The rapid evolution of new sublineages of H5N1 influenza poses the greatest challenge in control of H5N1 infection by currently existing vaccines. To overcome this, an MVAtor vector expressing three H5HA antigens A/Vietnam/1203/04, A/Indonesia/669/06 and A/Anhui/01/05 (MVAtor-tri-HA vector) was developed to elicit broad cross-protection against diverse clades by covering amino acid variations in the major neutralizing epitopes of HA among H5N1 subtypes.7 TCID50/animal of MVAtor-tri-HA vector. The immunogenicity and cross-protective immunity of the MVAtor-tri-HA vector was evaluated against diverse clades of H5N1 strains.BALB/c mice and guinea pigs were immunized i.m. with 8×1050 of a divergent clade of H5N1 infection (clade 7). Importantly, the serological surveillance of post-vaccinated guinea pig sera demonstrated that MVAtor-tri-HA vector was able to elicit strong cross-clade neutralizing immunity against twenty different H5N1 strains from six clades that emerged between 1997 and 2012.The results showed that mice immunized with MVAtor-tri-HA vector induced robust cross-neutralizing immunity to diverse H5N1 clades. In addition, the MVAtor-tri-HA vector completely protected against 10 MLDThe present findings revealed that incorporation of carefully selected HA genes from divergent H5N1 strains within a single vector could be an effective approach in developing a vaccine with broad coverage to prevent infection during a pandemic situation. The continuing evolution of highly pathogenic H5N1 avian influenza in Asia and the recent emergence of H7N9 avian influenza in humans in Eastern China are increasing the threat of the next influenza pandemic. As of January 2014, the World Health Organization confirmed 650 human cases of H5N1 infection with 386 deaths Progressively developing recombinant vector technologies can efficiently deliver multiple genes in a single vector, which allows the cost-effective production of large quantities in a single manufactured product. Vectored vaccines based on adenovirus and poxviruses are among the several human viruses that have been extensively exploited for the development of multivalent vector-based vaccines In order to obtain proof of concept, selected HA genes from three H5N1 strains were inserted into recombinant MVAtor vector in a single insertion site (MVAtor-tri-HA) and the cross-protective efficacy of the vaccine candidate was evaluated in a mouse model. Additionally, serological surveillance was conducted to evaluate the neutralizing efficacy of post-vaccinated guinea pig sera against various clades of H5N1 strains that circulated worldwide during 1997–2012.All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Temasek Life Sciences Laboratory, Singapore. . All challenge experiments were conducted in a animal BSL3 containment facility in compliance with CDC/NIH and WHO recommendations.H5N1 virus A/Chicken/Cambodia/008LC1/2011 (clade 1.1) was obtained from the National Influenza Centre, Institute Pasteur in Cambodia. The hemagglutinin (HA) and neuraminidase (NA) genes of H5N1 viruses from clades 0, 1, 2, 4, 7 and 9  were previously selected in a three-step approach: mapping of neutralizing epitopes of hemagglutinin by using neutralizing monoclonal antibodies (mAbs), an analysis of the distribution of identified neutralizing epitopes among all H5N1 lineages, and the selection of vaccine strains to cover the variations within the neutralizing epitopes of H5N1 subtypes For the construction of the MVAtor-tri-HA vector the sequences were optimized for minimal homology and the three HAs from A/Vietnam/1203/04, A/Indonesia/CDC669/06 and A/Anhui/01/05 were fused to the PsynI, PsynII and H5 promoters, respectively within one DNA fragment (synthesized by Geneart) . The comThe shuttle vectors vEM99 and vEM106 were generated by direct insertion of HA genes in deletion III within the MVA genome. CEF cells were infected with MVAtor vector with a multiplicity of infection of 0.05 for one hour at room temperature. Then the cells were transfected with shuttle vector vEM99 or vEM106 using FuGene HD . The homologous recombination resulted in MVAtor-mono-HA or MVAtor-tri-HA vector. After plaque selection on CEF cells the recombinant MVAtor constructs were analyzed by PCR to verify HA gene insertion and lack of residual empty vector. To generate virus stocks/POC material the viruses were amplified in CEF cells and purified by ultracentrifugation through a sucrose cushion and reconstituted in 25 mM Tris buffer (pH 9.0). Original stock of the control virus MVA-LacZ was kindly provided by Prof. Gerd Sutter .2. Cells were incubated for another 20–24 hours. After fixation, cells were permeabilized with 0.1% Triton X-100 and stained using the HA specific mAbs or vaccinia virus specific antibodies. The cells were then incubated with Alexa Fluor-conjugated anti-mouse antibody  and DAPI  and analysed with a fluorescence microscope.HeLa cells infected with MVAtor-mono-HA or MVAtor-tri-HA vector were analysed by immunofluorescence using monoclonal antibodies (mAbs) specific for each HA, anti-AH for Anhui HA , 3H11 7 TCID50 of MVAtor-tri-HA vector or MVA-LacZ control vector on day 0 and day 28. In separate studies, the reference control group (n = 40 mice) was immunized subcutaneously (s.c.) with 100 µL of 256 HA units of inactive whole H5N1 virus (A/Indonesia/CDC669/06), emulsified with Montanide ISA563 adjuvant. The H5N1 virus was inactivated with binary ethylenimine as described previously 7 TCID50 of monovalent MVAtor-HA (A/Vietnam/1203/04) vector. Blood samples were collected on days 27 and 47 for serum hemagglutination inhibition (HAI) assay and virus micro-neutralization (VMN) assay.Female BALB/c mice aged 6–7 weeks were purchased from Biological Resource centre, A Star Research institute, Singapore and housed at the Animal Holding Unit of the Temasek Life Sciences Laboratory, Singapore. Mice (40 mice per group) were immunized intramuscularly (i.m.) with 100 µL of 8×1050 of A/Vietnam/1203/04 (clade 1) or A/chicken/Shanxi/2/06 (clade 7) H5N1 strain on day 49. Mice were observed daily to monitor body weight, clinical signs of disease  and mortality. Mice were humanely euthanized with CO2 inhalation if their body weight dropped to 75% of baseline weights. For determination of lung viral titers after challenge, five mice from each vaccinated group were euthanized by CO2 inhalation on day 3 post-challenge. Whole lungs were homogenized in 1 mL Dulbecco's Minimal Essential Medium to enable 10-fold serially diluted suspensions of lung samples. The homogenized suspensions were titrated on monolayers of MDCK cells. The viral titers were calculated 10 TCID50/mL ± S.E. The limit of virus detection was 1.5 log10 TCID50/mL of lung tissue specimen.To assess the protective efficacy of the vaccines, twenty mice from the MVAtor-tri-HA or MVA-LacZ vector or the inactive H5N1 vaccinated group were anesthetized intraperitoneally with ketamine (100 mg/kg)/Xylazine (20 mg/kg) and intranasally challenged with 50 µL (25 µL per naris) of 10 MLDReceptor-destroying enzyme  treated sera were serially diluted two fold in V-bottom 96-well plates. Four hemagglutination units of each influenza viral antigen was added to each well of the 96-well plate and incubated with the serum for 30 min and 1% chicken red blood cells were added, and incubated for 40 min at room temperature 50) of H5N1 virus and incubated at room temperature for 1 h. The virus antibody mixture was then added to the monolayer of MDCK cells and incubated for 72 h at 37°C. The neutralizing antibody titer was expressed as the highest dilution of serum which showed no cytopathic effect.Serum VMN antibody titer was performed as described previously 7 TCID50/animal of MVAtor-tri-HA vector. Additionally, two animals were immunized with MVA-LacZ as a negative control. After the final immunization, all the sera were individually tested for neutralizing antibody titer. The sera were pooled into large aliquots and stored at −20°C for further serological surveillance. Sera were tested for antibodies by HAI and VMN titers against clade 1, 2.1, 2.2, 2.3, 4, 7, 7.1 and 9, that emerged and circulated worldwide since 1997  were immunized i.m. three times on day 0, 28 and 49 with 100 µL of 8×10nce 1997 .t-test was performed to determine the level of significance in the difference between means of two groups. The level of significance was expressed as P<0.05.The data were expressed as geometric or arithmetic mean as indicated ± standard deviation (SD) standard error (SE). The unpaired two tailed Student's 7 TCID50/mL. The purified stock was analyzed by PCR (data not shown) and titrated in CEF cells (Recombinant MVAtor vector expressing the Vietnam HA gene (MVAtor-mono-HA) and the three HA proteins (MVAtor-tri-HA) were generated by integration of the HA expression cassettes into the deletion III site of the MVA genome by homologous recombination . The MVAEF cells . Also, tEF cells . In contHemagglutination inhibition titers measure the antibody response to inhibit HA function. As shown in 50 of different clades of H5N1 strains on day 47 showed that vaccination with MVAtor-tri-HA vector led to a significant increase (P<0.001) in neutralizing antibody titers against homologous H5N1 strains  compared to mice immunized with MVAtor-mono-HA vector (A/Vietnam/1203/04) or monovalent inactive H5N1 (A/Indonesia/CDC669/06) vaccine. Importantly, mice immunized with MVAtor-tri-HA vector showed cross-neutralizing antibodies against different clades of H5N1 strains   . In contclade 9) .50 of highly pathogenic homologous, clade 1 (RG-A/Vietnam/1203/04) or heterologous, clade 7 (RG-A/chicken/Shanxi/2/06) H5N1 virus. Mice immunized with MVAtor-tri-HA vector obtained complete protection from weight loss and death against clade 1 challenge while adjuvanted inactive whole H5N1 virus (A/Indonesia/CDC669/06) vaccinated mice rapidly lost body weight and showed only 30% protection  on day 3 and regained their body weight rapidly  of H5N1 strains. In addition, MVAtor-tri-HA vector elicited potent cross-clade neutralizing antibody titers (>1∶735) against distinct clades of H5N1 strains compared to monovalent vaccines. In our previous study, three mixtures of HA expressed on the baculovirus with adjuvant showed comparatively lower neutralizing antibody titers (1∶220–240) of against similar clades of H5N1 strains 50 of homologous or heterologous H5N1 challenge. However, our previous pilot study showed that mice immunized with MVAtor-mono-HA (A/Vietnam/1203/04) vector provided only moderate (66.6%) protection against 10 MLD50 homologous H5N1 strain (data not shown). In this study, mice immunized with adjuvanted inactive whole virus also showed rapid decline in body weight and showed insufficient protection against distinct H5N1 clades. This might be due to the variations in the neutralizing epitopes of hemagglutinin among the H5N1 strains. Also, we report that replication-deficient MVAtor vector expressing selected HAs induced robust neutralizing antibody response after two immunizations. These data indicate that pre-existing anti-vector immunity does not interfere with protective immune responses after immunization using the same MVA vector.The protective efficacy of the vaccine candidate was evaluated by challenging the immunized mice with a homologous (clade 1.0) and an antigenically distinct heterologous clade (clade 7.0) of H5N1 strains. The results demonstrated that mice immunized with MVAtor-tri-HA vector provided complete protection against 10 MLDAlso, the serological surveillance of post-immunized guinea pig sera against distinct H5N1 demonstrated that MVAtor-tri-HA induced robust cross-clade immunity against 20 heterologous clades or sub clades  of H5N1 strains, including the circulating strains from clade 1.1, 2.2.1.1 and 2.3.2.1. This observation shows the ability of the MVAtor-tri-HA vector against the genetic drift from 2010 to 2012 in clade 1.1 and 2 sublineages of currently circulating strains in Asia and Egypt . ImportaIn summary, the combination of three carefully selected HAs in a single vaccine candidate has efficiently induced neutralizing antibody titers against distinct clades of H5N1 strains isolated between 1997 and 2012. The use of a single multivalent MVA vector is the major advantage of this approach allowing cross-protection against a broad range of H5N1 strains with a single manufactured product. Thus, further investigations of the efficacy of multivalent MVAtor-HA vaccine candidates in ferrets and human are warranted.In conclusion, trivalent MVA vector expressing three carefully selected HAs exhibited a robust cross-neutralizing antibody response that mediates protection against distinct H5N1 challenge. Further serological surveillance of post-vaccinated guinea pig sera demonstrated that MVAtor-tri-HA vector efficiently neutralized six distinct clades/subclades that emerged worldwide by natural evolution. Therefore it seems likely that the demonstrated broadly neutralizing efficacy of the MVAtor-tri-HA vector could also induce some degree of cross-protection against yet unknown pandemic H5N1 strains. The strategy for vaccine strain selection and vaccine candidate design indicates the feasibility of the development of a broadly protective ‘universal’ H5N1 vaccine for pandemic preparedness.


