Looptail mutant mouse, which harbors a missense mutation in the vangl2 gene, have been essential for studies of planar polarity and linking the function of the core planar cell polarity proteins to other developmental signals. Originally described as having dominant phenotypic traits, the molecular interactions underlying the Looptail mutant phenotype are unclear because Vangl2 protein levels are significantly reduced or absent from mutant tissues. Here we introduce a vangl2 knockout mouse and directly compare the severity of the knockout and Looptail mutant phenotypes by intercrossing the two lines and assaying the planar polarity of inner ear hair cells. Overall the vangl2 knockout phenotype is milder than the phenotype of compound mutants carrying both the Looptail and vangl2 knockout alleles. In compound mutants a greater number of hair cells are affected and changes in the orientation of individual hair cells are greater when quantified. We further demonstrate in a heterologous cell system that the protein encoded by the Looptail mutation (Vangl2S464N) disrupts delivery of Vangl1 and Vangl2 proteins to the cell surface as a result of oligomer formation between Vangl1 and Vangl2S464N, or Vangl2 and Vangl2S464N, coupled to the intracellular retention of Vangl2S464N. As a result, Vangl1 protein is missing from the apical cell surface of vestibular hair cells in Looptail mutants, but is retained at the apical cell surface of hair cells in vangl2 knockouts. Similarly the distribution of Prickle-like2, a putative Vangl2 interacting protein, is differentially affected in the two mutant lines. In summary, we provide evidence for a direct physical interaction between Vangl1 and Vangl2 through a combination of in vitro and in vivo approaches and propose that this interaction underlies the dominant phenotypic traits associated with the Looptail mutation.Experiments utilizing the  Drosophila wing. Taken together, several recent studies indicate that Fz and Dvl proteins form a complex on one side of the hair cell that is opposite to Vangl and Pk proteins on the other Drosophila, vertebrate PCP proteins also coordinate planar polarity between cells so that, as in the case of hair cells, the stereocilia bundles of neighboring cells are oriented in the same direction.Planar polarity is the polarized organization of cells and cellular structures within the plane of a tissue, perpendicular to the apical-basolateral cell axis vangl2 (Entrez Gene ID: 93840) mutant line Looptail (Lp)Lp is a missense mutation of vangl2 resulting in a Serine to Asparagine substitution at amino acid position 464, which is located in the cytoplasmic domain of the protein. In Lp/Lpvangl2 mutants, hair cell stereocilia are properly formed and polarized, yet individual hair cells are frequently misoriented relative to neighboring cells in the cochlea and utricle vangl1 (Entrez Gene ID: 229658) and vangl2 demonstrating functional redundancy of the two homologous genes Lp/Lpvangl2 mutants have craniorachischisis, which is a severe neural tube defect (NTD). The iconic looped tail of the Lp line is due to a milder neural tube phenotype in heterozygous mice. Craniorachischisis is a class of NTD specifically resulting from disrupted planar polarity during convergent extension of the embryo prior to neurulation, and is a common feature of vertebrate PCP mutants. Other mouse lines that develop craniorachischisis and hair cell planar polarity defects include fz3/6 double knockouts CELSR mutants spin cycle and crashscribble mutants ptk7 knockout mice vangl1 and vangl2 have been linked to neural tube defects in humans An early demonstration the functional significance of planar polarity and PCP signaling mechanisms in hair cells came from analysis of the Lp mouse has served as an important animal model for studying vertebrate planar polarity Lp line classified the Lp mutation as having a partially penetrant and dominant phenotype ex vivo, heterozygous tissue develops pathologic characteristics similar to mutant tissue Lp mutation was mapped to vangl2S464N protein stability and expression in Lp/Lpvangl2 mutants Lp as a null allele with a heterozygous phenotype resulting from haploinsufficiency. The Lp line has implicated PCP signaling in a number of developmental events such as axon pathfinding not originally associated with planar polarity Lp and other mutant lines suggest that other signaling pathways, including those functional in cilia, incorporate Vangl2 Lp mutation.The Lp mutation inhibits Vangl2 function by disrupting Vangl2 protein trafficking through the endoplasmic reticulum (ER). Consistent with this hypothesis, mutations in sec24b, which encodes an ER transport protein that is required for Vangl2 movement to the plasma membrane, result in craniorachischisis similar to LpS464N is not loaded into ER vesicles indicating that the mutated region of the Vangl2 cytoplasmic domain may be required for sorting by Sec24b. Furthermore, a second vangl2 point mutant (m1Jus) fails to enter ER vesicles m1Jus/m1Jusvangl2 mice have craniorachischisis similar to Lp/Lpvangl2in vitroLp/WTvangl2 mice is due to haploinsufficiency or if it is a partially penetrant, dominant phenotype as described in the original characterization of the line.One explanation is that the vangl2 knockout mice is more severe than the Lp and m1Jus mutations because remaining elements of vangl2 are not sufficient to encode a membrane spanning protein. Using the sensory hair cells of the inner ear, we compared the mutant phenotype of vangl2 knockout and Lp mutant mice to establish the phenotypic nature of the Lp mutation during hair cell development.We have generated a complementary mouse line in which a single exon encoding the Vangl2 transmembrane domains is disrupted by LoxP site addition and subsequent excision by Cre recombinase. At the chromosomal level, the mutation in these vangl2 knockout (KO) mouse was generated and compared to the established Looptail (Lp) mouse line to determine the effect of the S464N substitution on Vangl2 function. The vangl2 gene was modified by homologous recombination in ES cells to introduce tandem LoxP sites flanking exon 4, which encodes the four Vangl2 transmembrane domains  confirms a loss of Vangl2 protein in ΔTMs/ΔTMsvangl2 embryos . Therefore ΔTMsvangl2 is a complete null allele.To further evaluate Vangl2 function during sensory hair cell development, a  domains . Accurat domains . Followi domains . Since t embryos . A VanglΔTMsVangl2 mice were backcrossed to C57Bl6 for at least 5 generations before experimental intercrosses. On this genetic background the majority of ΔTMs/ΔTMsvangl2 mutants exhibited craniorachischisis, a severe NTD  present only with spina bifida, a milder NTD restricted to the posterior neural tube adjacent to the tail (ΔTMs/WTvangl2 mice (11/99) had the tail loops or kinks that are characteristic of the Lp line. Tail phenotypes were never observed in ΔTMs/WTvangl2 mice backcrossed for a minimum of 5 generations to the FVB (0/66 heterozygotes) or strain A/J (0/15 heterozygotes) inbred lines. Finally in some ΔTMs/ΔTMsvangl2 KOs, incomplete or failed eyelid closure was observed that was similar in appearance to that described for other PCP mutants ΔTMs/ΔTMsvangl2 mice than in Lp/Lpvangl2 mutants.vere NTD  that is the tail . SimilarΔTMs/ΔTMsvangl2 phenotype was assayed in greater detail in the inner ear because the sensory hair cells have distinct planar polarity that is readily visualized and quantified for direct comparison with the Lp mutant phenotype. The apical surface of a hair cell extends a bundle of stereocilia that is organized in a staircase pattern with the tallest stereocilia adjacent to single kinocilium. Planar polarity is apparent in the shared stereocilia∶kinocilium polarity of neighboring cells  of individual hair cells from all embryos using circular histograms  were grouped and analyzed together as the middle rows  Together these experiments suggest that the presence of the Vangl2ΔTMs/Lpvangl2 mutants than ΔTMs/ΔTMsvangl2 KOs is that altered Vangl2S464N trafficking disrupts the distribution of other polarity molecules. A likely candidate is Vangl1 because Vangl1 expression overlaps with Vangl2, and enhanced planar polarity defects are seen in mice lacking both vangl1 and vangl2DrosophilaS464N or Vangl2D255E may disrupt Vangl1 or Vangl2 delivery to the plasma membrane. This hypothesis was tested using a heterologous system in which hemagluttinin (HA) tagged Vangl2 constructs were co-expressed with EGFP-tagged Vangl1 or Vangl2. Specifically these experiments utilized 3XHA-tagged Vangl2, 3XHA-Vangl2S464N and 3XHA-Vangl2D255E constructs characterized by Merte et al. One explanation for the stronger phenotype that occurs in S464N or 3XHA-Vangl2D255E constructs were found predominantly in cytoplasmic compartments , the Vangl1 antibody appears specific for Vangl1 because there are no differences in Western blot analyses between protein lysates collected from WT and ΔTMs/ΔTMsvangl2 embryos  was assayed in ir cells , arrows.ir cells . Asymmetely lost  similar vangl2 knockout line and intercrossing those mice with Lp mutants, we have demonstrated the dominant nature of the Lp