JAK2V617F mutated cells due to persistence of phosphorylated serine residues of STAT5b, that conversely are targeted by PI3K and mTORC1 inhibitors. We found that PI3K/mTOR-dependent phosphorylation of STAT5b serine residues involves Protein Phosphatase 2A and its repressor CIP2A. The levels of CIP2A were found increased in cells harboring the JAK2V617F mutation, and we provide evidence of a correlation between clinical responses and the extent of CIP2A downregulation in myelofibrosis patients receiving the mTOR inhibitor RAD001 in a phase II clinical trial. To achieve maximal inhibition of STAT5 phosphorylation, we combined ruxolitinib with BKM120, a PI3K inhibitor, and RAD001, an mTOR inhibitor, obtaining improved efficacy in JAK2V617F mutated cell lines, primary patients’ cells, and JAK2V617F knock-in mice. These findings contribute to understanding the effectiveness of PI3K/mTOR inhibitors in MPN and argue for the rationale to develop combination clinical trials.Inhibition of the constitutively activated JAK/STAT pathway in JAK2V617F mutated cells by the JAK1/JAK2 inhibitor ruxolitinib resulted in clinical benefits in patients with myeloproliferative neoplasms. However, evidence of disease-modifying effects remains scanty; furthermore, some patients do not respond adequately to ruxolitinib, or have transient responses, thus novel treatment strategies are needed. Here we demonstrate that ruxolitinib causes incomplete inhibition of STAT5 in  JAK2V617F mutation [JAK2 wild-type patients, including JAK2 exon12 mutations in PV [MPL) [CALR) mutations in ET and PMF [Clonal proliferation of hematopoietic progenitor cells and dysregulated production of blood cells of different lineages are the main characteristics of chronic myeloproliferative neoplasms (MPN) . MPN incmutation , additions in PV  and throPV [MPL) , 6 and c and PMF . Additio and PMF , 10.The JAK1 and JAK2 inhibitor ruxolitinib  is approJAK2V617F mutation [In normal cells, PI3K-dependent signals contribute to the regulation of cell survival and proliferation through the activation of downstream kinases such as Akt and the mammalian target of rapamycin, mTOR; the latter is found in the different complexes, mTORC1 and mTORC2, and in turn controls downstream effectors including the transcription factor 4e binding protein 1 (4eBP1) and p70 s6 kinase (p70s6K). In normal hematopoiesis, the PI3K/mTOR pathway is essential for erythroid and megakaryocytic differentiation . The difmutation .JAK2V617F mutated cells remained poorly characterized. In this work, we show that signals originated by the activated JAK2 and PI3K pathway in JAK2V617F mutated cells specifically control the phosphorylation of different residues of STAT5. This might contribute to explain the persistence of activated STAT5 in cells exposed to JAK1/2 inhibitor ruxolitinib alone as well as the synergism exerted by combination of ruxolitinib with PI3K and mTOR inhibitors. Overall, these data provide mechanistic explanation for the involvement of PI3K/mTOR signaling in cells harboring the JAK2V617F mutation and might be translationally relevant for designing innovative strategies in MPN.Evidences favoring the relevance of targeting PI3K/mTOR pathway in MPN derive from cellular and animal models , 28; furJAK2V617F mutated cells was down-regulated after exposure to drugs targeting the PI3K/mTOR signaling [JAK2V617F mutated SET2 cells, we found that ruxolitinib dose-dependently reduced phosphorylated STAT5 Tyrosine-694 (Y694) that, together with Tyrosine-699 (not shown), is the immediate target of activated JAK2 in human STAT5a and STAT5b, respectively. Conversely, none of the PI3K and/or mTOR inhibitors tested were able to affect the phosphorylation of Y694. However, all the PI3K/mTOR inhibitors caused a dose-dependent reduction of the phosphorylation of Serine-731 (S731) and Serine-193 (S193) residues of STAT5b; of note, these serine residues on STAT5b were not affected by exposure to ruxolitinib. Since RAD001, an mTORC1 complex inhibitor, reduced phosphorylation of S731 and S193 at levels comparable to those obtained with BEZ235 and BKM120, that are PI3K inhibitors, we interpreted these findings as indicating that phosphorylation of serine residues of STAT5b is a RAD001 sensitive, mTORC1-dependent phenomenon. To support the hypothesis, we performed siRNA-mediated mTOR silencing in SET2 cells , and also prevented their de-phosphorylation when BKM120 was added to the cells  even though we succeeded in inducing only a 50–60% down-regulation of phosphatase expression levels . Taken together, these data illustrate a prominent role of PP2A in the de-phosphorylation of STAT5b serine residues induced by BKM120, and indirectly support a defective PP2A activity in cells harboring the JAK2V617F mutation.To address the mechanism(s) underlying STAT5 serine de-phosphorylation induced by PI3K/mTOR inhibitors in rylation –34, and rylation , we firss Figure . Notablys Figure . To asses Figure . These cJAK2V617F mutated cells, might result from excessive inhibition exerted by endogenous inhibitors. Therefore, we treated SET2 cells with different PI3K and mTOR inhibitors and with ruxolitinib and measured the levels of CIP2A and I2PP2A mRNA. Ruxolitinib did not change the baseline levels of CIP2A mRNA while PI3K inhibitor BKM120 and the double PI3K and mTOR inhibitor BEZ235 dramatically reduced CIP2A mRNA and protein levels  and I2PP2a (Inhibitor 2 of Protein Phosphatase 2A)   through s Figure . The mTOs Figure . Of notes Figure . On the s Figure , confirmd Figure . It was d Figure .JAK2V617F mutated cells, we measured CIP2A mRNA levels in granulocytes of MPN patients  and healthy controls (n = 15) , further supporting abnormally increased CIP2A levels as a mechanism for reduced PP2A activity in patients’ cells. The relevance of this observation was strengthened by the analysis of CIP2A mRNA levels in granulocytes from 14 patients with myelofibrosis who had received RAD001 in a phase II clinical trial [JAK2V617F mutated cells as the main reason for defective PP2A activity, that might be responsible for deregulated STAT5b serine residues phosphorylation, and mechanistically associate such an increase with abnormal signaling originated by the PI3K/mTOR pathway.To clarify a possible role of CIP2A in the constitutive phosphorylation of serine residues of STAT5 in primary ) Figure . We founal trial . The CIPal trial . InteresJAK2V617F mutated cells exposed to ruxolitinib indicate incomplete inhibition of phospho-STAT5-mediated signaling. Tyrosine phosphorylation allows STAT5 to dissociate from the receptor complex and promotes the formation of hetero- or homodimers and their translocation to the nucleus to activate target genes transcription [JAK2V617F mutated cells. For these studies we used as relevant drugs the pan PI3K inhibitor BKM120, that is being tested in a clinical trial in association with ruxolitinib [in-vitro models [The findings of persistent STAT5b serine residues phosphorylation in cription . On the cription , 44. Theolitinib , and theolitinib , and haso models .in-vitro models of JAK2V617F mutated cells, by measuring changes in cell proliferation, cell cycle and apoptosis  and SET2 cells (P < 0.05) (P < 0.05) than for the combination of BKM120 and ruxolitinib only. Furthermore, the combination of suboptimal doses of BKM120, RAD001 and ruxolitinib led to a stronger inhibition of the phosphorylation of tyrosine and serine residues of STAT5a and b in SET2 cells than that obtained with single drugs , and by 33.0%, 29.3% and 27.3%, respectively, in the presence of RAD001 (P < 0.04), BKM120 and ruxolitinib (P < 0.05) as single drugs. Finally, to assess the ability of treatment to preferentially target JAK2V617F mutated cells, we plated BM cells obtained from JAK2V617F knock-in and wild-type mice [JAK2V617F-positive colonies from 50% at baseline to 18% on day 4 (P < 0.05) , to ascertain possible synergistic activity of drugs combination. The IC < 0.05) , indicats Figure . In orden Figure . The colype mice , in a 1:) Figure .JAK2V617F knock-in mice to test the effects of treatment with the triple combination of BKM120, RAD001 and ruxolitinib; these mice develop a progressive myeloproliferative disease starting from the first months after birth, characterized by marked erythrocytosis, thrombocytosis and leukocytosis, and splenomegaly, that mimics PV and evolves to myelofibrosis at later stages [P < 0.01), 2, 2.3 and 3.4 respectively for BKM120, ruxolitinib (both P < 0.05) and RAD001 (P < 0.01). Histopathology showed a marked reduction of megakaryocytes and myeloid cells infiltrating the BM and the spleen in mice treated with the triple drug combination; in the spleen, the overall tissue architecture was improved towards normal appearance with splenic lymphoid follicles being restored , 50, 29 and 33 in mice receiving RAD001 (P < 0.01), ruxolitinib or BKM120 (P < 0.05), respectively  and RAD001 (a mTOR inhibitor), that when used together might prevent the well known rebound activation of AKT by mTOR inhibitors alone. The relevance of these data for the clinics is that both drugs are already being used in clinical trials, and they might be combined as well as with low doses of ruxolitinib. Finally, our findings also suggest that PP2A may serve as a novel therapeutic target for modulating the extent of STAT5 activation in MPN.These findings provide an explanation for the reported efficacy of PI3K/mTOR inhibitors in different models of MPN, as well as for the clinical efficacy of the mTOR inhibitor RAD001 in myelofibrosis, and offer a rationale for combination therapy. In fact, in our experiments, the concurrent blockade of JAK2 and PI3K/mTOR pathway signaling produced synergistic inhibition of STAT5 phosphorylation, resulting in effective prevention of cell proliferation In-vitro and in-vivo formulations and administration schedules are detailed in BKM120 (a selective pan class-I PI3-Kinase inhibitor), RAD001 , BEZ235 (a double PI3K and mTOR inhibitor) and ruxolitinib (a JAK1/JAK2 kinase inhibitor) were kindly provided by Novartis ; PP242 was purchased from Sigma-Aldrich . JAK2V617F mutated), K562 (BCR/Abl positive), SET2 (JAK2V617F mutated); and murine: Ba/F3 and Ba/F3-EPOR cells expressing JAK2 wild-type (wt) or JAK2V617F (VF) cell lines were used, as previously described [The following human: HEL  samples were obtained from PV or PMF patients, diagnosed according to the 2008 WHO criteria, under a protocol approved by Institutional Review Board of Azienda Ospedaliera-Universitaria Careggi and after obtaining a written informed consent. Mononuclear cells (MNCs) and Granulocytes from MPN patients or control subjects were isolated by Ficoll Hipaque (Lonza). MNCs were plated at 1×10JAK2V617F MPN patients. Fixed and permeabilized cells were incubated with relevant primary antibodies followed by anti-rabbit IgG Alexa Fluor-488 conjugated. Microscopic images were taken by a LSM 510 META Zeiss confocal microscope system  and analyzed by Confocor 2 (Zeiss) software (details in Granulocytes were isolated from Gene silencing was performed by siRNAs transfection according to Nucleofector technology (Lonza): mTOR, PP1, PP2A, CIP2A and non-targeting control siRNAs were from Cell Signaling Technologies .Cells were lysed in RIPA lysis buffer containing a proteinase inhibitor cocktail and assayed in western blotting analysis, as described in -DDCt method.Total RNA was extracted and reverse-transcribed to cDNA, then subjected to quantitative real-time PCR (qRT-PCR) according to standard protocols. Methods and primers for qRT-PCR analysis are listed in i.e., spleen weight/body weight ×100) was calculated.All animal procedures were performed according to Italian laws in an animal facility  under humanized conditions. KI mice were generated and experimental procedures were carried out as described . Three mP < 0.05. All P values were determined by unpaired two-tailed Student’s T test and confirmed by BootstRatio test [The level of significance from two-sided tests was tio test  on fold tio test  as repor


