Thirteen (12%) participants had detectable prepandemic IC50 titers, but only one reached a titer of 10. This proportion increased to 44% after the pandemic, when 39 participants had a titer of 10, and 67% of infected compared to 44% of noninfected had detectable IC50 titers (P < 0.001). The low levels of SR antibodies in prepandemic sera were not associated with subsequent H1N1pdm09 infection (P = 0.241), and the higher levels induced by H1N1pdm09 infection returned to prepandemic levels within 2 years. The findings indicate that natural infection induces only low titers of SR antibodies that are not sustained.The discovery of influenza virus broadly neutralizing (BrN) antibodies prompted efforts to develop universal vaccines. Influenza virus stem-reactive (SR) broadly neutralizing antibodies have been detected by screening antibody phage display libraries. However, studies of SR BrN antibodies in human serum, and their association with natural infection, are limited. To address this, pre- and postpandemic sera from a prospective community cohort study in Vietnam were assessed for antibodies that inhibit SR BrN monoclonal antibody (MAb) (C179) binding to H1N1 pandemic 2009 virus (H1N1pdm09). Of 270 households, 33 with at least one confirmed H1N1pdm09 illness or at least two seroconverters were included. The included households comprised 71 infected and 41 noninfected participants. Sera were tested as 2-fold dilutions between 1:5 and 1:40. Fifty percent C179 inhibition (ICIMPORTANCE Universal influenza vaccines could have substantial health and economic benefits. The focus of universal vaccine research has been to induce antibodies that prevent infection by diverse influenza virus strains. These so-called broadly neutralizing antibodies are readily detected in mice and ferrets after infection with a series of distinct influenza virus strains. The 2009 H1N1 pandemic provided an opportunity to investigate whether infection with a novel strain induced broadly neutralizing antibodies in humans. We found that broadly neutralizing antibodies were induced, but levels were low and poorly maintained. This could represent an obstacle for universal vaccine development and warrants further investigation. The ability of variant influenza strains to repeatedly infect humans fueled speculation that broadly neutralizing (BrN) antibodies are lacking . However2–Few studies have assessed SR BrN antibody levels in human serum, the factors associated with their detection, or whether detection is associated with protection. In previous studies involving the Ha Nam community influenza cohort in Vietnam, we found that increasing age and prior seasonal H1N1 infection were associated with protection against pandemic H1N1 infection in the absence of detecting hemagglutination-inhibiting (HI) antibodies . TherefoThis study examined pre- and postexposure sera from people with and without subsequent infection to investigate associations between antibodies and protection or infection. The study utilized stored sera from the Ha Nam community influenza cohort, which has been described in detail previously . The coh°C with H1N1pdm09 virus . Twofold virus dilutions in carbonate-bicarbonate buffer, pH 9.6 , were compared, and 80 HA units was chosen as the optimal dilution for C179 binding. Virus-coated plates were blocked with 3% bovine serum albumin (BSA) in PBS-Tween and then incubated with serum dilutions for 2 h at 37°C before addition of 2 μg/ml of C179 MAb and incubation for a further 1 h. C179 binding was detected with goat anti-mouse IgG conjugated to horseradish peroxidase  and then o-phenylenediamine dihydrochloride . Each plate contained four virus control wells, as well as four background subtraction wells coated with BSA. Background absorbance was subtracted before calculating the percentage inhibition of C179 binding as follows: 100 − [(OD490 with serum/OD490 of virus control) × 100], where OD490 is optical density at 490 nm. Comparison of C179 MAb binding to plates coated with A/Cal/07/2009-like H1N1 versus that with A/Perth/16/09-like H3N2 virus (VIDRL) verified that the assay detects C179 binding to the former (A490 > 0.5) but not the latter (A490 < 0.07). Reference sera  were tested as a potential positive control and a specificity control for group 1 versus group 2 FP-reactive antibodies. A/Cal/07/09-specific reference sera, but not A/H3N2 (A/Victoria/361/2011 or A/Texas/50/2012)-specific reference sera, inhibited C179 binding to A/Cal/07/09-like virus  binding to an A/Cal/07/09-like (H1N1pdm09) virus. Ninety-six-well plates  were coated overnight at 4ke virus . C179 bihttp://www.who.int/influenza/gisrs_laboratory/2010_12_06_serological_diagnosis_of_influenza_by_microneutralization_assay.pdf). Briefly, 100× 50% tissue culture infective dose (TCID50) of virus  was mixed in equal volume with 2-fold serial dilutions of serum and incubated for 2 h at 37°C. MDCK cells (1.5 × 104/well) were added to the plates, followed by incubation at 37°C for 20 h. Cells were fixed, and viral antigen was detected by indirect ELISA with a primary mouse MAb against influenza A virus nucleoprotein  followed by goat anti-mouse IgG conjugated to horseradish peroxidase (as mentioned above) and then OPD (as mentioned above). A cutoff, or OD value at which 50% of the MDCK cells were infected, was determined using the following equation: cutoff = (average OD490 of virus control wells − average OD490 of cell control wells)/2. All serum dilutions with OD490 values below or equal to the cutoff were considered positive for neutralization activity. The reciprocal of the highest positive serum dilution was inferred to be the neutralizing antibody titer for that serum sample. PRNT was performed according to the protocol of Margine et al. (dK [dissociation constant] = 40 nM)  . HoweverThe Mann-Whitney test was used to compare antibody levels or titers and participant ages for infected versus noninfected or pre- versus postpandemic groups. Proportions were compared using the chi-square test. Pearson and Spearman correlation coefficients were used to indicate the correlation between two different antibody assays. Binary logistic regression was also used to assess the effect of the prepandemic FP-reactive antibody level on infection during the first pandemic wave. Generalized estimating equations were used to account for household clustering. Statistical analyses were performed with SPSS 19 statistical software.P < 0.001), consistent with a previous analysis of all households in the cohort (Thirty-three of 270 households in the Ha Nam cohort contained at least one participant with H1N1pdm09 illness (8 houses) or at least two H1N1pdm09 seroconverters (15 houses) or both (10 houses) and were selected for analysis of SR antibodies in serum. Of 129 participants residing in these houses, 112 provided prepandemic sera in June 2009, and 107 also provided postpandemic sera in April 2010. Seventy-one of the included participants were infected, including 23 with ILI, and 41 were not infected during the 2009 H1N1 pandemic. The age and gender distribution of selected households was similar to that of all households in the cohort . The med50) titers were low and did not exceed 10 in either pre- or postpandemic sera  than among noninfected  participants. The proportion of infected participants who had detectable postpandemic IC20 titers approached 100% (Sera were assessed over 2-fold dilutions from 1:5 to 1:40 to detect antibodies that inhibit binding of FP-reactive MAb (C179) to H1N1pdm09 virus. These antibodies are also referred to here as SR antibodies. Fifty percent inhibitory . Inhibition levels in postpandemic sera diluted 1:10 correlated with MN titers , for sera diluted 1:5 , and for correlations with IC50 titers . However, correlations were poor and nonsignificant for sera collected prepandemic . A total of 33.3% of infected participants had postpandemic H6 PRNT titers of 20 or more, compared to 9.7% of noninfected participants. H6 PRNT seroconversion was detected in only one participant, who was infected. PRNT titers against H1N1pdm09 rose among infected, but not noninfected, participants, as expected . Correlations were stronger when analysis was limited to the infected group  or to postpandemic sera  but not as strong as correlations between H6 and H1N1pdm09 PRNT results  or between C179 inhibition and H1N1pdm09 PRNT titer .H6 PRNT and C179 inhibition ELISA titers were only weakly correlated using the Spearman nonparametric test  induced following influenza virus infection or vaccination. ASC that make SR BrN antibodies are induced upon first exposure to antigenically novel viruses, such as H1N1pdm09 or H5N1, but rarely after exposure to seasonal influenza viruses, which favor ASC that react with strain-specific epitopes in the bulky head of HA , 8, 21. It is not known whether the higher levels of SR antibodies detected in response to novel strains would be sufficient for protection. Even so, boosted levels were not sustained, a finding of concern for the development of vaccines that target fusion-inhibiting epitopes to elicit broad protection. SR BrN antibodies are predominantly encoded by variable heavy (VH) chain genes, such as VH1-69, which encode long, compact complementarity-determining region 2 (CDR2) loops. These CDR2 loops enable binding to inaccessible hydrophobic epitopes such as FP , 8, 23. 50 titers were used, and another cutoff could be considered. The cutoff is also calculated based on the assumption that C179 binding can be fully inhibited, which did not occur. SR antibody detection could potentially be improved by using recombinant HA protein instead of whole virus (The current study has several limitations that impede interpretation of the effects of SR antibodies on infection. As discussed above, prepandemic SR antibody levels were low, and the levels required for protection have not been determined. The low levels of SR antibodies also meant that there was little resolution between sera if ICle virus , but thile virus . Alternale virus . It rema


