Our results form a structural basis for further development of potent and selective tankyrase inhibitors based on the WIKI4 scaffold.Recently a novel inhibitor of Wnt signaling was discovered. The compound, WIKI4, was found to act through tankyrase inhibition and regulate β-catenin levels in many cancer cell lines and human embryonic stem cells. Here we confirm that WIKI4 is a high potency tankyrase inhibitor and that it selectively inhibits tankyrases over other ARTD enzymes tested. The binding mode of the compound to tankyrase 2 was determined by protein X-ray crystallography to 2.4 Å resolution. The structure revealed a novel binding mode to the adenosine subsite of the donor NAD Subsequent additions of ADP-ribose units lead to a growing ADP-ribose polymer (PAR) attached to the target protein. Enzymes catalyzing this protein modification and sharing a homologous catalytic domain form a superfamily of 17 members in human (EC 2.4.2.30) Tankyrases are enzymes catalyzing a covalent modification of proteins, poly(ADP-ribosyl)ation or PARsylation. In the reaction the enzyme cleaves NADTNKS1 was discovered as an enzyme controlling the length of human telomeres The rationale for using tankyrase inhibitors in cancer therapy comes from its various functions within the cell. Tankyrases PARsylate TRF1, a shelterin complex protein protecting telomeres. The modification causes dissociation of TRF1 from the telomeres allowing extension of the telomere by a telomerase enzyme. Due to high telomerase activity, tumor cells escape cellular senescence by uncontrolled telomere extension. Inhibition of tankyrase catalytic activity in tumor cells prevents uncontrolled telomere extension, triggering cellular senescence Tankyrase 1 is also involved in mitosis as the protein is localized to spindle poles and its catalytic activity is essential for normal bipolar spindle structure Wnt signaling pathway is often overactivated in cancers. The identification of tankyrases as part of the β-catenin destruction complex has put tankyrases as one of the promising drug targets regulating Wnt signaling The first potent tankyrase inhibitor, XAV939, was discovered though the Wnt-responsive luciferase reporter assay Recently another novel inhibitor of the Wnt signaling pathway, Wnt Inhibitor Kinase Inihibitor 4 or WIKI4, was discovered using β-catenin reporter assays To further characterize the compound we first verified its high potency against TNKS1. We also report the profiling of the compound against many other human ARTD enzymes to verify that the compound is indeed selective for tankyrases over the other ARTD enzymes. Furthermore, we characterized the binding of WIKI4 to the catalytic domain of human TNKS2 using protein X-ray crystallography. This structural work elucidates how the small molecule binds to the protein and explains the selectivity within the ARTD family.basic-tag followed by a 6xHis-tag and a TEV-protease cleavage site (MHHHHHHSSGVDNKFNKERRRARREIRHLPNLNREQRRAFIRSLRDDPSQSANLLAEAKKLNDAQPKGTENLYFQ*SM). Constructs for the catalytic fragments of ARTD4 (residues 250–565) and ARTD12 (residues 489–684) were generous gifts of the SGC Stockholm. These constructs are also based on the pNIC28-Bsa4 vector. Other expression constructs used here have been described before cDNA for full length ARTD1/PARP1 was purchased from Source Bioscience and the codon optimized full length ARTD2/PARP2 was purchased from Genescript. Full-length ARTD1 was cloned by PCR extension cloning to pNIC28-Bsa4 vector containing an N-terminal 6xHis-tag and a TEV-protease cleavage site (MHHHHHHSSGVDLGTENLYFQ*SM). Full-length ARTD2 was cloned by PCR extension cloning to pNIC28-ZB vector with an N-terminal ZE. coli Rosetta 2 (DE3) competent cells. An overnight preculture was grown in LB broth medium with 50 µg/mL kanamycin and 34 µg/mL chloramphenicol at 37°C. The preculture was used to inoculate terrific broth auto-induction media (Formedium) with the same antibiotics. The culture was incubated at 37°C with shaking. When OD600 reached 0.4–0.5, 0.1 mM ZnSO4 was added. Temperature was lowered to 16°C at OD600 1 and the incubation was continued for 16 h. The cells were collected by centrifugation  and the cell pellet was resuspended in 1.5 mL/g lysis buffer  and stored at −20°C.ARTD1 plasmid was transformed into g, 45 min at 4°C). Supernatant containing ARTD1 was filtered through 0.45 µm filter and loaded on a HisTrap FF Crude column . The column was washed with 40 mL wash buffer 1  followed by 30 mL wash buffer 2  and 30 mL wash buffer 3 . ARTD1 was eluted with 10 mL of elution buffer .Cell suspension was thawed in warm water and supplemented with 250 U benzonase (Sigma-Aldrich) and 0.1 mM pefabloc (Sigma-Aldrich). Cells were lysed by sonication and the cell debris was cleared by centrifugation . Protein was purified with NaCl gradient elution using a BioLogic Duoflow chromatography system (Bio-Rad). ARTD1 was eluted at 600 mM NaCl. 30 µg of ARTD1 was obtained per 1 L of cell culture.E. coli Rosetta 2 (DE3) competent cells. An overnight preculture supplemented with 50 µg/mL kanamycin and 34 µg/mL chloramphenicol was grown in LB broth medium at 37°C. The preculture was used to inoculate terrific broth auto-induction media (Formedium) supplemented with 0.8% glycerol and the same antibiotics. The culture was incubated at 37°C with shaking until OD600 reached 1.5. The temperature was lowered to 18°C and the incubation was continued for 22 hours. The cells were collected by centrifugation  and the cell pellet was resuspended in 1.5 mL/g lysis buffer  and stored at −20°C.ARTD2 plasmid was transformed into g, 45 min at 4°C). Supernatant containing ARTD2 was filtered through 0.45 µm filter and loaded on a 1 ml HisTrap FF Crude column . The column was washed with 30 mL of wash buffer , followed by 30 mL wash buffer 2 . ARTD2 was eluted with 15 mL of elution buffer . The 6xHis-tag with the Zbasic fragment was cleaved with TEV-protease (1∶30 ARTD2/TEV molar ratio) at 4°C for 24 hours. The protein was then loaded to the 1 mL HisTrap FF Crude column  and the flowthrough containing ARTD2 was collected. The yield of ARTD2 was 1 mg per one liter of cell culture.Cell suspension was thawed at room temperature and supplemented with 250 U benzonase (Sigma-Aldrich). Cells were lysed by sonication and the cell debris was cleared by centrifugation (31000×E. coli Rosetta 2 (DE3) competent cells. An overnight preculture supplemented with antibiotics (50 µg/mL kanamycin and 34 µg/mL chloramphenicol) was grown in LB medium at 37°C. The preculture was inoculated in terrific broth autoinduction media (Formedium) supplemented with the same antibiotics and glycerol (8 g/L). The culture was grown at 37°C until OD600 reached 1 and the incubation was continued overnight at 18°C. The cells were collected by centrifugation  and the cell pellet was resuspended in lysis buffer  and stored at −20°C.ARTD4 construct was transformed into g, 20 minutes at 4°C). After filtering the supernatant with 0.45 µm filter, sample was loaded on a HisTrap FF Crude column  pre-equilibrated with the loading buffer . After loading the sample, the column was washed with the same buffer. Protein was eluted with the elution buffer (loading buffer containing 500 mM imidazole) on Äkta purifier . Fractions were pooled together and further purified with Superdex 200 HR 10/30 pre-equilibrated with gel filtration buffer . 2 mg of ARTD4 was obtained per 1 L of cell culture.Cell suspension was quickly thawed in warm water and supplemented with 250 U benzonase, Complete, EDTA-free protease inhibitor tablet (Roche) and 0.1 g of lysozyme. Cell suspension was sonicated and the cell debris was cleared by centrifugation (35000×E. coli Rosetta 2 (DE3) competent cells. An overnight preculture was grown in LB medium with 50 µg/mL kanamycin and 34 µg/mL chloramphenicol at 37°C. The preculture was inoculated into terrific broth auto-induction media (Formedium) with the same antibiotics. The culture was incubated at 37°C with shaking. When OD600 reached 1 the temperature was lowered to 18°C and the incubation was continued overnight. The cells were collected with centrifugation  and the cell pellet was resuspended in 1.5 mL/g lysis buffer  and stored at −20°C.ARTD12 plasmid was transformed into g, 1 hour at 4°C). Supernatant containing ARTD12 was filtered through 0.45 µm filter and loaded on a HisTrap FF Crude column . The column was washed with 40 mL of lysis buffer followed by 30 mL wash buffer . ARTD12 was eluted with 12 mL of elution buffer  and concentrated to 0.8 mL. The concentered protein was further purified with HiLoad 16/60 Superdex 75 pg pre-equilibrated with 30 mM HEPES, 300 mM NaCl, 10% glycerol, 0.5 mM TCEP, pH 7.5. 0.1 mg of ARTD12 was obtained per 1 L of cell culture.Cell suspension was thawed in warm water and supplemented with 250 U of benzonase (Sigma-Aldrich) and 1 Complete, EDTA-free protease inhibitor tablet (Roche). Cells were lysed by sonication and the cell debris was cleared by centrifugation . Assay buffer consisted of 50 mM Bis-Tris propane pH 7, 0.5 mM TCEP, and 0.01% Triton-X-100. After the enzymatic reaction 20 µL of 20% acetophenone in ethanol and 20 µL of 2 M KOH were added and the plate was incubated for 10 minutes. After adding 90 µL of formic acid the plate further incubated for 20 minutes and the fluorescence intensity was measured using Tecan Infinity M1000 with excitation/emission wavelengths of 372 nm and 444 nm, respectively.Assays were conducted as reported earlier The profiling assays were carried out similarly, but in order to get a robust answer, the reaction time was set to achieve at least 45% substrate consumption for each enzyme. The reactions were done in triplicates and protein, DMSO, and inhibitor controls were added to exclude the effects of autofluorescence and DMSO on protein activity. The detailed conditions including buffers and incubation times used for the profiling assays are shown in Crystallization of TNKS2 catalytic domain is done as previously reported Diffraction data were collected on beam line I04-1 at Diamond Light source, Didcot, UK. Data were processed and scaled with XDS package in vitro inhibitor potency of WIKI4 to TNKS1 was measured with a fluorescence based activity assay (50 131 nM) 50 11 nM) 50 25 nM) 50 8 nM) The ty assay . The IC550 114 nM) We profiled the inhibition of WIKI4 against 8 ARTDs, including polymerases  and monotransferases  . WIKI4 dfree of 20.2%/25.0%  to accommodate the 1,8-naphthalimide moiety of the inhibitor and Tyr1050 moves out from the binding site (Cαapo-CαWIKI4 5.2 Å) to accomodate the triazole moiety of the inhibitor  The 1,8-naphthalimide moiety  linked t+ binding site. The large 1,8-naphthalimide moiety extends out of the adenosine binding site and clashes with the regulatory domains of ARTD1-3 (WIKI4 is a new, recently reported inhibitor scaffold for tankyrases with high potency towards both TNKS isoforms. IC50 towards TNKS1 is 26 nM and the inhibitor profiling showed that WIKI4 is equally potent towards TNKS2 . It also ARTD1-3  [28]–[30 ARTD1-3 .The structure-activity studies of WIKI4 analogs by James and co-workers Both WIKI4 and IWR-1 bind to the adenosine site of TNKS2. Despite that both compounds were soaked to preformed crystals and induce the opening of the D-loop, they show completely different conformation of the loop . This inWIKI4 and JW74 analog G007-LK both contain a core triazole moeity linking three groups together. However, the binding of G007-LK induces similar structural changes in the D-loop as the binding of IWR-1. The conformation of His1048, which allows the parallel π-π stacking with WIKI4 and IWR-1 is present also in G007-LK, where it forms a parallel π-π stacking with the benzylamine group . The con+ binding groove explain both the high potency and selectivity of the compound. As such the compound is an excellent biological probe for the evaluation of the effects of tankyrase inhibition in different cancer cell lines. The structural characterization of the binding mode may also help in developing metabolic stability and pharmacokinetics of WIKI4 in the future.The identification of WIKI4 as a Wnt signaling antagonist and its high potency in several cell lines made it a promising hit compound as a tankyrase inhibitor We have analyzed the binding mode, potency and selectivity of WIKI4, a novel Wnt antagonist that acts through tankyrase inhibition. WIKI4 is selective for tankyrases over 6 other ARTDs tested and inhibits tankyrases with high potency. The inhibitor binds to the adenosine binding site of tankyrases and has a distinct binding mode compared to other inhibitors binding to this site. The compound uniquely extends towards and interacts with the G-loop, and retains critical hydrogen bonding and stacking interactions as observed in other TNKS inhibitors. Our results form a structural basis for the high potency and selectivity of WIKI4 and allow further rational development of WIKI4 based inhibitors.