Ms2 in common wheat has facilitated the release of hundreds of breeding lines and cultivars in China. Here, we describe the map-based cloning of the Ms2 gene and show that Ms2 confers male sterility in wheat, barley and Brachypodium. MS2 appears as an orphan gene within the Triticinae and expression of Ms2 in anthers is associated with insertion of a retroelement into the promoter. The cloning of Ms2 has substantial potential to assemble practical pipelines for recurrent selection and hybrid seed production in wheat.Male sterility is a valuable trait for plant breeding and hybrid seed production. The dominant male-sterile gene  Ms2 has facilitated the release of hundreds of wheat-breeding lines and cultivars in China. Here Ni et al. describe the cloning of the Ms2 gene, which appears as an orphan gene in the Triticinae, and show that Ms2 can be used to confer male sterility in wheat, barley and Brachypodium.The dominant male-sterile gene  Ms2 confers 100% male sterility regardless of genetic background, phytohormones and environmental conditions16Ms2 confers genetically dominant male sterility in hexaploid and tetraploid wheat1920Ms2 has no other phenotypes, and Taigu pistils are readily cross-pollinated. Consequently, Ms2 seems suitable for developing hybrids and for recurrent selection in multiple autogamous cropset al.Rht-D1c and Ms2 (RMs2). By 2010, RMs2-based systems had produced 42 wheat cultivars planted on a total of 12.3 mha, with an increased grain yield of 5.6 million tons. Today both Ms2- and RMs2-based recurrent selection systems are widely used in wheat-breeding programs in China during the recurrent selection phase. New wheat varieties are often bred in a ten or more generation process that includes recombination of parental lines , selection of recombinants during the ‘recurrent selection' phase, followed by five or more generations of selfing.In wheat, there are at least five 23' refs , 15. Ms2Ms2, and the Ms2-based transcriptome and interaction network. The cloned Ms2 could be used to improve global food security by facilitating breakthroughs in plant breeding and hybrid seed production.In this study, we report the map-based cloning of wheat male-sterile gene RMs2, sterile anthers ceased growth at S2 when meiosis occurs, remained the same size through the S3 time period, and decreased in size during the S4 time period. Taigu sterile anthers were initially pale green but faded to pale white or greyish by the end of the S4 time period. At the S4 stage, fertile anthers of wild-type wheat bore germinable pollen; Taigu lines have a ‘non-pollen type' male sterilityTaigu lines have small anthers  and abnoMs2. We mapped Ms2 using four BC1F1 populations, including popA, popD, popE and popF (Aegilops (Ae.) tauschii mapMs2 to the Xsdauw8-Xsdauw36 interval in popA . Xsdauw24 is in a pseudogene  and Xsdauw32 is near ribosomal protein S25 . Six potential genes PG5 mRNA could be detected in immature spikes of Taigu lines CSRMs2, LM15RMs2 and XY6Ms2, but not in male-fertile CS, LM15 and XY6 (RPS25) mRNA was detected in developing flowers of both Taigu and male-fertile lines; we did not detect expression of the other hypothetical genes  that is completely linked to male sterility in Taigu lines and the P1076-type PG5  from male-fertile LM15. Although the mRNA of PG5P1076F was not detected in LM15, it is expressed somewhat in the S2-stage anthers of LM15RMs2, albeit at 23% (mean) ±8.8%  of the mRNA of PG5P1593S in LM15RMs2 as indicated by RNA-seq reads (158 reads of PG5P1593S versus 28 reads of PG5P1076F), 5′- and 3′-terminal cDNA clones (10 clones of PG5P1593S versus 4 clones of PG5P1076F) and internal cDNA clones (199 clones of PG5P1593S versus 22 clones of PG5P1076F). Both PG5P1593S and PG5P1076F have eight exons. PG5P1593S encodes a 293-aa protein  with no annotated domains or protein homologues in plants except wheat.We constructed a bacterial artificial chromosome (BAC) library of LM15t genome . We asseLM15RMs2  and prim and XY6 . In contal genes . Thus, PPG5P1593S and PG5P1076F alleles of LM15RMs2 have seven SNPs in the coding region; the first six SNPs cause six amino acid changes and the seventh encodes a stop codon in PG5P1076F  retrotransposon integrated at nucleotide position −314..−310 . Taigu_P1593-1 is associated with two identical LTRs , a 630-bp internal domain, and a 5-bp target site duplication (TSD). We postulated that the Taigu retroelement acts as a transcription enhancer that activates transcription of PG5P1593S rather than an inactivator of a repressive cis-element in the promoter region.The G5P1076F . We foun14..−310 . This LTMs2 is dominant for male sterility (MS), in keeping with the dominant expression of PG5P1593S in Taigu lines; all Taigu lines are heterozygous for Ms2 because two Taigu lines cannot cross. We chemically mutagenized LM15RMs2 and CSRMs2 to determine if any EMS mutants had (1) a male-fertile (MF) phenotype and if so, (2) a variation in their PG5P1593S that would explain the transformation from MS to MF. We generated 2,266 M1 plants and examined 37,471 spikes, of which 254 had MF anthers and viable pollen , 2.9% of the primary tillers had a TILLING mutation in PG5P1593S, compared to a 40% mutation rate in PG5P1593S in the induced MF tillers (n=20). A χ2 test negated the independence between PG5P1593S mutations and male fertility in LM15RMs2 , that is, EMS-induced mutation(s) in PG5P1593S are associated with the loss of male sterility. Among all the EMS-treated CSRMs2 M1 sampled , we inspected 34,333 spikes and identified 234 MF spikes in 149 M1 plants. On average, each CSRMs2 M1 plant produced 32 tillers, but only one to eight tillers were MF, indicating a common phenomenon of chimeracism in the M1 generation  and the terminator region (936 bp) of the gene. PC976 was introduced into wheat, barley and Brachypodium  cDNA , PG5P1076F (PC971) and PG5P1593S:GFP (PC972) using the maize ubiquitin promoter in wheat, barley and Brachypodium  expression was not detected in early and late in anther development, but was detected in anthers at the S2 stage when meiosis occurs (MS2 mRNA in other tissues (ms2 (=PG5P1076F) was also limited to the S2-stage anthers. The transgenic Ms2 in wheat and barley also was detected at the S2 stage in anthers  and LM15 groups (MS2 in nine accessions (Ms2 has a Taigu retroelement (inserted at nucleotide position −314..−310), but the male-fertile ms2 does not; the only difference between these two alleles is the Taigu retroelement. Taigu has two identical LTRs , suggesting a recent formation of Ms2 within the CS lineage. There were seven exonic SNPs between CS and LM15; SNP3404 caused a premature stop codon (L284*) in the LM15 group. Both Ae. tauschii and common wheat had two additional variants: one in the promoter (at nucleotide position −673..−364) and one in the second intron (at nucleotide position 902..905).We compared the 5 groups . We thencessions . In the MS2 gene is associated with at least 18 haplotypes, three in the CS group , five in the LM15 group , seven in the hybrid group , and three in the null group   . Overalln=546). A χ2 revealed no significant difference among HT1, HT3 and HT4, but HT2 was associated with a higher number of ‘non-AB' accessions than those in either HT1, HT3 or HT4 . Therefore, the polymorphic site −314..−310 (HT2) is the most variable and might be a region that is more likely to be involved in genetic change. We conclude that Ms2 (haplotype A2) was formed when the retrotransposon Taigu inserted into the A1 haplotype.Of the four regions investigated, we compared the number of accessions in three genotype groups  originated in ‘O261,' a line in PopA that has an intragenic recombination in MS2. Ms2O261S inherited its promoter and first two exons from the dominant Ms2 in XY6Ms2, but the rest of the gene comes from a recessive ms2 in SW41 , we detected 72,499 active genes in anthers, leaves, and/or pistils . Using higher stringency, we then extracted 2,991 sterile-anther-enriched genes (SAEGs) and 939 fertile-anther-enriched genes (FAEGs)  . Among t (FAEGs) .P=4 × 10−10; hereafter in this paragraph: the P value is based on the Fisher's statistical test, and a FDR-adjusted P value is also indicated in the P=4 × 10−76), and ‘translation'  were overrepresented. Among molecular function GO terms, the ‘structural constituent of ribosome' was also significantly overrepresented in the FAEG group . The cellular component GO terms ‘macromolecular complex', ‘membrane', ‘thylakoid', ‘ribosome' and ‘intracellular non-membrane-bounded organelle' were overrepresented. In the SAEG group, there were also many genes involved in metabolism , oxidation reduction , and protein modification . Other GO terms overrepresented among SAEG genes include ‘cell wall organization'  and ‘cell wall macromolecule catabolic process' . Among molecular function GO terms, ‘catalytic activity'  was overrepresented and genes predicted to encode oxidoreductase, transferase, and chitinase were among this group. ‘Extracellular region'  was the only significantly overrepresented cellular component GO term among SAEG genes.In the FAEG group, genes with the GO terms ‘metabolism' , ‘ribonucleotide binding' , and ‘purine ribonucleotide binding'  for 17 entries (We screened for MS2-interacting proteins in the S2-stage spikes using yeast two-hybrids (Y2H) analysis, and identified 312 positive clones encoding for 142 proteins . Analysi entries  and 10,  entries . Among t entries .Ms2, Ms3, and Ms4 (ref. Ms2 is on chromosome arm 4DSAe. tauschii) has low DNA diversity, which complicated positional cloning of genes in this genome. Synthetic hexaploid wheat (derived from Ae. tauschii) has considerable diversity when compared to the D genome in common wheat. However, the MS2 region is partially or completely deleted in 40% of Ae. tauschii accessions tested here. Consequently, the particular selection of synthetic wheat was critical for the cloning of the Ms2 gene. For example, the deletion of the Ms2 region  represents a new family of non-autonomous LTR retrotransposon. First, Taigu_P1593-1 is a TRIM retroelementTaigu_P1593-1 is associated with two identical LTRs and a perfect TSD, suggesting a recent origin of this retroelement. Second, the Taigu retroelements only emerged in the Triticeae lineage; there are 50 repetitive sequences in common wheat and 28 repetitive sequences in Ae. tauschii that are similar to part or to a complete Taigu element. Third, there are no significant hits of Taigu in the Triticeae repeat sequence databaseTaigu into the MS2 promoter results in Ms2 expression in anthers of Taigu lines. There is one somewhat analagous case in Citrus sinensisTaigu retroelement acts as a transcription enhancer or its insertion destroys a repressive cis-element. In LTR-type retrotransposons, the 5′ LTR usually contains a promoter for producing template RNAscis-element in the promoter region of the MS2 gene, other sequence variants of the cis-element would likely also activate the MS2 gene. For example, the H2 and H3 haplotypes  line  line . AlthougMs2 offers a straightforward method to use cisgenics or transgenics transiently in either wheat or barley breeding programs. Here, we propose a high-throughput hybrid production system using any wheat that can be transformed with Ms2 plus an aleurone-specific gene for pigmented kernels (MSC for Ms2 colour wheat)  . The MSCMs2 in the middle layer and the male reproductive cells.This work sheds light on plant male sterility. In wild-type anthers, the middle layer and tapetum  eventualMs2 gene has played pivotal roles in wheat breeding, enabling the release of hundreds of breeding lines/cultivars in common wheat. Remarkably, Ms2 homologues are only found in wheat and its close relatives, and are absent from other crops outside the Triticeae tribe. The cloned Ms2 gene provides high value for breeding and producing hybrid wheat, and potentially for other major crops.In conclusion, we report molecular characterization of a male sterility gene in wheat. The current 1F1 populations, including those of common wheat  and synthetic hexaploid wheat  (MS2 diversity (We used 18 plant accessions and four BCn=6x=42) . Additioiversity . Plants DIC521-2B', and three SHW lines  were received from Dr J.D. Faris at the USDA-ARS, Fargo, ND, USA. The remaining SHW lines were provided by Dr T. Payne at the International Maize and Wheat Improvement Center, El Batán, Texcoco, México. All Ae. tauschii accessions except for ‘AL8/78' were provided by Dr H.E. Bockelman at the USDA-ARS, Aberdeen, ID, USA.