By intramuscular injection, species D vaccines were markedly weaker than species C vaccines. In contrast, the species D vaccines were equally efficient as species C when delivered mucosally by the intranasal route. Intranasal adenovirus vaccine doses as low as 108 virus particles per mouse induced complete protection against a stringent lethal challenge dose of influenza. These data support translation of species D adenoviruses as mucosal vaccines and highlight the fundamental effects of differences in virus tropism on vaccine applications.Seasonal and pandemic influenza remains a constant threat. While standard influenza vaccines have great utility, the need for improved vaccine technologies have been brought to light by the 2009 swine flu pandemic, highly pathogenic avian influenza infections, and the most recent early and widespread influenza activity. Species C adenoviruses based on serotype 5 (AD5) are potent vehicles for gene-based vaccination. While potent, most humans are already immune to this virus. In this study, low seroprevalent species D adenoviruses Ad26, 28, and 48 were cloned and modified to express the influenza virus A/PR/8/34 hemagglutinin gene for vaccine studies. When studied  Influenza virus infections impose a significant burden on our society. Annually, 5-15% of the world’s population is affected by epidemics and have upper respiratory tract infections, 3 to 5 million have severe illness and 250,000 to 500,000 cases result in death [While these seasonal epidemics are certainly of concern, pandemic influenza outbreaks are of substantially higher concern. During the past century there have been several severe pandemics . In 1918Trivalent Inactivated Vaccine (TIV) is the standard for influenza vaccination and has protected millions of humans from influenza morbidity and mortality. While somewhat effective, TIV has limitations that support the development of alternate vaccine platforms. Some of the limitations to using TIV include the following: The use of embryonated eggs is a fundamental problem with this vaccine platform. A large fraction of humans are allergic to egg ovalbumin and cannot take this vaccine. Generating influenza vaccines in eggs is time consuming and labor intensive and relies on the availability of embryonated eggs. TIV only provides short-term immunity  and thisFluMist is a live-attenuated cold-adapted influenza virus platform that is administered intranasally. While this new platform has the advantage of being needle-free and induces a low level of cellular immunity it still has many limitations that support the study of alternative vaccine platforms. These limitations include cost, egg allergies, the vaccine is restricted to certain age groups, priming requires two doses of vaccine, it cannot be administered to immunocompromised patients, and, importantly, studies have shown that the protective immune responses induced by Flumist decline more rapidly than the TIV –19 indicAdenoviruses are non-enveloped linear double-stranded DNA viruses. Human adenoviruses have been classified into 57 unique types that are defined into 7 species (A-G) . AdenoviOne of the most studied and used Ad vectors was derived from the species C adenovirus type 5 (Ad5). Unfortunately, epidemiological studies show that Ad5 is one of the most seroprevalent of these human viruses and is therefore more likely to be less effective in human vaccinees –23. BeyoTrivalent Inactivated Vaccine (TIV) is a protein-based vaccine and is the standard for seasonal influenza vaccination. The 2009 monovalent inactivated vaccine (MIV) is also a protein-based vaccine, but in this case directed against only one strain of influenza (A/CA/07/09) .10-106 virus particles (vp)/mouse. These doses correspond to delivery of 0.75 to 7500 ng of Ad protein. It should be noted, that this protein concentration is made up of Ad virus proteins and not influenza protein antigens, but is provided simply for comparison to the MIV vaccine.To assess the utility of Ad vectored vaccines against influenza, we compared a standard replication-defective Ad5 (Ad5-RD) expressing the HA from the 2009 H1N1 swine flu to a traditional protein-based MIV vaccine. In 2009-2010, human vaccinees received 15 µg doses of the 2009 MIV by the i.m. route. For a 70 kg human, this corresponds to approximately 0.2 µg/kg doses. Mice were immunized with 4, 40, or 1,500 ng of MIV by the i.m. route, which corresponds approximately to 0.2, 2.0, and 75 µg/kg doses. Parallel groups of mice were immunized with 10-fold dilutions of Ad5 expressing codon-optimized hemagglutinin (HA) from a 2009 H1N1 strain A/TX/05/09 (Ad5-TX). The MIV and Ad5-TX vaccines both contain isolates from the 2009 swine flu pandemic. The HA protein sequences of these two isolates are 99.5% identical. The doses ranged from 1050 of mouse-adapted influenza A/PR/8/34 to model a virus challenge heterologous to both of the vaccines. Under these conditions, the MIV vaccine did not provide any detectable cross-protection against A/PR/8/34 as assessed by weight loss and survival  protected mice completely against both weight loss and death. Cross protection by Ad expressed HA genes had been observed previously. Ad5 expressing full-length A/PR/8/34 HA was shown to provide cross-protection against A/FM/1/47 and A/CA/04/09 [BALB/c mice were immunized intramuscularly with varying doses of the two vaccines in order to titrate the level of protection induced by the two vaccine platforms . The micsurvival . In contll doses . A dose CA/04/09 .αv integrins and species D viruses use integrins, CD46, and/or sialic acid as receptors for virus entry. Each viral genome was cloned and replication-competent or replication-defective Ad vaccines were constructed by replacement of the E3 or E1 regions with an expression cassettes for the influenza hemagglutinin (HA) gene.Ad5 is a potent vaccine vector. However, most humans are immune to the virus and this immunity would likely blunt vaccine efficacy. Given this, we cloned the genome for Ad6. Ad6 is a lower seroprevalent species C virus  similar in vitro analysis of HA expression by the vectors showed that HA was expressed in a similar amount regardless of Adenovirus type or location of the transgene  to three species D vaccines  as replication-competent vectors with intact E1 genes and HA replacing the E3 domain. An ransgene . BALB/c /PR/8/34 . Under t/PR/8/34 . In cont/PR/8/34 . Ad48 in/PR/8/34 . Statist10 vp/mouse. This route stimulates the mucosa in a manner similar to that of the licensed FluMist vaccine. Three weeks after immunization the mice were challenged intranasally with 100MLD50 of influenza A/PR/8/34  and replication-defective Ad5 (Ad5-RD) expressing the same hemagglutinin were tested by the intranasal route . With th, Ad5-RD  showed m, Ad5-RD . There w10 vp/mouse of Ad28 expressing GFPLuc. Sera were collected 6 weeks after immunization and tested for anti-Ad26 and anti-Ad28 neutralizing antibodies . Groups of mice were challenged with 100 MLD50 of influenza A/PR/8/34 3 days and 3 weeks post-immunization with vectors expressing GFPLuc. When mice were challenged 3 days post-immunization there was no change in weight loss and survival as compared to the control group . We propose that vaccine studies using these transgenic mice will provide a more accurate evaluation of their vaccine efficacy. If these Ad D vaccines are transducing via a secondary receptor intranasally, the restoration of the CD46 receptor may allow for even greater levels of transduction that results in the induction of protection at much lower doses than this study indicates.7 vp/mouse [9 vp/mouse for complete protection against morbidity.The vaccine immunogen used in these studies, HA from A/PR/8/34, was modified to have a deletion of the cleavage, fusion and transmembrane domain (HA-ΔCFT). This was done in order to create a secreted protein that was resistant to proteolytic cleavage so that it would remain intact. We hypothesized that an intact secreted HA would induce greater humoral immunity. However, upon characterization we found that the full-length version was capable of generating protective immune responses at a lower dose. Dose response studies using Ad5 expressing the full-length A/PR/8/34 gene showed complete protection at 10vp/mouse . However10 vp/mouse. This dose may be too much to be effectively translated to a human dose equivalent. Further studies will determine if the vaccine dose could be titrated down to a more usable dose.In order to determine if a species D Ad vaccine could be effective in the presence of pre-existing immunity, we established anti-Ad neutralizing antibodies by immunizing with Ad28 expressing GFPLuc. We tested the sera at 6 weeks post-immunization and found high anti-Ad28 neutralizing antibodies. We found that mice that had high levels of pre-existing immunity against Ad28 could still be protected against a lethal influenza challenge using a homologous Ad28-PR vaccine. This is promising since it demonstrates an effective ability to reuse the same vaccine even in the presence of high anti-vector immunity. One caveat to this observation is that the second immunization with the homologous Ad28-PR vaccine was at a high dose of 10In this study we have investigated the use of species D Ad viral vectors as vaccines for influenza. One of the biggest advantages of using these viral vectors over other Ad species is that they have a low seroprevalence in humans. In addition, they exhibit low levels of pathogenicity, can be isolated from asymptomatic patients, and are not associated with any known severe disease. Importantly, we have shown that the route of immunization is critical when using species D Ad vaccines. The species D vaccines were more than 100-fold more effective when delivered mucosally. This is an important find since other researchers are investigating the use of species D Ad vaccines as well as other non-human primate Ad vaccines for use against HIV –41. In aFemale BALB/c mice (6-8 weeks old) were purchased from Charles River Laboratories  and housed in the Mayo Clinic