MicroRNAs (miRNAs) are non-coding RNA molecules that play pivotal role in modulating the expression of multiple target genes at the post-transcriptional level. Single Nucleotide Polymorphisms (SNP) in pre-miRNAs can alter miRNA expression, and polymorphism in target molecules can affect binding to target mRNA. Studies have shown an association between miR-146a polymorphisms and autoimmune disease. Taking into account that interleukin-1 receptor-associated kinase-1 (IRAK-1) is a target of miR-146a, we studied the association between SNPs of miRNA-146 and its target IRAK-1 with susceptibility to Juvenile Idiopathic Arthritis-Enthesitis Related Arthritis (JIA-ERA).One hundred and fifty patients with JIA-ERA (ILAR criteria) were included in the study. 216 blood donors  with a mean age of 30.5 years served as controls. miR-146a (C/G) (rs2910164) and it’s target IRAK-1(C/T) (rs1059703) at Exon 12 region and IRAK-1 (A/C) (rs3027898) at 3’UTR polymorphisms were analyzed using PCR-RFLP method.Among 150 patients, 134 were males and the mean age at onset of disease was 11 (4-16) years, mean disease duration was 4.5 (0.3-12) years. 22 had uveitis and 21 had positive family history. 73 had enthesitis and 75 had inflammatory back pain and all had arthritis. 116 were HLA B27 positive.Genotype frequency of miR-146a gene was in Hardy Weinberg equilibrium in healthy controls whereas in IRAK-1 genotype the frequency was contrary owing to its presence on chromosome X.The genotype frequency for miR-146a were different in controls and patients IRAK-1 (rs1059703) allelic frequencies in controls and patients were similar . IRAK-1 (rs3027898) allelic frequencies were also similar among control and patients [CC (70.1% vs 76.1%) and AA (29.8% vs 23.9%)].The CC genotype of the miR-146a rs2910164 polymorphism was significantly associated with the susceptibility to JIA-ERA.


Withania somnifera, induces cell death (apoptosis/necrosis) in multiple types of tumor cells, but the molecular mechanism underlying this cytotoxicity remains elusive. We report here that 2 μM WA induced cell death selectively in androgen-insensitive PC-3 and DU-145 prostate adenocarcinoma cells, whereas its toxicity was less severe in androgen-sensitive LNCaP prostate adenocarcinoma cells and normal human fibroblasts (TIG-1 and KD). WA also killed PC-3 cells in spheroid-forming medium. DNA microarray analysis revealed that WA significantly increased mRNA levels of c-Fos and 11 heat-shock proteins (HSPs) in PC-3 and DU-145, but not in LNCaP and TIG-1. Western analysis revealed increased expression of c-Fos and reduced expression of the anti-apoptotic protein c-FLIP(L). Expression of HSPs such as HSPA6 and Hsp70 was conspicuously elevated; however, because siRNA-mediated depletion of HSF-1, an HSP-inducing transcription factor, reduced PC-3 cell viability, it is likely that these heat-shock genes were involved in protecting against cell death. Moreover, WA induced generation of reactive oxygen species (ROS) in PC-3 and DU-145, but not in normal fibroblasts. Immunocytochemistry and immuno-electron microscopy revealed that WA disrupted the vimentin cytoskeleton, possibly inducing the ROS generation, c-Fos expression and c-FLIP(L) suppression. These observations suggest that multiple events followed by disruption of the vimentin cytoskeleton play pivotal roles in WA-mediated cell death.Withaferin A (WA), a major bioactive component of the Indian herb  Withania somnifera, induces cell death in many tumor cells . WA. WAWithaor cells . WA suppor cells . WA causor cells , and indor cells .WA binds directly to vimentin by covalently modifying a cysteine residue (Cys328), causing vimentin filaments to aggregate and colocalize with F-actin and thereby disrupting the vimentin cytoskeleton , 11]. W. W11]. WThe oncogenic transcription factor c-Fos regulates gene expression, in association with Jun or other basic leucine-zipper proteins, as a component of the activator protein 1 (AP-1) dimer complex. Although c-Fos exerts anti-apoptotic functions, recent reports suggest that it may also promote apoptosis , 17]. A. A17]. AThe pathogenesis of prostate tumors is initially androgen-dependent; however, many patients progress to metastatic castration-resistant (androgen-independent) PCa (mCRPC) . AndrogeIn this study, we sought to investigate whether WA can kill mCRPCs but not normal cells, and if so, to identify the essential molecular target of WA. We found that WA (2 μM) selectively induced cell death in androgen-independent PC-3 and DU-145 PCa cells, but not in LNCaP androgen-responsive PCa cells or TIG-1 normal fibroblasts. DNA microarray and western analyses revealed novel molecular targets of WA that may define the distinct responses of these cell lines to WA. Because WA killed PC-3 cells in spheroid-forming cultures, we propose that WA may serve as a starting molecule for the development of drugs that induce cell death in cancer stem cells (CSCs).We first found that WA effectively induced cell death in PC-3 and DU-145, but LNCaP were less sensitive to WA treatment . To deteAfter treatment with 2 μM WA, viability of PC-3 was reduced at 4, 8, and 24 h, whereas DU-145 became inviable only at 24 h  rescues cell death in PC-3 . Hence, c-Fos exerts its proapoptotic function in PC-3 and LNCaP cells in part by repressing expression of c-FLIP(L), which we will refer to as ‘FLIP’ hereafter . The FLIHSPA6, DNAJA4, HMOX1, HSPA1B, HSPA1L, HSPA1A, DNAJB1, DNAJB4, HSPH1, and SERPINH1) were among the 45 most up-regulated genes in PC-3  genes , the major transcription factor involved in up-regulation of the HSF genes , using aFour of the early growth response (EGR) genes were ranked among the top 40 WA-induced genes . The levXenopus laevis A6 kidney epithelial cells [WA induces apoptosis mediated by ER stress in human renal carcinoma cells  and Xenoal cells . Gene OnAshwagandha leaf extract containing WA causes breast cancer cells to generate ROS, which is a stress response initiated at mitochondria [tert-butyl hydroperoxide (TBHP), an inducer of ROS generation. All tested cells showed cytoplasmic signals (green) derived from ROS production  or wortmannin, which suppresses canonical autophagy, to PC-3 cells in the presence (+) or absence (−) of 4 μM WA treatment. Western analysis indicated that WA induced LC3 expression regardless of the presence of 3-MA or wortmannin . MoreoveBecause BAG3 activates noncanonical autophagy elicited by proteasome inhibitors such as MG132 , we inveWestern blot analysis showed that the intensity of the ~70 kDa band of BAG3 arrow in  was decrWA associates with vimentin and causes rapid reorganization of vimentin intermediate filaments (VIF) into perinuclear aggregates in BJ-5ta human fibroblasts . AlthougImmunostaining at 2, 4, and 8 h after 4 μM WA treatment caused vimentin aggregation around the plasma membrane in TIG-1 ; BAG3 (OriGene); BiP, Caspase-3 (8G10), c-Fos, CHOP, EGR-3, eIF2α, eIF2αx-pS51, FLIP (D16A8), FosB, HSF-1, HSP40, HSP70, IRE1α, PARP, PAR-4, PERK, TAZ, and YAP ; EGR-1 and Withaferin A (Santa Cruz Biotechnology); GAPDH ; HSPA6 (Abcam); LC3 (MBL); and vimentin (ARP).2 at 35°C in DMEM supplemented with FBS (10%), penicillin (100 U/mL), and streptomycin (100 μg/mL). For PC-3, DU-145, and LNCaP, DMEM was replaced with RPMI-1640 medium.Human cells were acquired from the indicated suppliers: PC-3 and DU-145 (RIKEN Bioresource Center), TIG-1 (Japanese Cancer Research Resources Bank), KD (Japanese Collection of Research Bioresources Cell Bank) and LNCaP (ATCC). TIG-1 was maintained in 5% COCGUACGCGGGAAUACUUCGADdTdT (Gene Design); siHSPA6, GGCAGAGAAGGAGGAGUAUGAGCdAdT (OriGene); siHSF-1, GAGUGAAGACAUAAAGAUCCGCCdAdG (OriGene); siFos, GCAUUAACUAAUCUAUUGGGUUCdAdT (OriGene); siBAG3, CCUGAUGAUCGAAGAGUAUUUGAdCdC (OriGene); and CHOP, AGCGUAUCAUGUUAAAGAUGAGCGG (OriGene). These siRNA duplexes were transfected using Oligofectamine (Invitrogen).The following synthesized siRNA duplexes were used for siRNA-mediated knockdown: siGL2, S1 FigCell viability was measured at 4, 24, 48, 72, and 96 h after 2 μM WA treatment. NT, non-treated. Bars represent means ± SEM of three independent measurements.(TIF)Click here for additional data file.S2 FigThe y-axis shows the log value of hybridization signal intensity obtained from the microarray data for WA-treated TIG-1, LNCaP, PC-3, and DU-145 (see yellow arrows in (TIF)Click here for additional data file.S3 FigFACS analysis of PC-3 cells transfected with siFos or siGL2 (negative control) after treatment with WA (4 μM) or DMSO (solvent) alone, using the GFP-Certified Apoptosis/Necrosis Detection System. Apoptosis was detected by Annexin V–EnzoGold, and necrosis by 7-AAD-Red. Upper left, necrosis; upper right, late apoptosis; bottom right, early apoptosis.(TIF)Click here for additional data file.S4 FigWestern blot analysis for EGR-1, EGR-3, PAR-4, and GAPDH in PC-3 cells either untreated (NT) or 4, 8, or 24 h after treatment with 4 μM WA.(TIF)Click here for additional data file.S5 Figp-value), i.e., the taller the bar, the higher the significance of the overlap.(A) Genes differentially expressed between PC-3 cells treated with DMSO or 2 μM WA see  were sub(TIF)Click here for additional data file.S6 FigWestern blot analysis of CHOP, caspase 3, and GAPDH (loading control) in TIG-1 and PC-3 at 8 h after 4 μM WA treatment in the presence (+) or absence (-) of the indicated siRNAs. The arrow and arrowhead indicate the CHOP and caspase 3 band, respectively.(TIF)Click here for additional data file.S7 Fig(A) Typical fluorescence images of TIG-1 (i), KD (ii), LNCaP (iii), PC-3 (iv), and DU-145 (v) that were cultured on glass coverslips and treated with TBHP for 90 min to induce ROS generation. Merged images are shown of cytoplasmic ROS signals (green) and nuclear DNA signals stained with Hoechst33342 (blue). Black bar, 10 μm. (B) Typical fluorescence images of KD cells that were incubated with 4 μM WA for 24 h and then treated with ROS detection reagents using the Image-iT LIVE Green ROS Detection Kit. To highlight the weak ROS signals (i), the image of nuclear DNA signals stained with Hoechst33342 (ii) is separately shown. Green bar, 100 μm.(TIF)Click here for additional data file.S8 FigTypical images obtained by immuno-electron microscopy of TIG-1  and PC-3  cultured for 4 h in the absence  or presence  of 4 μM WA. Multiple magnifications are shown (i–iii). Scale bars all indicate 1 μm in the corresponding images.(TIF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 TableGenes were arranged in descending order of fold change in PC-3; only the genes with a fold change > 4 in DU-145 are listed. HSP-related, EGR-related, Fos-related, and BAG genes are highlighted in red, green, blue, and pink font, respectively.(TIF)Click here for additional data file.


