protein extract (50 μg) were denatured with SDS/PAGE (10% gel). Protein abundance of GAPDH  served as a control for protein loading. Each sample was treated with rabbit monoclonal anti-PTEN (phosphatase and tensin homolog deleted on chromosome ten) , rabbit polyclonal anti-p-Akt , rabbit polyclonal anti-Akt , rabbit polyclonal anti-Bcl-2 , rabbit polyclonal anti-BAX , rabbit polyclonal anti-caspase-3 , primary antibodies at 4°C overnight. Membranes were incubated with secondary antibody, HRP-conjugated rabbit/mouse anti-IgG (LI-COR Biosciences), diluted at 1:16000 in TBST, for 2 h at room temperature. Protein bands were detected by Odyssey infrared imaging system (LI-COR Biosciences).The Annexin V–FITC/PI apoptosis detection kit was used to determine the cell apoptosis, according to the manufacturer’s instructions. After treatment, cells were harvested, washed twice with PBS, resuspended with 500 μl binding buffer, and stained with 5 μl of FITC–Annexin-V  and 10 μl of propidium iodide  for 15 min at room temperature protected from light. Cells were analyzed by flow cytometry using the Aria cell sorter . This experiment was repeated in triplicate.Morphological detection of apoptotic cells was observed using Hoechst 33342 staining. H9C2 cells were plated in six-well plates. After transfection, cells were harvested and washed twice with PBS. Ten microliter of Hoechst 33342  was added to each well and incubated for 30 min at 37°C protected from light, and then imaged using an inverted fluorescence microscope. Each treatment was performed in triplicate.miR-19a-binding site in the PTEN 3′-UTR was predicted by miRNA target prediction databases, including Miranda, TargetScan, and PicTar. dsDNA oligonucleotides containing the miR-19a-binding sequence (wild-type) or a mismatch sequence (mutant) of the 3′-UTR of PTEN mRNAs and the HindIII and SpeI restriction site overhangs were amplified using PCR method. After annealing, double-stranded oligonucleotides were inserted into the pMIR-REPORT plasmid, downstream of the firefly luciferase reporter. Twenty-four hours before transfection, H9C2 cells were plated in the 96-well plates at 1.5 × 104 cells/well in triplicate. pMIR-REPORT constructs (100 ng) together with 1 ng of Renilla luciferase plasmid phRL-SV40 (Promega) and 50 nM of miR-19a were transfected by Lipofectamine 2000 (Invitrogen). Thirty hours after transfection, cells were lysed and luciferase activity was measured by the dual-luciferase reporter assay system (Promega). Firefly luciferase activity for each condition was normalized by dividing to Renilla internal control and then compared with empty vector pMIR-REPORT.For luciferase assays, the potential Cells were fixed with 4% paraformaldehyde, blocked with 10% normal goat serum (Gibco Laboratories), and incubated with antibody anti-PTEN (dilution 1:100) or anti-p-Akt (dilution 1:100) overnight at 4°C in a humidified container. Immunolabeling was revealed by PE-conjugated or FITC-conjugated secondary antibodies. Immunofluorescence was observed under a laser scanning confocal microscope .P-value of <0.05 was considered statistically significant. The statistical analysis was performed with SPSS version 20.0 .All experiments were performed in triplicate at a minimum. The data were presented as the means ± S.D. and analyzed by one-way ANOVA, followed by all pair-wise multiple comparison procedures using Bonferroni’s test. A miR-19a in myocardial I/R injury, we measured the expression of miR-19a in H9C2 cells after 24-h hypoxia and 3-h reoxygenation. Expression of miR-19a was overtly reduced by reaching a inhibition of 40.48% in H9C2 after H/R treatment compared with normoxia group (P<0.05)  . This fimiR-19a up-regulation in H/R-induced cardiomyocyte injury. CCK-8 assay was used to test cell growth and LDH release is a sign of cellular injury. We observed that cell viability was decreased in the H/R group, as shown in P<0.05), whereas cells treated with the miR-19a mimics efficiently promoted H9C2 cell growth and exhibited an amelioration of the cell loss induced by H/R injury (#P<0.05) (miR-19a) group had a higher percentage of viable cells compared with the H/R group . The results of the CCK-8 assay confirmed the anti-H/R-induced cardiomyocyte injury role of miR-19a.We examined the role of #P<0.05) A. As shoP<0.05) (miR-19a up-regulation group significantly decreased LDH release (#P<0.05 compared with H/R + NC group). Together, these data indicated that miR-19a ameliorates H/R-induced cardiomyocyte injury.Cell death rate was measured by LDH release. After 24-h exposure to hypoxia and 3-h reoxygenation, the LDH release was increased in the culture medium B, the reP<0.05) B, and thmiR-19a expression was inhibited by H/R in H9C2 cells and we observed that cell viability was decreased in the H/R group, whereas overexpression of miR-19a inhibited the reduction in the cell induced by H/R injury, we wondered whether miR-19a protected cardiomyocyte against H/R-induced cell apoptosis. Flow cytometry revealed greater apoptosis with H/R than control treatment in H9C2 cells; transfection with miR-19a mimic significantly decreased the apoptosis rate induced by H/R as compared with H/R alone .As similar to flow cytometry results, with Hoechst 33342 staining, changes in cell morphology indicating apoptosis were observed in cells. Treatment with H/R resulted in the characteristic morphologic indicators of apoptosis, including nuclear condensation and fragmentation B, but thP<0.05 compared with control group), which was reduced to 1.46-fold when the cells were treated with the miR-19a mimics (#P<0.05 compared with H/R+NC group). In addition, H/R stimulation increased the expression of Bax and down-regulated the expression of Bcl-2. Overexpression of miR-19a markedly reversed the effect on the expression levels of Bcl-2 and Bax in the cells exposed to H/R. miR-19a + H/R) group than in the (H/R + NC) group (P<0.05).To further investigate the molecular mechanisms involved, the apoptosis-related proteins Caspase-3, Bcl-2, and Bax were detected by Western blotting. As shown in miR-19a. We found that PTEN contained theoretical miR-19a-binding site in its 3′-UTR . We also constructed a vector in which miR-19a-binding sites were all mutated; miR-19a mimics failed to decrease the activity of luciferase gene with mutant 3′-UTR  were 120-fold higher than control group and reached the level of a highly detectable. Therefore, there is a close association of circulating miR-19a levels with susceptibility to AMI, and has a highly predictive and distinguishing ability.Here, we explored the expression changes of g et al.  revealedmiR-19a after 24-h hypoxia and 3-h reoxygenation as compared with controls. Therefore, miR-19a is an H/R-induced myocardial cell injury related miRNA in cardiomyocyte.In our studies, it was found that H/R injury resulted in reduction by 47.5% of the expression of miR-19a in H/R-induced myocardial cell injury. We investigated the protective effect of miR-19a against H/R-induced cell injury by CCK-8 assay and LDH assay. Results from the experiments showed that H/R treatment significantly decreased cell viability and increased LDH release in the culture medium, whereas overexpression of miR-19a efficiently promoted cell growth and decreased LDH release. Our study demonstrated that H/R-induced injury of H9C2 cells was significantly inhibited by miR-19a pretreatment.Further, we explored the potential role of Accumulating investigation indicates that cardiomyocyte apoptosis contributes critically to cardiac H/R injury . In addimiR-19a pretreatment reversed these effects. So, our results showed that H/R injury resulted in increase in apoptosis and overexpression of miR-19a may be responsible for decreasing apoptosis and protecting cardiomyocyte during H/R injury, which is consistent with the results of other studies [In the present study, we found that the protein levels of caspase-3 were increased and the proportion of Bcl-2/Bax was decreased by H/R injury. Moreover, flow cytometry revealed that the H/R-treated cells showed greater apoptosis than control, whereas  studies ,38.Signal transduction pathways involving PTEN, PI3K, and p-Akt are known to be associated with apoptosis and play an important role in the control of cardiomyocyte survival and function . ActivatMoreover, the activation of Akt may produce its anti-apoptotic function via activating the downstream substrates such as Bcl-2, Bax, caspase, eNOs, and p70s6k . While tPTEN is a frequently inactivated tumor suppressor in human cancer, depresses the PI3K pathway, and