Fractalkine is an unusual C-X3-C motif chemokine that is important in neuron-microglial communication, a co-receptor for HIV infection, and can be neuroprotective. To assess the effects of fractalkine on opiate-HIV interactive neurotoxicity, wild-type murine striatal neurons were co-cultured with mixed glia from the striata of wild-type or Cx3cr1 knockout mice ± HIV-1 Tat and/or morphine. Time-lapse digital images were continuously recorded at 20 min intervals for up to 72 h using computer-aided microscopy to track the same cells repeatedly.Fractalkine/CX3CR1 mimicked the toxic effects of morphine plus Tat, but did not add to their toxicity; while fractalkine failed to protect wild-type neurons co-cultured with 3cr1Cx-/--null glia against morphine and Tat toxicity. Exogenous fractalkine also normalized microglial motility, which is elevated by Tat and morphine co-exposure, presumably limiting microglial surveillance that may lead to toxic effects on neurons. Fractalkine immunofluorescence was expressed in neurons and to a lesser extent by other cell types, whereas CX3CR1 immunoreactivity or GFP fluorescence in cells cultured from the striatum of Cx3cr1-/- (Cx3cr1GFP/GFP) mice were associated with microglia. Immunoblotting shows that fractalkine levels were unchanged following Tat and/or morphine exposure and there was no increase in released fractalkine as determined by ELISA. By contrast, CX3CR1 protein levels were markedly downregulated.Co-exposure to Tat and morphine caused synergistic increases in neuron death, dendritic pruning, and microglial motility as previously reported. Exogenous fractalkine prevented synergistic Tat and morphine-induced dendritic losses and neuron death even though the inflammatory mediator TNF-α remained significantly elevated. Antibody blockade of CX3CR1 signaling contribute to the synergistic neurotoxic effects of opioids and Tat. Importantly, exogenous fractalkine can selectively protect neurons from the injurious effects of chronic opioid-HIV-1 Tat co-exposure, and this suggests a potential therapeutic course for neuroAIDS. Although the cellular mechanisms underlying neuroprotection are not certain, findings that exogenous fractalkine reduces microglial motility and fails to protect neurons co-cultured with Cx3cr1-/- mixed glia suggest that fractalkine may act by interfering with toxic microglial-neuron interactions.The results suggest that deficits in fractalkine-CX Opioid drugs can increase HIV replication and modify HIV pathogenesis through direct interactions with opioid receptor-expressing cells in the immune system -5. We fo3CL1) i caused by excitotoxic levels of glutamate . The pr wild-typCx3cr1 -/- mice. Neuron cultures and neuron-glia co-cultures were similarly maintained in Neurobasal medium supplemented with B27, 0.5 mM L-glutamine, 0.025 mM glutamate, and allowed to mature for about 1 wk prior to the start of the experiments.Striatal neurons were prepared from wild-type E15-E16 ICR (CD-1) mouse embryos. Striata were dissected, minced, and incubated with 10 ml trypsin (2.5 mg/ml) and DNase (0.015 mg/ml) in Neurobasal medium  with 25 μM glutamate . Tissue was triturated, resuspended in 10 ml medium, and cells filtered twice through 70 μm pore nylon mesh. Isolated wild-type neurons were (i) plated alone on poly-D-lysine coated coverslips or (ii) co-cultured directly on a bedlayer of mixed glia derived from wild type or 2; PeCon Instruments, Houston, TX), to repeatedly track the same neurons o: extracellular calcium; CX3CR1: fractalkine receptor; CX3CL1: fractalkine; GFAP: glial fibrillary acidic protein; iNOS: inducible nitric oxide synthase; IL-1β: interleukin-1β; [Ca+2]i: intracellular calcium; Iba-1: ionized calcium binding adaptor molecule 1; HAND: HIV-associated neurocognitive disorders; HIV-1: human immunodeficiency virus-type 1; MAP-2: microtubule-associated protein type-2; MCP-1: monocyte chemoattractant protein-1; MAPK: mitogen-activated protein kinase; NK cells: natural killer cells; neuroAIDS: neuro-acquired immunodeficiency syndrome; NADPH: nicotinamide adenine dinucleotide phosphate oxidase; PI3K: phosphoinositide 3-kinase; RANTES (CCL5): regulated upon activation, normal T-cell expressed, and secreted; Tat: transactivator of transcription; TNF-α: tumor necrosis factor-α; TACE (or ADAM 17): TNF-α converting enzyme.ADAM: a disintegrin and metalloproteinase; ANOVA: Analysis of variance; RGD domain: Arg-Gly-Asp domain; CCL2 (MCP-1): chemokine (C-C motif) ligand 2 or monocyte chemoattractant protein-2; CCL5 (RANTES): chemokine (C-C motif) ligand 5; ELISA: enzyme-linked immunosorbent assay; ERK: extracellular signal-regulated kinases; [CaThe authors declare that they have no competing interests.MS, JLS, DHC, PEK, and KFH designed the experiments. MS, SZ, NE-H, Y-KH, and MES conducted the experiments. MS, JLS, DHC, PEK, and KFH analyzed and interpreted the data and wrote the manuscript. KFH coordinated the research and supervised the project. All authors have read and approved the final manuscript.


Previous studies showed that mesothelin (MSLN) plays important roles in survival of pancreatic cancer (PC) cells under anchorage dependent/independent conditions as well as resistance to chemotherapy. The recent success of intratumorally-injected adeno-encoded, chemo/radiation-inducible-promoter driven hTNF-α, (TNFerade) + gemcitabine in pre-clinical models of PC have renewed interest in use of TNF-α as a therapeutic component. To help find additional factors which might affect the therapy, we examined the resistance of MSLN-overexpressing pancreatic cancer cell lines to TNF-α-induced growth inhibition/apoptosis.Stable MSLN overexpressing MIA PaCa-2 cells (MIA-MSLN), stable MSLN-silenced AsPC-1 cells (AsPC-shMSLN) and other pancreatic cells  were used. NF-κB activation was examined by western blots and luciferase reporter assay. TNF-α induced growth inhibition/apoptosis was measured by MTT, TUNEL assay and caspase activation. IL-6 was measured using luminex based assay.75) BAD, and activated (p-Ser70) Bcl-2. Constitutively activated NF-κB and Akt were evident in MIA-MSLN cells that could be suppressed by MSLN siRNA with a resultant increase in sensitivity of TNF-α induced apoptosis. Blocking NF-κB using IKK inhibitor wedelolactone also increased sensitivity to TNF-α-mediated cytotoxicity with concomitant decrease in Mcl-1. Blocking Akt using PI3K inhibitor also had a likewise effect presumably affecting cell cycle. MIA-MSLN cells produced increased IL-6 and were increased furthermore by TNF-α treatment. SiRNA-silencing of IL-6 increased TNF-α sensitivity of MIA-MSLN cells.Compared to low endogenous MSLN-expressing MIA PaCa-2 and Panc 28 cells, high endogenous MSLN-expressing Capan-1, BxPC3, PL 45, Hs 766T, AsPC-1, Capan-2, Panc 48 cells were resistant to TNF-α induced growth inhibition. Stable MSLN overexpressing MIA-PaCa2 cells (MIA-MSLN) were resistant to TNF-α-induced apoptosis while stable MSLN-silenced AsPC1 cells (AsPC-shMSLN) were sensitive. Interestingly, TNF-α-treated MIA-MSLN cells showed increased cell cycle progression and cyclin A induction, both of which were reversed by caspase inhibition. We further found that MIA-MSLN cells showed increased expression of anti-apoptotic Bcl-XL and Mcl-1; deactivated (p-SerOur study delineates a MSLN-Akt-NF-κB-IL-6-Mcl-1 survival axis that may be operative in PC cells, and might help cancer cells' survival in the highly inflammatory milieu evident in PC. Further, for the success of TNFerade + gemcitabine to be successful, we feel the simultaneous inhibition of components of this axis is also essential. The importance of mesothelin (MSLN) as a biomarker and preferred immunotherapeutic target is steadily growing for many cancers, including pancreatic cancer (PC) -3. The fα (TNF-α) is a vital member of the TNF-α super family, and plays roles in immunity, cellular remodeling, apoptosis and cell survival , Ct = threshold cycle. Primer sequences for the genes are as follows; hMSLN sense: 5'-CTCAACCCAGATGCGTTCTCG-3', hMSLN antisense: 5'-AGGTCCACATTGGCCTTCGT-3', hTNFR1 sense: 5'CCTGGTCATTTTCTTTGGTC TTTG-3', hTNFR1 antisense: 5'-GGGTGAAGCCTGGAGTGG-3', hTNFR2 sense: CCAAGCACCTCCTTCCTG, hTNFR2 antisense: CACCACTCCTATTATTAGTAGA CC, hGAPDH sense: 5'-TGCACCACCAACTGCTTA GC-3', hGAPDH antisense: 5'-GGCATGGACTGTGGTCATGAG-3'.MSLN, TNFR1, TNFR2, and GAPDH mRNA levels were analyzed by real-time RT-PCR using the SYBR Green supermix kit (Bio-Rad) using 40 cycles at 95°C for 20 sec and 60°C for 1 min. Target gene mRNA was normalized to GAPDH mRNA level. Relative mRNA level was presented as unit values of 2^. Bars denote s.d. of duplicate data. *, # denote p < 0.05, **, ## denote p < 0.01, compared with controls by using t test.Click here for fileAdditional Figure S3: TNFR1 expression levels in MIA-V/MIA-MSLN cells. (A). Real-time PCR analysis of TNFR1 relative mRNA levels. Results denote GAPDH-normalized mRNA levels as 2^[Ct(GAPDH)-Ct(TNFR1)]. The bars denote s.d. of duplicate data. (B). Western blot data showing TNFR1 protein expression in MIA-V/MIA-MSLN cells.Click here for fileAdditional Figure S4: Sub-confluent MIA-V/MIA-MSLN cells were serum starved for 24 h and then treated with 10/20 ng/ml of TNF-α for 72 h. Cells were collected and fixed, PI-stained, and analyzed for cell cycle phase distribution (percentage of cells) with FACS. Percentage of cells in G0/G1, S, G2/M phase are shown against the respective peaks in the histograms.Click here for file


Myocardial phase velocity mapping studies have generally been acquired using Cartesian k-space coverage and respiratory gating ,2. AcquiThe sequence (TR=30ms) consists of five interleaved spiral k-space paths (23ms). Reference and 3-directional velocity encoded data  are acquired in consecutive cardiac cycles following a single dummy cycle (breath-hold duration 21 cardiac cycles). A black-blood suppression pulse (6ms) is output on alternate phases to reduce blood flow artefacts and retrospective gating allows full coverage of the cardiac cycle. Forty phases are reconstructed  with an acquired spatial resolution of 2.4 x 2.4 x 8mm (reconstructed 1.2x1.2mm). Basal, mid and apical short axis slices were acquired in 6 healthy volunteers on a Siemens Skyra 3Tesla scanner. Radial, circumferential and longitudinal velocities were calculated pixel-wise and as an average over six semi-automatically segmented regions of the left ventricle.Figure We have developed a phase velocity mapping technique which allows 3D myocardial velocities to be acquired in a single breath-hold. The spiral k-space coverage results in reasonable temporal resolution (33ms) and the implementation of retrospective ECG-gating allows analysis of the entire cardiac cycle, including atrial contraction. Future work will include implementing an on-line correction map to reduce off-resonance blurring and parallel imaging to reduce the breath-hold duration.Heart Research UK grant: RG2584.