These cells represent an orthologous cell type for expanded cells from human cord or placenta. Persistence, engraftment, expansion and anti-tumor efficacy of these fetal liver derived intermediate NK(FLiNK) cells were evaluated in allogeneic or syngeneic recipient mice after adoptive transfer. This study supports the possibility of clinical therapeutic application of ex vivo generated NK cells from HSC sources.Clinical studies have demonstrated that adoptive transfer of allogeneic Natural Killer (NK) cells can play a therapeutic role in hematological malignancies and potentially solid tumors. We established and characterized + cells (2X105) or Renca-luciferase+ cells were injected into recipient mice via tail vein to establish pulmonary metastasis. Tumor bearing mice received 2.5Gy irradiation followed by 5-10X106 NK cells by tail vein injection and administration of rIL-2 . Tumor growth was measured by IVIS, lung weight, total body weight obtained, and tumor number recorded.HSC were isolated from C57BL/6(B6) fetuses (day 18-20 of gestation) using a Hematopoietic Stem Cell isolation kit and cultured with a cytokine cocktail in three stages over 21 days. FLiNK cells were harvested and characterized prior to administration. MC38-luciferase+CD16-NK1.1-CD34- NK progenitor cells were successfully isolated from mouse fetal livers, and propagated over 21 days to give rise to FLiNK cells, which are CD3-NK1.1+NKp46+CD94+NKG2D+KLRG1+CD244+. Adoptive transfer of syngeneic FLiNK cell significantly inhibits development of mouse lung metastasis, while allogenic FLiNK showed slightly inhibitory effects. Compared with conventional splenic NK cells, FLiNK cells exhibited higher in vitro cytolytic activity against MC38 group (n=17) to 50 days in FLiNK recipients(n=15), (p < 0.05). Using allogeneic Balb/c Renca tumor bearing mice, B6 FLiNK also demonstrated anti-tumor effects, with control (no NK) survival of 25 days(n=5) compared with 30 days in the FLiNK group (n=5).c-kitex vivo protocol to obtain NK1.1+ cells from c-kit+CD16-NK1.1-CD34-NK progenitor cells from the mouse fetal liver. These FLiNK cells, on adoptive transfer, inhibit development of syngeneic and allogenic mouse lung metastasis. Proposed clinical trials of allogeneic NK cells in patients with pulmonary metastatic renal cancer are in preparation.This study successfully established an


In vitro C-ter-J28+-loaded DCskewed CD3+ T-cells toward Th1 polarization. C-ter-J28+-DC-vaccinations selectively enhanced cell immunoreactivity to Panc02, as demonstrated by CD4+- and CD8+-T-cell activation, increased percentages of CD4+- and CD8+-T-cells and NK1.1+ cells expressing granzyme B, and T-cell cytotoxicity. Prophylactic and therapeutic C-ter-J28+-DC-vaccinations reduced ectopic Panc02-tumor growth, provided long-lasting protection from Panc02-tumor development in 100% of micebut not from melanoma, and attenuated progression of orthotopic tumors as revealed by MRI. Thusmurine DC loaded with pancreatic tumor-specific glycoepitope C-ter-J28+ induce efficient anticancer adaptive immunity and represent a potential adjuvant therapy for patients afflicted with PDAC.Oncofetal fucose-rich glycovariants of the pathological bile salt-dependent lipase (pBSDL) appear during human pancreatic oncogenesis and are detected by themonoclonal antibody J28 (mAbJ28). We aimed to identify murine counterparts onpancreatic ductal adenocarcinoma (PDAC) cells and tissue and investigate the potential of dendritic cells (DC) loaded with this unique pancreatic tumor antigen to promote immunotherapy in preclinical trials. Pathological BSDLs purified from pancreatic juices of patients with PDAC were cleaved to generate glycosylated C-terminal moieties (C-ter) containing mAbJ28-reactive glycoepitopes. Immunoreactivity of the murine PDAC line Panc02 and tumor tissue to mAbJ28 was detected by immunohistochemistry and flow cytometry. C-ter-J28 Pancreatic ductal adenocarcinoma (PDAC) remains one of the most universally lethal diseases, with a 5-year survival rate below 5% . Pancrea+)  and 11±0.58 in non-vaccinated mice [n = 6]; two-way ANOVA F2,30 = 0.7151, P = 0.4973 for interaction; F1,30 = 9.694, P = 0.0040 for treatment effect; F2,30 = 27.29, P < 0.0001 for time effect; Bonferroni post hoc test *P < 0.05 for group comparison at d17-18)  and reduced secondary abdominal disease   . MRI shobladder) . Pleural+, we turned our attention towards bile salt-stimulated lipase (BSSL) present in human milk, which differs from BSDL only in glycosylation pattern  containing antigens. Cultures were carried out in triplicates or quadruplicates.Nine days after immunization, a cell suspension was prepared from draining lymph nodes (LN). T cells were purified using a negative isolation kit (Invitrogen) when indicated. After labeling with carboxyfluorescein succinimidyl ester (CFSE) , cells were plated at 2-4 × 10). At days 2 and 4, supernatant was removed and replenished with fresh DC media. IL-4 (20 ng/ml) was added when indicated. At days 5-6, DC were loaded with antigens. Maturation was induced with a combination of LPS  and 3T3 murine fibroblasts transfected with murine CD40L at 1/5 ratio (3T3/DC). At days 6-7, expression of surface membrane markers was controlled by cytometry analysis.DC were generated from C57Bl/6 mouse bone marrow according to Inaba's protocol , and culCulture supernatants were collected for cytokine detection by ELISA  and by RayBio® Mouse Cytokine Antibody Array I (RayBiotech), before being revealed using Gbox system .Cytotoxicity was assessed by lactate dehydrogenase (LDH) release assay using the CytoTox 96 Nonradioactive Cytotoxicity kit  following the manufacturer's protocol.in vivo on days 7-8, 9-10 and 17 or 18 after orthotopic tumor implantation by serial MRI. Twelve tumor-bearing and two control mice were imaged at very high field on an 11.75 T vertical Bruker AVANCE 500 WB wide-bore MR system  [3, 20 contiguous slices. Total acquisition time was 8 to 12 minutes per plane depending on the respiration rate. Tumor growth, morphology and spread were evaluated by two trained MRI scientists (ATP-B and AV) blinded to the treatment conditions on 20 contiguous slices in three orthogonal planes covering the entire primary tumor, using manual volumetry and a disease progression score inspired by the Tumor-Node-Metastasis (TNM) staging system [DC-induced response was assessed Germany) , with a g system .P < 0.05 were considered significant; P < 0.1, borderline significant. The survival curves were determined using the Kaplan-Meier method. The log-rank test was used to compare curves between study and control groups. Principal component analysis (PCA) on variances of MRI parameters was performed with JMP 9 software.The statistical analysis was performed using the two-way analysis of variance (ANOVA) test followed by a Bonferroni test, and the Mann-Whitney, the Kruskall-Wallis and the Fisher's exact tests. Values are reported as means ± SEM. Values of


In both sexes, 12-hydroxy-NVP levels were negatively correlated with ApoA1 . In men, ApoA1 was positively correlated with aspartate aminotransferase , while in women ApoA1 was associated  with alanine aminotransferase (ALT). These results show sex differences in NVP-induced ApoA1 synthesis. The higher ApoA1 levels in women might stabilize SULT2B1 [Nevirapine (NVP) is associated with severe liver and skin toxicity through sulfotransferase (SULT) bioactivation of the phase I metabolite 12-hydroxy-NVP . The fem


C. elegans intestine provides a simple system for studying how a single progenitor, the E blastomere, builds an epithelial tube of 20 cells. As the E descendants divide, they form a primordium that transitions between different shapes over time. We used cell contours, traced from confocal optical z-stacks, to build a 3D graphic reconstruction of intestine development. The reconstruction revealed several new aspects of morphogenesis that extend and clarify previous observations. The first 8 E descendants form a plane of four right cells and four left cells; the plane arises through oriented cell divisions and VANG-1/Van Gogh-dependent repositioning of any non-planar cells. LIN-12/Notch signaling affects the left cells in the E8 primordium, and initiates later asymmetry in cell packing. The next few stages involve cell repositioning and intercalation events that shuttle cells to their final positions, like shifting blocks in a Rubik’s cube. Repositioning involves breaking and replacing specific adhesive contacts, and some of these events involve EFN-4/Ephrin, MAB-20/semaphorin-2a, and SAX-3/Robo. Once cells in the primordium align along a common axis and in the correct order, cells at the anterior end rotate clockwise around the axis of the intestine. The anterior rotation appears to align segments of the developing lumen into a continuous structure, and requires the secreted ligand UNC-6/netrin, the receptor UNC-40/DCC, and an interacting protein called MADD-2. Previous studies showed that rotation requires a second round of LIN-12/Notch signaling in cells on the right side of the primordium, and we show that MADD-2-GFP appears to be downregulated in those cells.Genetic and molecular studies have provided considerable insight into how various tissue progenitors are specified in early embryogenesis, but much less is known about how those progenitors create three-dimensional tissues and organs. The  C. elegans as a model system to address how progenitor cells form a three-dimensional organ. The fully formed intestine is a cylindrical tube of only 20 epithelial cells, and all of these cells are descendants of a single cell, the E blastomere. The E descendants form a primordium that changes shape over time as different E descendants divide and move. Cells in the primordium must continually adhere to each other during these movements to maintain the integrity of the primordium. Here, we generated a 3D graphic reconstruction of the developing intestine in order to analyze these events. We found that the cell movements are highly reproducible, suggesting that they are programmed by asymmetric gene expression in the primordium. In particular, we found that the conserved receptor LIN-12/Notch appears to modulate left-right adhesion in the primordium, leading to the asymmetric packing of cells. One of the most remarkable events in intestinal morphogenesis is the circumferential rotation of a subset of cells. We found that rotation appears to have a role in aligning the developing lumen of the intestine, and involves a conserved, UNC-6/netrin signaling pathway that is best known for its roles in the guided growth of neurons.This report uses the intestine of the nematode  C. elegans digestive tract provides a simple genetic model system for studying epithelial cell polarization and tube morphogenesis .In an earlier report, we noticed what appeared to be only minor defects in the intestine primordium of mutants defective in axon guidance molecules such as UNC-6/netrin and its receptors UNC-40/DCC and UNC-5 . As suital rings . Howeverelopment –46. Brie) allele . We obtaunc-6, unc-40, madd-2, and unc-5 strains expressing fluorescent membrane reporters to examine intestinal twist. unc-5 mutants embryos appeared to have normal intestinal twist. However, almost none of the unc-6, unc-40, or madd-2 mutant embryos showed normal rotation of all three of the int2-4 rings  background, then shifted the mutant embryos to restrictive temperature at the E8 stage. Each of 23 embryos lacked int ring rotation and showed symmetrical expression of MADD-2::GFP in R and L cells . The “wild-type” strain used for imaging was JJ2360, which is N2 Bristol containing reporters for intestine-specific membranes, general membranes, and