Animal Facility under the Association for Assessment and Accreditation of Laboratory Animal Care (AALAC) guidelines with animal use protocols approved by the Mayo Clinic Animal Use and Care Committee (protocol A110). All animal experiments were carried out according to the provisions of the Animal Welfare Act, PHS Animal Welfare Policy, the principles of the NIH Guide for the Care and Use of Laboratory Animals, and the policies and procedures of Mayo Clinic.In order to create new recombinant adenoviral vectors the genomes of Ad6, Ad26, Ad28 and Ad48 were cloned individually into a single low copy plasmid. The Ad5 vaccine was generated using the stratagene shuttle plasmids as previously described . The E1 Wild-type (wt) Ad6 strain Tonsil 99 (ATCC VR-1083), Ad26 strain BP-2 (ATCC VR-224), Ad28 strain BP-5 (ATCC VR-226), and Ad48 strain T85-884 (ATCC VR-1406)  were amplified in low passage 293 cells . Viral genomic DNA (gDNA) was purified using a PureLink™ Viral RNA/DNA Mini Kit .5’-GCGATCGCCATCATCAATAATATACCTTATTTTGGATTG-3’), Ad6 Cla1 R (5’- GGGGGCTGTAATGTTGTCTCTACGCCTGCACATAATCTAACACAAACTCCTCACCCTC-3’) and Ad6 AvrII F (5’- GAGGGTGAGGAGTTTGTGTTAGATTATGTGCAGGCGTAGAGACAACATTACAGCCCCC-3’). The 3’ end of Ad6 Cla1 R contained 28 nucleotides (nt) of homology to the right arm of the Ad6 gDNA from nt 35,208 to 35,235 and the 5’ end of Ad6 AvrII F contained 30 nt of homology to the left arm of the Ad6 gDNA from nt 979 to 1,008 (R) and the pBR322 origin of replication (pBR322 ori) from Ad-Easy (Stratagene) was PCR amplified using Platinum Taq Supermix High Fidelity and cloned into TOPO-XL  using the following primers: Kan ori SgfI-F (5’- GCGATCGCTAATTAACATGCATGGATCCATATGCGGTGTG-3’) and Kan ori SgfI-R (5’- GCGATCGCTTAATTAAGAATTAATTCGATCCTGAATGGCGAATGG-3’) . A unique SgfI restriction enzyme site was added just outside of the left and right ITRs. The following primers were used to amplify the PCR product: Ad6 SgfI ITR  . The Kan6-cloner . Ad6 gDN6-cloner  and the 6-cloner . Finally6-cloner . The finATTTAATTAACATCATCAATAATATACCCCACAAAGTAAACAAAAG, Ad26-Clone Frag-F 5’-AATGAGAGCAGGAGTGATGAATATGAATTCTATTACCATCTTCGTCCCTTAGCAA, Ad26-Clone Frag-R 5’-TTGCTAAGGGACGAAGATGGTAATAGAATTCATATTCATCACTCCTGCTCTCATT. The two overlapping PCR products were used to create a final full-length PCR product that fused the two PCR fragments together at the EcoRI site  injection with ketamine (140 mg/kg)/kylazine (5.55 mg/kg) and were immunized by intramuscular (i.m.) or intranasal (i.n.) routes. Mice immunized by the i.m. route received the Ad vaccine or the monovalent inactivated virus (MIV) H1N1 2009 (BEI Resources) in two 25 µl injections into each quadricep muscle . Mice immunized by the i.n. route received the Ad vaccine in two 10 microliter instillations into each nare .10 vp/mouse of adenovirus expressing the HA-∆CFT codon-optimized gene from influenza A/PR/8/34. The mice were bled three weeks post-immunization and serum was separated using Becton Dickinson microtainer tubes. Two-fold dilutions of sera in 50 microliters of DPBS were added to a 96-well, nonsterile, nontissue culture–treated, round bottom microtiter plate. Four hemagglutinating units (HAU) of influenza virus in 50 µl was added to the diluted sera and incubated at room temperature (RT). After 1 hr., 50 µl of a 1.0% chicken RBC solution was added and incubated at RT for 1 hr. The HI titer was determined to be the highest serum dilution to inhibit hemagglutination.The HI assay was performed as previously described . Briefly50  of mouse adapted influenza virus A/PR/8/34 by two 10 microliter instillations into each nare . The mice were observed for morbidity and weight loss over subsequent days and were euthanized if weight loss reached 25% of initial body weight.Mice were challenged intranasally three weeks after immunization. The mice were anesthetized and challenged with 100 MLD10 vp/mouse of Ad28 expressing the eGFP/Luciferase fusion protein. Six weeks post immunization the mice were bled by the submandibular method and the sera was tested for anti-adenovirus neutralizing antibodies (NAbs). NAbs were determined as previously described [8 Ad-GL vp/ml. Naïve mouse serum was used as negative controls and was the maximum luciferase activity reference. The plates were incubated at 37°C for 1 h. Fifty µl of A549 cells at 1 X 105 cells/ml was added to each well and the plates were incubated for 20 h at 37°C and 5% CO2 before readout. The luciferase activity was determined using the reporter lysis 5X buffer and luciferase assay reagent (LAR)  as described by the manufacturer protocol. Briefly, 25 µl of 5X lysis buffer was added to each well, mixed and frozen at -80C. The plates were thawed at RT and 50 µl of LAR was added. The plate was shaken on an orbital shaker and luciferase activity was measured using a Beckman Coulter DTX 880 Multimode Detector. Neutralizing antibodies titers were determined as the reciprocal dilution to inhibit 50% of luciferase activity as compared to no serum controls and are expressed as the geometric mean titer (GMT).Groups of mice were immunized intramuscularly with 10escribed . BrieflyData was evaluated using GraphPad Prism 4 software. Unpaired, two-tailed TTests, ANOVA with Bonferroni post test and Log-rank (Mantel-Cox) tests were used to determine statistical significance. P values ≤ 0.05 were considered statistically significant.Figure S1The cloning strategy for Adenovirus type 6.An overlapping PCR product that fuses the left and right regions of the Ad6 genome at the ClaI and AvrII restriction sites (A and E). This PCR product was ligated to the low copy origin of replication and kanamycin resistance gene (C) to create the cloning plasmid (D). The genomic DNA of Ad6 is digested with Cla I and AvrII and ligated into the pAd26 cloner to create the pAd6 ClaI/AvrII plasmid (F). The AvrII genomic fragment was then ligated in to create the pAd6 gDNA correct plasmid (G).(TIFF)Click here for additional data file.Figure S2The cloning strategy for Adenovirus type 26.An overlapping PCR product that fuses the left and right regions of the Ad26 genome at the unique EcoRI restriction site (A and E) was ligated to the low copy origin of replication and kanamycin resistance gene (C) to create the cloning plasmid (D). The genomic DNA of Ad26 is digested with EcoRI and ligated into the pAd26 cloner to create the plasmid pAd26 gDNA correct (F).(TIFF)Click here for additional data file.Figure S3The cloning strategy for Adenovirus type 28.An overlapping PCR product that fuses the left and right regions of the Ad28 genome at the unique EcoRI and NheI restriction sites (A and E) was ligated to the low copy origin of replication and kanamycin resistance gene (C) to create the cloning plasmid (D). The genomic DNA of Ad28 was digested with EcoRI and NheI (E). The digested genomic DNA was ligated into the pAd28 cloner plasmid to create the plasmid pAd28 complete 4.5Kb kan ori (F).(TIFF)Click here for additional data file.Figure S4The cloning strategy for Adenovirus type 48.An overlapping PCR product that fuses the left and right regions of the Ad48 genome at the unique EcoRI and MluI restriction sites (A and E) is ligated to the low copy origin of replication and kanamycin resistance gene (C) to create the cloning plasmid (D). The genomic DNA of Ad48 was digested with EcoRI and ligated into the pAd48 cloner to create the plasmid pAd48 gDNA correct (F).(TIFF)Click here for additional data file.Figure S5Construction of shuttle plasmids to modify Adenoviral genomes.First, primers are designed to produce an overlapping PCR product that contains a unique AscI site between the overlapping products (A). The overlapping PCR products are designed to amplify the regions outside the area specified for recombination (C). To illustrate this design we used the Ad26 E3 deletion shuttle plasmid. 500 nucleotides of both the Ad26 17.5K and 14.7K gene were PCR amplified with primers with homology designed into the 3’ end of the 17.5K product and the 5’ end of the 14.7K product. The two PCR reactions were combined and extended to produce a fused PCR product (B). The final fused PCR product was designed to possess a unique AscI site between the two PCR product (B). The Ad26-dE3-PCR-AscI-PmeI PCR product was cloned into the Topo-pCR8 cloning plasmid and sequenced. The transgene was prepared by cloning the transgene (HA-∆CFT) into a CMV-PolyA expression cassette (D). To insert the selection marker, the EM7 promoter and zeocin gene were PCR amplified with homology to the transgene expression cassette 5’ to the PolyA. The zeocin gene was flanked by FRT sites to facilitate removal by FLP recombinase at a later time point if needed. Next the entire expression cassette and the selection marker was amplified using primers designed with AscI sites engineered into the 5’ and 3’ ends (D). The final plasmid, pCR8-Ad26-dE3-HA-dCFT-FZF was created by cloning the AscI flanked transgene into the shuttle plasmid for recombination into the E3 region.(TIFF)Click here for additional data file.Figure S6Recombination into the Adenoviral gDNA.The shuttle plasmid was digested with PmeI (A) and cotransfected into BJ5183 cells to recombine into the complete Ad26 genome plasmid (B). The final recombined pAd26-dcE3 PR-dCFT-FZF plasmid was transformed into XL-1 cells and maxiprepped (C).(TIFF)Click here for additional data file.Figure S7Analysis of HA expression by recombinant Adenoviral Vectors.293 and A549 cells were infected with 1000 vp/cell with each of the Ad-HA expressing viruses. The cells were harvested at 24h. Lysates from the infected cells were electrophoresed and transferred to PVDF membranes. After blocking, the membranes were probed with polyclonal anti-HA donkey sera (BEI Resources) and monoclonal anti-actin-HRP . The anti-HA donkey antibody was detected by rabbit anti-donkey-HRP (BioRad). After washing, the blot was developed using Super Signal West Dura substrate and imaged using a Kodak In Vivo F Imaging detector. Densitometry was performed using the Kodak Molecular Imaging Software v 4.0.4.(TIFF)Click here for additional data file.