Saccharomyces cerevisiae, the SUP70 gene encodes the CAG-decoding tRNAGlnCUG. A mutant allele, sup70-65, induces pseudohyphal growth on rich medium, an inappropriate nitrogen starvation response. This mutant tRNA is also a UAG nonsense suppressor via first base wobble. To investigate the basis of the pseudohyphal phenotype, 10 novel sup70 UAG suppressor alleles were identified, defining positions in the tRNAGlnCUG anticodon stem that restrict first base wobble. However, none conferred pseudohyphal growth, showing altered CUG anticodon presentation cannot itself induce pseudohyphal growth. Northern blot analysis revealed the sup70-65 tRNAGlnCUG is unstable, inefficiently charged, and 80% reduced in its effective concentration. A stochastic model simulation of translation predicted compromised expression of CAG-rich ORFs in the tRNAGlnCUG-depleted sup70-65 mutant. This prediction was validated by demonstrating that luciferase expression in the mutant was 60% reduced by introducing multiple tandem CAG (but not CAA) codons into this ORF. In addition, the sup70-65 pseudohyphal phenotype was partly complemented by overexpressing CAA-decoding tRNAGlnUUG, an inefficient wobble-decoder of CAG. We thus show that introducing codons decoded by a rare tRNA near the 5′ end of an ORF can reduce eukaryote translational expression, and that the mutant tRNACUGGln constitutive pseudohyphal differentiation phenotype correlates strongly with reduced CAG decoding efficiency.In  Gene regulation is frequently exerted at the level of transcription, although it is well understood that translational control, as well as regulation of mRNA and protein stability, also play important roles in setting the steady-state level of protein expression for any given gene. Many examples of translational control operate by controlling the ability of an mRNA to recruit ribosomal subunits during the translation initiation process. In both prokaryotes and eukaryotes, secondary structure elements within the 5′ untranslated region (5′UTR), often subject to specific binding by RNA-binding proteins, regulate ribosome access to the AUG codon and subsequently, the open reading frame  cascade or the cyclic AMP-dependent Protein Kinase A (PKA) pathway known to signal pseudohyphal growth  and the corresponding wild-type MLD17 (both generous gifts from Prof. R.A. Singer) were grown on either solid rich (YPD) or minimal media (SLAD), the latter containing limiting ammonium sulphate and known to trigger pseudohyphal differentiation in competent yeast strains  which allowed us to quantitatively compare chain formation under different conditions. Using this index, it was apparent that the degree of pseudohyphal-type differentiation in the mutant was significantly greater in N-limiting SLAD medium than that measured in SD or YPD media . The detection of chain formation in the S288C-derived strain was despite the absence of functional Flo8p, another indication that the sup70-65 pseudohyphal growth form was atypical. Both strains were then transformed with an additional plasmid-borne copy of constitutively active RAS2Val19, a dominant mutation that constitutively activates the yeast RAS-cAMP pathway  suppressor activity is detectable only when overexpressed on a multi-copy plasmid  in homozygous form and is thus tryptophan (Trp) auxotrophic. Trp prototrophic transformants identified sup70 nonsense suppressor alleles. Sequencing of these alleles revealed that almost all the amber suppressor mutants defined nucleotide substitutions in the anticodon stem and loop , due to the leakiness of the UAG codon used, and the natural propensity of SUP70 tRNA to decode UAG via first base wobble .The nonsense suppressor phenotype of these novel alleles was quantified using a dicistronic readthrough assay in strain BY4743  in which the substituted mutant nucleotide was preserved, but stem base pairing was restored, for example creating a, A31–U39 pair to create allele sup70-65c . Using these novel alleles, the role of nucleotide pair identity, and pair strength on the pseudohyphal growth phenotype could be tested.In order to characterize the impact of the up70-65c A. Other sup70-65 allele is an amber suppressor. Plasmids carrying the alleles were transformed into strain MLD17, which carries the amtrp1-1 UAG allele, and suppression assayed on medium lacking tryptophan  mutant exhibited no suppressor activity. Thus nucleotide identity, as well as the existence of an intact stem, appears to affect anticodon presentation and thus first base wobble.First, we assessed the ability of the new alleles to suppress the UAG stop codon, since the yptophan B. As expSUP70 strain background , then reconstituting poor decoding of CAG codons, or inefficient tRNACUGGln charging, via mutation at position 33 should also generate a pseudohyphal growth phenotype. The results did indeed confirm this; although the U33G mutant did not differ from the wild-type, both U33C and U33A mutants generate significantly more pseudohyphal cell chains than the wild-type  gene knockout in yeast, because the mcm5-s2-modified UUG anticodon has nonetheless some weak ability to third-base wobble-decode the CAG codon C) on a multi-copy plasmid could suppress constitutive pseudohyphal growth.Third base U-G wobble is thought to be restricted in eukaryotes, thus the CAA-decoding tRNAsup70-65/sup70-65) and the wild-type counterpart MLD17 (SUP70/SUP70) were therefore transformed with the multi-copy plasmid ptRNA-UUG carrying tQ(UUG)C and were assessed for pseudohyphal growth  and MLD17 (SUP70/SUP70) strains (data not shown) revealed that in the mutant, charging levels are slightly reduced from 67% in the wild-type MLD17 strain, to 50% in the sup70-65 mutant  or acceptor (sup70-33) stems, possibly enhanced by altered patterns of tRNA nucleoside modification. The combination of reduced overall tRNA level and reduced charging would reduce the overall level of charged tRNA available for CAG decoding in the sup70-65 mutant to 19% of wild-type , derived from a genome-wide application of the model . The modelling of translation in the strain carrying the sup70-65 gene was achieved by reducing the simulated levels of tRNACUG fivefold according to the results of the Northern analysis  experimentally measured in the sup70-65. The reason why very low levels of tRNACUGGln can affect expression of 5 × CAA is because of the presence of the seven internal CAG codons scattered across the native luc reading frame.Using the computer model, we simulated the effect of a range of tRNACUGGln levels. However, because of the complex dynamics of ribosome transit through runs of tandem codon repeats, the relationship between tRNACUG concentration and luciferase 5 × CAG-luciferase expression was indeed non-linear , probably arising through instability of the mutant form of the tRNA  in a homozygous SUP70 deletant diploid carrying a single plasmid borne gene copy of sup70-65, clear evidence of haplo-insufficiency  signal to the N-starvation pathway , MLD15 (MATa/MATα sup70-65/SUP70 trp1-1/trp1-1 ura3-52/ura3-52 leu2-3112/LEU2 his3-11/his3-11 ade1/ADE1), MLD14 (MATa/MATα sup70-65/sup70-65 trp1-1/trp1-1 ura3-52/ura3-52 leu2-3112/LEU2 his3-11/his3-11 ade1/ADE1), LMDWU (MATa/MATα SUP70/SUP70 ura3-52/ura3-52 leu2-3112/LEU2 ade1-1/ADE1), LMD651U (MATa/MATα sup70-65/sup70-65 ura3-52/ura3-52 leu2-3112/LEU2 ade1-1/ADE1) and LMD6533LU (MATa/MATα sup70-33/sup70-33 leu2-3112/ leu2-3112 ura3-52/ ura3-52) were used to investigate the causes of the pseudohyphal growth phenotype. These strains were provided by Dr L. Murray and Prof. R.A. Singer . Strain BY4743 (MATa/MATα his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met15Δ0/MET15 ura3Δ0/ura3Δ0) was employed as a wild-type S288C-derivative strain.SUP70 knockout diploid strain MLD17 ΔSUP70 (MATa/MATα sup70::kanMX/sup70::natMX ade1/ade1 his3-11/his3-11 trp1-1/trp1-1 ura3-52/ura3-52 [pAK01]) supported by a plasmid-borne copy of SUP70 was generated as follows. Using primers sup70-S1 and sup70-S2 . Using primers preS1 (situated in the upstream URA10 gene) and postS2  carrying the SUP70 allele was then transformed into the MLD17 sup70 heterozygous disruptant. Finally, primers preS1 and postS2 were used to amplify a natMX disruption cassette with SUP70 long flanking regions using BY4743 sup70::natMX DNA as a template; this was transformed into the MLD17 heterozygous sup70 deletant to generate strain MLD17 ΔSUP70. Antibiotic resistance, resistance to 5-fluoroorotic acid and diagnostic PCR were all used to verify successful deletion of both SUP70 gene copies.A sup70-S2 , heterozSUP70 homozygous knockout strain supported by a plasmid-borne copy of SUP70 was generated using strain BY4743. One allele of SUP70 was deleted using a SUP70-kanMX knockout cassette as described above and support plasmid pAK01 transformed into the strain. The diploid strain was then sporulated. Two spores were selected that had geneticin-resistance, and 5-fluoroorotic acid-sensitivity (indicating dependence on pAK01 [URA3 SUP70]). These were mated to create IS700 (MATa/MATα sup70::kanMX/sup70::kanMX his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 lys2Δ0/lys2Δ0 met15Δ0/met15Δ0 ura3Δ0/ura3Δ0 [pAK01]).A second −1, 100 μg ml−1 and 0.5 mg ml−1 respectively.Strains were grown at 30°C on either solid or liquid form of either nutrient-rich YPD medium , appropriate synthetic-defined (SD) selective dropout media  or nitrogen-poor selective SLAD media . Where required, 5-fluoroorotic acid, geneticin and nourseothricin were used at final concentrations of 200 μg ml600 of 0.6 was reached. At least 200 cells were counted using a haemocytometer. Total cell counts, as well as chain length counts were recorded. In order to produce an index of chain forming ability (CFI), cell counts were binned according to their length of chain. Bin counts for chains greater than three cells were used to calculate a cumulative total that described what proportion of the population had chain length ≥ L.Pseudohyphal growth in liquid medium was assessed as follows. From an initial overnight culture, a second 5 ml culture was grown for 16 h until an ODpL= total percentage of the population contained within chain length larger than or equal to L.SC was then calculated as follows:A cumulative sum SC represents the integrated area under the cumulative sum curve. An exemplar plot of the cumulative sum showing these areas is shown in The greater a proportion of cell population that exists in long chains, the larger this ‘sum of cumulative counts’ will be. Essentially the value of SC value for a given mutant was then expressed as a proportion of the corresponding wild-type SC to produce a chain formation index, or CFI, allowing quantitative comparison of the chain formation capability.The HIS3, CEN) carrying SUP70, psup70-33  carrying sup70-33 and psup70-65  carrying sup70-65 were created by PCR-amplifying the relevant SUP70 allele using primers sup70-S3 and sup70-S4  carrying the SUP70 gene, pSUP70-2μ  carrying SUP70 and pSUP70-65-2μ  carrying sup70-65 were created by amplifying the relevant cloned SUP70 allele with primers pRS-forward and pRS-reverse  carries the sup70  allele (sup70-65c) generated using oligonucleotides atRNA-std_fw2, atRNA-std_rv2 and sup70-65c; pAK12  carries the sup70 [C39T] allele (sup70-65i) generated using atRNA-std_fw2, atRNA-std_rv2 and sup70-65inv; pAK13  carries the sup70  allele (sup70-33c) generated using atRNA-7033_fw, atRNA-7033_rv and sup70-33c; pAK14  carries the sup70 [C5T] allele (sup70-33i) generated using atRNA-7033_fw, atRNA-7033_rv and sup70-33inv; pAK15  carries the sup70 [T33C] allele (sup70-C33) generated using atRNA-std_fw2, atRNA-std_rv2 and sup70-U33C; pAK16  carries the sup70 [T33A] allele (sup70-A33) generated using atRNA-std_fw2, atRNA-std_rv2 and sup70-U33A; and pAK17  carries the sup70 [T33G] allele (sup70-G33) generated using atRNA-std_fw2, atRNA-std_rv2 and sup70-U33G. For each plasmid, the oligonucleotides listed first and second were used in a 10-cycle annealing and polymerization reaction. The product of this reaction was used as PCR template in a second reaction using the second and third primers, to create a given tRNA allele ready for cloning.Plasmid pAK11 , used for quantifying stop codon readthrough, directs the expression of an in-frame translational fusion of lacZ and luc genes, separated by an in-frame UAG stop codon  is identical to pJR5 but with the in-frame stop codon separating lacZ and luc genes replaced by a glutamine CAG codon.Plasmid pJR5 (TRP1 CEN) that encode constitutively expressed firefly luciferase with tandem 5, or 10, tandem CAA, or CAG codons immediately downstream of the AUG translation initiation codon. They were created using a homologous recombination gap repair strategy in which a 5′ segment of firefly luciferase was amplified using a forward primer carrying a 5′ overhang encoding the tandem CAA or CAG codons. Using these PCR fragments, YCplac22-FL1  mutagenic target. The amplified, mutagenized library of sup70 fragments was co-transformed into MLD17 (trp1-1) with NotI-gapped pRS413. The vector sequences flanking the tRNA gene directed homologous recombination, and thus gap repair in vivo of pRS413, creating a mutagenized tRNA library.The template . The PCRet al., et al., To quantify readthrough of the UAG stop codon, yeast strains were transformed with either pJR5 or the control vector pJR7, which express β-galactosidase-luciferase fusion proteins. Dicistronic assays for stop codon readthrough were performed essentially as described . For quantification of total tRNA, 100 pmol of tRNA preparation was slot-blotted onto Amersham Hybond-N membrane using a hybridization manifold and then immediately UV-cross-linked as above.tRNA preparation was performed according to standard protocols  and sup70-65 probe (5′-ttg ttc gga tca gaa tc-3′). tRNAGlnUUG was detected using the probe 5′-ttg tcc gga tca aaa cc-3′, and tRNAHis detected using the probe 5′-ttt cat cgg cca caa cg-3′. Washing was carried out using sequential 15 min. treatments with wash solution I , II  and III . The blot was exposed for 24–72 h to a phosphoimager screen and hybridization quantified using a Fuji FLA-3000 phosphoimager and Aida/2D v 2.0 densitometry software.The probes to the respective tRNAs were end-labelled using polynucleotide kinase and standard methods  of 0.8 had been reached. Assays were performed using the Bright-Glo luciferase assay kit (Promega). qRT-PCR was carried out on five independent mRNA samples  following cDNA synthesis using Quantitect reverse transcriptase (Qiagen). Then, using SYBR Green methodology in a PCR reaction  using a Roche LightCycler 480 RT-PCR machine, the following were performed; using primers FLuc-f and FLuc-r , amplifiet al., et al., An extended Totally Asymmetric Simple Exclusion Process (TASEP) model of translation that had been previously developed  were recorded. The model is freely available from the authors.


