Caenorhabditis elegans, the LIN-2/7/10 protein complex regulates the activity of signalling proteins. We found that inhibiting lin-7 function, and also lin-2 and lin-10, resulted in enhanced C. elegans survival after infection by Burkholderia spp., implicating a novel role for these genes in modulating infection outcomes. Genetic experiments suggested that this infection phenotype is likely caused by modulation of the DAF-2 insulin/IGF-1 signalling pathway. Supporting these observations, yeast two-hybrid assays confirmed that the LIN-2 PDZ domain can physically bind to the DAF-2 C-terminus. Loss of lin-7 activity also altered DAF-16 nuclear localization kinetics, indicating an additional contribution by hsf-1. Unexpectedly, silencing lin-7 in the hypodermis, but not the intestine, was protective against infection, implicating the hypodermis as a key tissue in this phenomenon. Finally, consistent with lin-7 acting as a general host infection factor, lin-7 mutants also exhibited enhanced survival upon infectionby two other Gram-negative pathogens, Pseudomonas and Salmonella spp.In  Caenorhabditis elegans has been used as a model system to dissect host–pathogen interactions for many microbes, including Burkholderia, Pseudomonas and Salmonella spp. , TGF-β and DAF-2 insulin/IGF-1 signalling pathways , we selected 81 genes reported to be expressed in nematode intestinal cells (E. coli HT115 clones producing gene-targeting double-stranded RNA (dsRNA) . We specifically identified genes that, when silenced, resulted in prolonged C. elegans survival during infection. This strategy eliminates potential confounding factors due to general sickness caused by specific RNAi treatment, which might result in nematodes exhibiting decreased survival during infection.Since al cells . Using Ral cells , we systlin-7, myo-3, rfp-1 and rrt-2) conferred significant enhancements of survival (data not shown). Among these, we selected lin-7, encoding a cell junction protein  loss-of-function mutant  mutants are vulvaless and produce a characteristic ‘bag of worms’ phenotype , lin-7 (e1443) and lin-7 (n106)  (P < 0.0001), lin-7 (e1413) (P < 0.0001) and lin-7 (n106)  mutants was comparable to that of sterile wild-type nematodes grown on non-pathogenic E. coli  mutants (B. thailandensis loads by quantifying colony-forming units (cfu) from infected nematodes – there was no significant difference between the abilities of the wild-type nematode and the lin-7 mutant to limit intestinal pathogen growth  (P < 0.0001) and lin-10 (n1402) (P < 0.0001) mutants  loss-of-function mutant did not exhibit enhanced survival when compared with wild-type nematodes upon B. thailandensis infection , while LIN-2 has a type II PDZ domain which binds Φ-X-Φ motifs, where Φ is a hydrophobic residue  domains that can interact with the C-terminals of receptors  or wild-type nematodes pre-treated with gene-specific dsRNA (gpc-2 and ina-1) to B. thailandensis  mutants survived significantly longer than wild-type nematodes upon B. thailandensis infection , raising the possibility that the DAF-2 C-terminus may potentially bind to the LIN-2 PDZ domain. To experimentally test this romoters C. Confirromoters C. These daf-2 (e1370) mutants exhibited enhanced survival when exposed to B. thailandensis and that the LIN-2 PDZ domain bound to the DAF-2 C-terminus suggested that the LIN-2/7/10 complex could potentially modulate the DAF-2 signalling pathway. We thus tested the extent to which perturbations in DAF-2 signalling might affect the lin-7-mediated infection phenotype. Upon B. thailandensis infection, daf-2 (e1370) mutants died at highly similar rates irrespective of lin-7 status  mutants carrying gain-of-function mutations in the PI3K kinase suppressed the infection phenotype conferred by lin-7 RNAi treatment . Thus to assess the influence of lin-7 on DAF-16 nuclear localization, we implemented a heat shock regimen at 35°C for 30 min, previously shown to induce DAF-16::GFP nuclear localization , lin-7 dsRNA-treated nematodes exhibited slower DAF-16::GFP nuclear localization kinetics when compared with those without RNAi; upon 30 min of heat shock, lin-7 dsRNA-treated nematodes predominantly exhibited ‘unlocalized’ DAF-16::GFP localization patterns . These data show that lin-7 can significantly affect the kinetics of DAF-16 nuclear localization during heat shock, further corroborating a connection between LIN-7 and the DAF-2 signalling pathway. In addition, these results raised the possibility that perhaps the increased nuclear export of DAF-16::GFP in lin-7 dsRNA-treated nematodes could be attributed to hsf-1, given that reducing hsf-1 activity suppressed this phenomenon. Henceforth, our observations also suggest a functional relationship between LIN-7, DAF-2 and HSF-1.In contrast, lin-7 is known to function in non-intestinal tissues as well  mutants carrying the wild-type rde-1 transgene expressed either under the intestine-specific promoter pnhx-2 . As a further control for tissue specificity, we also treated these rde-1 (ne219) mutants with elt-2 dsRNA. Consistent with reports that elt-2 is specifically expressed in the intestine and protects against several bacterial pathogens  mutants carrying the intestinal pnhx-2::rde-1 transgene hypersensitive to B. thailandensis  mutants carrying the hypodermal plin-26::rde-1 transgene  mutants carrying the hypodermal plin-26::rde-1 transgene exhibited enhanced survival when treated with lin-7 dsRNA  mutants carrying the intestinal pnhx-2::rde-1 transgene with lin-7 dsRNA did not confer protection during infection  mutants carrying the hypodermal plin-26::rde-1 transgene also exhibited enhanced survival upon B. thailandensis infection . Subsequent genetic and biochemical assays linked LIN-7 to DAF-2, a major regulator of the insulin/IGF-1 signalling pathway. Tissue-specific RNAi experiments revealed that lin-7 likely functions in the hypodermal cells of C. elegans to affect infection outcomes. Collectively, our results suggest that during infection, LIN-7 regulates the DAF-2 signalling pathway in nematode hypodermal tissues and this modulation of DAF-2 signalling becomes detrimental to the infected host.In this study, we identified a novel role for LIN-7 during bacterial infection. Loss of C. elegans can be modulated by the removal of germline signals  nematodes, fertile lin-7 (e1449) mutants as well as sterile lin-7 (e1449) mutants , followed by 1 h primary antibody incubations using monoclonal antibody 3015 IgG1 . Images are representative of at least 50 nematodes from three independent assays.Nematodes infected by E. coli HT115 (DE3) strain or E. coli HT115 clones expressing gene-specific dsRNA  and seeding on isopropyl β-d-1-thiogalactopyranoside (IPTG)-containing modified NGM agar. Nematode embryos, generated by hypochlorite treatment, were propagated on these seeded plates until the L4 stage. Nematodes were subsequently transferred to pathogen-containing lawns.Unless specified otherwise, RNAi assays were carried out at 20°C by feeding nematodes with parental cdc-25.1 dsRNA at late embryogenesis till day 1 adult stage, before transferring them to pathogen-containing lawns. Under such conditions, cdc-25.1 RNAi resulted in nematodes with an Emb phenotype  to remove bacteria present on the exterior of the nematode. Nematodes were then washed thrice with M9 buffer only to remove trypsin-EDTA, and subsequently lysed by vortexing with 400 g silicon-carbide sharp particles  and 0.2% sodium dodecyl sulfate. Lysates were diluted appropriately in M9 buffer and plated on LB agar supplemented with gentamicin to select for B. thailandensis. After 2-day incubation at 37°C, amount of live bacteria per nematode was determined by cfu counts. At least 25 nematodes were harvested per nematode strain and experiments were performed in triplicates.Nematodes were infected with daf-2, or at NdeI and XmaI sites for lin-2, in-frame into the GAL4 DNA-binding domain (DNA-BD) vector pGBKT7 or GAL4 activation domain (AD) vector pGADT7  respectively. Reagents provided by the manufacturer included: (i) positive controls – murine p53 and SV40 large T antigen, as fusion proteins with the same GAL4 DNA-BD and GAL4 AD, respectively, and (ii) negative control – human lamin C, fused to the GAL4 DNA-BD.Plasmids and primers used in this assay are listed in E. coli TG1 strain, followed by direct sequencing of plasmids isolated from TG1 using the following pairs of oligonucleotides: daf2-check-F (TgACgATTCAgAAgCACTgg) and daf2-check-R (CATCTTgTCCACCACgTgTC); lin2-check-F (AgTggCAggTTTgACgAgAC) and lin2-check-R (AgTTgTCggAgT TCCAATgC).Y2HGold reporter and Y187 mating haploid yeast strains were transformed individually with either the GAL4 DNA-BD or AD construct, according to the manufacturer's instructions. Successfully transformed haploids were selected on plates lacking tryptophan or leucine accordingly. Six pairwise combinations of transformed Y2HGold and Y187 haploid strains see C were alE. coli HT115 strain or E. coli HT115 clones expressing lin-7 or hsf-1 dsRNA. Nematodes were grown at 20°C for parental HT115 and lin-7 dsRNA, or at 15°C for hsf-1 dsRNA until the L4 stage. Acute heat shock was performed at 35°C for intervals of 15 min by placing sealed plates into a water bath. Nematodes before and immediately after heat shock were visualized using an Olympus MVX10 dissecting microscope under 125× or 250× total magnification and images were analysed by DP controller software (version 3.1.1.267). Nematodes were classified as exhibiting either uniformly distributed DAF-16::GFP , nuclear DAF-16::GFP restricted to the 28–32 nuclei of the intestinal epithelial cells only  or nuclear DAF-16::GFP in all cell types . At least 50 nematodes were counted per RNAi treatment and experiments were performed in triplicates.Embryos from DAF-16::GFP nematodes, generated by hypochlorite treatment, were propagated on plates containing parental P values < 0.05 were considered significantly different from the control. Student's t-test was used to analyse nematode bacteria loads and DAF-16::GFP nuclear localization results.Survival curves were analysed using the PRISM (version 5.0) software. Kaplan-Meier survival curves with


ALVAC-HIV prime/HIV-1 Env protein boost regimens have shown HIV-specific neutralizing antibody (NAb) and cell-mediated immune responses, but the impact of protein subunit schedule and adjuvant requires further definition.A Phase 1 trial was conducted in two parts. In Part A, (open-label) 19 volunteers received oligomeric gp160 MN/LAI-2 (ogp160) with a dose escalation . In Part B, 72 volunteers  received placebo or recombinant canarypox expressing HIV-1 antigens,  prime with different doses and schedules of ogp160MN/LAI-2 in alum or polyphosphazene (PCPP).The vaccines were safe and well tolerated with no vaccine-related serious adverse events. Cumulative chromium release CTL frequency was 37%, and 54% of volunteers showed proliferative responses to HIV antigens. Lymphoproliferative CD4+, HIV-specific responses were seen in 53% of ogp160 only and 57% of prime-boost recipients, respectively. Induced binding antibody to ogp160 was dose-dependent. NAb responses to vaccine homologous Tier 1 HIV-1 MN were seen in 99% of vaccine recipients. While NAb to the heterologous Tier 2 US-1  pseudovirus was negative in all volunteers tested using TZM-bl cells, in a PBMC-based assay, US-1 primary isolate Nab was induced in 2/19 (10.5%) recipients of ogp160 protein alone and in 5/30 (16.7%) prime-boost volunteers who received ogp160 in PCPP. Primary isolate neutralization was observed more frequently overall in recipients of ogp160 in PCPP, as compared with alum (p=0.027). Using an intracellular p24 flow-cytometry assay, sera from an ALVAC-HIV/ogp160 recipient demonstrated 94% neutralization of US-1.A small percentage of vaccine recipients developed Nab to heterologous primary isolates, responses that to our knowledge have not been previously described. These results constitute proof of concept that Tier 2 NAb can be elicited by vaccination in humans, and underscore the importance of further optimization of prime-boost vaccination and adjuvanting strategies for HIV-1 prevention.