Maturity-Onset Diabetes of the Young (MODY) is a form of monogenic diabetes characterized by autosomal dominant inheritance, young age of onset and pancreatic beta-cell dysfunction without autoimmune cause. To date 13 genes have been identified associated with MODY phenotype, with four of them  being responsible for over 95% of cases. Recently, our group has initiated molecular genetic screening for MODY using Sanger's sequencing method.To report novel mutations related to MODY 2 (GCK) and 3 (HNF1A) of a large cohort of Brazilian diabetic families in a new MODY diagnostic center.A total of 126 subjects from 63 Brazilian families were screened for GCK or HNF1A mutations: 30 families with clinical suspicion of MODY 2 and 33 families with MODY 3 phenotype. All exons and adjacent intronic regions of GCK and HNF1A genes were studied.We found 16 GCK mutations  in 30 families (53%). Five of these variants were novel: c.580-3C>A (IVS5); c.505A>G/p.K169E; c.110T>C/p.M37T; c.326_326delC/p.I110Sfs*6 and c.1247_1247delA/p.H416Pfs*15. HNF1A mutations were found in 6 families (18%): 2 nonsynonymous, 1 nonsense and 3 frameshift-insertion. One was a novel mutation: c.1558C>T/p.Q520*. All 6 novel variants were absent in databases of healthy controls (1KG/ESP-6500) and were predicted to be damaging using in silico analysis . In variants p.K169E and p.M37T, the substitution occurred in a codon already associated with MODY. All 6 subjects with novel variants have diabetes onset before age of 25 and BMI below 25. Five of them have family history of diabetes and one have parents without impaired fasting glucose. Familial co-segregation analysis was possible in 3 of the 6 probands, in which the variant segregated in family members with diabetes. All of them had detectable fasting C-peptide  3 yrs. after diagnosis of diabetes, and negative beta cell pancreatic antibodies .This molecular study has identified 6 unpublished mutations in subjects with clinical features of MODY, expanding the number of variants associated to this phenotype.


Cellular senescence is described to be a consequence of telomere erosion during the replicative life span of primary human cells. Quiescence should therefore not contribute to cellular aging but rather extend lifespan. Here we tested this hypothesis and demonstrate that cultured long-term quiescent human fibroblasts transit into senescence due to similar cellular mechanisms with similar dynamics and with a similar maximum life span as proliferating controls, even under physiological oxygen conditions. Both, long-term quiescent and senescent fibroblasts almost completely fail to undergo apoptosis. The transition of long-term quiescent fibroblasts into senescence is also independent of HES1 which protects short-term quiescent cells from becoming senescent. Most significantly, DNA damage accumulates during senescence as well as during long-term quiescence at physiological oxygen levels. We suggest that telomere-independent, potentially maintenance driven gradual induction of cellular senescence during quiescence is a counterbalance to tumor development. In vivo, quiescence is considered to limit the uncontrolled proliferation of cells, especially stem cells, whose proliferation has to be controlled properly in order to maintain tissue function, therefore contributing to tissue homeostasis Many cells within our bodies, including fibroblasts, hepatocytes, lymphocytes, stem cells and germ cells, are in the state of quiescence, defined as a reversible cell cycle arrest with temporary absence of proliferation vice versa, cells with depleted p21 show impaired entry into quiescence. Quiescence is not simply a downstream consequence of cell cycle exit. Specific inhibition of CDKs arrests the cell cycle, but this neither induces the quiescence-specific gene expression program nor resistance to terminal differentiation Quiescent cells show low expression of cyclins and cyclin dependent kinases (CDKs) in vivo). After months of quiescence, MRC-5 and WI-38 cells still display HES1 up-regulation, but this is unable to protect the cells against transition into senescence. Only a few percent of the cells in the population are able to return into the cell cycle.In order to be reversible, quiescence must grant the return into the cell cycle. Consequently, quiescent cells repress transition into terminal differentiation in which cell cycle arrest is irreversible In vivo, cellular senescence is thought to significantly contribute to the aging process Cellular senescence is a cell cycle arrested state in which normal diploid primary cells have lost their proliferative potential Telomere shortening as a basic concept for aging assumes that each successive cell division acts as a mitotic counting mechanism inducing replicative senescence In a first set of experiments, quiescence was induced (three times for a period of 9 days each) by contact inhibition in replicatively aging MRC-5 fibroblasts (at population doublings (PDs) 36, 44 and 56) or WI-38 cells  . ContactIn cells having experienced periods of quiescence, p21 was up-regulated . [27] whTaken together, we observed that the longer times in culture of the cells having been in quiescence (3 times 9 days) compared to control cells, influence cellular properties. Next, we studied MRC-5 and WI-38 cells during and after longer periods of quiescence.2. After release from long-term quiescence, fibroblasts were able to undergo only three (after 100 days of quiescence) or one (after 150 days) further PD(s) (MRC-5 and WI-38 fibroblasts were subjected to long-term quiescence induction by contact inhibition for 100 or 150 consecutive days at 20% Oer PD(s)  and S3. er PD(s) ., whereaer PD(s) .The influence of long-term quiescence on WI-38 and MRC-5 cells was further analyzed by measuring the levels of markers for proliferation and cell cycle arrest. For both cell types, the proliferation marker Ki-67 strongly decreased reaching lowest values in the senescent state and S3. TAfter long-term quiescence induction, the percentage of SA-β Gal positive cells, when plotted versus time in culture, were found to be similar to controls for WI-38 cells, and only slightly delayed for intermediate time periods for MRC-5 cells  and S3. High-throughput RNA sequencing of MRC-5 cells at PD 32 showed up-regulation of p16, p27, Cyclin D2, Ki-67 and γH2A.X mRNAs after 150 days compared to 9 days of quiescence (unpublished data), correlating with corresponding protein levels. This suggests that up-regulation of the abundance of these proteins during quiescence is due to transcription. We observed that during long-term quiescence MRC-5 or WI-38 fibroblasts cells do not suffer from telomere shorting (data not shown), in agreement with previous reports 2 levels from 20% down to physiological 3% 2 , respectively  increased after release from 100 or 150 days of quiescence compared to proliferating controls of corresponding PDs  compared to those at 20% O2 (cumulative PD = 72). Parallel to the MRC-5 growth curve  quiescent MRC-5 fibroblasts (PD 35) for 96 hours with 7.5 µM Etoposide or 2.0 µM Staurosporine revealed 16% and 13% apoptotic cells, respectively ., as indWe observed that long-term quiescent fibroblasts transit into senescence by a similar rate as do normally proliferating cells  and S4. Several independent signals  induce cellular quiescence, a reversible cell cycle arrest, by up-regulation of the CDK inhibitor p21. Each of these signals causes regulation of a unique set of genes known to terminate growth and division in vivo, remains to be shown). We induced short-term and long-term quiescence in MRC-5 and WI-38 fibroblasts. After short-term (9 day) quiescence we observed p21 and p27 up-regulation but no induction of p16  during quiescence as well as during normal proliferation, similar cellular processes within these cells might rule this transition. We therefore compared the gene expression profile of young with that of long-term quiescent fibroblasts, and determined which genes are either up- or down-regulated when cells are in long-term quiescence. Interestingly, we found DNA repair pathway genes down-regulated, explaining accumulation of DNA damage in long-term quiescent cells. These differentially expressed genes were compared to those recently found when comparing young and senescent fibroblast cells Our observations suggest the following model for fibroblasts in cell culture. Cells proliferate with the rate “r” in vitroin vivoFibroblast cells can divide up to about 70 times before stably arresting and becoming senescent We speculated that high oxygen levels might induce the increasing amount of DNA damage. We reduced the extracellular oxygen levels from 20% to physiological 3% and detected no or only minor quantitative differences. Thus, the increasing amounts of DNA damage and the increase of SA-β Gal positive cells with time in culture are not mainly due to high (20%) oxygen levels; instead we consider internal cellular processes like metabolic effects responsible for this. Furthermore, when high oxygen stress (inducing DNA damage and not telomere shortening) would trigger senescence in quiescent cells, lower oxygen concentrations should delay the transition into senescence 2 atmosphere at 37°C. For subculturing procedures, 1x PBS (pH 7.4)  and trypsine/EDTA  were used. Fibroblast cultures approaching confluency were splitted at a ratio of 1∶4. For replicative senescence, fibroblasts were maintained until the end of their lifespan . Quiescence induction was performed by contact inhibition or serum starvation. For contact inhibition, quiescence was induced either repetitively (3 times for 9 days during the culture span of the fibroblasts) or in long-term (100 or 150 days of consecutive quiescence induction). In MRC-5 fibroblasts, short-term quiescence was induced during PDs 36, 44 and 56 whereas in WI-38 fibroblasts, short-term quiescence was induced at PDs 33, 43 and 51. Once the cells were confluent at their respective PDs, they were left in the confluent state for 9 days with media change (containing 10% FBS) every 3 days (contact inhibition). Then the cells were split and transferred back to normal culture conditions with subsequent canonical splitting. With respect to the long-term quiescence induction, the cells were allowed to reach confluence at early PDs and were left in a confluent state for 100 or 150 days. In this instance, media (containing 10% FBS) was changed every 3 days. Otherwise, quiescence was induced for 8 days by incubation of cells with serum-deprived medium, DMEM supplemented with 0.5% FBS (serum starvation). After addition of normal growth medium, cells resumed proliferation.Primary human lung fibroblasts MRC-5  and WI-38  were obtained from ATCC  at young population doublings (PD) of 26-28. Cells were cultured in Dulbeccos modified Eagles medium (DMEM) with L-Glutamine, low glucose , supplemented with 10% fetal bovine serum (FBS) . Cell culture was carried out under normal air conditions in a 9.5% COSA-β Gal activity was determined as described by 4 cells/µl were used per lane. Immunodetection was performed using 5%-powdered milk in PBS-T  for blocking . Primary antibodies, anti-p21 mouse antibody , anti-p16 mouse antibody , anti-p27 rabbit antibody , anti-γH2AX , anti-Cyclin D1 rabbit antibody , anti-Cyclin D2 mouse