for pharyngeal marginal cell nuclei. The general membrane reporter, ItIs44, encodes mCherry linked to a pleckstrin homology domain, which binds to phosphoinositide lipids at the plasma membrane  [JJ2482 [efn-2(ev658) and JJ2369], JJ2483 [vab-2(e96) and JJ2360], JJ2484 [vab-1(dx31) and JJ2360], JJ2486 [efn-4(bx80) and JJ2360], JJ2492 [lin-12(n941)/unc-32(e189) and JJ2360], JJ2502 [EFN-4-GFP built from CZ1566 (lin-15B(n765) juIs109) and OD70],::PAR-6)  (gift fr-1::GFP]  (gift frJJ2515 , JJ2521 , JJ2528 [madd-2(zu475) and JJ2360], JJ2529 [unc-5(e53) and JJ2360)], JJ2530 [unc-40(e1430) and JJ2360], JJ2531[tol-1(nr2013) and JJ2360)], JJ2532 , JJ2534 [unc-6(ev400) and JJ2360],], JJ2536 [RP835(MADD-2-GFP) gift from Peter Roy and OD70], and JJ2537 . Other mutant alleles examined are as follows: apx-1(zu347ts)V, efn-1/vab-2(e96)IV, efn-2(ev658)IV, efn-2(ev658)IV; efn-3(ev696)X, efn-4(bx80)IV; him-5(e1490)V, lin-12(n941), mab-20(ev574), madd-2(tr162)V; tris25;rrf-3(pk1426)II (gift from Peter Roy), madd-2(zu475)V, mab-20(ev574) (gift from Joe Culotti), smp-1(ev715)I; jcIs1 IV; him-5(e1490)V, smp-2(ev709)I; jcIs1 IV; him-5(e1490)V, plx-2(ev773); him-5(e1490)V, plx-1(ev724)IV; plx-2(ev773)II, plx-1(nc37) IV; him-5(e1490)V, plx-2(ev773) II; him-5(e1490) V, sax-3(ky123)X, smp-1(ev715) I; jcIs1 IV; him-5(e1490) V, smp-2(ev709) I; jcIs1 IV; him-5(e1490) V, slt-1(eh15)X, unc-5(e53)IV, unc-6(ev400)X, unc-40(e1430)I, vab-1(dx31)II, vab-2(ju1) efn-2(ev658)IV; efn-3(ev696)X, tol-1(nr2013)I.in437-578 gift froEmbryos selected for imaging were dissected from adult hermaphrodites and allowed to settle in dH20 onto a microscope dish  that was coated with polylysine (Sigma). This technique avoids compression and the severe distortion of the embryo associated with conventional mounting methods between an agar pad and a coverslip . Time-lahttps://www.blender.org/). We developed a “shadow” technique to estimate cell contact areas within the reconstructed primordium, based on the sophisticated light path tracking available with the Cycles Render option in Blender 2.6  mutant was isolated in a pilot screen from the progeny of a pool of 100 F1, JJ2360 adults following standard EMS  mutagenesis . Bars = 5 microns.(TIF)Click here for additional data file.S2 Fig(A) Oblique, surface views of the reconstructed primordium at 393 minutes. RaL contacts, but no LaR contacts, are visible between the int4 and int3 rings (left panel) and between the int6 and int5 rings (right panel). (B) Same views as panel A, but after removing the 3L and 5L cells. Note that int4, but not int6, had an additional, internal LaR contact (4L to 3R). (C) Quantitation of intestinal cell volumes over time. Previous studies showed that intestinal cell volumes increase sometime before hatching , which m(TIF)Click here for additional data file.S3 Fig(A-B) Supporting evidence that int ring rotation involves basal protrusions that extend transiently over complementary cells. Panels A and B are orthogonal projections of the same primordium shown in (TIF)Click here for additional data file.S4 Fig(A-E) Supporting data for (TIF)Click here for additional data file.S1 VideoThe top half of the frame shows the dorsal surface of the primordium and the bottom half shows the left surface; color coding as in (MOV)Click here for additional data file.S2 VideoThe first frame shows the left side of the intestine, dorsal up. Note that cells such as 3R and 4R are hexagons.(MOV)Click here for additional data file.S3 VideoThe first frame shows the right side of the E8 primordium; 2/5R has constricted dorsally, and 1/3R has engulfed a cell death (MSpaapp). 5R intercalation begins at 273 minutes; note the dorsal constrictions of 3R (beginning at 265 minutes) and 4R (beginning at 297 minutes).(MOV)Click here for additional data file.S4 VideoThe embryo is mounted without compression, so 8R is not visible in the initial frames. The reporter expressed in pharyngeal nuclei become visible at about 297 minutes, and were used for spatial and temporal reference throughout this study. The nuclei have threefold, radial symmetry relative to the lumen, with a dorsal right row (shown), a dorsal left row, and a ventral row. The focal plane selected shows the lateral protrusion (arrow at 289 minutes) that leads 2R intercalation. 2R reaches the dorsal roof of the primordium at 345 minutes, but undergoes little longitudinal expansion at the perimeter until 393 minutes, by which time the intestine and body are elongating. Similarly, 2L shows little longitudinal expansion by 417 minutes.(MOV)Click here for additional data file.S5 VideoThe first frame shows a dorsal view of the primordium, then flips to show a lateral view, followed by rotation of the primordium to show all surface contacts.(MOV)Click here for additional data file.S6 VideoThe movie shows that the int1 cells spread ventrally, then divide without rotating. The 2R and 2L intercalate and close together to form the int2 ring , then begin a clockwise rotation. The int3 cells begin rotation at 329 minutes. Note that the posterior int rings show no rotation over this period.(MOV)Click here for additional data file.


Bone marrow has recently been recognized as a novel source of stem cellsfor the treatment of wide range of diseases. A number of studies on murine bone mar-row have shown a homogenous population of rare stage-specific embryonic antigen 1(SSEA-1) positive cells that express markers of pluripotent stem cells. This study focuseson SSEA-1 positive cells isolated from murine bone marrow in an attempt to differentiatethem into insulin-secreting cells (ISCs) in order to investigate their differentiation potentialfor future use in cell therapy. This study is an experimental research. Mouse SSEA-1 positivecells were isolated by Magnetic-activated cell sorting (MACS) followed by characteriza-tion with flow cytometry. Induced SSEA-1 positive cells were differentiated into ISCs withspecific differentiation media. In order to evaluate differentiation quality and analysis,dithizone (DTZ) staining was use, followed by reverse transcription polymerase chainreaction (RT-PCR), immunocytochemistry and insulin secretion assay. Statistical resultswere analyzed by one-way ANOVA.Pdx1 (pancreatic transcription factors), Ngn3 (endocrine progenitor marker), Insulin1and Insulin2 (pancreaticβ-cell markers). Additionally, our results demonstrate expressionof Pdx1 and Glut2 protein and insulin secretion in response to a glucose challenge inthe differentiated cells. The results achieved in this study reveal that mouse bone marrow contains apopulation of SSEA-1 positive cells that expresses pluripotent stem cells markers such asSSEA-1, octamer-binding transcription factor 4 (OCT-4) detected by immunocytochem-istry and C-X-C chemokine receptor type 4 (CXCR4) and stem cell antigen-1 (SCA-1)detected by flow cytometric analysis. SSEA-1 positive cells can differentiate into ISCscell clusters as evidenced by their DTZ positive staining and expression of genes suchas Our study clearly demonstrates the potential of SSEA-1 positive cellsto differentiate into insulin secreting cells in defined culture conditions for clinical ap-plications. Diabetes is a global health problemand one ofthe leading causes of morbidity and mortality inmany countries, and its frequency has beensteadilyincreasing worldwide . TransplA number of studies have suggested that bonemesenchymal stem cells (BMSCs) can differentiateinto insulin secreting cells in Dulbecco’sModified Eagle Medium (DMEM) supplementedwith certain growth factors and induceragents , 2. HoweHowever, experimental evidence from severalstudies indicates that the contribution of bonemarrow derived stem cells to organ/tissue regenerationmay be explained not by BMSCs butrather by the presence of SSEA-1 positive stemcells in bone marrow , 6. ThisThis study is an experimental research. Allanimals were treated according to guidelines,approved by Tehran University of Medical SciencesEthics Committee under the code number90-03-33-15668. Bone marrow aspirate wascollected from the femur and tibia of 2–4 weeksold C57BL/6 mice  byvigorous flushing with DMEM . Erythrocytes were eliminated using erythrocytelysing solution . Magneticactivatedcell sorting  wasused to separate the suspension of bone marrowmononuclear cells (BMMNCs) according to theexpressed cell surface marker SSEA-1. BMMNCswere resuspended in cold MACS buffer  and incubated with microbeadconjugatedanti-SSEA-1 antibody  for 45 minutes at 4˚C on a plateshaker.The cells were then washed three timeswith MACS buffer.For the sorting of labeled SSEA-1 positivecells, were loaded on to a sterile LS column installed in a magneticfield . The columnwas rinsed several times with MACS bufferand negative unlabelled cells (flow-throughor negative fraction) were passed through andcollected. Trapped cells (positive or labelledfraction) were washed with MACS buffer afterremoval of the column from the magneticfield. To achieve higher purity, the positivefraction was reloaded on a second column andthe positive selection repeated, with the samplefinally being collected by centrifugation.Viability and purity of the isolated SSEA-1positive cells were evaluated by Trypan Blue exclusion and flow cytometryanalysis  respectively. For flowcytometry analysis, SSEA-1 positive cells werewashed with phosphate buffered saline  and resuspended inPBS plus 2 % fetal calf serum . The antibody stainingwas performed following standard protocols.250,000 cells were washed, incubated for 45-60 minutes with primary conjugated antibodies,washed three times, and analyzed using Becton-Dickinson flow cytometer . For everysample at least 20,000 events were counted andthe acquired data analysed using WinMDI 2.9software. The following antibodies were used:anti-CXCR4 , anti– SCA-1 and and anti-cluster determinant45 . Primary antibodieswere directly conjugated with fluorescein isothiocyanate.Undifferentiated SSEA-1 positive cells werecultured on a feeder layer of mouse embryonicfibroblasts  inDMEM with a low concentration of fetal bovineserum .The expansion of SSEA-1 positive cellswascontinued for 9 dayswith a change of mediumevery 3-4 days. Following expansion, cellswere trypsinized and expanded SSEA-1 positivecellswere isolated from MEF cells by flowcytometry. SSEA-1 positive cellswere seeded at1×106 cells per well to gelatin  coated plates in DMEM/F-12 mediumsupplemented with 4 mM L-glutamine , 4.5 g/l glucose , 1% heat-inactivated FBSand 50 ng/ml of recombinant human Activin A. After 48 hours, the mediumwas exchanged and differentiation wascarried out in DMEM/F12 medium with 4 mML-glutamine, 4.5 g/l glucose, 5% heat-inactivatedFBS in the presence of N2 supplement , B27 supplement and 10mM Nicotinamide .Medium was changed every second day. Isletlikeclusters appeared after 21days of culture.DTZ  stock solutionwas prepared by dissolving 100 mg of DTZin 5 ml of dimethyl sulfoxide . Cells induced for 21dayswere washed with PBS buffer twice. Next 1 mLPBS buffer and 10 μL DTZ stock solutions wereadded and the cells were stored a 37˚C incubatorfor 15 minutes. The crimson-red-stainedclusters were examined with a phase-contrastmicroscope .Pdx1 (pancreatic transcription factors), Ngn3(endocrine progenitor marker), glucagon, Amylase(exocrine pancreas marker), Insulin1 andInsulin2 (pancreatic β-cell markers).SSEA-1 positive cells and new producedISCs were washed and immediately transferredto micro tubes containing TRIzolj reagent . Tubes were vortexed to lysethe cells and kept at -20˚C until analysis. TotalRNA was extracted using a Nucleospin RNAIIkit . DNase treatmentapplied to clean up any possible genomicDNA contamination. cDNA synthesis wasundertaken with Revert AidTMH Minus FirstStrand cDNA Synthesi