E7, (iii) expression of the transcript (E7-E1∧E4) that encodes E2 repressor protein and (iv) viral load, in human papillomavirus 16 (HPV16) related cervical cancer (CaCx) pathogenesis. The results revealed over-representation (p<0.001) of methylation at nucleotide 58 of E2BS-I among E2-intact CaCx cases compared to E2-disrupted cases. Bisulphite sequencing of LCR revealed overrepresentation of methylation at nucleotide 58 or other CpGs in E2BS-I/II, among E2-intact cases than E2-disrupted cases and lack of methylation at replication origin in case of both. The viral transcript (E7-E1∧E4) that produces the repressor E2 was analyzed by APOT (amplification of papillomavirus oncogenic transcript)-coupled-quantitative-RT-PCR (of E7 and E4 genes) to distinguish episomal (pure or concomitant with integrated) from purely integrated viral genomes based on the ratio, E7 CT/E4 CT. Relative quantification based on comparative CT (theshold cycle) method revealed 75.087 folds higher E7 mRNA expression in episomal cases over purely integrated cases. Viral load and E2 gene copy numbers were negatively correlated with E7 CT (p = 0.007) and E2 CT (p<0.0001), respectively, each normalized with ACTB CT, among episomal cases only. The k-means clustering analysis considering E7 CT from APOT-coupled-quantitative-RT-PCR assay, in conjunction with viral load, revealed immense heterogeneity among the HPV16 positive CaCx cases portraying integrated viral genomes. The findings provide novel insights into HPV16 related CaCx pathogenesis and highlight that CaCx cases that harbour episomal HPV16 genomes with intact E2 are likely to be distinct biologically, from the purely integrated viral genomes in terms of host genes and/or pathways involved in cervical carcinogenesis.This study was undertaken to decipher the interdependent roles of (i) methylation within E2 binding site I and II (E2BS-I/II) and replication origin (nt 7862) in the long control region (LCR), (ii) expression of viral oncogene  HPV16 appears to be the most common high risk HPV type identified in CaCx cases, precancerous cervical lesions, and in cytologically normal cervical samples E2 gene disruption is significantly overrepresented among CaCx cases compared to controls E2, despite the fact that HPV16 E2 protein negatively regulates transcription of the E6 and E7 genes E6/E7 mRNA expression even with the E2 gene intact, i.e. presence of concomitant viral genomes  or purely episomal viral genomes It is established that although the HPV genome exists in the episomal form in low-grade lesions, the viral genome gets integrated, with increasing grades of lesion E2 gene, we proposed that loss of E2 repressor activity in such cases, predominantly of the E-lineage, could be attributable to CpG methylation at nucleotide 58 within E2 binding site I (E2BS-I) next to the p97 promoter, thus attenuating the binding of E2 to this site E2-intact cases was significantly higher compared to those with disrupted E2 gene. This further prompted us to interpret that viral load in association with E2-status, might be of causal relevance in CaCx pathogenesis Based on a comparison between fifteen HPV16 positive cytologically normal and fifty-seven HPV16 positive CaCx cases with intact E7, (iii) expression of the transcript (E7-E1∧E4) that encodes E2 repressor protein and (iv) viral load, in HPV16 related CaCx pathogenesis. Our study provided novel insights into alternative mechanisms of loss of E2 repressor activity, which could be related to E2BS-I/II methylation, presence of the transcript (E7-E1∧E4) that encodes E2 repressor protein, high viral load and E7 expression among HPV16 positive CaCx cases with episomal (pure or concomitant with integrated) viral genomes and not among the cases with purely integrated viral genomes. In this study, we further identified by employing a novel technique, APOT-coupled-quantitative-RT-PCR, that there was immense diversity among the HPV16 positive CaCx cases both in terms of viral copy numbers and E7 expression levels.We undertook the present study, focusing on HPV16 positive E2 intact and E2 disrupted CaCx cases initially, followed by their classification into episomal and integrated forms, to determine the interdependent roles of (i) methylation within E2 binding sites I and II (E2BS-I/II) and replication origin in the LCR, (ii) expression of viral oncogene E2 disruption status by overlapping DNA-based PCR and estimation of viral load are described elsewhere E2 was 140, and that having disrupted E2 was 44.The samples used for this study were nested to an ongoing natural cohort study E2-intact cases, methylation status of the E2BS-I [50-ACCGAAACCGGT-61] and also the region from LCR to E6 (LCR-E6) were determined by restriction-digestion with HpaII/MspI and McrBC enzymes (New England Biolabs), respectively, and analyzed by PCR, following published protocols McrBC restriction digestion) cases were randomly selected from each of E2-intact (30/140) and E2-disrupted (9/44) categories for bisulphite sequencing, using the primer pair 5′-TAA GGT TTA AAT TTT TAA GGT TAA TTA AAT-3′ and 5′-ATC CTA AAA CAT TAC AAT TCT CTT TTA ATA-3′ covering positions 7748-115 In 2, 50 mM DTT], 10 mM dNTP, 0.1 M DTT, 20U RNase Inhibitor and 400 ng of (dT)17-P3 primer. The mixture containing RNA and primers was heated at 70°C (10 minutes) and chilled (10 minutes) before addition of 5X buffer, 0.1 M DTT and RNase inhibitor, and then, incubated at 25°C (2 minutes), followed by addition of reverse transcriptase and subsequent incubations at 25°C (10 minutes) and 42°C (60 minutes). The reaction was inactivated at 70°C (15 minutes).Total RNAs, from 41 cancer samples, were isolated, purified and treated with DNase I using the Qiagen RNeasy kit following the manufacturer's protocol. One microgram of total RNA was reverse-transcribed using 200U of M-MuLV reverse transcriptase (Fermentas) in a 20 µl reaction containing 5X RT buffer . The reaction mixture (10 µl) contained 2 µl cDNA, 50 ng primers, 0.1 µM probes and 2X TaqMan® Universal PCR Master Mix, (Applied Biosystems). Human ACTB Endogenous Control  (Applied Biosystems) (amplicon size = 171 bp) was used as normalizer. The PCR-program and the controls used were same as in APOT-coupled-quantitative-RT-PCR assay. The assays, with three replicates per sample, were repeated twice.E2 disruption-status among the CaCx samples. The k-means clustering algorithm (k = number of clusters) was used to categorize different forms of purely integrated viral genomes. All analyses were performed using software package, SPSS for windows v16.0.Kolmogorov-Smirnov test was performed to identify whether the test-variables like viral copy numbers, expressions of housekeeping gene and viral genes followed normal distribution. Independent 2-sample t-test and Mann-Whitney U test were used to identify association with disease phenotype, respectively, for variables that followed normal distribution and those that did not. Chi-squared analysis was performed to test for association of methylation within E2BS-I with HpaII/MspI) and subsequent PCR analysis revealed methylation at E2BS-I (nucleotide 58), proximal to p97 promoter within the LCR, to be significantly higher (p<0.001) among E2-intact cases  compared to E2-disrupted cases .Subsequent to our earlier study E2-intact cases (n = 30), methylation was more prominent  portrayed no methylation at the positions 7862 and 31 and lesser methylation at the other positions. Representative electropherograms are shown in Bisulphite-sequencing analysis of LCR DNA from 39 CaCx cases confirmed that among rominent  at nucleE2 and absence in cases harboring disrupted E2, our next objective was to confirm the presence of E7-E1∧E4 transcripts in the former to confirm the expression of repressor E2 transcripts in such cases in contrast to those cases having disrupted E2. We employed the APOT assay in which case, presence of the band size of 1050 bp upon electrophoresis of PCR products obtained with P2 and (dT)17-P3 primers in 1.5% agarose gel, , concomitant (n = 18) and integrated (n = 19) forms of viral genomes, which we confirmed subsequently by performing real-time PCRs (Taqman assay) using the PCR products obtained with P2 and (dT)17-P3 primers, corresponding to E7 and E4 expression, instead of Southern hybridization.Having confirmed the occurrence of methylation in CpGs within E2 binding sites adjacent to p97 promoter in cases harboring intact ose gel, , was conE7 and E4  of pUC19 plasmid vector with HPV16 reference sequence insert  data corresponding to 7 and E4  have beee insert . Presenct (GAPDH  and abseDH (TP53 .E7 CT/E4 CT. The CaCx samples (19/41) showing E7 and ACTB expression but no E4 expression (E7 CT/E4 CT = undetermined), were designated as cases with integrated viral genomes. CaCx samples (18/41) showing more expression of E7 than E4 (E7 CT/E4 CT≤1) along with ACTB expression, were regarded as cases portraying the presence of both episomal and integrated viral genomes (concomitant). There were four CaCx cases showing similar levels of expression of both E7 and E4 (E7 CT/E4 CT = 1) along with ACTB expression, and these were regarded as samples harboring episomal viral genomes. There was no significant  difference in ACTB mRNA expression between CaCx cases harboring episomal viral genomes (pure and concomitant) with mean±sd = 32.98±3.99 and purely integrated viral genomes with mean±sd = 34.99±3.26. Thus, 53.66% (22/41) of CaCx cases harboured intact viral genomes of which, 18.18% (4/22) had pure episomal genomes and 81.82% (18/22) had concomitant genomes  higher among the cases portraying episomal (pure and concomitant) viral genomes  compared to those with purely integrated viral genomes .Reanalysis of our data on E2BSI methylation at nt 58 in LCR on this subset of samples, also revealed that such methylation was significantly  by TaqMan-based qRT-PCR of cDNA products generated directly from the mRNAs, in a subset of 17 episomal  and 13 purely integrated cases.Our next objective was to investigate whether the two types of cancers, (i) those harboring episomal (pure and concomitant) and (ii) integrated viral forms, differed in viral oncogenic expression, by analyzing E7 mRNA expression (E7 CT/ACTB CT) was found to be normally distributed in both episomal  and integrated  cases. The ratio, E7 CT/ACTB CT, was significantly lower  among episomal (mean±sd = 0.84±0.15) than integrated (mean±sd = 1.12±0.18) cases indicating higher E7 expression in the former.T method also revealed the significant difference  between ΔCT (E7 CT – ACTB CT) values of cases with integrated viral genomes (median ΔCT = 1.26) and with episomal genomes (median ΔCT = −4.97). The fold-change analysis , depicted that E7 expression in cases with episomal (pure and concomitant) viral genomes was 75.087 folds higher than cases with purely integrated viral genomes.Relative quantification based on comparative CE7 CT/ACTB CT, was found to be significantly correlated with the viral load  within the episomal samples (pure and concomitant) (2 = 0.038)  per 100 ng genomic DNA were also significantly higher  among the 17 episomal cases  = 18.02 per 100 ng DNA) compared to the 13 purely integrated cases  = 9.28 per 100 ng DNA). The ratio, omitant) , but not= 0.038) .E2 mRNA (mean (E2 CT/ACTB CT)±sd = 0.92±0.18), while the purely integrated cases failed to do so. The ratio, E2 CT/ACTB CT, was found to be significantly correlated  (E2 gene copy numbers (median ln (E2 load) = 15.41 per 100 ng DNA) among such cases harboring episomal (pure and concomitant) viral genomes.The episomal (pure and concomitant) cases showed simultaneous expression of = 0.621)  with E2 E7 expression with