Two of the three triazol groups in each ttmb ligand link the ZnII atoms, forming a looped-chain structure along [0-11]. The lattice water molecule shows half-occupancy due to disorder around an inversion centre.In the title complex, {[Zn(NCS) DOI: 10.1107/S1600536812039256/ds2211Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


To test whether BOLD-CMR can detect endothelium-dependent coronary vasodilatation.Endothelial function is important in the pathogenesis of atherosclerosis. Whereas endothelial function of peripheral circulation can be assessed non-invasively with Flow-Mediated Dilatation, no method exists for coronary circulation. BOLD-CMR utilizes inherent contrast, where the signal intensity (SI) is linearly correlated with the regional tissue level of deoxygenated hemoglobin. This technique may have potential to assess coronary endothelial function non-invasively.1) was performed at baseline and during graded ACh infusion in the LCX . Scan parameters were: field-of-view 228 x 280 mm; matrix size 125 x 192; in-plane resolution 1.8 x 1.6 mm; slice thickness 5 mm; TR/TE 5.8 ms/2.9 ms; flip angle 90°; readout bandwidth 275 Hz/Px; signal averages 1; typical breath-hold 16 seconds. CMR imaging was repeated with 10 µg ACh after pharmacological blockage of endothelial NO-synthetase by Nω-nitro-L-arginine methyl ester (L-NAME). Coronary flow, coronary sinus (CS) oxygen saturation (SvO2) and BOLD-CMR imaging were performed simultaneously. Images were analyzed using clinically validated software .In 6 anesthetized dogs, after instrumentation of the left circumflex coronary (LCx) artery with a coronary infusion catheter and an extravascular flow probe, the coronary endothelium was activated with acetylcholine (ACh). Using a clinical 1.5-T MRI  mid short axis cine SSFP BOLD sequence , which was accompanied by significant increases in CS SvO2  and in BOLD-CMR SI  . Compared to baseline, after L-NAME administration, flow response to the highest level of ACh was blunted (20%) and no significant increase of BOLD-CMR signal intensity was found (1.9%). Figure BOLD-sensitive CMR allows for non-invasive imaging of changes in myocardial oxygenation induced by endothelium-dependent vasodilatation. Further studies should address the utility of BOLD-CMR in the clinical setting of endothelial function testing.


Zdhhc13 nonsense mutation causing a Zdhcc13 deficiency develop alopecia, amyloidosis and osteoporosis. Our goal was to investigate the pathogenic mechanism of osteoporosis in the context of this mutation in mice. Body size, skeletal structure and trabecular bone were similar in Zdhhc13 WT and mutant mice at birth. Growth retardation and delayed secondary ossification center formation were first observed at day 10 and at 4 weeks of age, disorganization in growth plate structure and osteoporosis became evident in mutant mice. Serial microCT from 4-20 week-olds revealed that Zdhhc13 mutant mice had reduced bone mineral density. Through co-immunoprecipitation and acyl-biotin exchange, MT1-MMP was identified as a direct substrate of ZDHHC13. In cells, reduction of MT1-MMP palmitoylation affected its subcellular distribution and was associated with decreased VEGF and osteocalcin expression in chondrocytes and osteoblasts. In Zdhhc13 mutant mice epiphysis where MT1-MMP was under palmitoylated, VEGF in hypertrophic chondrocytes and osteocalcin at the cartilage-bone interface were reduced based on immunohistochemical analyses. Our results suggest that Zdhhc13 is a novel regulator of postnatal skeletal development and bone mass acquisition. To our knowledge, these are the first data to suggest that ZDHHC13-mediated MT1-MMP palmitoylation is a key modulator of bone homeostasis. These data may provide novel insights into the role of palmitoylation in the pathogenesis of human osteoporosis.ZDHHC13 is a member of DHHC-containing palmitoyl acyltransferases (PATs) family of enzymes. It functions by post-translationally adding 16-carbon palmitate to proteins through a thioester linkage. We have previously shown that mice carrying a recessive  Palmitoylation is a post-translational lipid modification involving the addition of a 16-carbon palmitate on specific cysteine residues of proteins through a thioester linkage DHHC2 and DHHC11 were reported to relate to cancers DHHC gene functions has been gleaned in the context of neurological development Zdhhc5 gene-trap mice show a reduction in contextual fear Zdhhc8 knockout mice manifest a schizophrenia phenotype Zdhhc21 show abnormalities of skin homeostasis and hair defects Zdhhc13 deficient mice show the most severe phenotype with amyloidosis, alopecia, and osteoporosis A family of proteins with palmitoyl acyltransferase (PAT) activity was recently identified in yeast Zdhhc13 deficient mice. We aimed to understand how a palmitoylation enzyme, Zdhhc13, can affect bone homeostasis with the hope of providing new insights into the biological functions of palmitoylation and the pathogenic mechanisms of human osteoporosis.Our goal in this study was to investigate the pathogenic mechanisms underlying osteoporosis in the Zdhhc13 mutant mice were generated by ENU mutagenesis as described previously 2 for 15–20 minutes. All the animals and protocols (IACUC number: 11-05-187) used in this study were approved by the Institutional Animal Care and Utilization Committee of Academia Sinica.The Newborn mice were skinned and eviscerated. The remaining skeletons were fixed overnight in 1% acetic acid and 95% ethanol, then stained with Alcian blue 8GX (0.05%) for 72 hours, followed by dehydration in 95% ethanol for 24 hours. The solution was changed to 1% KOH until the bone became visible. The skeletons were stained overnight with Alizarin red (0.005%). Specimens were cleared, dehydrated in 70% ethanol/glycerol (1∶1), and finally stored in 100% glycerol.Tissues were fixed in 4% paraformaldehyde overnight and transferred to 70% alcohol. The microCT scan was performed as described previously 2O2. After blocking with 5% normal goat serum, tissues were incubated with primary antibodies in 4°C, overnight. The following rabbit polyclonal antibodies against mouse were used, anti-VEGF , anti-PECAM (Abcam), and anti-Osteocalcin . Sections were then incubated with anti-rabbit secondary antibody  and developed with 0.1% 3, 39-diaminobenzidine. Images were captured using standard light microscopy  and quantified using Image-Pro Plus software . Data from three independent mice staining were used for statistical analysis.Tissues were fixed in 4% paraformaldehyde and decalcified in 10% EDTA. Paraffin sections were stained with Masson’s trichrome stain for morphological analysis. For IHC, antigens of de-paraffinized sections were retrieved by 0.05% trypsin or hyaluronidase (10 mg/ml) and treated with 3% Hin situ hybridizations were performed on paraffin sections as previously described Zdhhc13  was inserted into pGEM-T Easy vector . Digoxigenin (DIG)-labeled single-strand sense and anti-sense RNA probes were prepared with a DIG-RNA labeling kit . The hybridized probe-RNA was then detected by anti-DIG-AP  (Roche) and visualized by NBT/BCIP (Roche) with a developing time of 3 hours.RNA Gapdh for the fold change calculation. Primers: Zdhhc13 F- CAGCAGCATCCATCTGGCGGT; R- GCCGATAGCATGAGCGGCGT; Gapdh F-TGCCAAGGCTGTGGGCAAGG; R-TCTCCAGGCGGCACGTCAGA.Total RNA was extracted from tissue using the RNeasy kit . cDNA was synthesized from 2 μg total RNA using the SuperScript III First-Strand cDNA Synthesis Kit  and 15 ng of the cDNA was amplified in a final volume of 15 μl for Q-PCR. Q-PCR was performed using Power SYBER green PCR Master Mix with ABI PRISM 7700 Sequence Detection System . The level of gene expression was normalized to 2 fragments and digested in collagenase type II solution  for 4 hours. After washing with DMEM, the digested bone fragments were placed in culture dishes or chamber slides for following experiments. Primary epiphyseal chondrocytes were isolated as previously described HEK293 and MC3T3E1 cells were purchased and cultured as described by the American Tissue Culture Collection (ATCC). ATDC5 chondrocytic cells were purchased from RIKEN and cultured using F12/DMEM medium  with 10% FBS, 1% penicillin and streptomycin. The primary osteoblasts were isolated by modifying of a previous method Tissue or cell lysates were separated by SDS-PAGE gel and transferred to PVDF membrane (Millipore). After blocking with 5% milk, the same antibodies used for VEGF and osteocalcin IHC were used for WB detection. Signals were developed by film after incubating with appropriate horseradish peroxidase (HRP) - conjugated secondary antibodies (Millipore). Densitometry quantification was performed using software in BioSpectrum Imaging System . Intensity for all selected bands was all normalized to their actin (loading control) intensity then calculated as fold change to vector group. Data from three independent repeat experiments were used for statistical analysis.Mouse Zdhhc13 cDNA was cloned into a pcDNA4/myc-His expression vector (Invitrogen); mouse MT1-MMP was cloned into a pcDNA3.1/V5-His TOPO TA Expression Vector (Invtrogen). These vectors were co-transfected into HEK293 cells using Lipofectamine 2000 (Invitrogen). 