E17K mutation in the pleckstrin homology domain of AKT1 occurs in approximately 0.6–2% of human lung cancers. Recently, we have demonstrated that AKT1E17K transforms immortalized human bronchial cells. Here by use of a transgenic Cre-inducible murine strain in the wild type Rosa26 (R26) locus (R26-AKT1E17K mice) we demonstrate that AKT1E17K is a bona-fide oncogene and plays a role in the development of lung cancer in vivo. In fact, we report that mutant AKT1E17K induces bronchial and/or bronchiolar hyperplastic lesions in murine lung epithelium, which progress to frank carcinoma at very low frequency, and accelerates tumor formation induced by chemical carcinogens. In conclusion, AKT1E17K induces hyperplasia of mouse lung epithelium in vivo and cooperates with urethane to induce the fully malignant phenotype.The hotspot AKT1 Lung cancer is a leading cause of cancer-related deaths, being associated with a 5-year worldwide survival rate of less than 15% . Mutatio, [The AKT kinases  represent the primary downstream end-point of the phosphoinositide 3-kinase (PI3K) pathway, regulating proliferation, survival, metabolism and invasion. AKT1 and AKT2 are frequently activated in human cancer –18 follo,  or in AK, . In lung, ,12,23–25E17K shows increased affinity for PIP2, enhanced plasma membrane recruitment and constitutive activation [E17K mutant protein detected in lung cancer cells shows enhanced membrane localization [AKT1tivation ,26. Endolization , which rlization ,22.E17K in epithelial tumorigenesis remains unclear because studies in cellular models in breast and lung cells have produced discordant results [E17K mutation has been generated so far.The role of mutant AKT1 results –30. In aE17K in the transformation of lung epithelial cells in vivo. Herein, we demonstrate that AKT1E17K promotes hyperplasia in the mouse lung and cooperates with other genetic lesions to induce the full malignancy.These considerations prompted us to address the role of AKT1R26-AKT1E17K transgenic mice human AKT1 cDNA was amplified by PCR from human blood mononuclear cells by PCR using primers 5’-ATGAGCGACGTGGCTATT-3’ and 5’-TCAGGCCGTGCCGCTGGC-3’. The PCR product was cloned into a shuttle plasmid (pBlueScript) using standard cloning techniques. Mutant AKT1E17K cDNA was generated by Quick Change Site-Direct Mutagenesis (Stratagene) and sub-cloned into the pROSA26 vector [E17K, which consisted of 5' and 3' homology arms of the ROSA26 locus, FRT/FRT-flanked neomycin resistance (Neo) cassette and loxP/loxP-flanked triple polyadenylation (tpA) sequence. See To generate 6 vector –33 to obE17K plasmid was linearized with SfiI restriction enzyme  and electroporated into mouse embryonic stem cells (mESC). G418-resistant mESC clones were isolated and screened for correct targeting of the locus by PCR  amplifying a 3.9 kb fragment. PCR positive G418-resistant clones were subsequently screened by Southern blot probing for the 5’ recombination junction. Once identified, targeted mESC clones were transfected with Flp-expressing plasmid (pFlpE-IRES-puro) for the excision of the Neo cassette. Subsequently, Neo-deleted mESC clones were isolated, screened by PCR  and injected into B6 blastocysts, which were subsequently transferred into pseudo-pregnant females to produce chimeric animals at the Embryonic Stem Cell Facility of IRGS . The generated chimeras were bred to establish germline transmission of the human allele. Genotypes of R26-AKT1E17K mice were determined by PCR using genomic DNA isolated from the tail tips with the following primers: 5'ARMF, 5'-AACTGCAGACTTGTGGGATAC-3'; 3'ARMR, 5'-ATATTAGTCCACCTCACTCCT-3'; mE17KR5 5'-GCCAACCCTCCTTCACAATA-3'. The R26-AKT1E17K line was mated with Ttf1-Cre line , to generate R26-AKT1E17K; Ttf1-Cre; mice. The Ttf1-Cre mice were genotyped by using the following primers: FP, 5'-CCTGATGGACATGTTCAGGGACA-3': RP, 5'-GCCAGATCTCCTGTGCAGCATGT-3'. R26-AKT1E17K mice were back-crossed on B6 background. In all experiments littermates were used as controls.30μg of the R26-AKT1Animal experimentation was approved by the local ethical committee “Comitato Etico per la Sperimentazione Animale” (CESA) of IRSG and conform to regulations and guidelines of Italy and the European Union. All efforts were made to minimize animal suffering . Mice weTRF, 5'-GGATCGACGGTATCGTAGAGTCGAGGCCG-3';L2R 5'-GCCAATGAAGGTGCCATCATTCTTGAGGAGGAAG-3'.E17K in mESCs transfected with pCre-IRES-puro was determined by quantitative RT-PCR.Expression of AKT1A standard protocol for Southern blot was used. Genomic DNA (30 μg) was digested with EcoRV. A 500bp probe on the 5’ side of the targeted insertion site (from CTTGAAAGTGGAGTAACTAC to TCAGAAGCTTTGAACTAGAA in the ROSA26 locus) was labelled with DIG-dUTP, using PCR DIG Probe Synthesis Kit . This probe identifies a 11.5 kb fragment in wild type ROSA26 locus and a 4.3kb fragment in targeted ROSA26 locus.Whole tissue protein extracts were prepared with NP-40 buffer  containing protease inhibitors . Western blot analysis was carried out by standard methods. Anti-phTotal RNA was prepared as described . RT-PCR AKT1R26 forward 5'-CACACCACCTGACCAAGATG-3';AKT1R26 reverse 5'-AATCAAGGGTCCCCAAACTC-3'.R26 and R26-AKT1E17K mice were infected with 106 or 107 pfu of Adenoviruses expressing Cre (Ad-Cre) . Mice between 6 and 12 weeks of age were anesthetized with isoflurane and Ad-Cre was administered intranasally. Mice received 120 μl of Ad-Cre, in two doses of 60 μl each, with a break between the two doses, to allow the mice to recover a normal breathing. Control mice received equal amount of buffer phosphate saline solution (Control solution .R26 or R26-AKT1E17K littermates were either infected with Ad-Cre (106−107 PFU) or treated with solvent alone before receiving intra-peritoneal injection with a single dose of 1 mg/g of body weight of urethane, an ethyl ester of carbamic acid extensively used in murine models of carcinogenesis [ogenesis , dissolvLung tissues were collected and fixed with 10% formalin, and embedded in paraffin using standard procedures. Sections (5μm) were mounted on slides and stained with hematoxylin and eosin to be evaluated by light microscopy by veterinary (OP and HF) or human (CM) pathologists. A combined score of hyperplastic lesions was calculated by adding a score measuring the number of epithelial layers (Layer score) to a score measuring percentage of hyperplasia present in the whole section . The Layer score was defined as follows: score 1, presence of 1–2 layer(s), score 2, presence of 2–3 layers, score 3, presence of >4 layers. The Global hyperplasia score was defined as follows: score 1, when hyperplasia was present in 1–20% of total sections analysed ; score 2, when hyperplasia was fond in 21–60% of total sections analysed ; score 3, when hyperplasia was found in 61–100% of total sections analysed .Immunostaining with anti-phospho-AKT was performed with standard protocols using the Vectastain Universal Quick Kit and DAB Peroxidase Substrate Kit  according to the manufacturer’s instructions. Antigen retrieval was carried out with microwave treatment in antigen unmasking solution  for 10 min. Rabbit anti-phospho-AKT (Ser473)  was purchased from Cell Signaling Technology.Immunostaining for Ki67 was performed with standard protocols using Bond™ Polymer Refine Detection  according to the manufacturer’s instructions. Rabbit anti-Ki67  was purchased from Fisher Scientific . Immunostaining for p21 was performed with standard protocols using EnVision™ Detection Systems Dako, according to the manufacturer’s instructions. Rabbit polyclonal Ab-5; anti-p21/WAF1 antiserum (1:50) was purchased from Oncogene Research Products . Ki67 and p21 positivity was determined by counting positive nuclei in 10 fields at X400 magnification.Microdissection was performed using the Laser Microdissector Leica LMD6 & LMD7 . 5-μm tumor tissue sections onto glass slides were inserted into the Laser Microdissector for dissection of lung tumors. Selected areas were captured with infrared laser pulses onto CapSure Macro LCM Caps.Microdissected samples were lysed by SB LysePrep Kit  according to manufacturer' instructions. Aliquots of lysis solution (2.5 μl) were used as template for DNA amplification with the following primers: mKrasF-TCCTAC AGGAAACAAGTA and mKras R-TTATTTATGGCAAATACA. The amplification program used was: 95°C/5 min; 35x  for denaturation, annealing, and extension; 72°C/7 min to terminate extension, followed by cooling to 15°C. PCR products were separated on 2% agarose gels and purified by the QIAquick PCR purification kit (Qiagen). Purified PCR products were analyzed on 3500 Genetic Analyzer . For all analyses, data were aligned to the consensus sequence obtained from the BLAST GenBank database.Deparaffinized sections were hydrated in decreasing ethanol gradient solutions and rinsed in wash solution . Antigen retrieval was performed with citrate buffer pH 6 for 30 minutes at 98°C followed by washing in phosphate-buffered saline . Sections were incubated with antibodies against SP-C  or CC-10  for 60 min, followed by incubation with secondary Alexa Fluor 488-conjugated anti-goat IgG antibodies  for 60 min. The cells were counterstained with DAPI , mounted using anti-fade mounting medium  and observed at Fluorescence Microscopy (Leica Microsystems).n independent assays or replicates as indicated in the text. Continuous variables were analyzed by Student’s t-test or ANOVA test, while categorical variables were analysed by χ2 or Fisher’s exact tests. Significance was calculated by Log-rank (Mantel-Cox) test .Data presented are the means ± SD of R26-AKT1E17K) that conditionally expresses human mutant AKT1 in the lung by use of a knock-in system  that harbours loxP-flanked transcriptional stop sequences upstream of mutant AKT1 cDNA  or mating of Cre mice ,39. The elium [,  whereas R26-AKT1E17K mice with Ttf1-Cre. R26-AKTE17K; Ttf1-Cre mice; R26-AKTE17K; Ttf1-Cre mice compared to R26-Ttf1-Cre littermates. We measured AKT1 expression by RT-PCR in five additional tissues  derived from R26-AKTE17K; Ttf1-Cre and R26-Ttf1-Cre littermate mice and the results are shown in In the first set of experiments, we crossed the Ttf1-Cre mice showed no evidence of disease up to 12 months of age (n = 7). Conversely, histo-pathological analysis revealed the presence of hyperplastic lesions in the lung of all 12 month-old R26-AKT1E17K;Ttf1-Cre mice , 6 (n = 8) and 12 (n = 11) months of age were sacrificed as end-points. Cre mice . These mCre mice .E17K transgene by intranasal administration of Ad-Cre  in 6–12 weeks-old R26-AKT1E17K mice. After 6, 9 and 18 months  mice were sacrificed and lungs were analysed. We did not observe lung abnormalities in R26-AKT1E17K mice treated with solvent alone (PBS) (n = 7)  . Conversstration .R26-AKT1E17K mice, we evaluated lung hyperplasia by analysing the number of epithelial layers in the bronchial epithelium and the percentage of hyperplasia on the whole area of 3 serial sections derived from representative R26-AKT1E17K mice after 6 (n = 3) and 9 (n = 3) months from Ad-Cre administration (mice code #7a-#12a). As controls we made use of the same number of R26-AKT1E17K mice treated with solvent alone (mouse code #1a-#6a), respectively. The quantification of epithelial layers and of hyperplastic areas was performed as described in Materials and Methods. Results are summarised in To further characterise the lesions detected in transgenic R26-AKT1E17K mice Layer score was 1 for 1/6 mice and 2 for 5/6 mice and Global hyperplasia score was 1 for 2/6, 2 for 2/6 and 3 for 2/6 mice. Finally, we found that R26-AKT1E17K mice presented a Combined score of hyperplasia of 3.8±0.4 that was significantly higher than that in control mice (2.5±0.3) .Among the control mice Layer score was 1 for 4 out of 6 mice and 2 for 2 out of 6 mice. Global hyperplasia score