viral load, among CaCx cases with integrated viral genomes, pointed towards the possibility of existence of heterogeneity among such CaCx cases with respect to E7 expression. We thus further analyzed our data by performing a cluster analysis (k-means clustering) based on viral load and E7 CT (APOT-coupled-quantitative-RT-PCR). Such analysis could optimally classify the cases with disrupted viral genomes  into four clusters , or E7 CT values (APOT-coupled-quantitative-RT-PCR) .The lack of correlation of clusters  as depicE7 CT±sd = 37.91±1.416) included 6 samples (31.60% of purely integrated samples). Overall, these had low viral load and low E7 expression. Cluster 2  also included 6 samples (31.60% of purely integrated samples), which had high viral load and low E7 expression. Among these samples, ACTB transcription  did not correlate with viral load . Cluster 3  included 5 samples (26.32% of purely integrated samples) and these portrayed moderate viral load and moderate E7 expression. Cluster 4  included 2 samples (10.56% of purely integrated samples), which revealed low to moderate viral load and very high E7 expression. The distances of samples from the centers of their corresponding clusters are provided in E7 expression and the remaining two depicted low E7 expression on the basis of APOT-coupled-quantitative-RT-PCR assays. Mean CT-values for ACTB expression did not differ among the four clusters .Of these four clusters, Cluster 1  with intact E2 gene.Opposed to the prevailing concept of HPV16 E2, compared to HPV16 positive controls E2-intact, compared to E2-disrupted cases. We confirmed this finding subsequently in HPV16 positive CaCx cases harboring episomal (pure and concomitant) viral genomes with intact E2 gene as compared to those with purely integrated viral genomes with disrupted E2 gene. Sequence analysis of a subset of the samples further revealed that methylation was also prominent among E2-intact cases at CpGs in positions 31, which is SpI binding site, 37 and 43, which are within E2BS-II, and 52, which is within the E2BS-I. This observation further substantiates the role of E2BS-I and II methylation in blocking E2 repressor activity in cases harboring intact E2. This is supported by the in vitro analysis that HPV16 E2 binding site methylation prevents E2 binding et al., 2009, and Brandsma et al., 2009, also made similar observations Earlier, we identified that E2BS-I methylation at nucleotide 58 within LCR, was significantly higher among HPV16 positive CaCx cases harboring intact E7-E1∧E4) derived from episomal viral DNA , in a subset of the CaCx cases selected randomly, by an improved APOT assay. This assay involved coupling of the APOT assay with quantitative RT-PCR for E7 and E4 (nested to E2 gene) transcript confirmation, instead of employing Southern hybridization. On the basis of the ratio, E7 CT/E4 CT, derived from APOT-coupled-quantitative-RT-PCR, the viral genomic forms could be categorized into pure episomal, concomitant  and pure integrated. This assay revealed E7 expression in all cases irrespective of E2-status, but E4 expression in only a subset of cases, which we considered to harbor episomal (pure episome or concomitant) viral genomes. It also indicated that E7 expression was quantitatively several folds higher among CaCx cases with episomal HPV16 genomes over those with integrated viral genomes  cases compared to the purely integrated cases.In this study, we also confirmed the presence of the major viral transcript  HPV16 genomes, as opposed to those with purely integrated viral genomes. This ensured the expression of E2 from all viral genomes that were episomal and harbored intact E2 genes. A number of other studies have also observed the expression of E2 from E2-intact viral genomes in vitro and in vivo experiments, suggesting that E2 could also be translated in tumor samples et al., 2009, based on chromatin immunoprecipitation (ChIP), also showed that the E2 viral protein could not bind to the methylated E2 binding sites within the LCR region, thereby resulting in E6 and E7 overexpression and this could be reversed by a DNA demethylating agent (5-aza-29-deoxycytidine) E2 expression concomitant with the expression of E7, further strengthens the biological plausibility of E2BS-I and II methylation, specifically at nucleotide 58 adjacent to the p97 promoter, as a key alternative mechanism of loss of E2 repressor activity in CaCx cases harboring episomal viral genomes with intact E2 genes.Our finding is further consolidated by the observations from another study suggesting no significant increase in E6 and E7 in CaCx cases harboring episomal HPV16 genomes with intact E2 could likely be the ability to mount high viral copy numbers. It may further be speculated that a high copy number in the episomal form also essentially ensures that each dividing cell gets its share of virus, in contrast to the integrated form within host genome, where each cell gets its share of the virus along with the host chromosomes, despite low viral load.Apart from functioning as a transcriptional regulatory factor for viral oncogene expression, E2 also acts in assisting the assembly of E1 on the viral origin, thereby facilitating viral genome replication and segregation by tethering the viral genomes to host mitotic chromosomes E2 ORF with deletion of few nucleotides downstream E6/E7 transcripts from integrated DNA capture 3′ cellular polyadenylation signals thereby increasing viral mRNA stability E7 expression and viral copy numbers among the CaCx cases having integrated viral genomes, as revealed by application of k-means cluster analysis considering E7 CT from the APOT-coupled-quantitative-RT-PCR assay, in conjunction with viral load. As depicted in E7 expression) could be speculated as harboring single-copy integrated forms of HPV16. The cluster 2 samples  could be interpreted as concatenated forms of HPV16. Cluster 3 samples , on the other-hand, could be speculated as representing many single-copy integrated viral forms cumulatively expressing E7 mRNA. Likewise, the cluster 4 samples  could be speculated as harboring viral genomes that were single copy integrates at few sites and likely to be under the effect of strong host promoters accounting for the enhanced E7 mRNA expression. There was no difference in the ACTB mRNA expression among the four clusters of samples.Viral integration, by non-homologous end-joining recombination, mainly involves the hinge region of Thus, based on APOT-coupled-quantitative-RT-PCR assay along with viral load, it has been possible not only to distinguish between episomal and integrated CaCx cases, but also to identify variations in the oncogene expression levels of these two groups of samples. Moreover, this is the first report of its kind to reveal that amongst the CaCx cases harboring purely integrated HPV16 genomes, there exists immense diversity in terms of viral physical status and viral load as well. Worth noting is the fact that 60% of the CaCx samples harboring purely integrated viral genomes appeared to represent the single-copy integrations at few sites (similar to SiHa cell line) and concatenated forms (similar to CaSki cell line), while the remaining appeared to deviate from such canonical forms that have mostly been reported in earlier studies. This assay further revealed that majority of CaCx cases with episomal viral genomes harboured concomitant forms (81.81%) .E2 ORF in the region that codes for ‘hinge region’ of the E2 protein E4-specific primer-probe set was designed in a way such that the amplicon (nucleotides 3439–3556) encompasses the 3′end of the hinge (nucleotides 3418-3542). This ensured amplification of E4, only in presence of intact hinge (that is intact repressor-coding E2). In the traditional APOT assay, the E4 probe (nucleotides 3449–3472) used for Southern Blot does not encompass the 3′ end of the hinge and cannot identify integrate derived fusion transcripts resulting from partial E4 disruption E7 and E4 transcripts, either to reveal the presence of E7-E1∧E4 splice variant (that produces repressor E2) or disruption of E1∧E4 (that fails to produce repressor E2) and hence appears to be highly specific over the traditional APOT assay.Integration of viral genomes into host genome is mostly associated with the disruption of E7 and E4 real time PCRs, we designed duplex PCRs with differential fluorochrome tags for E7 (FAM-tagged) and E4 (VIC-tagged) probes. To account for the inter-sample variation, CT values from RT-PCR (TaqMan) of ACTB gene were compared between episomal (pure or concomitant) and purely integrated cases and were found to be similar. ACTB gene expression also did not correlate with either viral load or E7 expression (APOT-coupled-quantitative-RT-PCR) among the cases harbouring purely integrated viral genomes. ACTB mRNA expression also did not differ significantly among the four clusters of purely integrated cases obtained from k-means clustering. Thus, nested PCR based APOT-coupled-quantitative-RT-PCR assay increased the sensitivity of capturing the entire spectrum of E7 mRNA expression level (ranging from CT = 2.08 to CT = 39.34 among the 19 purely integrated samples), which otherwise could not be detected by the E7 expression assay using the cDNAs directly from the corresponding mRNAs  Our study further revealed that detection of integration status of viral genomes on the basis of DNA analysis only E7 expression in CaCx cases having episomal (pure episomes or concomitant) viral genomes with intact E2 could be attributable to loss of E2 repressor activity due to E2BS-I/II methylations. High viral load among CaCx cases having episomal (pure and concomitant) viral genomes as compared to those with purely integrated viral genomes could be attributable to the potential of E2 in maintenance of replication and segregation of viral genomes in such cases. The underlying mechanism of cervical carcinogenesis under the impact of oncogenic HPV is mediated through protein-protein interactions of the host and virus. We therefore speculate that HPV16 positive CaCx cases that harbour episomal viral genomes with intact E2 are likely to be distinct biologically, from the purely integrated viral genomes in terms of host genes and/or pathways involved in cervical carcinogenesis. Moreover, the novel technique, APOT-coupled-quantitative-RT-PCR assay, together with viral load could be used to classify CaCx samples into various groups based on different viral genomic forms. Such findings therefore prompt towards undertaking studies to decipher the role of host genomic underpinnings, if any, in influencing the status of the viral genomes within the host cells and their impact on disease prognosis.This study has provided novel insights into HPV16 related CaCx pathogenesis. High Figure S1Representative electropherograms (bisulphite sequencing) showing methylation status of CpGs within viral LCR regions. Nucleotide (nt) position 31 is SpI binding site, nt 37 and 43 are E2BS-I and nt 52 and 58 are E2BS-II. The upper two panels show sequences of two E2-intact patient specimens (T241 & T256) and the lower two panels show sequences from E2-disrupted samples (T122 & SST30), where all Cs have converted to Ts.(TIF)Click here for additional data file.Figure S2Standard Curve Plots of E7 and E4 RT-PCR based on Absolute Quantification of HPV16 plasmid DNA. (A) E7 based qRT-PCR (B) E4 based qRT-PCR. Both E7 and E4 have similar slopes, which justify their similar efficiencies. The three standards used were respectively, 1.75×108, 1.75×106 and 1.75×104 copies of HPV16 plasmid (pUC19 plasmid vector with HPV16 reference sequence insert)(TIF)Click here for additional data file.