24 to 48 hours after transfection, cells were harvested and lysed with lysis buffer , 1% TritonX-100). Total Zdhhc13 and MT1-MMP proteins were immunoprecipitated using anti-V5 (Invitrogen) or anti-myc (Millipore) antibodies, respectively with protein G sepharose beads . The Co-IP results were analyzed by WB with the antibodies used for IP.in vivo analysis, the palmitoylation level was presented as percentage. It was calculated as streptavidin intensity divided by MT1-MMP intensity.WT and mutant mouse Zdhhc13 cDNA were subcloned into the p3XFLAG-CMV14 vector (Sigma-Aldrich). The MT1-MMP-V5-His plasmid was subsequently co-transfected with the WT Zdhhc13 or mutant Zdhhc13 construct into HEK293 cells using Lipofectamine 2000. Cells and tissues were harvested and lysed in lysis buffer containing 50 mM N-ethylmaleimide. Overexpressed MT1-MMP-V5 was purified from 500 μg total cell lysate by immunoprecipitation with anti-V5 antibody (Invitrogen) and protein G sepharose beads  in 4°C overnight. Endogenous MT1-MMP was purified from 1 mg epiphysis tissue lysate by immunoprecipitation with anti-MT1-MMP antibody (Abcam) and protein G sepharose beads  in 4°C overnight. The antibody-beads purified MT1-MMP was then used for the acyl-biotin exchange assay as described previously Cells were fixed with 4% paraformaldehyde in PBS followed by 0.1% Triton X-100 (in PBS) permeabilized for 10 minutes at room temperature. After blocking with 5% normal goat serum for 1 hour, cells were incubated with primary antibodies overnight. Antibodies and their dilutions used: rabbit anti-MT1-MMP . The secondary antibodies used here were anti-mouse Alexa Fluor 594 or anti-rabbit Alexa Fluor 488 correspondence to species of primary antibody . Nuclei were stained by DAPI using ProLong Gold Antifade Reagent (Invitrogen). Images were captured by UltraVIEW . Granularity and nuclear intensity were calculated by MetaMorph software . Granularity represented the average speckle number per cell. Nuclear intensity was quantified as average intensity of green fluorescence which was overlapped with blue (DAPI) fluorescence. The quantitative and statistic data was calculated from a total 140 primary cultured osteoblasts or 122 primary chondrocytes from three littermate pairs.P-value <0.05 was considered statistically significant Statistical significance was determined by two-tailed Student's t-test. A Zdhhc13 deficient mutant mice had severe osteoporosis at 26 weeks of age Zdhhc13 deficiency impacts postnatal not prenatal bone mass and bone accumulation. Similar bone changes were also observed in Zdhhc13 gene-trap mice  to P28 showed a delay in formation of the secondary ossification center (SOC) in mutant mice . From P0In situ hybridization was performed to analyze the expression pattern of Zdhhc13. Zdhhc13 expression signals in P14 WT distal femur  -deficient mice ry (IHC)  and Westry (IHC) , WT and Finally, co-immunoprecipitation (Co-IP) was performed using ZDHHC13-myc (ZDWT-myc) and MT1-MMP-V5 co-overexpressing HEK293 cells to examine whether ZDHHC13 interacted with MT1-MMP. MT1-MMP-V5 was successfully immunoprecipitated with anti-myc antibodies  left andZdhhc13 mutation in our mouse model. MT1-MMP-V5 was co-overexpressed with Flag vector, ZDHHC13-Flag-WT (ZDWT) or ZDHHC13-mutant-Flag (ZDK) in HEK293 cells. Results of ZDWT and ZDK were compared to Flag vector control which indicating basal palmitoylation level. The palmitoylation level of MT1-MMP-V5 was quantified to be nearly 2 fold higher in the ZDWT co-expresssing group than in the vector control or ZDK (mutant mimicking construct) co-expressing groups  to evaluate the palmitoylation level of MT1-MMP. A ZDHHC13-mutant-Flag (ZDK) expression vector, without the DHHC domain, was constructed to mimic the g groups . Proteing groups . Abolishg groups . Finallyant mice . These rZdhhc13 mutant primary osteoblasts  palmitoylation. The C574 was converted to alanine (MT1-MMP C574A-V5) or serine (MT1-MMP C574S-V5). MT1-MMP WT, C574A, C574S and V5 vector control were transfected into a mouse chondrocyte cell line, ATDC5. VEGF expression was analysed using Western blot. VEGF level was significantly elevated 1.5 fold in the WT MT1-MMP overexpressing group comparing to vector control. The elevation was abolished in C574A and C574S overexpressing groups. Palmitoylation of MT1-MMP WT was inhibited using 4-hour 25 μM of 2-BP treatment. 2B-P inhibition of MT1-MMP WT palmitoylation resulted in greater inhibition of VEGF expression . To mimiin vivo, VEGF and osteocalcin IHC was performed on WT and mutant epiphyses. Reduced VEGF level was observed in mutant hypertrophic chondrocytes near cartilage canals  on clathrin-mediated MT1-MMP endocytosis in osteoblasts  eoblasts . We did in vitro chondrocytic ATDC5 cell system. The reduction of VEGF was also observed and quantified to be 50%- 60% of WT in Zdhhc13 mutant hypertrophic chondrocytes. A 75% less vascularity was further shown by PECAM IHC in mutant epiphysis. The vascularity also revealed to be only 50% of WT in HZ-TB region of Zdhhc13 mutant mice. Besides, osteocalcin expression was found to be associated with ZDHHC13-mediated MT1-MMP palmitoylation in MC3T3 cells and in cartilage-bone interface. Collectively, these suggested that Zdhhc13-mediated MT1-MMP palmitoylation was a novel regulator in skeletal vascularity and endochondral ossification. These also highlighted the potential diverse roles of ZDHHC13-mediated MT1-MMP palmitoylation in modulating bone homeostasis.MT1-MMP regulates the skeletal system in diverse ways. Manduca, et al. reported the critical temporal regulation of MT1-MMP in governing osteogenesis and mineralization in osteoblasts A PAT enzyme, Zdhhc13, may have numerous downstream substrates. A matrix metalloproteinase, MT1-MMP, may also have diverse substrates or interacting molecules. In this complex regulatory network we revealed a novel potential pathway involving regulation of bone formation, development and structure by palmitoylation. The involvement of other ZDHHC13 palmitoylation targets or MT1-MMP substrates is worthy of further investigation.Zdhhc13, in epiphyseal maturation and endochondral bone formation. This study revealed a novel pathogenic mechanism of osteoporosis governed by palmitoylation.To the best of our knowledge, this is the first report showing involvement of a DHHC PAT enzyme, Figure S1Postnatal growth retardation of Zdhhc13 deficient mutation mice. Appearance of WT and mutant mice at age P10, P14, P28, and P70.(TIF)Click here for additional data file.Figure S2Bone length of P0, P14 and P28 Zdhhc13 WT and mutant mice. Femurs were dissected and their lengths were measured. The values from 3 WT and 3 mutant were shown as mean±SD. The square is in 1 mm×1 mm of size. Statistical significance was determined by two-tailed Student's t-test. A P-value <0.05 was considered statistically significant . The yellow color of P0 femurs was resulted from fixation in Bouin's solution.(TIF)Click here for additional data file.Figure S3Bone phenotype in Zdhhc13 gene-trap mice. MicroCT (A) 3D images and (B) bone volume/ tissue volume (BV/TV) ratio of trabecular bone in Zdhhc13 WT and gene-trap (Gt) (5 month of age). The method for generation of this gene-trap model was described in our previous paper (TIF)Click here for additional data file.Figure S4Comparable MT1-MMP expression in WT and mutant epiphysis and primary chondrocytes. MT1-MMP IHC of P10 (A-C) WT and (D-F) mutant distal femoral epiphyseal sections. High power view of (B) WT and (E) mutant mice MT1-MMP expression in chondrocytes around cartilage canal. MT1-MMP expression in (C) WT and (F) mutant growth plate chondrocytes. Yellow star indicted the canal that will contribute to future SOC formation. (G) Expression of MT1-MMP in P14 epiphysis tissue by WB and quantitative results from three WT and mutant littermate pairs (below).(TIF)Click here for additional data file.Figure S5Abolishment of palmitoylation of mutant MT1-MMP  and 25 μM, 50 μM, 100 μM 2-Bromopalmitate treatment. Palmitoylation level was examined by acyl-biotin exchange. MT1-MMP-V5 WT or C574A or C574S were overexpressed in HEK293 cells. Palmitate was switched to biotin and detected by streptavidin-HRP. Purified MT1-MMP was analyzed by V5 antibody. Red arrows indicate the palmitoylation signals of WT MT1-MMP which was not detected in C574 mutation constructs and 2-BP treatment groups. The 2-BP concentration of 25 μM was used in further experiment. HA: hydroxylamine; 2BP: 2-bromopalmitate.(TIF)Click here for additional data file.Figure S6Clathrin-mediated MT1-MMP trafficking in WT and Zdhhc13 mutant primary osteoblast. (A) Localization patterns of MT1-MMP in primary osteoblast (OB) from P14 WT and mutant femur. MT1-MMP staining (green), Clathrin staining (red), and merge of MT1-MMP and Clathrin  in WT (upper panel) and mutant (lower panel) primary OB. White arrows indicated non-clathrin colocalized speckles. (B) Colocalization MT1-MMP and clathrin quantitative data. *P-value <0.05, t-test.(TIF)Click here for additional data file.Table S1Quantitative microCT results of WT and mutant femur trabecular bone and bone mineral density (BMD) at P10 and P14. 3 WT and 3 mutants were analyzed at each time point. Data was presented as average ± standard deviation. Statistical significance was determined by two-tailed Student's t-test. A P-value <0.05 was considered statistically significant . BV/TV: bone volume/ tissue volume; Tb.Th: trabecular bone thickness; Tb.Sp: trabecular separation; Tb.N: trabecular bone number; SMI: structure model index.(DOCX)Click here for additional data file.


Wk 48 efficacy/safety data from TRANxITION study are presented here.Wk 24 TRANxITION study data showed patients transitioned from immediate release nevirapine (NVP IR) twice daily (BID) to NVP extended release (NVP XR) once-daily (QD) demonstrated non-inferior efficacy to patients continuing on IR NVP BID . SimilarOpen label, randomized (2:1), non-inferiority, parallel group study comparing NVP XR 400 mg QD with NVP IR 200 mg BID in HIV-1 patients >18 years receiving IR NVP plus one of three NRTI combinations, with viral load (VL) <50 copies/mL. Patients remained on their previous background therapy for treatment duration. Sustained virologic response (VL <50 copies/mL) was assessed at Wk 48 using a time-to-loss of virologic response (TLOVR) algorithm.3 and 569.7 cells/mm3, respectively. 48 Wk data are reported in Table 426 patients completed 48 wks of treatment. 94.9% of NVP XR and 91.9% of NVP IR patients. Mean baseline CD4+ counts: 557.7 cells/mmAt Wk 48, non-inferiority between the NVP XR 400 mg QD and NVP IR 200 mg BID groups was sustained. No unexpected AEs were observed at Wk 48. These data support transition from NVP IR to NVP XR in patients stable on the former formulation.