mutant phenotype during inner ear development. This was completed through a detailed quantification and comparison of the planar polarity phenotypes of hair cells in ΔTMs/Lpvangl2 and ΔTMs/ΔTMsvangl2 mice. In addition we propose a mechanism for the dominant effect of the Lp mutation in which disrupted trafficking of Vangl2S464N alters the distribution of Vangl1, Vangl2 and Pk2. Together these findings will guide interpretation of studies employing the Looptail line, particularly when Looptail mice are used for molecular dissections of the PCP signaling pathway.By generating a novel S464N is due to disrupted trafficking of the mutant protein and its retention in the ER S464N with Vangl1 in a heterologous system also prevents Vangl1 delivery to the cell surface. Since vangl1 and vangl2 regulate planar polarity in many tissues including the inner ear ΔTMs/ΔTMsvangl2 vestibular hair cells that maintain apical localization of Vangl1 than in Lp/Lpvangl2 hair cells where apical Vangl1 is lost. We also demonstrate that Vangl2 forms oligomers with Vangl1 and Vangl2 in heterologous cells. This is similar to findings from Drosophila where Van Gogh oligomerizes in vivoS464N protein encoded by the Lp mutation exerts dominant effect on planar polarity by inhibiting the membrane trafficking and localization of Vangl1 and Vangl2, likely leading to their degradation.At a molecular level, the decreased protein stability and cell-surface presentation of Vangl2Lp/WTvangl2 heterozygotes if the presence of Vangl2S464N reduces but does not eliminate all Vangl1/2 protein from the cell surface. By comparison the uterine epithelium of the female reproductive track (FRT) is more sensitive to the Lp mutation than the neural tube. In a recent study where embryonic FRT tissues were cultured in vitro, similar developmental deficits were observed explants from Lp/WTvangl2 and Lp/Lpvangl2 embryos, further demonstrating the dominant effect of LpLp/WTvangl2 heterozygotes that was restricted to OHC3 and was only present in the less developed apical turns  and coding sequence exons located within the homologous arms were sequenced to check for PCR induced mutations. Following electroporation into the TC1 ES cell line and positive selection with G418, homologous recombination was validated in surviving clones by PCR and Southern blot . The vector contained a 4.2 kb 5′ arm that was modified by inserting LoxP and Hind3 sequences into a unique Xmn1 restriction site located in the intron upstream of ern blot . A singlACTB-FlpE) to remove the NeoR gene. The resulting allele (LoxP vangl2) was not viable when homozygosed because the positions of the remaining LoxP sites disrupted vangl2 expression. As a result, LoxPvangl2 was crossed to transgenic mice ubiquitously expressing Cre recombinase (ACTB-Cre) to permanently delete exon 4 in the germ line and produce the ΔTMsvangl2 knockout line.FlpE recombinase . C57Bl6 and FVB mice were purchased from Charles River Laboratories  and the ACTB-FLPe mice were obtained from S. Dymecki .For general colony maintenance, ΔTMsVangl2 mice were genotyped using a common 5′ primer (5′ATCACCTCACTTGGCTGGAATAGATG 3′) paired with 3′ primers that were either KO-specific (5′GAAGTTATAAGCTTTGTTCCAG 3′) or WT-specific (5′GGCGAATGGGAGAAAGGCAGAC 3′). Following ΔTMsvangl2 and Lp experimental intercross, the Lp mutation was genotyped by PCR amplification with primers flanking the mutation followed by DNA purification and sequencing of the amplified product. Lp genotyping primers are (5′ATATTTGGCTGCTGGACCCACCATCC3′) and (5′TGCAGCCGCATGACGAACTTATGTGA3′).Immunofluorescent labeling of auditory and vestibular hair cells was completed using E18.5 inner ears fixed for 2 hours in a solution of 4% paraformaldehyde prepared in Sorenson's phosphate buffer (pH7.4). Utricles and cochleae were subsequently removed, dissected to expose the surface of the sensory epithelia, permeabilized and blocked using blocking solution  supplemented with Triton X-100 to 0.5%. Primary antibodies and phalloidin Alexa488 (Invitrogen A12379) were diluted in blocking solution supplemented with Tween-20 to 0.1%, and incubated with the tissue overnight at 4°C. Tissue was washed thoroughly with PBS-T  followed by incubation with species-specific, Alexa Fluor (Invitrogen) or DyLight (Jackson ImmunoResearch) conjugated secondary antibodies. Tissue was subsequently washed with PBS-T, mounted using Fluoro-Gel (Electron Microscopy Sciences), and imaged using a Zeiss LSM510 confocal microscope. Image frames were limited to 1.5 microns in the Y-dimension and a stack of images 3–6 microns containing the stereocilia and apical surface of the hair cells was collected. A single image was generated by Z-projection using maximum or averaged pixel intensities. For preparations in which the striola region was marked using Gata3 antibodies, a second stack positioned deeper in the tissue was necessary to image Gata3 positive nuclei. The following commercial antibodies were used in this study: Actin (Millipore mAB1501), GFP (Invitrogen A11122), Gata3 (R&D AF2605), HA (Covance MMS-101P),Acetylated Tubulin (Sigma T7451), Vangl1 (Sigma A36455), Vangl2 (Santa Cruz SC46561), Oncomodulin (Santa Cruz SC7446), Pericentrin (Covance PRB432C). The Pk2 antibody was a gift from M. Scott and J. Axelrod (Stanford Univ.) and has been previously characterized For auditory hair cell analyses the organ of Corti was imaged by confocal microscopy at three positions corresponding to 25%, 50% and 75% of the length of the cochlea measured from the base. The orientation of individual hair cells was measured using the ImageJ (NIH) angle measurement tool. The short arm of each angle was drawn from the pericentrin labeled kinocilium and across the center of the hair cell. The long arm of each angle was drawn parallel to three adjacent hair cells within the same row. This yields a measurement range of 0 to 180 degrees in which a perfectly aligned hair cell has a raw measurement of 90 degrees. For those cells determined to have reversed bundles  the orientation was calculated with the formula x = (360-y) where x is orientation and y is the measured angle. This calculation and additional analyses were completed using Microsoft Excel. These absolute measurements of auditory hair cell orientation were assembled as a circular histograms using Oriana circular graphing software (Kovach Computing Services). Planar polarity phenotypes were further quantified by averaging the mean absolute deviation of hair cell polarities from an arbitrary reference (defined as 0°) for each animal as illustrated in For vestibular hair cell analyses the utricular maculae was imaged by confocal microscopy at two positions spanning the Gata3-positive or Oncomodulin-positive striola and images were combined based upon regions of overlap. As outlined in ΔTMs/ΔTMsvangl2 KOs, only KOs with craniorachischisis were included in measurements of hair cell planar polarity. All ΔTMs/Lpvangl2 and Lp/Lpvangl2 mutants collected had craniorachischisis. Hair cells in which the bundle polarity or pericentrin labeling could not be visualized were not included in the auditory or vestibular analyses.See 7 cells with 30 ug plasmid DNA  in a 2 mm gap electroporation cuvette, followed by electroporation at 260 V with 950 uF capacitance and 25 Ohm resistance using a BTX ECM630 electroporation box. MDCK cells were plated into single wells of a Lab-Tek II 8-well Chamber Slide (Thermo Scientific) and incubated 72 hours to allow adherence junctions to form at cell boundaries before the distribution of EGFP-tagged proteins was assayed by immunofluorescent labeling. HEK293 cells were transfected using Lipofectamine reagent as per the manufacturer's recommendation, cultured for 24–48 hours and harvested for Western blot analysis. For these experiments a plasmid expressing EGFP-Vangl1 was generated by PCR amplification from mouse cDNA and cloned into the pEGFP-C1 vector (Invitrogen) to generate an amino-terminus EGFP fusion. EGFP-Vangl2 MDCK cells (SIGMA 84121903) were grown in EMEM supplemented with 2 mM L-Glutamine, 1× non-essential amino acids, 10% FBS and 1× Penn/Strep. HEK293 cells (American Type Culture Collection ATCC-CRL-1573) were grown in DMEM, 10% FBS and 1× Penn/Strep. MDCK cells were transfected via electroporation, by combining 1×10For labeling, MDCK cells were fixed with 4% paraformaldehyde prepared in PBS, permeabilized and blocked as described previously. Cells were immunolabeled with anti-HA and anti-GFP antibodies diluted in 1% BSA/PBS followed by Alexa Fluor conjugated secondary antibodies, and then examined by fluorescent microscopy using a Nikon E600 compound microscope. For protein localization assays only cells expressing both HA-tagged and EGFP-tagged constructs were evaluated, and the distribution of EGFP-tagged proteins were scored as membrane associated, cytoplasmic or present in both locations.Protein lysates were prepared from tissues, MDCK or HEK293 cells using a lysis buffer consisting of 25 mM Tris pH 7.4, 1% Triton X-100, 50 mM NaCl and 1× protease inhibitor cocktail (SIGMA P8340) and quantified by UV absorption at 280 nm using a NanoDrop. Following SDS-PAGE electrophoresis proteins were transferred