Kit .A sample of 2.5 ml of cDNA, 16PCRbuffer , 200 mM dNTPs,1.5 mM MgCl2, 0.5 mM each of forward andreverse primers and 1 unit Taq DNA polymerase was used toprepare reactionmixtures for PCR. PCR wasperformed with an optimized programme foreach primer. Amplified DNA fragments wereelectrophoresed on 2% agarose gel. The gelswere stained with ethidiumbromide (10 mg/ml) and the amplified fragments photographedunder Ultraviolet (UV) transilluminator . β-actin amplification was used as aninternalgene control. RNA from mouse insulinomacell line (MIN6B1) was used as positivecontrol. Negative controls were mock RT withdiethyl pyrocarbonate treated water instead of RNA or cDNAsamples. In table 1, the primer pairs used foramplification of following genes are listed:Pdx1,1:400 dilution, Abcam, USA) and anti-GlucoseTransporter2  primary antibodies overnight at 4˚C. Afterfive sequential three minute washes in PBS,appropriate secondary antibodies, FITCconjugatedanti-goat IgG  andPE-conjugated anti-rabbit IgG  were used. After three washes with PBS,nuclei were counterstained with 4´, 6-diamidino-2-phenylindole .Slides were mounted with 50% (v/v) glycerolin PBS and checked under an Olympus BX 60fluorescent microscope.SSEA-1 positive cells that had differentiatedinto insulin secreting cells were cytospinnedon to glass slides and allowed to dryfor 15 minutes. Cells were then washed withPBS and fixed with 4% icecold paraformaldehydefor 10 minutes.The next step in the caseof intracellular markers, was permeabilizationby 0.4% Triton X-100 .Non-specific binding sites were blockedby incubation of cells with 10% serum containingsecondary antibody species. After two morewashes in PBS, cells were incubated with optimaldilutions of primary antibodies overnightand with secondary antibodies for 1 hour. Brieflythe SSEA-1 positive cells were stained withantibodies to SSEA-1 , OCT-4 . SSEA-1 positive cells inducedinto ISCs were incubated with rabbit polyclonalanti-pancreatic-duodenal homeobox 1 . After3 hours, the medium was removed and stored at–20˚C. Cells were then washed three times withPBS and incubated for 3 hours in DMEM-highglucose(25 mM glucose). Thereafter, the mediumwas stored at –20˚C. The stored media werethen analyzed for insulin content using a directmouse insulin enzyme-linked immunosorbentassay kit  according to the manufacturer’s instructions. Non-inducedSSEA-1 positive cells were used as a control.Differentiated cells were seeded at 1×10Data are expressed as mean ± SD. Comparisonsbetween groups were carried out with one-wayANOVA. P<0.05 was considered statistically significant.In the current study, SSEA-1 positive cells wereisolated from bone marrow mononuclear cell suspensionderived from 2-4 weeks old C57BL/6 miceusing anti-SSEA-1 antibody and MACS. Immunofluorescencestaining showed that SSEA-1 positivecells expressed embryonic stem cell markersOCT-4  and SSEASSEA-1 positive cells at passages 3-5 were treatedwith differentiated medium and their morphologiccharacteristics were detected every day. UndifferentiatedSSEA-1 positive cells were sphericalin shape . Over 14Pdx1, Ngn3, glucagon, Amylase,Insulin1 and Insulin2. However, glucagonand Amylase genes expression became silent inthe cells that expressed of Pdx1, Ngn3, Insulin1and Insulin2 indicating more differentiatedbeta-like precursor cells. These results indicatethat SSEA-1 positive cells derived clusters possessthe potential to differentiate into functionalinsulin-producing cells.To determine whether the SSEA-1 positivecells had differentiated into insulin producingcells, the expression of genes related to the pancreaticendocrine development and function wasexamined by RT-PCR analysis. As a positivecontrol, the expression of β-actin was detectedindicating our experimental system being wasintact. As shown in Pdx1 and Glut2 expression wasexamined by immunofluorescence staining, representedas green dots in the immunofluorescenceassay.Immunofluorescence staining showed thatthe Pdx1 and Glut2 expression was positive or high (25 mMglucose) glucose and assayed culture supernatantsfor mouse insulin, as described earlier. As shownin In vitro generation of ISCs, responsible for insulinsynthesis, storage and release, from adult stemcells is an important advance for use in future therapiesfor diabetes mellitus . It isimportant for the genesis of nicotinamide adeninedinucleotide phosphate  and playInsulin secreting celldifferentiation and increase β-cell quantity  and the expressionof MafA a necessary switch in maturation ofISCs (Pdx1 appears also to play a role in thefate of endocrine cells, encoding for insulin andsomatostatin, two pancreatic endocrine products,while repressing glucagon. Thus, Pdx1 expressionapparently favors the production of ISCs ratherthan glucagon secreting cells (Pdx1 is required forISCs survival. Cells with reduced Pdx1 have anincreased rate of apoptotic programmed cell death(Our results revealed that turation : develop ofISCs . Pdx1 apng cells . In addill death.Glut2 incells after 21 days of induction, using immunofluorescence.Glut2, a glucose transporter withthe lowest affinity and the highest capacity forglucose, is expressed in pancreatic β-cell (Glut2 catalyzes glucose uptake by β-cells. Thisis the first step in signaling a cascade, leading toglucose-stimulated insulin secretion (Glut2is present in β-cell, but not in the other islet endocrinecells (We also detected the expression of c β-cell .Glut2 cecretion . Glut2inecells .These findings suggested that SSEA-1 positivecells might have the ability to secrete insulin in responseto glucose. We tested this latter predictionby analyzing the secretion of insulin in response toglucose stimulation, which is an important indicatorof functional pancreatic β-cells. Therefore, it wasdemonstrated that SSEA-1 positive cells derivedISCs are able to respond to glucose concentrationand behave as matured insulin producing cells.in vivoevaluation of glucose levels in a diabetic model.Our study clearly demonstrates the potential ofSSEA-1 positive cells to differentiate into insulinsecreting cells in defined culture conditions. Thisfinding has the potential for clinical application inthe form of a new procedure for diabetes stem celltherapy. However, further research is needed to testthe differentiated cells we obtained in an


The CpG island methylator phenotype (CIMP) of clear cell renal cell carcinomas (ccRCCs) is characterized by accumulation of DNA methylation at CpG islands and poorer patient outcome. The aim of this study was to establish criteria for prognostication of patients with ccRCCs using the ccRCC-specific CIMP marker genes.DNA methylation levels at 299 CpG sites in the 14 CIMP marker genes were evaluated quantitatively in tissue specimens of 88 CIMP-negative and 14 CIMP-positive ccRCCs in a learning cohort using the MassARRAY system. An additional 100 ccRCCs were also analyzed as a validation cohort.FAM150A, ZNF540, ZNF671, ZNF154, PRAC, TRH and SLC13A5, for discrimination of CIMP-positive from CIMP-negative ccRCCs were larger than 0.95. Criteria combining the 23 CpG units discriminated CIMP-positive from CIMP-negative ccRCCs with 100% sensitivity and specificity in the learning cohort. Cancer-free and overall survival rates of patients with CIMP-positive ccRCCs diagnosed using the criteria combining the 23 CpG units in a validation cohort were significantly lower than those of patients with CIMP-negative ccRCCs . Patients with CIMP-positive ccRCCs in the validation cohort had a higher likelihood of disease-related death  than those with CIMP-negative ccRCCs.Receiver operating characteristic curve analysis showed that area under the curve values for the 23 CpG units including the 32 CpG sites in the 7 CIMP-marker genes, i.e. The established criteria are able to reproducibly diagnose CIMP-positive ccRCCs and may be useful for personalized medicine for patients with ccRCCs.The online version of this article (doi:10.1186/1471-2407-14-772) contains supplementary material, which is available to authorized users. Clear cell renal cell carcinoma (ccRCC) is the most common histological subtype of adult kidney cancer . In geneFAM150A, GRM6, ZNF540, ZFP42, ZNF154, RIMS4, PCDHAC1, KHDRBS2, ASCL2, KCNQ1, PRAC, WNT3A, TRH, FAM78A, ZNF671, SLC13A5 and NKX6-2, which are hallmarks of CIMP in ccRCCs  and those determined by the present MassArray analysis were confirmed . (PPTX 263 KB)Additional file 6: Figure S2: Scattergrams of DNA methylation levels determined by Infinium assay and those determined by MassArray analysis. Additional file 7: Table S5: Differences of DNA methylation levels at all examined 193 CpG units including 299 CpG sites of 14 CpG island methylator phenotype (CIMP) marker genes between CIMP-negative and CIMP-positive clear cell renal cell carcinomas (ccRCCs) in the learning cohort. (PDF 75 KB)Additional file 8: Table S6: Eighty-six CpG units showing area under the curve (AUC) values larger than 0.9 in receiver operating characteristic curve analysis for discrimination of CpG island methylator phenotype (CIMP)-positive clear cell renal cell carcinomas (ccRCCs) from CIMP-negative ccRCCs in the learning cohort. (PDF 40 KB)P = 1.01 × 10−3) and overall  survival rates of patients with CIMP-positive grade 3/4 ccRCCs were significantly lower than those of patients with CIMP-negative grade 3/4 ccRCCs (log-rank test). The cancer-free  and overall  survival rates of patients with CIMP-positive stage III/IV ccRCCs were significantly lower than those of patients with CIMP-negative stage III/V ccRCCs (log-rank test). Patients who underwent curative resection are included in panels A and C. (PDF 94 KB)Additional file 9: Figure S3: Kaplan–Meier survival curves of patients with CIMP-positive and -negative high-grade (grades 3 and 4) and high-stage (stages III and IV) clear cell renal cell carcinomas (ccRCCs) in the validation cohort. The cancer-free (Panel A,


In vivo interaction of miR-183/96/182 and Rac1 mRNA in retina was confirmed using miR-CATCH. Additional miRNAs  were both predicted to target Rac1 mRNA and enriched by Rac1-miR-CATCH. Other Rac1-miR-CATCH-enriched miRNAs  were not predicted to target Rac1. Furthermore, levels of ~25% of the retinal Rac1 interactors were determined by LC-MS/MS; expression of Rap1gds1 and Cav1 was elevated. Our data suggest significant utilisation of miRNA-based regulation in retina. Possibly more than 30 miRNAs interact with Rac1 in retina, targeting both UTRs and coding regions.miRNA dysregulation is a hallmark of many neurodegenerative disorders, including those involving the retina. Up-regulation of miR-1/133 and miR-142, and down-regulation of miR-183/96/182 has been described in the RHO-P347S mouse retina, a model for a common form of inherited blindness. High-throughput LC-MS/MS was employed to analyse the protein expression of predicted targets for these miRNAs in RHO-P347S mouse retinas; 133 potential target genes were identified. Pathway over-representation analysis suggests G-protein signaling/visual transduction, and synaptic transmission for miR-1, and transmembrane transport, cell-adhesion, signal transduction and apoptosis for miR-183/96/182 as regulated functions in retina. Validation of miRNA-target mRNA interactions for miR-1, miR-96/182 and miR-96 targeting Ctbp2, Rac1 and Slc6a9, respectively, was demonstrated  In silico predictions reveal that each miRNA can in principle target many (hundreds) mRNAs, while individual mRNAs can also be targeted by many miRNAs31578989MicroRNAs (miRNAs) are small (~22-nt in mature form) non-coding RNAs involved in post-transcriptional regulation of gene expression in eukaryotes and virusesSimilar to many other human conditionsThe genes encoding miR-183/96/182 are clustered