‘Langdon', ‘LDNWe inspected wheat anthers at four stages: when the auricle distance between the flag and penultimate leaves was from −3 to −5 cm , two tetraploid wheat , and two diploid accessions . To extract the A and B-specific SNP markers, we discarded non-specific or low quality SNP markers by performing a three-step elimination: (1) those with any missing data or any heterozygous genotypes within Langdon or LDNDIC521-2B; (2) those with any genotype data within CIae 9 or PI 569536; and (3) those with any missing data or any heterozygous genotypes within the six parental lines. We used a similar procedure to extract the genome D-specific SNP markers, but step one was for CIae 9 or PI 569536 and step two was for Langdon or LDNDIC521-2B. Using genome-specific SNP markers , we calculated the proportion of polymorphic SNP markers for 15 pairwise comparisons between the parental lines.We used the wheat 90 K iSelect SNP arrayMs2 gene, we used three Taigu lines  and three SHW lines   . The thrAe. tauschii mapMs2 gene to the Xsdauw8-Xsdauw36 interval, which aligns to the AT4D3406-AT4D3410 region in Ae. tauschii . The recombination frequency was transformed into centimorgans (cM) using the Kosambi function.We initially developed nine PCR markers using the tauschii . AT4D340tauschii . We seleMI225K13 . Coinciding BACs  to narroRMs2. Briefly, high-molecular weight genomic DNA was extracted from leaf tissues, partially digested by the restriction enzyme HindIII, and ligated into the BAC vector pIndigoBAC536-S−1 chloramphenicol, 80 mg l−1 X-gal and 100 mg l−1 IPTG. White colonies were individually picked into 384-well microtiter plates. In total, 706,176 BAC clones were arranged in 1,839 384-well plates, representing a 5.1-fold coverage of the wheat genome (∼17 Gb) (Using standard prototols42(∼17 Gb) .RMs2 BAC library. In brief, a 384-well stock was duplicated and pooled to extract a primary DNA pool using the ZR BAC DNA Miniprep Kit . A super DNA pool was then prepared by mixing ten primary DNA pools. In total, we prepared 1,839 primary DNA pools and 184 super DNA pools. We screened the BAC library using Xsdauw11, Xsdauw20, Xsdauw25, Xsdauw33 and Xsdauw35, and recovered twelve BAC clones in this region . The BAC DNA was processed into a paired-end (PE) DNA library. In brief, DNA was acoustically fragmented using the Covaris instrument , end-repaired and 3′ adenylated using the NEBNext Sample Reagent Set , ligated to Illumina adaptors, and separated on a 2% agarose gel to select fragments about 400–500 bp. Adaptor specific primers were used to amplify the ligation products. The final library was evaluated by quantitative RT-PCR with the StepOne Plus Real-Time PCR system . PE reads (150 bp) were obtained using the Illumina HiSeq2500 .Q≤5 or unknown bases accounting for over 10% of any PE reads), and then removed any vector or E. coli. sequence using the cross_match tool in Phrapde novo assembly on clean reads  using the ABySS 1.5.2 programmeMs2 and ms2 regions. In the Xsdauw24-Xsdauw32 interval, we identified several Ms2 candidates and compared their transcription in Taigu and transgenic plants , and conducted the rapid amplification of cDNA ends (RACE) to recover the 5′ and 3′ untranslated-regions of PG5 using the SMARTer RACE cDNA Amplification Kit . The 5′ RACE utilized the PG5-specific primers P100 and P101, as well as the 3′ RACE with P102 and P103 (PG5 cDNA using the conserved primers P104 and P100 (TaqI (New England Biolabs). PCR products of the PG5P1593S clone were cut into two bands (398 bp and 42 bp), but only one band (440 bp) in the PG5P1076F clone, which was scored in a 1.5% Agrose gel  and the BC1F1 seeds of CSRMs2/2*SW7 and CSRMs2/2*SW8 (the CSRMs2 set) for chemical mutagenesis. Using ethyl methane sulfonate (EMS), we prepared the mutant populations. Briefly, lots of 400 seeds (M0) were soaked in 100 ml ethyl methane sulfonate  solution (87.4 μM in water), incubated on a shaker at 150 r.p.m. at 25 °C for 10 h, and washed with running water at room temperatures for 4 h. After germination, vigorous seedlings with roots were grown in greenhouse and field conditions. Because the populations of mutagenized seeds had an expected 1:1 segregation of dwarf, male-sterile RMs2:wild-type male-fertile rms2, at the flag-leaf stage, we screened the M1 plants for a dwarf phenotype (≤60 cm) and the presence of a PG5P1593S allele using the HT5 marker  exons 1–2 between P133 and P134, (2) exons 3–4 between P135 and P136 and (3) exons 6–8 between P137 and P138  native expression of PG5:GFP, (3) overexpression of PG5 and (4) overexpression of PG5:GFP. For native expression, we used a 10,592-bp genomic fragment (−5578..4078..+936) of the PG5P1593S gene, which includes the native promoter , the genomic coding region  and the native terminator . We isolated a 10,592-bp fragment from LM15RMs2 (or BAC clones) using PCR primers P139 and P140 (NP1::PG5P1593S) (NP1::PG5P1593S:GFP) , PC971 (Ubi::PG5P1076F), and PC972 (Ubi::PG5P1593S:GFP)  native expression of and P140 , and ass5P1593S) . By usin93S:GFP) .The KOD  to P151 , and ass93S:GFP) . These bAgrobacterium-mediated transformation in barleyBrachypodiumUsing standard methods on plant transformation and tissue culture, we performed biolistic bombardment in wheat49BAR, P133 and P141 for PG5P1593S and PG5P1593S:GFP  for PG5P1593S:GFP, P142 and P143 for ACTIN  and PC983 (35S::GFP:Ms2), and their respective controls PC134 (Ubi::GFP) and pMDC43 (35S::GFP) , and soaked the remaining tissue in a fresh enzyme solution , 0.1% BSA and 2 mM CaCl2; Sangon Biotech, Shanghai, China) for 2 h at room temperature. The protoplast suspension was inspected under a Leica TCS SP5 II laser scanning confocal microscope . Three views were captured for each target cell: (1) a brightfield, (2) a GFP channel and (3) a chlorophyll autofluorescence (ChlAF).To study cellular localization, we used four constructs: PC972 (5S::GFP) . Each coRMs2, pistils of LM15 and LM15RMs2, and flag leaves of LM15 and LM15RMs2. Tissues were stored in liquid nitrogen during collection and then at −80 °C for storage. There were three biological replicates per tissue-genotype.We collected four types of samples from plants grown in greenhouses to the S2 stage: fertile anthers of LM15, sterile anthers of LM15ca. 500 bp per insert) and performed high-throughput sequencing (125 bp PE reads) using HiSeq2500. We pre-processed raw reads using the FastQC algorithm (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and then performed quality trimming and adaptor removal by TrimmomaticPG5 gene, specific reads were indicated by a perfect match to PG5P1593S or PG5P1076F of LM15RMs2 using the STAR programmeWe extracted total RNA using TRIzol (Invitrogen) and evaluated the RNA integrity with an Agilent 2100 Bioanalyzer . The Berry Genomics Company prepared the sequencing libraries (On the basis of the current wheat genomehttp://plants.ensembl.org), we assembled the unique mapped reads using htseq-count script (v.0.6.1) with the union mode in the HTSeq packageP value (Padj<0.01). Furthermore, we divided the anther-related DEGs into two groups: (1) sterile-anther-enriched genes (SAEG): those associated with higher transcription in sterile anthers compared to the fertile anthers; (2) fertile-anther-enriched genes (FAEG): those associated with higher transcription in fertile anthers compared to the sterile anthers. The following rules were used to define the final list in each group: (1) genes active in only one type of anther with a count value >the 10th percentile (3.29) of the cleaned data  genTriticum aestivum transcript (v2.2) using the singular enrichment analysis (SEA). To prepare heat maps of the anther-enriched DEGs, their expression values were first lg transformed , and then organized in an expression matrix. A hclust function and ‘average' method in R was used to cluster the expression matrix using the correlation distance. We then created heatmaps using the Heatmap.2 of the R package gplots (http://www.inside-r.org/packages/cran/gplots/docs/heatmap.2).The 3,930 anther-enriched DEGs were analysed for GO frequencies. All genes were first assigned identities with the agriGO toolkitMs2 (=PG5P1593S) and ms2 (=PG5P1076F) were targeted for Y2H analyses. The coding sequence of each gene was amplified from LM15RMs2 using PCR primers P153 and P150 (ADH1::AD:Ms2) and PC987 (ADH1::BD:Ms2), PC988 (ADH1::AD:ms2), and PC989 (ADH1::BD:ms2)  . Similarin genes .RMs2. Aliquots of 1 ml with a titre value of 9 × 107 c.f.u. ml−1 were prepared and stored in a −80 °C freezer. Both Ms2 and ms2, as bait proteins, were used to screen the cDNA library on the QDO and TDO media, respectively. Positive clones from the library screening were tested for their autoactivity and pairwise interactions with Ms2 and ms2. Selected clones were sequenced using a T7 primer (The Y2H assay included the use of the double dropout medium (DDO: SD/-Trp/-Leu), the triple dropout medium (TDO: SD/-Trp/-Leu/-His), and the quadruple dropout medium (QDO: SD/-Trp/-Leu/-His/-Ade) (Clontech). We first performed autoactivity tests . We then7 primer . The ide7 primer .L44F, Ms2T109M, Ms2A153V and Ms2E248K , the GTP-binding protein (GTPase), and the ubiquitin-conjugating enzymes Ubc4B . We alsoMs2E248K , which cMs2E248K 59.For MS2-interacting proteins, we performed GO analysis using SEA and the parametric analysis of gene set enrichment (PAGE)in situ hybridization was performed according to a standard protocolRMs2 anthers at different stages were harvested and fixed in FAA  for 12 h at 4 °C. Probe templates were amplified from the Ms2 cDNA using PCR primers P146 and P147. Both sense and antisense RNA probes were prepared independently using the digoxigenin RNA labelling kit .Tissue preparation and MS2 gene, we genotyped 575 germplasm, including 469 common wheat lines, 36 SHW lines and 70 Ae. tauschii accessions . Their PCR products were separated on a 1.5–2% agarose gel.To assess genetic diversity of the cessions . For theMs2 cDNA), KX533930 (ms2 cDNA), KX585234 (Ms2-associated BAC sequence) and KX585235 (ms2-associated BAC sequence). The RNA-seq data have been deposited in the NCBI Sequence Read Archive under accession number SRP092366. Other data supporting the findings of this study are either within the article and the The DNA sequence data have been deposited in GenBank: KX533929 .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables and Supplementary ReferencesPCR markers used in linkage analysisOther PCR primers used in this studyMS2 geneHaplotypes of the PCR markers used in haplotype analysisP1593SPG5 gene in male-fertile mutantsStructural variations and point mutations in the 0 and T1 plants with the P1593SPG5 genePutative transgenic TMS2 region in common wheat and Ae. tauschiiHaplotypes of the 2-cM MS2 haplotype analysesGermplasm used for the Enriched GO terms in male-sterile anthers (SAEG & Y2H) and male-fertile anthers (FAEG) in the S2 stageMS2 interacting-proteins from cDNA library screening


