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Sample records for 5hmc 5-formylcytosine 5fc

  1. Bisulfite-free, base-resolution analysis of 5-formylcytosine at the genome scale.

    PubMed

    Xia, Bo; Han, Dali; Lu, Xingyu; Sun, Zhaozhu; Zhou, Ankun; Yin, Qiangzong; Zeng, Hu; Liu, Menghao; Jiang, Xiang; Xie, Wei; He, Chuan; Yi, Chengqi

    2015-11-01

    Active DNA demethylation in mammals involves oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). However, genome-wide detection of 5fC at single-base resolution remains challenging. Here we present fC-CET, a bisulfite-free method for whole-genome analysis of 5fC based on selective chemical labeling of 5fC and subsequent C-to-T transition during PCR. Base-resolution 5fC maps showed limited overlap with 5hmC, with 5fC-marked regions more active than 5hmC-marked ones. PMID:26344045

  2. Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution

    NASA Astrophysics Data System (ADS)

    Booth, Michael J.; Marsico, Giovanni; Bachman, Martin; Beraldi, Dario; Balasubramanian, Shankar

    2014-05-01

    Recently, the cytosine modifications 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) were found to exist in the genomic deoxyribonucleic acid (DNA) of a wide range of mammalian cell types. It is now important to understand their role in normal biological function and disease. Here we introduce reduced bisulfite sequencing (redBS-Seq), a quantitative method to decode 5fC in DNA at single-base resolution, based on a selective chemical reduction of 5fC to 5hmC followed by bisulfite treatment. After extensive validation on synthetic and genomic DNA, we combined redBS-Seq and oxidative bisulfite sequencing (oxBS-Seq) to generate the first combined genomic map of 5-methylcytosine, 5hmC and 5fC in mouse embryonic stem cells. Our experiments revealed that in certain genomic locations 5fC is present at comparable levels to 5hmC and 5mC. The combination of these chemical methods can quantify and precisely map these three cytosine derivatives in the genome and will help provide insights into their function.

  3. Base pairing and structural insights into the 5-formylcytosine in RNA duplex

    PubMed Central

    Wang, Rui; Luo, Zhipu; He, Kaizhang; Delaney, Michael O.; Chen, Doris; Sheng, Jia

    2016-01-01

    5-Formylcytidine (f5C), a previously discovered natural nucleotide in the mitochondrial tRNA of many species including human, has been recently detected as the oxidative product of 5-methylcytidine (m5C) through 5-hydroxymethylcytidine (hm5C) in total RNA of mammalian cells. The discovery indicated that these cytosine derivatives in RNA might also play important epigenetic roles similar as in DNA, which has been intensively investigated in the past few years. In this paper, we studied the base pairing specificity of f5C in different RNA duplex contexts. We found that the 5-formyl group could increase duplex thermal stability and enhance base pairing specificity. We present three high-resolution crystal structures of an octamer RNA duplex [5′-GUA(f5C)GUAC-3′]2 that have been solved under three crystallization conditions with different buffers and pH values. Our results showed that the 5-formyl group is located in the same plane as the cytosine base and forms an intra-residue hydrogen bond with the amino group in the N4 position. In addition, this modification increases the base stacking between the f5C and the neighboring bases while not causing significant global and local structure perturbations. This work provides insights into the effects of 5-formylcytosine on RNA duplex. PMID:27079978

  4. Base pairing and structural insights into the 5-formylcytosine in RNA duplex.

    PubMed

    Wang, Rui; Luo, Zhipu; He, Kaizhang; Delaney, Michael O; Chen, Doris; Sheng, Jia

    2016-06-01

    5-Formylcytidine (f(5)C), a previously discovered natural nucleotide in the mitochondrial tRNA of many species including human, has been recently detected as the oxidative product of 5-methylcytidine (m(5)C) through 5-hydroxymethylcytidine (hm(5)C) in total RNA of mammalian cells. The discovery indicated that these cytosine derivatives in RNA might also play important epigenetic roles similar as in DNA, which has been intensively investigated in the past few years. In this paper, we studied the base pairing specificity of f(5)C in different RNA duplex contexts. We found that the 5-formyl group could increase duplex thermal stability and enhance base pairing specificity. We present three high-resolution crystal structures of an octamer RNA duplex [5'-GUA(f(5)C)GUAC-3']2 that have been solved under three crystallization conditions with different buffers and pH values. Our results showed that the 5-formyl group is located in the same plane as the cytosine base and forms an intra-residue hydrogen bond with the amino group in the N4 position. In addition, this modification increases the base stacking between the f(5)C and the neighboring bases while not causing significant global and local structure perturbations. This work provides insights into the effects of 5-formylcytosine on RNA duplex. PMID:27079978

  5. Tissue-Specific Differences in DNA Modifications (5-Hydroxymethylcytosine, 5-Formylcytosine, 5-Carboxylcytosine and 5-Hydroxymethyluracil) and Their Interrelationships

    PubMed Central

    Starczak, Marta; Modrzejewska, Martyna; Olinski, Ryszard

    2015-01-01

    Background Replication-independent active/enzymatic demethylation may be an important process in the functioning of somatic cells. The most plausible mechanisms of active 5-methylcytosine demethylation, leading to activation of previously silenced genes, involve ten-eleven translocation (TET) proteins that participate in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxylcytosine. Recently, 5-hydroxymethylcytosine was demonstrated to be a relatively stable modification, and the previously observed substantial differences in the level of this modification in various murine tissues were shown to depend mostly on cell proliferation rate. Some experimental evidence supports the hypothesis that 5-hydroxymethyluracil may be also generated by TET enzymes and has epigenetic functions. Results Using an isotope-dilution automated online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry, we have analyzed, for the first time, all the products of active DNA demethylation pathway: 5-methyl-2′-deoxycytidine, 5-hydroxymethyl-2′-deoxycytidine, 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine, as well as 5-hydroxymethyl-2′-deoxyuridine, in DNA isolated from various rat and porcine tissues. A strong significant inverse linear correlation was found between the proliferation rate of cells and the global level of 5-hydroxymethyl-2′-deoxycytidine in both porcine (R2 = 0.88) and rat tissues (R2 = 0.83); no such relationship was observed for 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine. Moreover, a substrate-product correlation was demonstrated for the two consecutive steps of iterative oxidation pathway: between 5-hydroxymethyl-2′-deoxycytidine and its product 5-formyl-2′-deoxycytidine, as well as between 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine (R2 = 0.60 and R2 = 0.71, respectively). Conclusions Good correlations within

  6. Differential Stabilities and Sequence-Dependent Base Pair Opening Dynamics of Watson–Crick Base Pairs with 5-Hydroxymethylcytosine, 5-Formylcytosine, or 5-Carboxylcytosine

    PubMed Central

    2016-01-01

    5-Hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) form during active demethylation of 5-methylcytosine (5mC) and are implicated in epigenetic regulation of the genome. They are differentially processed by thymine DNA glycosylase (TDG), an enzyme involved in active demethylation of 5mC. Three modified Dickerson–Drew dodecamer (DDD) sequences, amenable to crystallographic and spectroscopic analyses and containing the 5′-CG-3′ sequence associated with genomic cytosine methylation, containing 5hmC, 5fC, or 5caC placed site-specifically into the 5′-T8X9G10-3′ sequence of the DDD, were compared. The presence of 5caC at the X9 base increased the stability of the DDD, whereas 5hmC or 5fC did not. Both 5hmC and 5fC increased imino proton exchange rates and calculated rate constants for base pair opening at the neighboring base pair A5:T8, whereas 5caC did not. At the oxidized base pair G4:X9, 5fC exhibited an increase in the imino proton exchange rate and the calculated kop. In all cases, minimal effects to imino proton exchange rates occurred at the neighboring base pair C3:G10. No evidence was observed for imino tautomerization, accompanied by wobble base pairing, for 5hmC, 5fC, or 5caC when positioned at base pair G4:X9; each favored Watson–Crick base pairing. However, both 5fC and 5caC exhibited intranucleobase hydrogen bonding between their formyl or carboxyl oxygens, respectively, and the adjacent cytosine N4 exocyclic amines. The lesion-specific differences observed in the DDD may be implicated in recognition of 5hmC, 5fC, or 5caC in DNA by TDG. However, they do not correlate with differential excision of 5hmC, 5fC, or 5caC by TDG, which may be mediated by differences in transition states of the enzyme-bound complexes. PMID:25632825

  7. In Vitro and In Vivo Effect of 5-FC Combined Gene Therapy with TNF-α and CD Suicide Gene on Human Laryngeal Carcinoma Cell Line Hep-2

    PubMed Central

    Chai, Li-Ping; Wang, Zhang-Feng; Liang, Wei-Ying; Chen, Lei; Chen, Dan; Wang, An-Xun; Zhang, Zhao-Qiang

    2013-01-01

    This study was aimed to investigate the effect of combined cancer gene therapy with exogenous tumor necrosis factor-alpha (TNF-α) and cytosine deaminase (CD) suicide gene on laryngeal carcinoma cell line Hep-2 in vitro and in vivo. Transfection of the recombinant eukaryotic vectors of pcDNA3.1 (+) containing TNF-α and/or CD into Hep-2 cells resulted in expression of TNF-α and/or CD gene in vitro. The significant increase in apoptotic Hep-2 cells and decrease of Hep-2 cell proliferation were observed using 5-FC treatment combined with TNF-a expression by CD/5-FC suicide system. Moreover, bystander effect was also observed in the TNF-α and CD gene co-expression group. Laryngeal squamous cell carcinoma (LSCC) mice model was established by using BALB/c mice which different transfected Hep-2 cells with pcDNA3.1 (+) containing TNF-α and/or CD were applied subcutaneously. So these mice are divided into four groups, namely, Hep-2/TIC group; Hep-2/CD group; Hep-2/TNF-α group; Hep-2/0 group. At day 29 after cell inoculation, volume of grafted tumor had significant difference between each two of them (P<0.05). These results showed that the products of combined CD and TNF-α genes inhibited the growth of transplanted LSCC in mice model. So by our observed parameters and many others results, we hypothesized that 5-FC combined gene therapy with TNF-αand CD suicide gene should be an effective treatment on Laryngeal carcinoma. PMID:23593411

  8. 5-Formylcytosine Could Be a Semipermanent Base in Specific Genome Sites.

    PubMed

    Su, Meng; Kirchner, Angie; Stazzoni, Samuele; Müller, Markus; Wagner, Mirko; Schröder, Arne; Carell, Thomas

    2016-09-19

    5-Formyl-2'-deoxycytosine (fdC) is a recently discovered epigenetic base in the genome of stem cells, with yet unknown functions. Sequencing data show that the base is enriched in CpG islands of promoters and hence likely involved in the regulation of transcription during cellular differentiation. fdC is known to be recognized and excised by the enzyme thymine-DNA-glycosylase (Tdg). As such, fdC is believed to function as an intermediate during active demethylation. In order to understand the function of the new epigenetic base fdC, it is important to analyze its formation and removal at defined genomic sites. Here, we report a new method that combines sequence-specific chemical derivatization of fdC with droplet digital PCR that enables such analysis. We show initial data, indicating that the repair protein Tdg removes only 50 % of the fdCs at a given genomic site, arguing that fdC is a semipermanent base. PMID:27561097

  9. Cytosine modifications in neurodevelopment and diseases

    PubMed Central

    Yao, Bing; Jin, Peng

    2013-01-01

    DNA methylation has been studied comprehensively and linked to both normal neurodevelopment and neurological diseases. The recent identification of several new DNA modifications, including 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), has given us a new perspective on the previously observed plasticity in 5mC-dependent regulatory processes. Here we review the latest research into these cytosine modifications, focusing mainly on their roles in neurodevelopment and diseases. PMID:23912899

  10. 5-hydroxymethylcytosine-mediated DNA demethylation in stem cells and development.

    PubMed

    Sun, Wenjia; Guan, Minxin; Li, Xuekun

    2014-05-01

    The pursuit of DNA demethylation has a colorful history, but it was not until 2009 that the stars of this story, the Ten-eleven-translocation (Tet) family of proteins, were really identified. Tet proteins convert 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), which can be further oxidized to 5-formylcytosine and 5-cyboxycytosine by Tet proteins to achieve DNA demethylation. Recent studies have revealed that 5hmC-mediated DNA demethylation can play essential roles in diverse biological processes, including development and diseases. Here, we review recent discoveries in 5hmC-mediated DNA demethylation in the context of stem cells and development. PMID:24400731

  11. Effects of Tet-induced oxidation products of 5-methylcytosine on Dnmt1- and DNMT3a-mediated cytosine methylation.

    PubMed

    Ji, Debin; Lin, Krystal; Song, Jikui; Wang, Yinsheng

    2014-07-01

    We investigated systematically the effects of Tet-induced oxidation products of 5-methylcytosine on Dnmt1- and DNMT3a-mediated cytosine methylation in synthetic duplex DNA. We found that the replacement of 5-methylcytosine at a CpG site with a 5-hydroxymethylcytosine, 5-formylcytosine, 5-carboxylcytosine or 5-hydroxymethyluracil resulted in altered methylation of cytosine at both the opposite and the neighboring CpG sites. Our results provided important new knowledge about the implications of the 5-methylcytosine oxidation products in maintenance cytosine methylation. PMID:24789765

  12. Various cytosine/adenine permease homologues are involved in the toxicity of 5-fluorocytosine in Saccharomyces cerevisiae.

    PubMed

    Paluszynski, John P; Klassen, Roland; Rohe, Matthias; Meinhardt, Friedhelm

    2006-07-15

    5-Fluorocytosine (5-FC), a medically applied antifungal agent (Ancotil), is also active against the model organism Saccharomyces cerevisiae. 5-FC uptake in S. cerevisiae was considered to be mediated by the FCY2-encoded cytosine/adenine permease. By applying a highly sensitive assay, a low-level but dose-dependent toxicity of 5-FC in fcy2 mutants was detected, whereas cells deficient in the cytosine deaminase (encoded by FCY1), which is essential for intracellular conversion of 5-FC to 5-fluorouracil, display strong dose-independent resistance. Thus, an alternative, Fcy2-independent access pathway for 5-FC exists in S. cerevisiae. A genome-wide search for cytosine permease homologues identified two uncharacterized candidate genes, designated FCY21 and FCY22, both of which exhibit highest similarity to FCY2. Disruption of either FCY21 or FCY22 resulted in strains displaying low-level resistance, indicating the functional involvement of both gene products in 5-FC toxicity. When mutations in FCY21 or FCY22 were combined with the FCY2 disruption, both double mutants displayed stronger resistance when compared to the FCY2 mutant alone. Disruptions in all three permease genes consequently conferred the highest degree of resistance, not only towards 5-FC but also to the toxic adenine analogon 8-azaadenine. As residual 5-FC sensitivity was, however, even detectable in the fcy2 fcy21 fcy22 mutant, we analysed the relevance of other FCY2 homologues, i.e. TPN1, FUR4, DAL4, FUI1 and yOR071c, for 5-FC toxicity. Among these, Tpn1, Fur4 and the one encoded by yOR071c were found to contribute significantly to 5-FC toxicity, thus revealing alternative entry routes for 5-FC via other cytosine/adenine permease homologues. PMID:16845689

  13. Identification of Sequence Specificity of 5-Methylcytosine Oxidation by Tet1 Protein with High-Throughput Sequencing.

    PubMed

    Kizaki, Seiichiro; Chandran, Anandhakumar; Sugiyama, Hiroshi

    2016-03-01

    Tet (ten-eleven translocation) family proteins have the ability to oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC). However, the oxidation reaction of Tet is not understood completely. Evaluation of genomic-level epigenetic changes by Tet protein requires unbiased identification of the highly selective oxidation sites. In this study, we used high-throughput sequencing to investigate the sequence specificity of mC oxidation by Tet1. A 6.6×10(4) -member mC-containing random DNA-sequence library was constructed. The library was subjected to Tet-reactive pulldown followed by high-throughput sequencing. Analysis of the obtained sequence data identified the Tet1-reactive sequences. We identified mCpG as a highly reactive sequence of Tet1 protein. PMID:26715454

  14. The TET2 interactors and their links to hematological malignancies

    PubMed Central

    Pan, Feng; Weeks, Ophelia; Yang, Feng-Chun; Xu, Mingjiang

    2016-01-01

    Ten-eleven translocation family proteins are dioxygenases that oxidize 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine in DNA, early steps of active DNA demethylation. TET2, the second member of TET protein family, is frequently mutated in patients with hematological malignancies, leading to aberrant DNA methylation profiling and decreased 5hmC levels. Located in the nucleus and acting as a DNA-modifying enzyme, TET2 is thought to exert its function via TET2-containing protein complexes. Identifying the interactome network of TET2 likely holds the key to uncover the mechanisms by which TET2 exerts its function in cells. Here, we review recent literature on TET2 interactors and discuss their possible roles in TET2 loss-mediated dysregulation of hematopoiesis and pathogenesis of hematological malignancies. PMID:26099018

  15. Charting oxidized methylcytosines at base resolution

    PubMed Central

    Wu, Hao; Zhang, Yi

    2015-01-01

    DNA cytosine methylation (5-methylcytosines) represents a key epigenetic mark and is required for normal development. Iterative oxidation of 5mC by TET family of DNA dioxygenases generates three oxidized nucleotides, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC), in the mammalian genome. Recent advances in genomic mapping techniques for these oxidized bases suggest that 5hmC/5fC/5caC are not only functionally relevant to the process of active reversal of 5mC, but may also possess unique regulatory functions. This perspective highlights the potential gene regulatory functions of these oxidized cytosine bases in the mammalian genome, and discusses the principles and limitations of recently developed base-resolution mapping technologies. PMID:26333715

  16. Fluorogenic Labeling of 5-Formylpyrimidine Nucleotides in DNA and RNA.

    PubMed

    Samanta, Biswajit; Seikowski, Jan; Höbartner, Claudia

    2016-01-26

    5-Formylcytosine (5fC) and 5-formyluracil (5fU) are natural nucleobase modifications that are generated by oxidative modification of 5-methylcytosine and thymine (or 5-methyluracil). Herein, we describe chemoselective labeling of 5-formylpyrimidine nucleotides in DNA and RNA by fluorogenic aldol-type condensation reactions with 2,3,3-trimethylindole derivatives. Mild and specific reaction conditions were developed for 5fU and 5fC to produce hemicyanine-like chromophores with distinct photophysical properties. Residue-specific detection was established by fluorescence readout as well as primer-extension assays. The reactions were optimized on DNA oligonucleotides and were equally suitable for the modification of 5fU- and 5fC-modified RNA. This direct labeling approach of 5-formylpyrimidines is expected to help in elucidating the occurrence, enzymatic transformations, and functional roles of these epigenetic/epitranscriptomic nucleobase modifications in DNA and RNA. PMID:26679556

  17. TET proteins and 5-methylcytosine oxidation in the immune system

    PubMed Central

    Tsagaratou, Ageliki; Rao, Anjana

    2015-01-01

    DNA methylation in the form of 5-methylcytosine (5mC) is essential for normal development in mammals and influences a variety of biological processes including transcriptional regulation, imprinting and the maintenance of genomic stability. The recent discovery of TET proteins, which oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), has changed our understanding of the process of DNA demethylation. Here, we summarize our current knowledge of the roles of DNA methylation and TET proteins in cell differentiation and function. The intensive research on this subject has so far focused primarily on ES cells and neurons. Here we summarize what is known about DNA methylation in T cell function. PMID:24619230

  18. Development of inhalable hyaluronan/mannitol composite dry powders for flucytosine repositioning in local therapy of lung infections.

    PubMed

    Costabile, G; d'Angelo, I; d'Emmanuele di Villa Bianca, R; Mitidieri, E; Pompili, B; Del Porto, P; Leoni, L; Visca, P; Miro, A; Quaglia, F; Imperi, F; Sorrentino, R; Ungaro, F

    2016-09-28

    Flucytosine (5-fluorocytosine, 5-FC) is a fluorinated analogue of cytosine currently approved for the systemic treatment of fungal infections, which has recently demonstrated a very promising antivirulence activity against the bacterial pathogen Pseudomonas aeruginosa. In this work, we propose novel inhalable hyaluronic acid (HA)/mannitol composite dry powders for repositioning 5-FC in the local treatment of lung infections, including those affecting cystic fibrosis (CF) patients. Different dry powders were produced in one-step by spray-drying. Powder composition and process conditions were selected after in depth formulation studies aimed at selecting the 5-FC/HA/mannitol formulation with convenient aerosolization properties and drug release profile in simulated lung fluids. The optimized 5-FC/HA/mannitol powder for inhalation (HyaMan_FC#3) was effectively delivered from different breath-activated dry powder inhalers (DPI) already available to CF patients. Nevertheless, the aerodynamic assessment of fine particles suggested that the developed formulation well fit with a low-resistance DPI. HyaMan_FC#3 inhibited the growth of the fungus Candida albicans and the production of the virulence factor pyoverdine by P. aeruginosa at 5-FC concentrations that did not affect the viability of both wild type (16HBE14o-) and CF (CFBE41o-) human bronchial epithelial cells. Finally, pharmacokinetics of HyaMan_FC#3 inhalation powder and 5-FC solution after intratracheal administration in rats were compared. In vivo results clearly demonstrated that, when formulated as dry powder, 5-FC levels in both bronchoalveolar lavage fluid and lung tissue were significantly higher and sustained over time as compared to those obtained with the 5-FC solution. Of note, when the same 5-FC amount was administered intravenously, no significant drug amount was found in the lung at each time point from the injection. To realize a 5-FC lung concentration similar to that obtained by using HyaMan_FC#3

  19. Study of the metabolism of flucytosine in Aspergillus species by sup 19 F nuclear magnetic resonance spectroscopy

    SciTech Connect

    Chouini-Lalanne, N.; Malet-Martino, M.C.; Martino, R.; Michel, G. )

    1989-11-01

    The metabolism of flucytosine (5FC) in two Aspergillus species (Aspergillus fumigatus and A. niger) was investigated by 19F nuclear magnetic resonance spectroscopy. In intact mycelia, 5FC was found to be deaminated to 5-fluorouracil and then transformed into fluoronucleotides; the catabolite alpha-fluoro-beta-alanine was also detected in A. fumigatus. Neither 5-fluoroorotic acid nor 5-fluoro-2'-deoxyuridine-5'-monophosphate was detected in perchloric acid extracts after any incubation with 5FC. 5FC, 5-fluorouracil, and the classical fluoronucleotides 5-fluorouridine-5'-mono-, di-, and triphosphates were identified in the acid-soluble pool. Two hydrolysis products of 5-fluorouracil incorporated into RNA, 5-fluorouridine-2'-monophosphate and 5-fluorouridine-3'-monophosphate, were found in the acid-insoluble pool. No significant differences in the metabolic transformation of 5FC were noted in the two species of Aspergillus. The main pathway of 5FC metabolism in the two species of Aspergillus studied is thus the biotransformation into ribofluoronucleotides and the subsequent incorporation of 5-fluorouridine-5'-triphosphate into RNA.

  20. Analysis of TET Expression/Activity and 5mC Oxidation during Normal and Malignant Germ Cell Development

    PubMed Central

    Nettersheim, Daniel; Heukamp, Lukas C.; Fronhoffs, Florian; Grewe, Marc J.; Haas, Natalie; Waha, Anke; Honecker, Friedemann; Waha, Andreas; Kristiansen, Glen; Schorle, Hubert

    2013-01-01

    During mammalian development the fertilized zygote and primordial germ cells lose their DNA methylation within one cell cycle leading to the concept of active DNA demethylation. Recent studies identified the TET hydroxylases as key enzymes responsible for active DNA demethylation, catalyzing the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine. Further oxidation and activation of the base excision repair mechanism leads to replacement of a modified cytosine by an unmodified one. In this study, we analyzed the expression/activity of TET1-3 and screened for the presence of 5mC oxidation products in adult human testis and in germ cell cancers. By analyzing human testis sections, we show that levels of 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine are decreasing as spermatogenesis proceeds, while 5-methylcytosine levels remain constant. These data indicate that during spermatogenesis active DNA demethylation becomes downregulated leading to a conservation of the methylation marks in mature sperm. We demonstrate that all carcinoma in situ and the majority of seminomas are hypomethylated and hypohydroxymethylated compared to non-seminomas. Interestingly, 5-formylcytosine and 5-carboxylcytosine were detectable in all germ cell cancer entities analyzed, but levels did not correlate to the 5-methylcytosine or 5-hydroxymethylcytosine status. A meta-analysis of gene expression data of germ cell cancer tissues and corresponding cell lines demonstrates high expression of TET1 and the DNA glycosylase TDG, suggesting that germ cell cancers utilize the oxidation pathway for active DNA demethylation. During xenograft experiments, where seminoma-like TCam-2 cells transit to an embryonal carcinoma-like state DNMT3B and DNMT3L where strongly upregulated, which correlated to increasing 5-methylcytosine levels. Additionally, 5-hydroxymethylcytosine levels were elevated, demonstrating that de novo methylation and active demethylation accompanies this transition

  1. Deficient methylation and formylation of mt-tRNA(Met) wobble cytosine in a patient carrying mutations in NSUN3.

    PubMed

    Van Haute, Lindsey; Dietmann, Sabine; Kremer, Laura; Hussain, Shobbir; Pearce, Sarah F; Powell, Christopher A; Rorbach, Joanna; Lantaff, Rebecca; Blanco, Sandra; Sauer, Sascha; Kotzaeridou, Urania; Hoffmann, Georg F; Memari, Yasin; Kolb-Kokocinski, Anja; Durbin, Richard; Mayr, Johannes A; Frye, Michaela; Prokisch, Holger; Minczuk, Michal

    2016-01-01

    Epitranscriptome modifications are required for structure and function of RNA and defects in these pathways have been associated with human disease. Here we identify the RNA target for the previously uncharacterized 5-methylcytosine (m(5)C) methyltransferase NSun3 and link m(5)C RNA modifications with energy metabolism. Using whole-exome sequencing, we identified loss-of-function mutations in NSUN3 in a patient presenting with combined mitochondrial respiratory chain complex deficiency. Patient-derived fibroblasts exhibit severe defects in mitochondrial translation that can be rescued by exogenous expression of NSun3. We show that NSun3 is required for deposition of m(5)C at the anticodon loop in the mitochondrially encoded transfer RNA methionine (mt-tRNA(Met)). Further, we demonstrate that m(5)C deficiency in mt-tRNA(Met) results in the lack of 5-formylcytosine (f(5)C) at the same tRNA position. Our findings demonstrate that NSUN3 is necessary for efficient mitochondrial translation and reveal that f(5)C in human mitochondrial RNA is generated by oxidative processing of m(5)C. PMID:27356879

  2. Effects of Tet-mediated Oxidation Products of 5-Methylcytosine on DNA Transcription in vitro and in Mammalian Cells

    NASA Astrophysics Data System (ADS)

    You, Changjun; Ji, Debin; Dai, Xiaoxia; Wang, Yinsheng

    2014-11-01

    5-methylcytosine (5-mC) is a well-characterized epigenetic regulator in mammals. Recent studies showed that Ten-eleven translocation (Tet) proteins can catalyze the stepwise oxidation of 5-mC to produce 5-hydroxymethylcytosine (5-HmC), 5-formylcytosine (5-FoC) and 5-carboxylcytosine (5-CaC). The exciting discovery of these novel cytosine modifications has stimulated substantial research interests about their roles in epigenetic regulation. Here we systematically examined the effects of the oxidized 5-mC derivatives on the efficiency and fidelity of DNA transcription using a recently developed competitive transcription and adduct bypass assay. Our results showed that, when located on the transcribed strand, 5-FoC and 5-CaC exhibited marginal mutagenic and modest inhibitory effects on DNA transcription mediated by single-subunit T7 RNA polymerase or multi-subunit human RNA polymerase II in vitro and in human cells. 5-HmC displayed relatively milder blocking effects on transcription, and no mutant transcript could be detectable for 5-HmC in vitro or in cells. The lack of considerable mutagenic effects of the oxidized 5-mC derivatives on transcription was in agreement with their functions in epigenetic regulation. The modest blocking effects on transcription suggested that 5-FoC and 5-CaC may function in transcriptional regulation. These findings provided new evidence for the potential functional interplay between cytosine methylation status and transcription.