to nitrocellulose filters and blotted with standard Western blot techniques using anti-GFP or anti-HA antibodies followed by chemiluminescent detection using BioRad Immun-Star HRP substrate. Immunoprecipitations were completed using Nynal protein G beads (Invitrogen) bound to rabbit anti-GFP or mouse anti-HA antibodies and combined with protein lysates from electroporated MDCK cells. Prior to immunoprecipitation, levels of recombinant protein expression were determined by Western blot analysis and equivalent amounts of recombinant protein were used from each electroporation condition. Target antigens were absorbed at 4°C, and washed using the manufacturers recommended protocols and solutions, then eluted from the beads using Laemmli 2× Gel-loading Buffer with DTT prior to denaturing SDS-PAGE and conventional Western blot analysis.Figure S1Gata3 and Oncomodulin immunolabeling marks the position of the striola in developing mouse utricle. (A) The transcription factor Gata3 (red) is expressed by multiple cells types located in the striola region of the developing utricle and Gata3 immunolabeling can be used to visualize this region. (B) The calcium binding protein Oncomodulin (red) is expressed exclusively by type1 hair cells located in the striola, and Oncomodulin immunolabeling can also be used to visualize this region. (A–B) Phalloidin (green) was used to assay stereocilia bundle orientation (arrows) and map the position of the line of polarity reversal . In mouse, the LPR is located along the lateral border of the striola.(TIF)Click here for additional data file.Figure S2Ectopic expression of tagged-Vangl1 or Vangl2 proteins does not disrupt cell junctions between adjacent MDCK cells. (A–E) The proper formation of adherens junctions between adjacent MDCK cells was determined by immunofluorescent labeling of endogenous E-cadherin (red) following electroporation with EGFP-Vangl1 , EGFP-Vangl2 , 3XHA-Vangl2 , 3XHA Vangl2D255E  or 3XHA Vangl2S464N  constructs. Asters mark transfected cells. Consistent with experiments utilizing E-Cadherin-EGFP Click here for additional data file.Figure S3Specificity of anti-Vangl1 antibody evaluated by Western blot. (A) Western blot against protein lysates from embryonic tissues demonstrate that the anti-Vangl1 antibody does not cross react with endogenous Vangl2 protein. Blots show a prominent band at the predicted 70 Kd mass in Wt and ΔTMs/WTvangl2 lysates and the labeling pattern is not altered in ΔTMs/ΔTMsvangl2 mutant tissue. Blots for Tubulin were used as a loading control. (B) Western blot against cell lysates from HEK293 cells transfected with EGFP-Vangl1 or EGFP-Vangl2 reveal that the anti-Vangl1 antibody can cross-react with recombinant Vangl2 protein when over-expressed. Serial dilutions of transfected cell lysates show the relative affinity of the anti-Vangl1 antibody and demonstrate that it can detect a 10-fold lower concentration of EGFP-Vangl1 than EGFP-Vangl2. The amount of total protein in each lysate was determined by UV absorption measured with a NanoDrop and equivalent amounts were loaded into each lane. The amount of total protein lysate per lane is indicated.(TIF)Click here for additional data file.Table S1Sample size summary for planar polarity phenotypic analysis. The number of experimental and control animals used for quantification of the averaged mean deviation of bundle orientations in vestibular assays and auditory assays completed at each of the three positions along the length of the cochlea. Different numbers of specimen were available at each location because some were excluded based upon dissection or labeling artifacts.(PDF)Click here for additional data file.


Young women with autoinflammatory diseases on long term IL-1 blockade are increasingly asking about the feasibility, safety and outcomes of pregnancy but few data are available. The FDA classes anakinra as pregnancy risk grade B and rilonacept and canakinumab as grade C. The manufacturers advise that there are no data on the outcome of pregnancy or on excretion into breast milk and that anakinra and canakinumab should only be taken if the benefits outweigh risk; for rilonacept the advice is to avoid in pregnancy. The literature contains only 3 reported cases of use of anakinra in pregnancy – all in adult onset Stills disease (AOSD) and with successful outcomes.To assess pregnancy outcomes in women who had received anti IL-1 therapies in pregnancy.We indentified women who have been exposed to anti IL-1 agents in completed or planned to complete pregnancies under our care. Data were collected on medication, pregnancy outcome, breast feeding and development.7 cases were identified; 5 completed, 1 first and 1 second trimester pregnancies. The underlying diseases were: 3 CAPS, 1 TRAPS, 1 FMF, 1 idiopathic pericarditis and 1 AOSD. 3 completed pregnancies  were on anakinra from preconception throughout the pregnancy, the 2 current pregnancies (CAPS) were on canakinumab pre conception, 1 switched to anakinra 8 weeks pre conception, the other stopped canakinumab 8 weeks after conception and currently (12 weeks later) is on no treatment. The patient with FMF also had multiple sclerosis with presumed prolonged febrile myalgia refractory to colchicine for which she received 12 weeks of anakinra from 22 weeks. The AOSD patient received anakinra and prednisolone from 22 to 33 weeks. Median maternal age was 30 years (25-38), all were first pregnancies. The 5 completed pregnancies resulted in 5 healthy boys; median gestation 38 weeks (35 to 41). One baby was delivered by caesarean section at 36 weeks for vaginal bleeding (FMF), the others were vaginal deliveries (2 induced); median birth weight 2.59 kg (2.02-3.94), 1 minute APGAR score was 8 in 1 case and 9 in the rest. All were normal on neonatal checks, one had evidence of unilateral reduced hearing at 6 weeks. One was breast fed, the others bottle fed. Follow up data is available on 3 beyond 6 months and their development remains normal.These 5 successful pregnancies more than double the number of known outcomes in anakinra treated mothers and provide reassurance to physicians caring for young women. Nonetheless the numbers remain very small and each pregnancy should be assessed and the risks and benefits of continued therapy individually discussed with the potential parents.None declared


The desmosome anchors keratin filaments in epithelial cells leading to the formation of a tissue wide IF network. Loss of the desmosomal plaque protein plakophilin3 (PKP3) in HCT116 cells, leads to an increase in neoplastic progression and metastasis, which was accompanied by an increase in K8 levels. The increase in levels was due to an increase in the protein levels of the Phosphatase of Regenerating Liver 3 (PRL3), which results in a decrease in phosphorylation on K8. The increase in PRL3 and K8 protein levels could be reversed by introduction of an shRNA resistant PKP3 cDNA. Inhibition of K8 expression in the PKP3 knockdown clone S10, led to a decrease in cell migration and lamellipodia formation. Further, the K8 PKP3 double knockdown clones showed a decrease in colony formation in soft agar and decreased tumorigenesis and metastasis in nude mice. These results suggest that a stabilisation of K8 filaments leading to an increase in migration and transformation may be one mechanism by which PKP3 loss leads to tumor progression and metastasis. Changes in cytoskeletal architecture and cell-cell adhesion are often observed in cells undergoing neoplastic transformation. Desmosomes are adherens type junctions that are required for cell-cell adhesion, especially in tissues that experience mechanical stress and anchor intermediate filaments (IF’s) leading to the generation of a tissue wide IF network (reviewed in The aberrant over-expression of K8 and K18 has been observed in a number of squamous cell carcinomas irrespective of their origin Metastasis in colon cancer often correlates with an increased expression of the Phosphatase of Regenerating Liver -3 (PRL-3) Plakophilin3 (PKP3) is a desmosomal plaque protein that belongs to the p120 catenin sub family of the armadillo family of proteins and is found in desmosomes in most epithelial tissues with the exception of hepatocytes Previous work from this laboratory has demonstrated that PKP3 loss in HCT116 cells leads to increased neoplastic progression and metastasis The two clones, S9 and S10, are generated using two different shRNA’s suggesting that the phenotypes observed are probably not due to off-target effects of the shRNA To determine if an increase in RNA levels led to the increase in K8 protein in the PKP3 knockdown clones, a reverse transcriptase coupled Real Time PCR analysis was performed. The mRNA levels of K8 and K18 in the PKP3 knockdown clones were not significantly altered as compared to the vector control (pTU6) , therefoTwo major sites of phosphorylation, S73 and S431, have been reported in K8 To further demonstrate that the change in migration and stability of K8 was due to changes in phosphorylation in K8, two phospho-site mutants (S73A and S431A) It has been reported previously that an increase in PRL3 levels leads to a decrease in K8 