within 4 kb on mouse chr6qA3. This cluster is referred to as ‘sensory organ-specific’, is highly expressed in retina and regulated by light8in silico computed targets for the above six miRNAs. An interesting validated target, i.e. Rac1, previously implicated in retinal degeneration2021There is clear evidence of a link between retinal dysfunction  and altered miRNA expression. Far less is known about the target genes, which are post-transcriptionally regulated by these miRNAs in retina and indeed elsewhere. As mentioned above, prior work highlighted miR-1, miR-133, miR-142 and miR-183/96/182 dysregulation in RP models1818http://www.biomart.org). The intersection between the retinal transcriptome and predicted miRNA targets was 4718 genes ; 1237 (~86%) of these genes were mapped to the retinal transcriptome library (11http://geneontology.org); 72.4% of proteins from retinal membrane protein extracts had ‘membrane’ among their GO terms , yet they were enriched in Rac1-miR-CATCH capture versus control samples  explored.Previously, six miRNAs with altered expression in R347 mouse retinas were identifiedin vitro UTR assays. For Rac1, miR-183/96/182 interactions were additionally demonstrated using in vivo miR-CATCH.As the retina is a complex tissue composed of many cell types, in some cases, it is possible that the determined miRNA1212As the retina is rich in membranes and associated proteins, beside standard protein extraction from whole retinas, proteins were extracted from enriched retinal membranes811 proteins exhibited differential expression between R347 and wt mouse retinas. To explore the possible function(s) of these proteins in retina, pathway over-representation analysis was performedUnless noted, according to miRTarBaseThe sensory organ-specific miR-183/96/182 cluster has been studied extensively in retinaIn vitro 3′UTR assays confirmed miR-96 targeting of Slc6a9, while miR-182 (a target site with lower conservation) did not suppress Slc6a9 n retina . We testin silico miRNA target predictions and Rac1-miR-CATCH data was explored to identify additional miRNAs, which may target Rac1 mRNA in retina. Apart from the conserved miR-96/182 target site, eight additional miR-183/96/182 target sites in Rac1 were predicted  transgenic mice (on 129 background)Target sites for mmu-miR-1a-3p (miR-1), miR-133a-1 (miR-133), miR-142a-3p (miR-142), miR-183-5p (miR-183), miR-96-5p (miR-96) and miR-182-5p (miR-182) were predicted employing Diana-microT (v.3.0)11http://geneontology.org) analysis was carried out using ‘membrane’ as query term. Pathway enrichment analysis in ConsensusPathDB (Release MM9)http://www.reactome.org), Wikipathways (http://www.wikipathways.org) and MouseCyc (http://mousecyc.jax.org) databases; the minimal overlap with the input list and the p value cut off were set to 2 and 0.05, respectively. The mouse Rac1 interactome was generated in InnateDBhttp://www.uniprot.org) and GeneCards (http://www.genecards.org), and modified for a number of genes, in particular for the ones with ‘unknown’ localization. Mouse orthologs of previously identified photoreceptor outer segment Rac1 interactors were added manuallyLC-MS-MS and label-free quantification were performed as describedMouse eyes were fixed in either 4% paraformaldehyde or 4% paraformaldehyde/2% acetic acid at 4 °C for 4 h. Eyes were washed, cryoprotected, embedded in PolyFreeze . 12 μm cryosections were processed for Api5 , Arcn1 , Cav1 , Ctbp2 , Dgke , Flot2 , Igf1r , Negr1 , Rac1 , Slc6a9 , Tagln3  immunohistochemistries using overnight incubation at 4 °C. Some sections were co-incubated with 20 μg/ml of lectin PNA-AlexaFluor-488 conjugate . Sections were then incubated with Cy3-conjugated secondary antibodies  for 2 h and counterstained with DAPI. Specimens were analysed with an Axiophot fluorescent microscope  and images acquired utilising AnalySIS^B software . Images taken with different filter sets were overlaid in Adobe Photoshop CS6 .5 HeLa cells/well were seeded in 24-well plates; 24 h later, cells were co-transfected with 400 ng 3′UTR plasmids and 16 pmoles of each pre-miRNAs using Lipofectamine 2000 . 24 h post-transfection, cells were analysed using a Dual-Glow luciferase assay system . ANOVA and post-hoc Tukey’s multiple comparison test were performed and p < 0.05 values were accepted as statistically significant.Mouse Rac1 (NM_009007), Ctbp2 (NM_009980) and Slc6a9 (NM_008135) 3′UTR sequences in dual firefly luciferase/Renilla luciferase expression vectors  were obtained from GeneCopoeia Inc. . Synthetic pre-miRNAs for mmu-miR-1a-3p (PM10617), mmu-miR-96-5p (PM10422), mmu-miR-182-5p (PM13088) and negative control were procured from Ambion (Thermo Fisher Scientific). 1.4 × 10To co-purify Rac1 mRNA with its endogenously bound miRNAs, miRNA capture affinity technology (miR-CATCH)Total RNA from retina and miR-CATCH samples was isolated using the miRCURY Cell & Plant kit . An on-column DNase step (1 h at 37 °C) was added to the protocol using RNase-free DNase . RT-qPCR was performed for Rac1 (NM_009007.2), Folr1 (XM_006507360.1), Plxna4 (NM_175750.3), Tmed5 (XM_006535240.1), Ttc21b (NM_001047604.1) and Actb (NM_007393.3) mRNAs; primer sequences are given in miRNA expression was analysed at Exiqon Services  and in-house. Exiqon miRNA PCR panel profiling (372 miRNA assays) was performed according to the company’s assay pipeline. RNA was reverse transcribed using the miRCURY LNA Universal RT microRNA PCR, Polyadenylation and cDNA synthesis kit (Exiqon). cDNA was assayed using the protocol for miRCURY LNA Universal RT microRNA PCR. Each miRNA was assayed once on the microRNA Ready-to-Use PCR, Mouse & Rat panel I (Exiqon rodent miRNA PCR panel) using ExiLENT SYBR Green master mix and LightCycler 480 Real-Time PCR System . As the miR-CATCH capture and scrambled control samples were expected to have different RNA compositions, no normaliser was used and comparison of the background-filtered raw expression data was performed. For in-house TaqMan microRNA assays (Applied Biosystems) 5 μl of miR-CATCH capture and scrambled RNA sample was reverse-transcribed using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). One μl of the RT reaction was analysed in triplicate in StepOnePlus real-time PCR system (Applied Biosystems) using TaqMan Universal Master Mix II (Applied Biosystems). Quantification was performed utilising the comparative Ct methodHow to cite this article: Palfi, A. et al. microRNA regulatory circuits in a mouse model of inherited retinal degeneration. Sci. Rep.6, 31431; doi: 10.1038/srep31431 (2016).


We wished to determine if a similar targeting mechanism operates to maintain DNA methylation at repetitive sequences.DNA methylation contributes to genomic integrity by suppressing repeat-associated transposition. In addition to the canonical DNA methyltransferases, several auxiliary chromatin factors are required to maintain DNA methylation at intergenic and satellite repeats. The interaction between Lsh, a chromatin helicase, and the Lsh−/− and Dnmt3b−/− somatic cells. DNA methylation is predominantly lost from specific genomic repeats in Lsh−/− cells: LTR -retrotransposons, LINE-1 repeats and mouse satellites. RNA-seq experiments demonstrate that specific IAP LTRs and satellites, but not LINE-1 elements, are aberrantly transcribed in Lsh−/− cells. LTR hypomethylation in Dnmt3b−/− cells is moderate, whereas IAP, LINE-1 and satellite elements are hypomethylated but silent. Repressed LINE-1 elements in Lsh−/− cells gain H3K4me3, but H3K9me3 levels are unaltered, indicating that DNA hypomethylation alone is not permissive for their transcriptional activation. Mis-expressed IAPs and satellites lose H3K9me3 and gain H3K4me3 in Lsh−/− cells.We mapped genome-wide DNA methylation patterns in de novo methylation to repeat sequences, which is subsequently maintained by Dnmt1 to enforce selective repeat silencing.Our study emphasizes that regulation of repetitive elements by Lsh and DNA methylation is selective and context dependent. Silencing of repeats in somatic cells appears not to be critically dependent on Dnmt3b function. We propose a model where Lsh is specifically required at a precise developmental window to target  The importance of DNA methylation is emphasised by the phenotypes of the DNA methyltransferase mouse knockouts: Dnmt1 knockouts are embryonic lethal between E9.5 and E10.5, and Dnmt3b knockouts between E14.5 and E16.5 : [GEO:GSM1090110] - Dnmt1+/+ MEFs (Bisulfite-seq); [GEO:GSM1090111] - Dnmt1−/− MEF (Bisulfite-seq). RNA-seq in Dnmt1+/+ and Dnmt1−/− MEFs [GEO:GSE44277]: [GEO:GSM1081738- GEO:GSM1081740] - Dnmt1+/+ MEF - (Replicates 1–3); [GEO:GSM1081741- GSM1081743] - Dnmt1−/− MEF - (Replicates 1–3). RNA-seq in WT and triple knockout DNA methyltransferase ES cells [GEO:GSE29413]: [GEO:GSM727427] - J1 WT (mRNA-seq); [GEO:GSM727428] - J1 DMNT TKO (mRNA-seq) [Raw and processed data were deposited into the Gene Expression Omnibus and is freely available [GEO:GSE52479]. Other datasets utilised in this study are as follows. DNA methylation in RNA-seq) .p53−/− | Dnmt1−/− double knockout MEFs; Dnmt: DNA methyltransferase; ERV: endogenous retrovirus; ES cells: embryonic stem cells; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; H3K27me3: histone H3 lysine 27 trimethylation; H3K4me2: histone H3 lysine 4 dimethylation; H3K4me3: histone H3 lysine 4 trimethylation; H3K9me3: histone H3 lysine 9 trimethylation; HELP-seq: HpaII tiny fragment enrichment by ligation-mediated PCR followed by tag sequencing; IAP: intracisternal A-type particle; LINE-1: long interspersed nuclear elements; LTR: long terminal repeat; MeDIP: methylated DNA immunoprecipitation using 5meC antibodies; MEF: mouse embryonic fibroblast; ORF1: open reading frame 1; qPCR: quantitative polymerase chain reaction; qRT-PCR: quantitative reverse transcription polymerase chain reaction; RNA-seq: messenger RNA sequencing; SINE: short interspersed nuclear elements; TKO: triple knockout; UTR: untranslated region; VLP: viral-like particle; WT: wild type.ChIP: chromatin immunoprecipitation; CpG: palindromic CG DNA dinucleotide; CGI: CpG island; DAPI: 4′,6-diamidino-2-phenylindole; DN: The authors declare that they have no competing interests.DSD, IRA and RRM conceived and designed the experiments. DSD performed the experiments with HC performing the immunofluorescence and TD the transmission electron microscopy. DSD, MS and JG performed the HELP-seq library preparation. DSD, RRM and IRA analysed the data. RA, CS and IRA contributed reagents, materials and analysis tools. DSD and RRM wrote the paper. All authors read and approved the final manuscript.IRA and RRM are joint senior authors.Included are seven supplemental figures  and one supplemental table (Table S1).Click here for file