  3. Effects of cytosine modifications on DNA flexibility and nucleosome mechanical stability

    PubMed Central

    Ngo, Thuy T. M.; Yoo, Jejoong; Dai, Qing; Zhang, Qiucen; He, Chuan; Aksimentiev, Aleksei; Ha, Taekjip

    2016-01-01

    Cytosine can undergo modifications, forming 5-methylcytosine (5-mC) and its oxidized products 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). Despite their importance as epigenetic markers and as central players in cellular processes, it is not well understood how these modifications influence physical properties of DNA and chromatin. Here we report a comprehensive survey of the effect of cytosine modifications on DNA flexibility. We find that even a single copy of 5-fC increases DNA flexibility markedly. 5-mC reduces and 5-hmC enhances flexibility, and 5-caC does not have a measurable effect. Molecular dynamics simulations show that these modifications promote or dampen structural fluctuations, likely through competing effects of base polarity and steric hindrance, without changing the average structure. The increase in DNA flexibility increases the mechanical stability of the nucleosome and vice versa, suggesting a gene regulation mechanism where cytosine modifications change the accessibility of nucleosomal DNA through their effects on DNA flexibility. PMID:26905257

  4. Deficient methylation and formylation of mt-tRNAMet wobble cytosine in a patient carrying mutations in NSUN3

    PubMed Central

    Van Haute, Lindsey; Dietmann, Sabine; Kremer, Laura; Hussain, Shobbir; Pearce, Sarah F.; Powell, Christopher A.; Rorbach, Joanna; Lantaff, Rebecca; Blanco, Sandra; Sauer, Sascha; Kotzaeridou, Urania; Hoffmann, Georg F.; Memari, Yasin; Kolb-Kokocinski, Anja; Durbin, Richard; Mayr, Johannes A.; Frye, Michaela; Prokisch, Holger; Minczuk, Michal

    2016-01-01

    Epitranscriptome modifications are required for structure and function of RNA and defects in these pathways have been associated with human disease. Here we identify the RNA target for the previously uncharacterized 5-methylcytosine (m5C) methyltransferase NSun3 and link m5C RNA modifications with energy metabolism. Using whole-exome sequencing, we identified loss-of-function mutations in NSUN3 in a patient presenting with combined mitochondrial respiratory chain complex deficiency. Patient-derived fibroblasts exhibit severe defects in mitochondrial translation that can be rescued by exogenous expression of NSun3. We show that NSun3 is required for deposition of m5C at the anticodon loop in the mitochondrially encoded transfer RNA methionine (mt-tRNAMet). Further, we demonstrate that m5C deficiency in mt-tRNAMet results in the lack of 5-formylcytosine (f5C) at the same tRNA position. Our findings demonstrate that NSUN3 is necessary for efficient mitochondrial translation and reveal that f5C in human mitochondrial RNA is generated by oxidative processing of m5C. PMID:27356879

  5. Simultaneous sequencing of oxidized methylcytosines produced by TET/JBP dioxygenases in Coprinopsis cinerea

    PubMed Central

    Chavez, Lukas; Huang, Yun; Luong, Khai; Agarwal, Suneet; Iyer, Lakshminarayan M.; Pastor, William A.; Hench, Virginia K.; Frazier-Bowers, Sylvia A.; Korol, Evgenia; Liu, Shuo; Tahiliani, Mamta; Wang, Yinsheng; Clark, Tyson A.; Korlach, Jonas; Pukkila, Patricia J.; Aravind, L.; Rao, Anjana

    2014-01-01

    TET/JBP enzymes oxidize 5-methylpyrimidines in DNA. In mammals, the oxidized methylcytosines (oxi-mCs) function as epigenetic marks and likely intermediates in DNA demethylation. Here we present a method based on diglucosylation of 5-hydroxymethylcytosine (5hmC) to simultaneously map 5hmC, 5-formylcytosine, and 5-carboxylcytosine at near–base-pair resolution. We have used the method to map the distribution of oxi-mC across the genome of Coprinopsis cinerea, a basidiomycete that encodes 47 TET/JBP paralogs in a previously unidentified class of DNA transposons. Like 5-methylcytosine residues from which they are derived, oxi-mC modifications are enriched at centromeres, TET/JBP transposons, and multicopy paralogous genes that are not expressed, but rarely mark genes whose expression changes between two developmental stages. Our study provides evidence for the emergence of an epigenetic regulatory system through recruitment of selfish elements in a eukaryotic lineage, and describes a method to map all three different species of oxi-mCs simultaneously. PMID:25406324

  6. Uracil-DNA Glycosylase UNG Promotes Tet-mediated DNA Demethylation.

    PubMed

    Xue, Jian-Huang; Xu, Gui-Fang; Gu, Tian-Peng; Chen, Guo-Dong; Han, Bin-Bin; Xu, Zhi-Mei; Bjørås, Magnar; Krokan, Hans E; Xu, Guo-Liang; Du, Ya-Rui

    2016-01-01

    In mammals, active DNA demethylation involves oxidation of 5-methylcytosine (5mC) into 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by Tet dioxygenases and excision of these two oxidized bases by thymine DNA glycosylase (TDG). Although TDG is essential for active demethylation in embryonic stem cells and induced pluripotent stem cells, it is hardly expressed in mouse zygotes and dispensable in pronuclear DNA demethylation. To search for other factors that might contribute to demethylation in mammalian cells, we performed a functional genomics screen based on a methylated luciferase reporter assay. UNG2, one of the glycosylases known to excise uracil residues from DNA, was found to reduce DNA methylation, thus activating transcription of a methylation-silenced reporter gene when co-transfected with Tet2 into HEK293T cells. Interestingly, UNG2 could decrease 5caC from the genomic DNA and a reporter plasmid in transfected cells, like TDG. Furthermore, deficiency in Ung partially impaired DNA demethylation in mouse zygotes. Our results suggest that UNG might be involved in Tet-mediated DNA demethylation. PMID:26620559

  7. Gadd45a promotes DNA demethylation through TDG.

    PubMed

    Li, Zheng; Gu, Tian-Peng; Weber, Alain R; Shen, Jia-Zhen; Li, Bin-Zhong; Xie, Zhi-Guo; Yin, Ruichuan; Guo, Fan; Liu, Xiaomeng; Tang, Fuchou; Wang, Hailin; Schär, Primo; Xu, Guo-Liang

    2015-04-30

    Growth arrest and DNA-damage-inducible protein 45 (Gadd45) family members have been implicated in DNA demethylation in vertebrates. However, it remained unclear how they contribute to the demethylation process. Here, we demonstrate that Gadd45a promotes active DNA demethylation through thymine DNA glycosylase (TDG) which has recently been shown to excise 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) generated in Ten-eleven-translocation (Tet)-initiated oxidative demethylation. The connection of Gadd45a with oxidative demethylation is evidenced by the enhanced activation of a methylated reporter gene in HEK293T cells expressing Gadd45a in combination with catalytically active TDG and Tet. Gadd45a interacts with TDG physically and increases the removal of 5fC and 5caC from genomic and transfected plasmid DNA by TDG. Knockout of both Gadd45a and Gadd45b from mouse ES cells leads to hypermethylation of specific genomic loci most of which are also targets of TDG and show 5fC enrichment in TDG-deficient cells. These observations indicate that the demethylation effect of Gadd45a is mediated by TDG activity. This finding thus unites Gadd45a with the recently defined Tet-initiated demethylation pathway. PMID:25845601

  8. A computational investigation on the substrate preference of ten-eleven-translocation 2 (TET2).

    PubMed

    Lu, Junyan; Hu, Lulu; Cheng, Jingdong; Fang, Dong; Wang, Chen; Yu, Kunqian; Jiang, Hualiang; Cui, Qiang; Xu, Yanhui; Luo, Cheng

    2016-02-14

    TET proteins iteratively convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) in a Fe(ii)/α-ketoglutarate-dependent manner. Our previous biochemical studies revealed that TET proteins are more active on 5mC than on 5hmC and 5fC. However, the source of the substrate preference of TET proteins still remains largely elusive. Here, we investigated the substrate binding and catalytic mechanisms of oxidation reactions mediated by TET2 on different substrates through computational approaches. In accordance with previous experimental reports, our computational results suggest that TET2 can bind to different substrates with comparable binding affinities and the hydrogen abstraction step in the catalytic cycle acts as the rate-limiting step. Further structural characterization of the intermediate structures revealed that the 5-substitution groups on 5hmC and 5fC adopt an unfavorable orientation for hydrogen abstraction, which leads to a higher energy barrier for 5hmC and 5fC (compared to 5mC) and thus a lower catalytic efficiency. In summary, our mechanical insights demonstrate that substrate preference is the intrinsic property of TET proteins and our theoretical calculation results can guide further dry-lab or wet-lab studies on the catalytic mechanism of TET proteins as well as other Fe(ii)/α-ketoglutarate (KG)-dependent dioxygenases. PMID:26799843

  9. Thymine DNA glycosylase exhibits negligible affinity for nucleobases that it removes from DNA

    PubMed Central

    Malik, Shuja S.; Coey, Christopher T.; Varney, Kristen M.; Pozharski, Edwin; Drohat, Alexander C.

    2015-01-01

    Thymine DNA Glycosylase (TDG) performs essential functions in maintaining genetic integrity and epigenetic regulation. Initiating base excision repair, TDG removes thymine from mutagenic G·T mispairs caused by 5-methylcytosine (mC) deamination and other lesions including uracil (U) and 5-hydroxymethyluracil (hmU). In DNA demethylation, TDG excises 5-formylcytosine (fC) and 5-carboxylcytosine (caC), which are generated from mC by Tet (ten–eleven translocation) enzymes. Using improved crystallization conditions, we solved high-resolution (up to 1.45 Å) structures of TDG enzyme–product complexes generated from substrates including G·U, G·T, G·hmU, G·fC and G·caC. The structures reveal many new features, including key water-mediated enzyme–substrate interactions. Together with nuclear magnetic resonance experiments, the structures demonstrate that TDG releases the excised base from its tight product complex with abasic DNA, contrary to previous reports. Moreover, DNA-free TDG exhibits no significant binding to free nucleobases (U, T, hmU), indicating a Kd >> 10 mM. The structures reveal a solvent-filled channel to the active site, which might facilitate dissociation of the excised base and enable caC excision, which involves solvent-mediated acid catalysis. Dissociation of the excised base allows TDG to bind the beta rather than the alpha anomer of the abasic sugar, which might stabilize the enzyme–product complex. PMID:26358812

  10. Isolation of Human Genomic DNA Sequences with Expanded Nucleobase Selectivity.

    PubMed

    Rathi, Preeti; Maurer, Sara; Kubik, Grzegorz; Summerer, Daniel

    2016-08-10

    We report the direct isolation of user-defined DNA sequences from the human genome with programmable selectivity for both canonical and epigenetic nucleobases. This is enabled by the use of engineered transcription-activator-like effectors (TALEs) as DNA major groove-binding probes in affinity enrichment. The approach provides the direct quantification of 5-methylcytosine (5mC) levels at single genomic nucleotide positions in a strand-specific manner. We demonstrate the simple, multiplexed typing of a variety of epigenetic cancer biomarker 5mC with custom TALE mixes. Compared to antibodies as the most widely used affinity probes for 5mC analysis, i.e., employed in the methylated DNA immunoprecipitation (MeDIP) protocol, TALEs provide superior sensitivity, resolution and technical ease. We engineer a range of size-reduced TALE repeats and establish full selectivity profiles for their binding to all five human cytosine nucleobases. These provide insights into their nucleobase recognition mechanisms and reveal the ability of TALEs to isolate genomic target sequences with selectivity for single 5-hydroxymethylcytosine and, in combination with sodium borohydride reduction, single 5-formylcytosine nucleobases. PMID:27429302

  11. 5-Carboxylcytosine is localized to euchromatic regions in the nuclei of follicular cells in axolotl ovary.

    PubMed

    Alioui, Anthony; Wheldon, Lee M; Abakir, Abdulkadir; Ferjentsik, Zoltan; Johnson, Andrew D; Ruzov, Alexey

    2012-01-01

    5-Methylcytosine (5-mC) is an epigenetic modification associated with gene repression. Recent studies demonstrated that 5-mC can be enzymatically oxidised into 5-hydroxymethylcytosine and further into 5-formylcytosine (5-fC) and 5-carboxylcytsine (5-caC). 5-caC has been found in embryonic stem cells and in mouse pre-implantation embryos but no detectable levels of this modification have been reported for somatic tissues to date. Whereas it has been suggested that 5-caC can serve as an intermediate in the process of active demethylation, the function of this form of modified cytosine remains obscure. Here we show that 5-caC is immunochemically detectable in somatic cells of axolotl ovary. We demonstrate that both 5-hmC and 5-caC are localized to the euchromatin in the nuclei of axolotl follicular cells with similar patterns of spatial distribution. Our results suggest that 5-carboxylcytosine may play a distinct functional role in certain biological contexts. PMID:23138778

  12. 5-Carboxylcytosine is localized to euchromatic regions in the nuclei of follicular cells in axolotl ovary

    PubMed Central

    Alioui, Anthony; Wheldon, Lee M.; Abakir, Abdulkadir; Ferjentsik, Zoltan; Johnson, Andrew D.; Ruzov, Alexey

    2012-01-01

    5-Methylcytosine (5-mC) is an epigenetic modification associated with gene repression. Recent studies demonstrated that 5-mC can be enzymatically oxidised into 5-hydroxymethylcytosine and further into 5-formylcytosine (5-fC) and 5-carboxylcytsine (5-caC). 5-caC has been found in embryonic stem cells and in mouse pre-implantation embryos but no detectable levels of this modification have been reported for somatic tissues to date. Whereas it has been suggested that 5-caC can serve as an intermediate in the process of active demethylation, the function of this form of modified cytosine remains obscure. Here we show that 5-caC is immunochemically detectable in somatic cells of axolotl ovary. We demonstrate that both 5-hmC and 5-caC are localized to the euchromatin in the nuclei of axolotl follicular cells with similar patterns of spatial distribution. Our results suggest that 5-carboxylcytosine may play a distinct functional role in certain biological contexts. PMID:23138778

  13. Transient accumulation of 5-carboxylcytosine indicates involvement of active demethylation in lineage specification of neural stem cells.

    PubMed

    Wheldon, Lee M; Abakir, Abdulkadir; Ferjentsik, Zoltan; Dudnakova, Tatiana; Strohbuecker, Stephanie; Christie, Denise; Dai, Nan; Guan, Shengxi; Foster, Jeremy M; Corrêa, Ivan R; Loose, Matthew; Dixon, James E; Sottile, Virginie; Johnson, Andrew D; Ruzov, Alexey

    2014-06-12

    5-Methylcytosine (5mC) is an epigenetic modification involved in regulation of gene activity during differentiation. Tet dioxygenases oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be excised from DNA by thymine-DNA glycosylase (TDG) followed by regeneration of unmodified cytosine via the base excision repair pathway. Despite evidence that this mechanism is operative in embryonic stem cells, the role of TDG-dependent demethylation in differentiation and development is currently unclear. Here, we demonstrate that widespread oxidation of 5hmC to 5caC occurs in postimplantation mouse embryos. We show that 5fC and 5caC are transiently accumulated during lineage specification of neural stem cells (NSCs) in culture and in vivo. Moreover, 5caC is enriched at the cell-type-specific promoters during differentiation of NSCs, and TDG knockdown leads to increased 5fC/5caC levels in differentiating NSCs. Our data suggest that active demethylation contributes to epigenetic reprogramming determining lineage specification in embryonic brain. PMID:24882006

  14. Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry.

    PubMed

    Abakir, Abdulkadir; Wheldon, Lee; Johnson, Andrew D; Laurent, Patrick; Ruzov, Alexey

    2016-01-01

    Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts. PMID:27585398

  15. Enzymatic DNA oxidation: mechanisms and biological significance

    PubMed Central

    Xu, Guo-Liang; Walsh, Colum P.

    2014-01-01

    DNA methylation at cytosines (5mC) is a major epigenetic modification involved in the regulation of multiple biological processes in mammals. How methylation is reversed was until recently poorly understood. The family of dioxygenases commonly known as Ten-eleven translocation (Tet) proteins are responsible for the oxidation of 5mC into three new forms, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Current models link Tet-mediated 5mC oxidation with active DNA demethylation. The higher oxidation products (5fC and 5caC) are recognized and excised by the DNA glycosylase TDG via the base excision repair pathway. Like DNA methyltransferases, Tet enzymes are important for embryonic development. We will examine the mechanism and biological significance of Tet-mediated 5mC oxidation in the context of pronuclear DNA demethylation in mouse early embryos. In contrast to its role in active demethylation in the germ cells and early embryo, a number of lines of evidence suggest that the intragenic 5hmC present in brain may act as a stable mark instead. This short review explores mechanistic aspects of TET oxidation activity, the impact Tet enzymes have on epigenome organization and their contribution to the regulation of early embryonic and neuronal development. [BMB Reports 2014; 47(11): 609-618] PMID:25341925

  16. Regulation of the Epigenome by Vitamin C

    PubMed Central

    Young, Juan I.; Züchner, Stephan; Wang, Gaofeng

    2015-01-01

    Emerging evidence suggests that ascorbate, the dominant form of vitamin C under physiological pH conditions, influences the genome activity via regulating epigenomic processes. Ascorbate serves as a cofactor for ten-eleven translocation (TET) dioxygenases that catalyze the oxidation of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), further to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), which are ultimately replaced by unmodified cytosine. The JmjC domain-containing histone demethylases also require ascorbate as a cofactor for histone demethylation. Thus, by primarily participating in the demethylation of both DNA and histones, ascorbate appears to be a mediator of the interface between the genome and environment. Furthermore, redox status has a profound impact on the bioavailability of ascorbate in the nucleus. In order to bridge the gap between redox biology and genomics, we suggest an interdisciplinary research field that can be termed “Redox Genomics” to study dynamic redox processes in health and diseases. This review examines the evidence and potential molecular mechanism of ascorbate in demethylation of the genome, while highlighting potential epigenetic roles of ascorbate in various diseases. PMID:25974700

  17. Effects of Tet-mediated oxidation products of 5-methylcytosine on DNA transcription in vitro and in mammalian cells.

    PubMed

    You, Changjun; Ji, Debin; Dai, Xiaoxia; Wang, Yinsheng

    2014-01-01

    5-methylcytosine (5-mC) is a well-characterized epigenetic regulator in mammals. Recent studies showed that Ten-eleven translocation (Tet) proteins can catalyze the stepwise oxidation of 5-mC to produce 5-hydroxymethylcytosine (5-HmC), 5-formylcytosine (5-FoC) and 5-carboxylcytosine (5-CaC). The exciting discovery of these novel cytosine modifications has stimulated substantial research interests about their roles in epigenetic regulation. Here we systematically examined the effects of the oxidized 5-mC derivatives on the efficiency and fidelity of DNA transcription using a recently developed competitive transcription and adduct bypass assay. Our results showed that, when located on the transcribed strand, 5-FoC and 5-CaC exhibited marginal mutagenic and modest inhibitory effects on DNA transcription mediated by single-subunit T7 RNA polymerase or multi-subunit human RNA polymerase II in vitro and in human cells. 5-HmC displayed relatively milder blocking effects on transcription, and no mutant transcript could be detectable for 5-HmC in vitro or in cells. The lack of considerable mutagenic effects of the oxidized 5-mC derivatives on transcription was in agreement with their functions in epigenetic regulation. The modest blocking effects on transcription suggested that 5-FoC and 5-CaC may function in transcriptional regulation. These findings provided new evidence for the potential functional interplay between cytosine methylation status and transcription. PMID:25394478

  18. Distributive Processing by the Iron(II)/α-Ketoglutarate-Dependent Catalytic Domains of the TET Enzymes Is Consistent with Epigenetic Roles for Oxidized 5-Methylcytosine Bases.

    PubMed

    Tamanaha, Esta; Guan, Shengxi; Marks, Katherine; Saleh, Lana

    2016-08-01

    The ten-eleven translocation (TET) proteins catalyze oxidation of 5-methylcytosine ((5m)C) residues in nucleic acids to 5-hydroxymethylcytosine ((5hm)C), 5-formylcytosine ((5f)C), and 5-carboxycytosine ((5ca)C). These nucleotide bases have been implicated as intermediates on the path to active demethylation, but recent reports have suggested that they might have specific regulatory roles in their own right. In this study, we present kinetic evidence showing that the catalytic domains (CDs) of TET2 and TET1 from mouse and their homologue from Naegleria gruberi, the full-length protein NgTET1, are distributive in both chemical and physical senses, as they carry out successive oxidations of a single (5m)C and multiple (5m)C residues along a polymethylated DNA substrate. We present data showing that the enzyme neither retains (5hm)C/(5f)C intermediates of preceding oxidations nor slides along a DNA substrate (without releasing it) to process an adjacent (5m)C residue. These findings contradict a recent report by Crawford et al. ( J. Am. Chem. Soc. 2016 , 138 , 730 ) claiming that oxidation of (5m)C by CD of mouse TET2 is chemically processive (iterative). We further elaborate that this distributive mechanism is maintained for TETs in two evolutionarily distant homologues and posit that this mode of function allows the introduction of (5m)C forms as epigenetic markers along the DNA. PMID:27362828

  19. TET proteins and 5-methylcytosine oxidation in hematological cancers

    PubMed Central

    An, Jungeun; Pastor, William A.; Ko, Myunggon; Rao, Anjana

    2015-01-01

    Summary DNA methylation has pivotal regulatory roles in mammalian development, retrotransposon silencing, genomic imprinting and X-chromosome inactivation. Cancer cells display highly dysregulated DNA methylation profiles characterized by global hypomethylation in conjunction with hypermethylation of promoter CpG islands (CGIs) that presumably lead to genome instability and aberrant expression of tumor suppressor genes or oncogenes. The recent discovery of Ten-Eleven-Translocation (TET) family dioxygenases that oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) in DNA has led to profound progress in understanding the mechanism underlying DNA demethylation. Among the three TET genes, TET2 recurrently undergoes inactivating mutations in a wide range of myeloid and lymphoid malignancies. TET2 functions as a bona fide tumor suppressor particularly in the pathogenesis of myeloid malignancies resembling chronic myelomoncytic leukemia (CMML) and myelodysplastic syndromes (MDS) in human. Here we review diverse functions of TET proteins and the novel epigenetic marks that they generate in DNA methylation/demethylation dynamics and normal and malignant hematopoietic differentiation. The impact of TET2 inactivation in hematopoiesis and various mechanisms modulating the expression or activity of TET proteins are also discussed. Furthermore, we also present evidence that TET2 and TET3 collaborate to suppress aberrant hematopoiesis and hematopoietic transformation. A detailed understanding of the normal and pathological functions of TET proteins may provide new avenues to develop novel epigenetic therapies for treating hematological malignancies. PMID:25510268

  20. Gadd45a promotes DNA demethylation through TDG

    PubMed Central

    Li, Zheng; Gu, Tian-Peng; Weber, Alain R.; Shen, Jia-Zhen; Li, Bin-Zhong; Xie, Zhi-Guo; Yin, Ruichuan; Guo, Fan; Liu, Xiaomeng; Tang, Fuchou; Wang, Hailin; Schär, Primo; Xu, Guo-Liang

    2015-01-01

    Growth arrest and DNA-damage-inducible protein 45 (Gadd45) family members have been implicated in DNA demethylation in vertebrates. However, it remained unclear how they contribute to the demethylation process. Here, we demonstrate that Gadd45a promotes active DNA demethylation through thymine DNA glycosylase (TDG) which has recently been shown to excise 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) generated in Ten-eleven-translocation (Tet)—initiated oxidative demethylation. The connection of Gadd45a with oxidative demethylation is evidenced by the enhanced activation of a methylated reporter gene in HEK293T cells expressing Gadd45a in combination with catalytically active TDG and Tet. Gadd45a interacts with TDG physically and increases the removal of 5fC and 5caC from genomic and transfected plasmid DNA by TDG. Knockout of both Gadd45a and Gadd45b from mouse ES cells leads to hypermethylation of specific genomic loci most of which are also targets of TDG and show 5fC enrichment in TDG-deficient cells. These observations indicate that the demethylation effect of Gadd45a is mediated by TDG activity. This finding thus unites Gadd45a with the recently defined Tet-initiated demethylation pathway. PMID:25845601

  1. Effects of cytosine modifications on DNA flexibility and nucleosome mechanical stability

    NASA Astrophysics Data System (ADS)

    Ngo, Thuy T. M.; Yoo, Jejoong; Dai, Qing; Zhang, Qiucen; He, Chuan; Aksimentiev, Aleksei; Ha, Taekjip

    2016-02-01

    Cytosine can undergo modifications, forming 5-methylcytosine (5-mC) and its oxidized products 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). Despite their importance as epigenetic markers and as central players in cellular processes, it is not well understood how these modifications influence physical properties of DNA and chromatin. Here we report a comprehensive survey of the effect of cytosine modifications on DNA flexibility. We find that even a single copy of 5-fC increases DNA flexibility markedly. 5-mC reduces and 5-hmC enhances flexibility, and 5-caC does not have a measurable effect. Molecular dynamics simulations show that these modifications promote or dampen structural fluctuations, likely through competing effects of base polarity and steric hindrance, without changing the average structure. The increase in DNA flexibility increases the mechanical stability of the nucleosome and vice versa, suggesting a gene regulation mechanism where cytosine modifications change the accessibility of nucleosomal DNA through their effects on DNA flexibility.

  2. Isocitrate Dehydrogenase 2 Dysfunction Contributes to 5-hydroxymethylcytosine Depletion in Gastric Cancer Cells.

    PubMed

    Chou, Nan-Hua; Tsai, Chung-Yu; Tu, Ya-Ting; Wang, Kuo-Chiang; Kang, Chi-Hsiang; Chang, Po-Min; Li, Guan-Cheng; Lam, Hing-Chung; Liu, Shiuh-Inn; Tsai, Kuo-Wang

    2016-08-01

    The isocitrate dehydrogenase (IDH) family of enzymes comprises of the key functional metabolic enzymes in the Krebs cycle that catalyze the conversion of isocitrate to α-ketoglutarate (α-KG). α-KG acts as a cofactor in the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). However, the relationship between 5hmC and IDH in gastric cancer remains unclear. Our study revealed that the 5hmC level was substantially lower and 5mC level was slightly higher in gastric cancer tissues; however, 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) levels did not change significantly in these tissues. We further examined the expression levels of IDH1 and IDH2 in gastric cancer tissues and observed that IDH2 levels were significantly lower in gastric cancer tissues than in the adjacent normal tissues. The ectopic expression of IDH2 can increase 5hmC levels in gastric cancer cells. In conclusion, our results suggested that IDH2 dysfunction is involved in 5hmC depletion during gastric cancer progression. PMID:27466503

  3. Screening for recombinants of Crambe abyssynica after transformation by the pMF1 marker-free vector based on chemical selection and meristematic regeneration.

    PubMed

    Qi, Weicong; Tinnenbroek-Capel, Iris E M; Salentijn, Elma M J; Schaart, Jan G; Cheng, Jihua; Denneboom, Christel; Zhang, Zhao; Zhang, Xiaolin; Zhao, Han; Visser, Richard G F; Huang, Bangquan; Van Loo, Eibertus N; Krens, Frans A

    2015-01-01

    The T-DNA region of pMF1 vector of marker-free system developed by Wageningen UR, has Recombinase R-LBD gene fusion and nptII and codA gene fusion between two recombination sites. After transformation applying dexamethasone (DEX) can activate the recombinase to remove the T-DNA fragment between recombination sites. The recombinant ought to be selected on 5-fluorocytocine (5-FC) because of codA converting 5-FC into 5-fluorouracil the toxic. A PMF1 vector was transformed into hexaploid species Crambe abyssinica. Two independent transformants were chosen for DEX-induced recombination and later 5-FC selection. In contrast to earlier pMF1 experiments, the strategy of stepwise selection based on meristematic regeneration was engaged. After a long period of 5-FC selection, recombinants were obtained successfully, but most of the survivors were wildtype and non-recombinant. The results revealed when applying the PMF1 marker-free system on C. abyssinica, 1) Increasing in the DEX concentration did not correspondingly enhance the success of recombination; 2) both of the DEX-induced recombination and 5-FC negative selection were apparently insufficient which was leading to the extremely high frequency in chimerism occurring for recombinant and non-recombinant cells in tissues; 3) the strategy of stepwise selection based on meristem tissue regeneration was crucial for successfully isolating the recombinant germplasm from the chimera. PMID:26358007

  4. Screening for recombinants of Crambe abyssynica after transformation by the pMF1 marker-free vector based on chemical selection and meristematic regeneration

    PubMed Central

    Qi, Weicong; Tinnenbroek-Capel, Iris E. M.; Salentijn, Elma M. J.; Schaart, Jan G.; Cheng, Jihua; Denneboom, Christel; Zhang, Zhao; Zhang, Xiaolin; Zhao, Han; Visser, Richard G. F.; Huang, Bangquan; Van Loo, Eibertus N.; Krens, Frans A.