P = 0.001), poor patient survival (P < 0.001) and a high Ki67 staining index (P = 0.008) in 187 human breast cancer specimens. Furthermore, we find that overexpressing E2F8 promotes, whereas silencing E2F8 suppresses, the proliferation and tumorigenicity of breast cancer cells both in vitro and in vivo. We further demonstrate that E2F8 transcriptionally upregulates CCNE1 and CCNE2 via directly interacting with their respective gene promoter, which accelerates the transition of G1 to S phase of breast cancer cells. Taken together, these findings uncover a novel biologic role and regulatory mechanism of E2F8 responsible for the progression of breast cancer, indicating E2F8 may represent a novel prognostic biomarker and therapeutic target against breast cancer.E2F transcription factors are involved in cell cycle regulation and synthesis of DNA in mammalian cells, and simultaneously play important roles in the development and progression of cancer when dysregulated. E2F8, a novel identified E2F family member, was found to be associated with the progression of several human cancers; however, the biological role and clinical significance of E2F8 in breast cancer remain to be further elucidated. Herein, we report that E2F8 is robustly elevated in breast cancer cell lines and clinical breast cancer tissue samples, respectively. The high expression level of E2F8 significantly correlates with clinical progression ( Sustaining proliferation is thought to be the most fundamental hallmark of cancer . General+/− mice significantly suppressed the development of pituitary tumors, extending the tumor-free lifespan of Rb1+/− mice [The E2Fs (E2F1-E2F8) are a large family of transcription factors containing one or more conserved DNA binding domains (DBDs) that bind to target promoters and regulate their expressions . E2F fam+/− mice –16. In c+/− mice . Moreove+/− mice –22. Henc+/− mice , 24 and +/− mice  and lymp+/− mice  in zebra+/− mice . Moreove+/− mice –30. Reim+/− mice . In addi+/− mice  and lungin vitro and in vivo. Furthermore, we demonstrate that E2F8 can promote entry into the G1/S phase of the cell cycle via transcriptionally upregulating cyclin E1 and cyclin E2 expression, thus contributing to cell proliferation and tumorigenicity in human breast cancer. Taken together, our findings indicate that E2F8 plays an important role in the progression of human breast cancer and suggest that E2F8 may be a potential target for human breast cancer treatment.In the present study, we show that E2F8 is markedly upregulated in human breast cancer and closely correlates with the clinicopathological features and prognosis of breast cancer. Overexpressing E2F8 dramatically promoted, whereas silencing E2F8 inhibited the proliferation and tumorigenicity of breast cancer cells both To investigate the clinical significance and biological role of E2F8 in breast cancer, we first analyzed the mRNA expression of E2F8 in breast cancer tissues with different molecular subtypes which show significant heterogeneity of breast cancer using published data from The Cancer Genome Atlas (TCGA) . As showP < 0.05, Figure 2 test revealed that E2F8 levels significantly correlated with the clinical stage, and TNM classifications in patients with breast cancer   in 187 paraffin-embedded, archived breast cancer tissues, including 33 cases of clinical stage I (17.6%), 95 cases of stage II (50.8%), 51 cases of stage III (27.3%) and 8 cases of stage IV breast cancers (4.3%) . Quantit < 0.05) , indicatP < 0.001; Figure n = 128, P = 0.001; Figure n = 59, P = 0.039; Figure Furthermore, Kaplan-Meier and log-rank tests for survival analysis revealed that patients with high E2F8 expression had a significantly poorer overall survival compared to patients with low E2F8 expression  and our IHC results (P < 0.001)   based on) Figure , suggestin vitro.We then evaluated the role of E2F8 in breast cancer cell proliferation by stably exogenously overexpressing, or endogenously knocking down of E2F8 expression via retrovirus infection Figure . An MTT Since E2F8 expression was correlated with the clinical staging and TNM classification of breast cancer , we furtin vivo. As shown in Figure in vivo.Moreover, the role of E2F8 in the tumorigenicity of breast cancer cells was further determined We then explored the mechanism underlying the promotion of cellular proliferation by E2F8. GSEA results indicated that high E2F8 expression was significantly correlated with the cell cycle-associated gene signatures, suggesting that E2F8 is involved in the cell cycle regulation Figure . MoreoveSince E2F8 is involved in the cell cycle regulation of breast cancer cells, we examined the expression of cell cycle regulators. As shown in Figure Interestingly, by analysis of the promoter regions of CCNE1 and CCNE2 using the ConSite  and Genor = 0.723, P = 0.018), cyclin E2 , and phosphorylation level of Rb . Collectively, these results further support the notion that upregulation of E2F8 contributes to uncontrolled cell proliferation and tumorigenecity, resulting in poor clinical outcome in breast cancer.Finally, we examined whether E2F8-mediated cyclin E1 and cyclin E2 activation in breast cancer cells was clinically relevant. As shown in Figure E2F proteins have been proved to be important regulators of many processes relevant to cancer. For instance, the most studied member E2F1 maintained centrosome amplification and inhibited the promoter activity of the tumor suppressor gene ARHI, contributing to the tumorigenesis of breast cancer , 19. Newin vivo tumor models. Thus, our findings suggest that E2F8 contributes to the proliferation capabilities in different breast cancer subtypes.Based on gene expression profiles and genomic characterization, four main breast cancer subtypes have been proposed: luminal A, luminal B, HER2-enriched and basal-like , 36. SubThe functions of E2F8 on cell cycle progression and tumorigenesis in breast cancer are still unclear. In the present work, overexpression of E2F8 induced the expression of multiple cell cycle regulators, and upregulated cylin E1 and cyclin E2 by directly targeting their promoter elements, leading to G1/S phase transition and high cell proliferation rate. The CDK2-cyclin E complex was well known as they play an important role in tumor development through regulation of the cell cycle –40. The The E2F proteins are conventionally considered to function as either activators (E2F1-3a) or repressors (E2F3b-8) of transcription during cell cycle regulation . E2F8 waIn summary, our study has revealed that E2F8 upregulation plays an important role in breast cancer progression and E2F8 is a critical cell cycle promoter by directly upregulating CCNE1, and CCNE2. Understanding the precise role of E2F8 in breast cancer pathogenesis and in the cell cycle regulation promises to increase our knowledge of the biological basis of cancer development and may also facilitate the development of new therapeutic strategies against breast cancer.2 atmosphere.Primary normal breast epithelial cells (NBEC1 and NBEC2) were established from the mammoplasty materials of two women at the Department of Plastic Surgery, the First Affiliated Hospital of Sun Yat-sen University, according to the rules and regulations relating ethical issues on research use of human subjects in China, as described previously . Breast A total of 187 paraffin-embedded breast cancer specimens were collected for this study, which had been diagnosed histopathologically at the Sun Yat-sen University Cancer Center from 2002 to 2007. Clinical information of the samples is described in detail in Western blotting analysis was performed as described previously . BrieflyImmunohistochemistry (IHC) analysis was performed on the 187 paraffin-embedded breast cancer tissue sections as previously described . The degt-test to compare the average MOD difference between different groups of tissues, and P < 0.05 was considered significant.The method of MOD was used to determine the immunostaining intensity of each tested specimen and performed as previously reported . Briefly5) were seeded and infected by retroviral generated by pMSCV-puro-E2F8 or pLKO-puro-E2F8-shRNA transfecting in 293FT for 3 days. All these cells were further transfected with pMSCV-neo-luci plasmid. The stable cell lines expressing E2F8-luci and E2F8-shRNA-luci were elected with 0.5 μg/mL puromycin and 250 μg/mL G418 for 10 days. The sequences of primers were provided in the Human CCNE1 and CCNE2 promoters were cloned, respectively, into the pGL3 luciferase reporter plasmid . Transfection of luciferase reporter plasmids was performed using the Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer's instruction. Human E2F8 cDNA was PCR-amplified and cloned into the pMSCV-puro-retro vector (Clontech). Two short-hairpin RNA (shRNA) against E2F8 in pLKO-puro vector were commercially purchased (Sigma-Aldrich). Luciferase cDNA was PCR-amplified and cloned into the pMSCV-neo-retro vector (Clontech). Cells -2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay as described previously . 2 × 1033 cells per well). The cell mixture was plated on top of a bottom layer with 0.66% agar completed medium mixture. At 10 days, viable colonies that were larger than 0.1 mm were counted.Cells were trypsinized and suspended in 2 mL of complete medium plus 0.3% agar (Sigma) on 6-well plate (5 × 10n = 6/group). One group of mice was inoculated subcutaneously with SK-BR-3/pMSCV-vector cells (5 × 106) in the left mammary fat pad and with SK-BR-3/E2F8 cells (5 × 106) in the right mammary fat pad per mouse. The other group of mice was inoculated subcutaneously with SK-BR-3/pSUPER-vector cells (5 × 106) in the left mammary fat pad and with SK-BR-3/E2F8-sh#1 cells (5 × 106) in the right mammary fat pad per mouse. 7 days later, kinetics of tumor formation was estimated by measuring tumor size at every 3 day interval. Tumor volume was calculated using the equation (L*W2)/2. On day 34, tumors were detected by an IVIS imagining system , then animals were euthanized, tumors were excised, weighed and paraffin-embedded. Serial 6.0 μm sections were cut and subjected to immunohistochemical and H&E staining. After deparaffinization, sections were IHC analyzed using an anti-Ki67  or H & E stained with Mayer's hematoxylin solution. Proliferation index was quantized by counting proportion of Ki67-positive cells among the total number of invasive cells in the area scored based on the recommendations from the International Ki67 in Breast Cancer working group [Female BALB/c-nu mice  were purchased from the Center of Experimental Animal of Guangzhou University of Chinese Medicine, and were housed in barrier facilities on a 12 h light/dark cycle. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University. The mice were randomly divided into two groups  were cultured in triplicate in 48-well plates for 24 h. 100 ng luciferase reporter plasmids or the control-luciferase plasmid, plus 1 ng pRL-TK Renilla plasmid (Promega), were transfected into cells using the Lipofectamine 3000 reagent (Invitrogen), according to the manufacturer's recommendations. Luciferase and Renilla signals were measured 24 h after transfection, using the Dual Luciferase Reporter Assay Kit (Promega), according to a protocol provided by the manufacturer.6) in a 100 mm culture dish were treated with 1% final concentration of formaldehyde to cross-link proteins to DNA, and the reaction was stopped by addition of glycine. The cell lysates were sonicated to shear DNA to sizes of 300–1000 bp. Equal aliquots of chromatin supernatants were incubated with 1 μg of anti-E2F8, anti-H3K27Ac (Sigma) or anti-immunoglobulin G antibodies  overnight at 4°C with rotation. After reverse cross-link of protein/DNA complexes to free DNA, PCR was performed. Specific primers for chromatin immunoprecipitation (ChIP) were presented in the Supplementary Materials and Methods.Cells (2 × 102 test. Bivariate correlations between study variables were calculated by Spearman's rank correlation coefficients. Survival curves were plotted with the Kaplan-Meier method and compared by the log-rank test. Survival data were evaluated using univariate and multivariate Cox-regression analyses. P < 0.05 in all cases was considered statistically significant.All statistical analyses were carried out using the SPSS 17.0 statistical software package. The relationship between E2F8 expression and the clinicopathological characteristics was tested by the χ


P2RX7 gene are associated with the risk of cervical cancer in Taiwanese women. P2RX7 253 T/C, 835 G/A, and 1513 A/C loss-of-function polymorphisms were genotyped in a hospital-based study of 507 women with cervical squamous cell carcinoma (CSCC) and 1619 age-matched healthy control women. The presence and genotypes of HPV in CSCC was determined. The frequency of 253 C/C genotype was found to increase significantly in patients with HPV-16 positive CSCC compared with controls . No significant associations were found for other 2 polymorphisms. Analysis of haplotypes also revealed no significant differences among women with CSCC, those with HPV-16 positive CSCC and controls. In conclusion, inheritance of the C/C genotype at position 253 in the P2RX7 gene may contribute to the risk of HPV-16 associated CSCC in Taiwanese women.Human papillomavirus (HPV) infection and the fate of HPV infected cervical epithelial cells are strictly associated with cervical cancer development. P2X7 receptor has been implicated in both the regulation of immune responses and apoptosis of cervical cancer cells. The study aims to investigate if polymorphisms in the  Cervical cancer is the fourth most common cancer in women worldwide. In Taiwan, cervical cancer also poses a major public health concern, with nearly 2700 women were diagnosed with cervical cancer each year . Human p2+, Na+, and K+ across the plasma membrane [The P2X7 receptor is a homotrimeric ligand-gated cation channel characterized by two transmembrane domains, an extracellular loop, an intracellular N terminus, and a long C-terminal chain containing 240 residues . This remembrane , 9, actimembrane , 11, posmembrane , inhibitmembrane , and themembrane . Furthermembrane , 16.P2RX7) contains 13 exons and is mapped to the 12q24 chromosomal region [P2RX7 gene have been reported to affect receptor function [P2RX7 SNPs have been studied in several types of cancers like breast cancer, chronic lymphocytic leukemia, nasopharyngeal carcinoma, and papillary thyroid cancer [P2RX7 SNPs and cervical cancer has not been determined. We therefore performed an association study of 507 cervical squamous cell carcinoma (CSCC) cases and 1619 controls to test whether specific P2RX7 SNPs are associated with susceptibility to CSCC.The human P2X7 receptor gene . We found the genotypes of 253 T/C SNP in controls differed significantly from CSCC patients (P = 0.01)  in CSCC patients did not survive the Bonferroni correction (cP = 0.06). For the other 2 polymorphic sites examined on the P2RX7 gene, no significant difference was found in relation to the risk of CSCC  , and the significance remained after Bonferroni correction (cP = 0.03). Pairwise calculations of linkage disequilibrium between SNPs showed that there were strong linkage disequilibriums in controls (D′ 0.86–1.00) and cases (D′ 0.91–1.00).By stratification analysis based on HPV-16 positivity, the combined effect of HPV-16 infection and 253 T/C, 835 G/A, and 1513 A/C SNPs were shown in Table The inferred haplotypes based on P2RX7 gene and their haplotypes and CSCC susceptibility in Taiwanese population. Our findings revealed that 253 C/C genotype frequency increased significantly in the subgroup of CSCC women infected with HPV-16 as compared with healthy controls. In addition, it is noteworthy that the frequency of homozygous risk allele of the 253 T/C SNP was very rare in controls, with only two was homozygous in the 1619 cohort  that is equally expressed in normal and cancer cervical cells [7-j and abrogated the P2X7-mediated apoptosis. Moreover, P2X7 activation by extracellular ATP induces lesser ratio of apoptosis in cancer cervical cells than in normal human ectocervical cells through the mitochondrial pathway [A few studies have reported that P2X7 expression was found in both normal and cancer cervical tissues and associated with the growth of cervical cells. A study reported by Li et al. showed that both protein and mRNA levels of the P2X7 were significantly lower in the cervical cancer than in the normal tissues . They alal cells . The low pathway . These fP2RX7 gene is highly polymorphic and a number of SNPs within the coding region have been reported to affect P2X7 receptor function. The C allele of 253 T/C SNP changes valine to alanine at residue 76 (V76A) in the extracellular loop and produces a partial loss of P2X7 function [835 G/A, resulting in a change of the amino acid arginine to histidine at residue 270 (R270H) was also found to have a reduced receptor function [1513 A/C polymorphism causes an amino acid change from glutamic acid to alanine at residue 496 (E496A) in the C-terminus of P2X7 receptor and abolishes the receptor function [1513 C polymorphism has been shown to associate linearly with the loss of P2X7 receptor function. There are other variants causing either loss-of-function such as 474 G/A, 853 G/A, 946 G/A, 1096 C/G, and 1729 T/A; or gain-of-function such as 489 C/T, 1068 G/A, and 1405 A/G [P2RX7 gene are clearly involved in the regulation of P2X7 receptor function.The function . Anotherfunction . The 151function . The all1405 A/G . OverallP2RX7 gene and HPV-16 positive CSCC risk are not addressed in this study. Nevertheless, based on our findings, it is conceivable to reason that impaired receptor function caused by 253 T/C polymorphism may lead to decreased immune cell function and apoptosis of cervical cancer cells when stimulated with exogenous ATP. This specific P2RX7 variant is therefore possible to lead to failure in clearance of HPV infections and the consequential CSCC development. If this correlation can be supported by other studies, women with this genotype should undergo cervical screening more frequently or receive prophylactic HPV vaccine.What molecular mechanisms lie beneath the association between 253 C/C genotype and harboring HPV-16 infection are more susceptible to CSCC in Taiwanese women. Future studies that explore the associations between other functional P2RX7 SNPs and CSCC should aid in defining the precise role of P2RX7 gene in CSCC pathogenesis.In conclusion, we showed for the first time that patients possessing the A hospital-based case-control study was conducted at the Mackay Memorial Hospital. Five hundred and seven Taiwanese women with pathologically proven CSCC (mean ± SD age at diagnosis: 54.5 ± 12.5 years) were enrolled in the study. Controls were 1619 women (mean ± SD age at sampling: 53.1 ± 13.3 years) selected randomly from a cervical cancer screening program. Exclusion criteria included abnormal Pap result, history of cervical neoplasia, skin or genital warts, immunocompromised conditions, other cancers, and previous operations on the uterine cervix. All the control subjects were frequency matched to the cases by age. The study was approved by the Institutional Review Board of Mackay Memorial Hospital, in accordance with the Helsinki Declaration, and all participants provided informed written consent for use of their surgical resections or cervical scrapings.Formalin-fixed, paraffin-embedded tissue blocks from CSCC patients were sectioned and dewaxed, and genomic DNA was then extracted using the Qiagen DNeasy Tissue Kit  according to the manufacturer's protocol. Genomic DNA of controls was extracted from cervical scrapings using Qiagen DNA extraction kit.HPV detection and genotyping were performed by polymerase chain reaction (PCR)-based amplification of a fragment of approximately 192 bp in the L1 region of the HPV genome with a pair of degenerate primers, GP6+/MY11 , 26. The253 T/C (rs17525809), 835 G/A (rs7958311), and 1513 A/C (rs3751143) SNPs were genotyped because of their potential functional significance. They were determined using the Pre-Developed TaqMan Allelic Discrimination Assay . Briefly, PCR were carried out in a 96-well GeneAmp PCR System 9700 (Applied Biosystems) with mixes consisting of 10 ng of genomic DNA, 5 μl of TaqMan Universal PCR Master Mix, 0.5 μl of 20× Assay Mix, and ddH2O to a final volume of 10 μl. Thermal cycle conditions were as follows: denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 92°C for 15 sec, and annealing and extension at 60°C for 1 min. After PCR, the TaqMan assay plates were transferred to the ABI PRISM 7000 Sequence Detection System (Applied Biosystems) where the endpoint fluorescence intensity in each well of the plate was read. The allele specific fluorescence data from each plate were analyzed using the SDS v.1.1 software (Applied Biosystems) to automatically determine the genotype of each sample.The P2RX7 haplotypes as well as linkage disequilibrium between paired SNPs in controls and cases were estimated using the Haploview 4.2 program [cP = 1 - (1-P)n, was used for multiple comparisons where cP is the corrected P value, P the uncorrected value, and n the number of comparisons [P value (cP) of <0.05 (2-tailed) was considered statistically significant. Using the Quanto Ver. 1.1 software , we designed the study to have >90% power at a 5% significance level to determine a genotype relative risk of 1.8 conferred by P2RX7 variants with an estimated prevalence of 360/100,000 [Statistical differences in frequency between each genotype, allele, and haplotype of CSCC, HPV-16 positive CSCC, and control groups were compared using chi-square test with Yates’ correction or Fisher's exact test (when the number of subjects in a cell was <5). Odds ratio (OR) and 95% confidence interval (CI) were calculated to determine the magnitude of associations. The Hardy-Weinberg equilibrium was assessed for each SNP in the control group by chi-square analysis. The frequencies of  program . Haplotyparisons . In thisparisons  but no cparisons  and 4 foparisons . A corre/100,000 .