Pseudomonas aeruginosa strain 18A is a clinical, nonclonal isolate retrieved from the sputum of a chronically infected cystic fibrosis patient. The genome of 18A was sequenced for comparison with environmental and clinical isolates to identify genes that might facilitate its persistence during infection. Pseudomonas aeruginosa is a major factor in the mortality of cystic fibrosis (CF) patients. Although several studies have adopted a genomic approach to identify potential virulence determinants of laboratory type strains  (A combination of the 454 FLX titanium system (Roche) and the paired-end Illumina genome analyzer GAII (Illumina) sequencing was used. We performed a hybrid assembly using all of the 454 reads and a subset of the Illumina reads in MIRA3 and obtained average coverage of 59× for 454 reads and 147× for Illumina reads for contigs with length greater than 500 bp. MIRA3 flags were set to perform accurate P. aeruginosa 18A yielded a total of 6,093,587 bp, with a 66.4% GC content, consistent with the GC content (66.2% to 66.6%) for P. aeruginosa strains (rbs operon), mannitol (mtl operon) (bkd-lpdV operon), gluconate (psl operon) (P. aeruginosa genome island 1 (PAGI-1) (The draft genome of  operon) . Ten gen operon) , a P. aePAGI-1) , and a c(PAGI-1) . The 18A , and a CAQZ01000001 through CAQZ01000179.The draft genome and annotation are accessible from the EMBL database under accession numbers


Gamete and embryo development are crucial for successful reproduction and seed set in plants, which is often the determining factor for crop yield. Proline accumulation was largely viewed as a specific reaction to overcome stress conditions, while recent studies suggested important functions of proline metabolism also in reproductive development. Both the level of free proline and proline metabolism were proposed to influence the transition to flowering, as well as pollen and embryo development.p5cs2 mutant plants were retarded in vegetative development and displayed reduced fertility under long-day conditions. Single mutant plants devoid of P5CS1 did not show any developmental defects. Simultaneous absence of both P5CS isoforms resulted in pollen sterility, while fertile egg cells could still be produced. Expression of P5C reductase (P5CR) was indispensable for embryo development but surprisingly not needed for pollen or egg cell fertility. The latter observation could be explained by an extreme stability of P5CR activity, which had a half-life time of greater than 3 weeks in vitro. Expression of P5CR-GFP under the control of the endogenous P5CR promoter was able to restore growth of homozygous p5cr mutant embryos. The analysis of P5CR-GFP-fluorescence in planta supported an exclusively cytoplasmatic localisation of P5CR.In this study, we performed a detailed analysis of the contribution of individual proline biosynthetic enzymes to vegetative development and reproductive success in Arabidopsis. In contrast to previous reports, we found that pyrroline-5-carboxylate (P5C) synthetase 2 (P5CS2) is not essential for sexual reproduction although P5CS1 alone does not affect development of non-stressed plants.Our results demonstrate that potential alternative pathways for proline synthesis or inter-generation transfer of proline are not sufficient to overcome a defect in proline biosynthesis from glutamate during pollen development. Proline biosynthesis through P5CS2 and P5CR is limiting for vegetative and reproductive development in Arabidopsis, whereas disruption of  Reproductive success in plants depends to a large extent on successful adaptation to frequently changing environmental conditions. One of the most prominent adaptive responses to various kinds of stress that result in decreased water availability is the accumulation of the amino acid proline  synthetase (P5CS) and P5C reductase (P5CR) . In animP5CS1 (At2g39800) transcription is strongly induced in response to stress, and p5cs1 T-DNA insertion mutants were viable but showed reduced basic levels of free proline and a delayed floral transition [p5cs1 mutants showed strongly reduced proline accumulation in response to stress, concomitantly with reduced root growth, enhanced production of reactive oxygen species in leaves, and a lower NADP+ to NADPH ratio [In Arabidopsis, as well as in many other plant species, two P5CS isoforms have been identified with different expression patterns and specific functions in primary metabolism and stress defence . P5CS1  transcripts and P5CS2-GFP fluorescence were mainly detected in meristematic and developing tissues [P5CS2 promoter was identified as an early target of CONSTANS, a transcriptional activator involved in floral transition [P5CS2 did not respond strongly to abiotic stress, they were enhanced in plants undergoing a hypersensitive response to avirulent Pseudomonas infection [P5CS2, an embryo lethal phenotype has been reported that could be overcome by ex vivo cultivation of developing seeds with proline feeding. However, homozygous p5cs2 mutant plants died before the onset of flowering, thus the specific role of P5CS2 in reproductive development could so far not be analysed [P5CS genes resulted in retarded growth, delayed flowering and reduced apical dominance [2 At3g556 transcriansition . While tnfection ,13. For 2 At3g556 transcriominance ,14.P5CR as the embryo-defective mutation emb2772[in vivo were available so far.The second and final step of proline biosynthesis is catalysed by P5CR, which is encoded by a single gene (At5g14800) in Arabidopsis. The SeedGenes project identified T-DNA insertions in n emb2772. Also inn emb2772. P5CR exn emb2772. Translan emb2772. The subn emb2772,20. Despp5cr mutants. Additionally, we report a comprehensive analysis of the requirement of P5CR and the two P5CS isoforms for vegetative and reproductive development in Arabidopsis, while previous reports focused mostly on a single step of proline biosynthesis. In contrast to previous reports, suitable cultivation conditions allowed us the generation of fertile homozygous p5cs2 mutants and we observed that p5cs2 but not p5cs1 mutants showed reduced growth and a delay in the onset of flowering. Infertility of p5cs1/p5cs2 double mutant pollen precluded the generation of double homozygous p5cs1/p5cs2 mutant plants even though double mutant female gametes were fertile. The absence of a functional P5CR gene had no influence on the fertility of either male or female gametes, but embryos were aborted after only a few cell divisions in presumably homozygous p5cr mutant seeds. Gamete development in the absence of de-novo P5CR gene expression could rely upon the extraordinary stability of the P5CR protein.In this study, we demonstrate that a P5CR-GFP fusion protein is localised exclusively in the cytosol and can fully complement the developmental defects of p5cs2 T-DNA insertion lines as embryo-lethal or conditionally embryo-lethal and non-fertile. To eliminate potential secondary mutations or ecotype-specific effects, we backcrossed the p5cs2-1 (GABI452_G01) and p5cs2-2 (FLAG_139H07) T-DNA insertion lines three and five times, respectively, to the Col-8 accession. In both lines, the segregation pattern was consistent with a single T-DNA insertion and premature abortion of homozygous seeds (data not shown). Homozygous p5cs2 mutant plants were then generated by in vitro cultivation of immature mutant seeds on MS plates containing 60 mM sucrose and 2 mM proline. We observed that also application of mild salt stress to heterozygous p5cs2-1 mutant plants allowed the formation of approximately 1% homozygous and fertile seeds in vivo. Most of the homozygous plants obtained in either way were phenotypically normal, showing only reduced growth compared to wildtype plants . Cultivation in long-day conditions increased the propensity of p5cs2 mutant plants to form abnormal leaves and decreased the proportion of fertile plants. In infertile p5cs2 flowers, the anthers were collapsed and the petals were elongated .With respect to floral transition, P5CS1 expression on growth and development, also the knockout mutant line p5cs1-4  was backcrossed three times to Col-8, and homozygous progeny was included in all experiments. Contrary to the observations of [p5cs1-4 mutant plants did not show any deviations from the wildtype with respect to growth, flowering time, inflorescence architecture or fertility under non-stressful cultivation conditions, irrespective of the light period and the light intensity  or BASTA (p5cs2-2) resistant progeny  that appeared to have originated from a p5cs1-4/p5cs2-2 double mutant pollen.To determine whether the transmission of P5CS1 or P5CS2 allele for male fertility prompted us to analyse knockout mutants for the second enzymatic step of proline biosynthesis as well. In Arabidopsis, P5CR is encoded by a single copy gene, and the two T-DNA insertion lines used in our experiments  are listed in the SeedGenes database as embryo-lethal [p5cr-1 plant, only heterozygous or wildtype plants were obtained from these seeds, indicating that the parent plant was not a homozygous p5cr mutant (data not shown). The p5cr-1 mutant line originally contained at least one additional T-DNA insertion that was eliminated by repeated crossing to the Col-8 wildtype. We determined the exact T-DNA insertion sites in both lines and confirmed that the embryo-lethal phenotype co-segregated with the insertion in the P5CR gene  were due to either poor expression of the resistance gene or mild defects in gamete development or fertility . De-novo expression of P5CR protein from an intact gene copy might be dispensable if the P5CR protein present in the pollen mother cell before the separation of the haploid microspores was still active during pollen maturation. To test the likelihood of this hypothesis, the stability of Arabidopsis P5CR was assessed in partially purified preparations from suspension-cultured cells or after heterologous expression in E. coli , isolated and tested under the same experimental conditions, lost more than 50% activity within 24 h. The time from microspore separation to anther opening in Arabidopsis is around 8 days, thus the stability of P5CR in vivo could be sufficient to enable the completion of pollen development after meiotic cytokinesis [Pollen fertility in the absence of okinesis .p5cr mutants with plants overexpressing a P5CR-GFP fusion protein under control of the Cauliflower Mosaic Virus (CaMV) 35S-promoter did not allow the generation of plants that were homozygous for the P5CR-inactivating insertion (data not shown). This failure may be attributable to the poor activity of the CaMV-35S promoter in embryonic tissue  Heynh., accession Col-8) and T-DNA insertion lines were obtained from the NASC  or from the INRA Versailles Resource Centre . Presence of the T-DNA and allelic status were verified by PCR and sequencing of the T-DNA flanking regions. Primer sequences are given in Additional file -2*s-1. To mimic the growth conditions described by [-2*s-1) at day/night temperatures of 24°C/20°C.Arabidopsis (A heterotrophic (white) cell culture was maintained in liquid MS medium supplemented with 3% (w/v) sucrose, 2x MS vitamin mix, 0.5 mg/l 2,4-dichlorophenoxyacetic acid, 0.5 mg/l benzylaminopurine and 0.2% (v/v) Plant Preservative Mixture  under constant agitation (100 rpm) in dim light.P5CR or a genomic fragment containing 1700 bp of 5’ upstream sequence and the entire P5CR gene without the stop codon were amplified by PCR from full-length ORF clone U13409  or from genomic Col-8 DNA, respectively. Sequences of PCR primers are given in Additional file P5CR to the GFP gene. Transformation of Arabidopsis plants by floral dip was performed according to [The open reading frame of rding to . Epifluording to . For conrding to . ProtoplFor analysis of developmental defects of embryos, immature seeds were isolated under a stereo microscope and cleared in an 8:3:1 (w:v:v) mixture of chloral hydrate:water:glycerol. Images were captured using differential interference contrast (Nomarski) optics and a black-and-white digital camera. Pollen was isolated by vigorously shaking open flowers in ice-cold 300 mM mannitol and centrifugation for 1 min at 12000 g. Viability staining was performed according to [-1 quartz sand or 2 g*(g cells)-1 alumina, respectively. The homogenate was resuspended in ice-cold extraction buffer , and centrifuged at 4°C for 10 min at 10000 g. Proteins were precipitated from the supernatant by adding solid ammonium sulphate to 70% saturation and then pelleted by centrifugation. The protein pellet was dissolved in a minimal amount of extraction buffer and desalted by passage through a Bio-Gel P6DG column . The sample was loaded onto a DEAE-Sephacel  column equilibrated with extraction buffer. Following extensive washing, proteins were eluted with increasing NaCl concentrations in extraction buffer.Plant and bacterial cells were homogenized in an ice-cold mortar with 1 g*(g cells)2, 2 mM DL-P5C, and 0.25 mM NADH in a final volume of 1 ml. A limiting amount of enzyme (0.05-0.25 nkat) was added to the prewarmed mixture, and the decrease in absorbance at 340 nm was determined at 35°C for up to 10 min against blanks from which P5C had been omitted. The activity was determined from the initial linear rate, with the assumption of an extinction coefficient for NADH of 6,220 M-1*cm-1. DL-P5C was synthesized by the periodate oxidation of δ-allo-hydroxylysine , and purified by cation-exchange chromatography on a Dowex AG50 (200–400 mesh) column.The physiological, forward