    2015-01-01

    The T-DNA region of pMF1 vector of marker-free system developed by Wageningen UR, has Recombinase R-LBD gene fusion and nptII and codA gene fusion between two recombination sites. After transformation applying dexamethasone (DEX) can activate the recombinase to remove the T-DNA fragment between recombination sites. The recombinant ought to be selected on 5-fluorocytocine (5-FC) because of codA converting 5-FC into 5-fluorouracil the toxic. A PMF1 vector was transformed into hexaploid species Crambe abyssinica. Two independent transformants were chosen for DEX-induced recombination and later 5-FC selection. In contrast to earlier pMF1 experiments, the strategy of stepwise selection based on meristematic regeneration was engaged. After a long period of 5-FC selection, recombinants were obtained successfully, but most of the survivors were wildtype and non-recombinant. The results revealed when applying the PMF1 marker-free system on C. abyssinica, 1) Increasing in the DEX concentration did not correspondingly enhance the success of recombination; 2) both of the DEX-induced recombination and 5-FC negative selection were apparently insufficient which was leading to the extremely high frequency in chimerism occurring for recombinant and non-recombinant cells in tissues; 3) the strategy of stepwise selection based on meristem tissue regeneration was crucial for successfully isolating the recombinant germplasm from the chimera. PMID:26358007

  5. Selective Delivery of a Therapeutic Gene for Treatment of Head and Neck Squamous Cell Carcinoma Using Human Neural Stem Cells

    PubMed Central

    Kim, Seung U; Song, Jae-Jun; Cho, Chang Gun; Park, Seok-Won

    2013-01-01

    Objectives Based on studies of the extensive tropism of neural stem cells (NSCs) toward malignant brain tumor, we hypothesized that NSCs could also target head and neck squamous cell carcinoma (HNSCC) and could be used as a cellular therapeutic delivery system. Methods To apply this strategy to the treatment of HNSCC, we used a human NSC line expressing cytosine deaminase (HB1.F3-CD), an enzyme that converts 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU), an anticancer agent. HB1. F3-CD in combination with 5-FC were cocultured with the HNSCC (SNU-1041) to examine the cytotoxicity on target tumor cells in vitro. For in vivo studies, an HNSCC mouse model was created by subcutaneous implantation of human HNSCC cells into athymic nude mice. HB1.F3-CD cells were injected into mice using tumoral, peritumoral, or intravenous injections, followed by systemic 5-FC administration. Results In vitro, the HB1.F3-CD cells significantly inhibited the growth of an HNSCC cell line in the presence of the 5-FC. Independent of the method of injection, the HB1.F3-CD cells migrated to the HNSCC tumor, causing a significant reduction in tumor volume. In comparison to 5-FU administration, HB1.F3-CD cell injection followed by 5-FC administration reduced systemic toxicity, but achieved the same level of therapeutic efficacy. Conclusion Transplantation of human NSCs that express the suicide enzyme cytosine deaminase combined with systemic administration of the prodrug 5-FC may be an effective regimen for the treatment of HNSCC. PMID:24069522

  6. Hydrogen-bonding patterns in 5-fluoro­cytosine–melamine co-crystal (4/1)

    PubMed Central

    Mohana, Marimuthu; Muthiah, Packianathan Thomas; Sanjeewa, Liurukara D.; McMillen, Colin D.

    2016-01-01

    The asymmetric unit of the title compound, 4C4H4FN3O·C3H6N6, comprises of two independent 5-fluoro­cytosine (5FC) mol­ecules (A and B) and one half-mol­ecule of melamine (M). The other half of the melamine mol­ecule is generated by a twofold axis. 5FC mol­ecules A and B are linked through two different homosynthons [R 2 2(8) ring motif]; one is formed via a pair of N—H⋯O hydrogen bonds and the second via a pair of N—H⋯N hydrogen bonds. In addition to this pairing, the O atoms of 5FC mol­ecules A and B inter­act with the N2 amino group on both sides of the melamine mol­ecule, forming a DDAA array of quadruple hydrogen bonds and generating a supra­molecular pattern. The 5FC (mol­ecules A and B) and two melamine mol­ecules inter­act via N—H⋯O, N—H⋯N and N—H⋯O, N—H⋯N, C—H⋯F hydrogen bonds forming R 6 6(24) and R 4 4(15) ring motifs. The crystal structure is further strengthened by C—H⋯F, C—F⋯π and π–π stacking inter­actions. PMID:27375887

  7. Structure of Naegleria Tet-like dioxygenase (NgTet1) in complexes with a reaction intermediate 5-hydroxymethylcytosine DNA

    PubMed Central

    Hashimoto, Hideharu; Pais, June E.; Dai, Nan; Corrêa, Ivan R.; Zhang, Xing; Zheng, Yu; Cheng, Xiaodong

    2015-01-01

    The family of ten-eleven translocation (Tet) dioxygenases is widely distributed across the eukaryotic tree of life, from mammals to the amoeboflagellate Naegleria gruberi. Like mammalian Tet proteins, the Naegleria Tet-like protein, NgTet1, acts on 5-methylcytosine (5mC) and generates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) in three consecutive, Fe(II)- and α-ketoglutarate-dependent oxidation reactions. The two intermediates, 5hmC and 5fC, could be considered either as the reaction product of the previous enzymatic cycle or the substrate for the next cycle. Here we present a new crystal structure of NgTet1 in complex with DNA containing a 5hmC. Along with the previously solved NgTet1–5mC structure, the two complexes offer a detailed picture of the active site at individual stages of the reaction cycle. In the crystal, the hydroxymethyl (OH-CH2-) moiety of 5hmC points to the metal center, representing the reaction product of 5mC hydroxylation. The hydroxyl oxygen atom could be rotated away from the metal center, to a hydrophobic pocket formed by Ala212, Val293 and Phe295. Such rotation turns the hydroxyl oxygen atom away from the product conformation, and exposes the target CH2 towards the metal-ligand water molecule, where a dioxygen O2 molecule would occupy to initiate the next round of reaction by abstracting a hydrogen atom from the substrate. The Ala212-to-Val (A212V) mutant profoundly limits the product to 5hmC, probably because the reduced hydrophobic pocket size restricts the binding of 5hmC as a substrate. PMID:26323320

  8. Structure of Naegleria Tet-like dioxygenase (NgTet1) in complexes with a reaction intermediate 5-hydroxymethylcytosine DNA

    SciTech Connect

    Hashimoto, Hideharu; Pais, June E.; Dai, Nan; Corrêa, Jr., Ivan R.; Zhang, Xing; Zheng, Yu; Cheng, Xiaodong

    2015-08-31

    The family of ten-eleven translocation (Tet) dioxygenases is widely distributed across the eukaryotic tree of life, from mammals to the amoeboflagellate Naegleria gruberi. Like mammalian Tet proteins, the Naegleria Tet-like protein, NgTet1, acts on 5-methylcytosine (5mC) and generates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) in three consecutive, Fe(II)- and α-ketoglutarate-dependent oxidation reactions. The two intermediates, 5hmC and 5fC, could be considered either as the reaction product of the previous enzymatic cycle or the substrate for the next cycle. Here we present a new crystal structure of NgTet1 in complex with DNA containing a 5hmC. Along with the previously solved NgTet1–5mC structure, the two complexes offer a detailed picture of the active site at individual stages of the reaction cycle. In the crystal, the hydroxymethyl (OH-CH2-) moiety of 5hmC points to the metal center, representing the reaction product of 5mC hydroxylation. The hydroxyl oxygen atom could be rotated away from the metal center, to a hydrophobic pocket formed by Ala212, Val293 and Phe295. Such rotation turns the hydroxyl oxygen atom away from the product conformation, and exposes the target CH2 towards the metal-ligand water molecule, where a dioxygen O2 molecule would occupy to initiate the next round of reaction by abstracting a hydrogen atom from the substrate. The Ala212-to-Val (A212V) mutant profoundly limits the product to 5hmC, probably due to the reduced hydrophobic pocket size restricts the binding of 5hmC as a substrate.

  9. Structure of Naegleria Tet-like dioxygenase (NgTet1) in complexes with a reaction intermediate 5-hydroxymethylcytosine DNA

    DOE PAGESBeta

    Hashimoto, Hideharu; Pais, June E.; Dai, Nan; Corrêa, Jr., Ivan R.; Zhang, Xing; Zheng, Yu; Cheng, Xiaodong

    2015-08-31

    The family of ten-eleven translocation (Tet) dioxygenases is widely distributed across the eukaryotic tree of life, from mammals to the amoeboflagellate Naegleria gruberi. Like mammalian Tet proteins, the Naegleria Tet-like protein, NgTet1, acts on 5-methylcytosine (5mC) and generates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) in three consecutive, Fe(II)- and α-ketoglutarate-dependent oxidation reactions. The two intermediates, 5hmC and 5fC, could be considered either as the reaction product of the previous enzymatic cycle or the substrate for the next cycle. Here we present a new crystal structure of NgTet1 in complex with DNA containing a 5hmC. Along with the previously solvedmore » NgTet1–5mC structure, the two complexes offer a detailed picture of the active site at individual stages of the reaction cycle. In the crystal, the hydroxymethyl (OH-CH2-) moiety of 5hmC points to the metal center, representing the reaction product of 5mC hydroxylation. The hydroxyl oxygen atom could be rotated away from the metal center, to a hydrophobic pocket formed by Ala212, Val293 and Phe295. Such rotation turns the hydroxyl oxygen atom away from the product conformation, and exposes the target CH2 towards the metal-ligand water molecule, where a dioxygen O2 molecule would occupy to initiate the next round of reaction by abstracting a hydrogen atom from the substrate. The Ala212-to-Val (A212V) mutant profoundly limits the product to 5hmC, probably due to the reduced hydrophobic pocket size restricts the binding of 5hmC as a substrate.« less

  10. Structural and mutation studies of two DNA demethylation related glycosylases: MBD4 and TDG

    PubMed Central

    Hashimoto, Hideharu

    2014-01-01

    Two mammalian DNA glycosylases, methyl-CpG binding domain protein 4 (MBD4) and thymine DNA glycosylase (TDG), are involved in active DNA demethylation via the base excision repair pathway. Both MBD4 and TDG excise the mismatch base from G:X, where X is uracil, thymine, and 5-hydroxymethyluracil (5hmU). In addition, TDG excises 5mC oxidized bases i.e. when X is 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) not 5-hydroxymethylcytosine (5hmC). A MBD4 inactive mutant and substrate crystal structure clearly explains how MBD4 glycosylase discriminates substrates: 5mC are not able to be directly excised, but a deamination process from 5mC to thymine is required. On the other hand, TDG is much more complicated; in this instance, crystal structures show that TDG recognizes G:X mismatch DNA containing DNA and G:5caC containing DNA from the minor groove of DNA, which suggested that TDG might recognize 5mC oxidized product 5caC like mismatch DNA. In mutation studies, a N157D mutation results in a more 5caC specific glycosylase, and a N191A mutation inhibits 5caC activity while that when X=5fC or T remains. Here I revisit the recent MBD4 glycos ylase domain co-crystal structures with DNA, as well as TDG glycosylase domain co-crystal structures with DNA in conjunction with its mutation studies.

  11. Structure of Naegleria Tet-like dioxygenase (NgTet1) in complexes with a reaction intermediate 5-hydroxymethylcytosine DNA.

    PubMed

    Hashimoto, Hideharu; Pais, June E; Dai, Nan; Corrêa, Ivan R; Zhang, Xing; Zheng, Yu; Cheng, Xiaodong

    2015-12-15

    The family of ten-eleven translocation (Tet) dioxygenases is widely distributed across the eukaryotic tree of life, from mammals to the amoeboflagellate Naegleria gruberi. Like mammalian Tet proteins, the Naegleria Tet-like protein, NgTet1, acts on 5-methylcytosine (5mC) and generates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) in three consecutive, Fe(II)- and α-ketoglutarate-dependent oxidation reactions. The two intermediates, 5hmC and 5fC, could be considered either as the reaction product of the previous enzymatic cycle or the substrate for the next cycle. Here we present a new crystal structure of NgTet1 in complex with DNA containing a 5hmC. Along with the previously solved NgTet1-5mC structure, the two complexes offer a detailed picture of the active site at individual stages of the reaction cycle. In the crystal, the hydroxymethyl (OH-CH2-) moiety of 5hmC points to the metal center, representing the reaction product of 5mC hydroxylation. The hydroxyl oxygen atom could be rotated away from the metal center, to a hydrophobic pocket formed by Ala212, Val293 and Phe295. Such rotation turns the hydroxyl oxygen atom away from the product conformation, and exposes the target CH2 towards the metal-ligand water molecule, where a dioxygen O2 molecule would occupy to initiate the next round of reaction by abstracting a hydrogen atom from the substrate. The Ala212-to-Val (A212V) mutant profoundly limits the product to 5hmC, probably because the reduced hydrophobic pocket size restricts the binding of 5hmC as a substrate. PMID:26323320

  12. Base-resolution profiling of active DNA demethylation using MAB-seq and caMAB-seq.

    PubMed

    Wu, Hao; Wu, Xiaoji; Zhang, Yi

    2016-06-01

    A complete understanding of the function of the ten-eleven translocation (TET) family of dioxygenase-mediated DNA demethylation requires new methods to quantitatively map oxidized 5-methylcytosine (5mC) bases at high resolution. We have recently developed a methylase-assisted bisulfite sequencing (MAB-seq) method that allows base-resolution mapping of 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), two oxidized 5mC bases indicative of active DNA demethylation events. In standard bisulfite sequencing (BS-seq), unmodified C, 5fC and 5caC are read as thymine; thus 5fC and 5caC cannot be distinguished from C. In MAB-seq, unmodified C is enzymatically converted to 5mC, allowing direct mapping of rare modifications such as 5fC and 5caC. By combining MAB-seq with chemical reduction of 5fC to 5hmC, we also developed caMAB-seq, a method for direct 5caC mapping. Compared with subtraction-based mapping methods, MAB-seq and caMAB-seq require less sequencing effort and enable robust statistical calling of 5fC and/or 5caC. MAB-seq and caMAB-seq can be adapted to map 5fC/5caC at the whole-genome scale (WG-MAB-seq), within specific genomic regions enriched for enhancer-marking histone modifications (chromatin immunoprecipitation (ChIP)-MAB-seq), or at CpG-rich sequences (reduced-representation (RR)-MAB-seq) such as gene promoters. The full protocol, including DNA preparation, enzymatic treatment, library preparation and sequencing, can be completed within 6-8 d. PMID:27172168

  13. Mechanistic insights into the recognition of 5-methylcytosine oxidation derivatives by the SUVH5 SRA domain

    PubMed Central

    Rajakumara, Eerappa; Nakarakanti, Naveen Kumar; Nivya, M. Angel; Satish, Mutyala

    2016-01-01

    5-Methylcytosine (5 mC) is associated with epigenetic gene silencing in mammals and plants. 5 mC is consecutively oxidized to 5-hydroxymethylcytosine (5 hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by ten-eleven translocation enzymes. We performed binding and structural studies to investigate the molecular basis of the recognition of the 5 mC oxidation derivatives in the context of a CG sequence by the SET- and RING-associated domain (SRA) of the SUVH5 protein (SUVH5 SRA). Using calorimetric measurements, we demonstrate that the SRA domain binds to the hydroxymethylated CG (5hmCG) DNA duplex in a similar manner to methylated CG (5mCG). Interestingly, the SUVH5 SRA domain exhibits weaker affinity towards carboxylated CG (5caCG) and formylated CG (5fCG). We report the 2.6 Å resolution crystal structure of the SUVH5 SRA domain in a complex with fully hydroxymethyl-CG and demonstrate a dual flip-out mechanism, whereby the symmetrical 5hmCs are simultaneously extruded from the partner strands of the DNA duplex and are positioned within the binding pockets of individual SRA domains. The hydroxyl group of 5hmC establishes both intra- and intermolecular interactions in the binding pocket. Collectively, we show that SUVH5 SRA recognizes 5hmC in a similar manner to 5 mC, but exhibits weaker affinity towards 5 hmC oxidation derivatives. PMID:26841909

  14. Stable 5-Hydroxymethylcytosine (5hmC) Acquisition Marks Gene Activation During Chondrogenic Differentiation.

    PubMed

    Taylor, Sarah Eb; Li, Ye Henry; Smeriglio, Piera; Rath, Madhusikta; Wong, Wing H; Bhutani, Nidhi

    2016-03-01

    Regulation of gene expression changes during chondrogenic differentiation by DNA methylation and demethylation is little understood. Methylated cytosines (5mC) are oxidized by the ten-eleven-translocation (TET) proteins to 5-hydroxymethylcytosines (5hmC), 5-formylcytosines (5fC), and 5-carboxylcytosines (5caC), eventually leading to a replacement by unmethylated cytosines (C), ie, DNA demethylation. Additionally, 5hmC is stable and acts as an epigenetic mark by itself. Here, we report that global changes in 5hmC mark chondrogenic differentiation in vivo and in vitro. Tibia anlagen and growth plate analyses during limb development at mouse embryonic days E 11.5, 13.5, and 17.5 showed dynamic changes in 5hmC levels in the differentiating chondrocytes. A similar increase in 5hmC levels was observed in the ATDC5 chondroprogenitor cell line accompanied by increased expression of the TET proteins during in vitro differentiation. Loss of TET1 in ATDC5 decreased 5hmC levels and impaired differentiation, demonstrating a functional role for TET1-mediated 5hmC dynamics in chondrogenic differentiation. Global analyses of the 5hmC-enriched sequences during early and late chondrogenic differentiation identified 5hmC distribution to be enriched in the regulatory regions of genes preceding the transcription start site (TSS), as well as in the gene bodies. Stable gains in 5hmC were observed in specific subsets of genes, including genes associated with cartilage development and in chondrogenic lineage-specific genes. 5hmC gains in regulatory promoter and enhancer regions as well as in gene bodies were strongly associated with activated but not repressed genes, indicating a potential regulatory role for DNA hydroxymethylation in chondrogenic gene expression. © 2015 American Society for Bone and Mineral Research. PMID:26363184

  15. Arsenite Targets the Zinc Finger Domains of Tet Proteins and Inhibits Tet-Mediated Oxidation of 5-Methylcytosine.

    PubMed

    Liu, Shuo; Jiang, Ji; Li, Lin; Amato, Nicholas J; Wang, Zi; Wang, Yinsheng

    2015-10-01

    Arsenic toxicity is a serious public health problem worldwide that brings more than 100 million people into the risk of arsenic exposure from groundwater and food contamination. Although there is accumulating evidence linking arsenic exposure with aberrant cytosine methylation in the global genome or at specific genomic loci, very few have investigated the impact of arsenic on the oxidation of 5-methylcytosine (5-mC) mediated by the Ten-eleven translocation (Tet) family of proteins. Owing to the high binding affinity of As(III) toward cysteine residues, we reasoned that the highly conserved C3H-type zinc fingers situated in Tet proteins may constitute potential targets for arsenic binding. Herein, we found that arsenite could bind directly to the zinc fingers of Tet proteins in vitro and in cells, and this interaction substantially impaired the catalytic efficiency of Tet proteins in oxidizing 5-mC to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-foC), and 5-carboxylcytosine (5-caC). Treatments with arsenite also led to a dose-dependent decrease in the level of 5-hmC, but not 5-mC, in DNA isolated from HEK293T cells overexpressing the catalytic domain of any of the three Tet proteins and from mouse embryonic stem cells. Together, our study unveiled, for the first time, that arsenite could alter epigenetic signaling by targeting the zinc fingers of Tet proteins and perturbing the Tet-mediated oxidation of 5-mC in vitro and in cells. Our results offer important mechanistic understanding of arsenic epigenotoxicity and carcinogenesis in mammalian systems and may lead to novel approaches for the chemoprevention of arsenic toxicity. PMID:26355596

  16. TET2-mediated 5-hydroxymethylcytosine induces genetic instability and mutagenesis.

    PubMed

    Mahfoudhi, Emna; Talhaoui, Ibtissam; Cabagnols, Xenia; Della Valle, Véronique; Secardin, Lise; Rameau, Philippe; Bernard, Olivier A; Ishchenko, Alexander A; Abbes, Salem; Vainchenker, William; Saparbaev, Murat; Plo, Isabelle

    2016-07-01

    The family of Ten-Eleven Translocation (TET) proteins is implicated in the process of active DNA demethylation and thus in epigenetic regulation. TET 1, 2 and 3 proteins are oxygenases that can hydroxylate 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC) and further oxidize 5-hmC into 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). The base excision repair (BER) pathway removes the resulting 5-fC and 5-caC bases paired with a guanine and replaces them with regular cytosine. The question arises whether active modification of 5-mC residues and their subsequent elimination could affect the genomic DNA stability. Here, we generated two inducible cell lines (Ba/F3-EPOR, and UT7) overexpressing wild-type or catalytically inactive human TET2 proteins. Wild-type TET2 induction resulted in an increased level of 5-hmC and a cell cycle defect in S phase associated with higher level of phosphorylated P53, chromosomal and centrosomal abnormalities. Furthermore, in a thymine-DNA glycosylase (Tdg) deficient context, the TET2-mediated increase of 5-hmC induces mutagenesis characterized by GC>AT transitions in CpG context suggesting a mutagenic potential of 5-hmC metabolites. Altogether, these data suggest that TET2 activity and the levels of 5-hmC and its derivatives should be tightly controlled to avoid genetic and chromosomal instabilities. Moreover, TET2-mediated active demethylation might be a very dangerous process if used to entirely demethylate the genome and might rather be used only at specific loci. PMID:27289557

  17. Phosphorylation of TET Proteins Is Regulated via O-GlcNAcylation by the O-Linked N-Acetylglucosamine Transferase (OGT)*

    PubMed Central

    Bauer, Christina; Göbel, Klaus; Nagaraj, Nagarjuna; Colantuoni, Christian; Wang, Mengxi; Müller, Udo; Kremmer, Elisabeth; Rottach, Andrea; Leonhardt, Heinrich

    2015-01-01

    TET proteins oxidize 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine and thus provide a possible means for active DNA demethylation in mammals. Although their catalytic mechanism is well characterized and the catalytic dioxygenase domain is highly conserved, the function of the regulatory regions (the N terminus and the low-complexity insert between the two parts of the dioxygenase domains) is only poorly understood. Here, we demonstrate that TET proteins are subject to a variety of post-translational modifications that mostly occur at these regulatory regions. We mapped TET modification sites at amino acid resolution and show for the first time that TET1, TET2, and TET3 are highly phosphorylated. The O-linked GlcNAc transferase, which we identified as a strong interactor with all three TET proteins, catalyzes the addition of a GlcNAc group to serine and threonine residues of TET proteins and thereby decreases both the number of phosphorylation sites and site occupancy. Interestingly, the different TET proteins display unique post-translational modification patterns, and some modifications occur in distinct combinations. In summary, our results provide a novel potential mechanism for TET protein regulation based on a dynamic interplay of phosphorylation and O-GlcNAcylation at the N terminus and the low-complexity insert region. Our data suggest strong cross-talk between the modification sites that could allow rapid adaption of TET protein localization, activity, or targeting due to changing environmental conditions as well as in response to external stimuli. PMID:25568311

  18. Behavioral and molecular neuroepigenetic alterations in prenatally stressed mice: relevance for the study of chromatin remodeling properties of antipsychotic drugs.

    PubMed

    Dong, E; Tueting, P; Matrisciano, F; Grayson, D R; Guidotti, A

    2016-01-01

    We have recently reported that mice born from dams stressed during pregnancy (PRS mice), in adulthood, have behavioral deficits reminiscent of behaviors observed in schizophrenia (SZ) and bipolar (BP) disorder patients. Furthermore, we have shown that the frontal cortex (FC) and hippocampus of adult PRS mice, like that of postmortem chronic SZ patients, are characterized by increases in DNA-methyltransferase 1 (DNMT1), ten-eleven methylcytosine dioxygenase 1 (TET1) and exhibit an enrichment of 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC) at neocortical GABAergic and glutamatergic gene promoters. Here, we show that the behavioral deficits and the increased 5MC and 5HMC at glutamic acid decarboxylase 67 (Gad1), reelin (Reln) and brain-derived neurotrophic factor (Bdnf) promoters and the reduced expression of the messenger RNAs (mRNAs) and proteins corresponding to these genes in FC of adult PRS mice is reversed by treatment with clozapine (5 mg kg(-1) twice a day for 5 days) but not by haloperidol (1 mg kg(-1) twice a day for 5 days). Interestingly, clozapine had no effect on either the behavior, promoter methylation or the expression of these mRNAs and proteins when administered to offspring of nonstressed pregnant mice. Clozapine, but not haloperidol, reduced the elevated levels of DNMT1 and TET1, as well as the elevated levels of DNMT1 binding to Gad1, Reln and Bdnf promoters in PRS mice suggesting that clozapine, unlike haloperidol, may limit DNA methylation by interfering with DNA methylation dynamics. We conclude that the PRS mouse model may be useful preclinically in screening for the potential efficacy of antipsychotic drugs acting on altered epigenetic mechanisms. Furthermore, PRS mice may be invaluable for understanding the etiopathogenesis of SZ and BP disorder and for predicting treatment responses at early stages of the illness allowing for early detection and remedial intervention. PMID:26756904

  19. Ecology of dermatophytes and other keratinophilic fungi in swimming pools and polluted and unpolluted streams.

    PubMed

    Ali-Shtayeh, M S; Khaleel, Tayseer Kh M; Jamous, Rana M

    2002-01-01

    The biodiversity and richness of keratinophilic fungal communities including dermatophytes were assessed in three stream sites and three swimming pools in the Nablus district in Palestine, using hair baiting (HBT) and surface dilution plate (SDP) techniques, over 8- and 6-month periods, respectively. The effect of wastewater effluent and selected ecological factors on these fungi in relation to species diversity and population densities were also considered. Fifty keratinophilic fungal species were recovered from the aquatic habitats studied, of which 42 were recovered from stream sites and 22 from swimming pools. Of these fungi 6 were either dermatophytes (Microsporum gypseum, and Trichophyton mentagrophytes) or dermatophyte related species (Chrysosporium merdarium, Ch. tropicum, Ch. keratinophilum and T. terrestre). The most frequently isolated species in the three pools were Acremonium strictum and Cladosporium cladosporioides, using Sabouraud dextrose agar medium (SDA). The most abundant species were Acr. strictum, and Aspergillus flavus. However, only 4 species were isolated using the SDA medium amended with 5-flurocytosine (5-FC). The most frequent and abundant species in the three stream sites using SDA medium were Geotricum candidum, and Penicillium chrysogenum. The most frequent species in the three sites using the 5-FC medium, was Paecilomyces lilacinus. Using HBT, the most abundant and frequent species in the three stream sites were G. candidum, and Pa. lilacinus, on SDA medium, and Pa. lilacinus, and Gliocladium nigrovirens on the 5-FC medium. The 5-FC medium was more suitable for the isolation of dermatophytes and closely related species than the SDA medium; 6 were recovered on 5-FC, whereas only one on the SDA medium. Variation in the levels of keratinophilic fungal populations from the three stream sites sampled 5 times over an 8-month period, followed comparable fluctuation patterns. Wastewater affected fungal population densities with the highest

  20. Mutation of Escherichia coli cytosine deaminase significantly enhances molecular chemotherapy of human glioma.

    PubMed

    Kaliberov, S A; Market, J M; Gillespie, G Y; Krendelchtchikova, V; Della Manna, D; Sellers, J C; Kaliberova, L N; Black, M E; Buchsbaum, D J

    2007-07-01

    Combined treatment using adenoviral (Ad)-directed enzyme/prodrug therapy and radiation therapy has the potential to become a powerful method of cancer therapy. We have developed an Ad vector encoding a mutant bacterial cytosine deaminase (bCD) gene (AdbCD-D314A), which has a higher affinity for cytosine than wild-type bCD (bCDwt). The purpose of this study was to evaluate cytotoxicity in vitro and therapeutic efficacy in vivo of the combination of AdbCD-D314A with the prodrug 5-fluorocytosine (5-FC) and ionizing radiation against human glioma. The present study demonstrates that AdbCD-D314A infection resulted in increased 5-FC-mediated cell killing, compared with AdbCDwt. Furthermore, a significant increase in cytotoxicity following AdbCD-D314A and radiation treatment of glioma cells in vitro was demonstrated as compared to AdbCDwt. Animal studies showed significant inhibition of subcutaneous or intracranial tumor growth of D54MG glioma xenografts by the combination of AdbCD-D314A/5-FC with ionizing radiation as compared with either agent alone, and with AdbCDwt/5-FC plus radiation. The results suggest that the combination of AdbCD-D314A/5-FC with radiation produces markedly increased cytotoxic effects in cancer cells in vitro and in vivo. These data indicate that combined treatment with this novel mutant enzyme/prodrug therapy and radiotherapy provides a promising approach for cancer therapy. PMID:17495948

  1. Dihydropyrimidine Dehydrogenase Is a Prognostic Marker for Mesenchymal Stem Cell-Mediated Cytosine Deaminase Gene and 5-Fluorocytosine Prodrug Therapy for the Treatment of Recurrent Gliomas

    PubMed Central

    Chung, Taemoon; Na, Juri; Kim, Young-il; Chang, Da-Young; Kim, Young Il; Kim, Hyeonjin; Moon, Ho Eun; Kang, Keon Wook; Lee, Dong Soo; Chung, June-Key; Kim, Sung-Soo; Suh-Kim, Haeyoung; Paek, Sun Ha; Youn, Hyewon

    2016-01-01

    We investigated a therapeutic strategy for recurrent malignant gliomas using mesenchymal stem cells (MSC), expressing cytosine deaminase (CD), and prodrug 5-Fluorocytosine (5-FC) as a more specific and less toxic option. MSCs are emerging as a novel cell therapeutic agent with a cancer-targeting property, and CD is considered a promising enzyme in cancer gene therapy which can convert non-toxic 5-FC to toxic 5-Fluorouracil (5-FU). Therefore, use of prodrug 5-FC can minimize normal cell toxicity. Analyses of microarrays revealed that targeting DNA damage and its repair is a selectable option for gliomas after the standard chemo/radio-therapy. 5-FU is the most frequently used anti-cancer drug, which induces DNA breaks. Because dihydropyrimidine dehydrogenase (DPD) was reported to be involved in 5-FU metabolism to block DNA damage, we compared the survival rate with 5-FU treatment and the level of DPD expression in 15 different glioma cell lines. DPD-deficient cells showed higher sensitivity to 5-FU, and the regulation of DPD level by either siRNA or overexpression was directly related to the 5-FU sensitivity. For MSC/CD with 5-FC therapy, DPD-deficient cells such as U87MG, GBM28, and GBM37 showed higher sensitivity compared to DPD-high U373 cells. Effective inhibition of tumor growth was also observed in an orthotopic mouse model using DPD- deficient U87MG, indicating that DPD gene expression is indeed closely related to the efficacy of MSC/CD-mediated 5-FC therapy. Our results suggested that DPD can be used as a biomarker for selecting glioma patients who may possibly benefit from this therapy. PMID:27446484

  2. Hypoxia imaging predicts success of hypoxia-induced cytosine deaminase/5-fluorocytosine gene therapy in a murine lung tumor model.