phosphorylation and an increase in K8 levels To determine whether inhibition of PRL3 could cause a decrease in the levels of K8 in the PKP3 knockdown clones, the cells were treated with either the vehicle control or increasing concentrations of PRL3 inhibitor. The levels of K8 decreased upon inhibition of PRL3 in the vector control and PKP3 knockdown clones suggesting that an increase in phosphorylation of K8 could account for the decrease in K8 stability . HoweverTo determine if there was a change in PRL3 localization in the cell or increased PRL3 localization on K8 filaments upon PKP3 knockdown, the vector control and knockdown cells were stained with antibodies to PRL3. As shown in To determine whether PRL3 shows an increased localization to K8 filaments upon PKP3 knockdown, HCT116 cells were transfected with a GFP-tagged PRL3 construct and stained with antibodies to PRL3. As shown in To determine the effects of K8 phosphorylation on filament formation, the amount of K8 in the soluble and insoluble fractions was measured in the vector control and PKP3 knockdown clones. Keratin solubility assays demonstrated that all the K8 was present in the insoluble fraction in both the vector control and PKP3 knockdown clones, even when a 100 µg of the soluble fraction was loaded . FurtherTo determine whether the elevated K8 levels were required for transformation upon PKP3 loss, we inhibited K8 expression using RNA interference using previously described shRNA constructs The PKP3 knockdown cells show a decrease in cell-cell adhesion and desmosome size and an increase in cell migration An increase in cell migration is often accompanied by an increase in actin dynamics and the formation of actin dependent structures such as lamellipodia and filopodia  showed high levels of K8 as compared to primary tumors derived from the double knockdown clones (8.21 and 8.28) . PrimaryLoss of the desmosomal plaque protein plakophilin3, leads to an increase in tumor progression and metastasis PKP3 loss leads to an increase in metastasis to the lungs . K8 lossAnother possible explanation for why an increase in K8 levels leads to increased metastasis might be the effect K8 has on cell migration. Loss of K8 in the PKP3 knockdown clones results in an inhibition of migration, reversing the phenotype observed upon PKP3 loss The GFP tagged phosphosite mutants show an increase in stability in comparison to the GFP tagged WTK8. These results suggest that the increase in stability observed upon PKP3 knockdown is not due to an increase in translation, given that the WT and mutant proteins are being expressed from a heterologous promoter with different 5′ and 3′ untranslated regions from the endogenous K8 gene. This is important because plakophilin family members, including PKP3, have been shown to regulate translation PRL3 has been reported to dephosphorylate K8 at both S73 and S431 It has been reported previously that phosphorylation of keratins leads to an increase in solubility and filament network disassembly The requirement for an intact filament network for transformation is supported by the data that knockdown of K18 does not phenocopy K8 knockdown, which might be due to the fact that another type I keratin is present in these cells . The incThe results described herein suggest that PKP3 loss leads to alterations in phosphorylation on K8, due to an increase in PRL3 levels. These result in increased K8 levels which promote increased migration, transformation and metastasis. Loss of K8 in the PKP3 knockdown clones leads to a reversal of these phenotypes, suggesting that an increase in K8 levels is required for the neoplastic progression and metastasis induced upon PKP3 loss. These results in conjunction with our data on the levels of phosphorylated keratin in human oral squamous cell carcinoma ad libitum. Mice were housed in the Individually Ventilated Cage (IVC) system  provided with autoclaved rice husk bedding material available locally. Protocols for the experiments were approved by the Institutional Animal Ethics Committee (IAEC) of the Advanced Centre for Treatment Reserach and Education in Cancer (ACTREC). The title of the project is “Mechanisms underlying tumor progression upon PKP3 loss in epithelial cells.” The project approval number is 13/2007 and approval was granted on the 27th of July, 2007.Maintenance of the animal facility is as per the national guidelines provided by the Committee for the Purpose of Control and Supervision of the Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government of India. The animals were housed in a controlled environment with the temperature and relative humidity being maintained at 23±2°C and 40–70% respectively and a day night cycle of 12 hrs each . The animals were received an autoclaved balanced diet prepared in-house as per the standard formula and sterile water The cloning of K8 shRNA constructs and the shRNA resistant K8 (GFP K8 Res) has been described elsewhere HCT116 cells, an epithelial cell line derived from a human colorectal carcinoma, were obtained from Dr. Bert Vogelstein  and ATCCThe high salt extraction for keratins was performed as previously described The primary antibodies for PKP3 , β Actin , K8 , K18 , phosphospecific antibodies for K8 Serine 73  or Serine 431 , p38 , rac  rhoA  and PRL3 (Abcam 1∶500) were used for Western blot analysis. Respective secondary antibodies were used at a dilution of 1∶1000 (Invitrogen) or 1∶5000 (Pierce). Protein samples were resolved on a polyacrylamide gel by SDS PAGE and then transferred to a nitro cellulose or PVDF membranes and processed for Western blot analysis as described The HCT116-derived PKP3 knockdown clone S9 and S10 and the vector control were grown on glass bottom plates and co-transfected with constructs expressing YFP K18 and CFP K8. 24 hours post transfection the cells were fed with DMEM lacking phenol red and placed on ice for 10 minutes prior to FRET to minimize mobility of the cells. FRET measurements were done using the acceptor photobleaching method in live cells efficiency = (Dpost−Bpost)−(Dpre−Bpre)/(Dpost−Bpost).FRET The nomenclature and equations for FRET calculations and the FRET protocol was obtained from the Centre for Optical Instrumentation Laboratory, Wellcome Trust Center for Cell Biology, University of Edinburgh.Wound healing assays were performed as previously described Staining for K8 was performed as described The knockdown clones were trypsinized and counted. 2,500 cells were plated in 0.4% soft agarose and the cells were maintained in the presence of the relevant antibiotic as previously described 6 cells of the HCT116 derived PKP3 knockdown or K8 PKP3 double knockdown clones were resuspended in DMEM medium without serum and injected subcutaneously in the dorsal flank of 6–8 weeks old NMRI Nude (Nu/Nu) 3) was calculated by the formula ½ LV2 where L is the largest dimension and V its perpendicular dimension, as previously reported Nude mice experiments were performed as described Four weeks after subcutaneous injection, the mice were sacrificed and the tumor and lung tissues were fixed in 10% formaldehyde (SIGMA) overnight and processed for histology. Five micron sections of paraffin embedded tissue were prepared and hematoxylin/eosin staining and immunohistochemical staining was performed according to standard methods Genomic DNA was purified from paraffin sections as previously described Total RNA was extracted from the HCT116 derived vector control (pTU6) and PKP3 knockdown clones  using Qiagen RNeasy kit and reverse transcribed using the Omniscript RT kit (Qiagen) as per the manufacturers instructions. PCR reactions for PKP3, PRL3 and GAPDH were performed on the cDNA using Taq polymerase (NEB) as per the manufacturers instructions. Real time PCR analysis using TaqMan gene expression assays (Applied Biosystems) were performed using gene specific probes for K8 and K18 from Applied Biosystems. A similar assay for GAPDH was performed to serve as an internal control for normalization. Fold changes in expression of K8 and K18 in the plakophilin3 knockdown clones were calculated relative to the expression levels in the pTU6 vector control cells as described previously Protein extracts were resolved on a ReadyStrip IPG strip (BioRad) pH range 5–8 according to the instructions supplied by the manufacturer. The proteins were then resolved in the second dimension on 10% SDS-PAGE gels followed by Western blotting with antibodies to K8.To detect the interaction between K18 and plakophilin3, HCT-116 cells were lysed with EBC buffer  and lysates were immunoprecipitated with anti K18 antibody. The immunoprecipitates were washed with NET-N buffer  for 3 times and run on SDS polyacrylamide gel electrophoresis followed by Western blots with antibodies to PKP3 and K18. The levels of O-linked N-acetylglucosamine (O-GlcNAc) on K8 were determined as previously described t test.Error bars represent standard error. Statistical analysis was performed using two tailed students Figure S1K8 and K18 organization in PKP3 knockdown cells. A. K18 forms a complex with PKP3 in HCT116 cells. EBC extracts prepared from HCT116 cells were incubated with a control antibody (IgG) or antibodies to K18 (K18). The reactions were resolved on SDS-PAGE gels and Western blots performed with antibodies to PKP3 and K18. (WCE = Whole cell extract. IP = Immunoprecipitation) B. K8 and K18 levels are altered upon PKP3 knockdown. High salt extracts from the vector control (pTU6) and PKP3 knockdown clones (S9 and S10) and the PKP3 and K8 double knockdown clone (8.21) were resolved on SDS-PAGE gels and the gel stained with coomassie blue to identify keratin bands. C. K8 and K18 form filaments in the PKP3 knockdown clones. The PKP3 knockdown clones or the vector control, were stained with antibodies against K8 or K18 . D. Vimentin levels are not altered upon PKP3 knockdown. Protein extracts from the vector control (pTU6), the PKP3 knockdown clones  and the double knockdown clones  were resolved on SDS-PAGE gels followed by Western blotting with antibodies to vimentin. None of the clones showed the presence of vimentin. AW13516 cells served as a positive control and a Western blot for actin served as a loading control.