In vitro knockdown of miR-193b in FaDu cancer cells substantially reduced cell proliferation, migration and invasion, along with suppressed tumour formation in vivo. By integrating in silico prediction algorithms with in vitro experimental mRNA profilings, plus mRNA expression data of clinical specimens, neurofibromin 1 (NF1) was identified to be a target of miR-193b. Concordantly, miR-193b knockdown decreased NF1 transcript and protein levels significantly. Luciferase reporter assays confirmed the direct interaction of miR-193b with NF1. Moreover, p-ERK, a downstream target of NF1 was also suppressed after miR-193b knockdown. FaDu cells treated with a p-ERK inhibitor (U0126) phenocopied the reduced cell proliferation, migration and invasion observed with miR-193b knockdown. Finally, HNSCC patients whose tumours expressed high levels of miR-193b experienced a lower disease-free survival compared to patients with low miR-193b expression. Our findings identified miR-193b as a potentially novel prognostic marker in HNSCC that drives tumour progression via down-regulating NF1, in turn leading to activation of ERK, resulting in proliferation, migration, invasion, and tumour formation.Despite improvements in therapeutic approaches for head and neck squamous cell carcinomas (HNSCC), clinical outcome has remained disappointing, with 5-year overall survival rates hovering around 40–50%, underscoring an urgent need to better understand the biological bases of this disease. We chose to address this challenge by studying the role of micro-RNAs (miRNAs) in HNSCC. MiR-193b was identified as an over-expressed miRNA from global miRNA profiling studies previously conducted in our lab, and confirmed in HNSCC cell lines.  The protocol was approved by the Animal Care Committee at the University Health Network (Protocol Number: 342.19).Patient samples were collected from a phase III randomized study (331 participants) of hyperfractionated radiotherapy conducted in 1988 to 1995 2, authenticated at the Centre for Applied Genomics  using the AmpF/STR Identifier PCR Amplification Kit (Applied Biosystems), and routinely tested for mycoplasma contamination using the Mycoalert detection kit (Lonza Group Ltd).The human hypopharyngeal HNSCC FaDu cell line was obtained from the American Type Culture Collection , and cultured according to the manufacturer’s specifications. The human laryngeal squamous cell lines, UTSCC-8 and UTSCC-42a  tm microRNA Power inhibitors Control A served as a negative control.The biological effects of miR-193b were investigated using a lock nucleic acid (LNA) probe containing a sequence specific antisense oligonucleotide targeting miR-193b, miRCURY LNA™ microRNA 193b Power inhibitor (Exiqon). A scrambled miRNA sequence, miRCURY LNATotal RNA was extracted from either cell lines, or primary tissues using the Total RNA purification kit (Norgen), then reverse transcribed using SuperScript II Reverse Transcriptase (Invitrogen Canada) according to specifications. MicroRNA profiles of three HNSCC cell lines  compared to NOE were generated with a TaqMan Low Density Array (Applied Biosystems) as previously described 2 for 8–12 days. The plates were then washed and stained with 0.1% crystal violet in 50% methanol, and the number of colonies was then counted. The fraction of surviving cells was calculated by comparison of LNA-193b with LNA-scrambled transfected cells.The viability of FaDu cells transfected with locked nucleic acid (LNA)-193b was determined using CellTiter 96 Non-Radioactive Cell Proliferation Assay (MTS) (Promega BioSciences), according to the manufacturer’s recommendations. The colony forming ability of FaDu cells transfected with LNA-193b was determined using clonogenic assay as previously described Cell cycle analysis was conducted on FaDu cells after LNA-193b transfection, as previously described 5 cells in 0.5% serum were added to the top of the chamber, and 20% serum added to the bottom chamber. Twenty-four hours later, inserts were fixed and stained with SIEMENNS DIFF-QUICK stain set , and the number of migrating and invading cells was counted using a light microscope.To assess the migration and invasion potential of FaDu cells after transfection with LNA-193b, BD BioCoat Control 8.0 um PET Membranes, were utilized to measure migration, and BD BioCoat Matrigel™ invasion chambers with a uniform layer of BD Matrigel basement membrane matrix were used to measure invasion. For both the migration and invasion assays, the chambers were placed in a 24-well plate, and 1.5×105 cells were suspended in 100 uL of media, and injected intramuscularly into the left gastrocnemius muscle of 8–10 week old female severe combined immunodeficient (SCID) BALB/c mice. Tumour growth was monitored by measuring the tumor plus leg diameter (TLD) two-three times per week; mice were sacrificed once the TLD reached 13 mm as a humane end-point.FaDu cells were transfected with LNA-scrambled or LNA-193b. Forty-eight hours later, viable cells were harvested and 2.5×10luciferase gene in the pMIR-REPORT vector . Another vector was constructed which carried a mutation of the NF1 3′UTR in the seed region of the miR-193b binding site using the indicated primers , with the firefly luciferase activity normalized to that of Renilla luciferase.The 3′ UTR of NF1 containing the predicted miR-193b binding site was amplified using AmpliTaq gold DNA polymerase (Applied Biosystems), using the indicated primers . Next, t primers . Twenty-FaDu cells were transfected with LNA-scrambled or LNA-193b, 72 hours post-transfection and lysed in 1 M Tris-HCl (pH 8), 5 M NaCl, and 1% NP40 plus the protease inhibitor cocktail (Roche Diagnostics). Protein concentration was assessed using the Bio-Rad Detergent-Compatible Protein Assay (Bio-Rad Laboratories). A total of 30 mg of protein was loaded onto 4–12% Tris-glycine protein gels (Invitrogen) for electrophoresis. The protein was transferred onto a PVDF (polyvinylidene fluoride) membrane using a mini Trans-Blot wet Transfer Cell (Bio-Rad). Next, membranes were blocked with Odyssey Blocking Buffer (Li-cor Biosciences). The membranes were probed with anti-NF1 rabbit polyclonal antibody , anti-Phospho-Erk rabbit monoclonal antibody , anti-Erk mouse monoclonal antibody , anti-tubulin mouse monoclonal antibody , anti-B-actin mouse monoclonal antibody , all followed by IRDye(r) Fluorescent secondary antibodies . Westerns blots were quantified using Odyssey Application Software 2.1 (Li-Cor Biosciences). When blotting for p-ERK at the 72-hour time point, the media was changed 24 hours after transfection with LNA-193b and LNA-scrambled since no protein otherwise would be detected.Cells were lysed in 1 M Tris-HCl (pH 8), 5 M NaCl, and 1% NP40 plus the protease inhibitor cocktail (Roche Diagnostics). Immunoprecipitations were preformed with glutathione-Sepharose 4 Fast Flow  coupled to GST-RBD (GST-fused to the Ras binding domain) FaDu cells were seeded in 6- or 96-well plates and 24 hours later, treated with 10 uM of U0126  or DMSO alone. MTS, migration and invasion assay were performed as described above.in situ hybridization on 5 um thick FFPE sections. A catalyzed signal amplification system method  was performed using 5′ biotin-labelled miR-193b miRCURY™ locked nucleic acid detection probe, or a scrambled negative control probe . Positive hybridization signals were visualized by adding the chromogenic substrate diaminobenzidine.Expression of miR-193b was evaluated in HNSCC patient samples using Expression of p-ERK was evaluated on primary HNSCC sections using microwave antigen retrieval in combination with the Level-2 Ultra Streptavidin System and anti-Phospho-Erk rabbit mAB .t test. Analysis and graphs were completed using Graphpad Prism Software .All experiments have been performed at least three independent times, and the data are presented as the mean ± SEM. The statistical significance between two treatment groups was determined using the Student’s vs. non-relapsed patients  approach. Corroboration of sustained reduction in miR-193b expression after LNA-193b was observed for up to 72 hours for all three HNSCC cell lines , with thin vivo using SCID mice injected intra-muscularly with FaDu cells transfected with LNA-193b or LNA-scrambled. Although not significant, suppression of miR-193b delayed tumour formation compared to the negative control , and invasion (35%) of FaDu cells compared to negative control . Tumorig control , which cin silico prediction algorithms, experimentally- determined data, and publicly available patient data To identify potential targets of miR-193b, we used a tri-modality approach previously described by our lab which incorporates bona fide miR-193b target  compared to control cells . This efb target . Similarb target . Thus, NNF1 is a GTPase which accelerates the conversion of active RAS-GTP into inactive RAS-GDP, thereby negatively regulating RAS signalling In situ hybridization was utilized to visually confirm miR-193b expression in primary HNSCC tissues. A miR-193b signal was observed in the cytoplasm of the tumour cells (vs. low (≤ median) expression, the former group experienced a worse disease-free survival compared to the latter ; although statistical significance was not attained due to the small cohort size  qRT-PCR analysis for miR-193b was conducted on 51 HNSCC primary tissue samples showing high expression in 43 relapsed vs. low expression for 8 non- relapsed tumors, relative to the expression of four normal larynx tissues. (B) qRT-PCR of miR-193b expression in HNSCC cell lines 24–72 hours after transfection with LNA-193b (40 nM) or LNA-scramble (40 nM). (C) Cell viability was assessed in UTSCC 42a and 8 cells by the MTS assay 24, 48 and 72 hours post transfection with LNA-193b (40 nM) or LNA-scramble (40 nM). (D) Clonogenic survival of UTSCC 42a and 8 cells was measured 10 to 12 days after transfection with LNA-193b (40 nM) or LNA-scramble (40 nM). (E) Cell cycle analysis was performed on FaDu cells using flow cytometry 72 hours post transfection with LNA-193b (40 nM) or LNA-scramble (40 nM). **P<0.005, ***P<0.0005, P = ns (not significant).(TIF)Click here for additional data file.Figure S2Identification of mRNA targets of miR-193b. (A) Venn diagram showing the tri-modality approach used to identify miR-193b targets. (B) NF1 transcript expression of UTSCC 42a and 8 cells was measured 72 hours post transfection with LNA-193b (40 nM) or LNA-scramble (40 nM). P = ns (not significant).(TIF)Click here for additional data file.Figure S3Identification of PER2 as a target of miR-193b. (A) PER2 transcript expression in FaDu cells was measured 72 hours post transfection with LNA-193b (40 nM) or LNA-scramble (40 nM). (B) PER2 transcript expression in UTSCC 42a was measured 72 hours post transfection with LNA-193b (40 nM) or LNA-scramble (40 nM). (C) Western blotting of PER2 in FaDu cells lines was determined 72 hours post transfection, images (above), quantification (below). (D) Relative luciferase activity of FaDu cells after co-transfection with pMIR-PER2 UTR (100 ng) or pMIR-PER2 Mutant (100 ng) vectors with LNA-193b (40 nM) or LNA-scramble (40 nM). *P<0.05, **P<0.005, P = ns (not significant).(TIF)Click here for additional data file.Figure S4MiR-193b targets the RAS signalling pathway in vitro and across HNSCC patient samples. (A) A representative image of miR-193b in situ hybridization of primary HNSCC biopsy samples, arrows indicate tumor cells exhibiting cytoplasmic staining. (B) Representative image of control in situ hybridization of primary HNSCC biopsy samples using a scramble probe. (C) Representative image of miR-193b in situ hybridization of primary breast cancer sample. (D) Representative image of immunohistochemical analysis of p-ERK expression in primary HNSCC biopsy samples (same patient as A), arrows indicate tumors exhibiting nuclear and cytoplasmic staining.(TIF)Click here for additional data file.Table S1(A) qRT-PCR primer design sequences (B) Cloning primer design sequences.(TIF)Click here for additional data file.