antibody , anti-Ki-67 mouse antibody , anti-HES1 rabbit antibody , anti-Bcl-2 , anti-Bax  and anti-tubulin mouse antibody  were diluted as indicated in 5%-powdered milk (in PBS-T) and incubated for one hour at room temperature. Washing steps were performed 3×10 min in 1×PBS-T. The secondary horseradish peroxidase-labeled antibodies (Jackson Immuno Research Lab) were incubated for 1 hr at room temperature. Detection of horseradish peroxidase was performed using ECL-detection system and radiographic film . After film development, quantification of signal intensities of the bands in the Western blots was carried out using Metamorph software.For Western blotting, 102. The fibroblasts were subjected to Etoposide or Staurosporine treatment after every 24 hrs of their span in culture. The percentage of SA-β Gal positive and apoptotic cells were investigated in the MRC-5 fibroblasts after different spans of treatment with Staurosporine and Etoposide.Two well established apoptotic inducers; Staurosporine 6/ml of MRC-5 fibroblasts cells treated with different concentrations of apoptotic agents were stained with Hoechst 33342 and propidium iodide staining to detect the percentage of apoptotic cells A BD FACS Canto II was used for flow cytometry. Ca. 10For quality check, total RNA was analyzed using Agilent Bioanalyzer 2100 (Agilent Technologies) and RNA 6000 Nano Kit (Agilent) to ensure appropriate RNA quality in terms of degradation. Total RNA was used for Illumina library preparation and next-generation sequencing. Around 2.5 µg total RNA was used for indexed library preparation using Illumina's TruSeq RNA Sample Prep Kit v2 following the manufacturer's instruction. Libraries were pooled and sequenced using Illumina HiSeq2000 and HiSeq2500 sequencing machines in single read mode with 50 cycles using sequencing chemistry v3. Reads were extracted in FastQ format using CASAVA v1.8.2 (Illumina).http://nemates.org/MA/progs/overlap_stats.html with a total number of genes of 16,035 representing the number of comparable genes between both datasets. Singular enrichment analysis was performed for the intersection of either up- and down-regulated DEGs found in both studies using DAVID Raw data sequencing results were received in FastQ format. Read mapping was performed using Tophat 2.0.6 Where appropriate, quantitative results were examined for statistical significance using paired two-sample type 2 Student's t-tests assuming equal variances; p values are presented where appropriate.The human fibroblast cell lines (MRC-5 and WI-38) used in this investigation was ordered from ATCC. A number of senescence related studies has been undertaken using MRC-5 and WI-38 S1 FigEffect of short term quiescence induction on protein expression levels of a number of cell cycle associated genes and a marker for DNA damage in MRC-5 fibroblasts. (A) The blots show the protein expression levels of p16, p21, p27, Cyclin D1, Cyclin D2 and γH2A.X in two MRC-5 fibroblast cell lines (control with no quiescence induction and a cell line where quiescence was induced 3 times separately for a span of 9 days) maintained at 20% O2 at different stages of their span in culture. The up or down-regulation was signified by the presence or absence of the bands in Western Blots.  Comparison of mean fold change of protein expression levels of p16 (B), p21 (C), p27 (D), Cyclin D1 (E), Cyclin D2 (F) and γH2A.X (G) in 3 times quiescence induced MRC-5 cell lines and control MRC-5 cell lines maintained in culture as triplicates. The bars indicate the mean ± S.D. ** p<0.01, *** p<0.001 - significantly different compared to fibroblasts with PD assigned 1. n = 3.(TIF)Click here for additional data file.S2 FigEffect of short term quiescence induction on protein expression levels of a number of cell cycle associated genes and a marker for DNA damage in WI-38 fibroblasts. (A) The blots show the protein expression levels of p16, p21, p27, Cyclin D1, Cyclin D2 and γH2A.X in two WI-38 fibroblast cell lines (control with no quiescence induction and a cell line where quiescence was induced 3 times separately for a span of 9 days) maintained at 20% O2 at different stages of their span in culture. The up or down-regulation was signified by the presence or absence of the bands in Western Blots.  Comparison of mean fold change of protein expression levels of p16 (B), p21 (C), p27 (D), Cyclin D1 (E), Cyclin D2 (F) and γH2A.X (G) in 3 times quiescence induced WI-38 cell lines and control WI-38 cell lines maintained in culture as triplicates. The bars indicate the mean ± S.D. ** p<0.01, *** p<0.001 - significantly different compared to fibroblasts with PD assigned 1. n = 3.(TIF)Click here for additional data file.S3 Fig2.Effect of long term quiescence induction (100 or 150 days) in WI-38 fibroblasts maintained at 20% O (A) Growth curve of 3 independent WI-38 fibroblast cell lines  maintained in culture at 20% O2 as triplicates from an early PD until senescence at late PDs. Each growth curve is measured in triplicate. Data points of all measurements are displayed (not the mean). (B & C) Percentage of SA-β gal positive cells at different time points of their growth in culture in the control WI-38 fibroblast cell line and in the cell lines where quiescence was induced for 100 or 150 days respectively. S3B and S3C Figs. are plotted with PDs and days in the y-axis respectively. Each curve is measured in triplicate, the mean value is displayed with error bar (± S.E). (D) The blots show the protein expression levels of p16, p21, p27, Cyclin D1, Cyclin D2, Ki-67 and γH2A.X in WI-38 fibroblast cell lines (subjected to different culture conditions of 100 or 150 days quiescence by contact inhibition and no quiescence induction) maintained in culture at 20% O2 until they approached senescence at late PD. The up or down-regulation was signified by the presence or absence of the bands in Western Blots.  Comparison of mean fold change of protein expression levels of p16 (E), p21 (F), p27 (G), Cyclin D1 (H), Cyclin D2 (I), Ki-67 (J) and γH2A.X (K) in WI-38 cell lines where quiescence was induced for 100 or 150 days by contact inhibition respectively compared to controls at corresponding span of time in culture. Cell lines were maintained at 20% O2 as triplicates. The bars indicate the mean ± S.D. ** p<0.01, *** p<0.001 - significantly different compared to fibroblasts with PD assigned 1. n = 3 specifies the number of samples except for γH2A.X .(TIF)Click here for additional data file.S4 Fig2.Effect of long term quiescence induction (100 or 150 days) in WI-38 fibroblasts maintained at 3% O (A) Growth curve of 3 independent WI-38 fibroblast cell lines  maintained in culture at 3% O2 as triplicates from an early PD until senescence at late PDs. Each growth curve is measured in triplicate. Data points of all measurements are displayed (not the mean). (B & C) Percentage of SA-β gal positive cells at different time points of their growth in culture in the control WI-38 fibroblast cell line and in the cell lines where quiescence was induced for 100 or 150 days respectively. S4B and S4C Figs. are plotted with PDs and days in the y-axis respectively. Each curve is measured in triplicate, the mean value is displayed with error bar (± S.E). (D) The blots show the protein expression levels of p16, p21, p27, Cyclin D1, Cyclin D2, Ki-67 and γH2A.X in WI-38 fibroblast cell lines (subjected to different culture conditions of 100 or 150 days quiescence by contact inhibition and no quiescence induction) maintained in culture at 3% O2 until they approached senescence at late PD. The up or down-regulation was signified by the presence or absence of the bands in Western Blots.  Comparison of mean fold change of protein expression levels of p16 (E), p21 (F), p27 (G), Cyclin D1 (H), Cyclin D2 (I), Ki-67 (J) and γH2A.X (K) in WI-38 cell lines where quiescence was induced for 100 or 150 days by contact inhibition respectively compared to controls at corresponding span of time in culture. Cell lines were maintained at 3% O2 as triplicates. The bars indicate the mean ± S.D. * p<0.05, ** p<0.01, *** p<0.001 - significantly different compared to fibroblasts with PD assigned 1. n = 3.(TIF)Click here for additional data file.S5 FigImpact of Staurosporine treatment in young and old PD MRC-5 fibroblasts. (A) Percentage of apoptotic cells in MRC-5 fibroblast cell lines (young PD = 34) treated with different concentrations of Staurosporine for different time spans (B) Percentage of SA-β gal positive cells in MRC-5 fibroblast cell lines (young PD = 34) treated with different concentrations of Staurosporine for different time spans (C) Percentage of apoptotic cells in MRC-5 fibroblast cell lines (old PD = 68) treated with different concentrations of Staurosporine for different time spans (D) Percentage of SA-β gal positive cells in MRC-5 fibroblast cell lines (old PD = 68) treated with different concentrations of Staurosporine for different time spans. In each instance, MRC-5 fibroblasts were maintained in culture at 20% O2 as triplicates. In A & C, the bars indicate the mean ± S.D. The mean values in B & D is displayed with error bar (± S.E). (E) The blots show the protein expression levels apoptotic protein Bax in MRC-5 fibroblast cell lines (young PD = 34 & old PD = 68) treated with 0.1 or 2.0 µM of Staurosporine for 96 hrs compared to controls (0 hrs). The MRC-5 fibroblasts were maintained in culture at 20% O2. The up or down-regulation was signified by the presence or absence of the bands in Western Blots. n = 3.(TIF)Click here for additional data file.S6 FigImpact of Etoposide or Staurosporine treatment in MRC-5 fibroblasts subjected to short or long term quiescence. (A) Percentage of apoptotic cells in MRC-5 fibroblast cell lines (untreated and treated with 7.5 µM Etoposide or 2.0 µM Staurosporine for 96 hrs) maintained at different culture conditions - after 9 (MRC-5 PD 35) or 150 days (MRC-5 PD 32) of quiescence induction by contact inhibition, fibroblasts of young PD = 34 and in fibroblasts of old PD = 68. (B) The blots show the protein expression levels of apoptotic protein Bax in MRC-5 fibroblast cell lines  treated with 2.0 (Staurosporine) or 7.5 µM (Etoposide) of apoptotic agents compared to controls (0 hrs). The MRC-5 fibroblasts were maintained in culture at 20% O2. The up or down-regulation was signified by the presence or absence of the bands in Western Blots. Values statistically different from their controls (t-test) are indicated with an asterix: * p<0.05, ** p<0.01, *** p<0.001. n = 3.(TIF)Click here for additional data file.S7 FigIntersection of the most commonly differentially regulated genes with age (both up and down) in IMR-90 fibroblasts subjected to replicative senescence  compared to genes retrieved from MRC-5 fibroblasts subjected to long term quiescence induction compared to their controls. Venn plots of DEGs in young vs. senescent IMR-90 fibroblasts  up-, (B) down-regulated. Both numbers of DEGs in the intersection are significant with regards to the expected overlap of two independent groups.(TIF)Click here for additional data file.S8 FigHeatmap showing scaled gene-set level changes for KEGG pathways identified using gene set enrichment analysis. The left column represents the comparison of young and senescent IMR-90 cells (TIF)Click here for additional data file.