(TIF)Click here for additional data file.Figure S2Post-translational modifications on K8 and K18 upon PKP3 knockdown. A. Migration of K18 in two-dimensional gel electrophoresis. Protein extracts from the vector control and PKP3 knockdown clones were resolved in two dimensional gels followed by Western blotting with antibodies to K18. Note that the levels of K18 are higher in the PKP3 knockdown clones but no difference in migration in two-dimensional gel electrophoresis was observed for K18. B. Densitometric analysis of K8 and phospho-K8 expression in the vector control and PKP3 knockdown clones. Expression of K8, phosphor-S73 and phosphor-S431 was normalized to that of β-actin and densitometry performed on Western blots using ImageJ software. The values for these are shown in the vector control (pTU6) and PKP3 knockdown clones (S9 and S10) as indicated. C. Testing the specificity of K8 phosphospecific antibodies. GFP tagged WTK8 or the phosphomutants (S73A and S431A) were transfected into HCT116 cells. 48 hours post transfection, protein extracts were resolved on SDS PAGE gels followed by Western blotting with either antibodies that recognize K8 or the phosphor-specific antibodies. Note that the phospho-specific antibodies do not recognize the phosphor-mutant constructs. D. p38 levels are not altered in the PKP3 knockdown clones. Protein extracts from the PKP3 knockdown clones or the vector control were resolved on SDSPAGE gels followed by Western blotting with antibodies to p38. Western blots for 14-3-3γ were performed as a loading control. E. O-linked glycosylation on K8 is not altered in the PKP3 knockdown clones. Protein extracts from the PKP3 knockdown clones or the vector control were immunoprecipitated with antibodies to K8 followed by Western blots with antibodies recognizing O-GlcNAc glycosylation or K8.(TIF)Click here for additional data file.Figure S3PRL3 localization in PKP3 knockdown cells. A. PRL3 localization in PKP3 knockdown cells. The vector control (pTU6) or PKP3 knockdown clones (S9 and S10) were stained with antibodies to PRL3 and imaged by confocal microscopy. Note that PRL3 levels increase in the PKP3 knockdown clones with an increase in perinuclear localization and a slight increase in staining at the cell border. . B. GFP-PRL3 colocalizes with endogenous PRL3. HCT116 cells were transfected with GFP-PRL3. The cells were fixed and stained with antibodies to PRL-3 and visualized by confocal microscopy. Note that the GFP-PRL3 signal overlaps with the endogenous PRL3 signal. . C. Inhibition of PRL3 results in a decrease in migration. Scratch wound healing assays were performed in the absence (dark grey bars) or presence (light grey bars) of a PRL3 inhibitor. The distance migrated is graphed on the Y-axis. The bar represents an average of three different experiments and the bars represent the standard deviation. Note that PRL3 inhibition resulted in a significant decrease in migration in all cell types. Statistical analysis was performed using two tailed students t test. D. Identification of an shRNA that inhibits PRL3 expression. HEK293 cells were transfected with GFP-PRL3 with the vector control or a vector expressing the PRL3 shRNA. Extracts from untransfected HEK293 cells served as controls. 60 hours post transfection, protein extracts were resolved on SDS-PAGE gels followed with Western blots with antibodies to GFP. Note that GFP-PRL3 expression is observed in the vector control but not in cells transfected with the shRNA expressing vector. Western blots for actin served as a loading control. E. Identification of PKP3 and PRL3 double knockdown clones. The PKP3 knockdown clone S10, was transfected with either the vector control (vector) or a construct expressing an shRNA targeting PRL3 (PRL3 KD). Stable clones were generated followed by Western blotting with antibodies to PRL3. Note that PRL3 levels were comparable between the cells transfected with the vector control and the PRL3 KD construct. Western blots for β-actin served as a loading control.(TIF)Click here for additional data file.Figure S4K8 and K18 filament formation in PKP3 knockdown clones. A. Localization of phosphorylated K8 in PKP3 knockdown clones. The vector control (pTU6) or PKP3 knockdown clones (S9 and S10) were stained with phospho-specific antibodies to PRL3 (S73 and S431) and imaged by confocal microscopy. Note that the intensity of the signal for pS73 increases due to an increase in K8 levels in the PKP3 knockdown clones while the intensity of the signal for pS431 remains the same in the PKP3 knockdown clones. . B. The interaction between K8 and K18 is not altered upon PKP3 knockdown. The vector control (pTU6) or PKP3 knockdown clones (S9 and S10) were transfected with YFP-K18 and CFP-K8. 48 hours post transfection, FRET experiments were performed as described. The region in the white rectangle was bleached in the YFP channel and a corresponding increase was observed in the CFP channel as expected. FRET efficiencies were calculated for 5 different regions in three cells and results plotted in a bar graph. The FRET efficiency for the K18 K8 pair is comparable between the vector control and the PKP3 knockdown clones as shown in the graph.(TIF)Click here for additional data file.Figure S5Characterization of K8 and PKP3 double knockdown clones. A. Generation of PKP3 and K8 double knockdown clones. Protein extracts from the S10 derived K8  knockdown clones or the vector alone (S10P3) or the vector control (pTU6) were resolved on SDSPAGE gels followed by Western blotting with antibodies to PKP3 and β-actin. B. The PKP3 K8 double knockdown clones or the PKP3 knockdown clone, were stained with antibodies against K8 . C. Protein extracts from the S10 derived K8 (8.21 and 8.28) or K18 knockdown clones (18.22 and 18.23) or the vector alone (S10P3) were resolved on SDSPAGE gels followed by Western blotting with antibodies to K8, K18, and β-actin. Note that K18 levels are low in the K8 knockdown clones while K8 levels are not altered substantially in the K18 knockdown clones. D. Scratch wound healing assays were performed on the double knockdown clones  or the vector alone (S10P3). At different time points the distance migrated was measured and plotted as shown. The bars represent the mean of three independent experiments and the error bars represent the standard deviation. Statistical analysis was performed using two tailed students t test. Note that the distance migrated is significantly lowered in the double knockdown clones at 10, 15 and 20 hour time points. E. Scratch wound healing assays were performed on the double knockdown clones  or the vector alone (S10P3). At different time points the distance migrated per minute was measured and plotted as shown. The bars represent the mean of three independent experiments and the error bars represent the standard deviation. Statistical analysis was performed using two tailed students t test. Note that the distance migrated is significantly lowered in the double knockdown clones at 10, 15 and 20 hour time points. F. Scratch wound healing assays were performed on the double knockdown clones 8.21, transfected with either GFP alone or GFP K8 res.(TIF)Click here for additional data file.Figure S6Transformation induced upon keratin loss in the PKP3 knockdown clones. A. The S10 derived K18 (18.22 and 18.23) knockdown clones or the vector alone (S10P3) were plated in soft agar and colony formation determined after 2–3 weeks. The number of colonies formed by the clones per 20 low power fields (10X) was counted in triplicate in each experiment and the mean and standard deviation of three independent experiments is plotted as shown. B. Densitometric analysis of K8 in tumor samples. Protein extracts derived from tumors generated upon injection of either the vector control (S10P3) or the double knockdown clones 8.21 and 8.28) were resolved on SDS PAGE gels followed by Western blotting Expression of K8 was normalized to that of β-actin and densitometry performed on Western blots using ImageJ software. Note that K8 levels are lower in the double knockdown clones when compared to the vector control.(TIF)Click here for additional data file.