Caenorhabditis elegans (C. elegans), the promotion of longevity by the transcription factor DAF-16 requires reduced insulin/IGF receptor (IIR) signaling or the ablation of the germline, although the reason for the negative impact of germ cells is unknown. FOXO/DAF-16 activity inhibits germline proliferation in both daf-2 mutants and gld-1 tumors. In contrast to its function as a germline tumor suppressor, we now provide evidence that somatic DAF-16 in the presence of IIR signaling can also result in tumorigenic activity, which counteracts robust lifespan extension. In contrast to the cell-autonomous IIR signaling, which is required for larval germline proliferation, activation of DAF-16 in the hypodermis results in hyperplasia of the germline and disruption of the surrounding basement membrane. SHC-1 adaptor protein and AKT-1 kinase antagonize, whereas AKT-2 and SGK-1 kinases promote, this cell-nonautonomous DAF-16 function. Our data suggest that a functional balance of DAF-16 activities in different tissues determines longevity and reveals a novel, cell-nonautonomous role of FOXO/DAF-16 to affect stem cells.In  Previous studies have shown that DAF–16/FOXO transcription factor promotes longevity and stress resistance and inhibits tumor progression in the absence of insulin signaling. Here we show that active DAF-16 in the epidermis can shorten lifespan by promoting a tumorous germline phenotype. In contrast to the known inhibitory effect of insulin signaling upon DAF-16, an active insulin and PI3K signaling are required for DAF-16–mediated signaling to the germline. In addition, AKT-1– and SHC-1–mediated JNK signaling antagonize AKT-2 and SGK-1 to affect the reproductive system. This is to our knowledge the first report about a detrimental effect of DAF-16 on lifespan. Furthermore it emphasizes that DAF-16 activity is highly dependent on the cellular context and communication between different tissues. Caenorhabditis elegans (C. elegans) to mammals C. elegans genome. DAF-16/FOXO proteins are inactivated by the insulin/IGF-1 signaling (IIS) through PI3K and the AGC kinases Akt/SGK which promote its cytosolic localization The forkhead box O (FOXO) subfamily of Forkhead transcription factors is conserved from The Akt/FOXO signaling network acts as a critical control mechanism at the intersection between cancer and stem cell biology. FOXO proteins have been considered as tumor suppressors, because of their ability to induce DNA damage repair, cell cycle arrest and apoptosis C. elegans, DAF-16 is a key regulator of development, longevity and stress response. Active DAF-16 resulting from reduced IIS leads to dauer formation, enhanced stress response and lifespan extension C. elegans. During larval development DAF-16 inhibits robust proliferation of the germline through both cell-autonomous (the germline) and -nonautonomous (the musculature) mechanism gld-1 mutants In C. elegans p52Shc homolog SHC-1 modulates DAF-16 activity through promoting its nuclear entry . SHC-1 negatively regulates IIS by inhibition of the insulin/IGF receptor DAF-2. SHC-1 also associates with MEK-1, the mitogen-activated protein kinase kinase 7 (MAPKK7), to activate a C. elegans JNK homolog JNK-1, thus affecting stress response and longevity C. elegans JNK homolog, KGB-1, upon heavy metal stress In a previous study we have shown that the C. elegans. The adapter protein SHC-1 modulates both IIS and JNK pathways to antagonize this hypodermal function of DAF-16. Our finding reveals a new aspect of DAF-16 activity besides its well known roles in the regulation of longevity, stress response and dauer formation. Our data show that these two aspects affect longevity differently and indicate that the capability of DAF-16 to extend lifespan is dependent on the balance of these two opposing effects.Here, we report a novel role of DAF-16 activity in epidermal cells affecting the reproductive system in a cell-nonautonomous manner, resulting in germline hyperplasia and disruption of the surrounding extracellular matrix of shc-1 mutant animals are generally healthy, grow at a normal rate, and produce normal numbers of offspring SHC-1 positively regulates the activity of DAF-16 by both inhibiting DAF-2 and activating JNK signaling pathways daf-16 acts downstream of shc-1, access of wild type DAF-16, in particular the proportion that escapes phosphorylation by active AKT-1/AKT-2/SGK-1, should compensate for the loss of shc-1 during the control of lifespan. We tested this hypothesis relying on the frequently used strain TJ356 which expresses the full length daf-16 isoform a fused to GFP in a wild type background daf-16 transgene did not extend, but further reduced the already short lifespan of shc-1 (shc-1(ok198);Is[daf-16::gfp] adult animals died within the first five days of adulthood. In addition, about half of the animals were sterile and the remaining fertile animals showed a strongly reduced brood size .Since, according to this model, of shc-1 . Remarkahenotype . We concshc-1(ok198);Is[daf-16::gfp] animals up to two fold ;Is[daf-16::gfp] may be linked to proliferation and, possibly, abnormal mitosis, and that this detrimental effect is blocked by FUDR.Unexpectedly, raising the adult animals in the presence of 5-fluoro-2′-deoxyuridine (FUDR), an inhibitor of DNA synthesis which blocks cell proliferation, suppressed the early lethality and extended the lifespan of two fold . This washc-1(ok198);Is[daf-16::gfp] animals. The GLP-1 mediated Notch signaling represses meiosis of the germ cells and keeps them in mitotic proliferation from the L3 larval stage. In glp-1 mutants the germ cells stop mitotic cell division and enter meiosis shc-1(ok198);Is[daf-16::gfp] animals is indeed caused by proliferation of the germ cells, inactivation of glp-1 should result in a similar lifespan extension as seen upon FUDR treatment. To test this assumption, we used the temperature sensitive allele glp-1(q231), because glp-1(q231) animals at the restrictive temperature 25°C do not have a pronounced difference of mean lifespan compared to wild type probably due to a dysfunction in the atrophy intestine, in contrast to other long lived glp-1(lf) alleles, such as e2141ts or q158 (shc-1(ok198);glp-1(q231);Is[daf-16::gfp] animals lived 240% longer than shc-1(ok198);Is[daf-16::gfp] when shifted at the L2 larval stage to the restrictive temperature  background extends lifespan of wild type animals, which require intestinal DAF-16 and the adaptor protein KRI-1. shc-1;Is[daf-16::gfp] animals. To validate this assumption, we further tested whether kri-1 knock-down could suppress the FUDR dependent lifespan extension. kri-1 RNAi treatment did not shorten the lifespan of shc-1;Is[daf-16::gfp] animals fed with FUDR (-1(ok198);Is[daf-16::gfp] animals is due to a negative input of germline proliferation rather than a general sickness of the strain.The germline is the only tissue that undergoes mitosis in adult animals, so we asked whether mitosis of the germline leads to the early lethality of  or q158  [22],  animals using DIC microscopy revealed that the reproductive system exhibited multiple defects (glp-1(q231) or glp-1(e2141) these extra cells were absent, indicating that they may be germ cells (shc-1(ok198);Is[daf-16::gfp] at different larval stages revealed that the gonad ruptured at the L3 larval stage at the proximal side next to the developing somatic gonad primordium. In animals in which germ cells leaked out into the pseudocoelom, a sharp gonad boundary indicative of an intact gonadal basement membrane, as seen in the wild type animals (shc-1(ok198);Is[daf-16::gfp] animals ;Is[daf-16::gfp] animals with an antibody against the germ cell specific protein PGL-1, a P-granule component. The released cells were PGL-1 positive, corroborating their identity as germ cells (shc-1(ok198);Is[daf-16::gfp] animals contained significantly more germ cells than in wild type (46±14 vs. 35±4) (shc-1(ok198);Is[daf-16::gfp] animals in which one gonad arm was still intact, we counted more germ cells in the disrupted gonad arm than in the intact one. In adult animals the volume of some of these released cells increased so that they looked like oocytes. However, they lacked the condensed diakinetic chromosomes that are characteristic for oocytes (shc-1(ok198);Is[daf-16::gfp] animals show abnormal larval germline proliferation and disruption of the gonadal basement membrane. In the following results we focus mainly on the disruption of gonad phenotype.In order to determine the identity of the extra-gonadal cells, we stained rm cells . We notis. 35±4) . In shc- oocytes . Instead oocytes . Taking daf-16 expression by RNAi significantly suppressed all phenotypic aspects  of shc-1(ok198);Is[daf-16::gfp] animals  mutant background. No gonad disruption was seen in either daf-16(mu86) or daf-16(mgDF50) animals  animals showed gonad disruption, yet with a delayed onset. Deletion of daf-16 almost fully suppressed this defect in shc-1(ok198) animals ;Is[daf-16::gfp] compared to Is[daf-16::gfp] animals vs. nuclear DAF-16::GFP would be the cause of the observed phenotype, then expression of a constitutively nuclear daf-16 mutant (daf-16(4A)::gfp) should not result in gonad disruption. Expressing daf-16(4A)::gfp in wild type animals caused only weak disrupted gonad phenotype (daf-16(4A)::gfp in a shc-1(−) background caused severe defects. We observed that DAF-16(4A)::GFP was nuclearly localized and this nuclear localization was not affected by either presence or absence of SHC-1 (shc-1 mutation did not affect the expression level of daf-16(4A)::gfp ::gfp ::gfp . We also4A)::gfp . Given takt-1, akt-2 or sgk-1 that activate DAF-16 also influence the integrity of the gonadal basement membrane, shc-1(ok198);akt-1(ok525), shc-1(ok198);akt-2(ok393) and shc-1(ok198);sgk-1(RNAi) were analyzed (akt-1(ok525), akt-2(ok393) single mutants nor sgk-1(RNAi) displayed gonad disruption. Inactivation of akt-2 or sgk-1 did not enhance the penetrance of the gonad defect of shc-1(ok198) animals, either. In contrast, 58.5±21.0% of first-day adult shc-1(ok198);akt-1(ok525) animals showed gonad disruption, providing further evidence that this phenotype is not an artificial effect of transgenic daf-16 since this strain does not harbor a daf-16 transgene. In addition, a loss-of-function allele of daf-16 suppressed this phenotype in shc-1;akt-1 animals, verifying its dependence on DAF-16  into shc-1(ok198) background. At 25°C both daf-2(e1370) and shc-1(ok198);daf-2(e1370) mutants formed constitutive dauer larvae with a developmentally arrested germline that prevented the analysis of adult animals. At 20°C, at which daf-2 is partially inactivated, daf-2(e1370) did not alter the defect in the gonadal integrity in shc-1 mutant (Since 1 mutant .daf-2(e1370) into shc-1(ok198);Is[daf-16::gfp] animals and quantified the percentage of animals displaying disrupted gonad at 15°C, since at 20°C shc-1(ok198);daf-2(e1370);Is[daf-16::gfp] showed development arrest due to excessive daf-16 transgene expression (daf-2(e1370) mutant is already reduced, indicated by an extended lifespan daf-2(e1370) significantly reduced instead of increased the penetrance of animals with gonad disruption (shc-1(ok198);Is[daf-16::gfp] animals was completely suppressed by daf-2 mutation and the brood size of the fertile animals was increased (shc-1(ok198);Is[daf-16::gfp] animals require active DAF-2.In order to explore whether DAF-2 inhibits a DAF-16 dependent effect on the germline, we crossed pression . At 15°Csruption , indicatncreased . Consistncreased . These dakt-1 and loss of daf-2 have divergent consequences for DAF-16. To address whether the daf-2 mutation could still suppress the defect in the basement membrane in akt-1;shc-1 animals, we examined shc-1(ok198);daf-2(e1370);akt-1(ok525) triple mutant at 15°C, since only at this temperature