was 1 for 5/6 and 2 for 1/6 mice. Conversely, of the E17K in the lung of transgenic mice are a result of increased cell proliferation.Subsequently, we investigated whether the hyperplasic lesions observed were proliferative or senescence-related by immonostaining of Ki67 and p21. Results are summarised in R26-AKT1+/E17K mice treated with Ad-Cre developed a single nodule each, which was diagnosed as bronchio-alveolar adenocarcinoma with papillary pattern consisting of cords and nests of epithelial cells surrounded by sparse fibrovascular stroma , a chemical compound present in smoke that has been extensively used to experimentally model multistage lung carcinogenesis [R26 or R26-AKT1E17K littermates were either infected with Ad-Cre (106−107 PFU) or treated by solvent alone before being exposed to urethane (1mg/g) and analysed after 6 or 9 months. Multiplicity and size of tumors/lung were found to be significantly higher in R26-AKT1E17K mice infected with Ad-Cre. Being relatively resistant to urethane, R26-AKT1E17K mice that had not been previously exposed to Ad-Cre developed nodules in 1 out of 6 mice at 6 months and in 1 out of 4 mice at 9 months after urethane administration, respectively  and 9 (n = 6) months of treatment. The average number of tumors developed by mutant mice treated with urethane and infected with Ad-Cre was 3/mouse at 6 months and 7/mouse at 9 months; conversely, mice treated only with urethane developed <0.15 tumor/mouse at 6 months and of 1.6 tumors/mouse at 9 months . The region spanning codon 61 in exon 3 of Kras gene was amplified and subjected to Sanger DNA sequencing. Sequence analysis of tumors demonstrated the presence of an A>G transition in codon 61 leading to Q61 replacement with R. See E17K may cooperate with the oncogenic events induced by urethane in mouse lung (i.e. Kras gain of function mutations), thus accelerating progression to overt tumors.Tumors generated by urethane in the  pattern . A finalstaining . FinallyIn the mouse, Clara cells line bronchiolar epithelium and express Clara cell-associated protein 10 (CC10) whereas alveolar type II (AT2) cells reside in the alveoli and express surfactant protein C (SPC) –44.E17K in mouse, we performed indirect immunofluorescence for SP-C and CC10 on FFPE lung sections derived from R26-AKT1E17K mice treated with Ad-CRE (4 mice of 9 months), urethane (2 mice of 9 months) and in R26-AKT1E17K treated with urethane followed by Ad-CRE administration (2 mice of 9 months).Therefore, to characterize the cells that constitute the lung lesions induced by AKT1In agreement with previous studies ,46, we fAKT1E17K in the lung were consistently positive for both SP-C and CC10. This immunophenotype is typical of i) a subset of variant Clara cells termed bronchioalveolar stem cells (BASCs) located at the bronchioalveolar duct junctions [+ bronchiolar epithelial cells, SBEC) that represents a SP-C+/CC10+ intermediate stage of Clara cell differentiation into AT2 cells [Conversely, hyperplastic lesions initiated by unctions –49 and cunctions  and ii) T2 cells .E17K confers proliferative and pro-migratory characteristics to immortalized human bronchial epithelial cells. However, so far no murine model that recapitulates the E17K mutation identified in human lung cancer has been generated.The hotspot E17K mutation in the PH domain of AKT1 occurs in approximately 0.6–2% of human NSCLC patients. Recently, we have demonstrated that AKT1Rosa26 locus (R26-AKT1E17K) that allows targeted expression of mutant AKT1E17K and studied its effects after activation in the lung. The most prominent findings are that in mice, AKT1E17K promotes hyperplasia in bronchi and bronchi-alveoli and strongly cooperates with chemical carcinogens to induce malignancy. We found that, coincident with increased AKT signalling in lung epithelial cells, expression of mutant AKT1E17K in the lung epithelium induced at high frequency moderate or severe hyperplasia of bronchi and/or terminal bronchioli that, in the long term, may progress to overt carcinoma in a very limited number of animals. It is of note that, hyperplastic lesions initiated by AKT1E17K in the lung were positive for both Clara cell (CC10+) and AT2 (SP-C+) markers, an immunophenotype shared with rare BASCs at the bronchioalveolar duct junctions [In this manuscript, we have genetically engineered a transgenic Cre-inducible murine strain in the unctions –49 and bunctions .E17K mutation has, to our knowledge, not been reported in premalignant lung lesions isolated from patients, AKT signalling is frequently activated in bronchial pre-malignancy as shown by immunohistochemical studies of AKT phosphorylation in bronchial dysplasia [Although AKT1ysplasia . Thus, tysplasia .G12D (3 weeks). In addition to this, in R26-AKT1E17K mice lung tumors appears with longer latency and lower incidence compared with mice expressing mutant Egfr (3–4 weeks from the activation of the oncogene) [Pik3caH1047R (12–60 weeks) [KrasG12D  [Pten knock-out mice showed contradictory results [Pten deficiency is not sufficient to induce histological abnormalities of the lung or to initiate lung tumorigenesis up to 12 months when induced by CCSP-driven Cre [Hyperplasia induced by mutant AKT1 in the mouse lung occurs later  compared with those induced by the oncogenic version of other genes within the PI3K pathway such as the upstream regulators EGFR (3–4 weeks) or KRASncogene) ,53, Pik30 weeks)  or KrasG of age) ,55,56. C results ,58 possi results . In factiven Cre  but is aR26-AKT1E17K mouse described here only partly recapitulates the human disease. On one hand, transgenic mice develop hyperplasia that in the long term (and infrequently) progresses to full-blown neoplastic lesions. This suggests that AKT1E17K mutation, which in the human seems to be a weak driver mutation considering its rarity and absence in cell lines, represents a weak oncogene for the lung epithelium also in the mouse. However, at difference with what occurs in human patients, where the mutant allele AKT1E17K is detected predominantly in SCC, the few tumors that develop in transgenic mice are ADC. This situation is reminiscent of mutant Pik3ca and/or Pten silencing both of which lead to ADC development in mice 41, 44, 45 but are preferentially detected in SCC in human.It is to be noted that the Kras, Egfr, Pik3ca, Pten etc.) promote development of ADC, raising the possibility that Ad-CRE allows expression of transgenes only in ADC precursor cells. On the other hand, we have recently reported that human immortalized bronchial cells (BEAS-2B) transduced with a lentiviral vector carrying the activated AKT1E17K mutant become tumorigenic and induce the appearance of poorly differentiated tumors [Possibly, the observed difference in tumor histotype between human and mouse is dependent on the type of cell in which the oncogene is expressed. In fact, most oncogenes whose expression has been induced by Ad-CRE in mouse lung  and are not selected for in human NSCLC cell lines.In summary, the conclusion drawn by the experiments reported here is that mutant AKT1S1 ARRIVE Checklist(PDF)Click here for additional data file.S1 Fig(TIFF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S3 Fig(TIFF)Click here for additional data file.S4 Fig(TIFF)Click here for additional data file.


Background: Characteristics of seed-competent Tau are unknown.Results: Native Tau aggregates have a higher seeding potency than recombinant Tau aggregates. Recombinant Tau acquires the conformation and potency of native Tau aggregates by seeded assembly.Conclusion: Conformation determines the seeding potencies of Tau aggregates.Significance: Understanding the properties of seed-competent Tau gives insight into disease mechanisms. 275VQIINK280 and 306VQIVYK311 abolished the seeding activity of recombinant full-length Tau, suggesting that its aggregation was necessary for seeding. We describe conformational differences between native and synthetic Tau aggregates that may account for the higher seeding activity of native assembled Tau. When added to aggregated Tau seeds from the brains of mice transgenic for P301S Tau, soluble recombinant Tau aggregated and acquired the molecular properties of aggregated Tau from transgenic mouse brain. We show that seeding is conferred by aggregated Tau that enters cells through macropinocytosis and seeds the assembly of endogenous Tau into filaments.Intracellular Tau inclusions are a pathological hallmark of several neurodegenerative diseases, collectively known as the tauopathies. They include Alzheimer disease, tangle-only dementia, Pick disease, argyrophilic grain disease, chronic traumatic encephalopathy, progressive supranuclear palsy, and corticobasal degeneration. Tau pathology appears to spread through intercellular propagation, requiring the formation of assembled “prion-like” species. Several cell and animal models have been described that recapitulate aspects of this phenomenon. However, the molecular characteristics of seed-competent Tau remain unclear. Here, we have used a cell model to understand the relationships between Tau structure/phosphorylation and seeding by aggregated Tau species from the brains of mice transgenic for human mutant P301S Tau and full-length aggregated recombinant P301S Tau. Deletion of motifs  MAPT, the Tau gene, through alternative mRNA splicing ,splicing . Alternasplicing , 4. The splicing . The isosplicing –8, whereplicing –. The assplicing ––13.MAPT cause familial forms of frontotemporal dementia, establishing that Tau protein dysfunction is sufficient to cause neurodegeneration and dementia  or hyperphosphorylated native Tau aggregates (from the brains of mice transgenic for human mutant P301S Tau), we show that pathological Tau was capable of entering cells, where it triggered the aggregation of soluble Tau in an aggregation-dependent manner. Sarkosyl-insoluble Tau from the brains of mice transgenic for human mutant P301S Tau was more effective at seeding than Sarkosyl-insoluble aggregates of unphosphorylated and hyperphosphorylated recombinant Tau. Using proteinase K digestion and guanidine hydrochloride disaggregation, we identified conformational differences that may account for the differing seeding activities of these preparations. The addition of soluble recombinant Tau to aggregated Tau seeds from the brains of mice transgenic for P301S Tau led to its assembly, with a seeding potency and resistance to disaggregation with guanidine hydrochloride like that of Sarkosyl-insoluble Tau from the transgenic mouse brain.Homozygous mice transgenic for human 0N4R Tau with the P301S mutation (TgP301S Tau) with a severe paraparesis  were kilg. Cleared lysates were prepared in 25 mm Tris-HCl, pH 7.4, 10 mm EDTA, 0.1 mm dithiothreitol (DTT), and 0.1 mm phenylmethanesulfonyl fluoride (PMSF) using a cell disruptor , followed by centrifugation at 18,000 × g for 20 min. The lysate was passed through a DE52 anion exchange