Here we identify a mutation (C19T) that converts an arginine to a tryptophan (R7W) in the TYRO3 protein tyrosine kinase 3 (Tyro3) gene, which resides within the anx critical interval, as contributing to the severity of anx phenotypes. Our observation that, like −/−Tyro3 mice, anx/anx mice exhibit abnormal secondary platelet aggregation suggested that the C19T Tyro3 variant might have functional consequences. Tyro3 is expressed in the hypothalamus and other brain regions affected by the anx mutation, and its mRNA localization appeared abnormal in anx/anx brains by postnatal day 19 (P19). The presence of wild-type Tyro3 transgenes, but not an R7W-Tyro3 transgene, doubled the weight and lifespans of anx/anx mice and near-normal numbers of hypothalamic Npy-expressing neurons were present in Tyro3-transgenic anx/anx mice at P19. Although no differences in R7W-Tyro3 signal sequence function or protein localization were discernible in vitro, distribution of R7W-Tyro3 protein differed from that of Tyro3 protein in the cerebellum of transgenic wild-type mice. Thus, R7W-Tyro3 protein localization deficits are only detectable in vivo. Further analyses revealed that the C19T Tyro3 mutation is present in a few other mouse strains, and hence is not the causative anx mutation, but rather an anx modifier. Our work shows that Tyro3 has prosurvival roles in the appetite regulatory circuitry and could also provide useful insights towards the development of interventions targeting detrimental weight loss.Severe appetite and weight loss define the eating disorder anorexia nervosa, and can also accompany the progression of some neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS). Although acute loss of hypothalamic neurons that produce appetite-stimulating neuropeptide Y (Npy) and agouti-related peptide (Agrp) in adult mice or in mice homozygous for the anorexia ( Summary: The receptor tyrosine kinase Tyro3 improves weight and survival in the anorexia mouse model, and is one of the few factors known to sustain appetite regulatory circuitry. Detrimental appetite and weight loss are the defining features of the restrictive subtype of the eating disorder anorexia nervosa (ANR) , and alsNeuronal circuitry that links the hypothalamus with extra-hypothalamic regions, including the parabrachial nucleus and the nucleus accumbens, monitors peripheral energy levels and regulates food intake  mutation restrict their eating and become severely emaciated  neurons begin to degenerate at postnatal day (P)10-12  and NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, assembly factor 1 (Ndufaf1), which reside in the anx critical interval 10-12 . By P19-d perish . In addianx mice , and theanx mice . Currentinterval . Ndufaf1anx/anx mice, we considered whether the TYRO3 protein tyrosine kinase 3 (Tyro3) gene, which has prosurvival roles in neuronal culture  receptors, is a member of the TAM family of receptor tyrosine kinases -expressing neurons into the hypothalamus is compromised , the weight and lifespans of anx/anx mice was doubled relative to that of their non-transgenic anx/anx littermates and hypothalamic Npy+ neuron degeneration was delayed. Finally, we show that in the cerebella of transgenic mice, R7W-Tyro3 protein appeared mislocalized. Additional sequencing analyses revealed that the R7W-Tyro3 variant is present in AKR/J, FVB/NJ and NZW/lacZ mice, and thus R7W-Tyro3 is not the primary anx causative event, but rather is a strain-specific modifier of anx phenotypes. Taken together, these studies identify Tyro3 as having important neuroprotective roles in the appetite regulatory circuitry.Here, we identify a point mutation (C19T) in the signal sequence of Tyro3 (R7W-Tyro3), and detect a deficit in secondary platelet aggregation in Tyro3 gene is located within the anx critical interval, which we had refined to a 3.5 Mb region between D2Mit484 and D2Mit3 by using simple sequence length polymorphisms (SSLPs) to analyze 335 progeny (670 meioses) from heterozygous intercrosses on C57BL6/J (n=111) and Mus molossinus/Ei (MOLF/EiJ) (n=224) strain backgrounds . Upon sequencing the exons and flanking intronic regions of Tyro3, we identified a cytosine-to-thymine (C19T) mutation in the signal sequence of Tyro3  and 17.5±5.9% (mean±s.e.m.)  of anx/anx platelets contained large vacuoles, which were never observed in +/+ or anx/+ platelets . Previously, Tyro3 mRNA had been detected in the subventricular zone and layers 2/3, 5 and 6 of the cerebral cortex, the CA1 region of the hippocampus, the median eminence of the hypothalamus, and granule cells of the cerebellum in the adult mouse brain , but also detected Tyro3 mRNA in areas affected by the anx mutation. Tyro3 was strongly expressed in the arcuate nucleus, ventromedial nucleus and, at lower levels, in the paraventricular nucleus of the hypothalamus and in ependymal cells lining the third ventricle . However, at P21, anx/anx brains showed marked differences in Tyro3 expression. At low magnification, mutated (C19T) Tyro3 expression appeared overall more diffuse in specific cell populations and throughout the brain, and mislocalized in some regions  and serotonergic neurons . In the arcuate nucleus and median eminence, a subset of Tyro3-GFP- and R7W-Tyro3-GFP-expressing neurons co-express Npy . In the hindbrain raphe nuclei, Tyro3-GFP- and R7W-Tyro3-GFP-expressing neurons express the 5-HT marker tryptophan hydroxylase 2 (Tph2) . +/+ and anx/+ mice transgenic for HuTyro3 or Tyro3-GFP were indistinguishable from non-transgenic +/+ and anx/+ littermates. Tyro3 transgenic wild-type mice showed no adverse effects with regards to viability, fertility and overall health.Next, we performed transgenic rescue experiments. Because phylogenetic sequence comparisons between n vector A. We genn vector B. The R72 (Tph2) , thus idanx/anx mice transgenic for Tyro3-GFP or HuTyro3 showed mild or no neurobiological anx-related behaviors such as ataxia, shaking and headweaving at P21, and survived at least until P35, as shown for all Tyro3-GFP and HuTyro3 transgenic lines (P<0.01). By contrast, the R7W-Tyro3-GFP transgene, even though expressed at levels similar to some of the rescuing Tyro3 transgenes , could not delay onset of the behavioral phenotypes by P12-15 or extend the lifespans of anx/anx mice past P21  and dorsomedial nucleus of the hypothalamus, as detected by silver-enhanced immunoperoxidase staining  . Signal sequence mutations can also affect translocation from the cytoplasm to the endoplasmic reticulum and post-translational modifications, including N-glycosylation, that occur during protein secretion. In accordance with predictive analysis, when we assessed proteolytic processing of in vitro transcribed and translated Tyro3 and R7W-Tyro3 proteins mixed with signal peptidase from canine pancreatic rough microsomes, the extent of signal sequence cleavage and glycosylation of the R7W-Tyro3 protein seemed equivalent to that of Tyro3 . Thus, the C19T mutation in Tyro3 does not impact signal sequence function in vitro.To understand the possible consequences of the C19T mutation on Tyro3 localization and function, we examined whether the mutation impinged on signal sequence function SignalP) , which eSignalP) , did notin vitro and in vivo. We detected no differences in Tyro3- or R7W-Tyro3-dependent Akt phosphorylation in the absence or presence of serum in transiently transfected COS7 cells, in stably transfected Neuro2A cells or in protein extracts from +/+ and anx/anx cerebella or hippocampi . Using immunofluorescence with an anti-Tyro3 antibody on cultured cortical and hippocampal neurons from anx/anx and +/+ littermates, endogenous R7W-Tyro3 and Tyro3 localization also appeared indistinguishable . However, when we used silver-enhanced immunohistochemistry with an anti-GFP antibody to survey Tyro3-GFP and R7W-Tyro3-GFP protein distribution in the brains of P10 transgenic +/+ mice, we detected distinctly different localization patterns in cerebella  with ANR  emerged as a candidate for ANR associated with increased depression and anxiety in a high-throughput sequencing study  or hexanucleotide repeat expansions in chromosome 9 open reading frame 72 (C9ORF72), is unknown. The observation that ALS shows great variability in disease onset and progression even within families , ciliary neurotrophic factor (CNTF), and insulin-like growth factor 1 (IGF-1) , which, in familial cases, is often caused by mutations in Cu/Zn superoxide dismutase (families  has fuelfamilies . As TAM  (IGF-1) . Moreove (IGF-1) . Given oAls2) gene exhibited a shorter life span than wild-type littermates when they were on a FVB/N than on a C57BL6/J background , survived twice as long on a pure C57BL/6N background than on the R7W-Tyro3-carrying FVB/N background . Moreoveckground . Thus, tin vivo. Often, the biochemical effects of signal sequence mutations that are associated with human disorders correlate well with the disease state, as shown for prepro-parathyroid hormone (PTH)  resulted in less protein at the plasma membrane, but the in vitro co-translational processing of the CASR mutant protein was unaffected  . Howeveraffected . Nonetheaffected . Thus, Cin vitro signal sequence function is that it impacts RNA-sequence-based functions. Although, conventionally, the main roles of signal sequences have been based on their peptide sequences, the RNA sequence of the signal sequence coding region of some secretory proteins can activate nuclear mRNA export  did not score disruption or creation of a binding site for a known RNA binding protein (RBP) by the C19T Tyro3 mutation. Moreover, the RNA binding profiles for some neuronally important RNA binding proteins (RBPs), such as FMRP1   . Many reFig. S9)  and Arc Fig. S9) , reside in vivo and that, given that these mice appear normal, this effect is not due to overtly compromised neuronal health. The molecular mechanism by which the R7W-Tyro3 variant disrupts Tyro3 function(s) remains unknown, and given that its effect is only apparent in vivo in the mouse and most striking in anx/anx mice, identification and validation of such a mechanism is more challenging.Regardless of mechanism, the mislocalization of R7W-Tyro3-GFP protein in the cerebella of wild-type mice does suggest that the R7W variant causes protein mislocalization anx phenotypes, and shown that normal Tyro3 acts protectively to sustain the appetite regulatory circuitry in anx/anx mice. Whereas predictive programs and in vitro analyses could not predict or detect appreciable differences, the biological consequences of both the R7W-Tyro3 variant and normal Tyro3 are apparent in vivo. Furthermore, our findings suggest the existence of subtle, quite possibly often missed, mechanisms that might regulate protein and/or RNA subcellular localization. To date platelet function has not been systematically examined in ANR. Our observations suggest that, in the future, systematic analyses of platelet function in eating disorders and possibly other psychiatric and neurodegenerative disorders might prove useful and help categorize these into specific biological subtypes. Overall, our observations highlight the importance of analyses in the mouse or other model organisms as a testbed during the search for human disease modifiers and for genetic variants with measurable biological effects. Finally, enhancing Tyro3 expression or signaling could provide a strategy for counteracting deficits in the appetite regulatory circuitry that might contribute – in a primary or secondary manner – to the ingestive dysfunctions seen in ANR and some neurodegenerative disorders.In conclusion, here we have identified a