reaction of P5CR was measured by following the P5C-dependent oxidation of NADH. The assay mixture contained 100 mM Tris–HCl buffer pH 7.4, 0.1 mM MgClThe authors declare that they have no competing interests.DF designed the study and performed or supervised the majority of the experiments. GW produced the confocal images, LB performed the analyses of growth and flowering time, GF isolated and assayed the P5CR enzymes. All authors have contributed to the manuscript and approved its final version.Figure S1. Developmental defects of homozygous p5cs2 mutants. A: Five-week-old plants cultivated in short-day conditions. B: Two-week-old seedlings cultivated axenically on half strength MS medium with 30 mM sucrose.Click here for fileFigure S2. Heredity diagram for p5cs1/p5cs2 double heterozygous mutants. A indicates wildtype P5CS2 allele; a indicates p5cs2 mutant allele; B indicates wildtype P5CS1 allele; b indicates p5cs1 mutant allele; green shading indicates herbicide resistance mediated by the T-DNA insertion in p5cs2 mutant alleles; pink shading indicates non-viable gamete or embryo; hatched pink shading indicates that the respective progeny will not be observed because of non-viable pollen. Note that the thick black lines delimit the heredity diagram for a heterozygous p5cs2 single mutant, while the red lines delimit the progeny of a heterozygous p5cs2 mutant that is additionally homozygous for a p5cs1 mutation.Click here for fileFigure S3. Expression of P5CR-GFP in embryos. Epifluorescense and brightfield images of isolated embryos displayed in false colour. A-D: Embryo of a homozygous p5cr-2 mutant plant complemented with pGWB4-P5CR, containing the native P5Cpromoter and gene fused with GFP. E-H: Embryo of a Col-8 wildtype plant transformed with pGWB5-P5CR, expressing the P5CR-cDNA fused to GFP under control of the CaMV-35S promoter. I-L: Embryo of a Col-8 wildtype plant. Scale bar = 20 μm.Click here for fileFigure S4. Subcellular localisation of P5CR-GFP and complementation of the p5cr-1 mutant. A-D: Epifluorescence images of homozygous p5cr-1 mutants expressing a P5CR-GFP fusion protein under control of the native P5CR promoter. A-C: Epidermal and spongy parenchyma cells; scale bar = 20 μm. A: GFP fluorescence. B: Overlay of GFP and chlorophyll fluorescence. C: Brightfield image of the same area. D: Comparison of a P5CR-GFP expressing root tip (left) and a non-transgenic root tip, overlay of a brightfield image with GFP fluorescence, scale bar = 50 μm. E: Schematic drawing of the DNA construct in pGWB4 that was used to complement the p5cr mutants in comparison to the wildtype P5CR gene. Arrows indicate the binding sites of the primers that were used to determine the genotype of the complemented mutants (see panel I). F-H: Epifluorescence images of epidermal and spongy parenchyma cells of a wildtype plant transformed with pEG103-P5CR. Note that overexpression with this construct resulted in the formation of cytosolic protein aggregates; scale bar = 20 μm. F: GFP fluorescence. G: Overlay of GFP and chlorophyll fluorescence. H: Brightfield image of the same area. I: PCR-genotyping of p5cr-1 mutants complemented by transformation with pGWB4-P5CR. See panel E for the primer binding sites in the genomic and the transgenic copy of P5CR. J: Schematic drawing of the constructs in pEG103 and pGWB5 for overexpression of P5CR-GFP.Click here for fileTable S1. PCR-primers used in this study.Click here for file


SLC35D3 and NHEG1. Here we report that the ros mutant mice harboring a recessive mutation in the Slc35d3 gene show obesity and MetS and reduced membrane dopamine receptor D1 (D1R) with impaired dopamine signaling in striatal neurons. SLC35D3 is localized to both endoplasmic reticulum (ER) and early endosomes and interacts with D1R. In ros striatal D1 neurons, lack of SLC35D3 causes the accumulation of D1R on the ER to impair its ER exit. The MetS phenotype is reversible by the administration of D1R agonist to the ros mutant. In addition, we identified two mutations in the SLC35D3 gene in patients with MetS, which alter the subcellular localization of SLC35D3. Our results suggest that the SLC35D3 gene, close to the D6S1009 locus, is a candidate gene for MetS, which is involved in metabolic control in the central nervous system by regulating dopamine signaling.Obesity is one of the largest health problems facing the world today. Although twin and family studies suggest about two-thirds of obesity is caused by genetic factors, only a small fraction of this variance has been unraveled. There are still large numbers of genes to be identified that cause variations in body fatness and the associated diseases encompassed in the metabolic syndrome (MetS). A locus near a sequence tagged site (STS) marker D6S1009 has been linked to obesity or body mass index (BMI). However, its genetic entity is unknown. D6S1009 is located in the intergenic region between  SLC35D3 gene) is linked to obesity or BMI in the Framingham Heart Study, but its genetic entity is unknown. Here we characterized the features of obesity and metabolic syndrome with reduced physical activity levels in a previously identified ros mouse mutant, carrying a homozygous Slc35d3 mutation. These ros phenotypes were caused by the intracellular accumulation of D1R mostly on ER in the striatal neurons, impairing D1R signaling and reducing energy expenditure. In addition, we identified two mutations of SLC35D3 in patients with metabolic syndrome which are subcellularly mislocalized. We propose that the SLC35D3 gene is likely a novel candidate gene for MetS and obesity.Genome-wide linkage analyses have revealed that an STS marker D6S1009 (about 55 kb from the  This has resulted in a significant increase in morbidity and mortality associated with the metabolic syndrome . Obesity and associated MetS or body mass index  are regarded as complex traits influenced by both additive genetic effects and environmental factors FTO gene as an obesity gene is an example of loci uncovered by genome-wide association or linkage studies The worldwide prevalence of obesity  and NHEG1 (867 bp apart at the telomere side) in the NCBI Map Viewer (http://www.ncbi.nlm.nih.gov/mapview/). NHEG1 (neuroblastoma highly expressed 1) is a predicted gene with unknown function. No association with obesity of this gene has been documented. SLC35D3  is predicted as an orphan nucleotide sugar transporter or a fringe connection-like protein with 10 transmembrane domains. Previous studies have characterized the recessively inherited ros-/− mutant mouse (ros hereafter), which has a spontaneous intracisternal A particle (IAP) insertion at the first exon of the Slc35d3 gene to disrupt its function ros mutant, suggesting that SLC35D3 is involved in the biogenesis of platelet dense granules Slc35d3 is specifically expressed in the brain as determined by multiple tissue Northern blots Slc35d3 expression was restricted to the striatonigral medium spiny neurons (MSNs) expressing dopamine receptor D1 (D1R) rather than the striatopallidal MSNs expressing dopamine receptor D2 (D2R) ros mice gained weight progressively. Here we have characterized the ros mutant as a mouse model of MetS and obesity. In addition, we found two MetS patients with mutations of the SLC35D3 gene. Our results suggest that SLC35D3 is a candidate gene for obesity-related MetS, which is involved in metabolic control in the central nervous system by regulating dopamine signaling.Genome-wide linkage analyses have revealed that a locus on chromosome 6q23-25 is linked to obesity in the Framingham Heart Study, with a major locus near the sequence tagged site (STS) marker D6S1009 ros mutant mice, we observed that adult ros mice became obese compared to sex and age-matched wild-type (WT) mice  mice . Growth 6 months . Based oWT males , while tcontrols .ros mice reflects body composition changes, we dissected and weighed two distinct fat pads, epididymal and perirenal white adipose tissue (WAT). Both epididymal and perirenal fat mass were enormously increased in ros mice  1,12 = 11.48, P = 0.05), suggesting the impact of the mutation is on the total amount of activity as well as the energy costs of locomotion.The decreased physical activity in ros mutant with an antibody to mouse SLC35D3 (http://www.brain-map.org/) shows a wider distribution, in either WT or mutant mice, we did not detect the SLC35D3 protein in other brain sub-regions and especially in the obesity-related brain tissues such as thalamus and hypothalamus (ros mice exhibited hyperplasia (increase in number), rather than hypertrophy (increase in size), to contribute mainly to the weight gain. The presence of SLC35D3 in non-neuronal tissues is only known so far in platelets and it plays a role in the biogenesis of platelet dense granules ros mice allow us to investigate the phenotypes related to its dysfunction in these neurons.The brain-specific expression pattern in the multiple Northern blots  SLC35D3 , althoug SLC35D3 . Althougthalamus , as wellthalamus . In addithalamus . Considethalamus , the adiSlc35d3 is specifically expressed in the striatonigral MSNs expressing D1R rather than the striatopallidal MSNs expressing D2R ros striatum (ros striatum than that (48.3%) in wild-type  production to detect functional D1R activation at the cell surface. Stimulation of D1R by the specific agonist SKF82958 (10 µM) produced an accumulation of cAMP in both groups. Consistently, cAMP production in controls , which m samples , indicatant mice , consistros neurons, we first examined the subcellular localization of mouse SLC35D3. Consistent with a recent report To ascertain the underlying mechanism of the accumulation of D1R within the ros striatal neurons. We performed immuno-EM by double-labeling with anti-D1R and anti-SEC61B (an ER marker) and found that the D1R particles co-residing with SEC61B in ros striatal neurons (19.6%) were significantly higher than that in wild-type (6.8%) (ros mice. Our OptiPrep gradient assays further confirmed the shift of D1R from plasma membrane (fractions 16–25) to intracellular fractions 2–10 mainly corresponding to ER in ros striatum compared with the wild-type (We then investigated the intracellular location of accumulated D1R in e (6.8%) . This ine (6.8%)  may moste (6.8%)  in ros mild-type .We then tested whether there is a physical interaction between SLC35D3 and D1R by co-immunoprecipitation. Indeed, we observed that Myc-SLC35D3 co-precipitated with Flag-D1R . ReciproTaken together, our results indicate that SLC35D3 is likely involved in the membrane trafficking of D1R on its ER exit, and that loss of SLC35D3 leads to the intracellular D1R retention mainly on ER, thus reducing the amount of plasma membrane D1R receptors and their signaling.ros mice prompted us to investigate whether there are mutations in the orthologous human SLC35D3 gene in patients with MetS. We screened 363 Chinese Han patients with MetS and 217 unaffected individuals by sequencing the two exons and adjacent exon/intron boundaries together with 1 kb untranslated sequence upstream of the start codon of the SLC35D3 gene.The above findings in SLC35D3 gene . In patient #1 , a heterozygous ΔK404 was identified  +/−ros mice at 5 months of age compared with WT littermates . The more severe weight gain in homozygous ros−/− mice at the same age is suggestive that the SLC35D3 mutation may have a gene dosage effect on D1R trafficking. It is unknown whether patients with homozygous or compound mutations may have more severe phenotypes. Unfortunately, we were not able to get access to the blood samples of the family members of these two patients, which precluded us to explore the penetrance of the mutations. This study suggests that mutant human SLC35D3 does not function properly in the ER exit of D1R, thus likely impairing the membrane trafficking of D1R and D1 signaling in the patients in a similar mechanism as revealed in ros mice.We did not find a second mutation in the ros mice is reversible by the treatment with a D1R agonist, adult male mice received a daily intraperitoneal injection of D1 receptor agonist SKF38393. Following the 12-day treatment period, we observed that body weight loss of ros mice (13%) was significantly higher than that of the wild-type (7%). In contrast, body weight changes of saline-treated wild-type mice or ros mice were not significant  increased internalization, 2) reduced reinsertion to the plasma membrane due to increased degradation, and 3) reduced trafficking or expression ros mice blocks the ER exit of D1R, thus leading to retention in the ER and reduced D1R distribution on the cell surface, thereby impairing D1R signaling. We have not completely excluded the possibility that D1R trafficking from early endosomes to plasma membrane is also blocked. Given that the ER-retained fraction of D1R accounts for the greatest proportion of reduced plasma membrane D1R  laid the foundation of our hypothesis that the MetS phenotype in ribution  is unaffD1r-knockout (KO) mouse lines as listed in the MGI database . However, reduced spontaneous locomotor activity was reported in a line of D1r-KO mutants ros mice is about 65% (34%/51.7%) of the WT mice  ros−/− and wild-type, we developed a PCR method of genotyping based on the nature of the insertional mutation in the Slc35d3 gene ad libitum. For other phenotypic analyses, males at 24 weeks of age were used except for those specified in figure legends or The SLC35D3 gene. Amplified PCR products were subjected to direct sequencing by an ABI PRISM 3700 automated