column, followed by a phosphocellulose cation exchange column. Tau fractions were eluted with 500 mm NaCl, precipitated using 25% ammonium sulfate, and run on a Superdex 200 HiLoad 16/60 column. The resulting fractions with the highest Tau content were passed through a Mono S 50/50 GL cation exchange column and eluted with a 50–275 mm NaCl gradient. Fractions were then pooled and dialyzed against 40 mm HEPES, pH 7.4, containing 0.1 mm DTT. Aliquots of recombinant Tau were snap-frozen and stored at −20 °C. To obtain monomeric Tau, purified recombinant Tau was centrifuged at 100,000 × g at 4 °C for 1 h, and the supernatant was used.Human P301S 0N4R Tau was expressed as described . Bacterim Tau was incubated for 6 h at 30 °C with 1 unit/ml cAMP-dependent protein kinase (PKA) (New England Biolabs) in 25 mm Tris-HCl, pH 7.4, 0.1 mm EGTA, 2 mm 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 10 mm magnesium acetate, 2 mm ATP, and 50 μg/ml heparin (Sigma). After 6 h, 1 unit/ml stress-activated protein kinase 4 (SAPK4) (Millipore) was added, and the reaction was allowed to proceed for an additional 6 h. The samples were then incubated for 5 min at 95 °C, before centrifugation for 15 min at 100,000 × g. The supernatants were used.Recombinant P301S Tau was phosphorylated as described previously . Two μm m) was assembled by incubation with 400 μg/ml heparin (Sigma) in 30 mm MOPS, pH 7.2, at 37 °C for 3 days, as described (Recombinant Tau (60 μescribed .m DTT and incubated at 37 °C in an 800-μl reaction volume with or without 5% (v/v) TgP301S Tau Sarkosyl-insoluble brain extract. An aliquot of the samples was added to 15 μm thioflavin T in a 384-well plate, and thioflavin T fluorescence was monitored over time with excitation and emission filters set to 444 and 485 nm, respectively, in an Envision (PerkinElmer Life Sciences) plate reader. Fluorescence readings were taken every 20 min, with agitation in between each reading. The aggregation reaction was stopped, by snap-freezing the samples, when the thioflavin T signal reached a plateau. Samples were Sarkosyl-extracted, as described below, and the pellets were washed and resuspended in 50 mm Tris-HCl, pH 7.4. Samples were run on SDS-PAGE and probed with total Tau antibody DA9. Equal amounts of Tau protein were then added to HEK Trex P301S 1N4R Tau-inducible cells following the seeding paradigm described below.Recombinant P301S Tau was diluted to 135 μg/ml in PBS plus 1 mm Tris-HCl, pH 7.4, 0.8 m NaCl, 1 mm EGTA, 5 mm EDTA, 10% sucrose), supplemented with protease and phosphatase inhibitors, followed by centrifugation at 13,000 × g at 4 °C for 20 min. The supernatants were retained and kept on ice, and the pellets were resuspended in 500 μl of A68 buffer and centrifuged for a further 20 min. Both supernatants were combined. Assembled recombinant Tau was diluted directly in A68 buffer. Samples were then incubated with 1% sodium lauroyl sarcosinate  at room temperature for 1 h in a flat rotating shaker at 700 rpm followed by centrifugation at 100,000 × g at 4 °C for 1 h. The pellets were washed and resuspended in 50 mm Tris-HCl, pH 7.4. Samples were used immediately or snap-frozen in liquid nitrogen and stored at −80 °C.Sarkosyl-insoluble Tau was prepared by diluting 5 mg of total protein of brain homogenate in 1 ml of A68 buffer (10 mTau was immunopurified from the brains of symptomatic TgP301S Tau mice (aged 4.5 months) as described  using anPhosphorylation-independent anti-Tau antibodies BR133 (amino terminus), BR304 , BR135 (repeat region), and BR134 (carboxyl terminus) have been described .235) (409) : total Tau, DA9 (amino acids 102–140)  and TG5 235)  and PG5 5) (409) .202/Thr(P)205) (212/Thr(P)214/Thr(P)217) r(P)205)  and AT10r(P)217)  as well HEK293T cells (ECACC) were transiently transfected with pcDNA3.1 constructs encoding human Tau using Lipofectamine 2000 (Invitrogen). The cells were split the following day and grown overnight. They were then seeded with Sarkosyl-insoluble Tau (at the concentrations stated in the figure legends) diluted in OptiMEM (Invitrogen). After 3 h, the seed/OptiMEM mix was removed and replaced with fresh complete medium ). Cells were then incubated for 3 days.For the production of an inducible cell line, HEK-TREx-293 cells (Invitrogen) were transfected with human 1N4R P301S Tau/pcDNA 4T/O using FuGene 6 (Roche Applied Science). Clones were selected in the presence of 5 μg/ml blasticidin and 200 μg/ml zeocin. Tau expression was induced in medium containing 1 μg/ml tetracycline for 24–48 h, and expression levels were evaluated by Western blotting with anti-Tau antibody DA9.g for 1 h at 4 °C, and the pellets were washed and resuspended in 15 μl of 50 mm Tris-HCl, pH 7.4. An aliquot of the supernatant was retained as the soluble fraction. The samples were resolved using 4–20% Tris-glycine gels. Tau protein was detected by enhanced chemiluminescence (ECL). The primary antibodies used were BR134, BR304, BR133, BR135 , AT8 , DA9 , and HT7 . Anti-GAPDH  was used as the loading control.Cell extracts were prepared in A68 buffer supplemented with protease and phosphatase inhibitors by lysis on ice for 30 min and a freeze-thaw cycle on dry ice. The samples were normalized to that with the lowest protein content, as determined by BCA. Total lysate samples were retained, and the remaining extracts were incubated with or without 1% Sarkosyl at room temperature for 1 h in a flat rotating shaker at 700 rpm. Samples were then centrifuged at 100,000 × versus insoluble analytes in absolute terms.The level of total and phosphorylated Tau were quantitated by AlphaScreen assays. The AlphaScreen technology is a sensitive and quantitative assay (similar to a sandwich ELISA) for the detection of molecules of interest in biological fluids . A biotiAlphaScreen assays (PerkinElmer Life Sciences) were carried out according to the manufacturer's instructions using specific anti-Tau antibodies. They used either biotinylated DA9 or biotinylated AT8 bound to donor beads and TG5 conjugated to acceptor beads (Ab-ACC). Ten μl/well of antibody mix consisting of Ab-ACC and biotinylated antibody were added to 384-well assay plates (Greiner), together with 5 μl/well of sample or standard diluted in AlphaScreen assay buffer . The plates were incubated overnight in the dark at 4 °C. Five μl of streptavidin-coated donor beads diluted in AlphaScreen buffer were then added to each well, and the plates were incubated in the dark with gentle agitation at room temperature for 4 h. They were read at excitation 680 nm and emission 520–620 nm using an Envision plate reader (PerkinElmer Life Sciences). The AlphaScreen counts or total Tau levels, relative to a standard of paired helical filaments purified from AD brain per μg of protein or per ml of sample, were calculated.-l-lysine-coated coverslips, fixed with 4% paraformaldehyde for 20 min, and probed by immunostaining. The antibodies used were AT100  or HT7 (1:500) with goat anti-mouse IgG secondary antibody conjugated to Alexa Fluor 488; PG5  with anti-IgG3 conjugated to Alexa Fluor 488; and 1N Tau (1:100) with anti-IgG1 conjugated to Alexa Fluor 568. Washings were carried out with PBS and antibody solutions prepared with either 0.1% Triton X-100 plus 1% bovine serum albumin (BSA) in PBS or 5% milk in PBS. DAPI or Hoechst staining was used to visualize nuclei. Confocal images were acquired using an Olympus Fluoview 1000 or Zeiss 710, with a 60× oil objective. High-content imaging was performed using a BD Pathway system (BD Biosciences).Cells were grown on polyTo reduce background staining, cells were incubated with 0.3% potassium permanganate for 5 min, washed, and incubated with 1% sodium metabisulfite plus 1% oxalic acid for 20–40 s . Cells wm ethylisopropyl amiloride (EIPA) (Sigma) or 300 nm latrunculin A (Sigma) for 30 min prior to and during the 3-h exposure to Tau seeds. For co-localization with dextran, cells were incubated for 1 h with 50 μg/ml dextran conjugated to Alexa Fluor 594 , plus 500 nm TgP301S Tau or synthetic Tau aggregates diluted in OptiMEM (Invitrogen). Cells were then washed with PBS and incubated with 0.0125% trypsin plus EDTA (Invitrogen) for 3 min at room temperature to remove extracellular Tau. Immunostaining with HT7 was as described.The uptake of TgP301S and synthetic P301S Tau seeds by cells was compared. The concentrations of both seeds were normalized by Western blotting before use. The cells were incubated with either 100 μm PMSF, and SDS-loading buffer was added. The samples were resolved on 4–12% bis-tris gels with MES running buffer, and Tau protein was detected by ECL. The primary antibody used was BR135 .Tau seeds were digested with 0.25–10 μg/ml proteinase K in PBS for 30 min at 21 °C. Digestion was stopped with 5 mm guanidine hydrochloride in 50 mm Tris HCl, pH 8, for 1 h at 21 °C. The samples were then centrifuged at 100,000 × g for 1 h at 4 °C. The pellets were washed and resuspended in 50 mm Tris-HCl, pH 7.4, and SDS-loading buffer was added. The samples were resolved on 4–20% Tris-glycine gels, and Tau protein was detected by ECL. The primary antibody used was HT7 .Tau seeds were disaggregated with 1–6 Tau seeds were placed on 400 meshed Formvar/carbon film-coated copper grids (Sigma) for 3 min, blocked with PBS plus 0.1% gelatin for 10 min, and incubated with anti-Tau antibody HT7 (1:50) for 1 h. Grids were then washed and incubated with secondary antibody 10-nm gold conjugate  for 1 h before being washed and stained with uranyl acetate. Images were taken on a Philips Spirit transmission electron microscope.m monomeric or aggregated recombinant Tau (assuming complete assembly) diluted in OptiMEM for 5–120 min prior to washing with cold PBS and detachment with trypsin/EDTA. This step also served to digest all extracellular Tau. Cells were resuspended in cold PBS and analyzed immediately (Sony Eclipse). Tau uptake was blocked by incubation with either 100 μm EIPA or 300 nm latrunculin A for 30 min prior to and during the 30-min exposure to Tau.Uptake of monomeric, aggregated full-length recombinant Tau or Tau immunopurified from TgP301S mice was measured by flow cytometry. Tau was labeled with DyLight 488 NHS-ester according to the manufacturer's guidelines (Thermo). Cells were exposed to 500 n202/Thr(P)205) . A dose-dependent increase in Sarkosyl-insoluble, AT8-positive 1N4R Tau was observed with increasing 0N4R seed concentrations, which was accompanied by a dose-dependent decrease in AT8-negative, Sarkosyl-soluble Tau . The use of two different isoforms (0N4R Tau seeds and expressed 1N4R Tau), in conjunction with specific antibodies, allowed the induced Tau aggregates to be distinguished from those added to the cells (A (bottom panel of Tau blots) and C). No inclusions were observed in HEK293 cells that were exposed to Tau seeds but did not express Tau.We examined the ability of Sarkosyl-insoluble Tau extracted from the brains of TgP301S Tau mice with a