While cold crystalloids have been used for induction of therapeutic hypothermia after cardiac arrest ,2, the en = 9) within 20 minutes using a Zoll Power Infuser, or 1,500 ml of 1°C cold Voluven (6% hydroxyethyl starch 130/0.4 in 0.9% NaCl) , or no infusion . The animals were observed for 90 minutes following infusion. Cerebral, rectal, intramuscular, pulmonary artery, and subcutaneous fat body temperatures (BT) were continuously recorded using GES 130 temperature probes and GMH 3250 digital thermometers . Data were analyzed with JMP 3.2 software  and are expressed as a mean ± SD. P < 0.05 was considered statistically significant.VF was electrically induced in 22 anesthetized domestic pigs (33 ± 2 kg). Defibrillation was attempted after 15 minutes CPR using the AutoPulse  and artificial ventilation. After spontaneous circulation was restored, the animals were randomized to receive either 1,500 ml of 1°C cold normal saline . At the time of finishing infusion there was a greater decrease in cerebral and pulmonary artery BT in group A compared to group B . Area under the curve analysis of the decrease in intracerebral BT revealed a more vigorous cooling effect in group A compared to B . There was also a higher calculated enthalpy for crystalloid solution compared to colloid in the time-point of maximal BT decrease .In total, 46.6 ± 3.2 ml/kg cold normal saline was infused in group A, and 45.7 ± 2.7 ml/kg cold colloid in group B. The animals treated with cold fluids achieved a significant decrease of BT in all measurement sites while there was a spontaneous increase in group C (Cold crystalloid infusions resulted in a more intense cooling effect than colloid infusions of the same temperature and infusion rate in this porcine model of cardiac arrest.


Accumulation of β-sheet-rich peptide (Aβ) is strongly associated with Alzheimer's disease, characterized by reduction in synapse density, structural alterations of dendritic spines, modification of synaptic protein expression, loss of long-term potentiation and neuronal cell death. Aβ species are potent neurotoxins, however the molecular mechanism responsible for Aβ toxicity is still unknown. Numerous mechanisms of toxicity were proposed, although there is no agreement about their relative importance in disease pathogenesis. Here, the toxicity of Aβ 1–40 and Aβ 1–42 monomers, oligomers or fibrils, was evaluated using the N2a cell line. A structure-function relationship between peptide aggregation state and toxic properties was established. Moreover, we demonstrated that Aβ toxic species cross the plasma membrane, accumulate in cells and bind to a variety of internal proteins, especially on the cytoskeleton and in the endoplasmatic reticulum (ER). Based on these data we suggest that numerous proteins act as Aβ receptors in N2a cells, triggering a multi factorial toxicity. Alzheimer's disease is the most widespread form of dementia worldwide. Characteristic pathological lesions are senile amyloid plaques, vascular amyloidosis and neurofibrillary tangles. The amyloid aggregates are formed by Aβ peptides of various amino-acid lengths Oligomers were produced and characterized in detail  with both untagged and EDANS -tagged Aβ 1–42 peptides . Time deAs confirmation, peptide internalization was investigated in the absence of the EDANS fluorophore after treatment of N2a cells with toxic Aβ 1–42 untagged oligomers. In this second experimental setting, Aβ 1–42 detection was performed by anti-Aβ 6E10 antibody (immunocytochemistry) using confocal microscopy; markers for peptide internalization were selected as follows: vimentin (cytoskeleton), cathepsin D (lysosomes), GRP-78 (endoplasmic reticulum and associated membranes) and Hoechst 33285 (nuclei). N2a cells were treated as before and after 6 hours the unlabeled peptide, detected by the 6E10 antibody, was clearly able to enter N2a cells since its fluorescence co-localized with vimentin and GRP-78 . UnexpecFollowing the suggestion that Aβ toxic oligomers were able to enter N2a cells, the possibility that these particles bind to intracellular proteins was investigated by using a high-throughput approach. N2a cells were lysed using a detergent-free solution in order to preserve membrane integrity; proteins were then separated by centrifugation into two fractions: (i) cytosol; (ii) membrane fragments and organelles. The resulting fractions were blotted onto nitrocellulose after SDS-PAGE fractionation and the membrane was washed overnight in TBST to promote the maximum of protein renaturation achievable A proteomic approach was then used to identify the proteins involved in Aβ binding. A particular type of 2D gel separation was selected In the set of experiments reported until now, only toxic oligomers from Aβ 1–42 peptides have been used. However, from the literature it is known that other aggregation forms of Aβ 1–42 as well as Aβ 1–40 peptides are implicated in the Alzheimer's pathology; all of them having different impact on neuronal toxicity Different Aβ preparations were assessed for their ability to alter N2a cells viability . N2a celvs Aβ 1–40 monomers  Dahlgren et al. In conclusion, our studies demonstrate that Aβ toxic species cross the plasma membrane and accumulate within N2a cells by docking to a variety of proteins. Therefore in our experimental conditions, our data dismiss the hypothesis of the presence of a single “death receptor” and put forward the idea that many proteins can be, under different extents, target of Aβ binding. The ability of Aβ toxic species to enter into cells and interact with many target proteins suggests the idea that a plurality of molecular alterations after massive protein binding could interfere generically, but concurrently, in numerous independent, physiological pathways. This may therefore explain the presence of a large number of molecular alterations associated with Aβ-induced toxicity in literature as well as many proposed Aβ receptors that, up to now, have always been considered independently.Aβ 1–40 and Aβ 1–42 were prepared by solid-phase peptide synthesis http://www.lgcstandards-atcc.org/LGCAdvancedCatalogueSearch/ProductDescription/tabid/1068/Default.aspx?ATCCNum=CCL-131&Template=cellBiology) grown on glass coverslips were treated with 30 µM EDANS -tagged Aβ 1–42, and incubated for 5 minutes or 6 hours. Cells were washed twice in PBS and coverglases were mounted onto glass slides using Fluor Save mounting medium . Slides were allowed to dry, before analysis using an Olympus fluorescence microscope  equipped with an Olympus U-RFL-T UV source and DAPI emission filter. All fluorescence images were acquired using the same exposure parameters. Merged images between bright-field and fluorescence were obtained with the Adobe Photoshop software CS 8.0 (Adobe System Incorporated).N2a cells . Cells were treated with 30 µM EDANS-tagged Aβ 1–42, and the wells were completely covered with mineral oil. Image recordings started 30 minutes after cell treatment using an Olympus time lapse microscope  equipped with a cell incubator box, Olympus U-RFL-T UV source and DAPI emission filter. Different positions were selected and images in bright-field and in fluorescence were recorded every 15 minutes for 16 hours. Single images were used to compose movies. Acquisition and movie composition were done with the Cell∧R 3.0 software .N2a cells were fixed with Carnoy's solution  and then incubated with Hoechst 33258 dye . Cells were then permeabilized with 0.1% Triton X-100 with 10% foetal bovin serum and incubated overnight (4°C) with mouse primary antibodies against the Aβ 1–42 peptide  in PBS containing 0.1% Triton X-100 and 10% foetal bovin serum. Cells were washed and incubated for 1 hour at room temperature with the secondary anti-mouse IgG conjugated with Alexa 546  in PBS containing 1% FBS. Cells were then washed and incubated overnight at 4°C with primary antibodies against GRP78 , vimentin  or cathepsin  in PBS containing 0.1% Triton X-100 and 10% foetal bovin serum. After washing, cells were incubated for 1 hour at room temperature with the secondary anti-rabbit or anti-goat IgG conjugated with Alexa 488  in PBS containing 1% FBS.2PO4, 5 mM NaHCO3, complete protease inhibitor cocktail and 1 mM sodium orthovanadate in 10 mM HEPES buffer. CLBi was added directly to the flask, and cells were scraped off and collected. Cell lysis was improved by using a 26G needle. Cell debris were eliminated by centrifugation (2×10 min centrifugation at 6.6 g) and the supernatant was collected as the post-nuclear fraction. This fraction was then centrifuged for 3 hours at 14 000 rpm at 4°C . The supernatant was collected as the cytosolic fraction; the pellet containing membrane/organelles was washed once and suspended in PBS containing complete protease inhibitor cocktail and 1 mM sodium orthovanadate. Protein was quantified by the BCA assay.Mild cell lysis for protein fractionation - slightly modified method from Proteins were diluted 1∶1 with the loading buffer containing 12% (w/V) SDS and 100 mM dithiothreitol in Tris-HCl buffer 0.5 M, pH 6.8, and immediately denatured at 100°C for 5 minutes. Samples were analyzed using Tris-glycine SDS-PAGE  and blotted in a semi-dry apparatus  onto nitrocellulose. The blotting membrane was blocked from 1 hour to overnight in 5% non-fat milk. After washing in TBST the membrane was incubated 2 hours with 100 ng/ml Aβ peptides in TBST and the membrane was processed as for Western Blot with the primary antibody 6E10  and anti-mouse HRP conjugated secondary antibody  using an ECL detection kit .For the experimental control the nitrocellulose membrane was incubated only with Aβ peptide and anti-mouse secondary antibody, without the 6E10 primary antibody.2. Blotting membranes were processed as for Far Western blot and gels were stained by Coomassie Instant blue . Images from gels and membranes were acquired and superimposed to select corresponding spots in the gel.2D gel electrophoresis was as described by Hartinger at al. Spots were excised and processed for mass spectrometry. Spots were destained in 40% ethanol and shrunk in acetonitrile. Proteins were then reduced for 1 hour at 37°C by 10 mM dithiothreitol and carbamidomethylated 20 min by 55 mM iodoacetamide. Proteins were digested overnight by the addition of 12.5 ng/µl sequence grade modified bovin trypsin . Trypsin digestion was blocked by addition of 0.1% TFA and peptides were spotted onto MALDI-TOF/TOF-TOF target plates using α-cyano-hydroxy-cinnamic acid as matrix. MS spectra were acquired using a Reflex III MALDI-TOF ; MS/MS spectra were acquired by using a 4700 MALDI-TOF-TOF  and the 6 most abundant precursor ions were selected for fragmentation. MS and combined MS-MS/MS data were submitted to the MASCOT database search engine with the following searching parameters: SwissProt 57.3 protein database; carbamidomethylation as variable modification and oxidation of methionine and carbamylation as fixed modifications; one missed cleavage, mass tolerance of 0.1 Da for the peptide mass values, 0.1 Da for the ion mass values.4 U/ml penicillin/streptomycin , 2 mM L-glutamine  and 10% foetal calf serum  at 37°C, in 5% CO2. For toxicity assays confluent cells were trypsinized, diluted in DMEM containing 1% FCS to minimize cell growth and plated at the concentration of 5×104 cells/ml in transparent, flat bottom 96-multiwell plates . Cells were treated 4 hours after plating by adding 10 µl of peptide solution to the culture medium. Cell viability was determined 72 hours after treatment using a MTT reduction assay , according to the manufacturer's directions.The murine neuroblastoma line N2a is frequently used when a cell line is needed which recapitulate neuronal features and it represents one of the common models for amyloid toxicity. N2a cells were grown in DMEM containing 4.5 g/l glucose  supplemented with 1×10Figure S1Aβ 1–42 untagged and EDANS-tagged peptides were aged as previously described (TIF)Click here for additional data file.Figure S2An Aβ 1–42 batch of lyophilized peptide was disaggregated as previously described to obtain a preparation closed to the monomeric form of the peptide (TIF)Click here for additional data file.Table S12/sec) for Aβ 1–40 peptide at different temperatures . Nuclear magnetic resonance (NMR) experiments were performed using a Varian 400-MHz Mercury  equipped with a z-axis gradient coil. Pulse field gradient NMR diffusion measurements give molecular size through the measurement of diffusion coefficients. 