The MnII cation is coordinated by two N-bonded selenocyanate anions, two bpa ligands and two O-bonded methanol molecules, within a slightly distorted octahedral geometry. The MnII cations and the non-coordinating N-donor ligands are located on centers of inversion while the coordinating N-donor co-ligands are located on a twofold rotation axis. In the crystal, the MnII cations are linked into chains along the c-axis direction by the bpa ligands. The chains are further connected via a non-coordinating bpa ligand into layers parallel to (3-10) via O—H⋯N hydrogen-bonding interactions.The reaction of manganese selenocyanate with 1,2-bis(pyridin-4-yl)ethane (bpa) leads to the title compound, {[Mn(NCSe) DOI: Click here for additional data file.10.1107/S1600536813007150/bt6898Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


Induced pluripotent stem cells (iPSCs) hold promise as a potential treatment for Duchennemuscular dystrophy (DMD). To determine the impact of the donor’s age onreprogramming, we generated iPSCs from muscle-derived fibroblasts (MuFs) of mdx mice aged 6weeks, 6 months, and 14 months. MuFs from 14-month-old mdx mice showed lower proliferativeactivity and lower reprogramming efficiency, compared with those from younger mdx mice.Furthermore, iPSCs derived from 14-month-old mdx mice (14m-MuF-iPSCs) gradually lost Nanogexpression, and regressed in conventional ES medium during passages. Interestingly,inhibition of TGF-β signaling and BMP signaling stabilized Nanog expression andpromoted self-renewal of 14m-MuF-iPSCs. Finally, rescued mdx-derived iPSCs efficientlydifferentiated into the skeletal muscle lineage. Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disorder caused by mutationsin the dystrophin gene . Satellite cells/myoblasts might be good candidates Recent studies show that the nature of the original cell has profound impacts on both theefficiency of reprogramming and the properties of established iPSCs. Therefore, it isimportant to choose the optimal cell type for establishment of safe, high-qualityiPSCsmdx mice, a widely used modelof DMD, at different ages. Muscle-derived fibroblasts (MuFs) from14-month-old mdx(14m-MuF-iPSCs) mice showed reduced replicative activity and lower reprogramming efficiencythan those of younger mdx mice. Furthermore, iPSCs from 14-month-old mdx micewere unstable. Interestingly, inhibition of TGF-b and BMP signaling enabled us to obtainstable 14m-MuF-iPSCs. Finally, rescued 14m-MuF-iPSCs as efficiently differentiated intoskeletal muscle lineage as iPSCs from younger mdx mice.In this report, to analyze the impact of aging on the generation efficiency and propertiesof iPSCs, we derived iPSCs from muscle fibroblasts of mdx mice, which were generated by backcrossing ofC57BL/6-background mdx to C57Bl/6 mice, were generously given by Dr. T. Sasaoka were maintained in our animalfacility. C57BL/6 and NOD/Scid mice were purchased from Nihon CLEA .All experimental procedures were approved by the Experimental Animal Care and Use Committeeat the National Institute of Neuroscience (No. 2008014).C57BL/6-background mdx mice. After removal of nerves, blood vessels, fatand connective tissues, muscle tissues were minced into small pieces by scissors, andplated onto culture dishes in MEF medium: Dulbeccos’ modified eagle medium  containing 15% fetal bovine serum , 1 mM sodium pyruvate(Invitrogen), 0.1 mM nonessential amino acids (NEAA) (Invitrogen), 2 mM L-glutamine(Sigma), 50 U/ml penicillin and 50 mg/ml streptomycin (Invitrogen). Mouse embryonicfibroblasts (MEFs) and tail-tip fibroblasts (TTFs) were prepared as described by Takahashiet al. Fibroblasts were isolated from whole hind limb muscles of 6-week-old (6w), 6-month-old(6m), or 14-month-old (14m) 6 cells per 60 mm collagen-coateddish (Iwaki). The next day, the cells were separately transfected with each retroviralvector plasmid by using FuGene 6 transfection reagent (Roche Diagnostics). Twenty fourhours after transfection, the medium was replaced with new mouse embryonic fibroblasts(MEF) medium. After another 24 hours, the medium was collected, and concentrated toone-tenth using an Amicon Ultra centrifugal filter (Ultra-15)(Millipore).  Plat-E packaging cells and pMX vectors were provided by Dr. T. Kitamura of the Universityof Tokyo 4 cells/well in 6-well plates or1.5-2.0x104 cells/dish in 60-mm dishes (IWAKI). The cells were maintained ina humidified atmosphere with 5% CO2 in air at 37°C. Two weeks aftertransduction, ES-like colonies were picked up and expanded. iPSCs were also maintained inthe presence of valproic acid (VPA) (0.5 mM) (Sigma), Bix01294 (1 mM) (Stemgent), RG108 (1mM) (Stemgent), PD0325901 (1 mM) (Stemgent), CHIR99021 (3 mM) (Stemgent), SB431542 (2.5mg/ml) (Sigma), recombinant (r) BMP-7 (100 ng/ml) (R&D Systems), recombinant BMP-4(100 ng/ml) (Stemgent), rNoggin (100 ng/ml)(R&D Systems), rTGF-β1 (100 ng/ml)(Stemgent), mouse rBMP-1a/ALK-3 Fc chimera protein (50 ng/ml), (R&D systems), humanrBMPR-1b/ALK-6 Fc chimera protein (50 ng/ml) (R&D systems), or mouse sTGF-βRII Fc chimera protein (50 ng/ml) (R&D Systems).The concentrated virus-containing medium was added to fibroblast cultures. Two dayslater, the medium was changed to ES medium: DMEM (Wako) containing 15% FBS (Hyclone), 1 mMsodium pyruvate (Invitrogen), 0.1 mM NAA (Invitrogen), 2 mM L-glutamine (Sigma), 50 U/mlpenicillin and 50 mg/ml streptomycin (Invitrogen), 0.11 mM 2-mercaptoethanol (2-ME)(Invitrogen), and 50 ng/ml recombinant mouse leukemia inhibitory factor (rLIF)(Millipore). Four days after retroviral transduction, fibroblasts were re-seeded onmitomycin C-treated MEFs at 1.0x10Eras, Fgf4, Oct4, Dax1, Nanog, Utf1, Cripto, Zfp296,Sox2, and Nat1, we referred to Takahashi et al. [Pax7, MyoD,Myogenin,Tgfbr2, Acvr1, Acvr2a, and Bmpr2 were amplified using the following primers:Pax7, 5’-catccagtgctggtaccccacag-3’  and5’-ctgtggatgtcacctgcttgaa-3’, MyoD,5’-aggctctgctgcgcgaccag-3’, and5’-tgcagtcgatctctcaaagc-3’, Myogenin,5’-tgagggagaagcgcaggctcaag-3’, and5’-atgctgtccacgatggacgtaagg-3’ Tgfbr2,5’-ggcttcactctggaagatgc-3’ and 5’-gggactgctggtggtgtatt-3’;Acvr1, 5’-cccaactctgaaacggacat-3’ and 5’-tgttgcatgggtaatggcta-3’; Acvr2a, 5’-gttacaccgaagccacccta-3’and 5’-acaggagggtaggccatctt-3’; Bmpr2,5’-ataggcgtgtgccaaaaata-3’ and 5’-attgtcaatggtgtgctgga-3’.PCR was performed on an iQ5 single color real-time PCR detection system (BIO-RAD). Total RNA was extracted from cells using MicroRNeasy Kit (Qiagen). Complementary DNA wassynthesized with QuantiTect reverse transcription kit (Qiagen). For primer sequences andconditions for RT-PCR analysis of i et al. . Pax7, M6 cells / 100ml in PBS containing 2% FBS, and incubated with CD31-PE (BD Bioscience), CD45-PE (BDBioscience), Sca-1-PE(BD Bioscience), PDGFRa-PE (eBiosciences), or integrin a7-PE  antibodies. Analyses and cell sorting were performed on aFACSAria flow cytometer (BD Bioscience). Cells were trypsinized, re-suspended at a concentration of 1.0x10For AP activity, cells were stained with an Alkaline Phosphatase Substrate Kit III. The image was recorded with a microscope BIOREVO BZ-9000(Keyence). For immunocytochemistry, cells were fixed with 4% paraformaldehyde for 5 min,permeabilized with 0.1% Triton-X in PBS for 10-30 min, blocked with 5% goat (Cedarlane) orhorse (Invitrogen) serum in 2%BSA for 15 min, and then incubated with anti-Nanog rabbitpolyclonal antibody (reproCELL), anti-sox2 antibody (6F1.2) (Millipore), anti-oct4antibody (C-10) (Santa Cruz Biotechnology), mouse monoclonal beta III tubulin , alpha-fetoprotein (AFP) (c-19) (Santa Cruz), mouse monoclonal cardiac troponin Tantibody , mouse monoclonal anti-Pax7 antibody ,rabbit anti-MyoD antibody (Santa Cruz), mouse monoclonal anti-myogenin antibody  or anti-MHC antibody . The specimens were thenincubated with secondary antibodies labeled with Alexa-Flour 488 or 568 (MolecularProbes). Images were photographed using a fluorescence microscope IX71  equipped with Orca2 air-cooled CCD camera (Hamamatsu Photonics) and AQUACOSMOSsoftware (Hamamatsu Photonics).In vitro differentiation was performed as described by Chang and colleagues 6) were subcutaneously injected into 6- to 8-week-oldNOD/Scid mice. Five weeks after injection, tumors were dissected and fixed in 15%formalin, embedded in paraffin, cut by a microtome, and stained with hematoxylin and eosin(H. & E.).iPSCs (1x10http://www.3d-gene.com). Total RNAs from iPSCs andESCs (E14) were labeled with Cy3- or Cy5- using the Amino Allyl MessageAmp II aRNAAmplification Kit (Applied Biosystems). The Cy3- or Cy5-labeled aRNA pools were thenhybridized with a 3D-Gene Mouse Oligo chip 24k in a buffer containing micro beads for 16h. Data were analyzed by GeneSpringTM software version 7.3.1 (SiliconGenetics).Microarray analysis was performed by Toray Industries, Inc. CD45(-)integrin a7(-)Sca-1(+) PDGFRa(+). In contrast, mouse embryonic fibroblasts (MEF) areheterogeneous in Sca1 expression, and negative for PDGFRa. Gene expression array analysisalso showed that MEF and muscle fibroblasts are quite different in gene expression profiles. Muscle fibroblasts from 14-month-old mdx mice(14m-MuFs) proliferated more slowly  than MuFs from younger mdxmice. Four reprogramming factors , Klf4, and c-Myc)and DsRed were introduced by retroviral vectors into MuFs. The infection efficiencyof 14m-MuFs (~30%) was much lower than those of 6w-MuFs (~60%), or 6m-MuFs (~60%). Alkalinephosphatase (AP) is an early marker of reprogramming. 14m-MuFs gave rise to fewerAP-positive colonies than those from younger mdx mice (data not shown). Next wesorted DsRed-positive cells by FACS and seeded them on feeders in 60 mm dishes, but the lowreprogramming efficiency did not change . Silencing ofretrovirally-induced transgenes occurs at a later stage of reprogramming Figure 1C, D).To determine the impact of aging on the iPSCs generation, we isolated muscle fibroblasts(MuFs) from 6-week-old, 6-month-old, and 14-month-old Figure2A). To further examine the properties of 14m-MuF-iPSCs, we picked up 50DsRed-negative (retrovirus silenced) colonies derived from six 14-month-old mdx miceand expanded them in vitro. However, 14m-MuF-iPSCs clones were found to be unstableafter 2-3 passages: many colonies regressed, and colony numbers dramatically decreased. AP staining of 14m-MuF-iPSCs became weak or faint after expansion. Similarly, Nanogexpression was gradually lost in 14m-MuF-iPSCs. In contrast, nearly all 6w-iPSC and 6m-iPSC lines tested werestable and remained Nanog positive after further passages . Theseresults indicate that the four reprogramming factors are capable of inducing a pluripotentES-like state in 14m-MuFs. However, in contrast to iPSCs from younger mice, the pluripotentstate of 14m-MuF-iPSCs was unstable. To select fully reprogrammed iPSCs, we picked up DsRed-negative colonies with ES-likemorphology. All clones expressed a battery of ES-specific genes (Supplementary 2=0.86-0.90) (n=3) than those between MEF-iPSCs and ESCs cells(r2=0.96) or 6m-iPSCs and ESCs (r2=0.95) . Theexpression levels of the genes involved in maturation and self-renewal of iPSCs datanot shown).To determine the molecular basis of the instability of 14m-MuF-iPSCs, three 14m-iPS clonesderived from different mice were selected, and global gene expression analysis wasperformed. The Pearson correlation coefficient between 14m-MuF-iPSCs and ESCs was lower than other iPSCs or ESCs after 2-3 passages , although there was no difference between the levels of these receptors in14m-MuFs and younger MuFs (data not shown).To obtain stable 14m-MuF-iPSCs, wechallenge several small molecules that were reported to improve reprogramming efficiency Figure 3B), but not on ESCs or iPSCs generated fromyoung mice (data not shown). We also added epigenetic modifiers to the culture medium of14m-MuF-iPSCs. VPA, Bix01294, and RG108 did not increase the numbers of Nanog-positivecolonies. A MEK inhibitor (PD0325901) and GSK3β inhibitor (CHIR99021), showed nobeneficial effects on stabilization of 14m-MuF-iPSCs . Interestingly, aTGF-β inhibitor, SB431542, which targets type I TGF-β receptors, Alk-4,Alk-5, and Alk-7 Figure3C). To confirm the effects of blockage of TGF-β or BMP signaling on14m-MuF-iPSCs, we added soluble fragments of BMPR-1a (sBMPR-1a), BMPR-1b (sBMPR-1b), orTGF-β RII (sTGF-βRII) to the culture medium of 14m-MuF-iPSCs. Exposure tothese soluble receptors resulted in an increase in the number of Nanog-positive colonies. The treated cells formed ES-like colonies, grew rapidly, and continuedto expressNanog . Then, ES-like colonies were picked up again and culturedin ES medium without SB431542 and Noggin. Surprisingly, even after removal of theinhibitors, the number of Nanog positive colonies did not decrease during passages.Interestingly, 14m-MuF-iPSCs exhibited higher mRNA levels ofmdx mice is due to the aging, we isolated tail-tip fibroblasts from 14-month-oldmdx mice (14m-TTFs). 14m-TTFs are CD31(-)CD45(-)integrin a7(-)Sca1(+) PDGFRa(+)(data not shown), and showed high viability (Supplementary figure 2A) andproliferation rate similar to mouse embryonic fibroblasts (MEFs). Importantly, 14m-TTFsshowed much higher reprogramming efficiency (Supplementary figure 2B) than 14m-MuFs.AP staining and Nanog staining (Supplementary figure 2C), and global gene expressionanalysis  confirmed that iPSCs derived from 14m-TTFs were fullyreprogrammed by four reprogramming factors. The established 14m-TTF-iPSCs were stable duringpassages (data not shown). These results suggest that the cellular senescence of14m-MuFs, rather than the age of the animal, is a main cause for the low efficiency ofreprogramming and the unstable state.To determine whether the low efficiency of reprogramming of fibroblasts from 14-month-oldSupplementary figure 3), and maintained in ES medium. To determine whether iPSCsderived from mdx mice were indeed pluripotent, we assessed the expression of pluripotentmarkers. All mdx-derived iPSCs were positive for Sox2, Oct4, and Nanog (Supplementaryfigure 3B). Next, we evaluated the in vitro differentiation capacity of iPSCs byformation of embryoid bodies (EBs) in suspension culture. All iPSCs tested formed EBs after6 days of culture and differentiated into different cell types as detected by the expressionof neuron-specific tubulin (Tuj1), toroponin T (cardiac), and endodermalα-fetoprotein (AFP) after attachment on Matrigel-coated plates (Supplementaryfigure 3C). Lastly, we tested the ability of these cells to generate teratomas inNOD/Scid mice. Six to eight weeks after injection, all iPSCs formed teratomascontaining complex tissue structures characteristic of all three germ layers(Supplementary figure 3D). After SB431642 and Noggin treatment, three sub-lines of 14m-MuF-iPSCs were established Figure5B). All mdx-derived iPSCs formed EBs and differentiated into multinucleatedmuscle fibers . Immunocytochemistry confirmed the expression of Pax7,MyoD, myogenin, and myosin heavy chain (MHC) . In this culture system, we found numerous Pax7-positivecells and Pax7-positive/MyoD-positive cells  two weeks after plating EBson Matrigel plates, indicating that myogenic stem/progenitor cells were successfully inducedin all mdx-derived iPSCs. To evaluated myogenic potential of mdx-derived iPSCs, we applied a directed skeletal muscledifferentiation protocol as described in M&M  from 14-month-old mdx mice showed lower proliferativeactivity and lower reprogramming efficiency, compared with those from younger mdxmice. In addition, 14m-MuF-iPSCs gradually lost Nanogexpression, and regressed inconventional ES medium during passages. In contrast, tail-tip fibroblasts prepared from14-month-old mdx mice were highly proliferative, and efficiently reprogrammed, andstable in culture. These observations suggest that poor reprogramming can not be simplyattributed to the age of the mice. Dystrophin deficiency causes continuousdegeneration/regeneration of skeletal muscle, and older mdx muscles show fibrosis. Interestingly, FACS analysis showed that musclefibroblasts are CD31(-)CD45(-)integrina7(-)Sca1(+)PDGFRa(+), suggesting that they aremuscle-resident fibro/adipogenic progenitors (FAPs), which were recently demonstrated toproliferate in response to damage and facilitate myogenesis but differentiate intoadipocytes and promote fibrosis in pathological conditions In this study, we examined the reprogramming efficiency and quality of iPSCs generated frommuscle-derived fibroblasts (MuFs) of data not shown). Previous studies showed that BMPs support self-renewal of mouseESCs Several chemicals have recently been reported to either enhance reprogramming efficienciesor substitute for specific reprogramming factors ex vivo gene transfer technique, patient-derived iPScells are a promising tool to combat degenerative muscle disorders.Importantly, we found mdx-iPSCs efficiently differentiate into myofibers. In the culture,we found numerous Pax7-positive cells and Pax7-positive/MyoD-positive cells in the muscleinduction system. This observation is promising, because Pax7 is a marker of muscle stem/progenitor cells and muscle satellite cells We thank all members of the Department of Molecular Therapy for technical support anddiscussion. We also appreciate Ryoko Nakagawa for technical assistance.This work was supported by a research grant for Neuromuscular Diseases (19A-7), a researchgrant for Nervous and Mental Disorders (20B-1), Health and Labor Sciences Research Grantsfor Translation Research ,Health Sciences Research Grants for Research on Psychiatry and Neurological Disease andMental Health (H18-kokoro-019) from the Ministry of Health, Labour and Welfare,Grants-in-Aid for Scientific Research  and a grant for the realizationof regenerative medicine from the Ministry of Education, Culture, Sports, Science andTechnology.The authors indicate no potential conflicts of interest.