    PubMed

    Lee, B-F; Lee, C-H; Chiu, N-T; Hsia, C-C; Shen, L-H; Shiau, A-L

    2012-04-01

    Tc-99m-HL91 is a hypoxia imaging biomarker. The aim of this study was to investigate the value of Tc-99m-HL91 imaging for hypoxia-induced cytosine deaminase (CD)/5-fluorocytosine (5-FC) gene therapy in a murine lung tumor model. C57BL/6 mice were implanted with Lewis lung carcinoma cells transduced with the hypoxia-inducible promoter-driven CD gene (LL2/CD) or luciferase gene (LL2/Luc) serving as the control. When tumor volumes reached 100 mm(3), pretreatment images were acquired after injection of Tc-99m-HL91. The mice were divided into low and high hypoxic groups based on the tumor-to-non-tumor ratio of Tc-99m-HL91. They were injected daily with 5-FC (500 mg kg(-1)) or the vehicle for 1 week. When tumor volumes reached 1000 mm(3), autoradiography and histological examinations were performed. Treatment with 5-FC delayed tumor growth and enhanced the survival of mice bearing high hypoxic LL2/CD tumors. The therapeutic effect of hypoxia-induced CD/5-FC gene therapy was more pronounced in high hypoxic tumors than in low hypoxic tumors. This study provides the first evidence that Tc-99m-HL91 can serve as an imaging biomarker for predicting the treatment responses of hypoxia-regulated CD/5-FC gene therapy in animal tumor models. Our results suggest that hypoxia imaging using Tc-99m-HL91 has the predictive value for the success of hypoxia-directed treatment regimens. PMID:22281757

  3. Cytosine Deaminase/5-Fluorocytosine Exposure Induces Bystander and Radiosensitization Effects in Hypoxic Glioblastoma Cells in vitro

    SciTech Connect

    Chen, Jennifer K.; Hu, Lily J.; Wang Dongfang; Lamborn, Kathleen R.; Deen, Dennis F. . E-mail: dennisdeen@juno.com

    2007-04-01

    Purpose: Treatment of glioblastoma (GBM) is limited by therapeutic ratio; therefore, successful therapy must be specifically cytotoxic to cancer cells. Hypoxic cells are ubiquitous in GBM, and resistant to radiation and chemotherapy, and, thus, are logical targets for gene therapy. In this study, we investigated whether cytosine deaminase (CD)/5-fluorocytosine (5-FC) enzyme/prodrug treatment induced a bystander effect (BE) and/or radiosensitization in hypoxic GBM cells. Methods and Materials: We stably transfected cells with a gene construct consisting of the SV40 minimal promoter, nine copies of a hypoxia-responsive element, and the yeast CD gene. During hypoxia, a hypoxia-responsive element regulates expression of the CD gene and facilitates the conversion of 5-FC to 5-fluorouracil, a highly toxic antimetabolite. We used colony-forming efficiency (CFE) and immunofluorescence assays to assess for BE in co-cultures of CD-expressing clone cells and parent, pNeo- or green fluorescent protein-stably transfected GBM cells. We also investigated the radiosensitivity of CD clone cells treated with 5-FC under hypoxic conditions, and we used flow cytometry to investigate treatment-induced cell cycle changes. Results: Both a large BE and radiosensitization occurred in GBM cells under hypoxic conditions. The magnitude of the BE depended on the number of transfected cells producing CD, the functionality of the CD, the administered concentration of 5-FC, and the sensitivity of cell type to 5-fluorouracil. Conclusion: Hypoxia-inducible CD/5-FC therapy in combination with radiation therapy shows both a pronounced BE and a radiosensitizing effect under hypoxic conditions.

  4. 5-Fluorocytosine combined with Fcy-hEGF fusion protein targets EGFR-expressing cancer cells

    SciTech Connect

    Lan, Keng-Hsueh; Shih, Yi-Sheng; Chang, Cheng Allen; Yen, Sang-Hue; Lan, Keng-Li

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer EGFR-expressing epithelial cancers account for significant portion of cancer deaths. Black-Right-Pointing-Pointer EGF-EGFR signaling pathway is validated as an important anticancer drug target. Black-Right-Pointing-Pointer EGF and Fcy fusion protein (Fcy-hEGF) can bind to EGFR and convert 5-FC to 5-FU. Black-Right-Pointing-Pointer Fcy-hEGF combined with 5-FC preferentially inhibits EGFR-expressing cells viability. -- Abstract: Human epithelial cancers account for approximately 50% of all cancer deaths. This type of cancer is characterized by excessive activation and expression of the epidermal growth factor receptor (EGFR). The EGFR pathway is critical for cancer cell proliferation, survival, metastasis and angiogenesis. The EGF-EGFR signaling pathway has been validated as an important anticancer drug target. Increasing numbers of targeted therapies against this pathway have been either approved or are currently under development. Here, we adopted a prodrug system that uses 5-fluorocytosine (5-FC) and human EGF (hEGF) fused with yeast cytosine deaminase (Fcy) to target EGFR-overexpressing cancer cells and to convert 5-FC to a significantly more toxic chemotherapeutic, 5-fluorouracil (5-FU). We cloned and purified the Fcy-hEGF fusion protein from Pichia pastoris yeast. This fusion protein specifically binds to EGFR with a similar affinity as hEGF, approximately 10 nM. Fcy-hEGF binds tightly to A431 and MDA-MB-468 cells, which overexpress EGFR, but it binds with a lower affinity to MDA-MB-231 and MCF-7, which express lower levels of EGFR. Similarly, the viability of EGFR-expressing cells was suppressed by Fcy-hEGF in the presence of increasing concentrations of 5-FC, and the IC{sub 50} values for A431 and MDA-MB-468 were approximately 10-fold lower than those of MDA-MB-231 and MCF-7. This novel prodrug system, Fcy-hEGF/5-FC, might represent a promising addition to the available class of inhibitors that specifically target EGFR

  5. [Establishment of lentivirus-mediated system of double suicide genes and its killing effects on K562 cells].

    PubMed

    Jiang, Yi-Rong; Liu, Chun-Sheng; Chen, Xue-Liang; Ma, Dao-Xin

    2004-02-01

    To establish lentivirus-mediated system of double suicide genes and explore its killing effects on K562 cells, lentivirus transfer vector for double suicide genes was constructed using molecular methods, three plasmids of lentivirus gene transfer vector system were transferred into packaging cell line 293T using lipofectine method, the transfer effect was observed through fluorescence microscopy, the lentivirus particles were observed by means of electron microscopy. High titer of lentivirus was harvested from the supernatant of virus-producing cell culture and concentrated by high-speed centrifugation with Poly-L-Lysine (PLL). The K562 cells were infected with the concentrated supernatant containing the virus with the double suicide genes. Fluorescence microscopy and RT- PCR confirmed the integration and expression of extraneous gene. The cytotoxicity to these transgenic cells treated with 5-FC and GCV was measured by MTT assays. The growth inhibition ratio (GIR) of cells and inhibition concentration 50 (IC(50)) were counted. After administration of GCV and 5-FC, the changes of those cells were observed through scanning electron microscope. The results showed that lentivirus transfer vector with double suicide genes was constructed successfully. The above-mentioned plasmids were effectively transferred into 293T cells. So much green fluorescence was observed through fluorescence microscope. A lot of lentivirus particles were observed through transmission electron microscope. Double suicide genes mediated by lentivirus were stably integrated and expressed in K562 cells after infection with the concentrated virus using fluorescence microscopy and RT-PCR. The GIR of K562 cells using GCV or 5-FC was 48.73% or 50.69% respectively and it was apparently higher than that of untransfected cells (P < 0.01). When using GCV and 5-FC together, the GIR was 87.69%, which was apparently higher than that of group using GCV or 5-FC alone (P < 0.01). In conclusion, lentivirus

  6. Pharmacodynamics of itraconazole against Aspergillus fumigatus in an in vitro model of the human alveolus: perspectives on the treatment of triazole-resistant infection and utility of airway administration.

    PubMed

    Al-Nakeeb, Zaid; Sudan, Ajay; Jeans, Adam R; Gregson, Lea; Goodwin, Joanne; Warn, Peter A; Felton, Timothy W; Howard, Susan J; Hope, William W

    2012-08-01

    Itraconazole is used for the prevention and treatment of infections caused by Aspergillus fumigatus. An understanding of the pharmacodynamics of itraconazole against wild-type and triazole-resistant strains provides a basis for innovative therapeutic strategies for treatment of infections. An in vitro model of the human alveolus was used to define the pharmacodynamics of itraconazole. Galactomannan was used as a biomarker. The effect of systemic and airway administration of itraconazole was assessed, as was a combination of itraconazole administered to the airway and systemically administered 5FC. Systemically administered itraconazole against the wild type induced a concentration-dependent decline in galactomannan in the alveolar and endothelial compartments. No exposure-response relationships were apparent for the L98H, M220T, or G138C mutant. The administration of itraconazole to the airway resulted in comparable exposure-response relationships to those observed with systemic therapy. This was achieved without detectable concentrations of drug within the endothelial compartment. The airway administration of itraconazole resulted in a definite but submaximal effect in the endothelial compartment against the L98H mutant. The administration of 5FC resulted in a concentration-dependent decline in galactomannan in both the alveolar and endothelial compartments. The combination of airway administration of itraconazole and systemically administered 5FC was additive. Systemic administration of itraconazole is ineffective against Cyp51 mutants. The airway administration of itraconazole is effective for the treatment of wild-type strains and appears to have some activity against the L98H mutants. Combination with other agents, such as 5FC, may enable the attainment of near-maximal antifungal activity. PMID:22615280

  7. Tumor-specific expression of shVEGF and suicide gene as a novel strategy for esophageal cancer therapy

    PubMed Central

    Liu, Ting; Wu, Hai-Jun; Liang, Yu; Liang, Xu-Jun; Huang, Hui-Chao; Zhao, Yan-Zhong; Liao, Qing-Chuan; Chen, Ya-Qi; Leng, Ai-Min; Yuan, Wei-Jian; Zhang, Gui-Ying; Peng, Jie; Chen, Yong-Heng

    2016-01-01

    AIM: To develop a potent and safe gene therapy for esophageal cancer. METHODS: An expression vector carrying fusion suicide gene (yCDglyTK) and shRNA against vascular endothelial growth factor (VEGF) was constructed and delivered into EC9706 esophageal cancer cells by calcium phosphate nanoparticles (CPNP). To achieve tumor selectivity, expression of the fusion suicide gene was driven by a tumor-specific human telomerase reverse transcriptase (hTERT) promoter. The biologic properties and therapeutic efficiency of the vector, in the presence of prodrug 5-fluorocytosine (5-FC), were evaluated in vitro and in vivo. RESULTS: Both in vitro and in vivo testing showed that the expression vector was efficiently introduced by CPNP into tumor cells, leading to cellular expression of yCDglyTK and decreased VEGF level. With exposure to 5-FC, it exhibited strong anti-tumor effects against esophageal cancer. Combination of VEGF shRNA with the fusion suicide gene demonstrated strong anti-tumor activity. CONCLUSION: The shVEGF-hTERT-yCDglyTK/5-FC system provided a novel approach for esophageal cancer-targeted gene therapy. PMID:27340350

  8. Suicidal gene therapy in the effective control of primary human hepatocellular carcinoma as monitored by noninvasive bioimaging.

    PubMed

    Sia, K C; Huynh, H; Chinnasamy, N; Hui, K M; Lam, P Y P

    2012-05-01

    Hepatocellular carcinoma (HCC) is usually refractory to the available treatments. For cancer gene therapy purposes, real-time imaging of therapeutic gene expression is of great importance because there are multiple factors that modulate the therapeutic gene expression in a complex tumor microenvironment. As a consequence, multiple doses of therapeutic viral vectors may be required for improved efficacy. In the present study, the luciferase reporter gene and the yeast cytosine deaminase (yCD) genes were bicistronically expressed using the foot-and-mouth disease virus 2A peptide under the regulation of the cytomegalovirus (CMV) promoter. The effectiveness of the yCD/5-FC (5-fluorocytosine) killing efficacy mediated by the herpes simplex virus type 1 (HSV-1) amplicon viral vector was shown using HCC and non-HCC cell lines in vitro. In addition, in vivo experiment also showed tumor regression of a primary HCC 26-1004 tumor xenograft in tumor expressing high levels of the yCD gene (as determined by noninvasive imaging) after intratumoral injection of 1.5 × 10(6) TU HGCX-L2C HSV-1 amplicon viral vector and 5-FC administration. The HSV-1 amplicon viral vector coupled with the yCD/5-FC prodrug activated suicide gene could potentially be of use in clinical gene therapy for HCC. PMID:21918545

  9. Safety and Efficacy of Suicide Gene Therapy with Adenosine Deaminase 5-Fluorocytosine Silmutaneously in in Vitro Cultures of Melanoma and Retinal Cell Lines

    PubMed Central

    Sakkas, Antonios; Zarogoulidis, Paul; Domvri, Kalliopi; Hohenforst-Schmidt, Wolfgang; Bougiouklis, Dimitris; Kakolyris, Stylianos; Zarampoukas, Thomas; Kioumis, Ioannis; Pitsiou, Georgia; Huang, Haidong; Li, Qiang; Meditskou, Soultana; Tsiouda, Theodora; Pezirkianidis, Nikolaos; Zarogoulidis, Konstantinos

    2014-01-01

    Local treatment as a treatment modality is gaining increased general acceptance over time. Novel drugs and methodologies of local administration are being investigated in an effort to achieve disease local control. Suicide gene therapy is a method that has been investigated as a local treatment with simultaneously distant disease control. In our current experiment we purchased HTB-70 (melanoma cell line, derived from metastatic axillary node) and CRL-2302 (human retinal epithelium) were from ATCC LGC Standards and Ancotil®, 2.5 g/250 ml (1 g/00ml) (5-Flucytosine) MEDA; Pharmaceuticals Ltd. UK. Adenosine Cytosine Deaminase (Ad.CD) was also used in order to convert the pro-drug 5-Flucytosine to the active 5-Fluoracil. Three different concentrations of 5-Flucytosine (5-FC) were administered (0.2ml, 0.8ml and 1.2ml). At indicated time-points (4h, 8h and 24h) cell viability and apoptosis were measured. Our concept was to investigate whether suicide gene therapy with Ad. CD-5-FC could be used with safety and efficiency as a future local treatment for melanoma located in the eye cavity. Indeed, our results indicated that in every 5-FC administration had mild cytotoxicity for the retinal cells, while increased apoptosis was observed for the melanoma cell line. PMID:24799955

  10. In vitro studies with combinations of 5-fluorocytosine and amphotericin B.

    PubMed

    Shadomy, S; Wagner, G; Espinel-Ingroff, E; Davis, B A

    1975-08-01

    Synergistic antifungal activity of 5-fluorocytosine (5-FC) and amphotericin B was studied using an abbreviated checkerboard titration scheme. 5-FC was titrated in twofold increments (100 to 0.05 mug/ml) in the absence and presence of graded increments of amphotericin B (1.0. 0.5, 0.1, 0.05, and 0.01 mug/ml) in buffered yeast nitrogen base. A limited number of experiments were performed using expanded dual titration checkerboard schemes and growth curve studies. Forty-eight isolates of yeastlike organisms were tested; two were inhibited by the buffer system. Evidence of synergy, as indicated by a fourfold or greater reduction of the minimal inhibitory concentration of 5-FC in the presence of subinhibitory concentrations of amphotericin B, was seen with 11 of 46 isolates, or 24%, at the fungistatic level and with three isolates, or 7% at the fungicidal level. Indifferent results were obtained for 44 and 74% of the isolates, respectively, at the fungistatic and fungicidal levels. Antagonism was observed with three isolates. PMID:1101814

  11. Design and selection of Toca 511 for clinical use: modified retroviral replicating vector with improved stability and gene expression.

    PubMed

    Perez, Omar D; Logg, Christopher R; Hiraoka, Kei; Diago, Oscar; Burnett, Ryan; Inagaki, Akihito; Jolson, Dawn; Amundson, Karin; Buckley, Taylor; Lohse, Dan; Lin, Amy; Burrascano, Cindy; Ibanez, Carlos; Kasahara, Noriyuki; Gruber, Harry E; Jolly, Douglas J

    2012-09-01

    Retroviral replicating vectors (RRVs) are a nonlytic alternative to oncolytic replicating viruses as anticancer agents, being selective both for dividing cells and for cells that have defects in innate immunity and interferon responsiveness. Tumor cells fit both these descriptions. Previous publications have described a prototype based on an amphotropic murine leukemia virus (MLV), encoding yeast cytosine deaminase (CD) that converts the prodrug 5-fluorocytosine (5-FC) to the potent anticancer drug, 5-fluorouracil (5-FU) in an infected tumor. We report here the selection of one lead clinical candidate based on a general design goal to optimize the genetic stability of the virus and the CD activity produced by the delivered transgene. Vectors were tested for titer, genetic stability, CD protein and enzyme activity, ability to confer susceptibility to 5-FC, and preliminary in vivo antitumor activity and stability. One vector, Toca 511, (aka T5.0002) encoding an optimized CD, shows a threefold increased specific activity in infected cells over infection with the prototype RRV and shows markedly higher genetic stability. Animal testing demonstrated that Toca 511 replicates stably in human tumor xenografts and, after 5-FC administration, causes complete regression of such xenografts. Toca 511 (vocimagene amiretrorepvec) has been taken forward to preclinical and clinical trials. PMID:22547150

  12. Safety and efficacy of suicide gene therapy with adenosine deaminase 5-fluorocytosine silmutaneously in in vitro cultures of melanoma and retinal cell lines.

    PubMed

    Sakkas, Antonios; Zarogoulidis, Paul; Domvri, Kalliopi; Hohenforst-Schmidt, Wolfgang; Bougiouklis, Dimitris; Kakolyris, Stylianos; Zarampoukas, Thomas; Kioumis, Ioannis; Pitsiou, Georgia; Huang, Haidong; Li, Qiang; Meditskou, Soultana; Tsiouda, Theodora; Pezirkianidis, Nikolaos; Zarogoulidis, Konstantinos

    2014-01-01

    Local treatment as a treatment modality is gaining increased general acceptance over time. Novel drugs and methodologies of local administration are being investigated in an effort to achieve disease local control. Suicide gene therapy is a method that has been investigated as a local treatment with simultaneously distant disease control. In our current experiment we purchased HTB-70 (melanoma cell line, derived from metastatic axillary node) and CRL-2302 (human retinal epithelium) were from ATCC LGC Standards and Ancotil(®), 2.5 g/250 ml (1 g/00ml) (5-Flucytosine) MEDA; Pharmaceuticals Ltd. UK. Adenosine Cytosine Deaminase (Ad.CD) was also used in order to convert the pro-drug 5-Flucytosine to the active 5-Fluoracil. Three different concentrations of 5-Flucytosine (5-FC) were administered (0.2ml, 0.8ml and 1.2ml). At indicated time-points (4h, 8h and 24h) cell viability and apoptosis were measured. Our concept was to investigate whether suicide gene therapy with Ad. CD-5-FC could be used with safety and efficiency as a future local treatment for melanoma located in the eye cavity. Indeed, our results indicated that in every 5-FC administration had mild cytotoxicity for the retinal cells, while increased apoptosis was observed for the melanoma cell line. PMID:24799955

  13. Quantification of 5-methylcytosine, 5-hydroxymethylcytosine and 5-carboxylcytosine from the blood of cancer patients by an Enzyme-based Immunoassay

    PubMed Central

    Chowdhury, Basudev; Cho, Il-Hoon; Hahn, Noah; Irudayaraj, Joseph

    2014-01-01

    Background Genome-wide aberrations of the classic epigenetic modification 5-methylcytosine (5mC), considered the hallmark of gene silencing, has been implicated to play a pivotal role in mediating carcinogenic transformation of healthy cells. Recently, three epigenetic marks derived from enzymatic oxidization of 5mC namely 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), have been discovered in the mammalian genome. Growing evidence suggests that these novel bases possess unique regulatory functions and may play critical roles in carcinogenesis. Methods To provide a quantitative basis for these rare epigenetic marks, we have designed a biotin-avidin mediated Enzyme-based Immunoassay (EIA) and evaluated its performance in genomic DNA isolated from blood of patients diagnosed with metastatic forms of lung, pancreatic and bladder cancer, as well as healthy controls. The proposed EIA incorporates spatially optimized biotinylated antibody and a high degree of horseradish-peroxidase (HRP) labeled streptavidin, facilitating signal amplification and sensitive detection. Results We report that the percentages of 5mC, 5hmC and 5caC present in the genomic DNA of blood in healthy controls as 1.025 + 0.081, 0.023 + 0.006 and 0.001 + 0.0002 respectively. We observed a significant (p<0.05) decrease in the mean global percentage of 5hmC in blood of patients with malignant lung cancer (0.013 + 0.003 %) in comparison to healthy controls. Conclusion The precise biological roles of these epigenetic modifications in cancers are still unknown but in the past two years it has become evident that the global 5hmC content is drastically reduced in a variety of cancers. To the best of our knowledge, this is the first report of decreased 5hmC content in the blood of metastatic lung cancer patients and the clinical utility of this observation needs to be further validated in larger sample datasets. PMID:25441900

  14. Role of glutamate 64 in the activation of the prodrug 5-fluorocytosine by yeast cytosine deaminase.

    PubMed

    Wang, Jifeng; Sklenak, Stepan; Liu, Aizhuo; Felczak, Krzysztof; Wu, Yan; Li, Yue; Yan, Honggao

    2012-01-10

    Yeast cytosine deaminase (yCD) catalyzes the hydrolytic deamination of cytosine to uracil as well as the deamination of the prodrug 5-fluorocytosine (5FC) to the anticancer drug 5-fluorouracil. In this study, the role of Glu64 in the activation of the prodrug 5FC was investigated by site-directed mutagenesis, biochemical, nuclear magnetic resonance (NMR), and computational studies. Steady-state kinetics studies showed that the mutation of Glu64 causes a dramatic decrease in k(cat) and a dramatic increase in K(m), indicating Glu64 is important for both binding and catalysis in the activation of 5FC. (19)F NMR experiments showed that binding of the inhibitor 5-fluoro-1H-pyrimidin-2-one (5FPy) to the wild-type yCD causes an upfield shift, indicating that the bound inhibitor is in the hydrated form, mimicking the transition state or the tetrahedral intermediate in the activation of 5FC. However, binding of 5FPy to the E64A mutant enzyme causes a downfield shift, indicating that the bound 5FPy remains in an unhydrated form in the complex with the mutant enzyme. (1)H and (15)N NMR analysis revealed trans-hydrogen bond D/H isotope effects on the hydrogen of the amide of Glu64, indicating that the carboxylate of Glu64 forms two hydrogen bonds with the hydrated 5FPy. ONIOM calculations showed that the wild-type yCD complex with the hydrated form of the inhibitor 1H-pyrimidin-2-one is more stable than the initial binding complex, and in contrast, with the E64A mutant enzyme, the hydrated inhibitor is no longer favored and the conversion has a higher activation energy, as well. The hydrated inhibitor is stabilized in the wild-type yCD by two hydrogen bonds between it and the carboxylate of Glu64 as revealed by (1)H and (15)N NMR analysis. To explore the functional role of Glu64 in catalysis, we investigated the deamination of cytosine catalyzed by the E64A mutant by ONIOM calculations. The results showed that without the assistance of Glu64, both proton transfers before and

  15. Oncosuppressive Suicide Gene Virotherapy “PVH1-yCD/5-FC” for Pancreatic Peritoneal Carcinomatosis Treatment: NFκB and Akt/PI3K Involvement

    PubMed Central

    Réjiba, Soukaina; Bigand, Christelle; Parmentier, Celine; Masmoudi, Ahmed; Hajri, Amor

    2013-01-01

    Peritoneal carcinomatosis is common in advanced pancreatic cancer. Despite current standard treatment, patients with this disease until recently were considered incurable. Cancer gene therapy using oncolytic viruses have generated much interest over the past few years. Here, we investigated a new gene directed enzyme prodrug therapy (GDEPT) approach for an oncosuppressive virotherapy strategy using parvovirus H1 (PV-H1) which preferentially replicates and kills malignant cells. Although, PV-H1 is not potent enough to destroy tumors, it represents an attractive vector for cancer gene therapy. We therefore sought to determine whether the suicide gene/prodrug system, yCD/5-FC could be rationally combined to PV-H1 augmenting its intrinsic oncolytic activity for pancreatic cancer prevention and treatment. We showed that the engineered recombinant parvovirus rPVH1-yCD with 5-FC treatment increased significantly the intrinsic cytotoxic effect and resulted in potent induction of apoptosis and tumor growth inhibition in chemosensitive and chemoresistant cells. Additionally, the suicide gene-expressing PV-H1 infection reduced significantly the constitutive activities of NFκB and Akt/PI3K. Combination of their pharmacological inhibitors (MG132 and LY294002) with rPVH1-yCD/5-FC resulted in substantial increase of antitumor activity. In vivo, high and sustained expression of NS1 and yCD was observed in the disseminated tumor nodules and absent in normal tissues. Treatment of mice bearing intraperitoneal pancreatic carcinomatosis with rPVH1-yCD/5-FC resulted in a drastic inhibition of tumor cell spreading and subsequent increase in long-term survival. Together, the presented data show the improved oncolytic activity of wPV-H1 by yCD/5-FC and thus provides valuable effective and promising virotherapy strategy for prevention of tumor recurrence and treatment. In the light of this study, the suicide gene parvovirotherapy approach represents a new weapon in the war against

  16. Role of Glutamate 64 in the Activation of the Prodrug 5-fluorocytosine by Yeast Cytosine Deaminase†

    PubMed Central

    Wang, Jifeng; Sklenak, Stepan; Liu, Aizhuo; Felczak, Krzysztof; Wu, Yan; Li, Yue; Yan, Honggao

    2012-01-01

    Yeast cytosine deaminase catalyzes the hydrolytic deamination of cytosine to uracil as well as the deamination of the prodrug 5-fluorocytosine (5FC) to the anticancer drug 5-fluorouracil. In this study, the role of Glu64 in the activation of the prodrug 5FC was investigated by site-directed mutagenesis, biochemical, NMR, and computational studies. Steady-state kinetics studies showed that the mutation of Glu64 causes a dramatic decrease in kcat and a dramatic increase in Km, indicating Glu64 is important for both binding and catalysis in the activation of 5FC. 19F-NMR experiments showed that binding of the inhibitor 5-fluoro-1H-pyrimidin-2-one (5FPy) to the wild type yCD causes an upfield shift, indicating that the bound inhibitor is in the hydrated form, mimicking the transition state or the tetrahedral intermediate in the activation of 5FC. However, binding of 5FPy to the E64A mutant enzyme causes a downfield shift, indicating that the bound 5FPy remains in an unhydrated form in the complex with the mutant enzyme. 1H and 15N NMR analysis revealed trans-hydrogen-bond D/H isotope effects on the hydrogen of the amide of Glu64, indicating that the carboxylate of Glu64 forms two hydrogen bonds with the hydrated 5FPy. ONIOM calculations showed that the wild type yCD complex with the hydrated form of the inhibitor 1H-pyrimidin-2-one is more stable than the initial binding complex, and in contrast, with the E64A mutant enzyme, the hydrated inhibitor is no longer favored and the conversion has higher activation energy as well. The hydrated inhibitor is stabilized in the wild-type yCD by two hydrogen bonds between it and the carboxylate of Glu64 as revealed by 1H and 15N NMR analysis. To explore the functional role of Glu64 in catalysis, deamination of cytosine catalyzed by the E64A mutant was investigated by ONIOM calculations. The results showed that without the assistance of Glu64, both proton transfers before and after the formation of the tetrahedral reaction

  17. Acute Infections, Cost per Infection and Turnaround Time in Three United States Hospital Laboratories Using Fourth-Generation Antigen-Antibody Human Immunodeficiency Virus Immunoassays.