1H spectra were acquired with 128, 160, 256 or 512 transients and 2 s recycle delay. Diffusion experiments were performed employing an array of 20 or 30 spectra for each experiment  varying the gradient strength from 3.33 to 19.4 G/cm2. The lengths of and delays between the gradient pulses were optimized depending on the experimental conditions and ranged between 0.002 and 0.005 s and 0.2–0.7 s, respectively. Data were fitted and diffusion coefficients determined with the Dosytoolbox software. Diffusion coefficients of disaggregated Aβ 1–40 peptide in solution as determined by STD-NMR. Expected (as standard curves) and obtained diffusion (m(DOC)Click here for additional data file.Supporting Information S1(DOC)Click here for additional data file.


Caulerpa genus, on guinea pig ileum. Our findings indicated that caulerpine inhibited phasic contractions induced by carbachol (IC50 = 7.0 ± 1.9 × 10−5 M), histamine (IC50 = 1.3 ± 0.3 × 10−4 M) and serotonin (IC50 = 8.0 ± 1.4 × 10−5 M) in a non-selective manner. Furthermore, caulerpine concentration-dependently inhibited serotonin-induced cumulative contractions (pD′2 = 4.48 ± 0.08), shifting the curves to the right with Emax reduction and slope of 2.44 ± 0.21, suggesting a noncompetitive antagonism pseudo-irreversible. The alkaloid also relaxed the ileum pre-contracted by KCl (EC50 = 9.0 ± 0.9 × 10−5 M) and carbachol (EC50 = 4.6 ± 0.7 × 10−5 M) in a concentration-dependent manner. This effect was probably due to inhibition of Ca2+ influx through voltage-gated calcium channels (CaV), since caulerpine slightly inhibited the CaCl2-induced contractions in depolarizing medium without Ca2+, shifting the curves to the right and with Emax reduction. According to these results, the spasmolytic effect of caulerpine on guinea pig ileum seems to involve inhibition of Ca2+ influx through CaV. However, other mechanisms are not discarded.In this work, we investigated the spasmolytic effect of caulerpine, a bisindole alkaloid isolated from marine algae of the  Natural products are a traditional source of drug molecules to treat infectious diseases, pain, and cancer, among others ,2. MarinAmong the class of secondary metabolites in marine organisms, alkaloids are the second most abundant . Indole b;5,6-b′]diindole-6,13-dicarboxylic acid dimethyl ester, called caulerpine or caulerpin  benzamide), sharing the indole nucleus, showed non-selective relaxant activity on guinea pig ileum diindole-6,13-dicarboxylic acid dimethyl ester, named caulerpine or caulerpin.® and diluted in distilled water. Carbamoylcholine chloride (carbachol), histamine dihydrochloride, serotonin hydrochloride were purchased from Sigma-Aldrich  and were dissolved and diluted in distilled water. The physiological solution was a freshly modified Krebs solution (pH 7.4) with the following composition (mM): NaCl (117.0), KCl (4.7), MgSO4 (1.3), NaH2PO4 (1.2), CaCl2 (2.5), glucose (11.0) and NaHCO3 (25.0). A high K+ isosmotic solution (pH 7.4) with the following composition was also used: NaCl (51.7), KCl (70.0), MgSO4 (1.3), NaH2PO4 (1.2), glucose (11.0) and NaHCO3 (25.0). These salts were obtained from Vetec  and Fmaia .Caulerpine was dissolved in CremophorCavia porcellus) of both sexes from the Professor Thomas George Bioterium of CBiotec/UFPB, weighing 368 ± 8 g, were used. The animals had free access to food and water, were kept in rooms at 21 ± 1 °C with a 12-h light–dark cycle and fasted for 18 h before the experiments. Measures to reduce pain, stress and any suffering were taken in accordance with the ethical guidelines for animal use. All experimental procedures were previously approved and performed in accordance with the Research Ethics Committee of the Federal University of Alagoas (UFAL) guidelines (protocol CEUA 039/2012).Adult guinea pigs (2 and 5% CO2). Segments of the ileum oriented along the longitudinal axis (2–3 cm in length) were suspended in a 5 mL organ bath, which contained modified Krebs solution, maintained under resting load of 1.0 g at 37 °C. The tissues were allowed to stabilize for 30 min. Isotonic contractions were recorded using isotonic levers coupled to kymographs and smoked drums  (Experiments 3.5.1 and 3.5.2). An isometric transducer (FORT-10) coupled to an amplifier (TMB4M), both from World Precision Instruments (EUA), connected to an analog/digital converter board  installed in a computer with BioMed© software version RV2 were used to record isometric contractions (Experiments 3.5.3 and 3.5.4).Animals were euthanized by cervical dislocation and exsanguination. The distal ileum was immediately removed, cleaned of adhering fat and connective tissue, immersed in modified Krebs solution at room temperature and continuously gassed with carbogen  [After a stabilization period, two phasic contractions were obtained for 10−5, 10−4, 3 × 10−4 and 10−3 M). The average amplitude of concentration-response curves for serotonin was considered to be 100% (control) and all contractions were assessed referring to it. Each preparation was exposed to only one caulerpine concentration. The antagonism exerted by caulerpine was evaluated based on the analysis of the Schild plot and their potencies with pD′2 values, which is defined as the negative logarithm to base 10 of molar concentration values of an antagonist that reduces the response to an agonist to 50% of its maximum effect (Emax), assessed through concentration-response curves in both absence (control) and presence of caulerpine [After a stabilization period, two similar cumulative concentration-response curves for serotonin were induced and caulerpine was incubated, in different preparations, in the absence of serotonin for 15 min in different concentrations as independent experiments. Afterwards, a new serotonin cumulative curve was obtained in the presence of caulerpine (3 × 10ulerpine .−5 M CCh. Contractile agents remained in contact with the preparation until a contraction plateau was reached (approximately 10 min), and the tissue was then washed. After 30 min, the process was repeated and caulerpine was added cumulatively (10−7 up to 10−3 M) at the plateau phase. Relaxation was expressed as the reverse percentage of initial contraction elicited by the contractile agents. The molar concentration of a substance that produces 50% of its maximal effect (EC50) was obtained graphically from the concentration-response curves [After a stabilization period, an isometric contraction was elicited by 40 mM KCl or 102+ solution for 45 min. Two similar CaCl2 cumulative concentration-response curves were obtained and caulerpine was incubated for 15 min in the absence of CaCl2. A third CaCl2 cumulative curve was obtained in the presence of caulerpine. The maximal contraction obtained with the first CaCl2 concentration-response curve was considered to be 100% (control), and all contractions were assessed in reference to it. Each preparation was exposed to a single caulerpine concentration.After the stabilization period, the modified Krebs solution was replaced by a depolarizing (with 70 mM KCl in equimolar exchange for NaCl) and nominally without Ca50 and IC50 values were determined by nonlinear regression [t-test or one-way ANOVA followed by Bonferroni’s test when appropriate. The significance level considered in all tests was p < 0.05. Schild plots were analyzed by linear regression. Antagonism was considered to be noncompetitive when the slope of the Schild plot was significantly different from unity and depression of the maximum response was observed. All values were obtained using Graph-Pad Prism® 5.01 software .Data are expressed as means and S.E.M. EC2+ influx through CaV. However, other mechanisms cannot be excluded.In conclusion, we demonstrated that caulerpine has a non-selective spasmolytic effect on guinea pig ileum. At the functional level, this effect is due in part to the inhibition of Ca


To identify the disease locus for autosomal recessive congenital cataracts in a consanguineous Pakistani family.All affected individuals underwent a detailed ophthalmologic examination. Blood samples were collected and genomic DNA was extracted. A genome-wide scan was completed with fluorescently-labeled microsatellite markers on genomic DNA from affected and unaffected family members. Logarithms of odds (LOD) scores were calculated under a fully penetrant autosomal recessive model of inheritance.Ophthalmic examination suggested that affected individuals have bilateral cataracts. Linkage analysis localized the critical interval to chromosome 8p with LOD scores of 3.19, and 3.08 at θ=0, obtained with markers D8S549 and D8S550, respectively. Haplotype analyses refined the critical interval to 37.92 cM (16.28 Mb) region, flanked by markers, D8S277 proximally and D8S1734 distally.Here, we report a new locus for autosomal recessive congenital cataract mapped to chromosome 8p in a consanguineous Pakistani family. Congenital cataracts are the principal cause of visual impairment in children worldwide and are responsible for about one third of cases of blindness in infants ,2. The pEPHA2), connexin50 (GJA8), glucosaminyl (N-acetyl) transferase 2 (GCNT2), heat-shock transcription factor 4 (HSF4), lens intrinsic membrane protein (LIM2), beaded filament structural protein 1 (BFSP1), alphaA-crystallin (CRYαA), betaB1-crystallin (CRYβB1), and betaB3-crystallin (CRYβB3) have been identified [Approximately one-third of congenital cataract cases are familial , and to-entified ,16,18-22Here, we report a consanguineous Pakistani family with multiple affected individuals. Linkage analysis localized the critical interval to chromosome 8p with a significant Lod scores and haplotype analyses refined the critical interval to a 37.92 cM (16.28 Mb), flanked by markers D8S277 