prevents Akt activation . PrevioumiR-19a. In our experiment, such a prediction was affirmed by a dual luciferase reporter assay. Therefore, we can assume that PTEN is a functional target gene of miR-19a involved in protecting cardiomyocyte injury in myocardial I/R. We verified that overexpression of miR-19a inhibited PTEN expression and initiated the PI3K/Akt pathway, resulting in down-regulation of apoptosis. Moreover, in the present study we found that the protein and mRNA levels of PTEN were increased and the p-Akt was decreased by H/R injury, whereas miR-19a pretreatment reversed these effects. So, our results showed that miR-19a can regulate via a PTEN/Akt pathway in H9C2 cells, which inhibits cardiomyocyte apoptosis during H/R injury.According to bioinformatic analyses, PTEN was regarded as a target of miR-19a can decrease cardiomyocyte apoptosis induced by H/R via inhibiting PTEN and increasing nuclear AKT. PTEN may be the target gene of miR-19a against H/R-mediated cardiomyocyte apoptosis. miR-19a may be a potential drug target for treating cardiomyocyte I/R injury. Further research in human clinical trials is necessary to pave the way for miR-19a ultimate application in the clinic to benefit myocardial I/R patients.In summary, the present study provided evidence that


B7-H6, a new member of B7-family ligand, also known as NCR3LG1, plays an important role in NK cells mediated immune responses. Many studies have shown that it is highly expressed in various human cancers, and its expression levels are significantly associated with cancer patients’ clinicopathological parameters and postoperative prognoses. But, still the exact role of B7-H6 expression in human glioma remains elusive. In the present study, we have characterized the B7-H6 expression in the human glioma tissues as well as glioma cell lines, U87 and U251. We observed that B7-H6 was highly expressed in the human glioma tissues, and its expression was significantly associated with cancer progression. By using the RNA interference technology, we successfully ablated B7-H6 expression in human glioma cell lines to further study its contribution towards various biological features of this malignancy. Our study identified that the B7-H6 knockdown in U87 and U251 glioma cells significantly suppressed cell proliferation, migration, invasion, and enhanced apoptosis along with induction of cell cycle arrest. It thus suggested that B7-H6 play an important role in the regulation of the biological behavior of these glioma cells. However, the detailed mechanism of B7-H6 mediated regulation of glioma cancer cell transformation and its prognostic value merits further investigation. Glioma, the most common tumor of the central nervous system, accounts for about 40–50% of the human adult brain tumors . The patNatural killer (NK) cells are an important component of innate immune system and usually act as the first line of defense against numerous pathogens as well as some cancers . The NK et al., has reported that reduced expression of NKp30 and the NK cells dysfunction in the peritoneal fluids of ovarian cancer patients, were significantly associated with the increased expression of soluble B7-H6 ligand in the peritoneal fluid [B7-H6, also known as NCR3LG1 or DKFZp686O24166, is a newly identified ligand in the B7 family . It bindal fluid . A previIn the present study, we have focused on analyzing the B7-H6 expression in human glioma tissues as well as human glioma cells lines, and to further understand its role in glioma progression using specific knockdown studies.The immunohistochemistry analysis showed positive staining for B7-H6, both on the membrane and in the cytoplasm of glioma cells to variant degrees Figure P<0.001), grade (P=0.003), and tissue type (P=0.001). However, we did not find any correlation with patient's gender. Thus, these results indicated that higher B7-H6 protein expression on glioma tumor cells may be involved in the progression of this malignancy.The correlation between patient's clinical parameters and glioma B7-H6 expression has been shown in Table P<0.01 and P<0.05, respectively, Figure To investigate, if B7-H6 has any specific role in the biological regulation of glioma cells, we first knocked down its expression using recombinant lentiviral GFP vector