mutation in the signal sequence of the Tyro3 tyrosine kinase receptor (R7W-Tyro3) as enhancing anx/+ mice maintained on a B6C3Fe a/a hybrid background were obtained from Jackson Laboratories  and crossed onto C57Bl6/J and Molf/EiJ backgrounds. To identify mice carrying the C19T-Tyro3 mutation, polymerase chain reaction (PCR)-based assay using primers 5′-GATGGCGCTGAGGCGGAGCATG and 5′-CGCGGCCGGAGGTCTGGCAG, and subsequent digestion with NlaIV was used. All animal experiments were performed in strict adherence to guidelines for experimentation with laboratory animals set by the Canadian Animal Care Center (CACC).6 platelets per mouse were incubated with 1 U thrombin (Sigma) for 10 min at room temperature (RT), labeled with FITC-conjugated anti-P-selectin antibody  and data analyzed using the Mann–Whitney Test.Platelet aggregation assays were performed as in anx/anx and three anx/+ and +/+ animals were analyzed. Data was analyzed using the Mann–Whitney Test.Isolated platelets were fixed with 4% paraformaldehyde and 2% gluteraldehyde in 0.1 M sodium cacodylate buffer pH 7.3. Sectioned samples were analyzed by Tecnai 20 transmission electron microscopy followed by data analysis using Excel Docu (Soft Imaging Systems). For morphologic analyses, 56-63 platelets from each of four anx/anx, anx/+ and +/+ mice was isolated using Trizol reagent (Invitrogen) according to manufacturer's instructions. cDNA was synthesized from total RNA using oligo-dT primers and the Superscript III First Strand Synthesis System (Invitrogen) according to manufacturer's instructions (2 µg of RNA used in 20 µl reverse-transcription reaction). Primers specific for Tyro3 (5′-AATGCCGAGATTTACAACTACCTCAT-3′ and 5′-GTTCCATTCGACAGACACGTGAAGCTT-3′) and GAPDH (5′-TGTGAACGGATTTGGCCGTAT-3′and 5′-CATGTAGACCATGTAGTTGAG-3′) were used. Reactions were run with SYBR green PCR master mix (Invitrogen) in an AB 7500 fast real-time PCR system and relative gene expression levels were calculated using Sequence Detection System 2.2 Software (Applied Biosystems). Expression values were normalized relative to sample Gapdh mRNA expression. RNA from five anx/+ and +/+ and three anx/anx animals was analyzed. Data were analyzed using nonparametric t-tests and Mann–Whitney test.RNA from P19 cortices from in situ hybridization on sections was performed on brains perfused with 4% paraformaldehyde in diethyl-pyrocarbonate-treated 1×PBS and embedded in optimal cutting temperature medium . Coronal sections of 14 µm were cut using the Leica VT1000 cryostat. RNA in situ hybridization was performed on tissue sections as described in HindIII fragment, containing the partial coding sequence of the Tyro3 intracellular kinase domain, cloned into pBluescript KSII. The sense probe was generated with XhoI digestion and T3 RNA polymerase transcription, the antisense probe upon EcoRI digestion and in vitro transcription with T7 RNA polymerase .RNA Tyro3 and R7W-Tyro3 cDNA was generated from RNA from +/+ and anx/anx brains using Superscript First-Strand Synthesis RT System (Invitrogen), amplified by real-time PCR, and inserted into pDONR201 with BP Clonase (Invitrogen). To generate an untagged human TYRO3 construct, human TYRO3 cDNA  was inserted into filled-in SalI sites of pDONR201. Using LR Clonase, these versions of Tyro3 were cloned into the T3Xpressn vector. The T3Xpressn vector consists of 9.5 kb BamHI-NotI fragment 5′ of and containing the transcriptional start site for signal sequence-containing exon 2c , and used to generate transgenic mice by standard pronuclear microinjection into FVBN/J oocytes. Positive transgenic mice were identified by Southern blot analysis and by a PCR-based assay using the following primers: GWTyro3-F1: GGGGACAAGTTTGTACAAAAAAGCAGGCTGCCGCCGATGGCGCTGAGGCGGAGCATGGGGTG and GWTyro3-R1: GGGGACCACTTTGTACAAGAAAGCTGGGTTAACTGCTACTGTGAGGCAGTAGCCCTTG. exon 2c  and 154 In all cases, a minimum of three mice of a given genotype and stage were analyzed. Adult brain cryosections were blocked in 10% normal donkey serum (NDS) in PBS with 0.2% Triton X-100 (PBST) for 1 h at RT. Antibodies were diluted in 2% NDS PBST and incubated overnight at 4°C. Samples were washed and incubated with Cy3 or Alexa Fluor 488 secondary antibodies  for 1 h at RT. Samples were mounted with Vectashield plus DAPI (Vector Labs).For immunofluorescence, primary neuronal culture samples were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 PBS for 5 min at RT. All samples were blocked in histoblock  for 1 h at RT, incubated overnight in primary antibodies. Secondary antibodies were applied and samples processed as described above.The following antibodies were used: anti-Npy , anti-Tyro3  and mouse anti-MAP2 , rabbit anti-calbindin , anti-5HT anx/anx and Tyro3-GFP; anx/anx mice for anti-Npy staining, and through cerebella of P19 and P10 Tyro3-GFP; +/+ and R7W-Tyro3-GFP;+/+ mice for anti-GFP staining. Immunoperoxidase staining was performed with the Vectastain kit (Vector Laboratories) and enhancement with silver nitrate was performed as described in anx/anx mice and as a more pronounced circular staining within the soma of anx/anx; Tyro3-GFP transgenic animals. Thus, it is possible that the numbers of Npy+ soma is underestimated in wild-type animals, and that the difference between wild-type and anx/anx Npy+ neurons is even greater than recorded here. Pairwise comparisons were performed using the Wilcoxon signed-rank test  .For silver nitrate enhanced immunoperoxidase staining, 20 µm sections were cut through hypothalami of P19 +/+, t-test. Ratios of Akt-P/Akt were 9.7±2.3% for Neuro2A (P=0.015), 29.9±7.0% for T3-RFP (P=0.451), and 37.1±6.1% for R7W-RFP (P=0.451).Tyro3-pDest47 and R7W-Tyro3-pDest47 plasmids were generated using the Gateway system (Invitrogen). Triple GFP-coding sequences in pDest47 were replaced with those of mRFP, and the Gateway system was used to generate Tyro3-RFP and R7W-Tyro3-RFP pDest47 constructs. After transfection into Neuro2A cells with Effectene transfection reagent (Qiagen), stable cell lines were generated by selecting with 500 µg/ml G418, and cell lines expressing equivalent amounts of Tyro3-RFP or R7W-Tyro3-RFP were used to assess Akt phosphorylation. Cells were serum-starved overnight, activated with serum for 15 min, harvested, lysed in RIPA buffer , incubated on ice for 10 min, and centrifuged at 4°C for 10 min. SDS-PAGE was carried out on 10% gels and transferred to nitrocellulose membranes. After blocking  at RT for 1 h, membranes were incubated with either anti-RFP , anti-Akt  and anti-phospho-Akt  in 5% BSA TBST at 4°C overnight, and immunoreactive antibodies were detected using the enhanced chemiluminescence (ECL) system (Perkin Elmer). Image J (NIH) was used to measure densitometry and subsequent statistical analysis was completed with GraphPad Prism software. A minimum of three separate cell lines expressing Tyro3-RFP or R7W-Tyro3-RFP were used and assays were performed at a minimum in duplicate. Data were analyzed using an unpaired anx/+ and anx/anx cerebella in RIPA buffer at 4°C, incubated on ice for 10 min and centrifuging at 10,000 g for 10 min at 4°C. 10 µg of protein was analyzed by immunoblot for Akt and phospho-Akt levels. Ratios of Akt-P/Akt were 119±16% for anx/+ (n=6) and 106±30% for anx/anx .Cerebellar lysates were generated by homogenizing P19 In vitro transcription/translation (TnT) with a Tyro3 and R7W-Tyro3 template containing all eight predicted N-glycosylation sites coupled with the addition of canine pancreatic microsomal membranes (CMM) and subsequent addition of endoglycosidase H was used to examine the cotranslational processing of R7W-Tyro3. Cell-free expression of Tyro3 and R7W-Tyro3 protein was performed using a TnT Coupled Reticulocyte Lysate System . DNA templates of Tyro3 and R7W-Tyro3 protein containing primary sequence for the first 410 amino acids of the extracellular domain, and, thus, all eight predicted N-glycosylation sites, were generated by digestion with NheI. TnT was performed in the presence and absence of Canine Pancreatic Microsomal Membranes (Promega) according to the manufacturer's instructions. To remove any N-acetylglucosamine residues, some products were denatured in denaturing buffer  at 37°C for 15 min and subsequently digested with 500 U of endoglycosidase H  at 37°C for 1 h. Samples were analyzed by 15% SDS-PAGE and autoradiography. β-lactamase from Escherichia coli was used to assay for signal peptidase activity, and α-factor from Saccharomyces cerevisiae to assay for core glycosylation activity.Primary hippocampal and cortical neurons were harvested from embryonic day-(E)17 mice and cultured for 10-14 days as described in  and Bou. For tra


Listeria monocytogenes  induces specific CD8+ and CD4+ T cell responses, and has been identified as a promising cancer vaccine vector. Cervical cancer is the third most common cancer in women worldwide, with human papillomavirus (HPV), particularly type 16, being the main etiological factor. The therapeutic HPV vaccines are urgently needed. The E7 protein of HPV is necessary for maintaining malignancy in tumor cells. Here, a genetically modified attenuated LM expressing HPV16 E7 protein was constructed. Intraperitoneal vaccination of LM4Δhly::E7 significantly reduced tumor size and even resulted in complete regression of established tumors in a murine model of cervical cancer. We provided evidence that recombinant LM strains could enter the tumor tissue and induce non-specific tumor cell death, probably via activation of reactive oxygen species and increased intracellular Ca2+ levels. LM4Δhly::E7 effectively triggered a strong antigen-specific cellular immunity in tumor-bearing mice, and elicited significant infiltration of T cells in the intratumoral milieu. In summary, these data showed LM4Δhly::E7 to be effective in a cervical cancer model and LM4Δhly::E7 induced an antitumor effect by antigen-specific cellular immune responses and direct killing of tumor cells, indicating a potential application against cervical cancer.Attenuated  Listeria monocytogenes , Lactobacillus lactis, and salmonella are the most common bacteria-based vectors for tumor immunotherapy  could augment specific immune responses and significant reduction of tumor growth  LM vaccine is safe and effectively retards tumor growth in a mouse model of cervical cancer. Furthermore, we provide evidence that the antitumor mechanism is due to induction of specific cytotoxic T lymphocyte (CTL) responses and direct killing of tumor cells.Previous studies have reported that attenuated LM was applied to deliver the HPV E7 gene via a plasmid-based complementation system, but there existed a high risk of plasmid loss and persistence of antibiotic resistance genes in clinical trials  peptide for the H-2Db restricted epitope was synthesized by Beijing Scilight Biotechnology, LLC . Virulent L. monocytogenes Yangzhou strain yzuLM4 (serotype 1/2a) was isolated and maintained at the Jiangsu Key Laboratory of Zoonosis, which was cultured in a Biosafety Level 2 laboratory. Shuttle vector pKSV7 was kindly provided by Dr. Guoqiang Zhu . Plasmid pNF8, containing the full-length gfp-mut1 gene was a gift from Prof. Nicolas Fortin, eau .HPV16 E72.C57BL/6 mice (7–8 weeks old) were purchased from the Comparative Medical Center of Yangzhou University. Animals were housed and used in accordance with the protocols approved by the Experimental Animal Center Institutional Committee of Yangzhou University. The TC-1 cell line, C57BL/6 lung tumor epithelial cells immortalized by HPV16 E6/E7 and transformed with the c-Ha-ras oncogene, was purchased from the Tumor Cell Center of the Chinese Academy