proximally and D8S1734 distally.A total of 125 consanguineous Pakistani families with nonsyndromic cataracts were recruited to participate in a collaborative study between the National Centre of Excellence in Molecular Biology, Lahore, Pakistan, and the National Eye Institute, Bethesda, MD, to identify novel loci associated with congenital cataracts. Institutional Review Board (IRB) approval was obtained from the both Institutes and all participating subjects gave informed consent consistent with the tenets of the Declaration of Helsinki. A detailed medical history was obtained by interviewing family members. Ophthalmic examinations were conducted with slit-lamp microscopy. Approximately 10 ml of blood samples were drawn from affected and unaffected members of the family and stored in 50 ml Sterilin® falcon tubes  containing 400 μl of 0.5 M EDTA. Blood samples were kept at −20 °C for long- term storage.DNA was extracted by a nonorganic method as described previously ,14. Brie2, and 0.2 U Taq DNA polymerase (Applied Biosystems). Initial denaturation was performed for 5 min at 95 °C, followed by 10 cycles of 15 s at 94 °C, 15 s at 55 °C, and 30 s at 72 °C and then 20 cycles of 15 s at 89 °C, 15 s at 55 °C, and 30 s at 72 °C. The final extension was performed for 10 min at 72 °C. PCR products from each DNA sample were pooled and mixed with a loading cocktail containing HD-400 size standards (Applied Biosystems). The resulting PCR products were separated in an ABI 3100 DNA Analyzer (Applied Biosystems) and genotypes were assigned with GeneMapper software (Applied Biosystems).A genome-wide scan was performed with 382 highly polymorphic fluorescent markers from the ABI PRISM Linkage Mapping Set MD-10  having an average spacing of 10 cM. Multiplex polymerase chain reaction (PCR) was completed in a GeneAmp PCR System 9700 thermocycler (Applied Biosystems). Briefly, each reaction was performed in a 5 μl mixture containing 40 ng genomic DNA, various combinations of 10 mM dye-labeled primer pairs, 0.5 ml 10× GeneAmp PCR Buffer (Applied Biosystems), 1 mM dNTP mix, 2.5 mM MgClLINKAGE Program Package, whereas the Maximum LOD scores were calculated with ILINK from the LINKAGE Program Package [Marshfield database and the National Center for Biotechnology Information (NCBI) chromosome 8 sequence maps. For the initial genome scan, equal allele frequencies were assumed, while for fine mapping allele frequencies were estimated from 96 unrelated and unaffected individuals from the Punjab province of Pakistan.Two-point linkage analyses were performed using the FASTLINK version of MLINK from the  Package ,24. AutoA large consanguineous family, PKCC144, consisting of four affected and four unaffected individuals was recruited from the Punjab province of Pakistan . The medInitially, linkage to all reported loci associated with autosomal recessive cataract loci were excluded by haplotype analyses with closely spaced fluorescently-labeled microsatellite markers (data not shown). Subsequently, a genome-wide scan was completed and during the genome-wide scan maximum two-point LOD scores of 3.19, and 3.08 at θ=0 were obtained with markers D8S550 and D8S549, respectively . AdditioVisual inspection of the haplotypes supported the results of linkage analysis . There iHere, we report a new locus for autosomal recessive congenital cataracts localized to chromosome 8p in a consanguineous Pakistani family. The medical records available to us suggest a congenial onset, whereas the slit lamp examination was suggestive of bilateral cataracts. The genome-wide scan localized the critical interval to chromosome 8p, which was further supported by haplotype analyses and places the critical interval in a 37.92 cM (16.28 Mb) region, flanked by markers D8S277 proximally and D8S1734, distally.While the maximum Lod score of 3.19 is only slightly higher than the traditional limiting value of 3.0, it represents the maximum value obtainable with this family with real allele frequencies. In addition, the lack of evidence for linkage during the genome-wide scan, except with markers D8S549 and D8S550 provides additional support for localization of the cataract locus to chromosome 8p. This is the first report associating chromosome 8p with autosomal recessive congenital cataracts and illustrates the heterogeneity of the disease.The critical interval is a gene rich region that harbors more than 100 genes according to the UCSC database. We have prioritized all annotated genes based on their known function and available expression data in the eye, and currently are sequencing each candidate in one affected and one unaffected individual. Identification of the pathogenic mutations that lead to congenital cataracts will increase our understanding of lens biology at a molecular level and will help in development of better treatments and therapeutics.


Studies in healthy volunteers and patients with chronic myelogenous leukemia or gastrointestinal stromal tumors have shown that therapeutic doses of nilotinib deliver drug levels similar to those of imatinib. The aim of this study was to compare the inhibitory effects of imatinib and nilotinib on proliferation, differentiation, adhesion, migration and engraftment capacities of human cord blood CD34+ cells.The availability of tyrosine kinase inhibitors (TKIs) has considerably changed the management of Philadelphia chromosome positive leukemia. The BCR-ABL inhibitor imatinib is also known to inhibit the tyrosine kinase of the stem cell factor receptor, c-Kit. Nilotinib is 30 times more potent than imatinib towards BCR-ABL After a 48-hour cell culture with or without TKIs, CFC, LTC-IC, migration, adhesion and cell cycle analysis were performed. In a second time, the impact of these TKIs on engraftment was assessed in a xenotransplantation model using NOD/SCID/IL-2Rγ (null) mice.in vitro inhibition of CFC formation due to inhibition of CD34+ cell cycle entry. Adhesion of CD34+ cells to retronectin was reduced in the presence of either imatinib or nilotinib but only at high concentrations. Migration through a SDF-1α gradient was not changed by cell culture in the presence of TKIs. Finally, bone marrow cellularity and human chimerism were not affected by daily doses of imatinib and nilotinib in a xenogenic transplantation model. No significant difference was seen between TKIs given the equivalent affinity of imatinib and nilotinib for KIT.TKIs did not affect LTC-IC frequencies despite These data suggest that combining non-myeloablative conditioning regimen with TKIs starting the day of the transplantation could be safe. Bcr-Abl gene. The Ph chromosome translocation is present in 95% and 20–30% of patients with chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL), respectively The Philadelphia (Ph) chromosome results from a reciprocal translocation between chromosomes 9 and 22 resulting in a chimeric in vitro activity in several Ph+ cell lines Imatinib  is a competitive inhibitor of ATP for binding to BCR-ABL + ALL. The majority of older adults with ALL are candidates for a reduced-intensity or a non-myeloablative conditioning regimen in vivoAllogeneic hematopoietic stem cell transplantation  is a potentially curative treatment for patients with Phin vitro, but does not interfere with engraftment of human hematopoietic stem cells in a xenogenic transplantation model in vitro and in vivo, the inhibitory effects of imatinib and nilotinib on proliferation, differentiation and engraftment capacities of human cord blood CD34+ HSCs.However, the impacts of imatinib and nilotinib on HSCs during the post-transplantation period are unknown. Reconstitution of a fully functional hematopoietic system is critical for transplantation outcomes. We have previously shown that imatinib inhibits progenitor cell growth After written informed consent of the mother, cord blood was collected according to the standard procedures of the Cord Blood Bank of the University Hospital of Liège. Mononuclear cells were isolated by centrifugation for 40 minutes at room temperature with Ficoll Paque™ plus density gradient  and washed twice in phosphate-buffered-saline (PBS)  supplemented with 1% Penicillin/Streptomycin (P/S) (Lonza).+ hematopoietic stem cells were isolated by magnetic separation according to the manufacturer’s instructions . First, cells were incubated during 30 minutes at 4°C with a primary anti-CD34 antibody. Cells were washed with PBS+P/S 1% and incubated for 30 minutes at 4°C with a secondary antibody coupled to magnetic beads. Cells were washed in PBS+P/S 1% and passed twice through a MS column (Miltenyi Biotech). CD34+ cells were collected after elution of unlabeled cells through the column. Cells were counted with Trypan Blue, washed in PBS and frozen in Fetal Bovine Serum (FBS)+Dimethylsulfoxide (DMSO) 10% .CD34+ cells was assayed by flow cytometry. A total of 50,000 collected cells were labeled for 30 minutes at 4°C with an allophycoerythrin (APC) conjugated anti-CD34 antibody  or with the isotype-matched control (BD Biosciences). Cells were washed twice with PBS+P/S 1% and resuspended in PBS+Formaldehyde 1%. Data acquisition was carried out on a FACSCanto II flow cytometer (BD Biosciences). In all experiments, the percentage of CD34+ cells in the starting cell population was higher than 95%.The purity of the CD34+ cells were thawed in Iscove’s MDM (IMDM) (Lonza) supplemented with bovine serum albumin, insulin, transferrin (BIT) 20% +P/S 1% and washed with PBS+P/S 1%. Cells were counted with Trypan Blue and resuspended in IMDM+BIT 20%+P/S 1% at a concentration of 10,000,000 cells/mL. A total of 1,000,000 CD34+ cells (100 µL) were seeded in 2.4 mL of IMDM+BIT 20%+P/S 1% supplemented with SCF (100 ng/mL), TPO (50 ng/mL) and FLT-3 (100 ng/mL) . TKIs were added from a stock solution of 10 mM in DMSO to the medium at a final concentration of 1 or 5 µM. Cells were incubated for 48 hours and then collected and lysed. Total proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lysis buffer contained 25 mM Hepes, 150 mM NaCl, 0.5% Triton X-100, 10% glycerol, 1 mM dithiothreitol, phosphatase inhibitors  and complete protease inhibitor mixture . Polyvinylidene fluoride membrane was incubated with 1∶1000 rabbit anti-human phospho-c-Kit . The membrane was then incubated with anti-rabbit horseradish peroxidase antibody at 1∶2000 . Goat anti-human actin conjugated to horseradish peroxidase  was used at 1∶400. Actin signal was used as an internal standard. Finally, the blot was developed using ECL Western Blot detection system .Human CD34+ cells were thawed in Iscove’s MDM (IMDM) (Lonza) supplemented with BIT 20%+P/S 1% and washed with PBS+P/S 1%. Cells were counted with Trypan Blue and resuspended in IMDM+BIT 20%+P/S 1% at a concentration of 50,000 cells/mL. A total of 5,000 CD34+ cells (100 µL) were seeded in 2.4 mL of MethoCult H4100® (Stem Cell Technologies) supplemented with FBS 30%, EPO 3 U/ml, 2-Mercaptoethanol 0.1 mM , L-Glutamine 2 mM (Lonza), P/S 1%, SCF (Stem cell factor) 50 ng/mL  and with conditioned medium of the 5637 cell-line. TKIs were added from a stock solution of 10 mM in DMSO to