Background. We aimed to investigate the distributive characteristics of SLC22A1 rs594709 and SLC47A1 rs2289669 polymorphisms and their influence on metformin efficacy in Chinese T2DM patients. Methods. The distributions of SLC22A1 rs594709 and SLC47A1 rs2289669 polymorphisms were determined in 267 T2DM patients and 182 healthy subjects. Subsequently, 53 newly diagnosed patients who received metformin monotherapy were recruited to evaluate metformin efficacy. Results. No significant difference was found between T2DM patients and healthy subjects in SLC22A1 rs594709 and SLC47A1 rs2289669 allele frequencies and genotype frequencies. After metformin treatment, SLC22A1 rs594709 GG genotype patients showed a higher increase in FINS (p = 0.015) and decrease in HOMA-IS (p = 0.001) and QUICKI (p = 0.002) than A allele carriers. SLC47A1 rs2289669 GG genotype patients had a higher decrease in TChol (p = 0.030) and LDL-C (p = 0.049) than A allele carriers. Among SLC22A1 rs594709 AA genotype, patients with SLC47A1 rs2289669 AA genotype showed a higher decrease in FBG (p = 0.015), PINS (p = 0.041), and HOMA-IR (p = 0.014) than G allele carriers. However, among SLC22A1 rs594709 G allele carriers, SLC47A1 rs2289669 AA genotype patients showed a higher decrease in TChol (p = 0.013) than G allele carriers. Conclusion. Our data suggest that SLC22A1 rs594709 and SLC47A1 rs2289669 polymorphisms may influence metformin efficacy together in Chinese T2DM patients.  Metformin is the most widely used oral antidiabetic drug, which could effectively lower blood glucose and reduce the risk of cardiovascular diseases . The cla in vivo. The gastrointestinal absorption, hepatic uptake, and renal excretion of metformin are mediated greatly by organic cation transporters (OCTs) and multidrug and toxin extrusion proteins (MATEs) [ SLC22A1 rs628031 (tagged to rs594709 in Asian) was reported to affect imatinib uptake in chronic myeloid leukemia (CML) cell line [ SLC47A1 rs2289669 was reported to be associated with metformin excretion [ SLC47A1 and SLC22A1, SLC47A1 and SLC22A2 had a significant influence on metformin's efficacy [ SLC47A1 rs2289669 and SLC22A1 rs622342 [ SLC22A1 rs594709 and SLC47A1 rs2289669 polymorphisms and SNP-SNP interaction on metformin efficacy in Chinese T2DM patients has not been widely evaluated.Metformin is not metabolized (MATEs) . It was  (MATEs) , 11–13. ell line . As we kxcretion  as well xcretion , 17. Besefficacy , 19. It rs622342 . However SLC22A1 rs594709 and SLC47A1 rs2289669 between T2DM patients and healthy subjects and the influences of SLC22A1 rs594709 and SLC47A1 rs2289669 polymorphisms on metformin's therapeutic efficacy in glucose lowering, serum lipids, and insulin sensitivity. Moreover, we are interested in whether there is a SNP-SNP interaction between SLC22A1 rs594709 and SLC47A1 rs2289669 on metformin's therapeutic efficacy.In this study, we aimed to investigate the distributive characteristics of SLC22A1 rs594709 and SLC47A1 rs2289669 polymorphisms were determined in 267 unrelated patients with T2DM and 182 healthy subjects. 267 T2DM patients  were recruited from the Second Xiangya Hospital of Central South University, the People's Hospital of Liuyang, and the First Hospital of Changsha. 182 healthy subjects  were recruited from Physical Examination Center of Xiangya Hospital of Central South University. T2DM was diagnosed according to DM diagnostic criteria of WHO 1999. Patients with type 1 diabetes, gestational diabetes, uncontrollable hypertension, severe liver, and kidney disease were not included in this study.Distributive characteristics of SLC22A1 rs594709 and SLC47A1 rs2289669 genotype individuals. Newly diagnosed type 2 diabetes was defined as being diagnosed with T2DM within 6 months before enrollment. All patients had not been treated with any insulin secretagogue and received metformin monotherapy for 3 consecutive months. Metformin doses ranged from 1000 mg/day to 2000 mg/day among individuals. The study was approved by the Ethics Committee of Xiangya School of Medicine, Central South University  (CTXY-110002-5), and was performed in accordance with the Helsinki Declaration. Written informed consent was provided by all subjects.Furthermore, 53 of 267 T2DM patients who were newly diagnosed and drug naïve  were enrolled to evaluate the effect of metformin among differentβ-cell function (HOMA-B), and quantitative insulin sensitivity check index (QUICKI) were calculated according to previous formulas [Detailed clinical information was collected from each patient. Body mass index (BMI), fasting blood glucose (FBG), postprandial blood glucose (PBG), fasting insulin (FINS), postprandial insulin (PINS), glycosylated haemoglobin (HbA1c), total cholesterol (TChol), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were evaluated before and after metformin treatment as previously described , 21. Homformulas , 23. SLC22A1 rs594709 polymorphism were 5′GGCGCTTCCCACACTCAT3′ (sense) and 5′GAGGAAAGCTCCACATGTAACC3′ (antisense), and the primers for SLC47A1 rs2289669 polymorphism were 5′CCTGACCTCTGTCCCTATT3′ (sense) and 5′GACTTCCGTGCCTGCT3′ (antisense). The PCR products were then sequenced using ABI 3700 automated sequencer .Genomic DNA was isolated from peripheral blood leukocytes using Promega DNA purification kit  according to the manufacturer's protocol and stored at −20°C until use. Genotyping was done using direct sequencing. The primers used to amplify t-test was used to compare baseline characteristics in T2DM patients and healthy subjects. The observed genotype frequencies in patients and controls compared with expected frequencies under Hardy-Weinberg equilibrium were evaluated by χ2 test. Comparisons of frequencies of genotypes and alleles were also analyzed by χ2 test.Independent-samples SLC22A1 rs594709 genotype was stratified for the SLC47A1 rs2289669 genotype and vice versa. Multivariate linear regression was used to analyze the interaction between SLC22A1 rs594709 and SLC47A1 rs2289669 genotype on metformin response in Chinese T2DM patients. All of the analyses were performed with SPSS software .We evaluated associations between each SNP and the change of clinical index before and after metformin therapy using multivariate linear regression by adjusting for sex, age, and BMI in additive model, recessive model, and dominant model, respectively. Besides, the analysis for the SLC22A1 rs594709 and SLC47A1 rs2289669 polymorphisms. The clinical characteristics of T2DM patients and healthy subjects were shown in 2 versus 23.98 ± 2.94 kg/m2, p < 0.001), waist circumference , waist-to-hip ratio , FBG , and TG  in T2DM patients were significantly higher than those of healthy subjects. Moreover, HDL-C level  in T2DM patients was significantly lower than that in healthy subjects. No significant differences were found in hip circumference, TChol, and LDL-C level between two groups.A total of 267 T2DM patients and 182 healthy subjects were enrolled to explore the allele frequencies and genotype frequencies of SLC22A1 rs594709 and SLC47A1 rs2289669 polymorphisms in T2DM patients and healthy subjects were shown in p > 0.05). Minor allele frequencies (MAFs) of SLC22A1 rs594709 in T2DM group and healthy control were 26.78% and 28.57%, respectively. MAFs of SLC47A1 rs2289669 were, respectively, 47.19% and 46.97% in T2DM patients and healthy subjects. No significant differences were found in allele frequencies  and genotype frequencies  between T2DM patients and healthy controls.The frequencies of the SLC22A1 rs594709 GG genotype patients had a higher increase in FINS (p = 0.015) and greater decrease in HOMA-IS (p = 0.001) and QUICKI (p = 0.002) than those of A allele carriers  and LDL-C (p = 0.049) level compared to GG genotype patients , PINS (p = 0.041), and HOMA-IR (p = 0.014) than GA or GG genotypes  than GA or GG genotypes  [ SLC22A1 rs594709 and SLC47A1 rs2289669, in T2DM patients and healthy subjects. However, no statistical difference was found between two groups.OCTs mediate the interactions between cells and their environment and are involved in transporting a broad spectrum of endogenous substrates and the detoxification of xenobiotics . NutrienProgram) . Therefo SLC22A1 gene is highly polymorphic, and several polymorphisms have been described to affect metformin responses [ SLC22A1 rs594709 locates in intron 4. Pairwise linkage disequilibrium (LD) analysis indicated that there were strong LDs between rs594709 and rs2282143, rs628031, rs3798168, and rs683369. Polymorphisms mentioned above could more or less affect the metabolizing and/or efficacy of OCT1 substrates. SLC22A1 rs2282143 variation led to a decrease of transport activity, but not completely abolished [ SLC22A1 rs628031  was identified in multiethnic groups worldwide [ in vitro [ SLC22A1 rs3798168, rs683369, and rs628031 could affect the response to imatinib therapy in chronic myeloid leukemia (CML) patients [ SLC22A1 rs594709 is the tagger SNP of rs3798168, rs683369, and rs628031 (r2 > 0.8), it is possible that the structural changes in SLC22A1 caused by any of these polymorphisms or even a combination effect of them may affect its transport ability of metformin.Humanesponses , 30, incesponses . SLC22A1bolished . SLC22A1in vitro . Howeverin vitro , 35, thoin vitro , 36. Recpatients . As SLC2 SLC47A1 gene was associated with the therapeutic efficacy of metformin [ SLC47A1 rs2289669 polymorphism on pharmacodynamics (n = 202) and pharmacokinetics (n = 28) of metformin was also evaluated in Chinese patients and showed that AA genotype patients had a better glucose lowering effect after one-year follow-up. As for pharmacokinetics parameters, AA genotype patients had higher AUC12h and lower renal clearance and renal clearance by secretion [ SLC47A1 rs2289669 polymorphism was correlated with metformin glucose lowering effect but was associated with the reduction of TChol and LDL-C. This result was similar to long-term metformin and sulphonylurea combination therapy study carried out in Slovenia [ SLC47A1 rs2289669 genotype was not associated with HbA1c levels but significantly associated with cholesterol levels in metformin-treated T2DM patients even after adjustment for renal function. In contrast to the previous studies, the sample size, period of metformin treatment, and different races were all possible reasons leading to different results. Replication studies in a relatively large population in a multi-ethnic diabetic cohort are needed to confirm SLC47A1 rs2289669 polymorphism effect on metformin efficacy both in glycemic control and in lipid profiles.Previously, Caucasians population-based study in diabetic patients revealed that rs2289669 G/A inetformin , 17. Theecretion . Thus, iecretion . HoweverSlovenia . Their r SLC47A1 rs2289669 and SLC22A1 rs594709, which affected the blood glucose, insulin level, insulin resistance improvement, and blood lipid after metformin treatment. Among SLC22A1 rs594709 AA genotype patients, SLC47A1 rs2289669 AA genotype patients showed a better glycemic control compared with G allele carriers, while, among SLC22A1 rs594709 G allele carriers, patients with SLC47A1 rs2289669 AA genotype showed a better metformin efficacy in blood lipid than GA or GG genotypes. Interaction between SLC22A1 and SLC47A1 polymorphisms and metformin response had been reported previously [ SLC47A1 rs2289669 polymorphism on the decrease of HbA1c by metformin was larger in incident users with the SLC22A1 rs622342 CC genotype than in incident users with the AA or AC genotype patients. Both of the rs622342 and rs594709 are located in intron regions of SLC22A1 gene. The interaction is possibly due to the genetic mutations in SLC22A1 and SLC47A1 altering the function of OCT1-mediated influx and MATE1-mediated efflux. As OCT1 transports metformin into the hepatocyte and MATE1 transports metformin out of the hepatocyte into the bile, altered OCT1 and MATE1 function may be able to influence the intracellular metformin concentrations and glucose lowering function of metformin. Further replication and mechanism studies are needed to illustrate the influences of SLC22A1 and SLC47A1 polymorphisms on metformin efficacy.Furthermore, we found that there was an interaction betweeneviously ; the eff SLC22A1 594709 and SLC47A1 rs2289669 polymorphisms may interact to affect the therapeutic efficacy of metformin in Chinese T2DM patients. From our data, T2DM patients with AA genotype of both SLC22A1 rs594709 and SLC47A1 rs2289669 may get maximum glucose lowering benefit from metformin monotherapy. The SLC22A1 and SLC47A1 gene-gene interaction further suggests the complexity of metformin efficacy in T2DM patients.In summary, we found that