    PubMed

    Wesolowski, Laura G; Nasrullah, Muazzam; Coombs, Robert W; Rosenberg, Eric; Ethridge, Steven F; Hutchinson, Angela B; Dragavon, Joan; Rychert, Jennifer; Nolte, Frederick S; Madory, James E; Werner, Barbara G

    2016-01-01

    Background.  To improve clinical and public health outcomes through early human immunodeficiency virus (HIV) detection, fourth-generation antigen/antibody immunoassay (4IA) and supplemental testing results must be returned rapidly. Methods.  We examined HIV testing data at Harborview Medical Center (HMC), Massachusetts General Hospital (MGH), and the Medical University of South Carolina (MUSC), which used 4IA and supplemental antibody and nucleic acid tests (NATs). At MGH and MUSC, HIV-1 Western blot (WB) and HIV-2 testing were conducted at a reference laboratory. We compared time from specimen collection to laboratory result for established (positive WB) and acute infections (reactive 4IA, negative/indeterminate WB, detectable NAT), and we calculated testing cost per positive-test result. Results.  From 3731 (MUSC) to 19 774 (MGH) tests were conducted; 0.01% (MGH) to 0.05% (HMC) were acute infections. Each laboratory had reactive 4IA, WB-negative, or indeterminate specimens without NAT (ie, potential acute infections). Time to result was 1.5 (HMC) to 5.2 days (MGH) for acute and 1.0 (HMC) to 5.2 days (MGH) for established infections. Costs were $1054 (MGH) to $1521 (MUSC). Conclusions.  Conducting supplemental testing in-house lowered turnaround times, which may be further reduced with rapid HIV-1/HIV-2 differentiation tests. Hospitals may benefit from quantitative NATs not requiring physician orders, so all potential acute infections receive NAT. PMID:26798766

  18. Analysis of a transgenic Oct4 enhancer reveals high fidelity long-range chromosomal interactions

    PubMed Central

    Cai, Mingyang; Gao, Fan; Zhang, Peilin; An, Woojin; Shi, Jiandang; Wang, Kai; Lu, Wange

    2015-01-01

    Genome structure or nuclear organization has fascinated researchers investigating genome function. Recently, much effort has gone into defining relationships between specific genome structures and gene expression in pluripotent cells. We previously analyzed chromosomal interactions of the endogenous Oct4 distal enhancer in pluripotent cells. Here, we derive ES and iPS cells from a transgenic Oct4 distal enhancer reporter mouse. Using sonication-based Circularized Chromosome Conformation Capture (4C) coupled with next generation sequencing, we determined and compared the genome-wide interactome of the endogenous and transgenic Oct4 distal enhancers. Integrative genomic analysis indicated that the transgenic enhancer binds to a similar set of loci and shares similar key enrichment profiles with its endogenous counterpart. Both the endogenous and transgenic Oct4 enhancer interacting loci were enriched in the open nucleus compartment, which is associated with active histone marks (H3K4me1, H3K27ac, H3K4me3 and H3K9ac), active cis-regulatory sequences (DNA hypersensitivity sites (DHS)), 5-hydroxymethylcytosine (5-hmc), and early DNA replication domains. In addition, binding of some pluripotency-related transcription factors was consistently enriched in our 4C sites, and genes in those sites were generally more highly expressed. Overall, our work reveals critical features that may function in gene expression regulation in mouse pluripotent cells. PMID:26435056

  19. Acute Infections, Cost per Infection and Turnaround Time in Three United States Hospital Laboratories Using Fourth-Generation Antigen-Antibody Human Immunodeficiency Virus Immunoassays

    PubMed Central

    Wesolowski, Laura G.; Nasrullah, Muazzam; Coombs, Robert W.; Rosenberg, Eric; Ethridge, Steven F.; Hutchinson, Angela B.; Dragavon, Joan; Rychert, Jennifer; Nolte, Frederick S.; Madory, James E.; Werner, Barbara G.

    2016-01-01

    Background. To improve clinical and public health outcomes through early human immunodeficiency virus (HIV) detection, fourth-generation antigen/antibody immunoassay (4IA) and supplemental testing results must be returned rapidly. Methods. We examined HIV testing data at Harborview Medical Center (HMC), Massachusetts General Hospital (MGH), and the Medical University of South Carolina (MUSC), which used 4IA and supplemental antibody and nucleic acid tests (NATs). At MGH and MUSC, HIV-1 Western blot (WB) and HIV-2 testing were conducted at a reference laboratory. We compared time from specimen collection to laboratory result for established (positive WB) and acute infections (reactive 4IA, negative/indeterminate WB, detectable NAT), and we calculated testing cost per positive-test result. Results. From 3731 (MUSC) to 19 774 (MGH) tests were conducted; 0.01% (MGH) to 0.05% (HMC) were acute infections. Each laboratory had reactive 4IA, WB-negative, or indeterminate specimens without NAT (ie, potential acute infections). Time to result was 1.5 (HMC) to 5.2 days (MGH) for acute and 1.0 (HMC) to 5.2 days (MGH) for established infections. Costs were $1054 (MGH) to $1521 (MUSC). Conclusions. Conducting supplemental testing in-house lowered turnaround times, which may be further reduced with rapid HIV-1/HIV-2 differentiation tests. Hospitals may benefit from quantitative NATs not requiring physician orders, so all potential acute infections receive NAT. PMID:26798766

  20. Cytotoxic effect of replication-competent adenoviral vectors carrying L-plastin promoter regulated E1A and cytosine deaminase genes in cancers of the breast, ovary and colon.

    PubMed

    Akbulut, Hakan; Zhang, Lixin; Tang, Yucheng; Deisseroth, Albert

    2003-05-01

    Prodrug activating transcription unit gene therapy is one of several promising approaches to cancer gene therapy. Combining that approach with conditionally replication-competent viral vectors that are truly tumor specific has been an important objective of recent work. In this study, we report the construction of a new conditionally replication-competent bicistronic adenoviral vector in which the cytosine deaminase (CD) gene and the E1a gene are driven by the L-plastin tumor-specific promoter (AdLpCDIRESE1a). A similar vector driven by the CMV promoter has also been constructed (AdCMVCDIRESE1a) as a control. We have carried out in vitro cytotoxicity in carcinomas of the breast, ovary and colon, and in vivo efficacy studies with these vectors in an animal model of colon cancer. While the addition of the AdLpCDIRESE1a vector to established cancer cell lines showed significant cytotoxicity in tumor cells derived from carcinomas of the breast (MCF-7), colon (HTB-38) and ovary (Ovcar 5), no significant toxicity was seen in explant cultures of normal human mammary epithelial cells (HMEC) exposed to this vector. The addition of 5-fluorocytosine (5FC) significantly increased the cytotoxicity in an additive fashion of both the AdLpCDIRESE1a and AdCMVCDIRESE1a vectors as well as that of the AdLpCD replication incompetent vector to established tumor cell lines. However, no significant cytotoxicity was observed with the addition of 5FC to explant cultures of normal human mammary epithelial cells that had been exposed to the L-plastin-driven vectors. Studies with mixtures of infected and uninfected tumor cell lines showed that the established cancer cell lines infected with the AdLpCDIRESE1a vector generated significant toxicity to surrounding uninfected cells (the "bystander effect") even at a ratio of 0.25 of infected cells to infected + uninfected cells in the presence of 5FC. The injection of the AdLpCDIRESE1a vector into subcutaneous deposits of human tumor nodules in the

  1. A Novel Armed Oncolytic Measles Vaccine Virus for the Treatment of Cholangiocarcinoma

    PubMed Central

    Lange, Sebastian; Lampe, Johanna; Bossow, Sascha; Zimmermann, Martina; Neubert, Wolfgang; Bitzer, Michael

    2013-01-01

    Abstract Cholangiocarcinoma (CC) is curable only in early stages by complete surgical resection. Thus, in advanced disease stages in which a complete removal of the tumor mass is no longer possible and palliative chemotherapy achieves only modest success, therapeutics employing new methods of action are desperately needed. Oncolytic viruses employed in clinical studies have been shown to spread preferentially in cancer cells. Beyond that, virotherapeutic cell killing can be enhanced by virus-based expression of suicide genes. We engineered a measles vaccine virus (MeV) vector expressing super cytosine deaminase (SCD), a fusion protein of yeast cytosine deaminase and uracil phosphoribosyltransferase, which converts the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) and subsequently to 5-fluorouridine-monophosphate. This novel vector was evaluated using three different human-derived CC cell lines. In vitro, all CC cell lines were found to be permissive to MeV infection. Partial blocking of MeV-mediated oncolysis could be overcome by employment of the SCD transgene together with administration of 5-FC. In vivo, intratumoral application of SCD-armed MeV together with a systemic 5-FC treatment showed a significant reduction in tumor size in a TFK-1 xenograft mouse model when compared with virus-only treatment. In a second animal experiment employing a HuCCT1 xenograft tumor model, an enhanced SCD-armed MeV vector, in which the SCD transgene was expressed from a different genomic position, led not only to reduced tumor volumes, but also to a significant survival benefit. On the basis of these encouraging preclinical data on employment of SCD-armed MeV for the virotherapeutic treatment of chemotherapy-resistant CC, a clinical virotherapy trial is set up currently. PMID:23550539

  2. Yeast Cytosine Deaminase Mutants with Increased Thermostability Impart Sensitivity to 5-Fluorocytosine

    PubMed Central

    Stolworthy, Tiffany S.; Korkegian, Aaron M.; Willmon, Candice L.; Ardiani, Andressa; Cundiff, Jennifer; Stoddard, Barry L.; Black, Margaret E.

    2008-01-01

    SUMMARY Prodrug gene therapy (PGT) is a treatment strategy in which tumor cells are transfected with a 'suicide' gene that encodes a metabolic enzyme capable of converting a nontoxic prodrug into a potent cytotoxin. One of the most promising PGT enzymes is cytosine deaminase (CD), a microbial salvage enzyme that converts cytosine to uracil. CD also converts 5-fluorocytosine (5FC) to 5-fluorouracil (5FU), an inhibitor of DNA synthesis and RNA function. Over 150 studies of cytosine deaminase-mediated PGT applications have been reported since 2000, all using wild-type enzymes. However, various forms of cytosine deaminase are limited by inefficient turnover of 5FC and/or limited thermostability. In a previous study we stabilized and extended the half-life of yeast cytosine deaminase (yCD) by repacking of its hydrophobic core at several positions distant from the active site. Here we report that random mutagenesis of residues selected based on alignment with similar enzymes, followed by selection for enhanced sensitization to 5FC, also produces an enzyme variant (yCD-D92E) with elevated Tm values and increased activity half-life. The new mutation is located at the enzyme's dimer interface, indicating that independent mutational pathways can lead to an increase in the temperature that induces protein unfolding and aggregation in thermal denaturation experiments measured by circular dichroism spectroscopy, and an increase in the half-life of enzyme activity at physiological temperature, as well as more subtle effect on enzyme kinetics. Each independently derived set of mutations significantly improves the enzyme's performance in PGT assays both in cell culture and in animal models. PMID:18291415

  3. A novel armed oncolytic measles vaccine virus for the treatment of cholangiocarcinoma.

    PubMed

    Lange, Sebastian; Lampe, Johanna; Bossow, Sascha; Zimmermann, Martina; Neubert, Wolfgang; Bitzer, Michael; Lauer, Ulrich M

    2013-05-01

    Cholangiocarcinoma (CC) is curable only in early stages by complete surgical resection. Thus, in advanced disease stages in which a complete removal of the tumor mass is no longer possible and palliative chemotherapy achieves only modest success, therapeutics employing new methods of action are desperately needed. Oncolytic viruses employed in clinical studies have been shown to spread preferentially in cancer cells. Beyond that, virotherapeutic cell killing can be enhanced by virus-based expression of suicide genes. We engineered a measles vaccine virus (MeV) vector expressing super cytosine deaminase (SCD), a fusion protein of yeast cytosine deaminase and uracil phosphoribosyltransferase, which converts the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) and subsequently to 5-fluorouridine-monophosphate. This novel vector was evaluated using three different human-derived CC cell lines. In vitro, all CC cell lines were found to be permissive to MeV infection. Partial blocking of MeV-mediated oncolysis could be overcome by employment of the SCD transgene together with administration of 5-FC. In vivo, intratumoral application of SCD-armed MeV together with a systemic 5-FC treatment showed a significant reduction in tumor size in a TFK-1 xenograft mouse model when compared with virus-only treatment. In a second animal experiment employing a HuCCT1 xenograft tumor model, an enhanced SCD-armed MeV vector, in which the SCD transgene was expressed from a different genomic position, led not only to reduced tumor volumes, but also to a significant survival benefit. On the basis of these encouraging preclinical data on employment of SCD-armed MeV for the virotherapeutic treatment of chemotherapy-resistant CC, a clinical virotherapy trial is set up currently. PMID:23550539

  4. Antifungal Susceptibility Patterns of Opportunistic Fungi in the Genera Verruconis and Ochroconis

    PubMed Central

    Samerpitak, K.; Rijs, A. J. M. M.; Melchers, W. J. G.; Mouton, J. W.; Verweij, P. E.; de Hoog, G. S.

    2014-01-01

    Species of Verruconis and species of Ochroconis are dematiaceous fungi generally found in the environment but having the ability to infect humans, dogs, cats, poultry, and fish. This study presents the antifungal susceptibility patterns of these fungi at the species level. Forty strains originating from clinical and environmental sources were phylogenetically identified at the species level by using sequences of the ribosomal DNA internal transcribed spacer (rDNA ITS). In vitro antifungal susceptibility testing was performed against eight antifungals, using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method. The geometric mean MICs for amphotericin B (AMB), flucytosine (5FC), fluconazole (FLC), itraconazole (ITC), voriconazole (VRC), and posaconazole (POS) and minimum effective concentrations (MECs) for caspofungin (CAS) and anidulafungin (AFG) across the Ochroconis and Verruconis species were as follows, in increasing order. For Verruconis species, the values (μg/ml) were as follows: AFG, 0.04; POS, 0.25; ITC, 0.37; AMB, 0.50; CAS, 0.65; VRC, 0.96; 5FC, 10.45; and FLC, 47.25. For Ochroconis species, the values (μg/ml) were as follows: AFG, 0.06; POS, 0.11; CAS, 0.67; VRC, 2.76; ITC, 3.94; AMB, 5.68; 5FC, 34.48; and FLC, 61.33. Antifungal susceptibility of Ochroconis and Verruconis was linked with phylogenetic distance and thermotolerance. Echinocandins and POS showed the greatest in vitro activity, providing possible treatment options for Ochroconis and Verruconis infections. PMID:24687495

  5. Improved negative selection protocol for Plasmodium berghei in the rodent malarial model

    PubMed Central

    2012-01-01

    An improved methodology is presented here for transgenic Plasmodium berghei lines that express the negative selectable marker yFCU (a bifunctional protein that combines yeast cytosine deaminase and uridyl phosphoribosyl transferase (UPRT)) and substitutes delivery of selection drug 5-fluorocytosine (5FC) by intraperitoneal injection for administration via the drinking water of the mice. The improved methodology is shown to be as effective, less labour-intensive, reduces animal handling and animal numbers required for successful selection thereby contributing to two of the "three Rs" of animal experimentation, namely refinement and reduction. PMID:22463060

  6. Photochemical internalization (PCI) enhanced nonviral transfection of tumor suppressor and pro-drug activating genes; a potential treatment modality for gliomas

    NASA Astrophysics Data System (ADS)

    Wang, Frederick; Zamora, Genesis; Sun, Chung-Ho; Trinidad, Anthony; Berg, Kristian; Madsen, Steen; Kwon, Young Jik; Hirschberg, Henry

    2014-03-01

    The overall objective of the research is to investigate the utility of photochemical internalization for the enhanced nonviral transfection of genes into cells. We have examined, in detail, the evaluation of photochemical internalization (PCI) as a method for the non-viral introduction of the tumor suppressor gene PTEN and the PCI mediated transfection of the cytosine deaminase (CD) pro drug activating gene into glioma cell monolayers and multi-cell tumor spheroids. Expression of the CD gene within the target cell produces an enzyme that converts the nontoxic prodrug, 5-fluorocytosine (5-FC), to the toxic metabolite, 5-fluorouracil (5-FU).

  7. Endothelial Progenitor Cells Combined with Cytosine Deaminase-Endostatin for Suppression of Liver Carcinoma.

    PubMed

    Chen, Rong; Yu, Hui; An, Yan-Li; Chen, Hua-Jun; Jia, ZhenYu; Teng, Gao-Jun

    2016-06-01

    Transplantation of gene transfected endothelial progenitor cells (EPCs) provides a novel method for treatment of human tumors. To study treatment of hepatocellular carcinoma using cytosine deaminase (CD)- and endostatin (ES)-transfected endothelial progenitor cells (EPCs), mouse bone marrow-derived EPCs were cultured and transfected with Lenti6.3-CD-EGFP and Lenti6.3-ES-Monomer-DsRed labeled with superparamagnetic iron oxide (SPIO) nanoparticles. DiD (lipophilic fluorescent dye)-labeled EPCs were injected into normal mice and mice with liver carcinoma. The EPCs loaded with CD-ES were infused into the mice through caudal veins and tumor volumes were measured. The tumor volumes in the EPC + SPIO + CD/5-Fc + ES group were found to be smaller as a result and grew more slowly than those from the EPC + SPIO + LV (lentivirus, empty vector control) group. Survival times were also measured after infusion of the cells into the mice. The median survival time was found to be longer in the EPC + SPIO + CD/5-Fc + ES group than in the others. In conclusion, the EPCs transfected with CD-ES suppressed the liver carcinoma cells in vitro, migrated primarily to the carcinoma, inhibited tumor growth, and also extended the median survival time for the mice with liver carcinoma. PMID:27319212

  8. Macrophage mediated PCI enhanced gene-directed enzyme prodrug therapy

    NASA Astrophysics Data System (ADS)

    Christie, Catherine E.; Zamora, Genesis; Kwon, Young J.; Berg, Kristian; Madsen, Steen J.; Hirschberg, Henry

    2015-03-01

    Photochemical internalization (PCI) is a photodynamic therapy-based approach for improving the delivery of macromolecules and genes into the cell cytosol. Prodrug activating gene therapy (suicide gene therapy) employing the transduction of the E. coli cytosine deaminase (CD) gene into tumor cells, is a promising method. Expression of this gene within the target cell produces an enzyme that converts the nontoxic prodrug, 5-FC, to the toxic metabolite, 5-fluorouracil (5-FU). 5-FC may be particularly suitable for brain tumors, because it can readily cross the bloodbrain barrier (BBB). In addition the bystander effect, where activated drug is exported from the transfected cancer cells into the tumor microenvironment, plays an important role by inhibiting growth of adjacent tumor cells. Tumor-associated macrophages (TAMs) are frequently found in and around glioblastomas. Monocytes or macrophages (Ma) loaded with drugs, nanoparticles or photosensitizers could therefore be used to target tumors by local synthesis of chemo attractive factors. The basic concept is to combine PCI, to enhance the ex vivo transfection of a suicide gene into Ma, employing specially designed core/shell NP as gene carrier.

  9. The growth of brain tumors can be suppressed by multiple transplantation of mesenchymal stem cells expressing cytosine deaminase.

    PubMed

    Chang, Da-Young; Yoo, Seung-Wan; Hong, Youngtae; Kim, Sujeong; Kim, Se Joong; Yoon, Sung-Hwa; Cho, Kyung-Gi; Paek, Sun Ha; Lee, Young-Don; Kim, Sung-Soo; Suh-Kim, Haeyoung

    2010-10-15

    Suicide genes have recently emerged as an attractive alternative therapy for the treatment of various types of intractable cancers. The efficacy of suicide gene therapy relies on efficient gene delivery to target tissues and the localized concentration of final gene products. Here, we showed a potential ex vivo therapy that used mesenchymal stem cells (MSCs) as cellular vehicles to deliver a bacterial suicide gene, cytosine deaminase (CD) to brain tumors. MSCs were engineered to produce CD enzymes at various levels using different promoters. When co-cultured, CD-expressing MSCs had a bystander, anti-cancer effect on neighboring C6 glioma cells in proportion to the levels of CD enzymes that could convert a nontoxic prodrug, 5-fluorocytosine (5-FC) into cytotoxic 5-fluorouracil (5-FU) in vitro. Consistent with the in vitro results, for early stage brain tumors induced by intracranial inoculation of C6 cells, transplantation of CD-expressing MSCs reduced tumor mass in proportion to 5-FC dosages. However, for later stage, established tumors, a single treatment was insufficient, but only multiple transplantations were able to successfully repress tumor growth. Our findings indicate that the level of total CD enzyme activity is a critical parameter that is likely to affect the clinical efficacy for CD gene therapy. Our results also highlight the potential advantages of autograftable MSCs compared with other types of allogeneic stem cells for the treatment of recurrent glioblastomas through repetitive treatments. PMID:20473873

  10. Adipose Tissue–derived Mesenchymal Stem Cells Expressing Prodrug-converting Enzyme Inhibit Human Prostate Tumor Growth

    PubMed Central

    Cavarretta, Ilaria T; Altanerova, Veronika; Matuskova, Miroslava; Kucerova, Lucia; Culig, Zoran; Altaner, Cestmir

    2009-01-01

    The ability of human adipose tissue–derived mesenchymal stem cells (AT-MSCs), engineered to express the suicide gene cytosine deaminase::uracil phosphoribosyltransferase (CD::UPRT), to convert the relatively nontoxic 5-fluorocytosine (5-FC) into the highly toxic antitumor 5-fluorouracil (5-FU) together with their ability to track and engraft into tumors and micrometastases makes these cells an attractive tool to activate prodrugs directly within the tumor mass. In this study, we tested the feasibility and efficacy of these therapeutic cells to function as cellular vehicles of prodrug-activating enzymes in prostate cancer (PC) therapy. In in vitro migration experiments we have shown that therapeutic AT-MSCs migrated to all the prostate cell lines tested. In a pilot preclinical study, we observed that coinjections of human bone metastatic PC cells along with the transduced AT-MSCs into nude mice treated with 5-FC induced a complete tumor regression in a dose dependent manner or did not even allow the establishment of the tumor. More importantly, we also demonstrated that the therapeutic cells were effective in significantly inhibiting PC tumor growth after intravenous administration that is a key requisite for any clinical application of gene-directed enzyme prodrug therapies. PMID:19844197

  11. Chemovirotherapy of malignant melanoma with a targeted and armed oncolytic measles virus.

    PubMed

    Kaufmann, Johanna K; Bossow, Sascha; Grossardt, Christian; Sawall, Stefanie; Kupsch, Jörg; Erbs, Philippe; Hassel, Jessica C; von Kalle, Christof; Enk, Alexander H; Nettelbeck, Dirk M; Ungerechts, Guy

    2013-04-01

    Effective treatment modalities for advanced melanoma are desperately needed. An innovative approach is virotherapy, in which viruses are engineered to infect cancer cells, resulting in tumor cell lysis and an amplification effect by viral replication and spread. Ideally, tumor selectivity of these oncolytic viruses is already determined during viral cell binding and entry, which has not been reported for melanoma. We engineered an oncolytic measles virus entering melanoma cells through the high molecular weight melanoma-associated antigen (HMWMAA) and proved highly specific infection and spread in melanoma cells. We further enhanced this oncolytic virus by inserting the FCU1 gene encoding the yeast-derived prodrug convertases cytosine deaminase and uracil phosphoribosyltransferase. Combination treatment with armed and retargeted MV-FCU1-αHMWMAA and the prodrug 5-fluorocytosine (5-FC) led to effective prodrug conversion to 5-fluorouracil, extensive cytotoxicity to melanoma cells, and excessive bystander killing of noninfected cells. Importantly, HMWMAA-retargeted MV showed antitumor activity in a human xenograft mouse model, which was further increased by the FCU1/5-FC prodrug activation system. Finally, we demonstrated susceptibility of melanoma skin metastasis biopsies to HMWMAA-retargeted MV. The highly selective, entry-targeted and armed oncolytic virus MV-FCU1-αHMWMAA may become a potent building block of future melanoma therapies. PMID:23223133

  12. A double suicide gene system driven by vascular endothelial growth factor promoter selectively kills human hepatocellular carcinoma cells

    PubMed Central

    WU, KAI; YANG, LIUCHENG; HUANG, ZONGHAI; ZHAO, HAIJUN; WANG, JIANJUN; XU, SHUAI

    2016-01-01

    The aim of the present study was to investigate the selective killing effect on hepatocellular carcinoma (HCC) cells of an adenovirus (Ad)-mediated cytosine deaminase (CD) in combination with thymidine kinase (TK) suicide gene system, driven by the vascular endothelial growth factor promoter (VEGFp), in vitro and in vivo. A double suicide gene system with VEGFp, named Ad-VEGFp-CDglyTK, was constructed and transfected into human HCC cells (BEL-7402 or HepG2; the latter cell type is deficient in VEGF) and human umbilical vein vascular endothelial cells (HUVEC). Green fluorescent protein expression was detected by fluoroscopy to verify transfection efficiency, and CDglyTK gene expression was detected by reverse transcription-polymerase chain reaction (PCR). The selective killing effect of Ad-VEGFp-CDglyTK was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry (FCM) in vitro and by xenograft studies in vivo. PCR revealed that the transgenic CDglyTK gene was expressed in BEL-7402 cells and HUVEC, but not in HepG2 cells. The cell survival rate significantly decreased in line with increasing concentrations of the prodrugs, ganciclovir (GCV) alone, 5-fluorocytosine (5-FC) alone or a combination of the two, in HUVEC and BEL-7402 cells with the transfected CDglyTK gene, but not in untransfected HUVEC or BEL-7402 cells, or in transfected or untransfected HepG2 cells. This result was additionally confirmed by FCM. GCV and 5-FC inhibited the HUVEC and BEL-7402 cells containing the transfected CDglyTK gene and also inhibited adjacent unmodified cells via the ‘bystander effect’. No similar results were observed in HepG2 cells. Compared with the control group, tumors with the transfected CDglyTK gene were smaller and the microvessel density of the tumor tissue was significantly decreased. It was concluded that a combination TK/GCV and CD/5-FC suicide gene system driven by VEGFp may provide a promising treatment strategy for HCC

  13. Spectroscopy of the archetype colliding-wind binary WR 140 during the 2009 January periastron passage

    NASA Astrophysics Data System (ADS)

    Fahed, R.; Moffat, A. F. J.; Zorec, J.; Eversberg, T.; Chené, A. N.; Alves, F.; Arnold, W.; Bergmann, T.; Corcoran, M. F.; Correia Viegas, N. G.; Dougherty, S. M.; Fernando, A.; Frémat, Y.; Gouveia Carreira, L. F.; Hunger, T.; Knapen, J. H.; Leadbeater, R.; Marques Dias, F.; Martayan, C.; Morel, T.; Pittard, J. M.; Pollock, A. M. T.; Rauw, G.; Reinecke, N.; Ribeiro, J.; Romeo, N.; Sánchez-Gallego, J. R.; Dos Santos, E. M.; Schanne, L.; Stahl, O.; Stober, Ba.; Stober, Be.; Vollmann, K.; Williams, P. M.