sequencer .We recruited 363 unrelated Chinese Han patients with MetS from The Affiliated Hospital of Qingdao University Medical College and The Affiliated Children's Hospital of Nanjing Medical University, and 217 unaffected individuals from Beijing Tongren Hospital of Capital Medical University. The patients were diagnosed as MetS according to the guidelines of International Diabetes Federation (IDF) Growth curves for males were obtained by measuring body weight once a month from 4 to 24 weeks of age. For determination of body length, mice were anesthetized and fully extended to measure the naso-anal distance. Epididymal and perirenal fat pads were harvested from male mice and weighed. Blood was collected by cardiac puncture after an overnight fast for measuring blood glucose, serum cholesterol and triglycerides by colorimetric kit assays  and analyzed using an automatic biochemical analyzer . Insulin was measured by a rat/mouse insulin enzyme-linked immunoassay (ELISA) Kit on non-fasted mice . For the insulin tolerance test (ITT) and glucose tolerance test (GTT), fasting plasma glucose levels were measured  using a glucosimeter . Then insulin  was injected intraperitoneally (1 U/kg) and blood glucose was measured again at 15, 30, 60, 90 and 120 min post injection. Alternatively, D-glucose  was injected intraperitoneally (2 g/kg body weight) and blood glucose was measured again at 15, 30, 60 and 120 min post injection.Mice were pre-exposed to the chamber before testing to allow environmental habituation, and activity was monitored under indirect dim light and sound-attenuated conditions. A single mouse was placed in a chamber (40 cm length×40 cm width×45 cm height) for 30 min. Total distance traveled, average velocity and total movement duration measured spontaneous activity. All these parameters were measured by JLBehv software . Behavioral testing was performed between 8:00 and 12:00 a.m.ad libitum were individually housed for 3 days to allow environmental habituation. Food was measured at 3:00 p.m. each day for 7 consecutive days.Before measurement of daily food intake, mice fed 2 production and physical activity (beam breaks) were recorded at 30-min intervals for 5 consecutive days (48 times a day). Volume of oxygen consumption (VO2) and carbon dioxide production (VCO2) were measured using electrochemical and spectrophotometric sensors respectively. Oxygen consumption data were converted to energy expenditure (Watts) using the measured RQ values using procedures outlined in Arch et al 2 and physical activity levels (Mice aged 8 to 12 weeks (prior to development of obesity) were measured using an indirect calorimetry system . Oxygen consumption, COy levels  and theny levels  as recomTwenty-four to 25-week-old male mice of each genotype received a daily intraperitoneal injection of dopamine D1 receptor SKF38393 (20 mg/kg) (Sigma-Aldrich) for a period of 12 days. Saline-treated mice served as controls. Body weight in all mice was measured on the fourteenth day. Blood glucose, serum cholesterol and triglycerides, and locomotor activities were measured as described above.2, 0.5 mM DTT, 0.1 mM ATP, 0.5 mM IBMX, and 10 µM D1 receptor agonists SKF82958 (Sigma-Aldrich). Reactions were terminated by placing the tubes into boiling water for 2 min. The cAMP concentrations were measured using the Direct cAMP Enzyme-linked Immunoassay Kit (Sigma-Aldrich) following the manufacturer's instructions. Optical density was measured at 405 nm by a microplate reader (Bio-Rad).The dorsal striatum of mice was dissected as above and homogenized in Buffer A  with 10% sucrose. Membranes were isolated by centrifugation  onto a cushion of Buffer A with 44.5% (w/v) sucrose. The membranes at the interface were transferred to a new tube and washed twice with Buffer A and collected by centrifugation . Protein concentrations were determined with Protein Assay . Adenylyl cyclase activity was determined by incubating membrane protein (20 µg) at 30°C for 10 min in 0.1 ml of buffer containing 10 mM imidazole (pH 7.4), 0.2 mM EGTA, 0.5 mM MgClThe polyclonal rabbit anti-mouse SLC35D3 antiserum (1∶1000) for immunoblotting (WB) was prepared using the purified C-terminal 322–422aa peptide as an antigen. The monoclonal mouse anti-GFP (1∶1000) and polyclonal rabbit anti-Myc (1∶1000) were obtained from Santa Cruz Biotechnology . Monoclonal mouse anti-Flag antibody (Sigma-Aldrich) was used for WB (1∶7000) and immunocytochemical (ICC) analysis (1∶3000). Mouse monoclonal antibody against calnexin (1∶200) for ICC was purchased from Abcam . Mouse monoclonal antibody against D1 receptor  was used for WB (1∶600) and ICC (1∶300). Polyclonal rabbit anti-D2 receptor (Millipore) was used for WB (1∶2000). Mouse polyclonal antibody against GM130 for ICC (1∶500) was a kind gift from Dr. S. Bao . Polyclonal rabbit anti-LAMP3 (Chemicon) was used for ICC (1∶200). Rabbit polyclonal anti-SEC61B antibody was purchased from Millipore and used for ICC (1∶600). Mouse monoclonal anti-EEA1 was from BD Biosciences  for ICC (1∶500). Rabbit polyclonal anto-GluR1 was from Millipore for WB (1∶1000). Goat polyclonal anti-BIP was from Santa Cruz Biotechnology for WB (1∶400). Mouse monoclonal anti-β-actin (Sigma-Aldrich) was used for WB (1∶10000). Alexa Fluor 594-conjugated donkey anti-mouse and donkey anti-rabbit IgG (H+L) were purchased from Molecular Probes .Cell lysates, immunoprecipitates or tissue lysates were combined with loading buffer and subjected to 8–12% SDS polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were blotted onto polyvinylidene difluoride membranes in phosphate buffer with 0.1% Tween-20 (PBST), blocked for 1 h in 5% non-fat dry milk/PBST and probed for 2 h with primary antibodies at room temperature. The membranes were rinsed three times (10 min each) with PBST prior to incubation with appropriate peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) and developed with enhanced chemiluminescence .Slc35d3 , D1r , D2r , and human SLC35D3  were subcloned into the pEGFP-C2 or -N2 vector (with GFP-tag), pCMV-tag2B vector (with Flag-tag) and pCMV-tag3B vector (with Myc-tag) as specified in the figures. The fragments of mouse SLC35D3 and mouse D1R specified in figure legends were generated by subcloning. The mutant human SLC35D3 constructs (ΔK404 and insL201) were generated by site-directed mutagenesis  using human wild-type SLC35D3 construct.We prepared constructs for co-immunoprecipitation and immunofluorescence experiments. Mouse entire coding regions of wild-type Transfected HEK-293T cells grew to confluency on 6-well plates. Cells were harvested and lysed in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and protease inhibitors. Cell lysates were centrifuged at 18,000 g for 10 min, and the supernatant was collected and recentrifuged. The supernatant was incubated overnight with 3 µg mouse monoclonal anti-FLAG M2 antibody (Sigma-Aldrich) and washed 6 times with ice-cold wash buffer . The samples were eluted with elution buffer (5 µg/µl 3× FLAG peptide) and subjected to SDS-PAGE and Western blotting with anti-Myc or anti-Flag antibody as described above.Mice were perfused through the heart with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) under deep pentobarbital anesthesia. The brains were removed, and 20 µm frozen sections in the coronal plane were prepared for hematoxylin and eosin (H–E) or immunohistochemical staining (IHC). The H–E staining followed routine procedures. A standard H–E staining protocol was applied to sections of adipose tissue, liver, pancreas and skeletal muscle. For IHC, the endogenous peroxidase activity was blocked by treatment with 0.3% hydrogen peroxide in methanol, sections were blocked with 0.01 M PBS containing 10% goat serum and were then incubated overnight at 4°C with the mouse monoclonal antibody against D1R (1∶400). Following 0.01 M PBS rinses, sections were incubated in a biotinylated secondary antibody  for an hour at room temperature, treated for another hour at room temperature with peroxidase-ligated streptavidin. The results were captured by a TS100 microscope .HEK293T cells transfected with EGFP-SLC35D3 (wild-type or mutant or other constructs as specified in the results or figure legends) were grown on glass cover slips in 24-well plates until 30–50% confluence. 18–20 hrs after transfection, they were fixed with freshly prepared 4% paraformaldehyde for 10 min. Cells were washed 3 times with 0.01 M phosphate buffer (pH 7.4). The permeabilization of cells was carried out in the presence of 0.3% Triton X-100 in PBS for 10 min. After blocking in 0.01 M PBS containing 1% BSA for 1 hr at 37°C, fixed cells were incubated with various antibodies as indicated in the results overnight at 4°C. Cells were then washed 3 times in 0.01 M PBS containing 0.1% Triton X-100 before incubating with Alexa Fluor 594-conjugated secondary antibody at 1∶2000 dilution for 1 h at 37°C. Cells were then washed 3 times before glass cover slips were mounted. Images were acquired with an ×100 lens on a D-ECLIPSE-si confocal microscope (Nikon).Mice were anesthetized with pentobarbital . Brains were separated and cut by a vibratome . The striatum was fixed with 2% paraformaldehyde, 2.5% glutaraldehyde and 0.1% tannic acid in 0.1 M natrium cacodylicum. Then sections were rinsed and postfixed with 1% osmium tetroxide for 30 min. After washing, the sections were dehydrated in an ascending series of dilution of acetone and impregnated in Epon 60°C, 24 hours. Ultrathin (70 nm) sections were collected on nickel grids, rinsed and incubated with mouse anti-D1R (1∶10) or rabbit anti-SEC61B (1∶50) in 1% BSA buffer overnight at 4°C, washed and incubated with 10 nm gold-anti-mouse IgG or 15 nm anti-rabbit IgG (1∶50). Sections were observed in JEM 2000 electron microscope (Japan). All the reagents were purchased from Electronic Microscope Science .The dissected striatum was immediately homogenized with 1 ml HB lysis buffer . The sample was placed onto the top of an 11 ml continuous 5%–20% Optiprep  gradient in HB buffer. The gradient was centrifuged at 200,000 g (34100 rpm) for 14 hours in a Beckman SW41 rotor. Twenty-six fractions (400 µl each) were collected from the top using auto-collector . Equal aliquots from each fraction were analyzed for immunoblotting.For the fractionation assay, the dissected striatum was immediately homogenized with 200 µl HB lysis buffer. 800 µl 20% Optiprep and 800 µl 5% Optiprep in HB buffer were placed into the tube constitutively. The tissue lysate was placed onto the top of the gradient. The sample was centrifuged at 28,000 rpm  for 14 hours. Eighteen fractions (100 µl each) were collected from the top. Based on the pilot Western assay, the fractions were combined as the 1–10th tube (mostly cytoplasm fraction) and the 11–18th tube (mostly plasma membrane fraction) respectively. Equal aliquots from each fraction were analyzed for immunoblotting.t-test. Data from the calorimetry system was tested by ANCOVA. Distribution of gold-labeled particles in immuno-EM pictures were counted and statistical significance was tested by Chi-square test. Intensities of immunofluorescence in cultured cells were analyzed using NIH Image J.cAMP ELISA was performed in duplicate and was repeated three or four times. The standard curves were generated using non-linear regression curve fitting. The specific protein bands on Western blots were scanned and analyzed using the software program NIH Image J. All data were obtained from at least three independent experiments. Data were expressed as mean ± SEM and statistical significance was tested by Student's All mouse procedures were approved by the Institutional Animal Care and Use Committee of IGDB (mouse protocol KYD2006-002). The study of human subjects was approved by the Bioethic Committee of IGDB, Chinese Academy of Sciences . The study was conducted according to the Declaration of Helsinki Principles. Written informed consents were obtained from all subjects.Figure S1ros mice were used as a control. β-actin was a loading control. No apparent expression level changes were noted in the blots. Two independent studies were performed.Expression of SLC35D3 in postnatal mouse striatum. Wild-type (WT) mouse striatum were dissected from mice at different postnatal ages  and the lysates were applied to Western blotting analyses. The striatum from 6-month-old (TIF)Click here for additional data file.Figure S2A) Typical pattern of oxygen consumption measured at 30 min intervals over a 5 day test period. (B) Simultaneous measurements of physical activity to the measurements of oxygen consumption shown in (A). (C) Histogram of the half hourly measurements in (A) and the cut-off used to determine the minimal (or resting) metabolic rate. (D) Plot of oxygen consumption against physical activity levels for the data in (A) and (B), showing the fitted regression and the estimated resting metabolism.Analysis strategy for oxygen consumption and energy expenditure measurements. ((TIF)Click here for additional data file.Figure S3A) No significant change of total level of D2R in striatum was observed between wild-type (WT) and ros mutant (P>0.05). Quantification of the intensities was calculated from three independent experiments. (B) Striatum lysates were fractionated into plasma membrane (PM) and cytoplasm (cyto) containing intracellular organelles. Distribution of D2R on the plasma membrane was normal in the ros mice compared to the wild-type (WT).D2R expression and distribution in striatum. ((TIF)Click here for additional data file.