Cauliflower mosaic virus (CaMV) 35S promoter (P35S) is a commonly used target for detection of genetically modified organisms (GMOs). There are currently 24 reported detection methods, targeting different regions of the P35S promoter. Initial assessment revealed that due to the absence of primer binding sites in the P35S sequence, 19 of the 24 reported methods failed to detect P35S in MON88913 cotton, and the other two methods could only be applied to certain GMOs. The rest three reported methods were not suitable for measurement of P35S in some testing events, because SNPs in binding sites of the primer/probe would result in abnormal amplification plots and poor linear regression parameters. In this study, we discovered a conserved region in the P35S sequence through sequencing of P35S promoters from multiple transgenic events, and developed new qualitative and quantitative detection systems targeting this conserved region. The qualitative PCR could detect the P35S promoter in 23 unique GMO events with high specificity and sensitivity. The quantitative method was suitable for measurement of P35S promoter, exhibiting good agreement between the amount of template and Ct values for each testing event. This study provides a general P35S screening method, with greater coverage than existing methods.The  Ever since the first genetically modified (GM) crop was commercially planted, genetically modified organisms (GMOs) have come under suspicion from governments and citizens because of potential safety risks1Cauliflower mosaic virus (CaMV) 35S RNA gene promoter (P35S) and the terminator of the Agrobacterium tumefaciens Ti plasmid nopaline synthase gene (TNOS)678Polymerase chain reaction (PCR) is the most generally accepted GMO detection technique. This is largely because of its ability to amplify specific DNA fragments from highly processed materialshttp://cera-gmc.org/index.php?action=gm_crop_database), we conducted preliminary statistical analyses into the presence of P35S and TNOS in GM crops. This revealed that 65.7% (67/102) of approved commercial GM events contain the P35S promoter, 53.49% (55/102) the NOS terminator, and 81.4 (83/102) either or both of these in their transgene constructs (Zea mays), only one  contained neither element, and in 16 commercial GMOs of rapeseed (Brassica napus) there were only two exceptions. Some transgenic events, such as GM maize Bt11, T25, and GM cotton Mon531 contained two copies of P35S in their transforming constructs. Consequently, the P35S promoter and NOS terminator are the most widely used GMO screening targets.Using the available GMO transformation information from the GM Crop Database (nstructs  online. http://gmdd.shgmo.org/). In the GMDD, some primer pairs are simultaneously used by both qualitative and quantitative assays, and some methods are actually repeats of the same method using different primer names. The P35S promoter is one of the most frequently modified elements in GMOs. The P35S promoter sequences in different GMOs and vectors may be different from each other owing to origination from different strains or from modification in vector construction or mutation during the breeding processet al. identified a single nucleotide polymorphism (SNP) in the primer binding site of the P35S sequence in TC1507 maize, leading to inefficient amplification of testing primer/probe sets9Because of the importance of the P35S promoter in screening detection of GMOs, a large variety of GMO screening tests have been established and published10111213141516171819202122232425262728293031323334353637383940414243441011131415162831323334et al. tested the suitability of five published P35S-based methods with eight maize reference materials, demonstrating that two methods had the flaws of poor linear regression parameters and multiple PCR amplicons in some of the testing materialsAlthough many P35S-based methods are available for the testing laboratories, only partial methods have gone through necessary validation processes and inter-laboratory studies against a small number of transgenic events13141924313443The human immunodeficiency virus (HIV) often generates drug resistance mutations owing to highly variable gene and drug selection pressure. During detection of HIV-1 drug resistance mutations, the genotyping assay, combining reverse transcription PCR with sequencing technology or high resolution melting (HRM) analysis, is commonly used to detect all the possible mutations in HIV genome47P35S-based methodologies play an important role during the GMO screening phase. Currently, GMO detection laboratories select methods from different sources, including published literature, ISO standards, databases, or in-house developed methods. This heterogeneity in methodology may result in divergent test results if the P35S sequence carried by the testing sample was altered during construction of the transforming vector or in the breeding process. The ILSI survey revealed that most participating laboratories were interested in adopting a standardized method, which could generate consistent testing results and lead to better inter-laboratory reproducibilityThe purpose of this study was (1) to isolate the P35S sequence from different transgenic events, (2) to analyze methodological flaws in existing P35S-based detection methods using sequence alignment between primers/probes and the P35S sequences from different GMOs and constructs, and to confirm the defects found by the experiments, and (3) to design a general qualitative and quantitative detection system that targets the conserved region of P35S for high-coverage GMO screening.http://www.cera-gmc.org/GMCropDatabase), of those, 23 unique GM events were available in the present study. After performing isolation of P35S promoters, nineteen P35S sequences were isolated from the following 16 transgenic events: GM soybean GTS-40-3-2, A5547-127; GM maize MON863, NK603, TC1507, Bt11, MON810, T25; GM cotton MON15985, MON88913, MON531, LLCotton 25, MON1445; GM rapeseed OXY235; and GM rice Kefeng 6 and KMD. The isolated sequences were submitted to the GenBank database, and accession numbers and sequences are summarized in A total of 67 GM events containing P35S have been collected by the GM crop database  from the patent database495051http://gmo-crl.jrc.ec.europa.eu/gmomethods/)143134Twenty-four different detection methods targeting the P35S promoter were identified from published papers and detection standards, and labeled as M1 to M24 . Of thes111016To investigate the sequence consistency of primer binding sites for methods M2, M7, and M12, the P35S conserved region from the CaMV genome, multiple transgenic events, and binary vectors harboring nucleotide alterations, together with primer/probe sets, were aligned . For theThe 24 collected P35S-based methods were used to detect P35S in MON88913. Since at least one primer lied outside the P35S region of MON88913, nineteen primer pairs failed to detect the P35S target when MON88913 genomic DNA was used as template; while five methods  successfully detected the P35S target . Sequenc2 and slope data of the standard curves are summarized in 2 values ranging from 0.994 to 0.997The binding sites of the primer/probe set contained SNPs for the M2, M7, and M12 methods . To evalThe TaqMan assays revealed that the SNP mutation in TC1507 generated large anomalies in the PCR performance of the M2, M7, and M12 methods, in agreement with the previous studyA fragment of approximately 195 bp between position 7148 and 7342 in the CaMV genome was relatively conserved across different P35S promoters, even though it contained multiple SNPs dividing it into smaller discrete segments. This conserved fragment was present in all collected P35S sequences in this study, furthermore, two copies were contained in double enhancer promoters such as the enhanced P35S promoter in pCambia-1381, maize NK603 and MON88017, cotton MON15985, MON88913 and MON531, and rice Kefeng6. Multiple candidate primer/probe sets were designed to anneal to conserved segments carrying no SNP. To select the best primer/probe set, all possible primer and probe combinations were tested for amplicon size and specificity using 0.1 ng genomic DNA from transgenic maize TC1507 as a template, which possesses a P35S promoter and has been observed to have inefficient amplification in some of the methods. The most effective, reliable, and robust primer/probe set was 35SEF/35SER/35SEP, yielding a 125 bp amplicon, and labeled as M25 . The binThe amplification stability of the primer pair 35SEF/35SER was tested using the genomic DNA from the 23 GMOs described above. Electrophoresis revealed that the unique 125 bp fragment was amplified from all samples containing a P35S promoter . SpecifiIn practice, DNA extracted from GM food or feed tends to be highly degraded or very low in quantity. To evaluate the detection sensitivity of the new qualitative detection system, genomic DNA from five transformants  containing the P35S promoter, representing the five major transgenic crops , were serially diluted to 100, 50, 20, and 10 copies per microliter and used as templates for PCR analysis. The lowest detectable template quantity required for maize TC1507 was estimated to be 20 copies; for soybean GTS40-3-2, 50 copies; for cotton MON1445, 20 copies; for rice KMD, 20 copies; and for rapeseed OXY235, 50 copies . These d2), slope, and amplification efficiency are summarized in 2 values ranged from 0.997 for TC1507 to 1.000 for GTS-40-3-2, this is significantly higher than the ENGL minimum requirement of 0.98. The slopes across the five events ranged from −3.411 in GTS-40-2 to −3.225 in KMD, this is within the acceptable range of −3.6 ≤ slope ≤ −3.1. Based on the slope of the standard curve, the efficiency of this P35S PCR method was estimated to be from 96.4% to 104.2%, close to the ideal efficiency of 100%. The real-time PCR assays verified that SNPs in conserved regions did not influence the amplification efficiency of the newly developed quantitative method. We therefore conclude that our real-time assay is suitable for quantifying the P35S promoter copy number in various GM crops.Variations in the DNA templates of different crops may affect test results during GMO screening by P35S-targeted methods. To evaluate the suitability of the newly developed P35S-based quantitative method for detecting various GM crops, the five events  were subjected to real-time PCR to analyze the performance of the quantitative PCR. The conserved regions in GTS-40-3-2 soybean, TC1507 maize, KMD rice, and MON1445 cotton all have SNPs present, while OXY235 rapeseed does not. A series of dilutions for each of the extracted DNAs was made, corresponding to 50000, 5000, 500, 50, and 10 copies/µL for events GTS-40-3-2, OXY235, TC1507, and KMD, and to 28000, 5000, 500, 50, and 10 copies/µL for event MON1445. The serial dilutions were used as calibrators to establish standard curves for P35S detection, and each dilution was assayed in triplicate. Standard curves were created by plotting Ct values against the logarithm of transgene copy numbers, good agreement was observed between the quantity of template and the Ct values for each event . The squTo determine the limits of detection (LOD) and quantification (LOQ) of the real-time PCR method, five genomic DNA templates  from the five major crops were each diluted to 80, 50, 40, 20, 10, 5, and 1 copies/µL to perform real-time PCR assays in 10 replicates. The discrepancy in the Ct values across the ten replicates became larger with decreased template copy number . All 10 NPTII gene, and 15 methods for Bar gene (http://gmdd.shgmo.org/). Method heterogeneity across testing laboratories can lead to adverse testing results during the screening phase of GMO detection, which can consequently cause problems for international trade. Due to the difficulty in detecting all the different transgene events, a single artificial sequence is recommended to be developed as a universal barcode, which would not be found in natural DNA sources. During the process of developing GMOs in future, all transgene developers could be required to include the single