the medium at a final concentration of 1 or 5 µM and cells were incubated for 14 days at 37°C under a 5% CO2 atmosphere. Colony forming cells (CFCs) were then counted.CD34+ cells were plated in limiting dilution in another 100 µL of LTC medium and maintained at 33°C in a 100% humidified atmosphere containing 5% CO2, with weekly half-medium change. After 6 weeks, medium was carefully aspirated from each well, followed by the addition of 200 µL of fully supplemented MethoCult. After an additional 2 weeks, wells were scored for the presence or absence of hematopoietic colonies, and the frequency of LTC-ICs was calculated using L-calc software (Stem Cell Technologies).Absolute frequencies of LTC-ICs in cell suspensions recovered after a 48-hour cell culture incubation with TKIs at a concentration of 1 or 5 µM, or in control medium, were determined by limiting dilution analysis over MS-5 feeder cells. Briefly, the MS-5 feeder cell line was cultured in RPMI 1640 with 10% FBS. Cells were irradiated at 50 Gy and then plated in 96-well plates at 20,000 cells per well in 100 µL long-term culture (LTC) medium consisting of α-MEM supplemented with 8% horse serum, 8% fetal bovine serum, 0.2 mM glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin , 0.2 mM inositol (Sigma-Aldrich), 0.1 mM 2-mercaptoethanol. Within a week, thawed CD34+ cells were counted with Trypan Blue and resuspended at a concentration of 1×106 cells/mL. A total of 1×105 cells/well (100 µL) was seeded in a 6-well plate, containing 2.4 mL of IMDM supplemented with BIT 20%, P/S 1%, SCF (100 ng/mL), TPO (50 ng/mL) and FLT-3 (100 ng/mL) (PeproTech). TKIs were added from a stock solution of 10 mM in DMSO at final concentrations of 1 µM or 5 µM. Flow cytometric cell cycle analyses of CD34+ cells cultivated during 48 hours with or without TKIs were performed using the CycleTEST™ Plus DNA Reagent Kit (BD Biosciences) as previously reported 2/M cells)/Total cells)×100.Thawed CD34+ cells were counted with Trypan Blue and resuspended at a concentration of 1×106 cells/mL. A total of 1×105 cells/well was seeded in a 6-well plate, containing 2.4 mL of IMDM supplemented with BIT 20%, P/S 1%, SCF (100 ng/mL), TPO (50 ng/mL) and with FLT-3 (100 ng/mL). TKIs were added at final concentrations of 1 µM or 5 µM. After 48 hours of culture, human CD34+ cells were washed twice with PBS+FBS 3%+P/S 1% and were then incubated with FITC-conjugated anti-VLA-4 (BD Biosciences) or FITC-conjugated anti-VLA-5 (BD Biosciences) in combination with PE-conjugated anti-CXCR-4 (BD Biosciences) antibodies for 30 minutes at 4°C in the dark. Cells were then washed twice with PBS+FBS 3% and finally resuspended in pure PBS. Data acquisitions  were performed on a FACSCanto II flow cytometer. Integrin density was expressed as the mean channel fluorescence ratio (MCFR) defined as the mean channel fluorescence (MCF) of CXCR-4 or integrin expression divided by MCF of fluorescence of the unstained control.Thawed CD345 CD34+ cells were plated in 100 µL of IMDM+BIT 20%+P/S 1%+SCF (100 ng/mL) in the upper chamber of the transwell. The bottom compartment was filled with IMDM supplemented with 20% BIT and 100 ng/mL stromal-derived factor-1 alpha (SDF-1α) (PeproTech). After incubation at 37°C during 4 hours, non-migrating and migrating cells were harvested by two standardized washes using PBS+FBS 3%+P/S 1%. Non-migrating and migrating cells were counted by flow cytometry using Trucount Tubes (BD Biosciences) after staining with an APC-conjugated anti-CD34 antibody. The percentage of non-migrating and migrating cells was calculated relative to the total number of harvested cells.Migration assays were performed in 6.5 mm diameter 5 µm pore transwells. A total of 1×10+ cells were counted with Trypan Blue and resuspended at a concentration of 1×106 cells/mL. An aliquot of 1.5×105 cells/well were seeded in a 12-well plate containing 1 mL IMDM supplemented with BIT 20%, P/S 1%, SCF (100 ng/mL), TPO (50 ng/mL) and FLT-3 (100 ng/mL). TKIs were added from a stock solution of 10 mM in DMSO at final concentrations of 1 µM or 5 µM. After 48 hours of culture, human CD34+ cells were washed twice with PBS+P/S 1%.Thawed CD342 during two hours at 37°C. Supernatant was aspirated and wells were then incubated with PBS+BSA 1%+P/S 1% for 30 minutes at room temperature. Wells were finally washed twice with PBS+Hepes 2%+P/S 1%.Adhesion assays were performed in a 12-well plate. Wells were first coated with retronectin  at a concentration of 9 µg/cm+ cells resuspended in IMDM+BIT 20%+P/S 1% were added in each well and incubated for 90 minutes at 37°C. Supernatants were collected in polypropylene tubes. Adherent cells were detached by using the non-enzymatic cell dissociation buffer (Sigma) and collected in new polypropylene tubes. Cells were finally stained with an APC-conjugated anti-CD34 antibody and counted by flow cytometry using Trucount Tubes. The percentage of adherent cells was calculated relative to the total number of harvested cells.A total of 150,000 CD34+ cell injection, NOD/SCID/IL-2Rγ (null) (NSG) mice  were irradiated with 2.5 Gy TBI using a 137Cs source. Human CD34+ cells were thawed in IMDM+FBS 10%+P/S 1% and washed in PBS+P/S 1%. Cells were counted with Trypan Blue and resuspended in PBS at a concentration of 3×106 cells/mL (6×105 cells/200 µL). Mice were inoculated intravenously with 6×105 CD34+ cells. Gavage with TKIs or a placebo was started at day 0. Imatinib was dissolved in sterile water and administrated at a dose of 150 mg/kg/day (50 mg/kg every morning and 100 mg/kg every evening) while nilotinib was prepared in 0.5% hydroxypropylmethyl cellulose  aqueous solution containing 0.05% Tween 80 and given at a concentration of 75 mg/kg/day (37.5 mg/kg every morning and evening). After 42 days, mice were sacrificed. Bone marrow cells from the two femurs were collected in sterile RPMI+FBS 10%+P/S 1%. Cells were counted with an HORIBA ABX® automatic cell counter . Cells were stained with anti-human CD45 (BD Biosciences) and anti-mouse CD45 (BD Biosciences) antibodies in order to determine the percentage of human chimerism by FACS analysis. Data acquisition was performed on a FACSCanto II flow cytometer on at least 20,000 mononuclear cells.Six hours before CD34ad libitum. Sterilized water supplemented with Baytril® 1%  was given from 3 days before to the end of the experiment. Water was change every 2–3 days. All euthanasia were performed under isoflurane anesthesia, and all efforts were made to minimize suffering.All experiments using NSG mice were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Liège (Permit Number: 712). Mice were maintained in top-filtered cages in a standard animal facility and provided sterilized food and water + cells in vitro. Percentages and numbers of human cells in NSG mice were compared with the unpaired Student’s T test.Statistical analyses were performed with the GraphPad® Prism 5.00 Software. The paired Student’s T test was used to assess the impact of TKI on human cord blood CD34+ cord blood HSCs were cultured in cytokine-supplemented medium for 48 hours with or without TKIs, washed and then lysed. Proteins were extracted on ice and dosed for western blotting analysis. The c-Kit phosphorylation levels in human CD34+ cells were decreased in a dose-dependent manner. Indeed at the highest concentration, both imatinib and nilotinib decreased dramatically the band intensity of phospho-c-Kit. No differences were seen between imatinib and nilotinib at a concentration of 1 µM and 5 µM . Human CD34and 5 µM Figure 1+ cells were cultured for 48 hours with or without TKIs, washed and then plated in cytokine-supplemented MethoCult for 14 days. Both imatinib and nilotinib significantly inhibited CFC formation. Indeed, imatinib, at a concentration of 1 or 5 µM, decreased CFC formation by a mean ± SD of 24.91±14.05% (p = 0.0415) and 49.66±30.19% (p = 0.0461) respectively, while nilotinib, at the same concentrations, reduced CFC numbers by 25.15±12.36% (p = 0.0268 and p = 0.8173 in comparison to imatinib) and 54.81±34.39% (p = 0.0498 and p = 0.1314 in comparison to imatinib), respectively  assays were carried out. In a first set of experiments (n = 4), CD34+ cells were seeded in MethoCult supplemented with or without imatinib or nilotinib at a final concentration of 1 or 5 µM for 14 days. As observed in the first series of experiments, TKIs significantly diminished CFC generation. Imatinib reduced colony formation by 41.67±14% (p = 0.0356) at a concentration of 1 µM and 79.42±9.309% (p = 0.0045) at 5 µM. Nilotinib decreased CFC formation by 19.64±1.90% (p = 0.0031 and p = 0.0898 in comparison to imatinib) and 78.28±5.27% (p = 0.0015 and p = 0.6733 in comparison to imatinib), respectively , human cord blood CD34+ cells were directly plated for LTC-IC assays and incubated in the presence/absence of TKIs at a final concentration of 1 or 5 µM. Inhibitors were also added at all weekly half-medium change. As observed above, neither imatinib nor nilotinib decreased absolute frequencies of LTC-ICs (In a second set of experiments (n = 3), CD34+ cell proliferation. HSC proliferation was markedly reduced in the presence of imatinib 1 µM  or nilotinib 1 µM , and even more with the presence of 5 µM of imatinib (p = 0.005) or nilotinib (p = 0.008 and p = 0.6746 in comparison to imatinib): HSC proliferation was decreased by 78.25±9.624% and 63.49±12.40% respectively (n = 3) (We assessed the impact of a 48-hour culture in the presence of imatinib or nilotinib on cord blood CD34 (n = 3) Figure 4+ cells was determined by flow cytometry after a cell culture containing TKIs (or not). Imatinib significantly decreased expression of VLA-4 by a mean ± SD of 10.457±3.058 (p = 0.0032) and 12.520±1.872 (p<0.0001) at a concentration of 1 and 5 µM, respectively. Nilotinib at the same concentration induced the same effect by decreasing MCFR values by a mean ± SD of 8.920±3.472 (p = 0.0088) and 11.247±2.336 (p = 0.0008), respectively (n = 3)  and 1.724±0.4219 (p = 0.0121) for imatinib at 1 and 5 µM, respectively. Nilotinib decreased VLA-5 MCFR values by a mean ± SD of 1.724±0.6833 (p = 0.0733) and 1.424±0.3931 (p = 0.0135) at the same concentration respectively (n = 3)   while imatinib 5 µM decreased it by 5.69±4.134% (p = 0.0135) (n = 10). Similarly, nilotinib had no significant effect on adhesion at 1 µM, while at 5 µM it significantly reduced adhesion by 3.51±5.933% was observed (p = 0.0432) (n = 10)   5.66±4.14×106 in control mice, 3.55±1.67×106 in mice treated with imatinib (p = 0.1982) and 4.17±2.74×106 in nilotinib-treated mice (p = 0.3731) (p = 0.4130), while engraftment of human CD34+ cells was slightly decreased  in mice treated with nilotinib  were injected intravenously with 6×10+ ALL, a 2–4 week TKI discontinuation may expose patients to early relapses. These data prompted us to evaluate the inhibitory effects of imatinib and nilotinib on the proliferation, differentiation and engraftment capacities of human cord blood CD34+ HSC.The aim of this project was to assess the impact of TKIs on hematopoietic stem cell function engraftment. Retrospective studies have