The second CuII atom is five-coordinated with a distorted square-pyramidal coordination sphere comprising a single bidentate chelating pyrimidine-2-carboxylate anion and three azide N anions, two of which doubly bridge centrosymmetric CuII centres, giving a two-dimensional network structure extending parallel to (010).In the title compound, [Cu DOI: 10.1107/S1600536810027030/zs2045Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Caenorhabditis elegans are typically generated by injecting DNA into the germline to form multi-copy extrachromosomal arrays. These transgenes are semi-stable and their expression is silenced in the germline. Mos1 transposon or microparticle bombardment methods have been developed to create single- or low-copy chromosomal integrated lines. Here we report an alternative method using ultraviolet trimethylpsoralen (UV/TMP) to generate single/low-copy gene integrations.Transgenic strains of vps-45 rescue fragment and negative selection based on benzimidazole sensitivity with a ben-1 rescue fragment. We confirmed that the integrants express transgenes in the germline. Quantitative PCR revealed that strains generated by this method contain single- or low-copy transgenes. Moreover, positive selection marker genes flanked by LoxP sites were excised by Cre recombinase mRNA microinjection, demonstrating Cre-mediated chromosomal excision for the first time in C. elegans.We successfully integrated low-copy transgenes from extrachromosomal arrays using positive selection based on temperature sensitivity with a Our UV/TMP integration method, based on familiar extrachromosomal transgenics, provides a useful approach for generating single/low-copy gene integrations. Caenorhabditis elegans, transgenic strains are typically generated by injecting DNA into the syncytial germ cells of the hermaphrodite gonad to form multi-copy extrachromosomal arrays [The development of methods to introduce exogenous DNA into animals has allowed for diverse genetic manipulations in many organisms. In l arrays . These tl arrays . Extrachl arrays , leadingl arrays . Althougl arrays ,5, integ4/bombardment) [Methods using microparticle bombardment were developed to create low-copy chromosomal integrated lines . The bioardment) . More reardment) . This teardment) . MosSCI ardment) . In the ardment) .In reverse genetic studies, ultraviolet trimethylpsoralen (UV/TMP), which induces a small deletion in the chromosomes, has been widely used to generate deletion mutants -11. In aIn the present study, we developed a technique using UV/TMP that produces single- or low- copy gene integrations from extrachromosomal arrays. In this method, we used a positive selection marker flanked by LoxP sites, allowing the marker to be excised by the Cre recombinase.vps-45 mutant phenotype. vps-45 mutants are unable to grow and reproduce normally at 20°C [vps-45 mutants carrying the positive selection marker, the vps-45 mini gene, grow and reproduce, allowing for easy identification of the transformants. To discriminate single- or low-copy integrations from extrachromosomal arrays and multi-copy integrations, we used ben-1 as a negative selection marker. Mutants of ben-1, which encodes a ß-tubulin of C. elegans, are resistant to an anti-tubulin drug benzimidazole: mutants grow paradoxically more quickly than wild-type animals, which are severely unhealthy, dumpy, and uncoordinated in movement on benzimidazole-containing selection media [ben-1 mutants not carrying the negative selection marker, the ben-1 gene, predominantly grow and reproduce on the selection media, enabling differentiation of low copy integrants from Ex arrays and multi-copy integrants that are highly likely to have the ben-1 gene  tag sequence to detect the insertion. The negative selection marker contained the ben-1 genomic sequence. Extrachromosomal (Ex) arrays were generated by microinjection of the positive and negative selection plasmids with an injection marker into tm234(ben-1);tm246(vps-45). To insert the extrachromosomal transgenes into the chromosomes, animals carrying Ex arrays were treated with UV/TMP to induce a small deletion in the chromosomes, leading to insertion during gap repair. UV/TMP-treated animals were cultured on benzimidazole-containing media at 20°C for positive-negative selection. To identify integrants that retained an intact insertion sequence, we conducted PCR#A and PCR#B, which amplify the 5' and 3' regions, respectively, of HBG-LoxP-vps-45-LoxP  as a tester plasmid. We co-injected Phsp-16.1::venus with selection markers and an injection marker, generating a parent Ex strain tm234;tm246;tmEx2677. Approximately 16,000 P0 animals were treated by UV/TMP and cultured for positive-negative selection as described above, resulting in 12 transformants. To identify integrated strains retaining the intact Phsp-16.1::venus sequence, we performed PCR#C, which amplifies the whole region of Phsp-16.1::venus ;tm246(vps-45)-background: tmIs840, tmIs841, tmIs843, tmIs844, and tmIs864 showed almost the same relative amount as N2, suggesting that these strains had two copies of the vps-45 gene, probably because they were homozygous for alleles carrying a single insertion site ;tm246(vps-45)-background: tmIs839, tmIs842 showed two times higher amounts, and one strain in the tm234(ben-1);tm246(vps-45)-background: tmIs852 showed three or four times higher amounts compared to N2  produced 410-bp bands with PCR#2 and no band with PCR#3, indicating complete excision of the LoxP sites  but showed a complete excision pattern, was also tested, and showed a linked chromosomal position at IV:1~14 . These results indicated that chromosomal insertion of the floxed vps-45 mini gene was excised by Cre recombinase.To determine the insertion sites and confirm chromosomal-excision  of the LoxP sites, we performed single nucleotide polymorphism (SNP) mapping, in which linkage between the HBG-loxP sequence (produced by Cre-mediated excision and detected by PCR#2) and SNPs. We examined C. elegans researchers. Our methods exhibited higher efficiency when compared to the microparticle bombardment system, and lower efficiency when compared to MosSCI III and vps-45(tm246)X, as described previously [stround, 5'-ACGTGGGAATGGAACCATGT-3', 5'-TCTCCATTTCCTCTTCCTCC-3'; tm234_2ndround, 5'-CTCCGGACATTGTAACGGAA-3', 5'-CCCTCCATTTGAAAGAGTCC-3'; tm246, 5'-CGCAATTGGATACTACTTGT-3', 5'-TCTCCTGCTCTACTTCTGCT-3'.chniques . The wilchniques  and ideneviously ,13. The pFX_HBG_Lw_vps-45) was constructed by subcloning the HBG1 sequence , wild-type LoxP sequence (ATAACTTCGTATAGCATACATTATACGAAGTTAT) [vps-45 mini gene (eft-3p::vps-45cDNA::unc-86 3'-UTR), into pBluescriptII MCS. The whole sequence is available upon request. The negative selection marker plasmid (pGEMT_ben-1(+)) was constructed by TA-cloning in which the ben-1 genome (2414 bp of the 5' upstream region followed by the coding sequence and 785 bp of the 3'-UTR) was subcloned into the pGEMT-easy vector. To generate the Phsp-16.1::venus plasmid (pFX_hs::venusT), 117 bp of the upstream genomic region of the hsp-16.1 gene was cloned into the 5' region of the Venus expression vector in-frame [tmEx2274 transgenic animals, pFX_HBG_Lw_vps-45, pGEMT_ben-1(+) (80 ng/μl) were co-injected with Pmyo-2::venus as an injection marker (20 ng/μl) into tm234(ben-1);tm246(vps-45). To generate tmEx2677 transgenic animals, pFX_HBG_Lw_vps-45, pGEMT_ben-1(+) and pFX_hs::venusT were co-injected at 67 ng/μl each along with Pmyo-2::venus as an injection marker (20 ng/ml) into tm234(ben-1);tm246(vps-45).The positive selection marker plasmid (AAGTTAT) , and vpsin-frame . To genetm234(ben-1);tm246(vps-45)-background Ex lines described above (cultured at 20°C) were collected from the NGM agar plates and incubated for 1 h at room temperature in the dark at 0.5 μg/ml of TMP. The animals were irradiated with 365 nm UV with a UV hand-monitor (UVP Inc.) at 200 J/cm2. The intensities of the UV light were calibrated with a UV luminometer (UVP Inc.) and controlled by the exposure time. UV/TMP-treated worms were plated on NGM agar dishes and allowed to lay eggs at 20°C. After 24 h, adults and larvae were washed off to remove all the animals fertilized before treatment, and incubated at 20°C for another 24 h, while F1 animals hatched. F1 animals were collected, washed three times with M9 buffer, and plated onto NGM agar plates containing 10 μg/ml of benzimidazole (Wako)  and cultured at 20°C for 6 to 7 days. Because tm246(vps-45) mutants exhibit a temperature-sensitive phenotype, only tm246 mutants carrying vps-45 rescue transgenes can survive at 20°C (positive selection). In contrast, because ben-1(tm234) mutants are resistant to benzimidazole, tm234 mutants carrying ben-1 transgenes are sensitive and unable to survive on benzimidazole-containing plates (negative selection). Transformed animals were cloned and further cultured on benzimidazole-containing plates over several generations. To ensure that each strain was established independently, only one transformant was picked up from each selection plate. Transformants were selected by PCR#A and PCR#B  was dissolved completely in acetone at a concentration of 0.3 mg/ml and diluted to 0.5 μg/ml in M9 buffer just before use. Mixtures of young adults and L4 larvae of tm234ben-;tm246(vpin vitro using a mMESSAGE mMACHINE T7 kit (Ambion). Synthesized RNA was purified by phenol/chloroform extraction followed by isoamyl alcohol precipitation. Poly-A tailing was performed using Yeast Poly(A) Polymerase . Poly-A tailed RNA was purified by phenol/chloroform extraction followed by ethanol precipitation. Cre recombinase mRNA was injected at 1 μg/ml along with Pmyo-2::venus as an injection marker (67 ng/ml) into integrant animals carrying a floxed vps-45 mini gene. F1 animals were genotyped by PCR#1 to detect Cre-mediated excision. F2 self-progenies from heterozygotes for the Cre-excised allele were genotyped to obtain homozygotes for the Cre-excised allele. Homozygous lines were further tested by PCR#2 to detect non-excised allele   and clonact-2 gene. Primers were designed within an exon for each gene using the Primer3 software. The primers used are listed in Additional file Genome DNA was isolated from adult animals using DNeasy Tissue & Blood kit (QIAGEN). Quantitative PCR was performed in a 7500 Real-time Thermal cycler (Applied Biosystems) using the Power SYBR master mix (Applied Biosystems) with the following parameters: 95°C for 10 min and 40 cycles of 95°C for 5 s, 55°C for 10 s and 72°C for 34 s. All data were normalized to the To map insertion sites, SNPs between the Hawaiian strain CB4856  and the Differential interference contrast and fluorescence images were obtained using a BX51 microscope equipped with a DP30BW CCD camera .EKN, HK and SM conceived and designed the UV/TMP integration method, carried out and supervised the experiments, and drafted the manuscript. OF carried out the molecular genetic studies. MO, KGA, SY and SH participated in the design of study and the construction of selection markers. All authors read and approved the final manuscript.Figure S1. A positive-negative selection scheme. The tm246(vps-45) mutants exhibited a temperature-sensitive phenotype. Only tm246 mutants carrying vps-45 rescue transgenes survived at 20°C (positive selection). The tm234(ben-1) mutants were resistant to benzimidazole. tm234 mutants carrying ben-1 rescue transgenes are sensitive and unable to survive on benzimidazole-containing plates (negative selection). Phenotypes of tm234;tm246 were rescued by transgenes, enabling the Ex line to survive at 20°C. The parent Ex animals were treated by UV/TMP, resulting in multi-copy insertion (Is), low-copy Is, and Ex arrays. Because multi-copy Is animals and Ex animals were highly likely to have the ben-1 transgene, only low-copy Is animals carrying the vps-45 and not the ben-1 transgene were selected.Click here for fileTable S1. Comparison of single- or low-copy integration methods.Click here for fileFigure S2. Heat-shock induction of Venus mRNA in integrant strains. Relative expression of venus mRNA determined by quantitative RT-PCR. Total RNA was extracted from a 100-μl pellet of worms collected from 0-min, 30-min, and 1-h culture at 20°C after heat-shock at 32°C for 1 h. The expression of mRNA  is presented as a ratio to heat-shock (-) control. tmIs893 and tmIs894 were examined.Click here for fileTable S2. PCR primers.Click here for file