artificial sequence that would make detection of all transgene events easier by one method. However, the implementation of this idea needs to obtain approval and support of both transgene developers and regulators.Screening methods are often used directly to make a preliminary judgment on whether or not samples are GMOs. Precise and accurate detection methods are a prerequisite for reliable control of GMOs, and the screening method should be suitable for a wide range of GMOs. The survey on the GMO detection methods revealed that multiple qualitative or quantitative methods were developed and published for the same one target sequence, for instance, 24 methods targeting P35S were established, 14 methods targeting TNOS, 11 methods for P35S are likely to remain an important component of GMO products at present and in the foreseeable future. Currently, 24 methods targeting different regions of the P35S sequence are reported and adopted by individual research and testing laboratories1011121314151617181920212223242526272829303132333435363738394041424344Most existing GMO labeling systems are based on transgenic content, but not all transformants have a corresponding event-specific detection method. If a real-time PCR method targeting general transgenic components could be used for GMO quantitation, then GMOs without an event-specific detection method can be quantified and labeled. However, the new detection methods still have problems that require addressing because the copy number of the detecting target is not the same for different single copy transformation events. In this analysis the detection target locates in the enhancer region, which is usually reused in the double enhancer promoter to enhance regulatory activity. For instance, the NK603, MON88017, MON15985, MON88913, MON531, and Kefeng 6 events all contain a double enhancer promoter. Furthermore, events such as Bt11, T25, and MON531 contain two copies of P35S in their transgene constructs, and for the MON531 event one copy is a double enhancer promoter. Hence, GMO content can be overestimated when using the P35S method for events harboring a double enhancer promoter or multiple copies of P35S. Therefore, when this new quantitative PCR method is used to estimate the exogenous gene copy number of samples, DNA extracted from the same sample should be used to construct the standard curve. In addition, if only P35S shows positive signal for test samples in practice, a subsequent experiment detecting other genomic sequence of CaMV, should be performed to rule out CaMV itself as contaminant in the plant DNA samples. In conclusion, the use of this new P35S method, which covers a wide range of GMOs, will lead to more consistent results of GMO detection during the screening phase among different testing laboratories.Transgenic materials, including seed powder of transgenic soybean (10% GTS 40-3-2) and transgenic maize , were purchased from the Institute for Reference Materials and Measurements . Seed powder of transgenic maize , cotton , and leaf DNA of soybean (A5547-127), maize (T25), cotton (LLcotton25), and rapeseed  were purchased from the American Oil Chemists' Society . Transgenic soybean (A2704-12), transgenic rice  and non-transgenic crop seeds were already available in our own laboratory.TM PicoGreen® dsDNA Assay Kit .A DNeasy Plant Mini Kit  was used to extract and purify genomic DNA from seeds or seed powders in accordance with manufacturer's instructions. DNA purity was checked using a NanoDrop 2000 spectrophotometer , and sample concentrations further quantified using a Versafluor Fluorometer  with a Quanti-iTEcoRV restriction enzyme site. Ligation products were transformed into Escherichia coli strain TOP10F , and positive clones screened. Plasmids containing the PCR products were sequenced using M13 forward and reverse primers .Primers used for isolating the P35S sequences from different transgenic events were designed using Primer Premier 5.0 software  according to the nucleotide sequences flanking the P35S promoter. Primers were synthesized by Sangon ; their sequences are given in http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq). Regions conserved in aligned P35S promoters were discovered based on the positions of different P35S promoters in the CaMV genome. The P35S region across the GMOs and binary vectors, together with the whole genome sequence of CaMV (NC_001497.1), were aligned using the AlignX program of the Vector NTI 9 software suite (Invitrogen).Pairwise comparison of nucleotide sequences was performed using the bl2seq program available from the National Center for Biotechnology Information (NCBI) , and the 3′ ends with the minor groove binder non-fluorescent quencher (MGBNFQ). All primers and fluorescent probes were synthesized by Sangon Biotech .2), 200 μM of each dNTP, 0.25 μM of each primer, 1 U EX Taq™ , and 20 ng genomic DNA in a total volume of 20 µL. The PCR used the following cycle conditions: initial denaturation for 120 s at 94°C; 35 cycles of 30 s at 94°C, 30 s at 60°C, and 30 s at 72°C; and terminal elongation for 2 min at 72°C. PCR products were size fractionated using 2% agarose gel electrophoresis in 1× TAE buffer, and visualized with ethidium bromide. The UV-fluorescent emission was recorded with a Gel Doc XR system using Quantity One software (Bio-Rad).Conventional PCR was run on a Bio-Rad C1000™ Thermal Cycler using an optimized conventional PCR mixture: 1× PCR buffer  in a final volume of 20 µL. The reaction mixture for the P35S promoter contained 1× TaqMan Universal PCR Master Mix , 400 nM primers, and 200 nM probe. All real-time PCRs were performed using the same program: pre-digestion at 50°C for 2 min; initial denaturation and uracil-N-glycosylase deactivation at 95°C for 10 min; and 50 cycles of 15 s at 94°C (denaturation) and 1 min at 60°C . Fluorescence was measured after each annealing and extension step using CFX Manager ver. 1.6 (Bio-Rad). Data analysis was performed using the CFX Manager ver. 1.6 (1.6.541.1028) software (Bio-Rad).G.W. designed this experiment and wrote the main manuscript; Y.W. designed this experiment, wrote the manuscript, and prepared figures; Y. Wang., J.L. and W.L. performed this experiment; L.Z. and Y.L. advised on the experimental design and commented on the manuscript; other authors, including X.L., L.Z. and J.L., reviewed the manuscript.Development of a general method for detection and quantification of the P35S promoter based on assessment of existing methods


MFRP/Mfrp) gene, specifically expressed in the retinal pigment epithelium (RPE) and ciliary body, cause nanophthalmia or posterior microphthalmia with retinitis pigmentosa in humans, and photoreceptor degeneration in mice. To better understand MFRP function, microarray analysis was performed on eyes of homozygous rd6Mfrp and C57BL/6J mice at postnatal days (P) 0 and P14, prior to photoreceptor loss. Data analysis revealed no changes at P0 but significant differences in RPE and retina-specific transcripts at P14, suggesting a postnatal influence of the rd6Mfrp allele. A subset of these transcripts was validated by quantitative real-time PCR (qRT-PCR). In rd6Mfrp eyes, a significant 1.5- to 2.0-fold decrease was observed among transcripts of genes linked to retinal degeneration, including those involved in visual cycle , phototransduction , and photoreceptor disc morphogenesis (Rpgrip1 and Fscn2). Levels of RPE65 were significantly decreased by 2.0-fold. Transcripts of Prss56, a gene associated with angle-closure glaucoma, posterior microphthalmia and myopia, were increased in rd6Mfrp eyes by 17-fold. Validation by qRT-PCR indicated a 3.5-, 14- and 70-fold accumulation of Prss56 transcripts relative to controls at P7, P14 and P21, respectively. This trend was not observed in other RPE or photoreceptor mutant mouse models with similar disease progression, suggesting that Prss56 upregulation is a specific attribute of the disruption of Mfrp. Prss56 and Glul in situ hybridization directly identified Müller glia in the inner nuclear layer as the cell type expressing Prss56. In summary, the rd6Mfrp allele causes significant postnatal changes in transcript and protein levels in the retina and RPE. The link between Mfrp deficiency and Prss56 up-regulation, together with the genetic association of human MFRP or PRSS56 variants and ocular size, raises the possibility that these genes are part of a regulatory network influencing postnatal posterior eye development.Mutations in the membrane frizzled-related protein ( MFRP mutations in humans are associated with nanophthalmia or posterior microphthalmia with autosomal recessive retinitis pigmentosa (RP), characterized by retinal spots, foveoschisis and optic nerve head drusen Mfrp in mice recapitulates central features of the human disease, including retinal spots and a slowly progressing retinal degeneration MFRP/Mfrp disease phenotype is variable in humans Mfrp mutant mice attractive for delineating the mechanism(s) that underlie MFRP/Mfrp-associated ocular disease and its genetic variability, which are poorly understood.MfrpWithin the eye, MFRP is exclusively localized to the apical surface of the retinal pigment epithelium (RPE) and the ciliary body epithelium rd6Mfrp and wildtype postnatal eyes. Microarray analysis revealed a moderate decrease in phototransduction, visual cycle, and gene transcripts associated with retinal degeneration in rd6Mfrp mutants. Most interestingly, an increased temporal expression of Prss56, a gene linked to posterior microphthalmia Prss56 upregulation appeared to be specific to rd6Mfrp mice, as it was not observed in other retinal degeneration models with similar disease progression. Broadly, our findings delineate a potential role of MFRP in postnatal development and/or maintenance of the posterior eye, and provide evidence that MFRP and PRSS56 participate in the same functional pathway.To obtain insight into the functional role of MFRP in the RPE and retina during normal eye development and disease, gene-profiling studies were carried out in Protocols using mice in this study were approved by The Jackson Laboratory Institutional Animal Care and Use Committee  in accordance with the "Guide for the Care and Use of Experimental Animals" established by the National Institutes of Health (NIH) .rd6/MfrpJ (rd6Mfrp) and C57BL/6J were bred and maintained in a vivarium with a 12 hour light and 12 hour dark cycle in the Research Animal Facility at The Jackson Laboratory. Autoclaved NIH31 diet (6% fat) and HCl acidified water (pH 2.8–3.2) were provided ad libitum.Mouse strains utilized in this study, B6.C3Ga-rd6Mfrp and C57BL/6J eyes at P0 and P14  was extracted with Trizol  according to the manufacturer's instructions. Two µg of total RNA was used for cDNA synthesis with the One-Cycle Target Labeling cDNA Synthesis Kit  and fragmented cRNA was synthesized using the GeneChip IVT Labeling kit  according to the manufacturer's protocol . Following cRNA fragmentation, 15 µg of cRNA were hybridized on a GeneChip Mouse Genome 430 v2.0 array at 45°C for 16 h and the microarray was scanned using the GeneChip Scanner 3000 . Average signal intensities for each probe set within the arrays were calculated by and exported from Affymetrix's Expression Console (Version 1.2) software using the RMA method, which incorporates convolution background correction, sketch-quantile normalization, and summarization based on a multi-array model fit using the median polish algorithm to generate gene expression data. For this experiment, six pairwise comparisons were used to statistically resolve gene expression differences between sample groups using the R/maanova analysis package. Specifically, differentially accumulated transcripts were detected by using Fs, a modified F-statistic incorporating shrinkage estimate of variance components from within R/maanova. Statistical significance levels of the pairwise comparisons were calculated by permutation analysis (1000 permutations) and adjusted for multiple testing using the false discovery rate (FDR) q-value threshold of 0.05. For each probe set, the raw intensities for all probes were log2-transformed. The log2-transformed intensities were quantile-normalized and a volcano plot was generated for each of the six pairwise comparisons. Differentially accumulated transcripts were classified into various biological pathways using the Ingenuity Pathway Analysis Systems