severe paraparesis to seed the assembly of Tau in HEK293T cells transiently transfected with P301S 1N4R Tau. Cells expressing Tau were exposed to Tau seeds for 3 h, washed, and then grown for a further 3 days. Expressed Tau was recruited into the Sarkosyl-insoluble fraction, as detected by antibodies DA9 (phosphorylation-independent) and AT8 (Ser(P)Seeding did not differ between 0N4R and 1N4R expressed Tau and was more efficient when P301S Tau rather than wild-type Tau was expressed (data not shown). Cells expressing P301S 1N4R Tau were used in all subsequent experiments unless otherwise stated.B). Sarkosyl-soluble Tau was phosphorylated at the MC6 (Ser(P)235) epitope. Following treatment with TgP301S Tau seed, there was a dose-dependent reduction in Sarkosyl-soluble total Tau ) and Tau phosphorylated at the MC6 (Ser(P)235) epitope ) and a parallel increase in Sarkosyl-insoluble total ) and MC6 (Ser(P)235)-positive Tau , suggesting that soluble Tau was being converted into insoluble Tau aggregates, consistent with the findings in Fig. 1A. In the Sarkosyl-soluble fraction, only a small amount of Tau was MC1 -positive (0.1 AlphaScreen units/μg); however, this species increased to 0.49 AlphaScreen units/μg (at the highest seed concentration) following seed treatment for 3 h and growth for 3 days ). In addition, this fraction was also AT8-positive following seed treatment but not upon soluble Tau expression and no seeding ). This could reflect an initial change in Tau conformation and subsequent phosphorylation upon seeding, followed by Tau becoming insoluble, as the concentration of these species increased, or the establishment of equilibria, whereby small misfolded and hyperphosphorylated soluble Tau species formed as a consequence of secondary nucleation events at the ends of filaments. We cannot completely exclude the possibility of an incomplete separation between Sarkosyl-soluble and Sarkosyl-insoluble material - and AT8 (Ser(P)202/Thr(P)205)-positive Tau in the Sarkosyl-soluble fractions were observed following 100,000 × g centrifugations at 30 min, 1 h, and 24 h). Following incubation with Tau seeds, a dose-dependent increase in the levels of MC1 - and AT8 (Ser(P)202/Thr(P)205)-positive Sarkosyl-insoluble Tau was observed .An AlphaScreen was used to measure the levels of different Tau species B. Sarkos409)-positive Tau (C).An inducible HEK293T cell line stably expressing P301S 1N4R Tau was used to further investigate seeded assembly. Cells were exposed to the Sarkosyl-insoluble fraction from the brains of TgP301S Tau mice 24 h after the induction of Tau expression and treated with 0.0125% trypsin to remove extracellular Tau. Exposure to 0.0125% trypsin/EDTA for 3 min at 21 °C completely removed extracellular, surface-bound Tau aggregates (data not shown). Tau aggregates were examined by fluorescence confocal microscopy. In cells expressing P301S 1N4R Tau, seeding gave rise to 1N- and PG5 (Ser(P)tive Tau C.202/Thr(P)205)-positive insoluble Tau, which was absent from cells seeded with Sarkosyl-insoluble material from non-transgenic mouse brain (A). We also examined the effect of varying the incubation times with seeds from 10 min to 3 h, followed by trypsin treatment and further growth for 3 days. Ten min of incubation was sufficient to seed the formation of AT8 (Ser(P)202/Thr(P)205)-positive insoluble Tau. Increasing the incubation time resulted in the formation of more insoluble Tau (B). These findings were confirmed by quantitative high-content imaging using antibody PG5 (Ser(P)409) to visualize the Tau inclusions (C).Following seed addition, we observed a time-dependent increase in AT8 (Ser(P)se brain A. We alsuble Tau B. These clusions C.202/Thr(P)205)-positive Tau determined by AlphaScreen assays. Sarkosyl treatment enriched for MC1 - and AT8 (Ser(P)202/Thr(P)205)-positive Tau relative to the total brain lysate  (A). We next compared the seeding abilities of these preparations. Whereas Sarkosyl-insoluble TgP301S Tau led to the accumulation of Sarkosyl-insoluble AT8 (Ser(P)202/Thr(P)205)-positive Tau, the total brain lysate failed to seed at this concentration (B).The Sarkosyl-insoluble fraction from the brains of TgP301S Tau mice was compared with total brain lysate. Tau levels were normalized, and the levels of MC1 - and AT8 (Ser(P) lysate) A. We nexntration B.A). Furthermore, the uptake of both Tau species over 30 min was greatly reduced at 4 °C and in the presence of 100 μm EIPA (B). A similar reduction by 300 nm latrunculin A was also observed (data not shown). This is consistent with endocytosis by macropinocytosis. Only aggregated Tau species were able to induce the aggregation of soluble Tau in the cytosol, giving rise to Sarkosyl-insoluble Tau composed of the expressed 1N Tau isoform. The latter failed to form in cells exposed to monomeric Tau (C).Both monomeric and aggregated recombinant full-length Tau were internalized by cells at 37 °C, with similar kinetics over 120 min, suggesting a common uptake mechanism A. Furthe μm EIPA B. A simieric Tau C.n = 50). Assembled recombinant P301S Tau comprised filaments with a mean length of 578 ± 363 nm (n = 50). To equate filament lengths, we sonicated the recombinant Tau filament preparation for 15 s  to produce filaments with a mean length of 179 ± 82 nm (n = 50) (A). Both preparations bound thioflavin T (data not shown).We compared Sarkosyl-insoluble Tau aggregates from the brains of TgP301S Tau mice with heparin-assembled full-length recombinant P301S 0N4R Tau. Immunoelectron microscopy with anti-Tau antibody HT7 showed that TgP301S human Tau existed as short, straight filaments with a mean length of 203 ± 83 nm ((n = 50) A. Both pB and C). Only the TgP301S Tau aggregates induced the formation of Sarkosyl-insoluble, hyperphosphorylated Tau from expressed protein. However, with increasing concentrations of recombinant P301S Tau aggregates, Sarkosyl-insoluble, hyperphosphorylated Tau formed from expressed protein, as seen by immunoblotting with HT7 (phosphorylation-independent) (B) and AT100 (Ser(P)212/Thr(P)214/Thr(P)217) and labeling with thioflavin S (C). Approximately 50 times more synthetic P301S Tau seed than TgP301S Tau seed was needed to produce equivalent amounts of Sarkosyl-insoluble Tau from expressed protein (B and C). Immunoelectron microscopy of the Sarkosyl-insoluble fraction from cells seeded with either TgP301S Tau aggregates or recombinant P301S Tau aggregates showed the presence of Tau filaments made of expressed 1N Tau (D). This indicates that, although both native and recombinant P301S Tau aggregates can seed the formation of Sarkosyl-insoluble, hyperphosphorylated Tau in cells expressing soluble P301S Tau, native Tau aggregates are more potent.We normalized the concentration of TgP301S Tau aggregates to the sonicated recombinant P301S Tau aggregates by immunoblotting and assessed their seeding potencies in the cell-based assay , B and Cpendent) B and AT1flavin S C. Approx protein , B and Cd 1N Tau D. This im TgP301S Tau aggregates and 500 nm recombinant Tau aggregates were taken up by cells within 3 h, as detected by immunohistochemistry (A). In order to quantify uptake, we immunopurified TgP301S Tau aggregates and confirmed that this material had characteristics similar to those of Sarkosyl-extracted TgP301S Tau (data not shown). This allowed us to directly label the Tau aggregates with DyLight 488 and to remove potential contaminating factors. We then applied equal concentrations of TgP301S Tau aggregates and recombinant P301S Tau aggregates to cells for 30 min and assessed uptake by measuring the mean fluorescence intensity of cells by flow cytometry . The mean fluorescence intensity of TgP301S Tau aggregate-treated cells was 77.6 ± 4.4% that of the recombinant P301S Tau aggregate-treated cells despite the TgP301S Tau aggregates displaying a significantly greater seeding potency (B and C). We therefore concluded that the different seeding potencies of TgP301S Tau aggregates and recombinant P301S Tau aggregates were not due to differences in uptake efficiency.We wanted to exclude the possibility that the difference in seeding between TgP301S Tau aggregates and recombinant P301S Tau aggregates was the result of the two preparations entering cells through different mechanisms. Both 500 nhemistry A. In ord potency , B and Cm) or latrunculin A (300 nm) for 30 min prior to and during the 30-min exposure to Tau aggregates, suggesting a shared uptake mechanism consistent with macropinocytosis (B). Treatment with EIPA reduced the mean fluorescence intensity of TgP301S Tau aggregate-treated cells by 59.2 ± 3.9% and recombinant P301S Tau aggregate-treated cells by 80.9 ± 0.7%. Similarly, treatment with latrunculin A reduced the mean fluorescence intensity of TgP301S Tau aggregate-treated cells by 65.6 ± 6.8% and recombinant P301S Tau aggregate-treated cells by 66.7 ± 2.7%. We conclude that the different seeding potencies of TgP301S Tau aggregates and recombinant P301S Tau aggregates were not caused by different uptake mechanisms. We also observed co-localization of TgP301S Tau aggregates and recombinant P301S Tau aggregates with the fluid phase endocytosis cargo dextran (C).Uptake of both TgP301S Tau aggregates and recombinant P301S Tau aggregates was significantly reduced by treating the cells with EIPA (100 μocytosis B. Treatm dextran C.A). Equivalent amounts of recombinant P301S Tau that had aggregated in the presence of heparin failed to induce the aggregation of recombinant P301S Tau (data not shown). When normalized for total Tau, the recombinant Tau aggregates seeded by aggregated TgP301S Tau in the absence of heparin were equally potent as aggregated TgP301S Tau in seeding assays (B and C).To examine the effects of aggregate conformation on the potency of Tau seeding and to control for the presence of heterologous components in the TgP301S aggregates, we seeded recombinant P301S Tau in the absence of heparin with 5% (v/v) Sarkosyl-insoluble fraction from the brains of mice transgenic for human mutant P301S Tau. Recombinant P301S Tau aggregated, as measured by thioflavin T binding over time A. Equivag assays , B and Cin vitro. Sequential phosphorylation of recombinant P301S Tau with PKA and SAPK4, in the presence of heparin, gave rise to 64-kDa hyperphosphorylated Tau, as described previously , the AT8 epitope (Ser(P)202/Thr(P)205; Fig. 8A, panel 4), and the AT100 epitope (Ser(P)212/Thr(P)214/Thr(P)217; Fig. 8A, panel 3). Hyperphosphorylation of these sites is characteristic of pathological Tau   .B) ..B) (50).C). As described previously, TgP301S Tau aggregates were more potent seeds than unphosphorylated recombinant P301S Tau aggregates, even at a 10-fold dilution. Hyperphosphorylated recombinant P301S Tau aggregates were no more potent than unphosphorylated recombinant P301S Tau aggregates .We compared the seeding abilities of hyperphosphorylated and unphosphorylated recombinant P301S Tau aggregates