containing, shRNA targeting B7-H6 gene (LV-B7-H6-shRNA virus) and the non-targeted shRNA sequence as control (LV-NC virus). The human glioma cell lines, U87 and U251 were infected with the lentiviral particles from these two vectors and were sorted using flow sorter  based on GFP expression. The efficiency of the infection was also confirmed by detecting GFP expression using fluorescence microscopy Figure . In paraP<0.05), while U251-LV-B7-H6-shRNA cells showed significantly lower proliferation rate only after 48 and 72 hrs than U251-LV-NC (P<0.05). This data suggested that reduced B7-H6 expression on human glioma cells could suppress their cell proliferation. In addition, we also tested the role of B7-H6 in a wound healing assay, and as shown in Figure P<0.05, Figure Next to identify the specific role of B7-H6 in the growth of human glioma cells, we compared the cell proliferation rate of U87 and U251 cell lines infected with either LV-B7-H6-shRNA or LV-NC, using CCK-8 assay As shown in Figure P<0.01 and P<0.05, respectively). These results again suggested that B7-H6 expression in human glioma cells indeed play a role in the regulation of tumor invasion. Moreover, the results from the siRNA method treated cells also confirmed that B7-H6 knockdown could significantly inhibit the invasive ability of glioma cells . Furthermore, the cell cycle analysis results showed that, the cells from the LV-B7-H6-shRNA group, in both U87 and U251 cell lines, displayed increased percentage of cells in the G1-phase and decreased percentage in G2/M phases, in comparison to cells from LV-NC group , which suggests the potential post-transcriptional regulation of B7-H6 in glioma progression. It has been proposed that up-regulation of B7-H6 expression on tumor cells is probably stress-induced and help in self-recognition by NK cells [et al. have also reported that B7-H6-specific chimeric antigen receptors lead to the tumor elimination and enhanced host antitumor immunity [It has been suggested that inhibition of NK cell mediated immunosurveillance, contributed importantly to the oncogenesis and cancer progression of human glioma . The glit glioma . Importat glioma . Thus itoversial . In our NK cells . Furtherimmunity .et al. reported that B7-H6 was over-expressed in ovarian cancer tissues, and the expression level was significantly associated with cancer progression and patient's prognosis [et al. reported that down-regulation of B7-H6 was associated with decreased B7-H6 reporter activity and reduced histone acetylation at the B7-H6 promoter, suggesting the novel immunotherapeutic strategy of combining B7-H6 targeting with histone deacetylase inhibitors [et al. also reported that B7-H6-specific chimeric antigen receptors could lead to the tumor elimination and enhance the host anti-tumor immunity [et al., demonstrated that tumor cells release B7-H6, the ligand for the activating NK-cell receptor NKp30, by ectodomain shedding, resulting in reduced surface level of its expression on tumor cells and subsequently reduced NKp30-mediated recognition by NK cells, and thus helping tumor cells escape NK cell mediated killing [et al. [However, there have been some additional linking B7-H6 expression with cancer progression and suggest it to be an important therapeutic target, indicating the involvement of different mechanism than immune cell regulation. Zhou hibitors . Wu et aimmunity . The stu [et al. .As alluded before, many members from B7 family ligands not only contributed to the regulation of cell mediated immune response, but have also been implicated in regulating the biological behaviors of cancer cells that eventually leads to cancer progression and metastasis . For insThe glioma tissue array, GL2083a was purchased from Alenabio Co., Ltd. , while another array, HBra-Gli060PG was purchased from Shanghai Outdo Biotech Co., Ltd. . From these arrays, the data from 249 cases of glioma tissues and 19 cases of normal control tissues was included in the final analysis. The confirmation of the tumor tissues as glioma was performed using hematoxylin and eosin (H&E) staining after surgical resection. The detailed clinical parameters of the patients are shown in Table Rabbit anti-human B7-H6 polyclonal antibody was purchased from Abcam , and APC labeled mouse anti-human