software . Microarray data (MIAME compliant) were submitted to the Gene expression Omnibus (GEO) database (http://www.ncbi.nlm.noh.gov/geo) under GEO Accession Number GSE53411 (http://www.ncbi.nlm.nih.gov/geo/info/linking.html).GeneChip Mouse Genome 430 v2.0 array (Affymetrix) was performed at The Jackson Laboratory Gene Expression Analysis Service (GES). Total RNA from www.ingenuity.com, Summer 2013 release, Qiagen, Valencia, CA, USA) was used to identify potential networks affected by the disruption of Mfrp across one dataset (rd6Mfrp versus C57BL/6J at P14). The dataset was derived from differential gene expression analysis resulting from the comparison of mRNA from whole eye tissue samples of rd6Mfrp versus C57BL/6J mice. Data uploaded into IPA consisted of Affymetrix Mouse Gene 430 2.0 probe sets as identifiers. Each identifier was mapped to its corresponding object in the IPA knowledge base (Summer 2013 release). Expression results were limited to genes having a q-value <0.05. Gene networks were algorithmically generated based on their connectivity to the uploaded data set. Networks pertaining to phototransduction and visual pathways were retained, and expression and significance values were overlaid onto networks of interest so as to identify differential patterns of up- and down-regulated genes. In each network, molecules are represented as nodes, and the biological relationship between two nodes is represented as an edge (line). All edges are supported by at least one reference from the literature, from a textbook, or from canonical information stored in the IPA Knowledge Base. The intensity of the node color indicates the degree of up- (red) or down- (green) regulation with regards to all expression values in the dataset. Nodes are displayed using various shapes that represent the functional class of the gene product; these are defined in the figure legends. Edges are displayed with various labels that describe the nature of the relationship between the nodes .Ingenuity pathway analysis software . The connective tissue, muscles and conjunctiva were carefully removed using iris scissors. A circumferential incision was made below the level of ciliary body and the anterior segment consisting of cornea, lens, iris and ciliary body was discarded. The neural retina was peeled from the RPE layer using surgical forceps and the remaining posterior eyecup consisting of the sclera, choroid and RPE was flash frozen in liquid nitrogen and stored at −70°C.T) and was quantified using 2C-ΔΔT with β-actin as an internal calibrator. Melting curve analysis was performed to validate accurate amplification of the target gene. The primers used for qRT-PCR were designed using mouse qPrimerDepot (mouseprimerdepot.nci.nih.gov). The primers spanned exon-exon borders that overlapped intron(s) . Total RNA was extracted from whole eyes, retina, and RPE cells using Trizol reagent  combined with the RNeasy kit  as per manufacturer's instructions. Genomic DNA contamination of RNA was prevented by on-column treatment with DNase I  according to the manufacturer's instructions . One µg of total RNA was reverse transcribed using the Retroscript kit . Quantitative real-time PCR (qRT-PCR) was performed with the SYBR Green Master Mix  and gene-specific primers using the ViiA7 Real Time PCR Cycler . Appropriate controls including no RT control was included in the assay to rule out genomic DNA contamination of the cDNA samples. A relative fold change in gene transcript was calculated using ViiA7 software V1.2.2 (Life Technologies), applying the comparative CT method (ΔΔCntron(s) .rd6Mfrp mice and placed in ice-cold PBS. The anterior portion of the eye including cornea, lens and ciliary body was excised. The neural retina was gently peeled from the underlying RPE layer, and the RPE layer including the choroid and sclera was flash frozen in liquid nitrogen and stored at −70°C. For preparation of tissue lysates, the aggregate RPE sample was combined with 100 µl of 1× SDS Laemmli sample buffer  containing 5% β-mercaptoethanol, and sonicated three times for 5 min using a probe sonicator 3000 Ultrasonic Liquid Processor  and stored in a −70°C freezer. The protein samples were suspended using a vortex mixer. Proteins were resolved by SDS-PAGE using a 10% Tris-glycine gel,  and transferred onto nitrocellulose  by semi-dry electroblotting using the Trans Blot Turbo Transfer System  according to the manufacturer's instructions. The membrane was washed briefly in PBS and blocked with Odyssey blocking buffer,  for 1 h at room temperature and subsequently incubated with anti-RPE65 antibody  at 1∶1000 dilution, overnight at 4°C. After four 5-min washes in 1× TBS containing 0.05% Tween20 (TBST), the blots were incubated with secondary antibodies, anti-mouse IRDye 700DX conjugated antibody  in blocking buffer for 45 min. Following four washes for 5 min in 1× TBST, the blots were scanned using the Odyssey Infrared Imaging system  at 700 nm. Blots were stripped and incubated with mouse β-actin antibody  at a 1∶10,000 dilution overnight at 4°C and processed as previously described. Odyssey Infrared Imaging System software 3.0 was used to quantify the protein bands using a standard curve after normalizing with the β-actin control. The normalized, relative change in protein concentration was expressed in arbitrary units.Whole eyes were dissected from C57BL/6J and rd6Mfrp mice was freshly harvested and fixed in 4% paraformaldehyde for 24 h at 4°C, immersed in 70% ethanol, dehydrated and embedded in paraffin blocks in situ hybridization. We performed in situ hybridization using both the QuantiGene ViewRNA ISH Tissue 1-Plex and 2-Plex assay according to the manufacturer's instructions . Briefly, PFA- fixed paraffin-embedded tissues were baked, dewaxed using xylene for 5 min with gentle agitation, twice, followed by two ethanol washes for 5 min each. A hydrophobic barrier was drawn on the glass slide using a template and the samples were heated at 90–95°C for 10 min, followed by protease treatment at 40°C using a Thermobrite Controlled Temperature Slide Processing System  for 10 min. Both heat and protease treatments were optimized for the eye tissue and to allow probe to access the specific RNA by unmasking the RNA. Mouse Prss56 Type 1 (FastRed) probe set (VB1-15207) and Mouse Glul Type 6 probe (FastBlue) set (VB6-16850) were custom designed  for target hybridization. Each probe set contained 20 oligonucleotide pairs that were specific for Prss56 or Glul and had a binding site for TYPE-specific sequences for signal amplification. Samples were incubated with Prss56 probe and/or Glul probe or no probe control at 40°C for 3 h using the slide processer. For signal amplification, a series of sequential hybridization steps were carried out using bDNA technology and signals were detected by addition of Fast Red substrate and/or Fast Blue substrate to the tissue section as per manufacturer's instructions . Target RNA was detected at specific sites within the tissue by chromogenic substrate deposition, which was visualized by using an SP5 confocal microscope  and fluorescent microscopy (Leica Microsystems Inc). Nuclei were visualized with Vectashield mounting medium with DAPI  . For FastRed substrate, we imaged using Cy3 filter and for FastBlue substrate, we imaged using the Cy5 filter. Images were processed using the Image J software  and Adobe Photoshop CS6  was used to create final images.Eye tissue from P14 C57BL/6J and homozygous The procedure for histological assays was previously described p-value) between two groups. P-values <0.05 were considered statistically significant.In this study, GraphPad Prism Version 5 software was used for statistical analyses. Student's t-test was used to calculate statistical significance  irrespective of the time point yielded a relatively small set of significantly accumulated probe sets  intensity level of the probe sets, where light blue represents a low level of hybridization to the probe set and dark blue a high level. Upregulated probe sets (5) with a relative fold change >5.0 are listed . The top five canonical IPA pathways that were altered significantly in rd6Mfrp mutant eyes were B cell development, allograft rejection signaling, autoimmune thyroid disease signaling, phototransduction, cytotoxic T lymphocyte-mediated apoptosis of target cells, and visual cycle. The genes identified in these pathways and results of statistical tests are given in rd6Mfrp mutant eyes. The rd6Mfrp allele, which is a loss of function mutation leading to the absence of MFRP protein from the RPE Rpe65 and Lrat, were decreased significantly in rd6Mfrp mice  of −1.2 to −2.0 in transcripts expressed in photoreceptor cells  pathway, identified additional transcripts that were differentially regulated in to −2.39 . These ird6 eyes , while trd6Mfrp mutants and C57BL/6J mice. Transcripts that were significantly increased in the microarray analysis are listed in , phototransduction pathway , and involved in disc morphogenesis , were also reduced as determined by qRT-PCR , Optc , Aqp5  .To validate the microarray data, qRT-PCR analysis was performed on whole eyes from isted in . Transcristed in , includi qRT-PCR . However q<0.05)  were not q<0.05) ; a PCR prd6Mfrp mutant was a non-specific effect of the disease process that occurs during retinal degeneration, we examined the levels of visual cycle and phototransduction pathway transcripts in two unrelated retinal degeneration models with mutations in the retina-specific gene, Tulp1 or the RPE-specific gene, Rpe65. Homozygous Tulp1 (tvrm124Tulp1) mutant mice model early onset retinal degeneration and have characteristically shorter OSs at P14 rd6Mfrp mutants. Homozygous null mutation of Rpe65 (tvrm148Rpe65) results in slow retinal degeneration and disorganized OS discs at P14 rd6Mfrp mice. These three retinal degeneration models show a similar extent of photoreceptor degeneration at P14  was used to control for antibody specificity. No RPE65 protein was detected in lysates from the Rpe65 mutant mice, whereas a 65 kDa protein was detected in all other samples  was observed in Prss56, a gene encoding a serine protease  and immune (Ctse and Ighm) function, were also upregulated . In the wild-type control at P14, confocal microscopy revealed a few cells in the retinal inner nuclear layer (INL) that showed expression of Prss56 transcript  and the level of significance . Differentially expressed probe sets  at and fold change greater than 2 are depicted in volcano plots in three pairwise comparisons. (A) B6 (C57BL/6J) P14 vs B6 (C57BL/6J) P0, (B) B6 (C57BL/6J) P14; rd6/rd6 (rd6Mfrp) P14 vs B6 (C57BL/6J) P0; rd6/rd6 (rd6Mfrp)P0 and (C) rd6/rd6 (rd6Mfrp) P0; rd6/rd6 (rd6Mfrp) P14 vs B6 (C57BL/6J) P0; B6 (C57BL/6J) P14.(TIF)Click here for additional data file.Figure S2Venn diagram depicting the overlapping and unique genes in the three data sets.rd6/rd6 (rd6Mfrp) P14 vs B6 (C57BL/6J) P14; rd6/rd6 (rd6Mfrp) P14 vs rd6/rd6 (rd6Mfrp)P0 and B6 (C57BL/6J) P14 vs B6 (C57BL/6J) P0.(TIF)Click here for additional data file.Figure S3Heat maps of differentially expressed genes in rd6/rd6Mfrp mice in comparison to age matched WT controls. (A) Upregulated genes in rd6/rd6 (rd6Mfrp) P14 vs B6 (C57BL/6J) P14, RFC>5.0, q<0.05 (B) Downregulated genes in rd6/rd6 (rd6Mfrp) P14 vs B6 (C57BL/6J) P14, RFC <−2.0, q<0.05 (C) Downregulated genes in rd6/rd6 (rd6Mfrp) P14 vs B6 (C57BL/6J) P14, RFC <−1.5 to −2.0, q<0.05. Asterisk denotes the genes that were validated by qRT-PCR analysis.(TIF)Click here for additional data file.Figure S4Outer segment degeneration at P14 in rd6Mfrp/rd6, tvrm124Tulp1/tvrm124, tvrm148Rpe65/tvrm148 mice compared to age matched controls visualized by light microscopy. Retinal sections at p14 were obtained from B6 (A), tvrm124Tulp1tvrm124/ (B) rd6Mfrprd6/ (C) and tvrm148Rpe65vrm148/t (D) and stained with hematoxylin & eosin. GC, gangalion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS, outer segments. Magnification: 20 x.(TIF)Click here for additional data file.Table S1Canonical pathways identified in rd6Mfrp mice.(DOCX)Click here for additional data file.Table S2Transcripts in retinal degeneration pathway that is downregulated in rd6Mfrp mutant mice at P14.(DOCX)Click here for additional data file.Table S3Transcripts in retinal degeneration pathway that is up-regulated in rd6Mfrp mutant mice at P14.(DOCX)Click here for additional data file.