the germline of the animals could proliferate after L3 stage. daf-2 mutation suppressed gonad disruption in shc-1(ok198);akt-1(ok525) animals completely ;akt-1(ok525) mutant  mutant , indicatshc-1;Is[daf-16::gfp] animals. We quantified number of the germ cells of shc-1;Is[daf-16::gfp] L3 larvae in daf-2, akt-1/akt-2 or sgk-1 mutant background. Knock-down of sgk-1 significantly reduced number of the germ cells ;akt-1(ok525) and shc-1(ok198);daf-18(e1375);akt-1(ok525) animals. daf-18 mutation did not enhance gonad disruption of akt-1(ok525) one day adult animals (daf-18 enhanced the defects in shc-1(ok198);akt-1(ok525) animals up to almost 100% and daf-16 mutation completely suppressed the defect in shc-1(ok198);daf-18(e1375);akt-1(ok525) animals ;Is[daf-16::gfp], jnk-1(gk7) Is[daf-16::gfp] and kgb-1(um3) Is[daf-16::gfp] animals. Inactivation of JNK-1 did not further enhance disruption of the gonadal basement membrane in Is[daf-16::gfp] animals (mek-1(ks54);Is[daf-16::gfp] animals died at early larval stages. The remaining animals could develop to adulthood. However, 95.6% of these animals displayed germ cells outside of the gonad ;Is[[daf-16::gfp] animals, fewer animals were sterile  Is[daf-16::gfp] animals died at larval stage. However, most of Is[daf-16::gfp] animals fed with kgb-1 RNAi could bypass the larval lethality and in 75% of these one day old adult animals germ cells in the body cavity were observed  and daf-16(mu86) null mutant backgrounds ::gfp resulted in a stronger phenotype compared to wild type daf-16::gfp, we used daf-16(4A) for this analysis. Transgenic expression of daf-16(4A)::gfp in the neurons, intestine, or musculature did not increase the weak shc-1(−) phenotype (daf-16(4A)::gfp in the hypodermis was sufficient to cause disruption of the gonad in 80% of one day adult shc-1(−) animals, suggesting that hypodermal DAF-16 causes disruption of the gonadal basement membrane cell-nonautonomously.In order to test how DAF-16 in different tissues promotes the phenotype and rule out an involvement of weak expression of the henotype . In contIn this manuscript we for the first time describe two qualities of DAF-16 function that affect longevity in opposite way. In contrast to the known lifespan extending effect, we discover that DAF-16 can shorten lifespan by inducing a tumor-like germline phenotype. It has probably affected the outcome of previously described experiments, however, to our knowledge this has not been reported before daf-2 +/+) animals by expressing transgenic copies of daf-16, unless the lifespan assays were performed in the presence of FUDR daf-16 differs from other key regulators of stress response and lifespan such as skn-1, which typically shorten lifespan when inactivated, and increase lifespan when being overexpressed as a transgene. Although daf-16 transgene rescued the short life-span of daf-2;daf-16 double mutants, it did not or only modestly increases lifespan in wild type background daf-2(+) background, AKT phosphorylation results in cytoplasmic retention of these extra copies of DAF-16, rendering them inactive daf-16 transgene  was nuclearly localized in both shc-1(+) and shc-1(−) animals (shc-1(−) background  mutation might only prevent AKT-1 mediated DAF-16 phosphorylation, daf-2(e1370) supposedly reduces both AKT-1, AKT-2, and SGK-1 phosphorylation, and this may result in controlling distinct downstream genes for long lifespan and gonad integrity, respectively. We consider this model less likely, since there is currently no evidence for C. elegans AKT-1 phosphorylating distinct sites in DAF-16 compared to SGK-1 or AKT-2 One possible explanation for the antagonistic effect of distinct IIs pathway mutants is that daf-2, akt-2, and sgk-1 mediated activation of DAF-16 affects different tissues than akt-1 knock-down mediated activation of DAF-16, suggesting that the germline reads out and responds to a balance of DAF-16 activities in somatic tissues. This way, DAF-2 could prevent DAF-16 from counteracting hypodermal DAF-16 in some tissues, e.g. the intestine. It has been previously shown that DAF-2 promotes larval germline proliferation via inactivating germline DAF-16 akt-2 nor sgk-1 caused disruption of the gonad in shc-1 mutant background ::gfp, the corresponding constructs were injected into wild type or shc-1(ok198) animals  was used as co-injection marker. GFP expression of the transgenic animals was confirmed using fluorescent microscopy.To generate transgenic animals carrying wild type A formaldehyde fixation procedure was used  for the Basement membranes were stained with MitoTracker Red CMXRos (invitrogen) by placing worms in a solution of 10 µM MitoTracker Red at 25°C for 2 hours. The worms were then allowed to recover for 30 minutes on an NGM agar plate and analyzed.E.coli 24 hours after the L4 larval stage. These were raised on the FUDR containing plates for four days and then transferred onto new plates without FUDR. FUDR was administrated to adult instead of L4 animals in order to exclude a possible interference from seam cell development to the reproductive system, since the epidermal seam cells undergo a final mitotic division at the L4 to adult molt and DAF-16 mediated signaling is sent from the hypodermis. All the lifespan assays were performed at 20°C with exception of those with glp-1(q231) and daf-2(e1370) mutants, as glp-1(q231) required 25°C to inactivate GLP-1 and daf-2(e1370);shc-1(ok198);Is[daf-16::gfp] could develop to adulthood only at 15°C. All of the lifespan assays were performed at least twice. The short lifespan of shc-1(ok198);Is[daf-16::gfp] can only be observed, when no censoring for adult animals dying due to the development defect is done. As reported previously by us , Is[daf-16::gfp] partially extends lifespan of shc-1(ok198) mutants, when adult animals dying due to the development defect are censored.Lifespan assays were initiated at the L4 larval stage. Synchronized animals were raised at 15°C prior to lifespan analysis. Then, L4 animals were transferred to the respective temperatures for the assays and examined every day. Animals that showed no response to touch were scored as dead. In assays without FUDR treatment animals were transferred every day onto new plates during the reproductive period. Animals died because of bagging of larvae were censored. In the lifespan assay with FUDR treatment, animals were transferred onto the agar plates containing 0.1 mg/ml FUDR and daf-2(e1370);shc-1(ok198);Is[daf-16::gfp] and daf-2(e1370);shc-1(ok198);akt-1(ok525), were raised at 20°C and analyzed via DIC microscopy 24 hours after L4 stage. Animals in which at least one germ cell was detected outside of the gonad were scored as positive. The percentage of positive one day old animals was calculated. Per test thirty animals were examined and each test was performed at least three times. shc-1(ok198);daf-2(e1370);Is[daf-16::gfp] and shc-1(ok198);daf-2(e1370);akt-1(ok525) animals were raised at 15°C and analyzed 24 hours after L4 larval stage.All animals in the tests except those with The numbers of anti-PGL-1 positive cells were quantified at the time point that vulva induction takes place.GraphPad Prism 4.0 software  was used to calculate mean value and to perform statistical analysis.Figure S1glp-1(q231) three day old adult animals display atrophy of the intestine and accumulation of undigested E. coli in the intestinal lumen. The arrow heads point to the intestinal cells in long-lived glp-1(e2141) animals. The arrows point to intestinal atrophy in glp-1(q231) animals. Animals are raised at 25°C from L2 larval stage to inactivate GLP-1.(DOCX)Click here for additional data file.Figure S2Knock-down of glp-1 extend lifespan of shc-1(ok198);Is[daf-16::gfp] animals at 20°C. Mean lifespan: N2: 17.6 days (n = 82); glp-1(q231): 16.8 days (n = 107); shc-1(ok198);Is[daf-16::gfp]: 6.4 days (n = 86); shc-1(ok198);glp-1(q231);Is[daf-16::gfp]: 22.7 days (n = 135). Animals were shifted to 25°C at the L2 larval stage to cease germline proliferation and shifted back to 20°C 24 hours after L4 stage. Adult lifespan was performed at 20°C. This Figure is related to the main (DOCX)Click here for additional data file.Figure S3shc-1;Is[daf-16::gfp] animals.Inactivation of GLP-1 suppresses tumorous germline phenotype in  A: shc-1(ok198);Is[daf-16::gfp] one day adult; B: shc-1(ok198) glp-1(q231);Is[daf-16::gfp] one day adult; C: shc-1(ok198) glp-1(e2141) ;Is[daf-16::gfp] one day adult. Animals were raised at 25°C from L2 larval stage. In both glp-1 alleles there were only somatic gonad and some sperms observed. This Figure is related to the main (DOCX)Click here for additional data file.Figure S4DTC migration defect in shc-1;Is[daf-16::gfp] animals.(DOCX)Click here for additional data file.Figure S5Staining of the gonadal basement membrane in shc-1;Is[daf-16::gfp] L3 animals. The white arrows point to the somatic gonad primordium. The white arrow heads donate the germ cells leaking out of the gonad.(DOCX)Click here for additional data file.Figure S6Residue expression of daf-16::gfp in shc-1;Is[daf-16::gfp] animals upon daf-16 RNAi knock-down. A: shc-1;Is[daf-16::gfp] on L4440 control; B: shc-1;Is[daf-16::gfp] on daf-16 RNAi, the arrows point to the residue GFP expression; C: quantification of fluorescent: shc-1(ok198) as background control: 9.67±3.58 Grey (n = 31), shc-1;Is[daf-16::gfp](L4440): 1876.02±231.78 Grey (n = 31), shc-1 daf-16(RNAi);Is[daf-16::gfp]: 90.62±40.15 Grey (n = 28), P<0.001 compared to shc-1;Is[daf-16::gfp](L4440).(DOCX)Click here for additional data file.Figure S7GFP intensity in shc-1(ok198) animals carrying daf-16 transgenes. The Figure A and Figure B  have the same exposure time. The exposure time was 42 ms for Figure C and 5,000 ms for Figure D, respectively. shc-1(ok198);Ex[daf-16::gfp] and shc-1(ok198);Ex[daf-16(4A)::gfp] animals display comparable phenotype in the basement membrane (45.5% vs. 57.2% of one day adults display disrupted gonad). Scale bar 10 µm. This Figure is related to the main (DOCX)Click here for additional data file.Figure S8DIC images of wild type and shc-1(ok198);daf-2(e1370);Is[daf-16::gfp] animals grown at 20°C. DIC images of wild type and shc-1(ok198);daf-2(e1370);Is[daf-16::gfp] animals were taken three days and six days after hatching, respectively. The gonad of wild type animals contained germ cells in mitotic (mito), meiotic (meiot) stages, oocytes (o), sperm (sp) in the spematheca and embryos (em) in the uterus. The gonad of shc-1(ok198);daf-2(e1370);Is[daf-16::gfp] animals contained less than 50 germ cells whereas vulva (v) induction still occurred. No oocyte or sperm could be identified in the gonad of shc-1(ok198);daf-2(e1370);Is[daf-16::gfp] animals. Scale bar 10 μm. This figure is related to the main (DOCX)Click here for additional data file.Figure S9shc-1(ok198);akt-1(ok525) and shc-1(ok198);akt-1(ok525);daf-2(e1370) animals.DIC images of  The arrow and arrowheads point to the ruptured gonad and germ cells outside the gonad, respectively. This figure is related to the main (DOCX)Click here for additional data file.Figure S10anti-PGL-1 antibody staining (I) of shc-1(ok198);Is[daf-16::gfp] and shc-1(ok198);sgk-1(ok538);Is[daf-16::gfp] L3 larvae. In shc-1(ok198);Is[daf-16::gfp] animals the anterior gonad is disrupted and contain more germ cells than the posterior intact one or that in shc-1(ok198);sgk-1(ok538);Is[daf-16::gfp] animals. This Figure is related to the main (DOCX)Click here for additional data file.Movie S1Z-stack image of PGL-1 antibody staining of shc-1(ok198);Is[daf-16::gfp] L3 animals.(MOV)Click here for additional data file.Table S1Lifespan with glp-1(q231).(DOCX)Click here for additional data file.Table S2Brood size and sterility.(DOCX)Click here for additional data file.Text S1Supplemental experimental procedure.(DOCX)Click here for additional data file.