as well as of 10-fold diluted TgP301S Tau aggregates C. As desA). Recombinant P301S Tau aggregates were more resistant to proteinase K digestion than TgP301S Tau aggregates.To further test the hypothesis that conformation determines the seeding potency of Tau aggregates, we examined the effect of aggregate digestion by proteinase K using immunoblotting with anti-Tau antibody BR135 (repeat region) A. RecombB and C). Recombinant P301S Tau aggregates were significantly more resistant to disaggregation by guanidine hydrochloride than TgP301S Tau aggregates. Moreover, treatment with low concentrations of guanidine hydrochloride increased the detection of TgP301S Tau aggregates by HT7 (phosphorylation-independent), probably through increased exposure of its epitope. Similar results were also obtained with DA9, another phosphorylation-independent anti-Tau antibody (data not shown). This was not observed for recombinant P301S Tau aggregates. Recombinant Tau seeded by TgP301S Tau aggregates in the absence of heparin displayed stability to guanidine hydrochloride similar to that of TgP301S Tau aggregates; it was significantly different from the guanidine stability of heparin-assembled recombinant Tau aggregates. Hyperphosphorylated heparin-assembled recombinant Tau aggregates did not show guanidine hydrochloride stability significantly different from that of unphosphorylated heparin-assembled recombinant Tau aggregates.We then used a guanidine stability assay to further examine conformational differences between TgP301S Tau aggregates and recombinant Tau aggregates. We also evaluated recombinant Tau seeded by TgP301S Tau aggregates in the absence of heparin and hyperphosphorylated recombinant Tau aggregates assembled in the presence of heparin , B and Cin vitro seeding.These findings suggest that there are significant conformational differences between native and heparin-assembled recombinant Tau aggregates, which are reflected in their respective seeding potencies. Moreover, unphosphorylated recombinant Tau can acquire the conformation of TgP301S Tau through A). HEK 293T cells expressing 0N4R Tau were exposed to the Sarkosyl-insoluble fraction from the brains of symptomatic TgP301S Tau mice for 3 h, followed by 3 days of growth. Seeding was evaluated by the detection of Sarkosyl-insoluble, hyperphosphorylated Tau. 0N4R Tau lacking the repeat region (amino acids 1–187) was incapable of being seeded (B), whereas its carboxyl-terminal half with the repeats (amino acids 188–383) could be seeded (C). Cells expressing full-length 0N4R Tau lacking residues 275VQIINK280 and 306VQIVYK311 (Δ275–280/Δ306–311), could not be seeded (D).We investigated the regions of expressed soluble full-length Tau required for seeding A. HEK 29g seeded B, whereae seeded C. Cells e seeded D.Using a cell-based assay, we compared the seeding activity of the Sarkosyl-insoluble fraction from the brains of symptomatic TgP301S Tau mice with that of total brain lysate. The Sarkosyl-insoluble fraction, which contained filamentous Tau, had a much greater seeding activity than the lysate, when normalized to total Tau. This indicates that the Tau species required for seeding are likely to be Sarkosyl-insoluble. Monomeric Tau was devoid of seeding activity, as was Sarkosyl-soluble Tau lacking aggregated Tau species (data not shown), showing the need for aggregation. Aggregation was also necessary for the ability of Tau to undergo seeding because Tau proteins incapable of aggregation could not be seeded. These findings using full-length Tau are in agreement with work showing that the seeding of tagged Tau repeats was aggregation-dependent . In the The Sarkosyl-insoluble fraction from TgP301S Tau mouse brain was enriched in aggregated and AT8-positive Tau. However, there are a number of potential Tau species, including small oligomers, which may contribute to the seeding activity. Further studies are required to characterize the full range of seed-competent species in the TgP301S Tau model.These findings are reminiscent of the behavior of prions consisting of conformationally modified prion protein , 62 and Here we show that, similar to synthetic prions, α-synuclein aggregates, and Aβ assemblies, synthetic Tau aggregates had a greatly reduced seeding activity compared with Sarkosyl-insoluble Tau from TgP301S Tau mouse brain. It has been suggested that the lower potency of synthetic prions, α-synuclein aggregates, and assembled Aβ could be the result of the presence of multiple molecular species, only some of which had seeding activity. We used sonication to create a homogeneous preparation of Sarkosyl-insoluble synthetic Tau filaments, as characterized by electron microscopy, reducing the likelihood of differences in filament length being the reason for the lower seeding ability. Synthetic Tau aggregates were more resistant to disaggregation by guanidine hydrochloride and digestion by proteinase K than Tau aggregates from TgP301S Tau mouse brain, consistent with the hypothesis that more stable aggregates possess lower seeding activity. These findings are in agreement with a study showing that Tau filaments produced by incubating recombinant human Tau with heparin were more stable than paired helical filaments from AD brain .Native and synthetic Tau aggregates may have distinct conformations. We used heparin to form synthetic aggregates; it compacts the repeats and induces dimerization of Tau, with filaments growing through monomer addition , 23. Thiin vitro and tested their seeding potency. Hyperphosphorylated recombinant P301S Tau aggregates were no more potent at seeding than unphosphorylated recombinant P301S Tau aggregates, demonstrating that phosphorylation was not necessary for seeding activity, although the seeded Tau aggregates were hyperphosphorylated and may have carried additional modifications. It is important to note that the recombinant Tau seeded with TgP301S Tau aggregates did not carry any post-translational modifications either but was able to seed as efficiently as TgP301S Tau aggregates. Moreover, seeding efficiency was not reduced in cell lines expressing Tau incapable of undergoing phosphorylation at disease-specific sites.We cannot rule out the possibility that post-translational modifications may reduce the stability of Tau aggregates because filaments from TgP301S Tau mouse brains were hyperphosphorylated  and may Macropinocytosis has been implicated in the uptake of aggregated Tau repeats into cells in culture . We founElucidation of the molecular mechanisms underlying aggregation and propagation is important for understanding the pathogenesis of the tauopathies, which appear to originate in defined brain regions, before spreading through the brain –29. This–66–73–Detailed characterization of the molecular species responsible for propagation is important for uncovering the mechanisms underlying seeding and spreading, for developing diagnostic tests, and for devising therapies that target these species .The fact that the conformation of aggregated Tau can determine its seeding characteristics may provide a molecular explanation for the tauopathies, which are defined by the presence of Tau inclusions in the brain but have distinct neuropathologies and rates of progression. It has been shown that the intracerebral injection of brain homogenates from human 4R tauopathies  into mice transgenic for one isoform of wild-type 4R human Tau seeded the formation of pathological inclusions like those seen in the human tauopathies . Future


Furthermore, we examined SCP3 expression in tumor specimens from 181 cervical cancer and 400 cervical intraepithelial neoplasia (CIN) patients by immunohistochemistry and analyzed the correlation between SCP3 expression and clinicopathologic factors or survival. Overexpression of SCP3 promoted AKT-mediated tumorigenesis both in vitro and in vivo. Functional studies using NIH3T3 cells demonstrated that the C-terminal region of human SCP3 is important for AKT activation and its oncogenic potential. High expression of SCP3 was significantly associated with tumor stage (P = 0.002) and tumor grade (P<0.001), while SCP3 expression was positively associated with pAKT protein level in cervical neoplasias. Survival times for patients with cervical cancer overexpressing both SCP3 and pAKT  were significantly shorter than for patients with low expression of either SCP3 or pAKT  as determined by multivariate analysis (P = 0.020). Our findings suggest that SCP3 plays an important role in the progression of cervical cancer through the AKT signaling pathway, supporting the possibility that SCP3 may be a promising novel cancer target for cervical cancer therapy.Synaptonemal complex protein 3 (SCP3), a member of Cor1 family, is up-regulated in various cancer cells; however, its oncogenic potential and clinical significance has not yet been characterized. In the present study, we investigated the oncogenic role of SCP3 and its relationship with phosphorylated AKT (pAKT) in cervical neoplasias. The functional role of SCP3 expression was investigated by overexpression or knockdown of SCP3 in murine cell line NIH3T3 and human cervical cancer cell lines CUMC6, SiHa, CaSki, and HeLa both  Tumor cells display a variety of antigens including anomalous expression of cancer/testis-associated antigens (CTAs). CTAs are restricted in normal tissues to germ cells of the testis, with occasional expression in female reproductive organs, and are expressed in histologically different types of malignant human tumors Cor1 family members such as X-linked lymphocyte regulated protein (XLR), Xlr-related meiosis regulated protein (XMR), and synaptonemal complex protein 3 (SCP3) are typical CTAs in vitro and in vivo experiments using both a murine cell line (NIH3T3) and human cervical cancer cell lines . Here, we report that overexpression of SCP3 induces phenotypic changes characteristic of transformation both in vitro and in vivo. Furthermore, we analyzed patterns of SCP3 and pAKT expression by immunohistochemistry in cervical tissue specimens from patients with cervical intraepithelial neoplasia (CIN) or invasive cervical carcinoma. The relationships between protein expression and clinicopathological parameters/survival of cervical cancer patients were also analyzed, and demonstrated that SCP3 expression is a prognostic factor for patients with cervical cancer.To elucidate the role of SCP3 in tumorigenesis, we examined the role of these members as potential oncoproteins by conducting a series of 2 balanced air at 37°C. The identities of cell lines were confirmed by short tandem repeat (STR) profiling by IDEXX Laboratories Inc. and used within 6 months for testing.Six- to eight-week-old female Balb/c Nude mice were purchased from Daehan Biolink . All animal procedures were performed under a protocol approved by the Korea University Institutional Animal Care and Use Committee (KUIACUC-2009-126). The murine fibroblast cell