    2011-11-01

    We present the results from the spectroscopic monitoring of WR 140 (WC7pd + O5.5fc) during its latest periastron passage in 2009 January. The observational campaign consisted of a constructive collaboration between amateur and professional astronomers. It took place at six locations, including Teide Observatory, Observatoire de Haute Provence, Dominion Astrophysical Observatory and Observatoire du Mont Mégantic. WR 140 is known as the archetype of colliding-wind binaries and it has a relatively long period (?8 yr) and high eccentricity (?0.9). We provide updated values for the orbital parameters, new estimates for the WR and O star masses and new constraints on the mass-loss rates and colliding-wind geometry.

  14. Comparison of the Sensititre YeastOne® dilution method with the Clinical Laboratory Standards Institute (CLSI) M27-A3 microbroth dilution reference method for determining MIC of eight antifungal agents on 102 yeast strains.

    PubMed

    Bertout, S; Dunyach, C; Drakulovski, P; Reynes, J; Mallié, M

    2011-02-01

    The Clinical Laboratory Standards Institute ([CLSI] formerly NCCLS) reference broth microdilution testing method (protocol M27-A3) was compared with a commercially available methods (Sensititre YeastOne(®)) by testing two quality control strains and 102 isolates of Candida sp. and Cryptococcus sp. against fluconazole, itraconazole, ketoconazole, posaconazole, voriconazole, flucytosin, amphotericin B and caspofungin. Minimal inhibitory concentrations (MIC) endpoints were determined after 24h of incubation for Sensititre YeastOne(®) and after 24 and 48 h for CLSI microdilution method. Essential agreements between methods vary from 70.6 to 92.2%. Categorical agreements vary from 94.1% for 5FC to 72.6% for AMB. Sensititre YeastOne(®) reading appears to be useful for avoiding very major errors and this confirms the interest of this method for evaluating new antifungals activity in vitro. PMID:20843616

  15. Correlating the Impact of Well-Defined Oligosaccharide Structures on Physical Stability Profiles of IgG1-Fc Glycoforms.

    PubMed

    More, Apurva S; Toprani, Vishal M; Okbazghi, Solomon Z; Kim, Jae H; Joshi, Sangeeta B; Middaugh, C Russell; Tolbert, Thomas J; Volkin, David B

    2016-02-01

    As part of a series of articles in this special issue describing 4 well-defined IgG1-Fc glycoforms as a model system for biosimilarity analysis (high mannose-Fc, Man5-Fc, GlcNAc-Fc and N297Q-Fc aglycosylated), the focus of this work is comparisons of their physical properties. A trend of decreasing apparent solubility (thermodynamic activity) by polyethylene glycol precipitation (pH 4.5, 6.0) and lower conformational stability by differential scanning calorimetry (pH 4.5) was observed with reducing size of the N297-linked oligosaccharide structures. Using multiple high-throughput biophysical techniques, the physical stability of the Fc glycoproteins was then measured in 2 formulations (NaCl and sucrose) across a wide range of temperatures (10°C-90°C) and pH (4.0-7.5) conditions. The data sets were used to construct 3-index empirical phase diagrams and radar charts to visualize the regions of protein structural stability. Each glycoform showed improved stability in the sucrose (vs. salt) formulation. The HM-Fc and Man5-Fc displayed the highest relative stability, followed by GlcNAc-Fc, with N297Q-Fc being the least stable. Thus, the overall physical stability profiles of the 4 IgG1-Fc glycoforms also show a correlation with oligosaccharide structure. These data sets are used to develop a mathematical model for biosimilarity analysis (as described in a companion article by Kim et al. in this issue). PMID:26869421

  16. Combined Alloreactive CTL Cellular Therapy with Prodrug Activator Gene Therapy in a Model of Breast Cancer Metastatic to the Brain

    PubMed Central

    Hickey, Michelle J.; Malone, Colin C.; Erickson, Kate L.; Lin, Amy; Soto, Horacio; Ha, Edward T.; Kamijima, Shuichi; Inagaki, Akihito; Takahashi, Masamichi; Kato, Yuki; Kasahara, Noriyuki; Mueller, Barbara M.; Kruse, Carol A.

    2013-01-01

    Purpose Individual or combined strategies of cellular therapy with alloreactive cytotoxic T lymphocytes (alloCTL) and gene therapy employing retroviral replicating vectors (RRV) encoding a suicide prodrug activating gene were explored for the treatment of breast tumors metastatic to the brain. Experimental Design AlloCTL, sensitized to the human leukocyte antigens of MDA-MB-231 breast cancer cells, were examined in vitro for anti-tumor functionality toward breast cancer targets. RRV encoding the yeast cytosine deaminase (CD) gene was tested in vivo for virus spread, ability to infect, and kill breast cancer targets when exposed to 5-fluorocytosine (5-FC). Individual and combination treatments were tested in subcutaneous and intracranial xenograft models with 231BR, a brain tropic variant. Results AlloCTL preparations were cytotoxic, proliferated and produced interferon-gamma when coincubated with target cells displaying relevant HLA. In vivo, intratumorally-placed alloCTL trafficked through one established intracranial 231BR focus to another in contralateral brain and induced tumor cell apoptosis. RRV-CD efficiently spread in vivo, infected 231BR and induced their apoptosis upon 5-FC exposure. Subcutaneous tumor volumes were significantly reduced in alloCTL and/or gene therapy treated groups compared to control groups. Mice with established intracranial 231BR tumors treated with combined alloCTL and RRV-CD had a median survival of 97.5 days compared with single modalities (50–83 days); all experimental treatment groups survived significantly longer than sham-treated groups (median survivals 31.5 or 40 days) and exhibited good safety/toxicity profiles. Conclusion The results indicate combining cellular and suicide gene therapies is a viable strategy for the treatment of established breast tumors in the brain. PMID:23780889

  17. Cytosine deaminase as a negative selectable marker for the microalgal chloroplast: a strategy for the isolation of nuclear mutations that affect chloroplast gene expression.

    PubMed

    Young, Rosanna E B; Purton, Saul

    2014-12-01

    Negative selectable markers are useful tools for forward-genetic screens aimed at identifying trans-acting factors that are required for expression of specific genes. Transgenic lines harbouring the marker fused to a gene element, such as a promoter, may be mutagenized to isolate loss-of-function mutants able to survive under selection. Such a strategy allows the molecular dissection of factors that are essential for expression of the gene. Expression of individual chloroplast genes in plants and algae typically requires one or more nuclear-encoded factors that act at the post-transcriptional level, often through interaction with the 5' UTR of the mRNA. To study such nuclear control further, we have developed the Escherichia coli cytosine deaminase gene codA as a conditional negative selectable marker for use in the model green alga Chlamydomonas reinhardtii. We show that a codon-optimized variant of codA with three amino acid substitutions confers sensitivity to 5-fluorocytosine (5-FC) when expressed in the chloroplast under the control of endogenous promoter/5' UTR elements from the photosynthetic genes psaA or petA. UV mutagenesis of the psaA transgenic line allowed recovery of 5-FC-resistant, photosynthetically deficient lines harbouring mutations in the nuclear gene for the factor TAA1 that is required for psaA translation. Similarly, the petA line was used to isolate mutants of the petA mRNA stability factor MCA1 and the translation factor TCA1. The codA marker may be used to identify critical residues in known nuclear factors and to aid the discovery of additional factors required for expression of chloroplast genes. PMID:25234691

  18. Targeting eradication of malignant cells derived from human bone marrow mesenchymal stromal cells

    SciTech Connect

    Yang, Yingbin; Cai, Shaoxi; Yang, Li; Yu, Shuhui; Jiang, Jiahuan; Yan, Xiaoqing; Zhang, Haoxing; Liu, Lan; Liu, Qun; Du, Jun; Cai, Shaohui; Sung, K.L. Paul

    2010-12-10

    Human bone marrow mesenchymal stromal cells (hBMSC) have been shown to participate in malignant transformation. However, hampered by the low frequency of malignant transformation of hBMSC, we do not yet know how to prevent malignant transformation of implanted hBMSC. In this study, in order to establish a model for the eradication of hBMSC-derived malignant cells, a gene fusion consisting of a human telomerase (hTERT) promoter modified with both c-Myc and myeloid zinc finger protein2 (MZF-2) binding elements and followed by the E. coli cytosine deaminase (CD) and luciferase genes was stably transferred into hBMSC via lentiviral transduction; n-phosphonacelyl-L-aspartic acid (PALA) selection was used to generate malignant cell colonies derived from transduced hBMSC after treatment with the carcinogenic reagent BPDE. Cells that were amplified after PALA selection were used for transplantation and 5-FC pro-drug cytotoxicity tests. The results showed that PALA-resistant malignant cells could be generated from hBMSC co-induced with lentiviral transduction and treatment with Benzo(a)pyrene Diol Epoxide (BPDE); the modification of c-Myc and MZF-2 binding elements could remarkably enhance the transcriptional activities of the hTERT promoter in malignant cells, whereas transcriptional activity was depressed in normal hBMSC; malignant cells stably expressing CD under the control of the modified hTERT promoter could be eliminated by 5-FC administration. This study has provided a method for targeted eradication of malignant cells derived from hBMSC.

  19. Effects of Cationic Microbubble Carrying CD/TK Double Suicide Gene and αVβ3 Integrin Antibody in Human Hepatocellular Carcinoma HepG2 Cells

    PubMed Central

    Li, Jiale; Zhou, Ping; Li, Lan; Zhang, Yan; Shao, Yang; Tang, Li; Tian, Shuangming

    2016-01-01

    Objective Hepatocellular carcinoma (HCC), mostly derived from hepatitis or cirrhosisis, is one of the most common types of liver cancer. T-cell mediated immune response elicited by CD/TK double suicide gene has shown a substantial antitumor effect in HCC. Integrin αVβ3 over expresssion has been suggested to regulate the biology behavior of HCC. In this study, we investigated the strategy of incorporating CD/TK double suicide gene and anti-αVβ3 integrin monoclonal antibodies into cationic microbubbles (CMBsαvβ3), and evaluated its killing effect in HCC cells. Methods To improve the transfection efficiency of targeted CD/TK double suicide gene, we adopted cationic microbubbles (CMBs), a cationic delivery agent with enhanced DNA-carrying capacity. The ultrasound and high speed shearing method was used to prepare the non-targeting cationic microbubbles (CMBs). Using the biotin-avidin bridge method, αVβ3 integrin antibody was conjugated to CMBs, and CMBsαvβ3 was generated to specifically target to HepG2 cells. The morphology and physicochemical properties of the CMBsαvβ3 was detected by optical microscope and zeta detector. The conjugation of plasmid and the antibody in CMBsαvβ3 were examined by immunofluorescent microscopy and flow cytometry. The binding capacities of CMBsαvβ3 and CMBs to HCC HepG2 and normal L-02 cells were compared using rosette formation assay. To detect EGFP fluorescence and examine the transfection efficiencies of CMBsαvβ3 and CMBs in HCC cells, fluorescence microscope and contrast-enhanced sonography were adopted. mRNA and protein level of CD/TK gene were detected by RT-PCR and Western blot, respectively. To evaluate the anti-tumor effect of CMBsαvβ3, HCC cells with CMBsαvβ3 were exposed to 5-flurocytosine / ganciclovir (5-FC/GCV). Then, cell cycle distribution after treatment were detected by PI staining and flow cytometry. Apoptotic cells death were detected by optical microscope and assessed by MTT assay and TUNEL

  20. Toxicity of Amphotericin B Deoxycholate-Based Induction Therapy in Patients with HIV-Associated Cryptococcal Meningitis

    PubMed Central

    Bottomley, Christian; Loyse, Angela; Brouwer, Annemarie E.; Muzoora, Conrad; Taseera, Kabanda; Jackson, Arthur; Phulusa, Jacob; Hosseinipour, Mina C.; van der Horst, Charles; Limmathurotsakul, Direk; White, Nicholas J.; Wilson, Douglas; Wood, Robin; Meintjes, Graeme; Harrison, Thomas S.; Jarvis, Joseph N.

    2015-01-01

    Amphotericin B deoxycholate (AmBd) is the recommended induction treatment for HIV-associated cryptococcal meningitis (CM). Its use is hampered by toxicities that include electrolyte abnormalities, nephrotoxicity, and anemia. Protocols to minimize toxicity are applied inconsistently. In a clinical trial cohort of AmBd-based CM induction treatment, a standardized protocol of preemptive hydration and electrolyte supplementation was applied. Changes in blood counts, electrolyte levels, and creatinine levels over 14 days were analyzed in relation to the AmBd dose, treatment duration (short course of 5 to 7 days or standard course of 14 days), addition of flucytosine (5FC), and outcome. In the 368 patients studied, the hemoglobin levels dropped by a mean of 1.5 g/dl (95% confidence interval [CI], 1.0 to 1.9 g/dl) following 7 days of AmBd and by a mean of 2.3 g/dl (95% CI, 1.1 to 3.6 g/dl) after 14 days. Serum creatinine levels increased by 37 μmol/liter (95% CI, 30 to 45 μmol/liter) by day 7 and by 49 μmol/liter (95% CI, 35 to 64μmol/liter) by day 14 of AmBd treatment. Overall, 33% of patients developed grade III/IV anemia, 5.6% developed grade III hypokalemia, 9.5% had creatinine levels that exceeded 220 μmol, and 6% discontinued AmBd prematurely. The addition of 5FC was associated with a slight increase in anemia but not neutropenia. Laboratory abnormalities stabilized or reversed during the second week in patients on short-course induction. Grade III/IV anemia (adjusted odds ratio [aOR], 2.2; 95% CI, 1.1 to 4.3; P = 0.028) and nephrotoxicity (aOR, 4.5; 95% CI, 1.8 to 11; P = 0.001) were risk factors for 10-week mortality. In summary, routine intravenous saline hydration and preemptive electrolyte replacement during AmBd-based induction regimens for HIV-associated CM minimized the incidence of hypokalemia and nephrotoxicity. Anemia remained a concerning adverse effect. The addition of flucytosine was not associated with increased neutropenia. Shorter AmBd courses

  1. Resistance Surveillance in Candida albicans: A Five-Year Antifungal Susceptibility Evaluation in a Brazilian University Hospital

    PubMed Central

    Peron, Isabela Haddad; Reichert-Lima, Franqueline; Busso-Lopes, Ariane Fidelis; Nagasako, Cristiane Kibune; Lyra, Luzia; Moretti, Maria Luiza

    2016-01-01

    Candida albicans caused 44% of the overall candidemia episodes from 2006 to 2010 in our university tertiary care hospital. As different antifungal agents are used in therapy and also immunocompromised patients receive fluconazole prophylaxis in our institution, this study aimed to perform an antifungal susceptibility surveillance with the C.albicans bloodstream isolates and to characterize the fluconazole resistance in 2 non-blood C.albicans isolates by sequencing ERG11 gene. The study included 147 C. albicans bloodstream samples and 2 fluconazole resistant isolates: one from oral cavity (LIF 12560 fluconazole MIC: 8μg/mL) and one from esophageal cavity (LIF-E10 fluconazole MIC: 64μg/mL) of two different patients previously treated with oral fluconazole. The in vitro antifungal susceptibility to amphotericin B (AMB), 5-flucytosine (5FC), fluconazole (FLC), itraconazole (ITC), voriconazole (VRC), caspofungin (CASP) was performed by broth microdilution methodology recommended by the Clinical and Laboratory Standards Institute documents (M27-A3 and M27-S4, CLSI). All blood isolates were classified as susceptible according to CLSI guidelines for all evaluated antifungal agents (MIC range: 0,125–1.00 μg/mL for AMB, ≤0.125–1.00 μg/mL for 5FC, ≤0.125–0.5 μg/mL for FLC, ≤0.015–0.125 μg/mL for ITC, ≤0.015–0.06 μg/mL for VRC and ≤0.015–0.125 μg/mL for CASP). In this study, we also amplified and sequenced the ERG11 gene of LIF 12560 and LIF-E10 C.albicans isolates. Six mutations encoding distinct amino acid substitutions were found (E116D, T128K, E266D, A298V, G448V and G464S) and these mutations were previously described as associated with fluconazole resistance. Despite the large consumption of antifungals in our institution, resistant blood isolates were not found over the trial period. Further studies should be conducted, but it may be that the very prolonged direct contact with the oral antifungal agent administered to the patient from which

  2. [Mesenchymal stem cells expressing cytosine deaminase inhibit growth of murine melanoma B16F10 in vivo].

    PubMed

    Krasikova, L S; Karshieva, S S; Cheglakov, I B; Belyavsky, A V

    2015-01-01

    The aim of this study was to estimate the efficacy of mesenchymal stem cell-based suicide gene therapy in mice bearing murine melanoma B16F10. Adipose mesenchymal stem cells (MSCs) were transfected with plasmid constructs expressing cytosine deaminase fused with uracil phosphoribosyltransferase (CDA/UPRT) or CDA/UPRT fused with HSV-1 tegument protein VP22 (CDA/UPRT/VP22). In this study, we demonstrate that direct intratumoral transplantation of MSCs expressing CDA/UPRT or CDA/UPRT/VP22 followed by systemic administration of 5-fluorocytosine (5-FC) results in a significant inhibition of tumor growth. There was a 53% reduction in tumor volume in mice treated with CDA/UPRT-MSCs and 58% reduction in tumor volume in mice treated with CDA/UPRT/VP22-MSCs as compared with control animals transplanted with B16F10 melanoma alone. Injection of CDA/UPRT-MSC and CDA/UPRT/VP22-MSC prolonged the life span of mice bearing B16F10 melanoma by 15 and 26%, respectively. The data indicate that in murine B16F10 melanoma model, MSCs encoding CDA/UPRT suicide gene have a significant antitumor effect. PMID:26710783

  3. Computational modeling and functional analysis of Herpes simplex virus type-1 thymidine kinase and Escherichia coli cytosine deaminase fusion protein

    SciTech Connect

    Zhang, Jufeng; Wang, Zhanli; Wei, Fang; Qiu, Wei; Zhang, Liangren; Huang, Qian . E-mail: qhuang@sjtu.edu.cn

    2007-08-17

    Herpes simplex virus type-1 thymidine kinase (HSV-1TK) and Escherichia coli cytosine deaminase (CD) fusion protein was designed using InsightII software. The structural rationality of the fusion proteins incorporating a series of flexible linker peptide was analyzed, and a suitable linker peptide was chosen for further investigated. The recombinant plasmid containing the coding regions of HSV-1TK and CD cDNA connected by this linker peptide coding sequence was generated and subsequently transfected into the human embryonic kidney 293 cells (HEK293). The Western blotting indicated that the recombinant fusion protein existed as a dimer with a molecular weight of approximately 90 kDa. The toxicity of the prodrug on the recombinant plasmid-transfected human lung cancer cell line NCIH460 was evaluated, which showed that TKglyCD-expressing cells conferred upon cells prodrug sensitivities equivalent to that observed for each enzyme independently. Most noteworthy, cytotoxicity could be enhanced by concurrently treating TKglyCD-expressing cells with prodrugs GCV and 5-FC. The results indicate that we have successfully constructed a HSV-1TKglyCD fusion gene which might have a potential application for cancer gene therapy.

  4. 2009: A Colliding-Wind Odyssey

    NASA Astrophysics Data System (ADS)

    Fahed, R.; Moffat, A. F. J.; Zorec, J.; Eversberg, T.; Chené, A. N.; Alves, F.; Arnold, W.; Bergmann, T.; Corcoran, M. F.; Correia Viegas, N. G.; Dougherty, S. M.; Fernando, A.; Frémat, Y.; Gouveia Carreira, L. F.; Hunger, T.; Knapen, J. H.; Leadbeater, R.; Marques Dias, F.; Martayan, C.; Morel, T.; Pittard, J. M.; Pollock, A. M. T.; Rauw, G.; Reinecke, N.; Ribeiro, J.; Romeo, N.; Sánchez-Gallego, J. R.; dos Santos, E. M.; Schanne, L.; Stahl, O.; Stober, Ba.; Stober, Be.; Vollmann, K.; Williams, P. M.

    2012-12-01

    We present the results from two optical spectroscopic campaigns on colliding-wind binaries (CWB) which both occurred in 2009. The first one was on WR 140 (WC7pd + O5.5fc), the archetype of CWB, which experienced periastron passage of its highly elliptical 8-year orbit in January. The WR 140 campaign consisted of a unique and constructive collaboration between amateur and professional astronomers and took place at half a dozen locations, including Teide Observatory, Observatoire de Haute Provence, Dominion Astrophysical Observatory, Observatoire du Mont-Mégantic and at several small private observatories. The second campaign was on a selection of 5 short-period WR + O binaries not yet studied for colliding-wind effects: WR 12 (WN8h), WR 21 (WN5o + O7 V), WR 30 (WC6 + O7.5 V), WR 31 (WN4o + O8), and WR 47 (WN6o + O5). The campaign took place at Leoncito Observatory, Argentina, during 1 month. We provide updated values of most of these systems for the orbital parameters, new estimates for the WR and O star masses and new constraints on the mass-loss rates and colliding wind geometry.

  5. Activation of multiple chemotherapeutic prodrugs by the natural enzymolome of tumour-localised probiotic bacteria.

    PubMed

    Lehouritis, Panos; Stanton, Michael; McCarthy, Florence O; Jeavons, Matthieu; Tangney, Mark

    2016-01-28

    Some chemotherapeutic drugs (prodrugs) require activation by an enzyme for efficacy. We and others have demonstrated the ability of probiotic bacteria to grow specifically within solid tumours following systemic administration, and we hypothesised that the natural enzymatic activity of these tumour-localised bacteria may be suitable for activation of certain such chemotherapeutic drugs. Several wild-type probiotic bacteria; Escherichia coli Nissle, Bifidobacterium breve, Lactococcus lactis and Lactobacillus species, were screened against a panel of popular prodrugs. All strains were capable of activating at least one prodrug. E. coli Nissle 1917 was selected for further studies because of its ability to activate numerous prodrugs and its resistance to prodrug toxicity. HPLC data confirmed biochemical transformation of prodrugs to their toxic counterparts. Further analysis demonstrated that different enzymes can complement prodrug activation, while simultaneous activation of multiple prodrugs (CB1954, 5-FC, AQ4N and Fludarabine phosphate) by E. coli was confirmed, resulting in significant efficacy improvement. Experiments in mice harbouring murine tumours validated in vitro findings, with significant reduction in tumour growth and increase in survival of mice treated with probiotic bacteria and a combination of prodrugs. These findings demonstrate the ability of probiotic bacteria, without the requirement for genetic modification, to enable high-level activation of multiple prodrugs specifically at the site of action. PMID:26655063

  6. Adenoviral-Mediated Imaging of Gene Transfer Using a Somatostatin Receptor-Cytosine Deaminase Fusion Protein

    PubMed Central

    Lears, Kimberly A.; Parry, Jesse J.; Andrews, Rebecca; Nguyen, Kim; Wadas, Thaddeus J.; Rogers, Buck E.

    2015-01-01

    Suicide gene therapy is a process by which cells are administered a gene that encodes a protein capable of converting a nontoxic prodrug into an active toxin. Cytosine deaminase (CD) has been widely investigated as a means of suicide gene therapy due to the enzyme’s ability to convert the prodrug 5-fluorocytosine (5-FC) into the toxic compound 5-fluorouracil (5-FU). However, the extent of gene transfer is a limiting factor in predicting therapeutic outcome. The ability to monitor gene transfer, non-invasively, would strengthen the efficiency of therapy. In this regard, we have constructed and evaluated a replication-deficient adenovirus (Ad) containing the human somatostatin receptor subtype 2 (SSTR2) fused with a C-terminal yeast CD gene for the non-invasive monitoring of gene transfer and therapy. The resulting Ad (AdSSTR2-yCD) was evaluated in vitro in breast cancer cells to determine the function of the fusion protein. These studies demonstrated that the both the SSTR2 and yCD were functional in binding assays, conversion assays, and cytotoxicity assays. In vivo studies similarly demonstrated the functionality using conversion assays, biodistribution studies, and small animal positron-emission tomography (PET) imaging studies. In conclusion, the fusion protein has been validated as useful for the non-invasive imaging of yCD expression and will be evaluated in the future for monitoring yCD-based therapy. PMID:25837665

  7. Dexamethasone Regulates EphA5, a Potential Inhibitory Factor with Osteogenic Capability of Human Bone Marrow Stromal Cells.

    PubMed

    Yamada, Tsuyoshi; Yoshii, Toshitaka; Yasuda, Hiroaki; Okawa, Atsushi; Sotome, Shinichi

    2016-01-01

    We previously demonstrated the importance of quality management procedures for the handling of human bone marrow stromal cells (hBMSCs) and provided evidence for the existence of osteogenic inhibitor molecules in BMSCs. One candidate inhibitor is the ephrin type-A receptor 5 (EphA5), which is expressed in hBMSCs and upregulated during long-term culture. In this study, forced expression of EphA5 diminished the expression of osteoblast phenotypic markers. Downregulation of endogenous EphA5 by dexamethasone treatment promoted osteoblast marker expression. EphA5 could be involved in the normal growth regulation of BMSCs and could be a potential marker for replicative senescence. Although Eph forward signaling stimulated by ephrin-B-Fc promoted the expression of ALP mRNA in BMSCs, exogenous addition of EphA5-Fc did not affect the ALP level. The mechanism underlying the silencing of EphA5 in early cultures remains unclear. EphA5 promoter was barely methylated in hBMSCs while histone deacetylation could partially suppress EphA5 expression in early-passage cultures. In repeatedly passaged cultures, the upregulation of EphA5 independent of methylation could competitively inhibit osteogenic signal transduction pathways such as EphB forward signaling. Elucidation of the potential inhibitory function of EphA5 in hBMSCs may provide an alternative approach for lineage differentiation in cell therapy strategies and regenerative medicine. PMID:27057165

  8. Strategies to Save 50% Site Energy in Grocery and General Merchandise Stores

    SciTech Connect

    Hirsch, A.; Hale, E.; Leach, M.

    2011-03-01

    This paper summarizes the methodology and main results of two recently published Technical Support Documents. These reports explore the feasibility of designing general merchandise and grocery stores that use half the energy of a minimally code-compliant building, as measured on a whole-building basis. We used an optimization algorithm to trace out a minimum cost curve and identify designs that satisfy the 50% energy savings goal. We started from baseline building energy use and progressed to more energy-efficient designs by sequentially adding energy design measures (EDMs). Certain EDMs figured prominently in reaching the 50% energy savings goal for both building types: (1) reduced lighting power density; (2) optimized area fraction and construction of view glass or skylights, or both, as part of a daylighting system tuned to 46.5 fc (500 lux); (3) reduced infiltration with a main entrance vestibule or an envelope air barrier, or both; and (4) energy recovery ventilators, especially in humid and cold climates. In grocery stores, the most effective EDM, which was chosen for all climates, was replacing baseline medium-temperature refrigerated cases with high-efficiency models that have doors.