A subset of EIA positive samples was further screened for rotavirus RNA through RT-PCR and 44 (49.43%) samples, out of total 89 EIA positive samples, were found positive. G and P type prevalence was found as follows: G1P Pakistan harbors high disease burden of gastro-enteric infections with majority of these caused by rotavirus. Unfortunately, lack of proper surveillance programs and laboratory facilities have resulted in scarcity of available data on rotavirus associated disease burden and epidemiological information in the country. We investigated 1306 stool samples collected over two years (2008–2009) from hospitalized children under 5 years of age for the presence of rotavirus strains and its genotypic diversity in Lahore. The prevalence rate during 2008 and 2009 was found to be 34% (n = 447 out of 1306). No significant difference was found between different age groups positive for rotavirus ( The triple layered capsid consists of 2 proteins in the outer shell (VP7 and VP4), the intermediate layer constitutes VP6 while VP2 forms the inner layer enclosing two proteins VP1 and VP3. Based on VP6 capsid gene, the virus has been classified into seven major genogroups while VP7 and VP4 are the basis of a binary-system for further classifying the genogroup A viruses into 27 G- and 35 P- genotypes respectively Diarrheal infections are considered as a significant cause of infant and childhood morbidity and mortality in both developing as well as developed countries Rotavirus strain identification is considered as the key component of epidemiological surveys, disease distribution, genotype prevalence studies, vaccine administration and efficacy monitoring programs. Many past studies have highlighted the significance of continued monitoring of circulating rotavirus strains in order to maintain sufficient population immunity Samples from hospitalized children suspected of rotavirus gastroenteritis were collected as per World Health Organization’s standard case definitions that describe a suspected case as a child <5 years of age, admitted to a designated sentinel hospital for treatment of gastroenteritis while a confirmed case is a suspected case in whose stool the presence of rotavirus is demonstrated by means of an enzyme immunoassay. The study concept and design was approved through the Pakistan’s National Institute of Health Internal Review Board. The samples were collected after informed and written consent from the patient’s parents/guardians.http://whqlibdoc.who.int/publications/2011/9789241502641_eng.pdf).A total of 1306 stool samples were collected from hospitalized patients at ‘The Children’s Hospital’ Lahore during January 2008 to December 2009. Majority of these samples were collected from children below than 5 years of age who were hospitalized with suspected rotavirus gastroenteritis. The collected stool samples were processed for the detection and confirmation of Rotavirus antigen. ELISA test was performed using the ProSpecT™ Rotavirus Microplate Assay  as per World Health Organization’s recommendations (A subset (20%) of EIA positive samples were transported to Department of Virology, National Institute of Health, Islamabad for rotavirus RNA detection through RT-PCR and further genotype determination on the basis of VP7 and VP4 gene segments using the protocol as described by Gouvea et al 1990 http://megasoftware.net/). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The GenBank accession numbers, country, year of sample collection and respective genotype information has been given where available. The VP7 sequences obtained in this study have been submitted to GenBank under the accession numbers KC896141-KC896157 and the VP4 sequences under accession numbers KC896127-KC896140.Phylogenetic analyses of VP7 and VP4 sequences were performed in comparison to the strains belonging to different geographical regions as retrieved from GenBank. Evolutionary tree and distances (number of base substitutions per site) were generated by Neighbor Joining method with Kimura-2 parameter using MEGA 4.0 (P>0.05) was found between different age groups positive for rotavirus through ELISA; however, high infection rate was observed among children between 12–17 and 24–59 months during 2008 and 2009 respectively. The rotavirus positive cases appeared to occur throughout the year with peaks during January to April and July to September  for the presence of rotavirus antigen yielded 447 (34.22%) samples positive for group A major inner capsid protein (VP6 antigen) common to all known rotavirus genotypes. The prevalence rates during 2008 and 2009 were 34.12% (n = 243 out of 712) and 34.34% (204 out of 594) respectively. No significant difference  samples were found positive for rotavirus through PCR. G and P types were found with the prevalence as G1P The genetic sequence of 18 viruses included in this study was determined for the VP7 gene. These samples were randomly selected on the basis of sample quantity and resource limitations which showed that all viruses are highly identical (99–100%) to each other within their respective genotypes; G1, G2 and G9. The G1 strains shared close sequence similarity with the previously reported viruses from Pakistan (JN001862) as well as to those from Belgium  and USA (JN258346). The VP7 sequences of G9 strains were found 100% identical among themselves as well as highly similar level of homology was found with the viruses from South Africa (GQ338887), Russia (FJ447573) and Australia (AY307090). These viruses also hold 98% similarity to previously reported G9 strains  from Faisalabad city of Pakistan (unpublished data). Likewise, the G2 viruses showed 99–100% identity among themselves and a similar genetic closeness was found among the G2 strains from Bangladesh  as well as the already identified G2 strains from Pakistan .The genetic relationships of rotavirus strains from this study were also determined on the basis of VP4 gene. 14 viruses were grouped among three genotypes P Phylogenetic reconstruction revealed clustering of all rotavirus strains from Children Hospital, Lahore with viruses  differences, in the neutralizing epitopes of VP7 and VP4 proteins, between vaccine and the circulating strains. We compared the VP7 aa sequences of Pakistani viruses, constituting the antigenic epitopes with those of the two available vaccines strains (RotaTeq™ and Rotarix™). The VP7 protein contains three antigenic epitopes defined as 7-1a, 7-1b and 7-2 which comprise of 29 amino acid residues from positions 87 to 291 based on rhesus Rotavirus strain numbering (GenBank accession No. AF295303) Analysis of Pakistani G1 strains showed that the 29 amino acid residues across the three antigenic sites are highly conserved when compared to Rotarix™ G1P The VP7 antigenic epitopes of G2 Pakistani viruses showed 3 variations (02 substitutions within 7-1a and 01 change in 7-1b) from the RotaTeq™ G2P Among the G9 viruses presented in this study, the VP7 protein was compared with Rotarix™ G1 strain as well as all constituent strains of RotaTeq™ vaccine (G1-4 with P The VP4 protein of rotavirus contains four antigenic regions defined as 8-1 to 8-4 spanning between aminoacid positions 88 to 196 based on rhesus Rotavirus strain (GenBank accession No. AY033150) When compared to Rotarix™ P A high degree of variation was observed for P Diarrhea is accountable for approx. 5% of all deaths among children below five years of age. Viral pathogens are the most common cause of gastroenteritis in both developing and developed countries Seasonality of rotavirus gastroenteritis in Pakistan has never been determined although the cases have been detected round the year et al.The higher percentage of samples positive for rotavirus was found among the children between 12 and 17 months of age. These findings are slightly variable than the previous study conducted in Karachi city of Pakistan During the last 15 years, very few reports about circulating genotypes of rotavirus are available from Pakistan In the present study, we compared the amino acid motifs constituting the neutralizing epitopes of VP7 and VP4 proteins between the circulating Pakistani rotaviruses and available vaccine strains. Multiple antigenic variations were noticed among both VP7 and VP4 epitopes highlighting the need for detailed antigenic mapping of prevalent rotavirus genotypes in the country. Complete genome sequencing will therefore be required to generate more comprehensive information on molecular epidemiology and evolutionary dynamics of rotaviruses on account of its segmented genome and the possible role of internal genes in immunity The vaccine introduction has significantly reduced the rotavirus disease burden in many countries like Australia, Austria, Belgium, Brazil, El Salvador, Nicaragua, Panama and the United States and both vaccines have been found to carry equivalent efficacy against G1, G3, G4 and G9 strains with P et al reported that most of the currently circulating G1 strains and the Rotarix™ vaccine RRV-S1 strain differ in their antigenic properties and mutations in these antigenic sites may ultimately cause vaccine failure The mutations in the antigenic regions of rotavirus play an important role in the outcome of vaccine response. When compared to the respective prototype strains, our data substantiates the previous findings Despite these variable epidemiological reports, it has been now established that rotavirus vaccines are equally effective against vast diversity of rotavirus genotypes by generating heterotypic immune response. In the recent vaccine trails, both Rotarix™ and RotaTeq™ have been proven as equally effective in regions with high child and adult mortality Our present findings conclude that the rotavirus genotypes circulating in different geographical areas of Pakistan are quite variable and large scale studies must be conducted to calculate the burden of disease as well as the epidemiological understanding of contributing viral genotypes. Similar diversity of prevalent rotavirus strains within the Indian subcontinent including Pakistan has been reported by Miles et al.,