Escape from cellular senescence induction is a potent mechanism for chemoresistance. Cellular senescence can be induced in breast cancer cell lines by the removal of estrogen signaling with tamoxifen or by the accumulation of DNA damage induced by the chemotherapeutic drug doxorubicin. Long term culturing of the hormone-sensitive breast cancer cell line MCF-7 in doxorubicin (MCF-7/DoxR) reduced the ability of doxorubicin, but not tamoxifen, to induce senescence. Two pathways that are often upregulated in chemo- and hormonal-resistance are the PI3K/PTEN/Akt/mTOR and Ras/Raf/MEK/ERK pathways. To determine if active Akt-1 and Raf-1 can influence drug-induced senescence, we stably introduced activated ΔAkt-1(CA) and ΔRaf-1(CA) into drug-sensitive and doxorubicin-resistant cells. Expression of a constitutively-active Raf-1 construct resulted in higher baseline senescence, indicating these cells possessed the ability to undergo oncogene-induced-senescence. Constitutive activation of the Akt pathway significantly decreased drug-induced senescence in response to doxorubicin but not tamoxifen in MCF-7 cells. However, constitutive Akt-1 activation in drug-resistant cells containing high levels of active ERK completely escaped cellular senescence induced by doxorubicin and tamoxifen. These results indicate that up regulation of the Ras/PI3K/PTEN/Akt/mTOR pathway in the presence of elevated Ras/Raf/MEK/ERK signaling together can contribute to drug-resistance by diminishing cell senescence in response to chemotherapy. Understanding how breast cancers containing certain oncogenic mutations escape cell senescence in response to chemotherapy and hormonal based therapies may provide insights into the design of more effective drug combinations for the treatment of breast cancer. Expression of activated Akt-1 alone was sufficient to increase the ICkt-1(CA) . SimilarWe also isolated MCF7/ΔAkt-1(CA) cells with increased resistance to chemotherapeutic drugs by culturing the cells in medium containing 25 nM doxorubicin. These cells were named MCF7/ΔAkt-1(CA)DoxR. The MCF7/ΔAkt-1(CA)DoxR were 5-fold more frequently recovered from MCF7/ΔAkt-1(CA) than the MCF7/DoxR were from MCF-7 cell line. The MCF7/ΔAkt-1(CA)DoxR cells were approximately 6.7-fold more resistant to doxorubicin than control MCF-7 cells .Interestingly, constitutive activation of Akt-1 in the drug resistant cells [MCF7/ ΔAkt-1(CA)DoxR] resulted in the complete abrogation of drug-induced senescence at all doses examined (Panel D), suggesting an interplay between active Akt-1 and other resistance mechanisms acquired by the MCF7/DoxR cells. We have observed that MCF7/DoxR cells overexpress activated ERK, but not activated Akt when cultured in medium containing doxorubicin. In addition, we have seen that MCF7/ΔAkt-1(CA) also overexpress activated ERK, suggesting the presence of both active pathways may serve to prevent drug-induced senescence.Since we observed elevated activated ERK1/2 expression in MCF7/DoxR cells, the activity of the Raf/MEK/ERK cascade was manipulated in MCF-7 cells in order to determine whether signals transduced by this pathway control the induction of drug-induced senescence. MCF-7 cells were stably transfected with a constitutively-active Raf-1 gene Raf-. ExpressConsistent with prior reports, activated Raf-1 expression increased baseline cellular senescence in the absence of stress by 3-fold, indicating that MCF-7 cells were undergoing oncogene-induced senescence (OIS) DoxR cells with increased resistance to chemotherapeutic drugs by culturing the cells in medium containing 25 nM doxorubicin. These cells are approximately 5-fold more resistant to doxorubicin than MCF7/ΔRaf-1(CA) cells. The MCF7/ΔRaf-1(CA)DoxR cells had a 5.8-fold higher baseline level of cellular senescence than MCF-7 cells . MCF-7 or MCF7/DoxR cells expressing constitutively-active Akt-1 or Raf-1 were treated with 10, 50 and 100 nM 4HT for six days then stained for β-galactosidase activity  after 4HT treatment, suggesting that mechanisms of drug resistance are independent of senescence inducted by 4HT. Interestingly, ΔAkt-1(CA) expression did not prevent cellular senescence induction by 4HT treatment in the unselected MCF7/ΔAkt-1(CA) cells (Panel B), but potently suppressed senescence in doxorubicin-resistant MCF7/ΔAkt-1(CA)DoxR cells (Panel B). In contrast, Raf-1 activation in doxorubicin-resistant MCF7/ΔRaf-1(CA)DoxR cells did not suppress senescence by 4HT treatment (Panel C). Together, these results suggest that presence activated PI3K/PTEN/Akt/mTOR pathway in the presence of acquired drug resistant Raf/MEK/ERK pathway activation is sufficient to block drug-induced senescence to both doxorubicin and 4HT.In our studies, we determined the effects of the chemotherapeutic drug doxorubicin and the hormonal drug tamoxifen (4HT) on the induction of cellular senescence in MCF-7 and derivative cell lines which varied in their levels of activated Akt-1 or Raf-1 expression. Moreover, we also examined the induction of cellular senescence in cells which were selected for drug resistance by prolonged culture in medium containing 25 nM doxorubicin to determine whether drug resistance would alter the induction of senescence after either doxorubicin or 4HT treatment. An advantage of our investigation was that all cells had the same basic genetic background (MCF-7) and differed only in the levels of activated Akt-1 or Raf-1, or their resistance to doxorubicin.A lower level of senescence was observed after 50 nM doxorubicin treatment of MCF7/DoxR than in MCF-7 cells. However, when the cells were treated with 100 nM doxorubicin, similar levels of cellular senescence were observed. These results demonstrated that the doxorubicin-resistant MCF7/DoxR wells were more resistant to the induction of senescence after doxorubicin treatment than unselected MCF-7 cells. However, the MCF7/DoxR doxorubicin-resistant phenotype did not prevent the induction of senescence in response to 4HT treatment. This indicates that acquired resistance to DNA damage-induced senescence does not protect against senescence induced by hormone blockade, and thus the two are likely to occur through independent signaling cascades.While previous studies have shown that overexpression of either activated Akt-1 or Raf-1 results in resistance to doxorubicin ,3,99,100Cip-1were induced in doxorubicin-resistant MCF-7 cells which also contain activated Akt-1[Cip-1levels may result in less cellular senescence in response to doxorubicin in the cells containing activated Akt-1 than in wild type MCF-7 cells.MCF-7 cells contain WT p53 and doxorubicin induces p53 in MCF-7 cells . Lower lted Akt-1. p53 plated Akt-1,110-120,ted Akt-1-123, celted Akt-1-143, chrted Akt-1-146, apoted Akt-1-151, sented Akt-1-154, radted Akt-1,155 tumoted Akt-1-158, metted Akt-1-161, celted Akt-1,162, hypted Akt-1,163,164,ted Akt-1,165,166 ted Akt-1 as well ted Akt-1,169, theted Akt-1, MDM2 anted Akt-1-180 and ted Akt-1-187 and ted Akt-1-173. Smated Akt-1,188-190.Cip1 can activate pRb which can regulate entry into replicative senescence [The target genes of p53 and other members of the p53 family are clearly important in regulating many diverse processes -200. p21nescence ,201. p53nescence ,40,44, hnescence -204. miRnescence ,190,205.Recently it has been demonstrated by Blagosklonny and colleagues that the mTOR complex plays a critical role in regulating senescence and quiescence ,40,206. Other studies by Blagosklonny's group demonstrated that knock down of TSC2, a negative upstream regulator of mTOR, could convert quiescent cells into senescent cells in the presence of Nutlin-3A . Nutlin-PTEN, NF1 or RB) has been associated with “oncogene induced senescence” (OIS) which can be viewed as a protective mechanism to prevent tumor development [The effects of Akt on the induction of cellular senescence remain controversial. Often abnormal oncogene expression or tumor suppressor gene deletion DoxR compared with MCF7/ΔAkt-1ER*(Myr+) cells which had not been selected for doxorubicin- or hormonal-resistance . Growth We have also shown that introduction of mutated forms of PTEN into MCF-7 cells which lacked lipid and protein phosphatase activity also suppressed the ability of doxorubicin to induce cellular senescence . These cDoxorubicin induces the Raf/MEK/ERK pathway in MCF-7 cells and higher levels of active ERK are detected in MCF7/DoxR cells than in drug sensitive MCF-7 cells. ERK can in some cases phosphorylate p53  and alter its activity. However, this increased ERK expression does not appear to prevent the induction of cellular senescence. In fact, a higher as a higher baseline level of cellular senescence was detected in MCF7/ΔRaf-1(CA) cells. Thus it can be argued that there is probably an additional modification(s) besides increased ERK expression in MCF7/DoxR which results in the decreased induction of senescence after doxorubicin treatment. While both MCF7/ΔAkt-1(CA) and MCF7/ΔRaf-1(CA) cells display increased resistance to doxorubicin compared with MCF-7 cells ,3,100, tThe ability of 4HT to induce cellular senescence in breast cancer is not well documented. 4HT likely induces reactive oxygen species (ROS) which in turn cause DNA damage and subsequently cellular senescence -213. SimInduction of cellular senescence in response to 4HT differed in these models which expressed activated Akt-1(CA) or Raf-1(CA). In the MCF7/ΔAkt-1(CA) cells that had not been selected in doxorubicin, there was more cellular senescence than in the MCF-7 cells in response to 4HT treatment  and MCF7/ΔRaf-1(CA)DoxR cells in response to 4HT treatment as was seen after doxorubicin treatment of these cells. Taken together with the results observed with MCF7/DoxR cells, which express high levels of activated ERK, it is likely that increased levels of the Raf/MEK/ERK pathway does not inhibit the induction of cellular senescence induced by either 4HT or doxorubicin.In some cases, the Ras/Raf/MEK/ERK pathway may induce cellular senescence ,214-218.PIK3CA and dysregulation of PTEN. Thus this pathway and downstream substrates such as the transcription factor Twist are critically involved in breast cancer and are targets for improved therapy [PIK3CA or PTEN mutations or gene silencing. PTEN can be mutated or silenced by various mechanisms in human cancer. Mutations occur which either delete the PTEN gene or alter its activity. These mutations which result in activated Akt expression may have effects on the induction of cellular senescence in response to chemotherapeutic drugs currently used to treat breast cancer patients. Our studies also indicate that just because a cell is drug resistant, that does not mean that it will be resistant to the induction of cellular senescence by a chemotherapeutic drug. Doxorubicin induced cell cycle arrest in the G2/M phase in both MCF7/ΔRaf-1 and MCF7/Akt-1 cells, yet these cells differed in the extent of cellular senescence after doxorubicin treatment. Clearly cell cycle regulatory proteins play key roles in regulation of cellular senescence [Cip-1are required in some cells for the induction of senescence but not for cell cycle arrest in response to HDAC inhibitors such as sodium butyrate [These results have clinical significance as the PI3K/PTEN/Akt/mTOR pathway is often activated in breast cancer by mutations at  therapy -222. Furnescence . Other sbutyrate . TogetheMCF-7 cells were obtained from the American Type Culture Collection (ATCC) . Cell culture medium for MCF-7 cells consisted of Roswell Park Memorial Institute-1640 (RPMI 1640) medium  supplemented with 10% (v/v) heat inactivated fetal bovine serum (FBS) as described .5 MCF-7 cells were plated into 6-well cell culture plates  and one day later infected with a retrovirus containing the various plasmid DNAs as described [5 × 10escribed ,100,109.Senescent cells were identified by a senescence associated (SA) β-galactosidase  assay as described .