The conventional MRI protocol for the characterization of atherosclerotic plaques involves a series of scans that provide multiple contrast weightings for resolving high-risk plaque characteristics [1]. However, mis-registration between image sets due to the inter-scan motion often compromises evaluation accuracy and image interpretation is expertise dependent [2]. The aim of this work was to develop a 3D MRI technique that acquires multiple image sets in a single scan with distinct contrast weightings.1 = 945 mTms2/m, FSD/T2 duration = 18/40 ms, centric reordering, iPAT = 2, scan time = 5-6 min. For comparison, spatially matched T1-w/T2-w TSE and TOF imaging were performed.The developed MATCH sequence utilizes a low-flip-angle gradient echo-based MRI acquisition combined with magnetization preparative schemes, and multiple co-registered 3D image sets are collected in an interleaved scan with 4 TRs per cycle: the 1st TR provides hyper T1-weighted (T1w) contrast by using a nonselective inversion pulse and a blood-suppressing flow-sensitive dephasing (FSD) preparation [3]; the 2nd TR provides gray-blood lumen arising from both blood T1-recovery and in-flow fresh blood; the 3rd TR is for signal recovery without readout events, followed by the 4th TR for T2-weighted (T2w) contrast by using a long-duration FSD preparation. The three contrasts aims to identify the intra-plaque haemorrhage (IPH), juxtaluminal calcification (CA), loose matrix (LM), and potentially lipid core, respectively. The technique was optimized based on computer simulations and healthy volunteer studies and then evaluated on patients (n = 8) with carotid plaques on a 3T system (Siemens Verio). Imaging parameters include: 55-62 segments per TR of 1200 ms, flip angle = 8°, in-plane resolution = 0.55-0.63 mm, slice thickness = 2 mm, 18 slices, CHESS fat saturation, inversion time delay = 460-480 ms, mA total of 12 locations with one of plaque components were assessed. With the MATCH acquisition, IPH Figure  arrows aMATCH is a promising technique for an expedite and accurate characterization of carotid plaques. A large-scale patient validation is currently underway, using histology specimens as reference. Further technical improvements in spatial resolution and imaging speed will strengthen its clinical value.NIH HL096119, AHH 11POST7650043.


Salt stress is one of most serious limiting factors for crop growth and production. An isobaric Tags for Relative and Absolute Quantitation (iTRAQ) approach was used to analyze proteomic changes in rice shoots under salt stress in this study. A total of 56 proteins were significantly altered and 16 of them were enriched in the pathways of photosynthesis, antioxidant and oxidative phosphorylation. Among these 16 proteins, peroxiredoxin Q and photosystem I subunit D were up-regulated, while thioredoxin M-like, thioredoxin x, thioredoxin peroxidase, glutathione S-transferase F3, PSI subunit H, light-harvesting antenna complex I subunits, chloroplast chaperonin, vacuolar ATP synthase subunit H, and ATP synthase delta chain were down-regulated. Moreover, physiological data including total antioxidant capacity, peroxiredoxin activity, chlorophyll a/b content, glutathione S-transferase activity, reduced glutathione content and ATPase activity were consistent with changes in the levels of these proteins. The levels of the mRNAs encoding these proteins were also analyzed by real-time quantitative reverse transcription PCR, and approximately 86% of the results were consistent with the iTRAQ data. Importantly, our data suggest the important role of PSI in balancing energy supply and ROS generation under salt stress. This study provides information for an improved understanding of the function of photosynthesis and PSI in the salt-stress response of rice. Salt stress is a major obstacle limiting plant growth and development . Under hCyanobacteria under salt stress . Bars: SD. The changes in transcript abundances at 12 h were compared with the iTRAQ data. (A) Overview of differentially expressed proteins involved in regulation. (B) Putative MAR binding filament-like protein 1 (gi|55296302). (C) Putative transcription factor (gi|24431603). (D) Carboxyvinyl-carboxyphosphonate phosphorylmutase (gi|77553225). (E) 26S proteasome regulatory particle triple-A ATPase subunit 4 (gi|11094192). (F) Auxin-repressed protein-like protein ARP1 (gi|77552436). (G) Putative CBS domain containing protein (gi|34015153). (H) Hydroperoxide lyase (gi|33358444). (I) Putative P-II nitrogen sensing protein (gi|50878396).The transcript abundances of mRNAs encoding proteins involved in regulation were analyzed at 0 h, 1 h, 3 h, 6 h, 12 h and 24 h following salt stress treatment. The mRNA levels at 12 h were compared with the iTRAQ data. Red indicates the proteins that were up-regulated and green indicates the proteins that were down-regulated. Significant differences were determined relative to each treatment using a student’s (TIF)Click here for additional data file.S3 Figt-test [P-values <0.05 (*) and <0.01 (**)]. Bars: SD. The changes in transcript abundances at 12 h were compared with the iTRAQ data. (A) Overview of differentially expressed proteins involved in RNA and protein synthesis. (B) Putative ribosomal protein L34 (gi|37805854). (C) Putative acidic ribosomal protein P1a (gi|50725625). (D) 26S proteasome regulatory particle triple-A ATPase subunit 4 (gi|11094192).The transcript abundances of mRNAs encoding proteins involved in RNA and protein synthesis were analyzed at 0 h, 1 h, 3 h, 6 h, 12 h and 24 h following salt stress treatment. The mRNA levels at 12 h were compared with the iTRAQ data. Red indicates the proteins that were up-regulated and green indicates the proteins that were down-regulated. Significant differences were determined relative to each treatment using a student’s (TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Figt-test [P-values <0.05 (*) and <0.01 (**)]. Bars: SD. The changes in transcript abundances at 12 h were compared with the iTRAQ data. (A) qRT-PCR analysis of mRNA encoding H2A protein (gi|6319146). (B) qRT-PCR analysis of mRNAs encoding up-regulated proteins OSJNBb0008G24.11 (gi|19571117), putative nuclear RNA binding protein A (gi|18461235), Actin (gi|34851127), putative Isocitrate lyase (gi|34393921) and nonspecific lipid-transfer protein 2 precursor (gi|77553487). (C) qRT-PCR analysis of mRNAs encoding down-regulated proteins CBS domain (gi|62701927), OSJNBa0029H02.25 (gi|70663913), putative isopentenyl pyrophosphate: dimethyllallyl pyrophosphate isomerase (gi|27260946) and ferritin (gi|21686526).The transcript abundances of mRNAs encoding the differentially expressed proteins were analyzed at 0 h, 1 h, 3 h, 6 h, 12 h and 24 h following salt stress treatment. The mRNA levels at 12 h were compared with the iTRAQ data. Red indicates the proteins that were up-regulated and green indicates the proteins that were down-regulated. Significant differences were determined relative to each treatment using a student’s (TIF)Click here for additional data file.