Here, we establish the temporal de novo appearance of Sup35 aggregates during such overexpression in relation to other cellular proteins. Fluorescently-labeled Sup35 initially forms one or a few dots when overexpressed in [+PIN] cells. One of the dots is perivacuolar, colocalizes with the aggregated Rnq1 dot and grows into peripheral rings/lines, some of which also colocalize with Rnq1. Sup35 dots that are not near the vacuole do not always colocalize with Rnq1 and disappear by the time rings start to grow. Bimolecular fluorescence complementation failed to detect any interaction between Sup35-VN and Rnq1-VC in [PSI+][PIN+] cells. In contrast, all Sup35 aggregates, whether newly induced or in established [PSI+], completely colocalize with the molecular chaperones Hsp104, Sis1, Ssa1 and eukaryotic release factor Sup45. In the absence of [+PIN], overexpressed aggregating proteins such as the Q/N-rich Pin4C or the non-Q/N-rich Mod5 can also promote the de novo appearance of [PSI+]. Similar to Rnq1, overexpressed Pin4C transiently colocalizes with newly appearing Sup35 aggregates. However, no interaction was detected between Mod5 and Sup35 during [+PSI] induction in the absence of [PIN+]. While the colocalization of Sup35 and aggregates of Rnq1 or Pin4C are consistent with the model that the heterologous aggregates cross-seed the de novo appearance of [PSI+], the lack of interaction between Mod5 and Sup35 leaves open the possibility of other mechanisms. We also show that Hsp104 is required in the de novo appearance of [+PSI] aggregates in a [PIN+]-independent pathway.Prions are self-perpetuating conformational variants of particular proteins. In yeast, prions cause heritable phenotypic traits. Most known yeast prions contain a glutamine (Q)/asparagine (N)-rich region in their prion domains. [ De novo prion appearance can be made very frequent in yeast by transient overexpression of the prion protein in the presence of heterologous prions or prion-like aggregates. Here, we show that the aggregates of one such newly induced prion are initially formed in a dot-like structure near the vacuole. These dots then grow into rings at the periphery of the cell prior to becoming smaller rings surrounding the vacuole and maturing into the characteristic heritable prion tiny dots found throughout the cytoplasm. We found considerable colocalization of two heterologous prion/prion-like aggregates with the newly appearing prion protein aggregates, which is consistent with the prevalent model that existing prion aggregates can cross-seed the de novo aggregation of a heterologous prion protein. However, we failed to find any physical interaction between another heterologous aggregating protein and the newly appearing prion aggregates it stimulated to appear, which is inconsistent with cross-seeding.Certain proteins can misfold into β-sheet-rich, self-seeding aggregates. Such proteins appear to be associated with neurodegenerative diseases such as prion, Alzheimer's and Parkinson's. Yeast prions also misfold into self-seeding aggregates and provide a good model to study how these rogue polymers first appear.  Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), were shown to infect a variety of mammals de novo conversion of a protein to amyloid. For example, Aβ accelerated the in vivo aggregation of tau in vitroA number of other neurodegenerative diseases are also associated with conversion of a soluble protein to amyloid. For example, amyloid-like forms of Aβ and Tau, α-synuclein, huntingtin, FUS/TLS, TDP-43 or SOD1 are linked respectively to Alzheimer's (AD) Several proteins in the simple eukaryote yeast have been shown to convert from soluble to amyloid. The amyloid forms of these proteins are self-propagating prions associated with transmissible phenotypes +MOD] prion, composed of Mod5, as well as PrP, Aβ and α-synuclein do not contain Q/N rich domains For both human and yeast proteins, only a portion of the protein, called the prion domain (PrD), converts to amyloid. This portion of the protein is required for prion induction and propagation +PSI], the prion form of Sup35. In its native form, Sup35 (release factor 3) works with Sup45 (release factor 1) to promote translational termination at stop codons +PSI] forms when Sup35 molecules assemble into amyloid-like aggregates, causing loss-of-function in translation termination, which leads to read-through of stop codons The most well-studied yeast prion is [-psi] to [+PSI] was determined to be ∼5.8×10−7de novo appearance of [+PSI] dramatically, presumably by increasing the chance of Sup35 prion domains to misfold and interact +PSI] requires either the presence of another prion +PIN], the prion form of the Rnq1 protein. Although [+PIN] dramatically enhances the appearance of [+PSI], it is not required for [+PSI] propagation The spontaneous appearance of prions in yeast is extremely rare. Indeed, the conversion of prion-free cells, [+PIN] enhances de novo induction of [+PSI] will help us understand analogous interactions between heterologous human disease proteins. Several models have been proposed  and Rnq1 lead to the efficient induction of [+PSI] in the presence of [+PIN], even without Sup35 overexpression +PIN] variants preferentially cause the genesis of different variants of [PSI+] Understanding how [+PIN] prion that they are not available to prevent the appearance of the new prion, [+PSI] The titration model postulates that cellular factors responsible for the disassembly of aggregates and the refolding of misfolded proteins are so busy working on the existing [+PSI] Molecular chaperones, which are normally involved in proper protein folding play a critical role in the maintenance of yeast prions . Sup35NM-GFP overexpressed in [+PIN][-psi] cells   : 30% of HSP42 tagged with GFP +PIN][-psi] HSP42-GFP cells first caused the occasional appearance of cells with 1-6 dots, one of which always colocalized with the Hsp42-GFP dot   , bottom.+PIN]-promoted Sup35-RFP aggregation, all Rnq1 dots perfectly overlapped newly induced Sup35 dots around the vacuole, but additional Sup35 dots (away from the vacuole) without overlapping Rnq1 sometimes existed in those cells. In contrast, in established [+PSI] cells, all Sup35 dots partially overlapped Rnq1 dots, but additional Rnq1 dots without overlapping Sup35 also existed.In summary, after 6 h of [+PIN] cells also expressing Rnq1-GFP from its own promoter. Sup35-RFP formed rings 70% of which colocalized with Rnq1-GFP  were scored for [+PSI], 6.5% of these cells formed pink or white colonies and were able to grow on media lacking adenine (SD-Ade), indicative of [+PSI]  colocalize with Sup35 during its aggregation in the presence of [f [PSI+] , S8 Fig.+PSI] +PSI] aggregates  Pin4C-RFP formed large fluorescent dots , which were previously shown to substitute for [-] cells , Table 3ent dots . As expein4C-RFP .de novo aggregation, we overexpressed Pin4C-RFP in [-pin] HSP42-GFP cells in the presence vs. absence of Sup35NM overexpression  suggesting that cross-seeding may not be universal for [+PSI] induction. Curiously, in [+PIN] cells, overexpression of Mod5-VN and Sup35NM-VC resulted in first diffuse fluorescence and then the formation of a single (near the vacuole) to multiple dots over time in 15% of cells  in [-pin] HSP104 cells, caused endogenous Sup35-GFP molecules to form dots and short lines in 6% of the cells (n≈1200)  formed Sup35-GFP lines. Furthermore, all these lines appeared to be at the cell membrane as opposed to those seen in the cytoplasm in HSP104 cells.The Hsp104 chaperone requirement for the maintenance of [+] PSI+] , Fig. 5A(n≈1200)  and this+PSI] induction, we used Pin4C overexpression to substitute for [+PIN]. We co-overexpressed Sup35NM-GFP and Pin4C-RFP in [-pin] hsp104Δ cells  as inclusions, +PIN] cells with overexpressed BiFC-tagged Sup35NM and Rnq1  are found to overlap newly appearing Sup35 rings  +PSI] by overexpressing Pin4C -psi][PIN+] and [-psi][pin-] versions of 74-D694 with RNQ1-CFP under its own promoter integrated into the genomic TRP1 . GF647 was constructed by replacing chromosomal RNQ1 with KanMX4 in 74D-694 -pin] versions of these cells, they were grown on YPD plates with 5 mM GuHCl for three passes Yeast plasmids and strains used in this study are listed in GAL1 promoter unless otherwise stated. p1951 (Sup35NM-GFP) was constructed by inserting the SUP35NM BamHI-NotI fragment, and the GFP NotI-SacI fragment in-frame into the pRS413GAL1 plasmid backbone. p1893 (Sup35NM-VN) was constructed by replacing the GFP NotI-SacI fragment in p1951 with the VN173 NotI-SacI fragment PCR-amplified from pFA6a-HIS3MX6-pGAL1-VN173GAL1-SUP35NM-VN XhoI-SacI fragment in p1893 to the pRS415 backbone. p2170 was constructed by replacing the SUP35NM BamHI-NotI fragment in p1893 with the MOD5 BamHI-NotI fragment PCR-amplified from the genome. p2171 was constructed by replacing the SUP35NM BamHI-NotI fragment in p1892 with the MOD5 BamHI-NotI fragment PCR-amplified from the genome. p1892 (Sup35NM-VC) was constructed by (1) replacing the GFP NotI-SacI fragment in p1951 with the VC155 NotI-SacI PCR-amplified from pFA6a-HIS3MX6-pGAL1-VC155GAL1-SUP35NM-VC155 XhoI-SacI fragment into the XhoI-SacI sites of pRS414. p1894 was constructed by replacing the SUP35NM BamHI-NotI fragment in p1892 with the RNQ1 BamHI-NotI fragment PCR-amplified from the genome. p1984 was constructed by cloning the XmaI-Sal1 fragment of p1156 into p742. p2036 was constructed by replacing the YFP SpeI-XhoI fragment in p1752 with the NM(TAA) SpeI-XhoI fragment PCR-amplified from the genome URA3 or LEU2 markers, respectively were constructed in a two-step cloning: (1) The GFP NotI-SacI fragment in p1951 was replaced with the RFP NotI-SacI fragment amplified from p1708. (2) Then, this GAL1-NM-RFP XhoI-SacI fragment was cloned into pRS416 (URA3) and pRS415 (LEU2) backbones, respectively. All other plasmids listed in All overexpression plasmids in this study were driven by the Yeast strains were cultivated using standard media and growth conditions de novo Sup35 aggregation. In time course experiments where time exceeded 48 h, cultures were diluted back to OD∼0.5 in a fresh growth media to keep cells in exponential phase.Yeast cells with Sup35 overexpression plasmids were grown in synthetic liquid selection media (SRaf) overnight. Unless otherwise stated, 2% Gal was added to the culture (OD∼0.5) to induce de novo [+PSI] was induced in [-psi][+PIN] cells, they were grown on synthetic dropout media with 2% dextrose (SD) for many generations to maintain [+PSI] (∼8 days). Then, they were grown in 0.05% Gal for 3-4 h to allow Sup35NM-YFP to decorate existing [+PSI] aggregates.After +PSI] induced de novo was scored in yeast as described previously +PSI] but not [-psi] causes read-through of the nonsense mutation, ade1-14. The ade1-14 mutation causes the accumulation of a red by-product in the adenine biosynthesis pathway, so [-psi] ade1-14 cells are red. In [+PSI] cells, when nonsense mutations are suppressed, ade1-14 cells become pink or white in rich media (YPD). In addition, the read-through of the ade1-14 allele in [+PSI] allows them to grow in media lacking adenine (SD-Ade), while [-psi] cells cannot grow on this medium + cells are of [+PSI] rather than suppressor mutants, they were mated with a tester strain  to look for multiple fluorescent dots formed by endogenous Sup35-GFP in only [+PSI] cells..To locate the vacuole, cells were stained with FM4-64 as described previously Aggregates formed in cells by fluorescently labeled proteins were examined with a Nikon Eclipse E600 fluorescent microscope (100X oil immersion) and/or an Olympus FV1000 confocal microscope . Colocalization was visualized by the confocal microscope using the channels of interest and by moving the focal plane up and down. Z-stacks were analyzed to confirm colocalization of proteins with 8-12 layers, with 0.5-1 µm increments. In dual color RFP/GFP studies, the RFP channel was always examined first to prevent visualization of activated GFP in the RFP channel.Upon the induction of overexpression of proteins tagged with VN or VC with galactose, cells were lysed as described previously S1 Figde novoSup35 aggregates in cells with endogenous SUP35-GFP. A. In [+PIN] cells, similar to Sup35NM (+PSI] (n≈550). B. In [-pin] control cells with endogenous SUP35-GFP (GF658), untagged Sup35NM was overexpressed from p2036 in 2% Gal, but there was no aggregation.Visualization of induced  Sup35NM , high le(PDF)Click here for additional data file.S2 Fig+PIN] cells with endogenous VPH1-GFP by growth in 2% Gal.Sup35 aggregates initially appear near the vacuole, and extend to the periphery thereafter. Sup35NM-RFP