suggested that treatment with TKIs before allo-HSCT did not preclude the outcome of engraftment and did not increase transplant-related toxicity The effects of TKIs were first assayed on LTC-ICs by limiting dilutions. Our data show that neither imatinib nor nilotinib had a significant impact on primitive progenitors. Indeed, as described before, LTC-IC frequencies were not affected by a 48 hour pre-culture or a 6-week incubation with TKIs + cells in the presence of TKIs. Our data demonstrate a significant decrease in HSC proliferation. These observations could be explain by the inhibition of others tyrosine kinases by imatinib and nilotinib. Indeed, Bartolovic et al. have shown that imatinib exerts growth inhibitory effect on normal CD34+ cells by the inhibition of SCF/c-kit pathway + patients receiving nilotinib for treatment of CML have demonstrated that nilotinib potently inhibited osteoblast proliferation through inhibition of the platelet-derived growth factor (PDGFR). Furthermore, inhibition of c-Abl could contribute to the growth inhibition of CFCs by TKIs since antisense strategies have demonstrated that inhibition of c-Abl leads to the accumulation of CD34+ cells in G0/G1 and to inhibition of CFU-GM formation + CML cells is anti-proliferative rather than pro apoptotic. Indeed, the anti-proliferative effect of TKIs on Ph+ CD34+ cells is mainly caused by the inhibition of BCR-ABL We also evaluated the effects of TKIs on CFCs. Unlike LTC-IC frequencies, CFC growth was significantly decreased in culture containing either imatinib or nilotinib. Moreover, their inhibitory effects were permanent since a 48-hour pre-culture with TKIs is sufficient to decrease significantly the CFC generation definitively. Two hypotheses could explain these observations: (a) increased apoptosis or (b) inhibition of cell cycle entry. Since we and others have previously showed that imatinib do not increase the apoptosis of committed progenitors + cells was not modified by imatinib + cells were seen. These apparent discrepancies could be explained by the cell source since in our previous publication + cells isolated from peripheral blood of mobilized healthy volunteers were investigated while, in this study, CD34+ cells from cord blood were used in all experiments. Indeed, despite a higher VLA-4 and VLA-5 expression, cord blood CD34+ cells exhibit a lower CXCR-4 cell surface expression and a higher capacity to regenerate LTC-IC per competitive repopulating unit (CRU) +. We then tested whether the decreased expression of VLA-4 and VLA-5 affected the capacity of CD34+ cells to adhere to retronectin in vitro. Adhesion was not affected by imatinib or nilotinib at physiological concentrations (1 µM) but decreased at higher doses (5 µM). CD34+ cell migration towards a SDF-1α gradient was not affected by TKIs. Inverse relationships between migration and adhesion capacities have often been observed. In HSCs, higher cell cycle activity is related with stronger adherence and decreased motility + cell proliferation, their effect on hematopoietic cell adhesion and migration appears to be independent of cell cycle activity. Additional studies will be necessary to investigate the impact of imatinib or nilotinib on tyrosine kinases implicated in adhesion and migration, such as the focal adhesion kinase or the related kinase PYK2 that is expressed in CD34+ cells Because VLA-4, VLA-5, and CXCR-4 play a major role in the homing of HSCs, we investigated the effect of TKIs on the expression of these surface receptors by flow cytometry. Despite our previous findings that the expression of VLA-4, VLA-5, and CXCR-4 of CD133+ cells between mice treated with imatinib or placebo. However, the engraftment of human HSCs was slightly decreased in mice treated with nilotinib. This might be explained by the high daily dose of nilotinib (75 mg/kg/day) used in these experiments. Our results are comparable to those of our previous study Finally, we assessed the impact of TKIs on engraftment in a xenotransplantation model. Numbers of cells in the bone marrow of the femurs were similar in mice treated with placebo, imatinib or nilotinib. Moreover, no significant differences were seen in the percentages of bone marrow human CD45+ ALL patients in order to maximize the graft-versus-leukemia effect could be possible with both nilotinib and imatinib.On the basis of our data, combining non-myeloablative conditioning with TKIs for Ph


Copeptin, a marker for stress mirroring vasopressin concentrations, has been shown to increase upon insulin-induced hypoglycaemia in patients after transsphenoidal surgery of pituitary adenomas. Patients with type 1 diabetes mellitus are prone to hypoglycaemia, but no data about copeptin levels upon hypoglycaemia are available. Furthermore, the perception of hypoglycaemia can vary from total unawareness to disabling episodes. The aim of this study was to investigate whether copeptin increases upon hypoglycaemia in patients with type 1 diabetes mellitus and is associated with the degree of hypoglycaemia awareness.In this prospective observational study, 17 patients with type 1 diabetes underwent a standardized insulin infusion test. Blood sampling for glucose and copeptin was performed at baseline and after 60 minutes (min). To assess hypoglycaemia associated symptoms the Mood and Symptom Questionnaire (MSQ) was conducted at baseline and after 60 min.During insulin infusion, blood glucose decreased from 5.1 (SD±0.2) to 3.0 (±0.5) mmol/L at 60 min (p<0.001). Copeptin concentrations increased from 3.2 (±1.7) to 3.8 (±1.9) pmol/L (p = 0.03). Mood and Symptoms Questionnaire scores increased from 14 (±3.0) to 18 (±5.8), (p = 0.006). Patients with good hypoglycaemia awareness had an increase in copeptin from 3.0 (±1.8) to 4.2 (±2.4) pmol/L (p = 0.03) in contrast to patients more unaware of hypoglycaemia who only showed an increase in copeptin from 3.3 (±1.6) to 3.6 (±1.4) pmol/L (p = 0.4). There was a trend to a larger copeptin increase in patients aware of hypoglycemia compared to patients unaware of hypoglycemia (p = 0.074).Copeptin increases in patients with type 1 diabetes upon insulin induced hypoglycaemia. Interestingly, the copeptin increase seems associated with the degree of hypoglycaemia awareness. This hypothesis warrants further verification.NCT00515801ClinicalTrials.gov  Hypoglycaemia is an acute complication of insulin therapy. Patients with type 1 diabetes are particularly prone to develop hypoglycaemia. Physiologically, a strong stress reaction is activated during hypoglycaemia to counteract the hypoglycaemic state. In healthy subjects this stress reaction consists of a down-regulation of insulin secretion, followed by the secretion of hormones such as glucagon, epinephrine, growth hormone, adrenocorticotropin and vasopressin In type 1 diabetes these neuroendocrine response mechanisms are often compromised and perception of hypoglycaemia is impaired. The secretion of insulin and glucagon deteriorates and the response of epinephrine is often delayed or blunted The objective of this proof of concept study was to analyse the response of copeptin to hypoglycaemia in type 1 diabetes. Furthermore, we evaluated the association between copeptin response and hypoglycaemia awareness.The protocol for this trial and supporting CONSORT checklist are available as supporting information; see In this prospective observational study, a total of 17 patients with diabetes mellitus type 1 were included. Inclusion criteria were age 18 to 50 years and stable metabolic control .The study protocol was approved by the institutional review board (Human Ethics Committee of Basel). Written informed consent was obtained from all participating patients. The study took place at the University Hospital Basel.2/min was administered for the duration of 60 min. Blood samples for glucose and copeptin measurements were obtained at baseline and after 60 min. Plasma glucose levels were assessed using an automated glucose analyser . Copeptin, a secondary endpoint of the study, was measured using the CT-pro AVP LIA  as described elsewhere All patients were admitted the night before beginning of the insulin infusion study. They were instructed to omit their basal insulin. Overnight blood glucose levels were adjusted between 5 and 8 mmol/l with a continuous intravenous insulin infusion with human insulin. Patients remained fasting until the end of the study. The next morning blood glucose levels were stabilized at 5 mmol/l one hour before the onset of the intravenous insulin infusion test. Thereafter the intravenous insulin infusion study was started. A standardized infusion with human insulin 30 mU/mContinuous variables are presented as mean (±standard deviation). Comparisons within subjects (at baseline and 60 minutes) were analysed using the paired t-test. Comparisons across subjects (unaware versus aware diabetics) were analysed using the unpaired t-test. The Kolmogorov-Smirnov test was applied to test normal distribution. Univariate and multivariate logistic regression models were used to analyse variables influencing copeptin increase. All tests were two tailed; p<0.05 was defined as significant. Data were analysed using statistical software .17 patients with diabetes mellitus type 1 , mean age 39.6 years (standard deviation±10.1), mean diabetes duration 13.1 years (±12.5) and mean HbA1c level 7.3% (±1.1) were included in the study .During insulin infusion, blood glucose levels decreased from a mean of 5.1 (±0.2) at baseline to 3.0 (±0.5) mmol/L at 60 min (p<0.001). The mean copeptin levels increased from 3.2 (±1.7) to 3.8 (±1.9) pmol/L (p = 0.03), whereas mean MSQ scores increased from 14 (±3.0) to 18 (±5.8) points (p = 0.006). Patients with poor hypoglycemia awareness, had an increase in copeptin levels from 3.3 (±1.6) to 3.6 (±1.4) pmol/L (p = 0.4), whereas patients with good hypoglycemia awareness had an increase from 3.0 (±1.8) to 4.2 (±2.4) pmol/L (p = 0.03) . CompariTo our knowledge this is the first study investigating copeptin in patients with type 1 diabetes mellitus upon insulin induced hypoglycaemia. Herein, we demonstrate a copeptin increase during hypoglycemia in type 1 diabetics. Interestingly, this finding was restricted to patients with good hypoglycaemia awareness.It is well known that hypoglycaemia activates the hypothalamic pituitary axis. Among many neuronal and humoral responses, neurons of the supraoptic and paraventricular nuclei are activated, subsequently releasing vasopressin In our study of patients with type 1 diabetes we were able to illustrate an increase of copeptin during hypoglycaemia. These findings agree with the results of previous studies Summarized, copeptin increases in patients with type 1 diabetes upon insulin induced hypoglycaemia. Furthermore, vasopressin regulation may be associated with hypoglycaemia awareness in type 1 diabetics. Importantly, due to the small sample size this hypothesis requires further verification in a larger population.Checklist S1CONSORT Checklist.(DOC)Click here for additional data file.Protocol S1Trial Protocol.(DOC)Click here for additional data file.