GBA gene have recently also been identified as major genetic risk factor for Parkinsonism. Disturbed metabolism of GlcCer may therefore play a role in neuropathology. Besides lysosomal GBA, cells also contain a non-lysosomal glucosylceramidase (GBA2). Given that the two β-glucosidases share substrates, we speculated that over-activity of GBA2 during severe GBA impairment might influence neuropathology. This hypothesis was studied in Niemann-Pick type C (Npc1-/-) mice showing secondary deficiency in GBA in various tissues. Here we report that GBA2 activity is indeed increased in the brain of Npc1-/- mice. We found that GBA2 is particularly abundant in Purkinje cells (PCs), one of the most affected neuronal populations in NPC disease. Inhibiting GBA2 in Npc1-/- mice with a brain-permeable low nanomolar inhibitor significantly improved motor coordination and extended lifespan in the absence of correction in cholesterol and ganglioside abnormalities. This trend was recapitulated, although not to full extent, by introducing a genetic loss of GBA2 in Npc1-/- mice. Our findings point to GBA2 activity as therapeutic target in NPC.The enzyme glucocerebrosidase (GBA) hydrolyses glucosylceramide (GlcCer) in lysosomes. Markedly reduced GBA activity is associated with severe manifestations of Gaucher disease including neurological involvement. Mutations in the  GBA gene causes Gaucher disease (GD). This lysosomal storage disorder is remarkably heterogeneous in onset, progression and nature of symptoms. In severely affected GD patients with marked reduction in GBA activity neurological symptoms develop. Of interest, mutations in the GBA gene have recently been associated with development of Parkinson disease (PD) and other Lewy bodies disorders . Do. Doin vidination .Npc1-/- mice with special attention to GBA and GBA2. As reported earlier by others . Bo. BoGba2 Npc1-/- mice (BALB/c background) were fed a diet calculated to deliver 1 or 5 mg/kg.bw/day of MZ-21 or MZ-31. Again, iminosugar-mediated reduction of GBA2 activity increased lifespan by 20–30 days  and 9 weeks  (P = 0.060) (Npc1-/- mice need significantly longer time than controls to make a single step during the non-perturbed (P < 0.001) and the fix-obstacle (P < 0.001) sessions  . Remarka= 0.060) . Only atsessions . Thus, aNpc1-/- mice on brain lipid composition and lysosomes. No significant differences between total brain cholesterol and ceramide levels of age-matched (85-day-old) Npc1+/+, treated or untreated Npc1-/- mice were detected , GlcCer (P < 0.05) and GlcSph (P < 0.001) levels were increased in the cerebellum of 75-day-old untreated Npc1-/- mice compared to age-matched wt animals. In end-stage (115-day-old) Npc1-/- mice, treatment did not affect ceramide levels and aggravated elevations in GlcCer and GlcSph. GalCer was reduced in cerebellum of Npc1-/- mice.Lipid analysis of dissected cerebella gave very similar results to total brain . CeramidNpc1-/- mice as a consequence of lysosomal hypertrophy. Iminosugar-treatment did not normalize these markers  points to an impairment of the enzyme in vivo. Secondly, we examined the outcome of introducing a genetic loss of GBA2 in Npc1-/- mice. This ablation led to a significant increase in lifespan and amelioration of motor coordination, reflected by prolonged survival of PCs. Next, we investigated whether the loss of the GBA2 protein as such or the absence of its activity exerted the beneficial effect. Employing extremely potent GBA2 inhibitors , we observed that administration of as little as 25 μg per mouse daily led to similar beneficial responses as observed with genetic loss of GBA2, such as increased maximal body weight, delayed PC loss and delayed onset of motor deficits. The genetic background of mice is known to influence severity of disease manifestation in Npc1-/- animals [Npc1-/- mice led to a relatively larger increase in life span  [A currently approved drug for NPC disease is N-butyl-1-deoxynojirimycin . N-alkylated iminosugars such as Zavesca inhibit GlcCer synthase (GCS), the enzyme catalyzing the first step in the biosynthesis of GSLs . Zavescactively) . Inhibitctively) ,43. The Npc1-/- mice despite persistent lysosomal abnormalities. Evidently, reduction of this enzymatic activity in Npc1-/- mice, or even genetic absence of the enzyme, does not completely prevent neurodegeneration. Nevertheless, our results suggest that pharmacological inhibition of GBA2 might result in a clinically significant delay of neurological complications. Given their advantageous inhibitory profile GBA2 inhibitors AMP-DNM (MZ-21) and l-ido-AMP-DNM (MZ-31) may serve as lead components for drug development for NPC.In conclusion, GBA2 inhibition suffices to significantly increase lifespan of S1 ARRIVE Checklist(PDF)Click here for additional data file.S1 Fig(A) and cerebellum (C) homogenates of Npc1+/+, Npc1+/- and Npc1-/- mice (seewt (Npc1+/+) control. GBA and GBA2 enzymatic activities (assayed with 4MU-β-D-Glc substrate) in brain (B) and cerebellum (D) homogenates of Npc1+/+, Npc1+/- and Npc1-/- mice (seeQuantification of scanned band intensity of ABP 1-fluorescently labelled (slab gel) and immuno-probed (Western blot) GBA and GBA2 in brain mice see. Intensimice see.(TIF)Click here for additional data file.S2 FigNpc1+/+, Npc1+/- and Npc1-/- mice.Ratio of GBA2 and GBA enzymatic activities (assayed with 4MU-β-D-Glc substrate) in homogenates of dissected pons-medulla, cortex, hippocampus, olfactory bulb, hypothalamus and remaining brain (rest) of 75-day-old (TIF)Click here for additional data file.S3 FigNpc1+/+ and Npc1-/- mice were single immunostained with anti-calbindin D-28K and anti-GFAP antibodies. Staining patterns in the anterior cerebellum indicate progressive loss of calbindin D-28K positive Purkinje cells and concomitant increase of GFAP positive astrogliosis in Npc1-/- mice . Sagittal cerebellar sections of 40-, 60- and 80-day-old Npc1+/+ and Npc1-/- mice were double immunostained with anti-GBA and anti-GBA2 antibodies. For the central cerebellar zones, in the region where only few calbindin D-28K positive Purkinje cells remain in Npc1-/- mice at day 80, staining patterns of anti-GBA and anti-GBA2 are each displayed separately  and as composite image . The arrows indicate Purkinje cells.Sagittal cerebellar sections of 40-, 60- and 80-day-old (TIF)Click here for additional data file.S4 FigNpc1-/- and Gba2-/- crossed animals (seewt (Npc1+/+/Gba2+/+) control.Quantification of scanned band intensity of ABP1-fluorescently labelled (slab gel) GBA and GBA2 and immuno-probed (Western blot) GBA2 in brain homogenates of end-stage mals see. Intensi(TIF)Click here for additional data file.S5 FigNpc1+/+, Npc1+/-and Npc1-/- iminosugar-treated and untreated 85- and 110-day-old mice (seewt (Npc1+/+) control.Quantification of scanned band intensity of ABP1-fluorescently labelled (slab gel) GBA and GBA2 and immuno-probed (Western blot) GBA2 in brain homogenates of mice see. Intensi(TIF)Click here for additional data file.S6 FigNpc1+/+ and Npc1-/- untreated mice and treated with 1 mpk MZ21 stained with HE and immunostained with anti-Iba-1 and -F4/80 antibodies. Scale bar = 100 μm. (B) Klüver-PAS staining of sagittal cerebellar sections of 85-days old Npc1+/+ and Npc1-/- untreated mice and treated with 1 mpk MZ21. Scale bar = 50 μm.(A) Sagittal cerebellar sections of 85-day-old (TIF)Click here for additional data file.S7 FigNpc1+/+, Npc1+/-and Npc1-/- mice untreated (75 days of age) and treated with 5 mpk MZ-21 (115 days of age).Levels of ceramide, GlcCer, GalCer and GlcSph in dissected cerebella of (TIF)Click here for additional data file.S8 FigNpc1+/+/Gba2+/+ mice and did not label PCs nor any other structures in cerebellum of Npc1-/-/Gba2-/- mice. Haematoxylin was used as counterstain. (B) Cerebellum of Npc1+/-/Gba2-/- mice, which do not suffer from NPC disease and maintain presence of PCs as shown in haematoxylin and eosin (HE) stained sections, is not stained by anti-GBA2. (C) Double immunostaining directed against either first GBA and then GBA2 or first GBA2 and then GBA on Npc1+/+/Gba2+/+ cerebellum showed detection of GBA2 in PCs, both in the cell body and in the dendrites, and of GBA mainly in a punctuated pattern in the PC cell body. Background staining with secondary AP- and HRP-conjugated antibodies or with substrates VectorBlue and AMEC-Red was negligible. Methyl Green was applied as counterstain. (D) Double immunostaining with anti-GBA and anti-GBA2 revealed only binding of anti-GBA on PCs of Npc1-/-/Gba2-/- mice.(A) Single indirect immunostaining using rabbit-anti-GBA2 primary antibody, HRP-conjugated Goat-anti-Rabbit secondary antibody, and DAB as substrate did detect Purkinje cells (PCs) in cerebellum of (TIF)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S1 Text(DOC)Click here for additional data file.


The cine steady-state free-precession (SSFP) is the standard sequence for left ventricular (LV) function evaluation. However, SSFP mandates uninterrupted RF excitations to maintain steady-state (SS) during suspended respiration. In patients who cannot perform breath-holds (BH), a respiratory triggered (RT) free breathing (FB) retrospectively cardiac gated cine SSFP sequence that drives the magnetization to SS before commencing cine acquisition may be an alternative [1]. In this work, we validate the RT FB SSFP sequence by comparing it to the BH SSFP sequence for LV function evaluation.This prospective study included 21 consecutive patients  undergoing clinically indicated CMR. MR Acquisition: Images covering the LV in short-axis (SA) were acquired on a 1.5 T MR scanner  using identical parameters for both BH and FB RT cine SSFP sequences - TR/TE/flip angle: 2.6/1.32/70 degrees; acquired voxel size: 1.7-2.0 × 1.6-2.0 × 8 mm3; SENSE acceleration factor: 1.3-1.9; 12-14 slices to cover the LV; temporal resolution: 30 - 40 ms. Data Analyses: Image sets were randomized and anonymized. Two fellowship-trained cardiac imagers blinded to the study design independently scored image quality for myocardium-to-blood pool contrast, endocardial edge definition and inter-slice alignment on a 3-point scale . Readers also performed independent quantitative volumetric analyses on both image sets by manually drawing the LV endocardial and epicardial contours.There was no statistically significant difference in the quantitative metrics of global LV function, EDV, ESV, and EF estimated from images acquired with FB and BH MR techniques Table . Image qThe FB RT-SSFP sequence generates diagnostic image sets with contrast and spatio-temporal resolution that is comparable to BH SSFP sequence at the expense of a modest time penalty. Given that the LV functional parameters obtained from the two sequences were in good agreement, the FB RT sequence offers a robust alternative method for LV function evaluation in patients with impaired breath-holding capacity.NA.