Genetic linkage maps have been essential tools to examine the inheritance of qualitative and quantitative traits, to carry out comparative mapping and to provide markers for molecular breeding applications. Linkage maps for species of Eucalyptus have been reported for several pedigrees using different molecular marker technologies . HoweverE.grandis and E.urophylla. Genomic representations of both parents and their F1 offspring were produced with the same complexity reduction method used to prepare the library (PstI/TaqI) generating the ‘targets’ for hybridizing to the arrays. Microarray imaging, data extraction polymorphism detection and marker scoring were carried out using DArTSoft v.7.44 (http://www.diversityarrays.com/). Polymorphic markers were filtered according to reproducibility, quality parameter (Q) and marker call rate as described earlier [Map construction was carried out using an F1 progeny of 177 individuals derived from a cross between  earlier . An inte earlier using a fA segregation ratio filtering (both for 1:1 and 3:1 markers) together with a relaxed call rate threshold > 50% were initially applied to include the largest number of potentially mappable markers. A total of 4,271 DArT markers segregated 1:1 and 1,572 segregated 3:1. The complete dataset with 6,065 markers  was submitted to a linkage analysis resulting in eleven groups at LOD ≥ 15 with a total of 2,484 markers. A subset of 1,032 markers positioned at high likelihood for ordering (864 DArTs and 168 microsatellites) provided a framework map with 1,176 cM and an average recombination distance between consecutive markers of 1.15 cM. When all the 2,484 mapped markers were fitted, the total recombining genome length increased to 1,303 cM with a resolution of 0.6 cM. A redundancy analysis of the 6,918 DArT probes for which sequences could be obtained resulted in 4,583 unique sequences (66%). The estimated redundancy of 34% represents a useful resource by providing alternative probes for detecting polymorphism in the same genomic region in different individuals. A total of 6,480 probes (93.7%) were confidently mapped onto the reference genome; 4,189 of them (65%) mapped to single positions while the remaining 2,291 probes mapped to a second position with a relatively high suboptimal alignment score and were therefore considered mapped at lower confidence. About 50% of these low confidence mapping  actually mapped to a sequence position containing a repeat element which might explain the mapping result. A total of 438 DArT probes could not be mapped to the current assembly. These DArT probes might further improve the current genome version by including some yet unassembled genome contigs. A total of 1,987 linkage mapped DArT markers for which sequence was available were positioned relative to the 41,208 gene models in the current genome, distributed in 500 kbp bins providing an average of 1.6 ± 2.4 DArT markers for the 34 ± 15 gene models present in each genome bin. Interestingly a total of 4,663 DArT probes (67%) mapped at less than ten basepairs from the nearest gene model and only 76 probes mapped at more than 10kbp. The largest distance from a DArT probe to the next gene was only 156 kbp and a modest although highly significant coincidence across the whole genome was seen between the number of DArT markers and the number of gene models (Spearman rank correlation R = 0.33 p<0.00000).Eucalyptus grandis genome currently available through the JGI Phytozome website. This map, together with other parallel mapping efforts that used this same genotyping platform have provided between 2,000 and 3,000 segregating markers irrespective of species of Eucalyptus. The mapping density achieved with the Eucalyptus DArT microarray provides unprecedented opportunities for comparative mapping across pedigrees, high resolution QTL analysis and molecular breeding applications across species of the genus. We have shown that not only the DArT marker sequences are highly enriched for genes, but that their distribution relative to the predicted gene models provides an extremely efficient tool to specifically tag genes at distances within the extent of LD in typical breeding populations. The use of this standardized high-throughput genotyping platform will therefore be instrumental to implement genome-wide selection strategies and positional cloning efforts in multiple Eucalyptus species.The linkage map presented in this work was used to aid the ordering of contigs during the assembly of the


Pattern formation in developing tissues is driven by the interaction of extrinsic signals with intrinsic transcriptional networks that together establish spatially and temporally restricted profiles of gene expression. How this process is orchestrated at the molecular level by genomic cis-regulatory modules is one of the central questions in developmental biology. Here we have addressed this by analysing the regulation of Pax3 expression in the context of the developing spinal cord. Pax3 is induced early during neural development in progenitors of the dorsal spinal cord and is maintained as pattern is subsequently elaborated, resulting in the segregation of the tissue into dorsal and ventral subdivisions. We used a combination of comparative genomics and transgenic assays to define and dissect several functional cis-regulatory modules associated with the Pax3 locus. We provide evidence that the coordinated activity of two modules establishes and refines Pax3 expression during neural tube development. Mutational analyses of the initiating element revealed that in addition to Wnt signaling, Nkx family homeodomain repressors restrict Pax3 transcription to the presumptive dorsal neural tube. Subsequently, a second module mediates direct positive autoregulation and feedback to maintain Pax3 expression. Together, these data indicate a mechanism by which transient external signals are converted into a sustained expression domain by the activities of distinct regulatory elements. This transcriptional logic differs from the cross-repression that is responsible for the spatiotemporal patterns of gene expression in the ventral neural tube, suggesting that a variety of circuits are deployed within the neural tube regulatory network to establish and elaborate pattern formation. The complex organization of tissues is established precisely and reproducibly during development. In the vertebrate neural tube, as in many other tissues, the interplay between extrinsic morphogens and intrinsic transcription factors produces spatial patterns of gene expression that delineate precursors for specific cell types. One such transcription factor, Pax3, defines the precursors of all sensory neuron subtypes and distinguishes them from precursors fated to give rise to the motor circuits. To gain insight into the molecular mechanisms by which the spinal cord is segregated into these two functional domains, we analysed the genomic regulatory sequences responsible for controlling Pax3 activity. We identified two regions of the genome, the coordinated activity of which establishes and refines Pax3 activity. We showed that the combination of activating signals from secreted Wnt factors together with Nkx family homeodomain repressors restrict Pax3 activity to the presumptive sensory region of the neural tissue. Subsequently, Pax3 acts to directly potentiate its own transcription and this autoregulation sustains Pax3 expression at later developmental stages. Together, our study reveals the way in which intrinsic and extrinsic signals are integrated by cells and converted into a sustained pattern of gene activity in the developing nervous system. Embryonic development relies on the coordinated and dynamic control of gene expression. This is achieved, in the main, by interactions between transcription factors (TFs) and the genomic cis-regulatory modules (CRMs) associated with regulated genes The specification of progenitor identity in the vertebrate neural tube is a well studied example of developmental patterning By contrast, less is known regarding the specification of sensory interneurons within the dorsal spinal cord Several studies indicate that a genomic interval immediately upstream of the mouse Pax3 promoter is sufficient to direct expression to the neural tube, however this region is not required for Pax3 expression Here we take advantage of lineage tracing analyses in mice and transgenic assays in chick and zebrafish embryos to dissect the molecular mechanism and regulatory logic of Pax3 expression in dorsal neural progenitors. We show that Pax3 expression is refined during neural tube patterning by the temporal activity of distinct regulatory elements. We provide evidence that in addition to Wnt signaling, Nkx family homeodomain (HD) containing repressors are critical for establishing the restricted expression of Pax3. Moreover, we demonstrate that autoregulation and positive feedback is required to maintain Pax3 expression in the neural tube.Rosa26-YFP or Rosa26-Tomato/GFP reporter strains  , all of which were located within the 4the gene . Candidaon (hpf)  before bon (hpf) . Maximumon (hpf) , after won (hpf)  and progon (hpf) , in agrepression . Howeverpression .The genomic interval corresponding to CNE3 is a highly conserved region of a previously defined CNS specific Pax3 CRM, termed both ECR2 In addition to CNE3, our functional assays identified CNE1 as a CNS specific Pax3 enhancer. CNE1 transient transgenic zebrafish exhibited Citrine expression in presumptive dorsal progenitors at 10 hpf  and 12 hWe sought to investigate the molecular basis of CNE3 activity in order to gain insight into the regulation of Pax3 expression in the CNS. The conservation of sequence across 12 vertebrate genomes was used to define 5 statistically enriched 15 bp motifs, predicted to contain the transcription factor binding sites (TFBS) that mediate enhancer activity . AnnotatTCFSiam transgenic zebrafish TCFSiam transgenic embryos assessed between 12 hpf and 18 hpf exhibited activated Wnt signaling within the tailbud and neural tube, however reporter expression was markedly decreased in the spinal cord of 24 hpf embryos . By contrast, deletion of Motif5 resulted in a significant increase in CNE3 activity along the entire D–V axis of the zebrafish spinal cord compare . EctopicTo investigate the binding of putative repressors to CNE3, we performed electrophoretic mobility shift assays (EMSAs) using nuclear extracts from dissected E3 chick spinal cords and a 48 bp DNA probe which spanned Motif3, 4 and 5. Supershift reactions were performed by the addition of antibodies raised against β-catenin, several candidate HD containing ventral fate determinants and FoxA2 . AdditioTogether, these data suggested that Nkx6.1, or a protein with a similar binding specificity, interacted with Motif5 of CNE3 to mediate transcriptional repression of Pax3 within the developing spinal cord. This is consistent with the observation that the ventral domain of the Pax3 lineage was mutually exclusive with Nkx6.1 expression in transgenic mice assessed at E9.75 . FurtherWe next sought to investigate the molecular basis of CNE1 activity by identifying statistically over represented conserved 15 bp motifs within this ∼170 bp enhancer . ControlAmongst the TFs that potentially interact with Motif3 , the SoxpairedExamination of the matrix represented by Motif1 revealed a 14 bp sequence similar to a Pax6 paired domain (PD) binding site . We wereWe assessed the ability of both Pax3 and Pax7 to bind Motif1 by EMSA, using a 33 bp DNA probe and in vitro synthesized proteins. These assays revealed that both TFs interacted with this sequence, verifying it as a functional PD binding site  FurthermPrevious binding and structural studies have shown that the PD is comprised of two helix-turn-helix subdomains, commonly termed PAI and RED, that interact with the 5′ and 3′ region of the binding site, respectively engrailed repressor domain to either Pax3 or Pax7 We next sought to assess the ability of Pax3 and Pax7 to induce CNE1 activity in the neural tube. Electroporation of a dominant active protein consisting of the DNA binding domain of human PAX3 fused to the transactivation domain of FOXO1A (PAX3FOXO1A) was sufficient to activate CNE1 mediated transcription in the ventral neural tube of chick embryos at E3 . MoreoveIn this study we provide evidence that the dynamic expression profile of Pax3 within the developing neural tube is achieved by the coordinated action of two distinct regulatory mechanisms, acting through separate CRMs. The combined activity of these enhancers converts transient inductive