DACT1 gene promoter and lower mRNA expression in bladder urothelial carcinoma tissue. The methylation status of 29 urothelial carcinoma samples and 29 normal tissue samples were examined by methylation-specific polymerase chain reaction (MSP). The DACT1 mRNA transcript levels and DACT1 protein levels in all samples were then evaluated to define the relationship between the methylation status of the DACT1 promoter and its expression at the transcriptional and translational levels. Decreased expression of DACT1 was detected in 89.66% of urothelial carcinomas . Promoter hypermethylation was found in 58.62% (17/29) urothelial carcinomas and 25% (7/29) normal tissues, respectively (P < 0.05). DACT1 expression was lower in tissues where the DACT1 gene promoter was hypermethylated than in unmethylated tissues . DACT1 gene hypermethylation was closely related to tumor size, grade and stage (P < 0.05). Our results indicate that silencing and downregulation of DACT1 mRNA may be implicated in carcinogenesis and the progression of bladder urothelial carcinoma, and may be a potential prognostic factor.The purpose of this study was to determine the relationship between hypermethylation of  Bladder cancer is the sixth most common cancer in the worldAberrant DNA methylation is now considered to be the most important epigenetic alteration in many cancersDACT1 gene was initially found as a signal regulation molecule in the clawed frogDACT gene is well conserved at the genomic level during evolution. The DACT1 gene maps to chromosome 14, at 14q23.1. Many studies have demonstrated that DACT1 is a key mediator in the negative regulation of the Wnt/β-catenin signaling pathway, whereas abnormal activation of this signaling pathway participates in the process of cancer progressionDACT gene in the pathogenesis of cancer. Several studies have reported aberrant expression of a number of DACT genes in cancers. The examples include DACT1 in stomach neoplasms, liver cancer and germ cell tumor, and DACT2 in endometrial adenocarcinomaThe DACT1 gene was examined in tissue from 29 normal bladders and tissue from 29 bladder urothelial carcinomas. We also examined the expression level of DACT1 mRNA and protein in all samples. Using methylation-specific polymerase chain reaction (MSP) and reverse transcription polymerase chain reaction (RT-PCR), a clear relationship was found between hypermethylation status and the low expression of DACT1 in bladder transitional cell carcinomas. DACT1 was hypermethylated in almost all human bladder cancers but unmethylated in normal tissues. We hypothesized that these changes could be of diagnostic value for the detection of bladder cancer and decided to measure the degree of DNA methylation in DACT1 in bladder transitional cell carcinoma tissues.In this study, the methylation status of the We obtained frozen tissue samples of 29 bladder carcinomas and 29 normal bladder tissues from the First Affiliated Hospital of Nanjing Medical University. The study was approved by the Institutional Human Investigations Committee. All patients signed the informed consent. The tumors were verified as transitional cell carcinomas by two experienced pathologists according to the WHO criteriaBladder cancer tissues and normal bladder tissues were obtained after surgical resection and snap-frozen and stored at -70°C. Genomic DNA was isolated by using a Promega Wizard DNA isolation kit. The tissues were incubated at 55°C in a homogenization buffer containing 50 mmol/L Tris (pH 8.0), 1 mmol/L EDTA, 0.5% Tween 20, and 5 mg/mL proteinase K for 3 h prior to genomic DNA isolation.DACT1 amplification were 5′-GCGAAGAGATGCTGGTITGT-3′ (sense) and 5′-AAATTGTGGTGTGGAGAGGG-3′ (antisense). The PCR included 2 min of denaturation at 95°C followed by 35 cycles of 30 s at 94°C, 30 s at 55°C and 30 s at 72°C, and a final extension for 7 min at 72°C. Primer sequences for β-actin amplification were 5′-GTGGGGCGCCCCAGGCACCA-3′ (sense); antisense: 5′-CTCCTTAATGTCACGCACGATTTC-3′ (antisense). A BLAST search confirmed that these primer sequences were identical to the endogenous human target genes. The PCR products were resolved by electrophoresis in a 1.5% agarose gel and stained with ethidium bromide.Total RNA was extracted with Trizol reagent , and cDNA was synthesized from total RNA with oligo-dT primer  following the manufacturer's instructions. Primer sequences for DACT1 for methylated reaction were as follows: 5′-ACTACTAATCAAAAACGCCCTACG-3′; (sense) and 5′-AATAGTCGTGTTTTATITTCGGGTAC-3′ (antisense). The sequences for the unmethylated reaction were 5′-AAAACTACTAATCAAAAACACCCTACAC-3′ (sense) and 5′-AT AGTTGTGTTTTATTTTTGGGTATGA-3′ (antisense).Genomic DNA was obtained by digestion with proteinase K , followed by phenol/chloroform (1:1) extraction. Briefly, 4 ng genomic DNA was denatured by NaOH and modified by sodium bisulfite. The DNA was purified using a Wizard DNA purification kit (Promega), desulfonated with NaOH, precipitated with ethanol, and resuspended in water. PCR was performed with bisulfite-treated DNA as a template using specific primer sequence for methylated and unmethylated forms of the genes. The primer sequences of 2+), 2 µL dNTP, 1 µL each PCR primer, 0.2 µL AmpliTaq polymerase, 2 µL bisulfite-modified DNA, and 16.8 µL diethyl-pyrocarbonate-treated water. The reaction was started by heating the samples at 95°C for 12 min followed by 35 cycles at 94°C for 30 s, 55°C (methylated) or 53°C (unmethylated) for 30 s, and 72°C for 30 s, followed by a final extension at 72°C for 7 min. The amplification products were separated on a 1.5% agarose gel and visualized by UV illumination. The results were confirmed by repeating bisulfite treatment and methylation-specific PCR for all samples.Step-down PCR was performed in 20-µL reaction buffer containing 10×loading buffer (including Mg2+ plus), dNTP mixture (2.5 µmol/L), forward primer (20 µmol/L), reverse primer (20 µmol/L), 2 µL cDNA template, and 20× Eva Green (1 µL). PCR primer sets were as follows: β-actin: sense 5′-GTGGGGCGCCCCAGGCACCA-3′ and antisense: 5′-CTCCTTAATGTCACGCACGATTTC-3′; DACT1: sense:5′-GCGAAGAGATGCTGGTITGT-3′ and antisense: 5′-AAATTGTGGTGTGGAGAGGG-3′. The PCR was run at 50°C for 2 min, 95°C for 10 min, 95°C for 15 s, and 60°C for 1 min. Melting curve analysis was used to monitor the accumulation of the PCR products. β-actin mRNA was used as internal control to standardize the results of different mRNA expression levels. Triplicate Ct values were analyzed in Microsoft Excel using the comparative CT (▵▵CT) method as described by the manufacturer . The results were normalized against the β-actin control.Real-time quantitative PCR analysis was performed on cDNAs, using the ABI7300 instrument. PCR samples were prepared in a final volume of 20 µL containing TagHS (5 U/µL), 10× buffer (MgTissue samples were homogenized in ice-cold lysis buffer (50 mmol/L Tris (pH 8.0), 100 mmol/L NaCl, 0.1% SDS, 1% NP-40, 0.5 mmol/L EDTA) containing a complete protease inhibitor cocktail . Proteins (20 µg) were boiled for 5 min and separated with 15% SDS-PAGE, and then transferred onto a nitrocellulose membrane , blocked in 5% skim milk in phosphate buffered saline (PBS), and probed with a goat-anti-human DACT1 antibody , followed by incubation with an anti-rabbit IgG secondary antibody.2 or Fisher's exact test. A Student's t-test was used for statistical comparison in RT-PCR analysis. P < 0.05 was considered statistically significant.All statistical analyses were performed by SPSS version 13.0 for Windows . The difference between unmethylated and hypermethylated tissues was assessed by χDACT1 gene was mostly methylated in bladder cancer tissues (DACT1 gene in 17 out of 29 (58.62%) cancer tissue specimens and in 7 out of 29 (25%) normal tissues was hypermethylated (P < 0.05) .DACT1 gene hypermethylation was closely associated with tumor size, grade and stage (P < 0.05), but not gender and age  than benign tumors , and virtually undetectable in healthy volunteers (0%) (P < 0.0001). Hypermethylation of tumor suppressor genes SFRP1 and SOCS-1 correlates with bladder carcinogenesis and developmentIn the United States, there were approximately 68,810 newly diagnosed bladder cancer cases and 14,100 deaths were expected in 2008DACT1 mRNA transcripts and protein. Hypermethylation was detected in bladder carcinomas by methylation specific PCR. The mono-alleles were detected in most bladder cancers. The DACT1 gene was hypermethylated in 58.62% (17 out of 29) urothelial carcinomas, and only 25% (7 of 29) in healthy bladder tissue (P < 0.05). These results indicate that the methylated type, which might occur either randomly or in mono-allelic manner, can be found in bladder cancer and methylation alone could repress DACT1 expression in bladder cancer. Although some cases  showed a hypomethylation status , the DACT1 expression levels proved different by RT-PCR assays. Based on these findings, we hypothesize that the ratio of unmethylated DACT1 is higher, and the expression level of DACT1 may increase. In other words, the methylation status in some genes determines their expression level in tissues. Western blot analysis showed that DACT1 expression is lower in bladder cancer and the trend was the same as the hypermethylation status of the DACT1 gene. Additionally, we analyzed the correlation between methylation status of the DACT1 gene and clinicopathological features. The results suggest that increasing DACT1 methylation is correlated with tumor growth and progression. We also detected methylation in some normal bladder tissue samples, suggesting that the presence of other mechanisms, such as histone modification, may also regulate the expression of DACT1. Together with the fact that methylation of the DACT1 CpG island is common in bladder cancer, these findings indicate that DACT1 hypermethylation is related to the progression of bladder transitional cell carcinomas. Our data also suggest that other molecular mechanisms contribute to lower DACT1 expression in the tumor.In the present study, we first examined the hypermethylation status of DACT1 in normal and bladder cancer tissue, as well as the levels of Paradoxical aberration of DNA methylation pattern is a hallmark of cancer. With a global loss of DNA methylation that coexists with regional hypermethylation of certain genesIn conclusion, our study showed that hypermthylation could regulate DACT1 expression in bladder transitional cell carcinomas. From a clinical point of view, DACT1 could potentially become a target for intervention. Better understanding of the mechanism of DACT1 expression may eventually lead to novel treatment for bladder transitional cell carcinomas.