was overexpressed (p2018) in [(PDF)Click here for additional data file.S3 FigA. Failure of colocalization in some [+PIN] cells. Sup35-RFP was overexpressed (p1678) in 2% Gal for 24 h in [+PIN] cells with p1730 expressing Rnq1-GFP from its own promoter. Rnq1-GFP remained diffuse (top), or as dots (bottom) in, respectively, 20% and 10% of cells with Sup35-RFP rings/lines  in 2% Gal for 24 h in [-pin] cells with p1730 expressing Rnq1-GFP. Neither of them aggregated in [-pin]. C. Control cells without Rnq1-GFP. Sup35-RFP was overexpressed (p1678) in 2% Gal for 24 h in [+PIN] cells without p1730 (Rnq1-GFP). Sup35-RFP showed rings, which did not show any fluorescence through the GFP filter.Failure of some Sup35 newly appearing aggregates to colocalize with Rnq1-GFP. ines see . B. Fail(PDF)Click here for additional data file.S4 FigA. Control [-pin] cells with integrated RNQ1-CFP and overexpressed Sup35NM-YFP. Sup35NM-YFP was overexpressed (p1753) in [-pin] RNQ1-CFP integrants. Both Rnq1-CFP and Sup35NM-YFP remained diffuse after 48 h. B. Control [+PIN] cells with integrated Rnq1-CFP and repressed Sup35NM-YFP. [+PIN] RNQ1-CFP integrants with the p1753 plasmid were grown in repressing media (Glucose) to inhibit Sup35NM-YFP overexpression. As expected, Rnq1-CFP formed dots, while Sup35NM-YFP displayed no signals after 48 h. C. Control [+PIN] cells with integrated Rnq1-CFP and overexpressed YFP. [+PIN] RNQ1-CFP integrants with the empty vector p1752 (YFP) were grown in 2% Gal. Rnq1-CFP showed only dots, indicative of [+PIN], while YFP always remained diffuse.Control experiments for colocalization of Sup35NM-YFP aggregates with Rnq1-CFP. (PDF)Click here for additional data file.S5 Fig+PIN] aggregates decorated by Rnq1-CFP and Sup35 aggregates induced by Sup35NM-YFP overexpression through different focal planes. A. Perfect colocalization of Sup35NM-YFP with Rnq1-CFP. [+PIN] RNQ1-CFP integrants were grown in 2% Gal for 48 h to overexpress Sup35NM-YFP (p1753). Z-stacks of 12 optical sections spaced ∼1 µm apart were collected. Rnq1-CFP perfectly overlapped Sup35NM-YFP in all sections. Representative pictures shown are from layers 5 through 8 out of 12 sections. B. Partial colocalization of Sup35NM-YFP with Rnq1-CFP. Representative pictures were taken from another group of cells with 8 z-stacks, layers 3 through 5 are shown. Yellow arrows and blue stars indicate colocalized rings and non-colocalized lines, respectively Click here for additional data file.S6 Fig+PSI< induction, but do not in established [+PSI]. Sup35NM-VN (p1893) and Rnq1-VC (p1894) in [+PIN][-psi] or [+PIN][+PSI] 74D-694 cells were co-overexpressed by growth in 0.2% Gal for the indicated times. (n≈600). Expression levels of Sup35NM-VN and Rnq1-VC (bottom) were detected by respectively, α-Sup35N and α-Rnq1 (a kind gift of S. Lindquist) in [+PIN][-psi] (left) or [+PIN][+PSI] (right) cells harboring p1893 and p1894, and grown in 0.2% Gal for 48 h. Pgk1 was used as an internal loading control.Rnq1 and Sup35 form a close physical interaction during [(PDF)Click here for additional data file.S7 FigA. Visualization of Rnq1-CFP in control [-pin] cells overexpressing untagged Sup35NM. Sup35NM was overexpressed (p2036) in [-pin] RNQ1-CFP integrants by growth in 2% Gal for 48 h. Rnq1-CFP always displayed diffuse fluorescence. B. Visualization of Rnq1-CFP in control [+PIN] cells without Sup35NM overexpression. [+PIN] RNQ1-CFP integrants lacking p2036 (Sup35NM) were grown in 2% Gal. Rnq1-CFP displayed only fluorescent dots by 48 h in 90% of the cells, while 10% of the cells contained diffuse fluorescence.Failure of Rnq1-CFP to form aggregates other than dots in control cells. (PDF)Click here for additional data file.S8 FigA. Sup35 aggregates colocalized with Ssa1 and Sis1 chaperones. [+PIN] cells with GFP-tagged SSA1 or SIS1 endogenously were induced to overexpress Sup35NM-RFP (p2017) for 48 h by growth in 2% Gal. Observed Sup35NM-RFP rings in SSA1-GFP cells  completely colocalized with Ssa1-GFP. Sup35NM-RFP rings  completely colocalized with Sis1-GFP in SIS1-GFP cells. B. Hsp104 did not form aggregates in [-pin] cells. Overexpressed Sup35NM-RFP (p2017) in [-pin] HSP104-GFP cells with 2% Gal for 48 h resulted in no aggregate formation.Colocalization of newly induced Sup35 aggregates with chaperones. (PDF)Click here for additional data file.S9 FigA. Q/N-rich proteins. Among the Q/N rich proteins that, when overexpressed, can facilitate overexpression of the Sup35 prion domain to form [+PSI] are Swi1, Cyc8, New1, Pin3 and Pin4 +SWI] and [+OCT] prions +NU] prion aka Lsb2), was shown not to form a prion, but to colocalize transiently with some Sup35 aggregates during Pin3-promoted [+PSI] induction presumably involving the actin cytoskeleton de novo induction of the [+PSI] prion +PSI] +PIN] cells with one of the Q/N rich proteins  labeled endogenously with GFP, were induced with 2% Gal to overexpress Sup35NM-RFP (p2017) for 48 h. Cyc8-GFP gave a nuclear diffuse signal while New1-GFP, Pin3-GFP and Pin4-GFP were cytoplasmic and diffuse. However, none of these Q/N rich proteins showed colocalization with the Sup35NM-RFP aggregates observed in ∼7% of the cells (n≈450) during [+PSI] induction. Your Favorite Gene.YFG: B. Non-Q/N-rich proteins. [+PIN] cells with endogenously GFP tagged proteins that influence [+PSI] induction, Mod5 Failure of newly induced Sup35 aggregates to colocalize with various proteins. (PDF)Click here for additional data file.S10 FigA. Pin4C-RFP does not form rings in the absence of Sup35NM-GFP overexpression, and Sup35NM-GFP does not form aggregates without Pin4C overexpression. 74D-694 rnq1Δ cells, which contained either p1708 or 1951, were grown in 2% Gal to separately overexpress respectively, Pin4C-RFP or Sup35NM-GFP for 72 h. Pin4C-RFP formed large fluorescence dots, while Sup35NM-RFP remained diffuse. B. Sup35NM overexpression changes the location of Pin4C relative to the Hsp42-GFP dot. Pin4C-RFP was overexpressed from p1708 in [-pin] HSP42-GFP cells in the presence (top) or absence (bottom) of Sup35NM overexpression (from p1893-2).Colocalization of Pin4C-RFP with Sup35NM-GFP and Hsp42-GFP. (PDF)Click here for additional data file.S11 FigA. Overexpression of neither Mod5-GFP nor Sup35NM-RFP individually formed aggregates in rnq1Δ. 74D-694 rnq1Δ cells, which contained either p2061 or 2018, were grown in 2% Gal to separately overexpress Mod5-GFP or Sup35NM-RFP, respectively for 48 h. Both Mod5-GFP and Sup35NM-RFP remained diffuse. B. Expression levels of Sup35NM-VC and Mod5-VN after 48 h of induction by 2% Gal were detected by α-Mod5  and Mod5-VC (from p2171) were co-overexpressed in [-pin] (top) and [+PIN] (bottom) for 48 h by growth on 2% Gal. In [-pin], Mod5 BiFC did not show any fluorescence, while in [+PIN], it showed diffuse fluorescence. Expression levels of Mod5 from BiFC constructs p2170 and p2171 were detected by α-Mod5 in [-pin][-psi] vs. [+PIN][-psi] cells grown in 2% Gal for 48 h. Note that a lower percentage (7% vs 10%) of SDS-PAGE was used to resolve Mod5-VN and Mod5-VC, which are only 9 kDa apart.Control experiments for the relationship between Mod5-GFP and Sup35NM-RFP. ells see . C. Mod5(PDF)Click here for additional data file.S12 FigHSP104, but were larger in the presence of [+PIN]. Sup35NM-GFP and Pin4C-RFP were respectively co-overexpressed from p1181 and p1708, by growing hsp104Δ , HSP104 [-pin] (L2910) or HSP104 [+PIN] (L1749) cells in 50 µM CuSO4 and 2% Gal. Pin4C-RFP aggregates were larger and more numerous in [+PIN] than [-pin] cells -pin] cells.Pin4C aggregates were not affected by the presence vs. absence of (PDF)Click here for additional data file.S1 Table+PIN] cells by growth in 0.2% Gal. Sup35NM-GFP in 74D-694 [+PIN][-psi] cells was overexpressed from p1951 by growth in 0.2% Gal.Visualization of Sup35NM-GFP aggregation in [(PDF)Click here for additional data file.S2 Table+PIN] cells. After 6 h of induction of Sup35-RFP (p1678) by growth of 74D-694 [+PIN][-psi] cells with p1730 expressing Rnq1-GFP from its own promoter in 2% Gal, 242 cells were seen to have Sup35-RFP dots out of 6000 cells counted. Among these 242 cells, 144 also showed Rnq1-GFP dots colocalized with Sup35-RFP, but the other 98 cells had diffuse Rnq1-GFP.Colocalization data of Rnq1-GFP with Sup35-RFP after 6 h of induction of Sup35-RFP in [(PDF)Click here for additional data file.S3 Table+PIN] cells. After 24 h of induction of Sup35-RFP (p1678) by growth of 74D-694 [+PIN][-psi] cells with p1730 expressing Rnq1-GFP on its own promoter in 2% Gal, 575 cells were seen to have Sup35-RFP lines/rings out of 4800 cells counted. Among these 575 cells, 401 also showed Rnq1-GFP rings/lines colocalized with Sup35-RFP rings/lines, while 57 had Rnq1-GFP dots and the other 117 had diffuse Rnq1-GFP.Colocalization data of Rnq1-GFP with Sup35-RFP after 24 h of induction of Sup35-RFP in [(PDF)Click here for additional data file.S4 Table+PIN] cells. After 24 h of induction of Sup35-RFP (p1678) by growth of 74D-694 [+PIN][-psi] cells with p1730 expressing Rnq1-GFP on its own promoter in 2% Gal, 480 cells were seen to have Rnq1-GFP lines or rings out of 6000 cells counted. Among these 480 cells, 474 also showed Sup35-RFP rings/lines colocalized with Rnq1-GFP, but the other 6 cells had diffuse Sup35-RFP.Colocalization data of Sup35-RFP with Rnq1-GFP after 24 h of induction of Sup35-RFP in [(PDF)Click here for additional data file.S5 Table+PIN] cells. After 24 h of induction of Sup35NM-YFP (p1753) by growth of 74D-694 [+PIN][-psi] RNQ1-CFP cells with p1753 in 2% Gal, 63 cells were seen to have Sup35NM-YFP dots, and 7 cells had Sup35NM-YFP rings/lines out of 900 cells counted. Among these 63 dot-bearing cells, 57 also showed Rnq1-CFP dots colocalized with Sup35NM-YFP, while the other 6 cells had diffuse Rnq1-CFP. However, among the 7 Sup35-RFP ring bearing cells, they all had Rnq1-CFP rings colocalized with Sup35-RFP.Colocalization data of Rnq1-CFP with Sup35NM-YFP after 24 h of induction of Sup35NM-YFP in [(PDF)Click here for additional data file.S6 Table+PIN] cells. After 48 h of induction of Sup35NM-YFP (p1753) by growth of 74D-694 [+PIN][-psi] RNQ1-CFP cells with p1753 in 2% Gal, 216 cells were seen to have Sup35NM-YFP lines/rings out of 2000 cells counted. Among these 216 cells, 162 also showed Rnq1-CFP lines/rings colocalized with Sup35NM-YFP, while 15 harbored Rnq1-CFP dots and the other 39 had diffuse Rnq1-CFP.Colocalization data of Rnq1-CFP with Sup35NM-YFP after 48 h of induction of Sup35NM-YFP in [(PDF)Click here for additional data file.S7 Table+PIN] cells. After 48 h of induction of Sup35NM-YFP (p1753) by growth of 74D-694 [+PIN][-psi] RNQ1-CFP cells with p1753 in 2% Gal, 375 cells were seen to have Rnq1-CFP lines or rings out of 5000 cells counted. Among these 375 cells, 371 also showed Sup35NM-YFP rings/lines colocalized with Rnq1-CFP, but the other 4 cells had diffuse Sup35NM-YFP.Colocalization data of Sup35NM-YFP with Rnq1-CFP after 48 h of induction of Sup35NM-YFP in [(PDF)Click here for additional data file.S8 Tablea. Sup35NM-VN (p1893) and Rnq1-VC (p1894) in 74D-694 [+PIN][-psi] or [-pin][-psi] cells were co-overexpressed by growth in 0.2% Gal. The control [-pin] cells showed no fluorescence at any time. BiFC fluorescence was checked by YFP filter. Percentages are based on n≈600.Data for overexpressed Sup35 and Rnq1 from the BiFC constructs Sup35NM-VN and Rnq1-VC, respectively by growing cells in 0.2% Gal(PDF)Click here for additional data file.S9 Table+PSI] in rnq1Δ cells in the presence of Sup35NM and Pin4C overexpression. To see if observed Sup35NM-GFP aggregates in the absence of Rnq1 when Pin4C-RFP was overexpressed (p1708) were associated with the appearance of [+PSI] Click here for additional data file.