line NIH3T3 and human cervical cancer cell lines CUMC6, SiHa, CaSki, and HeLa were purchased from American Type Culture Collection  and were grown in Dulbecco's Modified Eagle Medium (DMEM) in the presence of 10% fetal bovine serum (FBS). All cells were cultured in 5% COhttp://www.kgcb.or.kr). All tumor tissues were histologically reviewed and only specimens with a sufficient abundance of tumor cells were included in tissue microarray construction. Staging was performed according to the International Federation of Gynecology and Obstetrics (FIGO) staging system. Primary treatment for invasive cervical cancer consisted of a type 3 radical hysterectomy with pelvic lymph node dissection. Concurrent Platinum-based chemoradiation was given in cases with increased risk of recurrence, such as positive resection margins, positive lymph nodes, or parametrial invasion. Medical records were reviewed to obtain data including age, surgical procedure, survival time, and survival status. Response to therapy was assessed according to the Response Evaluation Criteria in Solid Tumors , either by computed tomography or magnetic resonance imaging Primary tumor specimens were obtained between 1996 and 2010 from 181 cases of cervical cancer, 301 cases of high grade cervical intraepithelial neoplasia (CIN), and 99 cases of low grade CIN undergoing primary surgery at Gangnam Severance Hospital, Yonsei University College of Medicine. All patients gave oral and written informed consent. Paraffin blocks for some of the patients were provided by the Korea Gynecologic Cancer Bank through the Bio & Medical Technology Development Program of the Ministry of Education, Science, and Technology, Korea , 1-80, 1-130, 81-236, and 131-236 fragments were subcloned into the pMSCV-puro vector (Clontech) at the Xho I and EcoR I restriction sites. Recombinant pMSCVs encoding SCP3 or its deletion mutants were confirmed by DNA sequence analysis.hSCP3 full and deletion mutants were created with a PCR-based strategy from a human testis cDNA library  with the following primers: hSCP3 1 forward BamHI-compatible) and reverse 5′- AGCTTTTCCAAAAAAGGAGAAGAATCATGATAATCTCTTGAATTATCATGATTCTTCTCCG-3′ (HindIII-compatible) oligonucleotides were ligated to BamHI/HindIII-digested pSilencer 3.1-H1 puro. The shRNA control employed for these studies was a scrambled DNA sequence that does not target any identified human coding sequence .To generate the Scp3 short hairpin RNA (shRNA) exoression construct human Scp3-shRNA annealed forward 5′ GATCCGGAGAAGAATCATGATAATTCAAGAGAT TATCATGATTCTTCTCCTTTTTTGGAAA-3′  , incubated for 30 min on ice, and then were scraped and centrifuged. Protein concentrations were determined by the coomassie plus protein assay . 50 µg of protein were solubilized in Laemmli buffer , boiled for 5 min, and then separated by SDS polyacrylamide gel electrophoresis. Separated gel was transferred to nitrocellulose membranes at 90 V for 1 hr on ice. Primary antibodies against phospho AKT (Ser473), AKT, and phospho ERK (T202/Y204) were purchased from Cell Signaling  and used at a dilution of 1∶1000. Likewise, primary antibodies against dual phospho p38 MAPK  and SCP3  were used at a dilution of 1∶1000, while antibodies against β-actin and Flag  were used at a dilution of 1∶10,000 in Tris-buffered saline (TBS)-T containing 5% BSA  at 4°C overnight, followed by 3 washes in TBST, 5 minutes per wash. Goat anti-mouse IgG-HRP, anti-rabbit IgG-HRP secondary antibodies (1∶5000) (Stressgen) were incubated for 1 hr at room temperature conjugated with horseradish peroxidase. Immunoreactive bands were visualized by enhanced chemiluminescence . Densitometry was performed using an image analyzer Fujifilm LAS-4000  and Multi Gauge V3.1 imaging software . To quantitate the intensity profile of protein band images, we used Image J densitometry software . The band images were scanned in grayscale at a resolution of at least 600dpi and expressed a percentage of the total size of all the measured peaks. To calculate a relative value, the intensity of each sample was divided by that of control. Numbers below western blots refer to the relative values of the intensity normalized to each control.For each experiment, a total of 5×104 cells per well for 3 days. The percentages of live cells were determined using a hematocytometer after 0.4% trypan blue staining. Measurements were made in triplicate for each of the cell lines and the experiments were repeated three times. For the soft-agar colony formation assay, 1×104 NIH3T3 cells, CaSki cells and HeLa cells were plated in 6-well culture plate as a suspension in 2 mL of DMEM containing 10% FBS and 0.4% agar on top of the base layer of 0.7% agar containing 2 mL of the same medium. Plates were incubated at 37°C for 2 weeks until colonies were formed. Two weeks later, >2 mm colonies were counted with an ocular micrometer on a microscope. All experiments repeated at least twice.For cell proliferation assays, the cells (n = 3) were grown in six well plates at a density of 1×10in vitro delivery, NIH3T3 cells on a 6-well vessel were transfected with 300 pmol of the synthesized siRNAs using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The siRNA-treated cells were collected 48 h after transfection for western blot analysis, cell proliferation assay, and colony forming assay.Synthetic small interfering RNA (siRNA) specific for green fluorescent protein (GFP) and mouse AKT were purchased from Invitrogen . The following sequences were used: GFP (non-specific target control) 5′-GCAUCAAGGUGAACUUCAA-3′ (sense), 5′-UUGAAGUUCACCUUGAUGC-3′ (antisense); AKT, 5′-GACAACCGCCAUCCAGACU-3′ (sense), 5′-AGUCUGGAUGGCGGUUGUC-3′ (antisense). For 5 cells/mouse), CaSki cells (1×106 cells/mouse), or HeLa cells (1×106 cells/mouse) were injected subcutaneously into Balb/c Nude mice. Tumor size was measured twice a week for 24 days after tumor cell injection. Each individual tumor size was measured with a caliper, and the tumor volume was calculated using the following formula: tumor volume (mm3) = [width×length2]/2 The transfected NIH3T3 cells  in molds. For immunofluorescent analyses, 10-µm cryosections were placed on poly-L-lysine–coated slides, fixed in 4% paraformaldehyde, and blocked in PBS with 10% normal goat serum (NGS) (NGS/PBS). Tissue sections were incubated with pAKT antibody  for overnight at 4°C. After washing, sections were incubated with the secondary Alexa555-conjugated goat anti–rabbit IgG  and then mounted in Fluorescent Mounting medium  on glass microscope slides. All images were acquired using a Zeiss LSM 5 Pascal confocal microscope . To quantitate the intensity profile of pAKT fluorescence image in tumor tissue sections, we used ImageJ densitometry software. The tumor tissues on slides were scanned in grayscale at a resolution of at least 600dpi. Tumor areas on scanned images were selected more than 10 times from each group with freehand tool and measured the mean grey value. To calculate a relative value, the intensity of each sample was divided by that of blank slides (without primary antibody).th section for the presence of tumor cells.Full-face sections of all donor blocks were stained with haematoxylin and eosin (H&E) and reviewed by a pathologist who marked representative tumor areas. The tissue microarray (TMA) was constructed using a Manual Tissue Arrayer MTA-1 . Four 1.0 mm diameter tissue cores consisting of matched tumor specimens and normal epithelial samples were extracted from selected regions of each donor block. The presence of tumor tissues on the TMA was verified with H&E staining. Multiple 5 µm thick sections were cut with a microtome and H&E staining of TMA slides were examined every 502O2 for 10 minutes. Antigen retrieval was performed in a steam pressure cooker  with prewarmed antigen retrieval buffer pH 9 (Dako) at 95°C for 10 minutes. To minimize nonspecific staining, sections were incubated with protein block (Dako) for 20 minutes. The sections were incubated with an anti-SCP3 antibody  overnight at 4°C or an anti-pAKT antibody  for 120 minutes at room temperature. EnVision FLEX+ and EnVision+ Dual Link System (Dako) were used for the detection of SCP3 and pAKT, respectively. Staining was visualized using 3,3′-Diaminobenzidine (DAB) and the sections were counterstained with hematoxylin. Finally, the slides were covered and observed under a light microscope . In this study, five conventional whole section slides were carefully selected to validate the immunohistochemical staining performed for SCP3 and pAKT. During this process, staining patterns were thoroughly compared, and some of the patterns matched those of conventional slides from the same case. Furthermore, in designing this study, stromal cells from the whole sections of cercival cancer were selected as internal positive control while the primary antibody was omitted from the negative control.TMA sections were deparaffinized and hydrated in xylene and descending gradient alcohol solutions. Endogenous peroxidase was blocked by incubation in 3% HSCP3 and pAKT staining results were scored based on (a) intensity  and (b) the percentage of positive stained epithelial cells  . An overP values<0.05 were considered statistically significant.All data are representative of at least 2 independent experiments. Non-parametric 1-way or 2-way ANOVA was performed with SPSS version 18.0 software . Comparisons between individual data points were assessed with Student's T-test. Statistical analyses of SCP3 and pAKT expression were performed using the Mann-Whitney test and the Kruskal-Wallis test. The Spearman nonparametric correlation test was used to analyze the relationship between SCP3 and pAKT. Survival curves were estimated by Kaplan-Meier analysis. The log-rank test was used to compare the differences of survival distribution between groups. The multivariate analysis with Cox proportional hazards model was performed to identify independent predictors of disease-free survival in adjustment with relevant clinical covariates such as age, FIGO stage, histologic type, tumor grade, tumor size, and lymph node status. The final model was chosen based on the result of univariable analysis as well as consideration of the clinical or biological importance of the variables. All statistical tests were two-sided, and P<0.005). The results of a soft agar colony formation assay with the same cell lines were consistent with those of the cell proliferation assay (P<0.005). We next subcutaneously injected hSCP3- or no insert-expressing NIH3T3 cell in the left flank of athymic Balb/c Nude mice . We next explored whether other genes from the murine Cor1 family such mXLR, mXMR and mSCP3, which share more than 60% homology with hSCP3, could have similar tumorigenic potential in the NIH3T3 model in vitro cell proliferation and soft agar colony formation as well as in vivo tumor formation in NIH3T3 cell line model, which was consistent with that of the hSCP3. Taken