Escherichia coli O157:H7 and Salmonella. The objective of this study was to examine the effects in vitro that varying concentrations of CBP in the powdered or pelleted variety have on the survival of Shiga-toxin Escherichia coli (STEC) serotypes O26:H11, O103:H8, O111:H8, O145:H28, and O157:H7 in bovine ruminal microorganism media. The O26:H11, O111:H8, O145:H28, and O157:H7 serotypes did not exhibit a change in populations in media supplemented with CBP with either variety. The O103:H8 serotype displayed a general trend for an approximate 1log10 reduction in 5% powdered CBP and 20% pelleted CBP over 6 h. There was a trend for reductions in populations of a variant form of O157:H7 mutated in the stx1 and stx2 genes in higher concentrations of CBP. These results suggest that variations exist in the survival of these serotypes of STEC within mixed ruminal microorganism fluid media when supplemented with CBP. Further research is needed to determine why CBPs affect STEC serotypes differently.Citrus byproducts (CBPs) are utilized as a low cost nutritional supplement to the diets of cattle and have been suggested to inhibit the growth of both  Escherichia coli (STEC) is capable of naturally colonizing within the gastrointestinal tract of cattle without causing illness [E. coli O157:H7. Due to increased surveillance and pre- and post-harvest intervention, the occurrence of O157:H7 infections in the United States has been reduced to ≤1 case per 100,000 people. However, there now appears to be an increase in the occurrence of foodborne outbreaks due to non-O157 STEC serotypes. According to the Center for Disease Control (CDC) an estimated 265,000 cases of STEC infections were reported a year; of these, approximately 67% are attributed to non-O157 STEC [Shiga-toxin producing  illness . Human c illness , 3. The 157 STEC . With instx1stx2 in bovine ruminal microorganism media.The production of citrus for various food and nonfood products generates byproducts, such as the pulp and peel from citrus fruit. These citrus byproducts (CBPs) have been utilized by dairy and beef cattle producers in some regions of the United States as an inexpensive nutritionally dense feed source . The inc2PO4, 6.0 g KH2PO4, 12.0 g (NH4)2SO4, 12.0 g NaCl, 2.5 g MgSO4·7H2O, 1.6 g CaCl2·2H2O, 0.04 g cysteine HCl; base medium was sterilized by autoclaving [2CO3 and 333 mL of ruminal fluid were added and homogenized by mixing. The pH was adjusted to 6.5 with a 1 M NaOH solution and bubbled with CO2. The fully prepared medium was incubated in an orbital shaker incubator at 140 rpm at 37°C for 12 h.Ruminal contents (1 L) were collected from the rumen ventral sac of a 544 kg cannulated steer at the Henry Leveck Animal Research Center at Mississippi State University. Rumen particles were separated from the ruminal fluid by passing contents through nylon paint strainers as previously described by others . After soclaving . To the E. coli included in this study were purchased from the American Type Culture Collection (ATCC): O157:H7 (ATCC 43895), O103:H8 (ATCC 23982), O145:H28 (BAA-2129), O26:H11 (BAA-1653), O111:H8 (BAA-179), and O157:H7 Δstx1stx2 (ATCC 43888). All E. coli strains were routinely cultured in Luria-Bertani medium  at 37°C. All strains were transformed with the bioluminescent pXEN-13 plasmid  as a means for identification after incubation. For transformation with the pXEN-13 plasmid, all strains were made competent by washing midlog cultures four times with ice cold 10% glycerol. Competent cells were then transformed with pXEN-13 by electroporation and cultured in LB supplemented with 100 μg/mL of ampicillin (AMP) using standard techniques [ Six strains of 600 = 0.05), after which cultures were pelleted, residual medium was removed, and cells were resuspended in 5 mL of mixed ruminal microorganism fluid medium supplemented with 0, 5, 10, or 20% CBP (w/v). Cultures were incubated at 37°C in a shaker incubator for 6 h. Aliquots (0.1 mL) were removed at 0, 2, 4, and 6 h after incubation in ruminal medium, serially diluted in 1X phosphate buffered saline (PBS), plated onto LB agar supplemented with AMP, and incubated overnight at 37°C. The pH values were measured from each strain at each time interval at the various CBP concentrations recorded.Isolates from fresh streaks of each strain transformed with pXEN-13 were used to inoculate a 5 mL starter culture of LB broth supplemented with AMP for 16 h at 37°C. Cultures were then diluted 1 : 100 in 5 mL LB broth and allowed to grow to mid-log phase . Experimental unit was defined as tube, and significance was declared at stx1stx2) suggested that all serotypes were capable of growing within mixed ruminal microorganism fluid media; however decreased populations of serotypes O103:H8 and O145:H28 were observed after 24 h in comparison to O157:H7 [in vitro. A study recently conducted by our group using the serotypes analyzed in this study  was analyzed within mixed ruminal microorganism fluid medium supplemented with powdered CBP (P < 0.11) within the powdered CBP, with an exception of a decrease in O26:H11 populations (P < 0.006) at 4 h in the presence of 20% powdered CBP. The O103:H8 serotype exhibited approximately a 1log10 reduction in populations over the 6 h study in the presence of 5% powdered CBP. The O157:H7 Δstx1stx2 and O157:H7 serotypes had decreased populations  in comparison to the other serotypes at 0 h. Although both O157 serotypes tended to grow similarly (P < 0.06) for 4 h, there was a difference observed at 6 h when O157:H7 Δstx1stx2 had significantly lower populations (P < 0.03) in comparison to O157:H7. When supplemented with 10% powdered CBP, the O157:H7 Δstx1stx2 strains displayed approximately a 1.5log10 reduction in populations, and at 20% powdered CBP there was approximately a 5log10 reduction in populations over 6 h.The growth of the various non-O157 STEC (logered CBP . The O26P < 0.07) in populations of O145:H28 from 0 h to 6 h. While O157:H7 had decreased populations (P < 0.02) at 0 h, there were no differences in populations observed between the O103:H8 and O157:H7 (P < 0.11) the remainder of the study. Populations of O103:H8 were decreased (P < 0.02) at 0 h, while populations tended to be similar (P < 0.06) to O111:H8 during the 6 h analyzed in this study. When O103:H8 was cultured in the mixed ruminal microorganism medium supplemented with 20% pelleted CBP, there was a general trend for approximately a 1log10 reduction observed over the 6 h study. The O26:H11 serotype populations decreased (P < 0.05) throughout the study. Escherichia coli O157:H7 Δstx1stx2 exhibited the lowest populations (P < 0.05) throughout the study. Variations were also observed in certain STEC serotypes when grown in the presence of mixed ruminal microorganism fluid medium supplemented with pelleted CBP . The pelstx1stx2 had reduced populations in mixed rumen microorganism fluid medium supplemented with powdered CBP, while O103:H8 had decreased populations within both varieties of CBP. These results are in accordance with previous studies that have suggested a decrease in O157:H7 populations using other varieties of CBP. A study conducted by Callaway et al. supplemented mixed ruminal microorganism fluid media with 0%, 0.5%, 1%, and 2% dried orange pulp and E. coli O157:H7 populations decreased according to increasing concentrations [E. coli O157:H7 populations have also been observed when sheep rations were supplemented with 5% or 10% orange peel [The O157:H7 Δtrations . Reductinge peel .stx1stx2 populations was observed, other STEC populations were not affected. In the presence of CBP in either pelleted or powdered form, the pH for all strains was reduced from ~5.0 to ~4.0, while in control groups the pH increased from ~6.6 to 7.3. Therefore, the reductions in populations must be attributed to the CBP, as the pH variations were consistent between all strains. Given that this study was only conducted for 6 h, the effects of CBP within the mixed ruminal microorganism fluid media and STEC serotypes may not had been fully observed within the short time frame. This is potentially due to the diffusion properties of CBP across the cell envelope of E. coli. Others studies indicate that CBPs decrease E. coli O157:H7 populations from 24 h to 72 h [The CBP was added to the mixed ruminal microorganism fluid media 2 h before the serotypes were added to the mixture (0 h). Although a decrease in O103:H8 and O157:H7 Δ to 72 h . This stE. coli O157:H7 populations. Although our research has reported a decrease in pH values with increasing CBP concentrations and an observed decrease in O103:H8 and O157:H7 Δstx1stx2 populations, this same trend was not observed within other STEC serotypes. Further research is needed to determine how the various STEC serotypes affect E. coli populations within mixed ruminal microorganism fluid media when supplemented with CBP.The essential oils within the CBP can permeabilize the bacterial cells walls and cytoplasm, leading to bacterial lysis, thus shifting the rumen environment leading to an increase in short-chain fatty acids while decreasing the pH. The acidic environment creates less favorable conditions for microbial populations to survive and replicate within, thus decreasing the possibility of


Burkholderia cepacia complex (BCC) is a group of related bacterial species that are commonly isolated from environmental samples. Members of the BCC can cause respiratory infections in cystic fibrosis patients and immunocompromised individuals. We report here the genome sequence of Burkholderia cenocepacia H111, a well-studied model strain of the BCC.The  Burkholderia cepacia complex (BCC) is a group of 18 closely related bacterial species, which can cause life-threatening disease in immunocompromised and cystic fibrosis (CF) patients  have been sequenced  (B. cenocepacia pathogenicity island cci (lxa (B. cenocepacia H111. Conversely, the facultative pC3 replicon is larger than that of B. cenocepacia J2315 at 1.04 Mb versus 0.88 Mb. The pC3 replicon codes for specific traits, such as virulence and stress resistance, and is less conserved among BCC members than chromosomes 1 and 2 (Chromosomes 1 and 2 in H111 have sizes of 3.57 Mb and 3.10 Mb, respectively, and are slightly smaller than those of the closely related land cci , the typ 1 and 2 , 21.B. cenocepacia genomes.The genome was annotated using the RAST pipeline . The resB. cenocepacia H111 have been deposited at DDBJ/EMBL/GenBank under the accession no. HG938370, HG938371, and HG938372.The sequences of the three replicons of