Opioid receptors are currently classified as Mu (μ), Delta (δ), Kappa (κ) plus the opioid related nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP). Despite compelling evidence for interactions and benefits of targeting more than one receptor type in producing analgesia, clinical ligands are Mu agonists. In this study we have designed a Mu-NOP agonist named DeNo. The Mu agonist component is provided by dermorphin, a peptide isolated from the skin of Phyllomedusa frogs and the NOP component by the endogenous agonist N/OFQ.2+ in cells coexpressing recombinant receptors and a the chimeric protein Gαqi5, (ii) stimulate the binding of GTPγ[35S], (iii) inhibit cAMP formation, (iv) activate MAPKinase, (v) stimulate receptor-G protein and arrestin interaction using BRET, (vi) electrically stimulated guinea pig ileum (gpI) assay and (vii) ability to produce analgesia via the intrathecal route in rats.We have assessed receptor binding profile of DeNo and compared with dermorphin and N/OFQ. In a series of functional screens we have assessed the ability to (i) increase Cai; 9.55) and NOP  and with reasonable selectivity. This translated to increased Ca2+ in Gαqi5 expressing cells , increased binding of GTPγ[35S]  and receptor-G protein interaction in BRET . cAMP formation was inhibited and arrestin was activated . For MAPK DeNo activated p38 and ERK1/2 at Mu but only ERK1/2 at NOP. In the gpI DeNO inhibited electrically-evoked contractions (pEC50 8.63) that was sensitive to both Mu and NOP antagonists. DeNo was antinociceptive in rats.DeNo bound to Mu  and N/OFQ (NOP). This molecule behaves essentially as the parent compounds in vitro. In the antonocicoeptive assays employed in this study DeNo displays only weak antinociceptive properties. Naloxone was purchased from Sigma-Aldrich Co. . Naltrindole (Delta antagonist) and -enkephalin  were purchased from Tocris . Antibodies and protein ladders were purchased from Cell Signalling . All tissue culture media and supplements were obtained from Invitrogen .The reference molecules, dermorphin, Leu-enkephalin, Dynorphin-A and N/OFQ were synthesised in house . Tritiated UFP-101   in anhydrous tetrahydrofuran (3 ml), 2% hydrazine in methanol (5 ml) was added. The reaction mixture was stirred under argon for 30 min, then filtered and the resin washed 3 times with dimethylformamide  and CH2Cl2 (5 ml). To a stirred solution of 4-butanoic acid (0.2 mmol) in (DMF) (5 mL) at 0°C,  (HATU) (0.2 mmol) and 4-methylmorpholine (0.2 mmol) were added. After 10 min, [Lys(free side chain)8]dermorpin-resin  was added and the reaction mixture stirred at room temperature for 4 h. The solution was then filtered and the resin washed 5 times with DMF (5 ml) and 3 times with CH2Cl2 (5 ml). The protected [Lys8]dermorphin-resin was treated with reagent B (trifluoroacetic acid (TFA) / H20 / phenol / triisopropylsilane 88: 5: 5: 2; v/v; 10 mL / 0.2 g of resin) for 1.5 h at room temperature [18]N/OFQ-NH2 was reacted with [Lys8]dermorphin following a procedure reported in literature and then purified by preparative HPLC to give the desired final product after lyophilisation  assays. Membranes were centrifuged at 20374 g for 10 min at 4°C. This process was repeated at least three times. The resulting pellet was resuspended in an appropriate amount of the necessary buffer and the protein concentration was determined by Lowry assay -DPN  or ~0.8nM [3H]-UFP-101 (for CHONOP cells), as well as varying concentrations (10 μM-1pM) of the reference ligand DeNo. Non-specific binding was determined in the presence of 10 μM naloxone for CHOhMu, CHOhDelta and CHOhKappa or 1μM of N/OFQ for CHONOP cells. Samples were incubated for 1 hr at room temperature and reactions were terminated by vacuum filtration, onto PEI-soaked Whatman GF/B filters, using a Brandel harvester. The concentration of displacing ligand producing 50% displacement was corrected for the competing mass of radioligand to yield pKi, a measure of its affinity . Varying concentrations of reference ligands  or DeNo (1 pM—10μM) was added prior to incubation. Non-specific binding was determined in the presence of unlabeled GTPγS (10μM). Samples were incubated at 30°C for 1 h with gentle agitation. Reactions were terminated by vacuum filtration through dry Whatman GF/B filters, using a Brandel harvester . For Mu; dermorphin and DeNo were added at 1μM concentrations. At NOP; N/OFQ, and DeNo were added at 1μM concentrations. Signalling was terminated via Lysis Buffer  followed by centrifugation , with the supernatant removed and added to an equal volume of 2XSDS buffer . Samples were denatured (heated 100°C for 5 minutes) and separated by 10% SDS-PAGE; transferred onto nitrocellulose paper in a semi-dry buffer  and blocked using conventional western blotting techniques. To detect phosphorylated ERK1/2 and phosphorylated p38 activity, pERK1/2 antibodies (1:6000 dilution) and p38 antibodies (1:3000dilution), diluted in TBS-T solution  with 0.01% (wt/vol) BSA, were used to probe the membrane and left overnight at 4°C. Horseradish peroxidase-conjugated secondary anti-rabbit antibodies  were used to visualise immune-reactive bands, followed by chemiluminescence detection using the ChemiDoc™ MP Imaging System (Bio-Rad). In order to ensure equal gel loading, membranes were stripped and reprobed for total ERK1/2 and total p38 MAPK. Membranes were incubated in Restore Plus™ (Fisher) for 15 minutes then thoroughly washed in TBS-T, following which they were blocked as previously described. The membrane was then probed using the specific antibody for ERK 1/2 , or p38 , overnight (4°C) followed by addition of the secondary antibody and chemiluminescence detection. Normalisation of total protein levels for each sample was then achieved by representing levels of pERK1/2 and p-p38 as a proportion of total ERK1/2 or total p38 protein. Data were analyzed using the “Origin 9” software and images analysed using Image Lab software  -DPN in a concentration dependent and saturable manner (i 9.55) demonstrated a significant increase in affinity at Mu, when compared to the parent compound dermorphin (8.69). At CHOhNOP, DeNo displaced [3H]-UFP-101 in a concentration dependent and saturable manner   demonstrated an increase in affinity compared to its parent compounds. Dermorphin displayed an affinity of 7.17, while N/OFQ failed to displace [3H]-DPN at the Delta receptor. Furthermore, DeNo (7.34) showed affinity for the Kappa receptor, whereas the parent compounds (dermorphin and N/OFQ) failed to displace [3H]-DPN at this receptor (In displacement binding studies at CHOe manner . DeNo (pe manner . DeNo (1 (10.69) , Table 1receptor , Table 1qi5 chimeric protein and the human Mu receptor, dermorphin produced a concentration-dependent stimulation of calcium mobilisation with high potency (pEC50 8.07) and maximal effects . DeNo showed similar maximal effect  but approximately 10-fold lower potency (pEC50 7.17)  and maximal effect . DeNo showed similar potency (pEC50 9.69) and maximal effect  to N/OFQ (In CHO cells stably expressing the Gα50 7.17) . In CHOhto N/OFQ .35S] in a concentration dependent and saturable manner at the Mu receptor (max 2.68) demonstrated a similar maximal response to that of dermorphin (2.63). The pEC50 values for DeNo (7.77) showed no significant difference to that of the parent compound, dermorphin (7.83)  similar to that of its parent compound, N/OFQ (2.57). The pEC50 value of 9.50 achieved by DeNo was similar to that of N/OFQ (9.05). (35S] in a concentration dependent and saturable manner in membranes expressing Delta receptors (max 1.84) produced a maximal response similar to that of the endogenous Delta peptide, Leu-enkephalin (1.90). However, the pEC50 value for DeNo (6.78) was significantly lower than that of Leu-enkephalin (8.50)  produced a maximal response similar to that of dynorphin-A (2.33). The pEC50 value of DeNo (5.91) was significantly lower than that of dynorphin-A (9.36) (Dermorphin and DeNo stimulated the binding of GTPγ) and downstream (cAMP) assays and in Ca2+ mobilisation experiments using chimeric G-protein constructs DeNo was a full agonist at both Mu and NOP. In BRET assays to assess receptor G-protein interaction and arrestin recruitment, DeNo also behaved as a full agonist. Moreover, DeNo activated ERK1/2 as a full agonist at Mu and NOP; there was no activation of p38 at NOP. In a more intact preparation DeNo inhibited contraction of the electrically stimulated gpI via simultaneous activation of NOP and Opioid receptors and was antinociceptive via the i.t. route in rats.In this study we have characterised a novel peptide bivalent Mu-NOP ligand, DeNo; created by combining dermorphin and N/OFQ. DeNo bound to Mu and NOP receptors and was ~5fold NOP selective. There was between 1 and 2 logs selectivity over Delta and Kappa receptors. At conventional upstream  and 331 fold at the chimeric G-protein (downstream). In contrast at NOP there was a 7 and 3 fold difference in affinity compared to potency. As the point of interrogation (assay) moved further down the signal transduction cascade the difference in potency at Mu and NOP increased. For example in GTPγ[35S] assay DeNo was 38 fold more potent at NOP and 331 fold more potent in the chimeric G-protein assay. In all assays DeNo was a full agonist. If there was amplification or a coupling reserve then the potency values should shift leftwards rather than rightwards. This rightward shift was more marked at Mu than NOP and could be explained by buffer composition and end point. It is well documented that the chimeric G-protein assay suffers from hemi-equilibrium problems and we have discussed this in detail previously N/OFQ(1–13)-NH2. DeNo may be a useful tool, particularly in vitro for investigating the consequences of the simultaneous activation of NOP and mu receptors.In conclusion we have created a novel bivalent Mu-NOP peptide agonist by combining dermorphin (Mu) and N/OFQ (NOP); this molecule behaves essentially as the parent compounds. Despite this promising profile, analgesic actions of DeNo are poor in the model employed here. As a mixed molecule, this ligand represents a useful addition to the non-peptides BU08028 , 54, the