  9. Mannan induces ROS-regulated, IL-17A–dependent psoriasis arthritis-like disease in mice

    PubMed Central

    Khmaladze, Ia; Kelkka, Tiina; Guerard, Simon; Wing, Kajsa; Pizzolla, Angela; Saxena, Amit; Lundqvist, Katarina; Holmdahl, Meirav; Nandakumar, Kutty Selva; Holmdahl, Rikard

    2014-01-01

    Psoriasis (Ps) and psoriasis arthritis (PsA) are poorly understood common diseases, induced by unknown environmental factors, affecting skin and articular joints. A single i.p. exposure to mannan from Saccharomyces cerevisiae induced an acute inflammation in inbred mouse strains resembling human Ps and PsA-like disease, whereas multiple injections induced a relapsing disease. Exacerbation of disease severity was observed in mice deficient for generation of reactive oxygen species (ROS). Interestingly, restoration of ROS production, specifically in macrophages, ameliorated both skin and joint disease. Neutralization of IL-17A, mainly produced by γδ T cells, completely blocked disease symptoms. Furthermore, mice depleted of granulocytes were resistant to disease development. In contrast, certain acute inflammatory mediators (C5, Fcγ receptor III, mast cells, and histamine) and adaptive immune players (αβ T and B cells) were redundant in disease induction. Hence, we propose that mannan-induced activation of macrophages leads to TNF-α secretion and stimulation of local γδ T cells secreting IL-17A. The combined action of activated macrophages and IL-17A produced in situ drives neutrophil infiltration in the epidermis and dermis of the skin, leading to disease manifestations. Thus, our finding suggests a new mechanism triggered by exposure to exogenous microbial components, such as mannan, that can induce and exacerbate Ps and PsA. PMID:25136095

  10. Suzaku monitoring of the Wolf-Rayet binary WR 140 around periastron passage: An approach for quantifying the wind parameters

    NASA Astrophysics Data System (ADS)

    Sugawara, Yasuharu; Maeda, Yoshitomo; Tsuboi, Yohko; Hamaguchi, Kenji; Corcoran, Michael; Pollock, Andy M. T.; Moffat, Anthony F. J.; Williams, Peredur M.; Dougherty, Sean; Pittard, Julian

    2015-12-01

    Suzaku observations of the Wolf-Rayet (W-R) binary WR 140 (WC7pd+O5.5fc) were made at four different times around periastron passage in 2009 January. The spectra changed in shape and flux with the phase. As periastron approached, the column density of the low-energy absorption increased, which indicates that the emission from the wind-wind collision plasma was absorbed by the dense W-R wind. The spectra can be mostly fitted with two different components: a warm component with kBT = 0.3-0.6 keV and a dominant hot component with kBT ˜ 3 keV. The emission measure of the dominant, hot component is not inversely proportional to the distance between the two stars. This can be explained by the O star wind colliding before it has reached its terminal velocity, leading to a reduction in its wind momentum flux. At phases closer to periastron, we discovered a cool plasma component in a recombining phase, which is less absorbed. This component may be a relic of the wind-wind collision plasma, which was cooled down by radiation, and may represent a transitional stage in dust formation.

  11. Reconstitution of Fusion Proteins in Supported Lipid Bilayers for the Study of Cell Surface Receptor-Ligand Interactions in Cell-Cell Contact.

    PubMed

    Ghosh Moulick, R; Afanasenkau, D; Choi, S-E; Albers, J; Lange, W; Maybeck, V; Utesch, T; Offenhäusser, A

    2016-04-12

    Bioactive molecules such as adhesion ligands, growth factors, or enzymes play an important role in modulating cell behavior such as cell adhesion, spreading, and differentiation. Deciphering the mechanism of ligand-mediated cell adhesion and associated signaling is of great interest not only for fundamental biophysical investigations but also for applications in medicine and biotechnology. In the presented work, we developed a new biomimetic platform that enables culturing primary neurons and testing cell surface-receptor ligand interactions in cell-cell contacts as, e.g., in neuronal synapses. This platform consists of a supported lipid bilayer modified with incorporated neuronal adhesion proteins conjugated with the Fc-domain of IgG (ephrin A5 Fc-chimera). We extensively characterized properties of these protein containing bilayers using fluorescence recovery after photobleaching (FRAP), quartz crystal microbalance with dissipation (QCM-D), and immunostaining. We conclude that the Fc-domain is the part responsible for the incorporation of the protein into the bilayer. The biomimetic platform prepared by this new approach was able to promote neuronal cell adhesion and maintain growth as well as facilitate neuronal maturation as shown by electrophysiological measurements. We believe that our approach can be extended to insert other proteins to create a general culture platform for neurons and other cell types. PMID:26986674

  12. Analysis of pH and buffer effects on flucytosine activity in broth dilution susceptibility testing of Candida albicans in two synthetic media.

    PubMed Central

    Calhoun, D L; Galgiani, J N

    1984-01-01

    We examined the influences of different pH levels and three different buffers on flucytosine activity against 12 isolates of Candida albicans in two synthetic media, yeast nitrogen base (YNB) and synthetic amino acid medium-fungal (SAAMF), using broth dilution techniques and measuring the endpoints of visual MICs and turbidimetric 50% inhibitory concentrations. The two media were originally prepared as follows: YNB, unbuffered, pH 5.6; SAAMF, buffered with morpholinepropanesulfonic acid-Tris, pH 7.4; the resultant geometric mean MIC and 50% inhibitory concentration of 5-FC were 78- and 32-fold higher, respectively, in SAAMF. Raising the pH of YNB or lowering the pH of SAAMF had virtually no effect on these differences in MIC and 50% inhibitory concentration in the two media. In contrast, virtually all of the discrepancy appeared to be due to morpholinepropanesulfonic acid-Tris, which exerted concentration-dependent inhibition of flucytosine activity not evident when N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid or phosphate buffer systems were substituted. In other turbidimetric studies, growth was slowed more than 50% in YNB as the pH was raised to 7.4, regardless of which buffer was used. Based on our studies, we recommend modifying the composition of SAAMF by substituting a nonantagonistic buffer if any buffer is to be used with SAAMF in the testing of flucytosine. With this modification, SAAMF warrants further study as a generally applicable medium for fungal-susceptibility testing. PMID:6391370

  13. Oncolytic virotherapy with an armed vaccinia virus in an orthotopic model of renal carcinoma is associated with modification of the tumor microenvironment

    PubMed Central

    Fend, Laetitia; Remy-Ziller, Christelle; Foloppe, Johann; Kempf, Juliette; Cochin, Sandrine; Barraud, Luc; Accart, Nathalie; Erbs, Philippe; Fournel, Sylvie; Préville, Xavier

    2016-01-01

    ABSTRACT Oncolytic virotherapy is an emergent promising therapeutic approach for the treatment of cancer. We have constructed a vaccinia virus (WR strain) deleted for thymidine kinase (TK) and ribonucleotide reductase (RR) genes that expressed the fusion suicide gene FCU1 derived from the yeast cytosine deaminase and uracil phosphoribosyltransferase genes. We evaluated this construct (VV-FCU1) in the orthotopic model of renal carcinoma (RenCa). Systemic administration of VV-FCU1 resulted in orthotopic tumor growth inhibition, despite temporary expression of viral proteins. VV-FCU1 treatment was associated with an infiltration of tumors by CD8+ T lymphocytes and a decrease in the proportion of infiltrating Tregs, thus modifying the ratio of CD8+/CD4+ Treg in favor of CD8+cytotoxic T cells. We demonstrated that VV-FCU1 treatment prolonged survival of animals implanted with RenCa cells in kidney. Depletion of CD8+ T cells abolished the therapeutic effect of VV-FCU1 while depletion of CD4+ T cells enhanced its protective activity. Administration of the prodrug 5-fluorocytosine (5-FC) resulted in a sustained control of tumor growth but did not extend survival. This study shows the importance of CD4+ and CD8+ T cells in vaccinia virus-mediated oncolytic virotherapy and suggests that this approach may be evaluated for the treatment of human renal cell carcinoma. PMID:27057460

  14. Redox-responsive polycation-functionalized cotton cellulose nanocrystals for effective cancer treatment.

    PubMed

    Hu, Hao; Yuan, Wei; Liu, Fu-Sheng; Cheng, Gang; Xu, Fu-Jian; Ma, Jie

    2015-04-29

    Carbon nanotubes have excellent penetrability and encapsulation efficiency in the fields of drug and gene delivery. Because of their excellent physicochemical properties, biocompatible rodlike cellulose nanocrystals (CNCs) were reportedly expected to replace carbon nanotubes. In this work, CNCs from natural cotton wool were functionalized with disulfide bond-linked poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) brushes for effective biomedical applications. A range of CNC-graft-PDMAEMA vectors (termed as CNC-SS-PDs) with various molecular weights of PDMAEMA were synthesized. Under reducible conditions, PDMAEMA chains can be easily cleaved from CNCs. The gene condensation ability, reduction sensitivity, cytotoxicity, gene transfection, and in vivo antitumor activities of CNC-SS-PDs were investigated in detail. The CNC-SS-PDs exhibited good transfection efficiencies and low cytotoxicities. The needlelike shape of CNCs had an important effect on enhancing transfection efficiency. The antitumor effect of CNC-SS-PDs was evaluated by a suicide gene/prodrug system (cytosine deaminase/5-fluorocytosine, CD/5-FC) in vitro and in vivo. This research demonstrates that the functionalization of CNCs with redox-responsive polycations is an effective method for developing novel gene delivery systems. PMID:25845425

  15. Evidence that the KIR2DS5 gene codes for a surface receptor triggering natural killer cell function.

    PubMed

    Della Chiesa, Mariella; Romeo, Elisa; Falco, Michela; Balsamo, Mirna; Augugliaro, Raffaella; Moretta, Lorenzo; Bottino, Cristina; Moretta, Alessandro; Vitale, Massimo

    2008-08-01

    In this study, after immunization with NK cells from a KIR2DS5(+) donor and screening on cell transfectants expressing different members of the killer immunoglobulin-like receptor (KIR) family, we generated a mAb, DF200, reacting with several KIR2D receptors including KIR2DL1/L2/L3, KIR2DS1/S2 and KIR2DS5. By the analysis of peripheral blood NK cells and in vitro derived NK cell clones, we have demonstrated for the first time that KIR2DS5 is expressed at the cell surface in discrete subsets of NK cells and, after DF200 mAb-mediated engagement, can induce both cytotoxicity and cytokine release. Using co-transfection and co-immunoprecipitation, we found that KIR2DS5 associates with the DAP12 signaling polypeptide. Finally, soluble KIR2DS5-Fc fusion protein does not bind to cell transfectants expressing different HLA-C alleles, suggesting that, if KIR2DS5 does recognize HLA-C molecules, this may only occur in the presence of certain peptides. PMID:18624290

  16. Dexamethasone Regulates EphA5, a Potential Inhibitory Factor with Osteogenic Capability of Human Bone Marrow Stromal Cells

    PubMed Central

    Yamada, Tsuyoshi; Yoshii, Toshitaka; Yasuda, Hiroaki; Okawa, Atsushi; Sotome, Shinichi

    2016-01-01

    We previously demonstrated the importance of quality management procedures for the handling of human bone marrow stromal cells (hBMSCs) and provided evidence for the existence of osteogenic inhibitor molecules in BMSCs. One candidate inhibitor is the ephrin type-A receptor 5 (EphA5), which is expressed in hBMSCs and upregulated during long-term culture. In this study, forced expression of EphA5 diminished the expression of osteoblast phenotypic markers. Downregulation of endogenous EphA5 by dexamethasone treatment promoted osteoblast marker expression. EphA5 could be involved in the normal growth regulation of BMSCs and could be a potential marker for replicative senescence. Although Eph forward signaling stimulated by ephrin-B-Fc promoted the expression of ALP mRNA in BMSCs, exogenous addition of EphA5-Fc did not affect the ALP level. The mechanism underlying the silencing of EphA5 in early cultures remains unclear. EphA5 promoter was barely methylated in hBMSCs while histone deacetylation could partially suppress EphA5 expression in early-passage cultures. In repeatedly passaged cultures, the upregulation of EphA5 independent of methylation could competitively inhibit osteogenic signal transduction pathways such as EphB forward signaling. Elucidation of the potential inhibitory function of EphA5 in hBMSCs may provide an alternative approach for lineage differentiation in cell therapy strategies and regenerative medicine. PMID:27057165

  17. Sensing Small Changes in Protein Abundance: Stimulation of Caco-2 Cells by Human Whey Proteins.

    PubMed

    Cundiff, Judy K; McConnell, Elizabeth J; Lohe, Kimberly J; Maria, Sarah D; McMahon, Robert J; Zhang, Qiang

    2016-01-01

    Mass spectrometry (MS)-based proteomic approaches have largely facilitated our systemic understanding of cellular processes and biological functions. Cutoffs in protein expression fold changes (FCs) are often arbitrarily determined in MS-based quantification with no demonstrable determination of small magnitude changes in protein expression. Therefore, many biological insights may remain veiled due to high FC cutoffs. Herein, we employ the intestinal epithelial cell (IEC) line Caco-2 as a model system to demonstrate the dynamicity of tandem-mass-tag (TMT) labeling over a range of 5-40% changes in protein abundance, with the variance controls of ± 5% FC for around 95% of TMT ratios when sampling 9-12 biological replicates. We further applied this procedure to examine the temporal proteome of Caco-2 cells upon exposure to human whey proteins (WP). Pathway assessments predict subtle effects due to WP in moderating xenobiotic metabolism, promoting proliferation and various other cellular functions in differentiating enterocyte-like Caco-2 cells. This demonstration of a sensitive MS approach may open up new perspectives in the system-wide exploration of elusive or transient biological effects by facilitating scrutiny of narrow windows of proteome abundance changes. Furthermore, we anticipate this study will encourage more investigations of WP on infant gastrointestinal tract development. PMID:26586228

  18. Antibody-based therapeutics to watch in 2011.

    PubMed

    Reichert, Janice M

    2011-01-01

    This overview of 25 monoclonal antibody (mAb) and 5 Fc fusion protein therapeutics provides brief descriptions of the candidates, recently published clinical study results and on-going Phase 3 studies. In alphanumeric order, the 2011 therapeutic antibodies to watch list comprises AIN-457, bapineuzumab, brentuximab vedotin, briakinumab, dalotuzumab, epratuzumab, farletuzumab, girentuximab (WX-G250), naptumomab estafenatox, necitumumab, obinutuzumab, otelixizumab, pagibaximab, pertuzumab, ramucirumab, REGN88, reslizumab, solanezumab, T1h , teplizumab, trastuzumab emtansine, tremelimumab, vedolizumab, zalutumumab and zanolimumab. In alphanumeric order, the 2011 Fc fusion protein therapeutics to watch list comprises aflibercept, AMG-386, atacicept, Factor VIII and Factor IX-Fc. Commercially-sponsored mAb and Fc fusion therapeutics that have progressed only as far as Phase 2/3 or 3 were included. Candidates undergoing regulatory review or products that have been approved may also be in Phase 3 studies, but these were excluded. Due to the large body of primary literature about the candidates, only selected references are given and results from recent publications and articles that were relevant to Phase 3 studies are emphasized. Current as of September 2010, the information presented here will serve as a baseline against which future progress in the development of antibody-based therapeutics can be measured. PMID:21051951

  19. Phenotypic characterization of virological failure following lopinavir/ritonavir monotherapy using full-length gag–protease genes

    PubMed Central

    Sutherland, Katherine A.; Mbisa, Jean L.; Ghosn, Jade; Chaix, Marie-Laure; Cohen-Codar, Isabelle; Hue, Stephane; Delfraissy, Jean-Francois; Delaugerre, Constance; Gupta, Ravindra K.

    2014-01-01

    Objectives Major protease mutations are rarely observed following first-line failure with PIs and interpretation of genotyping results in this context may be difficult. We performed extensive phenotyping of viruses from five patients failing lopinavir/ritonavir monotherapy in the MONARK study without major PI mutations by standard genotyping. Methods Phenotypic susceptibility testing and viral infectivity assessments were performed using a single-cycle assay and fold changes (FC) relative to a lopinavir-susceptible reference strain were calculated. Results >10-fold reduced baseline susceptibility to lopinavir occurred in two of five patients and >5-fold in another two. Four of five patients exhibited phylogenetic evidence of a limited viral evolution between baseline and failure, with amino acid changes at drug resistance-associated positions in one: T81A emerged in Gag with M36I in the protease gene, correlating with a reduction in lopinavir susceptibility from FC 7 (95% CI 6–8.35) to FC 13 (95% CI 8.11–17.8). Reductions in darunavir susceptibility (>5 FC) occurred in three individuals. Discussion This study suggests both baseline reduced susceptibility and evolution of resistance could be contributing factors to PI failure, despite the absence of classical PI resistance mutations by standard testing methods. Use of phenotyping also reveals lower darunavir susceptibility, warranting further study as this agent is commonly used following lopinavir failure. PMID:25096075

  20. Expression of Phosphocitrate-Targeted Genes in Osteoarthritis Menisci

    PubMed Central

    Sun, Yubo; Mauerhan, David R.; Steuerwald, Nury M.; Ingram, Jane; Kneisl, Jeffrey S.; Hanley, Edward N.

    2014-01-01

    Phosphocitrate (PC) inhibited calcium crystal-associated osteoarthritis (OA) in Hartley guinea pigs. However, the molecular mechanisms remain elusive. This study sought to determine PC targeted genes and the expression of select PC targeted genes in OA menisci to test hypothesis that PC exerts its disease modifying activity in part by reversing abnormal expressions of genes involved in OA. We found that PC downregulated the expression of numerous genes classified in immune response, inflammatory response, and angiogenesis, including chemokine (C-C motif) ligand 5, Fc fragment of IgG, low affinity IIIb receptor (FCGR3B), and leukocyte immunoglobulin-like receptor, subfamily B member 3 (LILRB3). In contrast, PC upregulated the expression of many genes classified in skeletal development, including collagen type II alpha1, fibroblast growth factor receptor 3 (FGFR3), and SRY- (sex determining region Y-) box 9 (SOX-9). Immunohistochemical examinations revealed higher levels of FCGR3B and LILRB3 and lower level of SOX-9 in OA menisci. These findings indicate that OA is a disease associated with immune system activation and decreased expression of SOX-9 gene in OA menisci. PC exerts its disease modifying activity on OA, at least in part, by targeting immune system activation and the production of extracellular matrix and selecting chondroprotective proteins. PMID:25525593

  1. Modulation of Cell Cycle Progression by 5-Azacytidine Is Associated with Early Myogenesis Induction in Murine Myoblasts

    PubMed Central

    Montesano, Anna; Luzi, Livio; Senesi, Pamela; Terruzzi, Ileana

    2013-01-01

    Myogenesis is a multistep process, in which myoblasts withdraw from the cell cycle, cease to divide, elongate and fuse to form multinucleated myotubes. Cell cycle transition is controlled by a family of cyclin-dependent protein kinases (CDKs) regulated by association with cyclins, negative regulatory subunits and phosphorylation. Muscle differentiation is orchestrated by myogenic regulatory factors (MRFs), such as MyoD and Myf-5. DNA methylation is crucial in transcriptional control of genes involved in myogenesis. Previous work has indicated that treatment of fibroblasts with the DNA-demethylating agent 5-azacytidine (AZA) promotes MyoD expression. We studied the effects of AZA on cell cycle regulation and MRFs synthesis during myoblast proliferation and early myogenesis phases in C2C12 cells. During the proliferation phase, cells were incubated in growth medium with 5µM AZA (GMAZA) or without AZA (GM) for 24 hours. At 70% confluence, cells were kept in growth medium in order to spontaneously achieve differentiation or transferred to differentiation medium with 5μM AZA (DMAZA) or without AZA (DM) for 12 and 24 hours. Cells used as control were unstimulated. In the proliferation phase, AZA-treated cells seemed to lose their characteristic circular shape and become elongated. The presence of AZA resulted in significant increases in the protein contents of Cyclin-D (FC:1.23 GMAZA vs GM p≤0.05), p21 (FC: 1.23 GMAZA vs GM p≤0.05), Myf-5 (FC: 1.21 GMAZA vs GM p≤0.05) and MyoD (FC: 1.20 GMAZA vs GM p≤0.05). These results propose that AZA could inhibit cell proliferation. During 12 hours of differentiation, AZA decreased the downregulation of genes involved in cell cycle arrest and in restriction point (G1 and G1/S phase) and the expression of several cyclins, E2F Transcription Factors, cyclin-dependent kinase inhibitors, specific genes responsible of cell cycle negative regulation. During 24 hours of differentiation, AZA induced an increment in the protein

  2. Modulation of cell cycle progression by 5-azacytidine is associated with early myogenesis induction in murine myoblasts.

    PubMed

    Montesano, Anna; Luzi, Livio; Senesi, Pamela; Terruzzi, Ileana

    2013-01-01

    Myogenesis is a multistep process, in which myoblasts withdraw from the cell cycle, cease to divide, elongate and fuse to form multinucleated myotubes. Cell cycle transition is controlled by a family of cyclin-dependent protein kinases (CDKs) regulated by association with cyclins, negative regulatory subunits and phosphorylation. Muscle differentiation is orchestrated by myogenic regulatory factors (MRFs), such as MyoD and Myf-5. DNA methylation is crucial in transcriptional control of genes involved in myogenesis. Previous work has indicated that treatment of fibroblasts with the DNA-demethylating agent 5-azacytidine (AZA) promotes MyoD expression. We studied the effects of AZA on cell cycle regulation and MRFs synthesis during myoblast proliferation and early myogenesis phases in C2C12 cells. During the proliferation phase, cells were incubated in growth medium with 5µM AZA (GMAZA) or without AZA (GM) for 24 hours. At 70% confluence, cells were kept in growth medium in order to spontaneously achieve differentiation or transferred to differentiation medium with 5μM AZA (DMAZA) or without AZA (DM) for 12 and 24 hours. Cells used as control were unstimulated. In the proliferation phase, AZA-treated cells seemed to lose their characteristic circular shape and become elongated. The presence of AZA resulted in significant increases in the protein contents of Cyclin-D (FC:1.23 GMAZA vs GM p≤0.05), p21 (FC: 1.23 GMAZA vs GM p≤0.05), Myf-5 (FC: 1.21 GMAZA vs GM p≤0.05) and MyoD (FC: 1.20 GMAZA vs GM p≤0.05). These results propose that AZA could inhibit cell proliferation. During 12 hours of differentiation, AZA decreased the downregulation of genes involved in cell cycle arrest and in restriction point (G1 and G1/S phase) and the expression of several cyclins, E2F Transcription Factors, cyclin-dependent kinase inhibitors, specific genes responsible of cell cycle negative regulation. During 24 hours of differentiation, AZA induced an increment in the protein

  3. Caffeic acid phenethyl ester enhances TRAIL-mediated apoptosis via CHOP-induced death receptor 5 upregulation in hepatocarcinoma Hep3B cells.

    PubMed

    Dilshara, Matharage Gayani; Jayasooriya, Rajapaksha Gedara Prasad Tharanga; Park, Sang Rul; Choi, Yung-Hyun; Choi, Il-Whan; Kim, Gi-Young

    2016-07-01

    Caffeic acid phenethyl ester (CAPE) exhibits various pharmaceutical properties, including anti-bacterial, anti-inflammatory, anti-viral, anti-cancer, and anti-oxidative activity. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been a promising anti-cancer agent that preferentially induces cancer cell apoptosis with negligible cytotoxicity toward normal cells. Therefore, the present study investigated whether CAPE promotes TRAIL-mediated cytotoxicity in hepatocarcinoma Hep3B cells. The present study demonstrated that CAPE sensitized TRAIL-mediated cell death in Hep3B carcinoma cells. The percentages of the apoptotic cells and annexin-V(+) cells significantly increased in combined treatment with CAPE and TRAIL (CAPE/TRAIL). Treatment with pancaspase inhibitor, z-VAD-fmk, attenuated CAPE/TRAIL-induced apoptosis, suggesting that the combined treatment triggers caspase-dependent apoptosis. Additionally, we found that CAPE stimulated the expression of death receptor 5 (DR5) and treatment with DR5/Fc chimera protein significantly blocked CAPE/TRAIL-induced apoptosis, which indicates that CAPE/TRAIL stimulated apoptosis through the binding of TRAIL to DR5. Moreover, expression of transcription factor C/EBP homologous protein (CHOP) markedly increased in response to CAPE and transient knockdown of CHOP abolished CAPE/TRAIL-mediated apoptosis. These results suggest that CHOP is a key regulator in CAPE/TRAIL-mediated apoptosis. Taken together, the present study found that CAPE significantly enhanced TRAIL-mediated apoptosis in Hep3B carcinoma cells and suggested that CAPE has promising potential in chemoprevention of hepatocellular carcinomas. PMID:27260301

  4. A novel platform for engineering blood-brain barrier-crossing bispecific biologics.

    PubMed

    Farrington, Graham K; Caram-Salas, Nadia; Haqqani, Arsalan S; Brunette, Eric; Eldredge, John; Pepinsky, Blake; Antognetti, Giovanna; Baumann, Ewa; Ding, Wen; Garber, Ellen; Jiang, Susan; Delaney, Christie; Boileau, Eve; Sisk, William P; Stanimirovic, Danica B

    2014-11-01

    The blood-brain barrier (BBB) prevents the access of therapeutic antibodies to central nervous system (CNS) targets. The engineering of bispecific antibodies in which a therapeutic "arm" is combined with a BBB-transcytosing arm can significantly enhance their brain delivery. The BBB-permeable single-domain antibody FC5 was previously isolated by phenotypic panning of a naive llama single-domain antibody phage display library. In this study, FC5 was engineered as a mono- and bivalent fusion with the human Fc domain to optimize it as a modular brain delivery platform. In vitro studies demonstrated that the bivalent fusion of FC5 with Fc increased the rate of transcytosis (Papp) across brain endothelial monolayer by 25% compared with monovalent fusion. Up to a 30-fold enhanced apparent brain exposure (derived from serum and cerebrospinal fluid pharmacokinetic profiles) of FC5- compared with control domain antibody-Fc fusions after systemic dosing in rats was observed. Systemic pharmacological potency was evaluated in the Hargreaves model of inflammatory pain using the BBB-impermeable neuropeptides dalargin and neuropeptide Y chemically conjugated with FC5-Fc fusion proteins. Improved serum pharmacokinetics of Fc-fused FC5 contributed to a 60-fold increase in pharmacological potency compared with the single-domain version of FC5; bivalent and monovalent FC5 fusions with Fc exhibited similar systemic pharmacological potency. The study demonstrates that modular incorporation of FC5 as the BBB-carrier arm in bispecific antibodies or antibody-drug conjugates offers an avenue to develop pharmacologically active biotherapeutics for CNS indications. PMID:25070367

  5. TBBPA exposure during a sensitive developmental window produces neurobehavioral changes in larval zebrafish.

    PubMed

    Chen, Jiangfei; Tanguay, Robert L; Xiao, Yanyan; Haggard, Derik E; Ge, Xiaoqing; Jia, Yinhang; Zheng, Yi; Dong, Qiaoxiang; Huang, Changjiang; Lin, Kuangfei

    2016-09-01

    Tetrabromobisphenol A (TBBPA), one of the most widely used brominated flame retardants (BFRs), is a ubiquitous contaminant in the environment and in the human body. This study demonstrated that zebrafish embryos exposed to TBBPA during a sensitive window of 8-48 h post-fertilization (hpf) displayed morphological malformations and mortality. Zebrafish exposed exclusively between 48 and 96 hpf were phenotypically normal. TBBPA was efficiently absorbed and accumulated in zebrafish embryos, but was eliminated quickly when the exposure solution was removed. Larval behavior assays conducted at 120 hpf indicated that exposure to 5 μM TBBPA from 8 to 48 hpf produced larvae with significantly lower average activity and speed of movement in the normal condition than in those exposed from 48 to 96 hpf. Specifically, 8-48 hpf-exposed larvae spent significantly less time in both activity bursts and gross movements compared to control or 48-96 hpf exposed larvae. Consistent with the motor deficits, TBBPA induced apoptotic cell death, delayed cranial motor neuron development, inhibited primary motor neuron development and loosed muscle fiber during the early developmental stages. To further explore TBBPA-induced developmental and neurobehavioral toxicity, RNA-Seq analysis was used to identify early transcriptional changes following TBBPA exposure. In total, 1969 transcripts were significantly differentially expressed (P < 0.05, FDR < 0.05, 1.5-FC) upon TBBPA exposure. Functional and pathway analysis of the TBBPA transcriptional profile identified biological processes involved in nerve development, muscle filament sliding and contraction, and extracellular matrix disassembly and organization changed significantly. In addition, TBBPA also led to an elevation in the expression of genes encoding uridine diphosphate glucuronyl transferases (ugt), which could affect thyroxine (T4) metabolism and subsequently lead to neurobehavioral changes. In summary, TBBPA exposure

  6. Therapeutic potential of stem cells expressing suicide genes that selectively target human breast cancer cells: Evidence that they exert tumoricidal effects via tumor tropism

    PubMed Central

    YI, BO-RIM; CHOI, KELVIN J.; KIM, SEUNG U.; CHOI, KYUNG-CHUL

    2012-01-01

    Breast cancer is the most prevalent cancer in women worldwide and is classified into ductal and lobular carcinoma. Breast cancer as well as lobular carcinoma is associated with various risk factors such as gender, age, female hormone exposure, ethnicity, family history and genetic risk factor-associated genes. Genes associated with a high risk of developing breast cancer include BRCA1, BRCA2, p53, PTEN, CHEK2 and ATM. Surgery, chemotherapy, radiotherapy and hormone therapy are used to treat breast cancer but these therapies, except for surgery, have many side-effects such as alopecia, anesthesia, diarrhea and arthralgia. Gene-directed enzyme/prodrug therapy (GEPT) or suicide gene therapy, may improve the therapeutic efficacy of conventional cancer radiotherapy and chemotherapy without side-effects. GEPT most often involves the use of a viral vector to deliver a gene not found in mammalian cells and that produces enzymes which can convert a relatively non-toxic prodrug into a toxic agent. Examples of these systems include cytosine deaminase/5-fluorocytosine (CD/5-FC), carboxyl esterase/irinotecan (CE/CPT-11), and thymidine kinase/ganciclovir (TK/GCV). Recently, therapies based on genetically engineered stem cells (GESTECs) using a GEPT system have received a great deal of attention for their clinical and therapeutic potential to treat breast cancer. In this review, we discuss the potential of GESTECs via tumor tropism effects and therapeutic efficacy against several different types of cancer cells. GESTECs represent a useful tool for treating breast cancer without inducing injuries associated with conventional therapeutic modalities. PMID:22736197

  7. Nanostructured self-assembly materials from neat and aqueous solutions of C18 lipid pro-drug analogues of Capecitabine—a chemotherapy agent. Focus on nanoparticulate cubosomes™ of the oleyl analogue

    SciTech Connect

    Gong, Xiaojuan; Moghaddam, Minoo J.; Sagnella, Sharon M.; Conn, Charlotte E.; Mulet, Xavier; Danon, Stephen J.; Waddington, Lynne J.; Drummond, Calum J.