of Medical Sciences . RAW264.7 murine macrophage-like cells were maintained in our laboratory. Cells were cultured in RPMI 1640, supplemented with 10% fetal calf serum, 1 mmol/L sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin in a 37°C incubator with 5% COhlya and downstream fragment of hlyb were amplified from the LM4 genome using the following primers: hlya (F) 5′-CCGGTACCAGGTTTGTTGTGTCAGGTAGAGC-3′, hlya (R) 5′-GTAGGTGTATCTCCATGTGAAATTGAATTTTCTTTAT-3′; hlyb (F) 5′-TGTTCTCAGAAACCACCAATCGAAAAGAAACACG-3′, hlyb (R) 5′-GGGTCTAGAACTTGAGATATATGCAGGAGG-3′; the BamHI and XhoI sequences are underlined. HPV16 E7 gene was amplified from pcDNA3.1-E7 using the following primers: E7 (F) 5′-ATAAAGAAAATTCAATTTCACATGGAGATACACCTAC-3′, E7 (R) 5′-CGTGTTTCTTTTCGATTGGTGGTTTCTGAGAACA-3′. hlya, hlyb and E7 were spliced by overlap extension PCR with the primers hlya (F) and hlyb (R). The fusion fragment aEb was cloned into the shuttle vector pKSV7 with restriction enzyme sites BamHI and XhoI. The resulting plasmid pKSV7-aEb was electroporated into electrocompetent LM4 cells. The E7 gene was cloned in-frame with the hly open reading frame and inserted downstream of the hly signal sequence by allelic exchange, and a 42 bp region was deleted from the N-terminal Pro-Glu-Ser-Thr (PEST)-like sequence of the hly gene. The recombinant plasmid- and antibiotic-resistance gene-free strain LM4Δhly::E7 was constructed to express and secrete LLO-E7 fusion protein, retaining a significant degree of attenuation. An irrelevant Listeria construct, LM4Δhly::esat-6 (LM control), which was based on the same method of construction, was included to account for the antigen-independent effects of Listeria on the immune system. Supernatants from the recombinant strain cultures were concentrated by trichloroacetic acid (TCA), and detected by Western blot using a monoclonal antibody against HPV16 E7 protein.The upstream fragment hly::E7 were cultured overnight in brain-heart infusion (BHI) medium. The culture supernatants were collected by centrifugation, and the same OD600 value was adjusted using PBS buffer. Seventy microliters of each supernatant were added to a 96-well plate, and then two-fold serial dilution was performed. Subsequently, 30 μL of 1% sheep erythrocytes were added, and hemolysis was observed after 1 h at 37°C. Hemolysis titers were expressed as the dilution of the supernatant that lysed 50% of total sheep erythrocytes.LM4 and LM4Δhly::E7 cells, resulting in construction of LM4(pNF8) and LM4Δhly::E7(pNF8). Subsequently, RAW264.7 cells cultured in 24-well plates were infected with LM4(pNF8) or LM4Δhly::E7(pNF8) at a multiplicity of infection of 1:20 for 1 h. The cells were incubated in medium containing 50 μg/mL of gentamicin for 1 h at 37°C to eliminate extracellular bacteria. The cells were fixed and stained with phalloidin (Invitrogen), which stains filamentous actin, by taking samples at 1 and 5 h. Intracellular bacteria were monitored by confocal-transmission electron microscopy.Firstly, pNF8 was electroporated into electrocompetent LM4 and LM4Δ5 TC-1 cells were subcutaneously injected into the left flank of C57BL/6 mice. After 1 week, tumor-bearing mice (tumor diameter reached 5 mm) were intraperitoneally administered with 0.1 50% lethal dose (LD50) of LM4Δhly::E7, LM4Δhly::esat-6 or PBS on days 7 and 14 after tumor cell injection. Tumors were monitored, and measured at the longest (L) and widest (W) points with calipers. Tumor volume was calculated as L × W2 × π/6. Experiments were initiated in the tumor-bearing mice to analyze the anti-tumor immune response.To establish the tumor model, 2 × 10hly::E7 on days 0, 7, and 14. Spleens, livers and tumors were homogenized on days 1 and 3 after each immunization. Bacterial numbers were determined by plating the cell suspensions on BHI agar.The tumor-bearing mice were intraperitoneally injected with LM4Δin vivo killing assay was performed as previously described  buffer, another was labeled with 0.25 μM CFSE (CFSElow). CFSEhigh cells were pulsed with the E7 peptide. CFSEhigh and CFSElow cells were mixed at a 1:1 ratio and injected intravenously into C57BL/6 mice at 7 days post-immunization. Fifteen hours later, spleens were harvested and processed into single cell suspensions. The splenocytes were analyzed by flow cytometry according to fluorescence density, and the specific lysis ratio was calculated with the following formula:100 – (100 × (% CFSEhigh immunized/%CFSElow immunized)/(% CFSEhigh control/% CFSElow control)).A standard 5/well. Cells were stimulated in triplicate with E749–57 peptide (10 μg/mL), with ConA  as a positive control or complete medium as a negative control, and incubated at 37°C for 48 h. Samples were then processed according to the manufacturer's instructions . The IFN-γ/IL-4-reactive spots were quantified by an automated ELISPOT Bioreader 5000 .The ELISPOT assay was performed as previously described  for 2 h at 37°C with agitation. Single cell suspensions were pooled through a nylon mesh.Cells were stained with anti-CD3-fluorescein isothiocyanate (FITC)  anti-CD4-phycoerythrin (PE)  and anti-CD8-allophycocyanin (APC)  monoclonal antibodies and then analyzed by flow cytometry.Tumor specimens on day 7 after the second immunization were fixed in 13% neutral buffered formalin and embedded in paraffin. Sections were cut at 5 μm thick, and incubated with anti-mCD8  or anti-mCD4  antibody overnight at 4°C. The anti-mCD8 or anti-mCD4 antibody was visualized with a HRP-DAB Cell and Tissue Staining Kit (R&D Systems).Tumor cell suspensions were obtained as described above. The cells were stained with FITC-labeled Annexin V (BD Pharmingen) and propidium iodide  for 15 min in the dark at room temperature. Subsequently, the rate of cell apoptosis and cell death were evaluated quantitatively by flow cytometry.The generation of intracellular ROS was measured using a dichloro-dihydro-fluorescein diacetate (DCFH-DA) dye assay. DCFH-DA penetrates the cells and is hydrolyzed to DCFH by non-specific esterases. In the presence of cellular oxidizing agents, DCFH is rapidly oxidized to the highly fluorescent compound dichlorofluorescein (DCF), which is trapped inside the cells. Therefore, the fluorescence intensity is proportional to the amount of ROS generated in the cells. In brief, tumor cell suspensions were obtained as described above. The cells were stained with DCFH-DA  at 37°C for 20 min, and then washed to remove extracellular DCFH-DA. Changes in DCF fluorescence were assayed by flow cytometry.2+ levels were detected by Fluo-3 acetoxymethyl ester . Intracellular Fluo-3 AM is deacetylated by esterases to yield Fluo-3, which is trapped inside the cells. Upon Ca2+ binding, Fluo-3 exhibits strong fluorescence. Briefly, the cells isolated from tumor tissue were incubated with 5 μM Fluo-3 AM at 37°C for 20 min, and then washed with PBS buffer to remove extracellular Fluo-3 AM. The fluorescence intensity of Fluo-3 was analyzed by flow cytometry.Changes in intracellular Cain vitro and in vivo experiments was carried out using GraphPad Prism Software . Student's t-test and one-way ANOVA were used for analysis of the comparisons between the groups. Statistical significance was established as P < 0.05 .Statistical analysis for hly::E7, which expresses and secretes the LLO-E7 fusion protein, was constructed by homologous recombination. In C57BL/6 mice, LM4Δhly::E7 was highly attenuated and exhibited a LD50 of 3.863 × 109 colony forming units (CFU) compared with the LD50 of wild type (wt) LM4, 5 × 104 CFU. The LLO-E7 fusion protein secreted by LM4Δhly::E7 was detected in Western blotting assay, which is 67 kDa in size  expressing green fluorescent protein were constructed and used to infect RAW264.7 cells, following which the multiplicity of these LM strains in RAW264.7 cells was determined. As shown in Figures hly::E7(pNF8) were increased  and LM4Δ Figures  and S1.hly::E7, spleens, livers and brains were harvested, along with tumors from the immunized mice at different time-points. Tissues were homogenized and CFU/organ was calculated. The results showed that the bacterial numbers in spleen and liver peaked on the 3rd day after the first immunization, and gradually decreased if further immunization doses were administered. As expected, LM was not found in brain homogenates. LM4Δhly::E7 was quantified in tumor tissues on the 1st day after the first immunization, and bacterial numbers started to increase, peaking at day 3 after the second immunization . Also, the number of IFN-γ-secreting cells were significantly expanded compared with IL-4-secreting cells in the LM4Δhly::E7-immunized group (**P < 0.01), which demonstrated that the immune response induced by LM4Δhly::E7 is skewed toward a Th1 type response in tumor-bearing mice.In order to investigate the cellular immune responses induced by LM4Δin vivo was determined using a CFSE-based assay. As shown in Figures hly::E7 generated a robust cytotoxic response with an E7-specific lysis ratio of 61.62 ± 6.2%, which was significantly higher than the control groups (**P < 0.01).CTL activity + and CD8+ T cells infiltrated into the tumor site in the LM4Δhly::E7 and LM4Δhly::esat-6 groups compared with the PBS group . However, there were no significant differences between the two rLM-immunized groups  and secretion by L. monocytogenes was found to significantly enhance anti-tumor immune responses in comparison to an LM vector that secreted only the tumor antigen   and suppress the recruitment and differentiation of Tregs in the tumor microenvironment  led to non-apoptotic cell death, the generation of intracellular ROS and elevated Ca2+ concentrations in vivo. Increases in intracellular ROS and Ca2+ levels were strongly associated with tumor cell death induced by LM-based vaccines. However, such tumor cell death induced by rLM strains was not influenced by delivery of specific antigen. Hence, these results indicated that LM4Δhly::E7 can induce antitumor efficacy via non-specific direct killing of tumor cells.CaListeria, which is a novel adjuvant with PAMP-like activity . Therefore, these properties suggest potential future clinical applications for this vector.LLO is necessary for intracellular proliferation of y Figure . Howeverhly::E7 successfully induced a robust cellular immunity. LM4Δhly::E7 effectively retards tumor growth in a murine model of cervical cancer. Furthermore, we observed that the antitumor mechanisms were dependent on antigen-specific CTL activity and direct killing of tumor cells. Future studies include evaluation of LM4Δhly::E7 as a candidate vaccine in a transgenic mouse model, in which immunological tolerance can be addressed. Furthermore, immunomodulatory agents such as Toll-like receptor 9 (TLR9) ligands and/or radiation therapy can be combined to further enhance their antitumor efficacy for application of LM-based vaccines in clinic. Taken in its entirety, our study provides strong evidence for future clinical translation of LM-based vaccines, as well as a basis for investigation into other HPV-related cancer models.In summary, although TC-1-induced tumors gradually grew along with the expansion of Treg suppressed cells, immunization with LM4ΔThe mice were housed, handled and immunized at the animal biosafety facilities and all procedures were approved by the institutional animal experimental committee of Yangzhou University.YJ, YY, and XJ designed the experiments. WT and FD performed the experiments and analyzed the data. YY, ZP, XC, and XJ contributed reagents/materials/analysis tools. YJ, YY, and XJ wrote and revised the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.