Unlike mammals, zebrafish exhibits extensive neural regeneration after injury in adult stages of its lifetime due to the neurogenic activity of the radial glial cells. However, the genes involved in the regenerative neurogenesis response of the zebrafish brain are largely unknown. Thus, understanding the underlying principles of this regeneration capacity of the zebrafish brain is an interesting research realm that may offer vast clinical ramifications.cxcr5 and analyzed the function of this gene during adult neurogenesis and regeneration of the zebrafish telencephalon. We found that cxcr5 was upregulated transiently in the RGCs and neurons, and the expression in the immune cells such as leukocytes was negligible during both adult neurogenesis and regeneration. We observed that the transgenic misexpression of cxcr5 in the ventricular cells using dominant negative and full-length variants of the gene resulted in altered proliferation and neurogenesis response of the RGCs. When we knocked down cxcr5 using antisense morpholinos and cerebroventricular microinjection, we observed outcomes similar to the overexpression of the dominant negative cxcr5 variant.In this paper, we characterized the expression pattern of cxcr5 imposes a proliferative permissiveness to the radial glial cells and is required for differentiation of the RGCs to neurons, highlighting novel roles of cxcr5 in the nervous system of vertebrates. We therefore suggest that cxcr5 is an important cue for ventricular cell proliferation and regenerative neurogenesis in the adult zebrafish telencephalon. Further studies on the role of cxcr5 in mediating neuronal replenishment have the potential to produce clinical ramifications in efforts for regenerative therapeutic applications for human neurological disorders or acute injuries.Thus, based on our results, we propose that  The amplicon (726 bp) was subcloned into pGEM T-Easy (Promega). A DIG or fluorescein-labeled mRNA probe was generated using an in vitro transcription kit . In situ hybridization was performed as described  was used , 243 aminoacids)  for the dominant-negative variant, and Tg(hsp:EGFP-T2A-FL-Cxcr5) for the full-length variant. We also used Tg(her4.1:GFP)[Tg(HuC:GFP)[For full-length (HuC:GFP) transgen2.2H2O, 0.33 mM MgSO4.7H2O, 0.0002% methylene blue, pH: 6.5), E3 preheated to 39°C was added in the tubes, which were incubated for 30 minutes at 38°C. After the heat shock, embryos were returned to Petri dishes filled with E3 and placed in a 28°C incubator. The embryos were analyzed with a fluorescent microscope at least 4 hours after the heat induction. Adult fish were transferred into the heat shock aquarium and received in total, four heat shocks on four consecutive days. Each day the water was heated to 37°C, then the heating elements switched off and the water passively cooled down to 28°C (this takes approximately 5 to 6 hours after the heat shock). The fish were analyzed with an Olympus MVX10 fluorescent microscope after anesthetization with MESAB (0.01%). Embryos were sorted and used for desired purposes.Heat-shock experiments were performed on embryos and adults. Embryos (at least 50% epiboly stage) were transferred into 2 ml tubes. After removal of the excess embryo medium  or a laser scanning confocal microscope (Zeiss 510 META). Quantifications were performed in an unbiased manner by counting the absolute number of cells in a minimum of eight different sections of the brain of every fish. For simplicity, results are depicted as the relative amount of cells. For statistical analysis, two-way analysis of variance (ANOVA) and Tukey’s post hoc test or paired t-test were used after determining whether the sample datasets conform to a normal distribution. P-values are indicated as follows: *ANOVA, analysis of variance; BCA-1, B-cell-attracting chemokine 1; BLR-1, Burkitt lymphoma receptor-1; BrdU, bromo-deoxyuridine; CVMI, cerebroventricular microinjection; cxcr5, chemokine (C-X-C motif) receptor 5; DAPI, 4,6-diamidino-2-phenylindole; dpf, days post-fertilization; dn, dominant negative; dpl, days post-lesion; dps, days post-sham; EDTA, ethylene-diamine-tetra-acetic acid; fl, full-length; EGFP, enhanced green fluorescent protein; FISH, fluorescent in situ hybridization; GFP, green fluorescent protein; her4.1, hairy-related 4.1 basic helix-loop-helix transcription factor; IHC, immunohistochemistry; ISH, in situ hybridization; MESAB, 3-amino-benzoic acid ethyl ester; PCNA, proliferating cell nuclear antigen; PFA, paraformaldehyde; qRT-PCR, quantitative real-time polymerase chain reaction; RGC, radial glial cell; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.The authors declare that there is no competing of interest.CK conceived and designed the experiments; CK, SD and AM generated the transgenic animals; CK, SD, NK, AM, JB carried out the experiments; VK contributed to the initial screen; CK wrote the manuscript; CK and MB edited the manuscript. All authors read and approved the final manuscript.Figure S1. Title: cxcr5 expression is overlapping to the proliferating ventricular cells in adult zebrafish telencephalon. (A) Lesion and bromo-deoxyuridine (BrdU) treatment paradigm. Fish were treated with BrdU 3 days post lesion (dpl) for 6 hours before the sacrifice. (B) cxcr5 in situ hybridization on lesioned telencephalon. (B’) High magnification of the dorsomedial region in (B). (B”) High-magnification of the dorsolateral region in (B). (C) BrdU immunohistochemistry on lesioned telencephalon. (C’) High-magnification of the dorsomedial region in (C). (C”) High-magnification of the dorsolateral region in (C). (D) Merged image of (B) and (C). (D’) Merged image of (B’) and (C’). (D”) Merged image of (B”) and (C”). Click here for fileFigure S2. Title: Neurons express cxcr5. (A) cxcr5 fluorescent in situ hybridization (FISH) coupled to HuC immunohistochemistry (IHC) on a section of unlesioned adult zebrafish telencephalon. Inset is magnified image. (B)cxcr5 FISH coupled to HuC IHC on a section of 3 days post lesion (dpl) adult zebrafish telencephalon. Inset is magnified image. cxcr5 is expressed in neurons before and after injury. (C) Quantification graph for cxcr5-expressing HuC-positive cells. (D)cxcr5 chromogenic in situ hybridization (ISH) coupled to NeuroD IHC in unlesioned telencephalon. NeuroD-positive differentiating neurons, which are several cell diameters away from the ventricle express cxcr5 (white asterisks). cxcr5 expression is weaker in NeuroD-positive neurons in comparison to cxcr5-positive cells closer to the ventricle (yellow asterisks). In a transition zone between strong NeuroD-positive (white asterisks) and NeuroD-negative cells (yellow asterisks), cells express NeuroD and cxcr5 weakly (blue asterisks). (E)cxcr5 chromogenic ISH coupled to NeuroD IHC in 3 dpl telencephalon. NeuroD-positive differentiating neurons are more numerous, dispersed inside the parenchyma distantly in comparison to unlesioned telencephalons and express cxcr5 (white asterisks). cxcr5 expression is weaker in NeuroD-positive neurons in comparison to cxcr5-positive cells closer to the ventricle (yellow asterisks). (F) Quantification graph for cxcr5-positive NeuroD-expressing cells. The number of NeuroD/cxcr5 double-positive cells increase upon injury in adult zebrafish telencephalon. Scale bars 25 μm; n = 4 telencephalons for every set of analyses. Click here for fileFigure S3. Title: cxcr5 is not expressed in L-Plastin-positive cells in the adult zebrafish telencephalon. (A) cxcr5 in situ hybridization coupled to immunohistochemistry for L-Plastin  and her4.1:green fluorescent protein (GFP)  in the unlesioned telencephalons. White asterisks indicate the L-Plastin cells. (A1-A3) Individual channels for L-Plastin, her4.1:GFP and cxcr5, respectively. (B) cxcr5 in situ hybridization coupled to immunohistochemistry for L-Plastin and her4.1:GFP in the lesioned telencephalons . White asterisks indicate the L-Plastin cells. (B1-B3) Individual channels for L-Plastin, her4.1:GFP and cxcr5, respectively. (C) cxcr5 in situ hybridization coupled to immunohistochemistry for L-Plastin and her4.1:GFP in the lesioned telencephalons . White asterisks indicate the L-Plastin cells. (C1-C3) Individual channels for L-Plastin, her4.1:GFP and cxcr5, respectively. Click here for fileFigure S4. Title: Larval phenotypes upon cxcr5 misexpression; and transgene activity in the adult zebrafish telencephalon. (A) Cxcr5 is a seven-span transmembrane protein with an extracellular receptor domain, seven transmembrane domains and a C-terminus intracellular domain. We generated full-length and dominant-negative versions of Cxcr5. Dominant negative variant lacks the last three transmembrane domains and the intracellular domain. Both variants are inserted into a transgenesis cassette that contains the coding sequence for enhanced green fluorescent protein (EGFP) and self-cleaving T2A peptide. The whole cassette is expressed under heat-inducible hsp70l promoter. (B) Non-transgenic sibling at 3 days post-fertilization (dpf). (B1) High-magnification image of the yolk-sac in B. (B2) High-magnification image of the yolk-tube extension in B. (B3) High-magnification image of the ventral fin fold in B. (C)Tg(hsp:egfp-T2A-dncxcr5) transgenic animals at 3 dpf after two heat shocks in gastrula. (C1) High-magnification image of the yolk-sac in C. (C2) High-magnification image of the yolk-tube extension in C. (C3) High-magnification image of the ventral fin fold in C. (D)Tg(hsp:egfp-T2A-FLcxcr5) transgenic animals at 3 dpf after two heat shocks in gastrula. (D1) High-magnification image of the yolk-sac in D. (D2) High-magnification image of the yolk-tube extension in D. (D3) High-magnification image of the ventral fin fold in D. (E) Quantification graph for the number of pigment cells in different regions of the non-transgenic, Tg(hsp:egfp-T2A-dncxcr5) and Tg(hsp:egfp-T2A-FLcxcr5) larvae. Note that dominant negative variant of cxcr5 significantly reduces while full-length cxcr5 significantly increases the number of pigment cells in comparison to the non-transgenic siblings. (F) Quantitative real-time PCR analyses at 3 dpf after the misexpression of cxcr5 with two heat shocks at gastrula. (G) Heat-shock scheme for adult zebrafish telencephalon expression. (H) GFP and DAPI staining in heat-shocked non-transgenic animals. (I) GFP and DAPI staining in heat-shocked Tg(hsp:egfp-T2A-dncxcr5) animals. (J) GFP and DAPI staining in heat-shocked Tg(hsp:egfp-T2A-FLcxcr5) animals. (K-M) GFP IHC on Tg(hsp:egfp).(K) Olfactory bulb at 1 day after heat-shock. (L) Olfactory bulb at 2 days after heat-shock. (M) Telencephalon at 1 day after heat-shock. (N) Telencephalon at 2 days after heat-shock. GFP protein as a result of heat shock paradigm reduces significantly after 24 hours after heat shock. Click here for fileFigure S5. Title: cxcr5 translation-blocking antisense morpholino is functional. (A) Uninjected 2 day post-fertilization (dpf) embryos. (B) Control morpholino-injected embryos show no morphological phenotypes at 2 dpf. (C)cxcr5 antisense morpholinoinjected embryos display severe anomalies in axial extension and head development. (D)cxcr5 mRNA rescues the knockdown phenotypes when co-injected with antisense morpholinos. (E) Quantitative real-time PCR analysis of genes regulated by cxcr5, upon control and antisense morpholino injections. All the genes tested are upregulated after knocking down cxcr5. Percentages in A-D represent the ratio of embryos with gross morphological defects to the whole clutch size. Click here for fileFigure S6. Title: Misexpression of cxcr5 does not lead to cell death. (A-I) TUNEL staining to detect the apoptotic cells in non-transgenic siblings, Tg(hsp:egfpt2a-FLcxcr5) and Tg(hsp:egfp-t2a-dncxcr5) animals pre-lesion, 3 days post-lesion (dpl) and 15 dpl time points. Red nuclei indicate the apoptotic cells. (J) Quantification graph shows the number of apoptotic nuclei in the lesioned hemisphere. Misexpression of cxcr5 does not alter the levels of cell death. Two telencephalons were used for every time point. Click here for fileTable S1. Title: qRT-PCR primers. Click here for file