Moreover, potent and selective inhibitors of PI3K were highly effective in reversing this phenotype, which is optimally revealed in 3-dimensional cell culture. In contrast, inhibition of Akt or mTOR exacerbated the invasive phenotype. Our results suggest that invasion is a core phenotype mediated by increased PTEN/PI3K pathway activity and that therapeutic agents targeting different nodes of the PI3K pathway may have dramatic differences in their ability to reverse or promote cancer metastasis.The PTEN/PI3K pathway is commonly mutated in cancer and therefore represents an attractive target for therapeutic intervention. To investigate the primary phenotypes mediated by increased pathway signaling in a clean, patient-relevant context, an activating PIK3CA mutation (H1047R) was knocked-in to an endogenous allele of the MCF10A non-tumorigenic human breast epithelial cell line.  Genetic deviations in the phosphatidylinoisitol 3′-kinase (PI3K) pathway have been detected in many human cancers Epithelial-mesenchymal transition (EMT) is a program of gene expression changes that leads to a dramatic shift in cell phenotype towards a more invasive and migratory behavior 3.In recent years gene targeting approaches have been utilized to introduce mutations into human cell lines in a disease relevant context 2. MCF10A cells were typically passaged and maintained in the presence of EGF and insulin. To detect differences with the p110α H1047R and parental isogenic pairs, EGF and insulin were absent from the media, except for those studies associated with 3-D culture. Breast tumor cell lines were obtained from the American Type Culture Collection (ATCC). Cell lines were tested and authenticated using gene expression and single nucleotide polymorphism genotyping arrays, as previously described 2.Parental and knock-in MCF10A clones (H1047R A and B) were first published by Di Nicolantonio and colleagues Thr308, phospho-AktSer473, total Akt, Akt1, Akt2, phospho-PRAS40Thr246, PRAS40, phospho-S6Ser235/236, phospho-GSK3βSer9, phospho-P70S6KThr389, phospho-IRS1Ser612 and mTOR were from Cell Signaling. The p110α antibody was from BD Biosciences and the BrdU Proliferation ELISA was obtained from Roche.GDC-0941, PI3Ki-A/D, PI103 and erlotinib were from Genentech, Inc. mTOR1/2i is from patent WO 2008/023159 A1 GGACAACTGTTTCATATAGTT) was from Genentech. The Akt siRNA inhibition kit, which inhibits the expression of both Akt1 and Akt2 was obtained from Cell Signaling. Transfection of siRNAs was accomplished using an Amaxa Nucleofector Kit as described by the manufacturer for MCF10A cells.mTOR and the non-targeting control siRNAs were from Dharmacon and the p110α siRNA .Cells were seeded at 2000 cells/well in 384-well plates. Diluted compounds were added to quadruplicate wells in 384-well cell plates. After 4 days incubation, relative numbers of viable cells were measured using CellTiter-Glo (Promega) and read with a Wallac Multilabel Reader (PerkinElmer). EC2 tissue culture plate overnight. Cells were treated with an EC50 concentration of inhibitors for the times indicated. Following treatment, cells were washed with cold PBS and lysed in 1× Cell Extraction Buffer (Biosource) supplemented with protease inhibitors (Roche), 1 mM PMSF, and Phosphatase Inhibitor Cocktails 1 and 2 (Sigma). For immunoblots, equal protein amounts were separated by electrophoresis through NuPage Bis-Tris 10% gradient gels (Invitrogen); proteins were transferred onto PVDF membranes using the Criterion system from Bio-Rad.Two million cells were seeded in a 10 cm3 Mass Strip Kit or a PI3K kinase ELISA assay (Echelon Biosciences) according to the manufacturer's instructions.The measurement of PIK3CA activity was carried out through a PIPCells were seeded at 10,000 cells per well in a 96-well plate overnight. After 48 hours of culture with the BrdU reagent, the BrdU ELISA was performed according to the manufacturer's instructions (Roche) and the plate was read on an EnVision Plate Reader (PerkinElmer).Gene expression analysis was carried out on RNA extracted from sub-confluent cell cultures using Qiagen RNeasy kits. RNA samples of sufficient quality were profiled on Affymetrix HGU133Plus_2.0 chips. Preparation of complementary RNA, array hybridizations, scanning and subsequent array image data analysis were done using the manufacturer's specified protocol. Gene expression data for experiments described in this study has been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE17785.Acinar growth and morphogenesis assays in 3D laminin-rich extracellular matrix (lrECM) were performed as described previously 2). This results in a value from 0 to 1, where 0 is a flat line and 1 is a perfect circle.Size and shape values were determined from multiple phase-contrast images as previously described Migration through matrigel was performed using Biocoat Matrigel Invasion Chambers containing FluoroBlok inserts (BD Biosciences). Cells were plated  and allowed to migrate through matrigel for 24 hours according to the protocol provided by the manufacturer. Number of invaded cells was detected and quantified using ImageXpress (Molecular Devices).3), a product of PI3K enzymatic activity  with small molecule inhibitors targeting PI3K, Akt, mTOR, and EGFR in the parental and knock-in clones in a growth inhibition format assay using CellTiter-Glo , the H1047R knock-in clones displayed increased PI3K pathway activity and proliferation, but oncogene addiction was not observed. We found that the H1047R mutation was linked to EMT by analysis of epidermal and mesenchymal gene signatures, rescue of EGFR inhibitor resistance by a PI3K inhibitor, and acinar morphologies in 3-D culture. PI3K inhibitors and siRNA reversed the morphologies in 3-D culture, but mTOR and Akt inhibitors and siRNA worsened the invasive phenotype. This was due to increased feedback to PIPThese data suggest that invasion may be the most relevant phenotype to assess for PI3K oncogene addiction. Consistent with this hypothesis is a previous in vivo study that showed knocking-out mutant PIK3CA in a fully tumorigenic colon cancer cell line had more pronounced effects on metastasis than on primary tumor growth In summary, the H1047R mutant of PIK3CA increases PTEN/PI3K pathway signaling and markers of EMT and cell migration phenotypes in isogenic breast epithelial cell lines. Notably, pathway targeted agents dramatically diverge in their ability to revert these phenotypes, with direct PI3K inhibitors representing the optimal intervention node for mutant PI3K cancers. These data suggest that invasion-based readouts and biomarkers may be the most appropriate end points to reveal the ‘gene-addiction’ responses to PI3K inhibitors in the clinic.Figure S1Differential mRNA expression of cell cycle genes in triplicate microarray samples. Error bars indicate ±SEM.(TIF)Click here for additional data file.Figure S2Phase-contrast images of the parental and knock-in clones in culture.(TIF)Click here for additional data file.Figure S3MCF10A cells with a PI3K mutation show gene expression patterns associated with invasiveness. Comparative gene expression analysis of the MCF10A parental and H1047R clone A. Microarray differences presented as a heat map are indicated as ratios  for genes commonly linked to invasiveness.(TIF)Click here for additional data file.Figure S4Horizon MCF10A H1047R knock-in cells show a more invasive phenotype in 3-D cell culture. Parental and H1047R knockin clones were cultured for 24 hours in Geltrex Reduced Growth Factor Basement Membrane Matrix (Invitrogen) in the presence or absence of GDC-0941 (1 µM). This type of matrigel was used to augment the invasive morphologies of the H1047R knockin clones.(TIF)Click here for additional data file.Figure S5GDC-0941 inhibits invasive morphologies in breast tumor cell lines. (A) BT20 cells were cultured for 24 hours in the presence or absence of inhibitors at EC50 viability concentrations (GDC-0941 (0.6 µM), PI103 (0.4 µM), PI3Ki-A/D (1.2 µM), AKT1/2i (3 µM), or mTOR1/2i (2 µM)). (B) MDA-MB-436 cells were cultured for 48 hours in the presence or absence of inhibitors at EC50 viability concentrations (GDC-0941 (0.8 µM), PI103 (0.5 µM), PI3Ki-A/D (1.5 µM), AKT1/2i (3.8 µM), or mTOR1/2i (1.7 µM)).(TIF)Click here for additional data file.Table S1Gene probe sets utilized for microarray analysis.(DOC)Click here for additional data file.


PTGS2(COX2) C-163G) to 0.479 ), and most of the variations were common (MAF ≥0.05 for 96 polymorphisms).In this article, we found errors in the genotype data for 5 single nucleotide polymorphisms (SNPs). We therefore corrected Tables P value for departures from the Hardy-Weinberg equilibrium was less than 0.05 for 15 polymorphisms. However, the only genotype for which the difference between the observed and expected frequencies exceeded 3% was the SLC30A8 Arg325Trp (C/T) heterozygote. As shown in Table 5, some polymorphisms demonstrated a considerable difference in MAF among the participating cohorts; for 27 of the 108 polymorphisms, including ABCC11 Arg180Gly (T/C), there was a highly significant difference in MAF among study areas (P < 0.001).”The P values less than 0.05.”Furthermore, the first sentence of the third paragraph of the Discussion should read: “Of the remaining 104 polymorphisms, 15 showed departures from the Hardy-Weinberg equilibrium, with The authors regret these errors.