Conceivably, using expired  CO2 as the input signal of closed-loop ventilation (Intellivent-ASV®) should optimize CO2 control compared to manual ventilator setting changes based on PaCO2 measurements.Both hypo- and hypercapnia can be deleterious to brain injured patients. Due to the variability of CO2 evolution over time during standard controlled ventilation and during Intellivent-ASV®.The aim of this study was to compare PaCO2 evolution during two sequential 2-hour periods of ventilation (standard ventilation and Intellivent-ASV® version VUP02.11c in the “brain injury setting”), applied in random-order. A one-hour washout period was inserted between both periods. Arterial blood gas analysis was performed every 30 minutes. The number of manual settings adjustments made on the ventilator and actions performed to reduce intracranial pressure (ICP) were also recorded. Due to the small number of patients in this preliminary dataset, no statistical differences were tested.Prospective interventional randomized study with a crossover design. Comparison of PaCO2, GCS at admission: 6 [4-6.5]. SAPS 2 score: 42 [32-50]. PaCO2 was 36 [33-37] mmHg (range: 28 and 43 mmHg) during standard ventilation and 36 [34-37] (range: 30 and 43 mmHg) during Intellivent-ASV®. DeltaPaCO2 between two consecutive PaCO2 measurements are illustrated in Figure (medians [IQR]):11 patients were included . Age: 52 [42-54] years. Body mass index: 25.5 [24.7-27.0] kg/m®. Four  and 2 (increase in sedation) actions were performed in order to decrease ICP during standard ventilation and Intellivent-ASV® respectively.During standard ventilation, 22 settings adaptations were performed whereas only two manual adjustments were made during Intellivent-ASV® CO2-regulated closed-loop ventilation mode can be safely used to deliver automated ventilation in brain injured patients. PaCO2 never reached extreme values and delta PaCO2 were very low during Intellivent-ASV®. Accordingly, fewer ventilator settings adaptations were required during Intellivent-ASV®. These preliminary results are promising and more patients must be included to evaluate the potential advantage of using Intellivent-ASV® to optimize the control of capnia during neuro-resuscitation.The Intellivent-ASV


The Ets-1 transcription factor is a candidate breast cancer oncogene that regulates the expression of genes involved in tumor progression and metastasis. Ets-1 signaling has also been linked to the development of a basal-like breast cancer phenotype. We recently described a nitric oxide (NO)-induced gene signature that is associated with poor disease outcome in estrogen receptor-negative (ER-) breast cancer and contains both stem cell-like and basal-like components. Thus, we examined the role of Ets-1 in NO signaling and NO-induced phenotypes in ER- human breast cancer cells.In vitro mechanisms were examined in human basal-like breast cancer cells lines. NO signaling effects were studied using either forced NOS2 expression or the use of a chemical NO-donor, diethlylenetriamine NONOate (DETANO).Promoter region analyses were performed on genes upregulated in inducible nitric oxide synthase (NOS2) high expressing tumors for Ets-binding sites. via a Ras/MEK/ERK signaling pathway by a mechanism that involved Ras S-nitrosylation. RNA knock-down of Ets-1 suppressed NO-induced expression of selected basal-like breast cancer markers such as P-cadherin, S100A8, IL-8 and αβ-crystallin. Additionally, Ets-1 knock-down reduced NO-mediated cellular proliferation, matrix metalloproteinase and cathepsin B activities, as well as matrigel invasion.Promoter region analysis of genes that are up-regulated in human ER-negative breast tumors with high NOS2 expression revealed that the Ets-binding sequence is the only common promoter element present in all of these genes, indicating that Ets-1 is the key transcriptional factor down-stream of oncogenic NOS2-signaling. Accordingly, both forced NOS2 over-expression and exposure to NO-donors resulted in significant Ets-1 transcriptional activation in ER- breast cancer cells. Functional studies showed that NO activated Ets-1 transcriptional activity These data show that Ets-1 is a key transcriptional mediator of oncogenic NO signaling that promotes the development of an aggressive disease phenotype in ER- breast cancer in an Ets-1 and Ras-dependent manner, providing novel clues of how NOS2 expression in human breast tumors is functionally linked to poor patient survival. Inducible nitric oxide synthase NOS2) is a pro-inflammatory enzyme generally with a key function in the innate immune response . However is a proEts-1 is an oncogenic transcription factor involved in the progression of breast cancer -21. Furtvia a Ras/mitogen-activated protein kinase (MEK)/ERK signaling axis by a mechanism that involved Ras S-nitrosylation (SNO). Finally, siRNA knock-down of Ets-1 also decreased NO-induced phenotypes of disease progression. Together, these data provide novel evidence that NO signaling promotes an aggressive breast cancer phenotype by activating the oncogenic Ets-1 transcription factor.Because both NOS2 and Ets-1 expression have oncogenic properties that advance the ER- disease, we investigated the functional relationship between them. This approach revealed that an Ets-binding sequence (EBS) is the only promoter element common to all genes in a previously described NOS2 expression signature for ER- breast tumors . Further2 and passaged two to three times per week and were authenticated by short tandem repeat profiling within the past six months (ATCC). Aminoguanidine (AG) and L-arginine (L-Arg) were purchased from Sigma-Aldrich . Farnesylthiosalicylic acid (FTS) and PD 184161 were purchased from Cayman Chemical . Gö6976 was purchased from EMD Chemicals . Recombinant human epidermal growth factor (EGF) was purchased from R&D Systems . Antibodies to αβ-crystalin, actin, Ets-1 and NOS2 were from Santa Cruz Biotechnology . Antibodies to phospho-ERK1/2 (thr 202/tyr 204), ERK1/2 and phospho-MEK1/2 (ser 217/221) were from Cell Signaling . Anti-Ras was from Thermo Scientific  and anti-phospho-Ets-1 (thr 38) was purchased from Invitrogen. DETANO was generously provided by Dr. Larry Keefer . DETANO stock solutions were made in 10 mM NaOH and concentrations were determined by absorbance at 250 nm (ε = 8000 M-1·cm-1) prior to every use.Human breast adenocarcinoma cell lines MDA-MB-231, MDA-MB-468 and SKBR3 (American Type Culture Collection (ATCC), Manassas, VA, USA) were cultured in RPMI medium  containing 10% fetal bovine serum  and 100 IU penicillin and 100 µg/ml streptomycin (Invitrogen). Cells were cultured at 37°C in 5% COet al. [The promoter sequence for each gene listed in Table 3 of Glynn et al.  was extrCells were transfected with 4 µg pCMV6-XL4 (empty vector) or pCMV6-XL4-human NOS2 (NM_000625)  by electroporation using the Amaxa Nucleofector kit V  and then grown for 48 hours under normal conditions before further treatment or analysis.3VO4 (10 mM), phenylmethylsulfonyl fluoride  and protease inhibitor cocktail ). Images were recorded on a FluoroChem SP system using AlphaEase FC software .Western blotting was performed by standard procedures. Cells were lysed on ice with cold lysis buffer (Tris-HCl pH 8.0 (50 mM), NaCl (150 mM), NP-40 (1%), ethylenediaminetetraacetic acid , NaF (50 mM), NaEts-1 transcriptional activity was performed by transiently transfecting cells with 750 ng of Ets-luciferase reporter plasmid expressing firefly luciferase  and 250 ng pGL4.70 plasmid expressing renella luciferase  using Lipofectamine LTX reagent for six hours at 37°C. After transfection, cell culture media was replaced with serum-free (D)MEM containing EGF (20 ng/mL), DETANO and inhibitors. Cells were incubated for 18 hours and luciferase activity was measured using the Dual-luciferase assay kit (Promega). Relative luminescent units (RLU) were measured using a Glomax 96-well plate luminometer (Promega) and data were normalized to fold change from untreated control cells. Data represent mean normalized RLU ± standard deviation (SD).Relative Ras activation was determined using the Ras binding domain (RBD)-pull-down assay kit (Thermo Scientific). Briefly, cell lysate was incubated with RBD-agarose beads. Immunoprecipitated active Ras was eluted by boiling in 4X-lithium dodecyl sulfate (LDS) sample buffer. Active Ras and total cellular Ras were measured by western blot. Activation of Ras is shown as mean fold increase compared to untreated cells ± SD. Ras was immunoprecipitated using Protein G-Dynabeads (Invitrogen) conjugated with monoclonal mouse anti-Ras and assayed with the S-Nitrosylated Protein Detection Kit  as instructed by the manufacturer. Procedures were performed under low ambient light to diminish Ras-SNO decomposition.6/100 µL) were transfected with 400 nM total siRNA by electroporation using the Amaxa Nucleofector Kit V. Cells were grown in RPMI + 10% FBS for 48 hours before further treatment or analysis. Human Ets-1 siGENOME SMARTpool (Thermo Scientific) oligonucleotide sequences: 5'-GAUAAAUCCUGUCAGUCUU-3'; 5'-GGACCGUGCUGACCUCAAU-3'; 5'-GGAAUUACUCACUGAUAAA-3' and 5'-GCAUAGAGAGCUACGAUAG-3'. Control siGENOME non-targeting siRNA pool (Thermo Scientific) sequences: 5'-UAGCGACUAAACACAUCAA-3'; 5'-UAAGGCUAUGAAGAGAUAC-3'; 5'-AUGUAUUGGCCUGUAUUAG-3' and 5'-AUGAACGUGAAUUGCUCAA-3'. Ets-1 knock-down was verified at the protein level by western blot.Cells (1 × 10Cells were treated with or without 0.5 mM DETANO in serum-free RPMI containing 20 µM bromodeoxyuridine (BrDU) for 24 hours. Using the BrDU ELISA kit , cells were then fixed, washed and BrDU incorporation was determined by incubating mouse anti-BrDU followed by anti-mouse-horseradish peroxidase (HRP) secondary. Absorbance data are normalized to fold-increase compared to untreated controls and are shown as mean fold change ± SD.Cathepsin B Activity Fluorometric Assay Kit  was used as instructed. Briefly, treated cells were lysed and samples were incubated with substrate Acetyl-arginine-arginine-amino-4-trifluoromethyl coumarin (Ac-RR-AFC). Released AFC was measured by fluorescence (400 nm excitation/505 nm emission). Data are normalized to fold change compared to untreated control cells and are shown as mean ± SD.MMP isoform expression was measured by spot-ELISA (R&D Systems) as instructed by the manufacturer. Briefly, conditioned medium was diluted and incubated in wells containing absorbed MMP antibodies. After washing, HRP-secondary antibody was applied and resulting spots were imaged by chemiluminescence as described above.Total MMP activity was measured by the MCa assay as previously described . BrieflyCellular invasion assays were performed as previously described . BrieflyP values less than 0.05 or 0.01 as stated in the figure legends.Data analyses were performed using Prism 4 software . Statistical significance was calculated using one-way analysis of variance (ANOVA) analyses with Dunnett's post-test or unpaired t-test. Significance was determined with Recently, we reported that NOS2 expression is significantly associated with poor survival in ER- breast cancer and that high NOS2 expression is associated with a distinct gene expression profile similar to the basal-like phenotype . FurtherTo evaluate Ets-1 activation by NO signaling, we examined the effect of forced NOS2 expression on Ets-1 (thr 38) phosphorylation in human basal-like cells. MDA-MB-468 cells, which do not express basal NOS2, were transfected with a human NOS2 expression plasmid and incubated with the NOS2 substrate L-Arg or the NOS2 inhibitor AG. NOS2 expression in the presence of L-Arg resulted in robust Ets-1 (thr 38) phosphorylation compared to cells transfected with empty vector control Figure . Ets-1  phosphorylation, we also examined the effect of NO signaling on Ets-1 activation in human ER- breast cancer cell lines treated with NO releasing compounds. Using the chemical NO-donor DETANO, the effect of NO on Ets-1 (thr 38) phosphorylation in MDA-MB-468, MDA-MB-231 and SUM159 cell lines was examined. The applied donor concentrations generate actual NO concentrations that are in the physiological nanomolar concentration range because of the slow release rate of NO from this donor [see Additional file 8 phosphovia NO signaling, increases Ets-1 transcriptional activity in ER- breast cancer cells.To examine the effect of NOS2 expression on Ets-1 transcriptional activity, MDA-MB-468 cells were transfected with a NOS2 expression plasmid and then transiently transfected with an Ets-luciferase reporter plasmid. Cells were then incubated in serum-free media supplemented with L-Arg or AG. NOS2 expression resulted in a significant increase in luciferase reporter activity when incubated with L-Arg; however, this effect was not observed in the presence of the NOS2 inhibitor AG, indicating that NO release resulted in Ets-1 transcriptional activation Figure . To examvia the MEK/ERK signaling pathway.Ets-1 is phosphorylated and activated by the MEK/ERK signaling pathway . Therefovia SNO post-translational modification [P < 0.01), whereas 0.1 mM DETANO induced an activation that was significantly lower than EGF, albeit still statistically significant above control levels [see Additional file Ras is a major activator of MEK/ERK signaling , therefofication ,34, Ras-fication . Similarvia a PKCα-independent mechanism.To examine the role of Ras in mediating the NO activation of the MEK/ERK/Ets-1 signaling pathway, MDA-MB-468 cells were treated with EGF or 0.5 mM DETANO with or without the Ras inhibitor FTS. FTS blocks Ras association with the cellular membrane and renders Ras protein susceptible to proteasomal degradation . EGF andvia a Ras/MEK/ERK signaling pathway and not via PKCα activation . Activation of Ets-1 by NO resulted in the increased expression of the basal-like markers P-cadherin, S100A8, IL-8 and αβ-crystallin, which mechanistically links two prognostic markers of poor basal-like patient survival ,23. FurtAG: aminoguanidine; cGMP: cyclic guanosine monophosphate; CTSB: cathepsin B; DETANO: diethlylenetriamine NONOate; (D)MEM: (Dulbecco's) modified Eagle's medium; EBS: Ets-binding sequence; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; ELISA: enzyme-linked immunosorbent assay; ER-: estrogen receptor-alpha negative; ERK: extracellular signal-regulated kinases; Ets-1: erythroblastosis virus E26 oncogene homolog 1; FTS: farnesylthiosalicylic acid; HRP: horseradish peroxidase; IL-8: interleukin-8; L-Arg: L-arginine; MEK: mitogen-activated protein kinase; MMP: matrix metalloproteinase; NAC: N-acetyl cysteine; NO: nitric oxide; NOS2: inducible nitric oxide synthase; PI3K: phosphatidylinositol 3-kinase; PLCα: protein kinase Cα; RBD: Ras-binding domain; RLU: relative luminescent units; SD: standard deviation; siRNA: small interfering RNA; SNO: S-nitrosylation; VEGF: vascular endothelial growth factor.The authors declare that they have no competing interests.CS conceived and designed experiments, acquired and analyzed data, and wrote and edited the manuscript. RC performed genomic analyses. LR measured steady state NO levels and assisted with MMP experiments. SG performed experiments and edited the manuscript. SA edited and rewrote the manuscript. DW helped with data analysis and funding for this study. All authors read and approved the final manuscript for publication.Table S1. Ets-binding sites in NOS2-associated gene signature. An Excel table listing the Ets-binding sites within the ER- NOS2 gene signature.Click here for fileFigure S1. Steady-state NO concentrations released from DETANO. A pdf file showing the concentration of NO, as measured by chemiluminescence, versus the concentration of DETANO over 24-hours.Click here for fileFigure S2. Response to EGF stimulation in ER- cell lines used in this study. A pdf file showing a western blot comparing relative phospho-(tyr1173) and total EGFR expression in EGF-treated MDA-MB-468, MDA-MB-231, SUM159 and SKBR3 cells.Click here for fileFigure S3. NO activation of Ets-1 in ER-/HER2+ SKBR3 cells. A pdf file showing a western blot of relative Ets-1 (thr 38), MEK1/2 (ser 217/221) and ERK1/2 (thr 202/tyr 204) phosphorylation in serum starved SKBR3 cells exposed to either EGF (10 ng/ml) or DETANO.Click here for fileFigure S4. Relative Ras activation of MDA-MB-468 cells. A pdf file showing Ras activation as calculated from densitometric analyses of active Ras normalized to total Ras. Activity is shown as mean fold compared to control. Significance  was determined by t-test.Click here for file