O. aries-tc/ZAF/11/OBP-115) isolated from experimentally vaccinated cattle. Full-genome sequencing and phylogenetic analyses show that the bluetongue virus 9 strain 9/B. taurus-tc/ZAF/15/Onderstepoort_B02b is a reassortant virus containing segments from both BTV-9 and BTV-8.This is a report of the complete genome sequences of plaque-selected isolates of each of the five virus strains included in a South African commercial trivalent bluetongue virus (BTV) attenuated live virus vaccine, a BTV-4 field strain isolated from Rustenburg, South Africa, in 2011, and a bluetongue reassortant (bluetongue virus 4 strain 4/ Culicoides biting midges (Diptera: Ceratopogonidae) . The genome of BTV is composed of 10 segments of double-stranded RNA (dsRNA) that collectively encode seven structural proteins and five nonstructural proteins . Sequencing templates were prepared using full-length amplification of cDNAs  is a noncontagious disease of ruminants that is transmitted by gonidae)   are available on GenBank as accession numbers KT317665 to KT317674. The full-genome sequences of the bluetongue virus 9 strain 9/B. taurus-tc/ZAF/14/Onderstepoort_B02b that is a reassortant between BTV-9 and BTV-8 has been deposited in GenBank as accession numbers KT885075 to KT885084.The full-genome sequences of bluetongue virus 3 strain 3/Labstr/ZAF/14/OBP-115, bluetongue virus 8 strain 8/Labstr/ZAF/14/OBP-115, bluetongue virus 9 strain9/Labstr/ZAF/14/OBP-115, bluetongue virus 10 strain10/Labstr/ZAF/14/OBP-115, and bluetongue virus 11 strain11/Labstr/ZAF/14/OBP-115 have been deposited in GenBank under the accession numbers


Our findings highlight an essential role of Paxillin and FAK in controlling cardiac contractility via the recruitment of Vinculin to mechano-sensitive sites in cardiomyocytes.An orchestrated interplay of adaptor and signaling proteins at mechano-sensitive sites is essential to maintain cardiac contractility and when defective leads to heart failure. We recently showed that Integrin-linked Kinase (ILK), ß-Parvin and PINCH form the IPP-complex to grant tuned Protein Kinase B (PKB) signaling in the heart. Loss of one of the IPP-complex components results in destabilization of the whole complex, defective PKB signaling and finally heart failure. Two components of IPP, ILK and ß-Parvin directly bind to Paxillin; however, the impact of this direct interaction on the maintenance of heart function is not known yet. Here, we show that targeted gene inactivation of Paxillin results in progressive decrease of cardiac contractility and heart failure in zebrafish without affecting IPP-complex stability and PKB phosphorylation. However, we found that Paxillin deficiency leads to the destabilization of its known binding partner Focal Adhesion Kinase (FAK) and  Loading controls included mouse anti-β-actin  and rabbit anti-pan cadherin . Signals were detected by chemiluminescence  using a luminescent image analyzer (Image Quant Las4000 mini).In-vitro synthesized Paxillin protein was generated following standard protocols  and used as positive control for western blot analysis.http://www.benegfx.de), then FS was calculated using the following formula:Still images were taken with an Olympus SZX 16 microscope and movies were recorded with a Leica DM IL LED microscope. The functional assessment of cardiac contractility was carried out as described . Fractiot-test, a value of P<0.05 was accepted as statistically significant.All results are expressed as mean ± s.d. and analysis were performed using unpaired Student's in vivo role of Paxillin in the vertebrate heart, we isolated the zebrafish orthologous sequence by BLAST analysis. Zebrafish Paxillin (GenBank: NM_201588.1) displays high amino acid identity compared to human and murine Paxillin (GenBank: U14588.1 and NP_035353.1), especially in the evolutionary conserved motifs and domains that are known to mediate protein-protein interactions, such as LD motifs, LIM domains, SH2 domain-binding sites and an SH3 domain-binding site (To study for the first time the ing site . By immuing site .paxillin (MO1-paxillin) or one targeting the splice-acceptor site of exon 2 (MO2-paxillin). As shown in paxillin injected embryos developed progressive heart failure characterized by reduced ventricular systolic force, severely reduced blood flow, blood congestion at the cardiac inflow tract and pericardial edema (paxillin morphant ventricles was reduced to 49.29% ± 4.39% compared to control morphants (FS: 69.3% ± 4.36%) at 72 hpf. By 96 hpf, the ventricular chambers of paxillin morphants became almost silent (FS: MO1-paxillin 4.23% ± 6.17% vs. MO1-control 74% ± 4.9%)  mediated gene knock-down using either a MO directed against the translational start site of al edema  Movies.  ± 4.9%) . MO1-paxpaxillin, we assessed Paxillin protein levels in morphant embryos and found that injection of MO1-paxillin resulted in severely reduced protein levels of Paxillin  led to a similar heart failure phenotype as injection of MO1-paxillin, 82.27% ± 7.9% of MO2-paxillin injected embryos developed contractile dysfunction, whereas only 9.5% ± 2.6% of control-injected embryos showed a comparable phenotype (paxillin 3.06% ± 5.78% vs. MO2-control 72.84% ± 5.25%)  to 58.6% ± 4.7% in rescued embryos (MO2-paxillin+mRNA) at 72 hpf  . Isolatisplicing . Further embryos . Whereas failure . FS meast 72 hpf . These fin vivo. Accordingly, PKB phosphorylation at Serine 473 downstream of the IPP-complex was not reduced in Paxillin morphants (The ILK-Parvin-PINCH- (IPP-) complex regulates contractility by granting phosphorylation and activation of PKB/AKT in cardiomyocytes. Loss of one of the IPP-complex components results in destabilization of the whole complex, defective PKB signaling and finally heart failure , 3. Firsorphants , furtherin vivo. Remarkably, western blot analysis revealed that ectopic expression of zebrafish paxillin mRNA in Paxillin morphants was able to restore FAK protein expression  protein levels, since it is known that Paxillin not only interacts with proteins of the IPP-complex but also binds to FAK . Interespression . By quan embryos , demonstfak1a and MO1-fak1b). At 72 hpf, 81.3% ± 4.6% of embryos co-injected with MO1-fak1a and MO1-fak1b developed heart failure similar to Paxillin-deficient zebrafish embryos. This phenotype was accompanied by progressive reduction of ventricular contractility, blood congestion at the sinus venosus and a pronounced pericardial edema (fak1a/fak1b morphant ventricular chambers was reduced to 63.57% ± 9.18% compared to corresponding 5bp-mismatch-MO injected embryos (FS: 70.33% ± 3.72%) at 48 hpf and to 2.6% ± 3.71% compared to control morphants (FS: 69.33% ± 4.36%) at 72 hpf  by MOs directed against the respective translational start sites  sites, at least in mouse embryonic fibroblasts [paxillin mRNA in MO2-paxillin injected embryos  and Vinculin localizing to sarcomeric Z-disks and costameres in cardiomyocytes is essential for the regulation of cardiac contractility. We found that heart failure in Paxillin-deficient zebrafish embryos was caused by the degradation of these interacting proteins. Furthermore, we show here that loss of Paxillin does neither impact on the stability of the Integrin-linked Kinase (ILK)-Parvin-PINCH (IPP)-complex nor on IPP-mediated Protein Kinase B (PKB) activation.paxillin in mice leads to early embryonic lethality around day E9.5 due to abnormal development of mesodermal derived organs such as the heart and somites [in vivo. We demonstrate here that loss of paxillin in zebrafish led to severe heart failure, characterized by reduced cardiac contractility. Paxillin is known to interact with the Z-disc-associated IPP-complex. Interestingly, mutations within IPP-complex components lead to destabilization of the complex, reduced phosphorylation of PKB and finally heart failure, similar to Paxillin morphants [Targeted knock-out of  somites , hinderiorphants , 3. Howein vivo [Vice versa, FAK absence leads to destabilization of Paxillin suggesting that Paxillin and FAK not only interact but also stabilize each other in vivo and that this functional interplay is essential for the regulation of cardiac contractility in the zebrafish embryo. Interestingly, in Paxillin deficient mice, FAK protein stability seems not to be severely affected but rather specifically its phosphorylation. However, in mice FAK-Paxillin complex stability might be preserved through up-regulation of the Paxillin orthologue Hic-5 that was shown, similar to Paxillin, to interact with FAK and Vinculin [The multi-adaptor protein Paxillin can also interact at mechanical integration sites with other proteins such as FAK. It was recently determined that Paxillin-FAK binding warrants their activation via the Proto-oncogene tyrosine-protein kinase Src (Src) and hence the recruitment of Vinculin to the costameres. Vinculin is a membrane-cytoskeletal protein that was shown to be important for transmitting mechanical forces and orchestrating mechanical signaling events , 23. Absin vivo  , mouse (mm) and human (hs) Paxillin, demonstrating high cross-species homology. Identical amino acids are shaded in black and shown as asterisks in the consensus line. Aminio acids with similar chemicals properties are shaded in gray. Highly homologous motifs such as the LD motifs and LIM domains are indicated.(PDF)Click here for additional data file.S2 Fig(A-C) Lateral view of MO1-paxillin (A) and MO1-paxillin+human paxillin mRNA (B) injected embryos at 72 hpf. (C) Bar graphs compare average of rescued embryos after co-injection of human paxillin mRNA compared to MO1-paxillin injected embryos . (D) Fractional shortening (FS) measurement of rescued embryos compared to Paxillin morphant embryos at 72 hpf (n = 5).  Lateral view of a (E) 5bp-mismatch MO (MO2-control) and (F) MO2-paxillin splice MO injected embryos at 72 hpf. The heart failure phenotype of MO2-paxillin splice morphants was identical to that of embryos injected with translation blocking paxillin MO (MO1-paxillin). (G) Bar graphs compare average of affected embryos after injection of MO2-paxillin compared to 5bp-mismatch MO (MO2-control) injected embryos at 72 hpf . (H) FS measurements of Paxillin morphant ventricles at 48, 72 and 96 hpf . (I) RT-PCR of control- and MO2-paxillin-injected embryos. Injection of MO2-paxillin caused partial and whole skipping of exon 2 (298 bp) leading to premature termination of Paxillin protein translation. Wild-type paxillin RNA was severely reduced in Paxillin morphants (527 bp product). (J) Western blot analysis of control- (MO1-control) and MO1-paxillin-injected embryos demonstrated high efficacy of MO-mediated paxillin gene knock-down. For each sample 50 embryos were pooled and 20 μg of protein lysate were loaded per lane. In vitro translated (IVT) zebrafish Paxillin protein was used as positive control.(PDF)Click here for additional data file.S3 Fig Lateral view of (A) control MO (MO2-control) and (B) MO2-fak1a/fak1b-injected embryos at 72 hpf. The heart failure phenotype of fak1a/fak1b splice morphants was identical to that of embryos injected with the translation blocking FAK MOs (MO1-fak1a/fak1b). (C) RT-PCR of control-, MO2-fak1a- and MO2-fak1b-injected embryos. Injection of MO2-fak1a and MO2-fak1b caused intron integration  leading to premature termination of FAK1a and FAK1b protein translation, respectively. Wild-type fak1a and fak1b RNA was severely reduced in the respective morphants . (D) Western Blot analysis of control and fak1a/fak1b morphant embryos with an antibody against FAK. For each sample 50 embryos were pooled and 20 μg of protein lysate were loaded per lane.(PDF)Click here for additional data file.S4 Fig Lateral view of MO-vinculin (A) and vinculin 5bp-mismatch-MO (MO-control) (B) injected embryos at 72 hpf. (C) Bar graphs compare average of affected embryos after MO injection . (D) Fractional shortening (FS) measurements of Vinculin morphant ventricles compared to control injected embryos at 48, 72 and 96 hpf. FS of Vinculin morphant ventricles was not affected compared to corresponding 5bp-mismatch-MO injected embryos (MO-vinculin 68.01% ± 3.52% vs. MO-control: 66.08% ± 3.12%) at 48 hpf. At 72 hpf, FS in Vinculin morphants was reduced to 42.97% ± 6.53% compared to control morphants (68.26% ± 1.5%), whereas ventricular chambers of Vinculin morphants became almost silent compared to controls at 96 hpf .(PDF)Click here for additional data file.S1 Movie(MOV)Click here for additional data file.S2 Movie(MOV)Click here for additional data file.S3 Movie(MOV)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.