together, these data clearly demonstrate the oncogenic potentials of SCP3 and other Cor1 members in NIH3T3 model.A previous study reported that human SCP3 (hSCP3) is expressed in various cancer cells and that its expression in human specimens is associated with oncogenesis on assay . NIH3T3/ude mice . ConsistP<0.001). Consistently, API2-treated NIH3T3/hSCP3 cells also formed smaller colonies, the number of which was almost 5-times less than colonies formed by DMSO-treated NIH3T3/hSCP3 cells. Thus, it is obvious that both proliferation and colony formation in these cells are dependent on the AKT signaling. To exclude potential off-target effects of API2, we performed in vitro cell proliferation and soft agar colony formation experiments using siRNA-targeting AKT (siAKT) to block the AKT pathway. siRNA targeting to irrelevant GFP (GFP siRNA) was used as a negative control. Consistent with API2 data, siAKT-treated NIH3T3/hSCP3 cells exhibited a decreased rate of cell proliferation  failed to increase phosphorylation of AKT  or hSCP3131-236 (P<0.003)]. Similarly, immunofluorescence staining for pAKT was increased substantially in histological sections of tumors ectopically expressing hSCP381-236 or hSCP3131-236 [no insert vs. hSCP381-236 (P<0.05) or hSCP3131-236 (P<0.003)] (As schematized in n of AKT . Convers of pAKT . Consisto insert . More im<0.003)] . Taken tin vitro data, the tumor-forming ability of CaSki cells was significantly increased after introducing SCP3 (in vitro cell growth rate and colony formation efficacies and in vivo tumor growth rate compared with shGFP control group (We further investigated the oncogenic role of SCP3 and its relationship with phosphorylated AKT (pAKT) in human cervical cancer cell lines. For this, the expression of SCP3 was measured in human cervical cancer cell lines CUMC6, SiHa, CaSki, and HeLa using western blot analysis. As shown in ing SCP3 . In contol group . These dHaving elucidated the molecular mechanism by which SCP3 activation promotes tumor growth via the AKT pathway, we next examined SCP3 and pAKT protein expression levels by immunohistochemistry in cervical tissue specimens from patients with CIN or invasive cervical cancer. P = 0.002), differentiation (P<0.001) and chemoradiation response (P = 0.005). However, there was no statistically significant difference between pAKT expression and clinicopathologic factors.Representative immunohistochemical staining of SCP3 and pAKT are shown in P<0.001) and cervical cancer   were available to confirm co-expression between SCP3 and pAKT, whereas 173 tumor samples (95.6%) were available to confirm co-expression between SCP3 and pAKT in cervical cancer. Notably, expression of SCP3 showed a positive linear correlation with that of pAKT both in CIN  .Taken toP = 0.001) and overall survival  compared with that of patients with low expression of SCP3 (P<0.001) and overall survival (P = 0.009) compared with the combined low SCP3 and low pAKT group. Interestingly, there were no deaths in combined low SCP3 and low pAKT patients (n = 37). We next examined the independent prognostic significance of SCP3 alone and high SCP3 combined with high pAKT, as well as other clinicopathological parameters, using the Cox proportional hazards model. According to multivariate analysis, FIGO stage was a significant risk factor for overall survival (P = 0.013), whereas lymph node metastasis remained a significant risk factor for disease-free survival (P = 0.024) , P = 0.023] and overall survival . Notably, the combination of high SCP3 and high pAKT expression was a significant risk factor for both disease free survival  and overall survival .To investigate the clinical relevance of SCP3, we examined the effect of SCP3 expression on patient outcomes. Five-year disease-free survival and overall survival were analyzed through Kaplan-Meier plots as shown in  of SCP3 . Further= 0.024) . High SCin vitro as well as increased tumor growth in vivo using a mouse model system. Furthermore, we observed activation of the AKT pathway in hSCP3-overexpressing cells. Interestingly, AKT activation appeared to be associated with hSCP3-mediated oncogenic potential, which was confirmed using an API-2 chemical inhibitor, siAKT treatment, and deletion mutant analysis.There is increasing evidence that hSPC3 may play a role in oncogenesis; however, the evidence has not been addressed at a cellular or molecular level. In this report, we provide the first compelling data that hSCP3 can play a key role in human tumorigenesis. We demonstrate that ectopic expression of hSCP3 in conventional non-tumorigenic murine NIH3T3 cells and a human cervical cancer cell line CaSki cells leads to increased cell proliferation and colony formation 1-80 deletion mutant exhibited a significant increase of colony number, proliferation, and tumor volume despite the lack of increased Akt phosphorylation in a NIH 3T3 tumor cell model as shown in The AKT pathway plays a pivotal role in transformation by inducing cell survival, growth, migration, and angiogenesis P<0.001). To the best of our knowledge, this is the first report to show that SCP3 protein expression increases according to severity of cervical lesions. Moreover, we observed that SCP3 protein expression was significantly associated with advanced tumor stage (P = 0.002), poor tumor grade (P<0.001), and poor response to chemoradiation therapy (P = 0.005), and exhibited the highest levels of expression in metastatic tissue specimens  and cervical cancer (P = 0.010) (P<0.001) and cancer specimens . Together, these data indicated that SCP3 mediates an oncogenic phenotype of cervical cancer cells through an AKT-dependent pathway. Collectively, the results of this study provide the first molecular and cellular evidence of AKT-dependent oncogenic properties of hSCP3, which may serve as a novel therapeutic target for cervical cancer therapy.Lastly, we examined the correlation of SCP3 expression with pAKT expression in CIN and invasive cervical cancer tissues. Consistent with our = 0.010) . Even whFigure S1Evaluation of SCP3 and pAKT IHC staining. Representative immunohistochemical staining images of the staining intensity (A) weak staining, 1; (B) moderate staining, 2; or (C) strong positive staining in most cells, 3 and the percentage of positive stained epithelial cells (D) 1–25% cells staining positive, 1; (E) 26–50% cells staining positive, 2; (F) 51–75% cells staining positive, 3; or (G) more than 75% cells staining positive, 4. Scale bar: 30 µm (A–C), 100 µm (D–G).(TIF)Click here for additional data file.Figure S2In vitro growth curves of NIH3T3 cells expressing each of the Cor1 members. NIH3T3/no insert cells were used as a control. The cells were counted after trypan blue staining to exclude dead cells. (C) Bar graph representing the number of colonies with a diameter greater than 2 mm in soft agar. (D) Representative colony images of each group . (E) Tumorigenicity of NIH3T3 cells expressing each of the Cor1 members. Balb/c Nude (n = 5) mice were inoculated subcutaneously with 1×105 cells/mouse using NIH3T3/no insert, NIH3T3/mSCP3, NIH3T3/mXMR, or NIH3T3/mXLR cells. Tumor volumes were measured beginning 28 days after tumor inoculation. (F) Representative tumor images of each group.Members of the Cor1 family have oncogenic potential. (A) NIH3T3 cells retrovirally transduced with pMSCV vector encoding either no insert (NIH3T3/no insert), mSCP3 (NIH3T3/mSCP3), mXMR (NIH3T3/mXMR), or mXLR (NIH3T3/mXLR) were analyzed for cellular expression of Cor1 members tagged with Flag by Western blot. (B) (TIF)Click here for additional data file.Figure S3In vitro growth curves and (E) in vitro soft agar colony formation of siGFP or siAKT-transfected NIH3T3/mXLR cells. Error bars represent the mean ± SD.Oncogenesis by Cor1 family is AKT-dependent. (A) Western blot analysis of levels of pAKT in NIH3T3/no insert, NIH3T3/mSCP3, NIH3T3/mXMR, or NIH3T3/mXLR cells. (B) Soft agar colony-forming capacity of NIH3T3/mXLR cells in the presence of API2 (Akt inhibitor), PD98059 (Erk inhibitor) or SB203580 (p38 inhibitor); (Left) Representative colony images of each group; (Right) Bar graph representing the number of colonies with a diameter greater than 2 mm in soft agar . (C) Western blot analysis of levels of pAKT in NIH3T3/hSCP3 cells transfected with siRNA targeting GFP or AKT (siGFP or siAKT) to confirm the reduction of protein levels of AKT. (D) (TIF)Click here for additional data file.Figure S4Relationship between the SCP3 and pAKT expression in CIN (left panel) and cancer (right panel) samples. The values of pAKT IHC expressions were plotted on the y-axis against the IHC scores of SCP3 on the x-axis.(TIF)Click here for additional data file.Table S1Characteristics of Patients.(DOCX)Click here for additional data file.Table S2Expression of SCP3 and pAKT in relation to clinicopathological characteristics in IHC analysis.(DOCX)Click here for additional data file.


Becker muscular dystrophy (BMD) is a severe genetic disease caused by an X-linked recessive mutation leading to reduced/abnormal dystrophin, resulting in a peripheral myopathy and a cardiomyopathy (CM). The purpose of this study is to evaluate the myocardial structure and function in BMD.From April 2012 to May 2014, 84 consecutive patients  with Becker muscular dystrophy (BMD) and 12 control subjects underwent cardiac magnetic imaging (MRI) on a 3-T clinical scanner . T1 maps and apparent extracellular volume fraction (appECV) pre- and post-Gd-DOTA injection was performed with a prototype modified Look-Locker sequence . T2 maps were generated with a T2 prepared TrueFISP sequence acquired with 3 preparation times . Late Gadolinium enhancement (LGE), T1, T2, appECV were assessed according to the classification of the AHA segmentation.t test for paired data. The abnormality cut-off values of GdT1 and T2 in the control group were respectively 400 ms and 53 ms. Combining both Gd T1 and T2 data , both fibrosis and inflammation in 1,2% vs 0.6 % (decreased T1 and increased T2).Mean left ventricular (LV) ejection fraction was 61±12% (range: 23-87 %), LV end-diastolic volume was 68±26 ml/m2 (range: 32-185 ml/m2) and LV mass 76±17 g/m2 (range: 48-122 g/m2). The values of native T1, GdT1, T2 and appECV of the LW l and the septum are summarized in table The LGE patterns are different in primitive CM and BMD: in primitive CM, LGE is usually mid-parietal and circumferential; in BMD, it is mainly lateral and sub-epicardial. Relaxometry data demonstrate that the main lesions are inflammatory and fibrotic. The coexistence of both inflammation and fibrosis is uncommon in the same segments. T2 mapping is a useful tool to differentiate fibrosis from both fibrosis and edema. Becker cardiomyopathy may be considered as a "chronic active genetic myocarditis". Further clinical and MRI investigations must explore if the relaxometry data may help to predict long-term survival in Becker dystrophy.N/A.