Vegfa gene gives rise to three major isoforms termed VEGF121, VEGF165 and VEGF189. VEGF165 binds the transmembrane protein neuropilin 1 (NRP1) and promotes the migration, survival and axon guidance of subsets of neurons, whereas VEGF121 cannot activate NRP1-dependent neuronal responses. By contrast, the role of VEGF189 in NRP1-mediated signalling pathways has not yet been examined. Here, we have combined expression studies and in situ ligand-binding assays with the analysis of genetically altered mice and in vitro models to demonstrate that VEGF189 can bind NRP1 and promote NRP1-dependent neuronal responses.The vascular endothelial growth factor  regulates neurovascular patterning. Alternative splicing of the  Summary: Although VEGF165 was thought to be the sole VEGF isoform acting through neuropilin 1 (NRP1), VEGF189 also binds to and signals through NRP1 in several types of developing mouse neurons. VEGFA) of eight exons that is alternatively spliced into isoforms, the major ones containing 121, 165 and 189 amino acid residues and therefore termed VEGF121, VEGF165 and VEGF189, respectively  is a potent inducer of blood vessel growth, but also has essential roles in neurodevelopment . In humaectively . The alt VEGF121 . Unexpec VEGF121 . By cont VEGF121 , promoti VEGF121  and enha VEGF121 .120/120Vegfa mice, which express VEGF120, the murine equivalent of VEGF121, but lack VEGF164 and VEGF188, corresponding to human VEGF165 and VEGF189, respectively -conjugated VEGF isoforms for g assays  to examiVegfa isoforms were expressed in these developmental contexts. For this experiment, we designed isoform-specific primers that can distinguish the Vegfa120, Vegfa164 and Vegfa188 mRNA species by reverse transcription (RT)-PCR . This analysis demonstrated that all three isoforms were co-expressed during relevant periods of VEGF/NRP1-dependent neurodevelopment in mice . Nrp1−/− hindbrains showed some defasciculation of these dorsolateral axons, but they were still clearly present in the mutant, suggesting that this are a suitable model to examine VEGFA isoform binding to NRP1.Because prior studies of VEGF binding to NRP1 have not examined whether VEGFfor NRP1  to confi121, VEGF165 and VEGF189, we fused each isoform to AP and performed in situ ligand binding assays on E12.5 hindbrains. As expected, all three isoforms bound vessels . These observations suggest that VEGF188, similar to VEGF164, can promote GnRH neuron survival. The ineffectiveness of VEGF120 agreed with the previously observed NRP1-dependent neuroprotection of GN11 cells and the fact that 120/120Vegfa mice have fewer GnRH neurons  and the following oligonucleotide pairs: 120-F 5′-GTAACGATGAAGCCCTGGAG-3′ and 120-R 5′-CCTTGGCTTGTCACATTTTTC-3′; 164-F 5′-AGCCAGAAAATCACTGTGAGC-3′ and 164-R 5′-GCCTTGGCTTGTCACATCT-3′; 188-F 5′-AGTTCGAGGAAAGGGAAAGG-3′ and 188-R 5′-GCCTTGGCTTGTCACATCT-3′.Total RNA was reverse transcribed using Superscript III (Life Technologies) and Open reading frames for the VEGF isoforms were amplified by PCR with the oligonucleotides 5′-AATAATGGATCCGCACCCATGGCAGAAGG-AG-3′ and 5′-TATATGCTCGAGCTCACCGCCTCGGCTTGTC-3′. The PCR products were cloned into pAG3-AP containing an upstream in-frame AP cassette. Binding assay were performed as described previously .in situ hybridisation, we used a digoxigenin-labelled Isl1 probe , goat anti-rat NRP1 , rabbit anti-mouse TUJ1  and mouse anti-rat ISL1 . Secondary antibodies used were: Alexa594-conjugated rabbit anti-goat Fab , Alexa488-conjugated donkey anti-rabbit Fab , Alexa488-conjugated goat anti-mouse  and biotinylated goat anti-rabbit . To detect blood vessels, we used biotinylated IB4 (Sigma) followed by Alexa633-conjugated streptavidin (Life Technologies). For l1 probe .164 in PBS (Preprotech) or VEGF188 (Reliatech). FBM neuron migration was measured with ImageJ (NIH) as the distance travelled from r5 to the leading group of cells in r6 in each hindbrain and normalised to the control side of each hindbrain.Hindbrain explants were cultured as previously described . Affi-GeDiI labelling was performed with fixed tissues as described previously . Briefly120, VEGF164 or VEGF188.Immunolabelled GnRH-positive cells were quantitated and GN11 survival assays performed as described previously . For sur


Mycobacterium tuberculosis from New Zealand (NZ3DS1) and compare it to a multidrug-resistant lineage 3 isolate (NZ3MDR1) with an identical 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat profile.We report here the draft whole-genome sequence of a drug-susceptible lineage 3 (East-African Indian) isolate of  Mycobacterium tuberculosis complex (Tuberculosis (TB) is a leading cause of infectious mortality worldwide, killing approximately 1.5 million people each year  by Burrows-Wheeler alignment  ; however, this mutation was reportedly detected in isoniazid-sensitive strains of M. tuberculosis, indicating low specificity for isoniazid resistance (embC) , although their specificities with respect to ethambutol resistance remain to be established. Interestingly, the latter M800I mutation was absent from NZ3MDR1. Nonsynonymous mutations were not detected with respect to genes associated with pyrazinamide resistance, i.e., Rv2043c (pncA), Rv1630 (rpsA), and Rv3601c (panD) (gyrA) . The S95T mutation, in particular, has been demonstrated to have low specificity with respect to fluoroquinolone resistance (Single-nucleotide variant (SNV) annotation was performed using snpEff , which isistance . In termc (panD) . Regardisistance .M. tuberculosis (Although the NZ3DS1 and NZ3MDR1 cases occurred in Indian-born individuals, as with most cases involving lineage 3 in New Zealand , there arculosis . In lighJZDR00000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number


Therefore we propose the term “subthreshold IKK activation”.Mast cell differentiation and proliferation depends on IL-3. IL-3 induces the activation of MAP-kinases and STATs and consequently induces proliferation and survival. Dysregulation of IL-3 signaling pathways also contribute to inflammation and tumorigenesis. We show here that IL-3 induces a SFK- and CaThis subthreshold IKK activation also primes mast cells for enhanced responsiveness to IL-33R signaling. Consequently, co-stimulation with IL-3 and IL-33 increases IKK activation and massively enhances cytokine production induced by IL-33.in vivo.We further reveal that in neoplastic mast cells expressing constitutively active Ras, subthreshold IKK activation is associated with uncontrolled proliferation. Consequently, pharmacological IKK inhibition reduces tumor growth selectively by inducing apoptosis Together, subthreshold IKK activation is crucial to mediate the full IL-33-induced effector functions in primary mast cells and to mediate uncontrolled proliferation of neoplastic mast cells. Thus, IKK2 is a new molecularly defined target structure. Mast cells are located in peripheral tissues and regulate innate and adaptive immune responses  by produThe most extensively characterized trigger for mast cell activation is crosslinking of the Fcε-receptor-I (FcεRI) resulting in the release of mediators including histamine, proteases, cytokines, and chemokines .Mast cells express TLR/interleukin-1 (IL-1) receptor (TIR) family members including TLR4 and the IL-33-receptor (IL-33R) . IL-33 iIn contrast, IL-3 is crucial to promote differentiation, survival, and proliferation of mast cells –19. IL-3Recent publications reported a crosstalk between FcεRI either with TLRs (including TLR4) or c-Kit resulting in increased NFκB-, NFAT- or JNK activation and cytokine production , 26. We IL-3 induced IKK activation results in anti-apoptotic signaling in hepatocytes . We founNext we used NFκB-EGFP-MC/9  mast celHaving shown that the IL-3-induced subthreshold IKK activation does not result in efficient cytokine production, we asked if IKKs mediate the activation of the mitogenic JNK signaling . Indeed,ΔIkk2-mice with poly(I:C) thymidin (1 μCi) was added for additional 18 h. Incorporated radioactivity was measured by using a β-scintillation counter (Perkin Elmer). The increase of mast cell proliferation is indicated as the stimulation index (SI). Thereby the basal CPM values were set to 1.BMMCs  and anti-B220 (clone RA3–6B2) and analyzed with the LSR II flow cytometer  and FlowJo .−/−Rag1-mice (10–18 weeks old) received 106 V2D1-cells in 100 μl subcutaneously into the shaved flanks. Animals were treated 3 times weekly with vehicle (1% DMSO/PBS) or the IKK-inhibitor VII  subcutaneously around the tumors. Tumor size was assessed 3 times weekly by using a Mitutoyo Quick Mini caliper (Mitutoyo). Measurements were recorded as tumor area (mm2) from groups of 10 mice. Tumor area (A) was calculated by the formula: [A = tumor height x tumor width]. Experiments were terminated after 6 weeks or if tumor size exceeded the ethically approved dimensions.DBA/1-p < 0,05 .All experiments were performed at least three times (shown is one representative experiment). Proliferation assays, and ELISAs were performed three times in at least a 6-fold determination. Cytokine concentration is indicated as the mean of measurements ± standard deviation. For proliferation assays and ELISA one representative experiment is shown. The statistical analysis was performed with IBM SPSS Statistics version 20.0 (IBM). Statistical significance was assessed by Mann-Whitney-U test. Statistical significance was accepted for


Dracaena cochinchinensis (Chinese dragon’s blood) yielded five new flavonoid oligomers, named cochinchinenins D-H (1–5), together with a known biflavonoid, cinnabarone (6), and a mixture of two known biflavonoids, socotrin-4′-ol (7) and homoisosocotrin-4′-ol (8). Of these new compounds, 1–3 were biflavonoids and 4 and 5 were triflavonoids. Their structures were determined on the basis of spectroscopic analysis. The isolated compounds were tested for cytotoxicity (Cdc25), antibacterial (PEPT) and antifungal (YNG) activities. A detailed chemical investigation of the red resins from Supplementary material is available for this article at 10.1007/s13659-012-0020-5 and is accessible for authorized users. Supplementary material, approximately 702 KB.