cues into a sustained domain of gene expression. CNE3 integrates inductive Wnt signaling and the repressive activity of HD transcription factors to initiate expression of Pax3 within the prospective dorsal neural tube. Subsequently, an autoregulatory loop acting via CNE1 is established that maintains Pax3 expression in the absence of activated Wnt signaling and HD mediated repression.I150 BAC stable line has been shown to recapitulate the expression profile of pax3a in the spinal cord Previously, the zebrafish Pax3-GFPThe initiation of Pax3 transcription in response to Wnt signaling appears to be mediated through CNE3, a small highly conserved region of the genomic interval that has previously been identified as ECR2 and IR1 The transcriptional activity of the Wnt pathway decreases in the neural tube as development progresses, as indicated by a downregulation of Wnt reporter transgene expression prior to the peak of Pax3 transcription in progenitors Motif3 within CNE1, comprising Fox and Sox TFBS, is required for the activity of this enhancer across the AP axis of the CNS. The reduction of CNE1 activity in constructs carrying mutations in the HMG box binding site of Motif3, together with the identification of Sox11 and Sox3 binding at CNE1 in neural progenitors, supports a positive role for SoxB proteins on Pax3 expression and might account for the neural specificity of this enhancer Together, these data provide a molecular framework that describes the regulatory logic of Pax3 expression in the developing spinal cord. Expression is initiated by Wnt induction acting through CNE3, however this induction is limited to prospective dorsal regions of neural tissue by the activity of ventrally induced Nkx class proteins . Pax3 prDrosophila development It is notable that at early developmental time points, both CNE1 and CNE3 appear to be active in Pax3 expressing cells. We propose they act in a cooperative manner to establish the Pax3 expression domain, thereby offering increased robustness and precision. For example, CNE1 mediated autoregulation could function not only to increase output from the Pax3 promoter but may also buffer fluctuations in CNE3 mediated transcription. This mechanism is reminiscent of the increased robustness and precision of gene expression achieved by the synergistic activity of multiple CRMs during The acquisition of unique molecular identities within defined progenitor populations requires the translation of transient inductive cues into discrete expression domains. In the ventral neural tube this process is achieved using a transcriptional circuit involving cross-repression between TFs downstream of the ventral morphogen, Shh. This mechanism functions both to establish and maintain expression domains, as well as confer robustness to fluctuating levels of intracellular signaling http://www.genome.ucsc.edu/) and uploaded to the Mulan alignment suite The genomic interval representing the Pax3 locus in each species was defined in the UCSC genome browser (http://www.ensembl.org/). The alignment of enhancer sequences across vertebrate genomes was produced and analysed using ClustalW2 The Human sequence representing each CNE was used as the query sequence for cross-species BLAST searches, performed within the Ensembl genome browser (Zebrafish embryos were collected within 15 minutes of laying according to established procedures and injected with injected with a mixture of plasmid DNA (20 ng/µl) and Tol2 transposase mRNA (14 ng/µl). Embryos were maintained at 28.5°C and sorted on the basis of Citrine expression before fixation with 4% paraformaldehyde (PFA) at the desired stage. Chick assays were performed in Hamburger and Hamilton stage 11–13 embryos pax3a (gift from Simon Hughes) were performed as described pairedWholemount in situ hybridizations for Zebrafish experiments were analysed by determining the number of embryos labeled by Citrine expression in spinal cord progenitors as a proportion of the total transgenic population. The activity of enhancer constructs in the chick neural tube was assayed against the presence of β-Galactosidase antibody staining, which was used as an internal control of transgenesis. The statistical significance of transgenic assays was determined using two-tailed Fisher's exact tests.Radiolabelled DNA probes were produced as described in Figure S1th intron of the Pax3 locus. (A) The full Mulan alignment of the Pax3 locus, summarised in Several functional CRMs are located within the 4(TIF)Click here for additional data file.Figure S2pax3a expression across the AP axis of the CNS and is restricted to the Pax3/7 domain of the dorsal spinal cord.CNE1 activity recapitulates Pax3 expression. (A) CNE1 stable transgenic embryos assessed at 10 hpf exhibit Citrine expression in the developing hindbrain (hb) and presumptive dorsal progenitors (dP) within the lateral regions of the posterior neural plate.  At 24 hpf, CNE1 activity recapitulates (TIF)Click here for additional data file.Figure S3CNE1 is the only p300 bound Pax3 enhancer at E11.5. UCSC genome browser view displaying the binding profile of the enhancer associated transcription co-factor p300 within mouse limb, forebrain and midbrain tissue, prepared at E11.5 (TIF)Click here for additional data file.Figure S4The conservation of CNE3 across vertebrates. ClustalW2 alignment of CNE3 across 12 vertebrate genomes, revealing multiple clusters of nucleotides that are conserved across the phyla. The location of motifs within CNE3, discovered by MEME analysis, is marked within the alignment.(TIF)Click here for additional data file.Figure S5Wnt pathway effectors directly bind CNE3. UCSC genome browser view displaying the binding profile of the Wnt pathway effector, Tcf3, across the Pax3 locus in mouse embryonic stem cells (mESC) (TIF)Click here for additional data file.Figure S6The conservation of CNE1 across vertebrate genomes. ClustalW2 alignment of detailing the conservation of CNE1 across 12 vertebrate genomes. The location of statistically overrepresented motifs within this sequence is marked within the alignment.(TIF)Click here for additional data file.Figure S7M3Mut1 transgenic zebrafish exhibit a reduction in CNE1 activity compared to controls (compare B to D) . A similar reduction in CNE1 activity is observed in M3Mut2 transgenics (compare B to G) . (F) Graphical summary of the effect of HMG binding site mutations upon CNE1 activity in zebrafish embryos. Experiments performed in chick reveal a reduction in CNE1 activity in both M3Mut1 (n = 3/7) and M3Mut2 (n = 2/5) electroporations, however this result did not reach statistical significance .Mutation of the HMG box site within Motif3 reduces CNE1 activity. (A) Schematic outlining the mutations introduced into the HMG box site of Motif3. (TIF)Click here for additional data file.Table S1Annotation of conserved TFBS within CNE3.(DOCX)Click here for additional data file.Table S2Annotation of conserved TFBS within CNE1.(DOCX)Click here for additional data file.Table S3List of oligonucleotides used in the study.(DOCX)Click here for additional data file.


Cardiac complications mainly related to myocardial iron overload (MIO) remain the main cause of morbidity and mortality in thalassemia major (TM). Thalassemia cardiomyopathy is treatable and partly reversible if appropriate chelation therapy is instituted in time. The validated multislice multiecho T2* Cardiovascular Magnetic Resonance (CMR) technique has permitted to quantify segmental and global myocardial iron burden detecting different patterns of iron overload. Aim of our study was to verify the risk of cardiac complications related to different patterns of MIO in a large cohort of TM patients.We considered 812 TM patients for who CMR and cardiac data were collected in a central data base. Three short-axis views  of the left ventricle were acquired using a multislice multiecho T2* sequence. Using a previously validated software the 16 segmental T2* values and the mean global heart T2* value were provided. A conservative cut off of 20 ms was considered the limit of normal for the segmental and global T2* values.We identified 4 groups of patients: group I (17%) with homogeneous MIO , group II (12%) with heterogeneous MIO (some segments with T2*<20 ms and others with T2*≥20 ms) and global heart T2*<20 ms; group III (29%) with heterogeneous MIO and global heart T2*≥20 ms; group IV (42%) with no MIO . The percentage of patients with cardiac complications was significantly different in the 4 groups . In particular, the percentage of patients with heart failure was significantly different in the 4 groups . No significant differences were found among groups in the percentage of arrhythmias and pulmonary hypertension. Odds Ratio for cardiac complications was 1.7  for patients with homogeneous MIO vs patients with no MIO. Odds Ratio for heart failure was 2.3  for patients with homogeneous MIO versus patients with no MIO and 2.2  for patients with heterogeneous MIO and global heart T2*<20 ms versus patients with no MIO.Homogeneous MIO predicts a significantly higher risk to develop cardiac complications, especially heart failure, suggesting an intensive chelation therapy in this group of patients.“No-profit” support by industrial sponsorships  and “Ministero della Salute, fondi ex art. 12 D.Lgs. 502/92 e s.m.i., ricerca sanitaria finalizzata anno 2006” e “Fondazione L. Giambrone”.


The germline mutations in BRCA1/2 genes are the most significant and well characterized genetic risk factors for breast and/or ovarian cancer. Detection of mutations in these genes is an effective method of cancer prevention and early detection. Different ethnic and geographical regions may have different BRCA1 and BRCA2 mutation spectrum and prevalence due to founder effect. The population of Lithuania has over several centuries undergone limited mixing with surrounding populations and is mostly of indigenous Baltic origin, which is different from Slavs. The aim of our study was to asses full BRCA1/2 mutational profile in Lithuanian population.We performed comprehensive mutation analysis of BRCA1/2 genes in 605 unrelated breast and/or ovarian cancer patients (with/without family history) and predictive unaffected patients (with family history) using high resolution melting (HRM) screening (Light Cycler 480/Light Scanner 384) followed by direct sequencing (ABI 3500) and MLPA for large genomic rearrangements (LGRs).Overall, we have identified 25 different mutations (16 in BRCA1 and 9 in BRCA2 genes). Seven frequent pathogenious mutations in BRCA1 gene  comprised 48%, 29%, 7,7%, 3,5%, 2,1%, 1,4% and 1,4% respectively of all BRCA1 mutations; a single BRCA2 mutation (c.658delGT) comprised 43% of all mutations in this gene. Five novel BRCA1  and 4 novel BRCA2 genes mutations  were identified; 3 different LGRs  were found in BRCA1. The most common c.4035delA  appears to be true Lithuanian  founder mutation and haplotype data confirmed ancient origin of this mutation c.a. 62 generations ago.Characterization of BRCA1/2 mutational profile in Lithuania enabled to develop screening protocol using HRM for 8 common BRCA1/2 point mutations, which comprise 88% of all mutations detected in our country. This knowledge will provide more efficient approach for the individualization of genetic testing affordable for all breast/ovarian patients and their relatives.