B7-H6 monoclonal antibody was purchased from R&D Systems . APC-conjugated mouse IgG1 Isotype control was purchased from eBioscience . The HRP-labeled goat anti mouse/rabbit secondary antibody  was purchased from Dako . The RNeasy Mini Kit was purchased from Qiagen , and SYBR Green Master Mix kits were purchased from TaKaRa . DMEM and fetal bovine serum (FBS) were purchased from Gibco .2, 37°C).Human glioma cell lines, U251 and U87 obtained from Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences, were cultured in DMEM, supplemented with 10% FBS, and incubated at standard culture conditions  solution, and the sections were later immersed in a 0.3% hydrogen peroxide solution for 30 min to block endogenous peroxidase activity. After rinsing with phosphate buffered saline (PBS) for 5 min, sections were then blocked with 3% BSA at room temperature for 30 min, and later incubated with purified rabbit anti-human B7-H6 antibody (1:150 dilution) at 4 °C overnight. The negative control did not have the primary antibody. Next, the sections were incubated with HRP-labeled goat anti rabbit secondary antibody, according to the manufacture's instruction, and followed by incubation with diaminobenzene as the chromogen, and hematoxylin as the nuclear counterstain. Finally, the sections were dehydrated, cleared and mounted.H-score method which has been described in the previous publication [All slides were examined independently by two senior pathologists who were not informed of the patients’ clinical parameters. The B7-H6 immunostaining densities were assessed according to the lication . BrieflyThe lenti-viral vector system was purchased from Clontech Laboratories Inc. . This vector system includes three plasmids: the pLVX-U6-GFP-puro vector, psPAX vector and pMD2G vector. The small hairpin RNA against the human gene B7-H6  was subcloned into the pLVX-U6-GFP-puro vector, which contained U6 promoter, GFP gene and puromycin gene (puro). The targeting siRNA sequence was 5’-CATCAAGAATATGGATGGCACATTT-3’, while the non-targeted control sequence was 5’-TTCTCCCCGAACAACAACGTGTCACCACCACGT-3’. The recombinant lentivirus shRNA targeting B7-H6 (LV-B7-H6-shRNA virus) as well as the non-targeted control lentivirus (LV-NC virus) were produced by transient transfection of HEK293 cells. All virus stocks were produced by lipofectamine-mediated transfection. After 48 h of post-transfection, cell supernatants containing viral particles were filtered using the 0.45-μm Steriflip vacuum filtration system  and concentrated by ultracentrifugation at 25000 rpm at 4˚C. The titer of the virus was tested according to the expression level of GFP. The day before infection, the U87 and U251 cells were seeded on dishes with a confluence of 30-40%. On the day of infection, the cells were infected by packaged lentiviral production. And subsequently propagated in selection medium containing puromycin (2 mg/ml) for at least 1 week. Finally, the infected cells from LV-B7-H6-shRNA group and LV-NC group, were analyzed by flow cytometry  and positive infected cells were sorted based on the GFP signal .The human glioma cell lines, U87 and U251 were used for B7-H6 RNAi knockdown studies. The small hairpin RNA against the human gene B7-H6  was cloned in the lentiviral gene transfer vector encoding green fluorescent protein (GFP). The targeting siRNA sequence was 5’-CATCAAGAATATGGATGGCACATTT-3’, while the non-targeted control sequence was 5’-TTCTCCCCGAACAACAACGTGTCACCACCACGT-3’. The recombinant lentivirus shRNA targeting B7-H6 (LV-B7-H6-shRNA virus) and the non-targeted control lentivirus (LV-NC virus) were prepared and the U87 and U251 cell lines were infected. The infected cells from both cell lines were categorized into LV-B7-H6-shRNA group and LV-NC group, based on the shRNA infection. Finally, the infected cells were analyzed by flow cytometry  and positive infected cells were sorted based on the GFP signal .Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to confirm the knockdown of B7-H6 mRNA expression. Total RNA from U87 and U251 cell lines was extracted using TRIzol (Invitrogen) reagent, and was then reverse transcribed into cDNA by using RT reaction kit (Promega). Real-time PCR was performed by using the ABI 7600 system  according to the manufacturer's instruction and SYBR Green was used as a DNA-specific fluorescent dye. Primer sequences for detection of the reference gene, GAPDH and the target gene B7-H6 were synthesized as follows, the human GAPDH, forward primer: 5’-TGACTTCAACAGCGACACCCA, and the reverse primer: 5’-CACCCTGTTGCTGTAGCCAAA-3’; the human B7-H6 forward primer: 5’-CTCCTGATT CTGCTGTGGGC-3’, and reverse primer: 5’-GTCGG AATGCCTCTTGGTGA-3’. The RT-PCR products for B7-H6 and GAPDH genes were also confirmed by using electrophoresis on 1.8% agarose gel containing 0.1% ethidium bromide. Images of the fluorescent bands were captured using Bio-Rad gel documentation system.4, respectively) were seeded into each separate well of a 96-well plate and cultured in 100μl of DMEM supplemented with 10% FBS. At the indicated time points, medium was replaced with CCK-8 reagent (10 μl CCK-8 and 90 μl DMEM), and the cells were further incubated for 1 h. Absorbance was measured for each well at a wavelength of 450 nm. Relative increase or decrease in the absorbance values at 450 nm in the experimental wells relative to the control wells, indicated cell growth or death, respectively. Cell growth was monitored every 24 hrs over 3 days. All experiments were independently repeated at least three times.The cell proliferation was evaluated by Cell Counting Kit-8 , according to the manufacturer's instruction. Briefly, the LV-B7-H6-shRNA and the LV-NC groups of U87 and U251 cells  with a calibrated eyepiece grid (1 mm / 20 μm graduation). Ten measurements were made at random intervals along the wound length and the data were averaged to express as a percent of the original width. The experiment was done in triplicate.Cell migration was evaluated by the wound scrape assay to determine if B7-H6 has some role in the regulation of migration ability of glioma tumor cells. Briefly, the cells from the LV-B7-H6-shRNA and the LV-NC group were incubated in 6-well plates. A small wound was made in the 90% confluent monolayer by using a 200 μl pipette tip in a lengthwise stripe. Cells were then washed twice with PBS and further incubated in serum-free DMEM medium at 37°C in a 5% CO® inserts with a 8 μm pore size and 6.5 mm diameter was coated with 20 μl matrigel diluted 1:3 in serum-free DMEM and incubated at 37 °C for 4 hours. In addition, LV-B7-H6-shRNA and LV-NC group cells were harvested by trypsinization after 12 hours of serum starvation, and were suspended in a serum-free DMEM medium at a dilution of 1×106/ml. The 200 μl of the cell suspension was added to the upper chamber of coated inserts placed in the wells of a 24-well plate with 600 μl DMEM medium containing 10% FBS in the bottom chamber as chemo attractant. After incubation at 37 °C for 24 hours in a 5% CO2 atmosphere the non-invading cells and matrigel were removed from the upper chamber with cotton tipped swabs. The insert were rinsed with PBS and cells on the filters were fixed with methanol for 30 minutes and stained with crystal violet solution (Sigma). The number of invading cells on the filters was counted in 5 random fields per filter at 100X magnification in triplicate wells of each group.The transwell culture system was used to evaluate the invasive ability of U87 and U251 cells ablated for B7-H6, as described in the previous study . In brie6 cells from each group were washed with phosphate-buffered saline (PBS) and resuspended in 100 μl binding buffer, and then incubated with 5 μl of APC-Annexin V and 5 μl of propodium iodide (PI) solutions. After 15 min incubation in the dark, the stained cells were analyzed by flow cytometry  using the FlowJo 10.0.6.software.Apoptosis-mediated cell death was examined using APC-Annexin V apoptosis detection kit  according to the manufacturer's instructions. Briefly, the U87 and U251 cells from LV-B7-H6-shRNA and LV-NC groups were harvested. 1×106, respectively) were first washed with PBS and then fixed with 70% ice-cold ethanol at 4°C for overnight. After washing twice with PBS, cells were then stained with a solution containing 200 μg/ml of propidium iodide, 0.1% sodium azide, 0.1% Triton-X100 and 10 μg/ml of RNAses for 2~4 hrs in the dark at room temperature. The stained cells were analyzed for subG1, S and G2 peaks by flow cytometry  using the Modfit software.The LV-B7-H6-shRNA and the LV-NC cells . The paired student's