    2014-09-24

    A series of prodrug analogues based on the established chemotherapy agent, 5-fluorouracil, have been prepared and characterized. C18 alkyl and alkenyl chains with increasing degree of unsaturation were attached to the N4 position of the 5-fluorocytosine (5-FC) base via a carbamate bond. Physicochemical characterization of the prodrug analogues was carried out using a combination of differential scanning calorimetry, cross-polarized optical microscopy, X-ray diffraction and small-angle X-ray scattering. The presence of a monounsaturated oleyl chain was found to promote lyotropic liquid crystalline phase formation in excess water with a fluid lamellar phase observed at room temperature and one or more bicontinuous cubic phases at 37 °C. The bulk phase was successfully dispersed into liposomes or cubosomes at room and physiological temperature respectively. In vitro toxicity of the nanoparticulate 5-FCOle dispersions was evaluated against several normal and cancer cell types over a 48 h period and exhibited an IC50 of -100 μM against all cell types. The in vivo efficacy of 5-FCOle cubosomes was assessed against the highly aggressive mouse 4T1 breast cancer model and compared to Capecitabine (a water-soluble commercially available 5-FU prodrug) delivered at the same dosages. After 21 days of treatment, the 0.5 mmol 5-FCOle treatment group exhibited a significantly smaller average tumour volume than all other treatment groups including Capecitabine at similar dosage. These results exemplify the potential of self-assembled amphiphile prodrugs for delivery of bioactives in vivo.

  8. Taphonomic perspectives on hominid site use and foraging strategies during Bed II times at Olduvai Gorge, Tanzania.

    PubMed

    Egeland, Charles P; Domínguez-Rodrigo, Manuel

    2008-12-01

    The faunal assemblages excavated by Mary Leakey in Bed II of Olduvai Gorge, Tanzania, have, like the more well-known Bed I assemblages, traditionally been interpreted as the result of hominid butchering activities in the lake margin and riverine settings of the paleo-Olduvai Basin. A reexamination of all of Leakey's Bed I sites has shown that hominids played little or no role in the formation of all but one of those faunal assemblages, a finding that prompted the reanalysis of the Bed II sites presented here. We expand upon a previous taphonomic study that provided systematic data for HWK East Levels 1-2, MNK Main, and BK. In addition to these assemblages, we provide data on HWK East Levels 3-5, FC West, TK, and SHK. Our data contradict previous interpretations of MNK Main as a hominid accumulation but uphold the contention that BK represents a primarily hominid accumulation reflecting early access to carcasses. The small and poorly preserved assemblages from FC West and TK are difficult to link unambiguously to either hominids or carnivores. Site MNK Main and HWK East Levels 3-5 appear to be death arenas where carcasses accumulated via natural deaths and/or serial predation. Site SHK is severely biased by selective retention and therefore little can be said of its formational history. Nevertheless, no hominid modifications were documented in this assemblage. Comparisons with other Olduvai sites indicate a more conspicuous hyena taphonomic signal during Bed II times than Bed I times, which appears to mirror the changing configuration of the large carnivore guild. These findings also beg the question of what activities were being carried out by hominids with the stone tools discarded at these sites. Although it seems clear that hominids were utilizing stone tools to carry out subsistence activities unrelated to carcass butchery, more excavation and techniques such as phytolith analysis should be employed to explore alternative explanations. PMID:18842286

  9. PSMA-Targeted Theranostic Nanoplex for Prostate Cancer Therapy

    PubMed Central

    Nimmagadda, Sridhar; Li, Cong; Banerjee, Sangeeta R.; Winnard, Paul T.; Artemov, Dmitri; Glunde, Kristine; Pomper, Martin G.; Bhujwalla, Zaver M.

    2014-01-01

    Theranostic imaging, where diagnosis is combined with therapy, is particularly suitable for a disease that is as complex as cancer, especially now that genomic and proteomic profiling can provide an extensive “fingerprint” of each tumor. With such information, theranostic agents can be designed to personalize treatment and minimize damage to normal tissue. Here we have developed a nanoplex platform for theranostic imaging of prostate cancer (PCa). In these proof-of-principle studies, a therapeutic nanoplex containing multimodal imaging reporters was targeted to prostate-specific membrane antigen (PSMA), which is expressed on the cell surface of castrate-resistant PCa. The nanoplex was designed to deliver small interfering RNA (siRNA) along with a prodrug enzyme to PSMA-expressing tumors. Each component of the nanoplex was carefully selected to evaluate its diagnostic aspect of PSMA imaging and its therapeutic aspects of siRNA-mediated down-regulation of a target gene and the conversion of a prodrug to cytotoxic drug, using noninvasive multimodality imaging. Studies performed using two variants of human PC3-PCa cells and tumors, one with high PSMA expression level and another with negligible expression levels, demonstrated PSMA-specific uptake. In addition, down-regulation of the selected siRNA target, choline kinase (Chk), and the conversion of the nontoxic prodrug 5-fluorocytosine (5-FC) to cytotoxic 5-fluorouracil (5-FU) were also demonstrated with noninvasive imaging. The nanoplex was well-tolerated and did not induce liver or kidney toxicity or a significant immune response. The nanoplex platform described can be easily modified and applied to different cancers, receptors, and pathways to achieve theranostic imaging, as a single agent or in combination with other treatment modalities. PMID:22866897

  10. Inflammation-driven malnutrition: a new screening tool predicts outcome in Crohn's disease.

    PubMed

    Jansen, Irene; Prager, Matthias; Valentini, Luzia; Büning, Carsten

    2016-09-01

    Malnutrition is a frequent feature in Crohn's disease (CD), affects patient outcome and must be recognised. For chronic inflammatory diseases, recent guidelines recommend the development of combined malnutrition and inflammation risk scores. We aimed to design and evaluate a new screening tool that combines both malnutrition and inflammation parameters that might help predict clinical outcome. In a prospective cohort study, we examined fifty-five patients with CD in remission (Crohn's disease activity index (CDAI) <200) at 0 and 6 months. We assessed disease activity (CDAI, Harvey-Bradshaw index), inflammation (C-reactive protein (CRP), faecal calprotectin (FC)), malnutrition (BMI, subjective global assessment (SGA), serum albumin, handgrip strength), body composition (bioelectrical impedance analysis) and administered the newly developed 'Malnutrition Inflammation Risk Tool' (MIRT; containing BMI, unintentional weight loss over 3 months and CRP). All parameters were evaluated regarding their ability to predict disease outcome prospectively at 6 months. At baseline, more than one-third of patients showed elevated inflammatory markers despite clinical remission (36·4 % CRP ≥5 mg/l, 41·5 % FC ≥100 µg/g). Prevalence of malnutrition at baseline according to BMI, SGA and serum albumin was 2-16 %. At 6 months, MIRT significantly predicted outcome in numerous nutritional and clinical parameters (SGA, CD-related flares, hospitalisations and surgeries). In contrast, SGA, handgrip strength, BMI, albumin and body composition had no influence on the clinical course. The newly developed MIRT was found to reliably predict clinical outcome in CD patients. This screening tool might be used to facilitate clinical decision making, including treatment of both inflammation and malnutrition in order to prevent complications. PMID:27546478

  11. The EphA8 Receptor Regulates Integrin Activity through p110γ Phosphatidylinositol-3 Kinase in a Tyrosine Kinase Activity-Independent Manner

    PubMed Central

    Gu, Changkyu; Park, Soochul

    2001-01-01

    Recent genetic studies suggest that ephrins may function in a kinase-independent Eph receptor pathway. Here we report that expression of EphA8 in either NIH 3T3 or HEK293 cells enhanced cell adhesion to fibronectin via α5β1- or β3 integrins. Interestingly, a kinase-inactive EphA8 mutant also markedly promoted cell attachment to fibronectin in these cell lines. Using a panel of EphA8 point mutants, we have demonstrated that EphA8 kinase activity does not correlate with its ability to promote cell attachment to fibronectin. Analysis using EphA8 extracellular and intracellular domain mutants has revealed that enhanced cell adhesion is dependent on ephrin A binding to the extracellular domain and the juxtamembrane segment of the cytoplasmic domain of the receptor. EphA8-promoted adhesion was efficiently inhibited by wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor. Additionally, we found that EphA8 had associated PI 3-kinase activity and that the p110γ isoform of PI 3-kinase is associated with EphA8. In vitro binding experiments revealed that the EphA8 juxtamembrane segment was sufficient for the formation of a stable complex with p110γ. Similar results were obtained in assay using cells stripped of endogenous ephrin A ligands by treatment with preclustered ephrin A5-Fc proteins. In addition, a membrane-targeted lipid kinase-inactive p110γ mutant was demonstrated to stably associate with EphA8 and suppress EphA8-promoted cell adhesion to fibronectin. Taken together, these results suggest the presence of a novel mechanism by which the EphA8 receptor localizes p110γ PI 3-kinase to the plasma membrane in a tyrosine kinase-independent fashion, thereby allowing access to lipid substrates to enable the signals required for integrin-mediated cell adhesion. PMID:11416136

  12. Adjunctive Interferon-γ Immunotherapy for the Treatment of HIV-associated Cryptococcal Meningitis: A Randomized Controlled Trial.

    PubMed Central

    Jarvis, Joseph N; Meintjes, Graeme; Rebe, Kevin; Williams, Gertrude Ntombomzi; Bicanic, Tihana; Williams, Anthony; Schutz, Charlotte; Bekker, Linda-Gail; Wood, Robin; Harrison, Thomas S

    2013-01-01

    Background Interferon-γ is of key importance in the immune response to Cryptococcus neoformans. Mortality related to cryptococcal meningitis (CM) remains high, and novel treatment strategies are needed. We performed an RCT to determine whether addition of IFNγ to standard therapy increased the rate of clearance of cryptococcal infection in HIV-associated CM. Methods Patients were randomized to: (1) Amphotericin-B 1mg/kg/day plus 5-FC 100mg/kg/day for 2-weeks (Standard therapy), (2) Standard therapy plus IFNγ1b 100μg days 1 and 3 (IFNγ 2-doses), or (3) Standard therapy plus IFNγ1b 100μg days 1, 3, 5, 8, 10 and 12 (IFNγ 6-doses). Primary outcome was rate of clearance of cryptococcus from the CSF (early fungicidal activity, EFA) calculated from serial quantitative cultures, previously shown to be independently associated with survival. Results Rate of fungal clearance was significantly faster in IFNγ containing groups than with standard treatment. Mean EFA (logCFU/ml/day) was −0.49 with standard treatment, −0.64 with IFNγ 2-doses, and −0.64 with IFNγ 6-doses. Difference in EFA was −0.15 (95%CI −0.02- −0.27, p=0.02) between standard treatment and IFNγ 2-doses, and −0.15 (95%CI-0.05- −0.26, p=0.006) between standard treatment and IFNγ 6-doses. Mortality was 16% (14/88) at 2 weeks and 31% (27/87) at 10 weeks, with no significant difference between groups. All treatments were well tolerated. Conclusions Addition of short-course IFNγ to standard treatment significantly increased the rate of clearance of cryptococcal infection from the CSF, and was not associated with any increase in adverse events. Two doses of IFNγ are as effective as 6 doses. PMID:22421244

  13. Inter-hemispheric EEG coherence analysis in Parkinson's disease: assessing brain activity during emotion processing.

    PubMed

    Yuvaraj, R; Murugappan, M; Ibrahim, Norlinah Mohamed; Sundaraj, Kenneth; Omar, Mohd Iqbal; Mohamad, Khairiyah; Palaniappan, R; Satiyan, M

    2015-02-01

    Parkinson's disease (PD) is not only characterized by its prominent motor symptoms but also associated with disturbances in cognitive and emotional functioning. The objective of the present study was to investigate the influence of emotion processing on inter-hemispheric electroencephalography (EEG) coherence in PD. Multimodal emotional stimuli (happiness, sadness, fear, anger, surprise, and disgust) were presented to 20 PD patients and 30 age-, education level-, and gender-matched healthy controls (HC) while EEG was recorded. Inter-hemispheric coherence was computed from seven homologous EEG electrode pairs (AF3-AF4, F7-F8, F3-F4, FC5-FC6, T7-T8, P7-P8, and O1-O2) for delta, theta, alpha, beta, and gamma frequency bands. In addition, subjective ratings were obtained for a representative of emotional stimuli. Interhemispherically, PD patients showed significantly lower coherence in theta, alpha, beta, and gamma frequency bands than HC during emotion processing. No significant changes were found in the delta frequency band coherence. We also found that PD patients were more impaired in recognizing negative emotions (sadness, fear, anger, and disgust) than relatively positive emotions (happiness and surprise). Behaviorally, PD patients did not show impairment in emotion recognition as measured by subjective ratings. These findings suggest that PD patients may have an impairment of inter-hemispheric functional connectivity (i.e., a decline in cortical connectivity) during emotion processing. This study may increase the awareness of EEG emotional response studies in clinical practice to uncover potential neurophysiologic abnormalities. PMID:24894699

  14. Advanced Running Performance by Genetic Predisposition in Male Dummerstorf Marathon Mice (DUhTP) Reveals Higher Sterol Regulatory Element-Binding Protein (SREBP) Related mRNA Expression in the Liver and Higher Serum Levels of Progesterone

    PubMed Central

    Brenmoehl, Julia; Walz, Christina; Ponsuksili, Siriluck; Schwerin, Manfred; Fuellen, Georg; Hoeflich, Andreas

    2016-01-01

    Long-term-selected DUhTP mice represent a non-inbred model for inborn physical high-performance without previous training. Abundance of hepatic mRNA in 70-day male DUhTP and control mice was analyzed using the Affymetrix mouse array 430A 2.0. Differential expression analysis with PLIER corrected data was performed using AltAnalyze. Searching for over-representation in biochemical pathways revealed cholesterol metabolism being most prominently affected in DUhTP compared to unselected control mice. Furthermore, pathway analysis by AltAnalyze plus PathVisio indicated significant induction of glycolysis, fatty acid synthesis and cholesterol biosynthesis in the liver of DUhTP mice versus unselected control mice. In contrast, gluconeogenesis was partially inactivated as judged from the analysis of hepatic mRNA transcript abundance in DUhTP mice. Analysis of mRNA transcripts related to steroid hormone metabolism inferred elevated synthesis of progesterone and reduced levels of sex steroids. Abundance of steroid delta isomerase-5 mRNA (Hsd3b5, FC 4.97) was increased and steroid 17-alpha-monooxygenase mRNA (Cyp17a1, FC -11.6) was massively diminished in the liver of DUhTP mice. Assessment of steroid profiles by LC-MS revealed increased levels of progesterone and decreased levels of sex steroids in serum from DUhTP mice versus controls. Analysis of hepatic mRNA transcript abundance indicates that sterol regulatory element-binding protein-1 (SREBP-1) may play a major role in metabolic pathway activation in the marathon mouse model DUhTP. Thus, results from bioinformatics modeling of hepatic mRNA transcript abundance correlated with direct steroid analysis by mass spectrometry and further indicated functions of SREBP-1 and steroid hormones for endurance performance in DUhTP mice. PMID:26799318

  15. Advanced Running Performance by Genetic Predisposition in Male Dummerstorf Marathon Mice (DUhTP) Reveals Higher Sterol Regulatory Element-Binding Protein (SREBP) Related mRNA Expression in the Liver and Higher Serum Levels of Progesterone.

    PubMed

    Ohde, Daniela; Moeller, Mark; Brenmoehl, Julia; Walz, Christina; Ponsuksili, Siriluck; Schwerin, Manfred; Fuellen, Georg; Hoeflich, Andreas

    2016-01-01

    Long-term-selected DUhTP mice represent a non-inbred model for inborn physical high-performance without previous training. Abundance of hepatic mRNA in 70-day male DUhTP and control mice was analyzed using the Affymetrix mouse array 430A 2.0. Differential expression analysis with PLIER corrected data was performed using AltAnalyze. Searching for over-representation in biochemical pathways revealed cholesterol metabolism being most prominently affected in DUhTP compared to unselected control mice. Furthermore, pathway analysis by AltAnalyze plus PathVisio indicated significant induction of glycolysis, fatty acid synthesis and cholesterol biosynthesis in the liver of DUhTP mice versus unselected control mice. In contrast, gluconeogenesis was partially inactivated as judged from the analysis of hepatic mRNA transcript abundance in DUhTP mice. Analysis of mRNA transcripts related to steroid hormone metabolism inferred elevated synthesis of progesterone and reduced levels of sex steroids. Abundance of steroid delta isomerase-5 mRNA (Hsd3b5, FC 4.97) was increased and steroid 17-alpha-monooxygenase mRNA (Cyp17a1, FC -11.6) was massively diminished in the liver of DUhTP mice. Assessment of steroid profiles by LC-MS revealed increased levels of progesterone and decreased levels of sex steroids in serum from DUhTP mice versus controls. Analysis of hepatic mRNA transcript abundance indicates that sterol regulatory element-binding protein-1 (SREBP-1) may play a major role in metabolic pathway activation in the marathon mouse model DUhTP. Thus, results from bioinformatics modeling of hepatic mRNA transcript abundance correlated with direct steroid analysis by mass spectrometry and further indicated functions of SREBP-1 and steroid hormones for endurance performance in DUhTP mice. PMID:26799318

  16. Death Receptor 5 Signaling Promotes Hepatocyte Lipoapoptosis*

    PubMed Central

    Cazanave, Sophie C.; Mott, Justin L.; Bronk, Steven F.; Werneburg, Nathan W.; Fingas, Christian D.; Meng, X. Wei; Finnberg, Niklas; El-Deiry, Wafik S.; Kaufmann, Scott H.; Gores, Gregory J.

    2011-01-01

    Nonalcoholic steatohepatitis is characterized by hepatic steatosis, elevated levels of circulating free fatty acids (FFA), endoplasmic reticulum (ER) stress, and hepatocyte lipoapoptosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 5 (DR5) is significantly elevated in patients with nonalcoholic steatohepatitis, and steatotic hepatocytes demonstrate increased sensitivity to TRAIL-mediated cell death. Nonetheless, a role for TRAIL and/or DR5 in mediating lipoapoptotic pathways is unexplored. Here, we examined the contribution of DR5 death signaling to lipoapoptosis by free fatty acids. The toxic saturated free fatty acid palmitate induces an increase in DR5 mRNA and protein expression in Huh-7 human hepatoma cells leading to DR5 localization into lipid rafts, cell surface receptor clustering with subsequent recruitment of the initiator caspase-8, and ultimately cellular demise. Lipoapoptosis by palmitate was not inhibited by a soluble human recombinant DR5-Fc chimera protein suggesting that DR5 cytotoxic signaling is ligand-independent. Hepatocytes from murine TRAIL receptor knock-out mice (DR−/−) displayed reduced palmitate-mediated lipotoxicity. Likewise, knockdown of DR5 or caspase-8 expression by shRNA technology attenuated palmitate-induced Bax activation and apoptosis in Huh-7 cells, without altering induction of ER stress markers. Similar observations were verified in other cell models. Finally, knockdown of CHOP, an ER stress-mediated transcription factor, reduced DR5 up-regulation and DR5-mediated caspase-8 activation upon palmitate treatment. Collectively, these results suggest that ER stress-induced CHOP activation by palmitate transcriptionally up-regulates DR5, likely resulting in ligand-independent cytotoxic signaling by this death receptor. PMID:21941003

  17. Increased sensitivity of glioma cells to 5-fluorocytosine following photo-chemical internalization enhanced nonviral transfection of the cytosine deaminase suicide gene.

    PubMed

    Wang, Frederick; Zamora, Genesis; Sun, Chung-Ho; Trinidad, Anthony; Chun, Changho; Kwon, Young Jik; Berg, Kristian; Madsen, Steen J; Hirschberg, Henry

    2014-05-01

    Despite advances in surgery, chemotherapy and radiotherapy, the outcomes of patients with GBM have not significantly improved. Tumor recurrence in the resection margins occurs in more than 80% of cases indicating aggressive treatment modalities, such as gene therapy are warranted. We have examined photochemical internalization (PCI) as a method for the non-viral transfection of the cytosine deaminase (CD) suicide gene into glioma cells. The CD gene encodes an enzyme that can convert the nontoxic antifungal agent, 5-fluorocytosine, into the chemotherapeutic drug, 5-fluorouracil. Multicell tumor spheroids derived from established rat and human glioma cell lines were used as in vitro tumor models. Plasmids containing either the CD gene alone or together with the uracil phosphoribosyl transferase (UPRT) gene combined with the gene carrier protamine sulfate were employed in all experiments.PCI was performed with the photosensitizer AlPcS2a and 670 nm laser irradiance. Protamine sulfate/CD DNA polyplexes proved nontoxic but inefficient transfection agents due to endosomal entrapment. In contrast, PCI mediated CD gene transfection resulted in a significant inhibition of spheroid growth in the presence of, but not in the absence of, 5-FC. Repetitive PCI induced transfection was more efficient at low CD plasmid concentration than single treatment. The results clearly indicate that AlPcS2a-mediated PCI can be used to enhance transfection of a tumor suicide gene such as CD, in malignant glioma cells and cells transfected with both the CD and UPRT genes had a pronounced bystander effect. PMID:24610460

  18. Ephrin-A5 acts as a repulsive cue for migrating cortical interneurons.

    PubMed

    Zimmer, Geraldine; Garcez, Patricia; Rudolph, Judith; Niehage, Ronny; Weth, Franco; Lent, Roberto; Bolz, Jürgen

    2008-07-01

    Cortical interneurons are born in the germinative zones of the ganglionic eminences in the subpallium, and migrate tangentially in spatially and temporally well-defined corridors into the neocortex. Because ephrin-A5 is expressed in the ventricular zone (VZ) of the ganglionic eminences at these developmental stages, we examined the possible effects of this molecule on interneuron migration. Double-immunocytochemistry of dissociated neurons from the medial ganglionic eminences (MGE) revealed that calbindin-positive cells express the EphA4-receptor. In situ, EphA4 is strongly expressed in the subventricular zone of the ganglionic eminences. Using different in vitro assays, we found that ephrin-A5 acts as a repellent cue for MGE neurons. We then examined interneuron migration in slice overlay experiments, where MGE-derived explants from enhanced green fluorescent protein-expressing transgenic mice were homotopically grafted into host slices from wild-type littermate embryos. In these in vitro preparations, interneurons recapitulated in vivo cell migration in several respects. However, interneurons in brain slices also migrated in the VZ of the ganglionic eminences, a region that is strictly avoided in vivo. In situ hybridizations revealed that ephrin-A5 became downregulated in the VZ in vitro. When recombinant ephrin-A5-Fc was added to the slices, it preferentially bound to the VZ, and migrating MGE neurons avoided the VZ as in vivo. The restoration of the normal migration pathway in slices required ephrin-A5 clustering and signalling of Src family kinases. Together, these experiments suggest that ephrin-A5 acts as an inhibitory flank that contributes to define the pathway of migrating interneurons. PMID:18662335

  19. MRI phenotypes with high neurodegeneration are associated with peripheral blood B-cell changes.

    PubMed

    Comabella, Manuel; Cantó, Ester; Nurtdinov, Ramil; Río, Jordi; Villar, Luisa M; Picón, Carmen; Castilló, Joaquín; Fissolo, Nicolás; Aymerich, Xavier; Auger, Cristina; Rovira, Alex; Montalban, Xavier

    2016-01-15

    Little is known about the mechanisms leading to neurodegeneration in multiple sclerosis (MS) and the role of peripheral blood cells in this neurodegenerative component. We aimed to correlate brain radiological phenotypes defined by high and low neurodegeneration with gene expression profiling of peripheral blood mononuclear cells (PBMC) from MS patients. Magnetic resonance imaging (MRI) scans from 64 patients with relapsing-remitting MS (RRMS) were classified into radiological phenotypes characterized by low (N = 27) and high (N = 37) neurodegeneration according to the number of contrast-enhancing lesions, the relative volume of non-enhancing black holes on T1-weighted images, and the brain parenchymal fraction. Gene expression profiling was determined in PBMC using microarrays, and validation of selected genes was performed by polymerase chain reaction (PCR). B-cell immunophenotyping was conducted by flow cytometry. Microarray analysis revealed the B-cell specific genes FCRL1, FCRL2, FCRL5 (Fc receptor-like 1, 2 and 5 respectively), and CD22 as the top differentially expressed genes between patients with high and low neurodegeneration. Levels for these genes were significantly down-regulated in PBMC from patients with MRI phenotypes characterized by high neurodegeneration and microarray findings were validated by PCR. In patients with high neurodegeneration, immunophenotyping showed a significant increase in the expression of the B-cell activation markers CD80 in naïve B cells (CD45+/CD19+/CD27-/IgD+), unswitched memory B cells (CD45+/CD19+/CD27+/IgD+), and switched memory B cells (CD45+/CD19+/CD27+/IgD-), and CD86 in naïve and switched memory B cells. These results suggest that RRMS patients with radiological phenotypes showing high neurodegeneration have changes in B cells characterized by down-regulation of B-cell-specific genes and increased activation status. PMID:26604134

  20. Evaluation of blue light exposure to beta brainwaves on simulated night driving

    NASA Astrophysics Data System (ADS)

    Purawijaya, Dandri Aly; Fitri, Lulu Lusianti; Suprijanto

    2015-09-01

    Numbers of night driving accident in Indonesia since 2010 are exponentially rising each year with total of loss more than 50 billion rupiah. One of the causes that contribute to night driving accident is drowsiness. Drowsiness is affected by circadian rhythm resulted from the difference of blue light quality and quantity between night and day. Blue light may effect on human physiology through non-visual pathway by suppressing melatonin hormone suppression that influence drowsiness. Meanwhile, the production of hormones and other activities in brain generate bioelectrical activity such as brainwaves and can be recorded using Electroencephalograph (EEG). Therefore, this research objective is to evaluate the effect of blue light exposure to beta brainwave emergence during night driving simulation to a driver. This research was conducted to 4 male subjects who are able to drive and have a legitimate car driving license. The driving simulator was done using SCANIA Truck Driving Simulator on freeform driving mode in dark environment. Subjects drove for total 32 minutes. The data collections were taken in 2 days with 16 minutes for each day. The 16 minutes were divided again into 8 minutes adaptation in dark and 8 minutes for driving either in blue light exposure or in total darkness. While driving the simulation, subjects' brainwaves were recorded using EEG EMOTIV 14 Channels, exposed by LED monochromatic blue light with 160 Lux from source and angle 45o and sat 1 m in front of the screen. Channels used on this research were for visual (O1; O2), cognition (F3; F4; P7; P8), and motor (FC5; FC6). EEG brainwave result was filtered with EEGLab to obtain beta waves at 13 - 30 Hz frequencies. Results showed that beta waves response to blue light varied for each subject. Blue light exposure either increased or decreased beta waves in 2 minutes pattern and maintaining beta waves on cognition and motor area in 3 out of 4 subjects. Meanwhile, blue light exposure did not maintain