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Sample records for target genes combining

  1. Il2rg gene-targeted severe combined immunodeficiency pigs.

    Science.gov (United States)

    Suzuki, Shunichi; Iwamoto, Masaki; Saito, Yoriko; Fuchimoto, Daiichiro; Sembon, Shoichiro; Suzuki, Misae; Mikawa, Satoshi; Hashimoto, Michiko; Aoki, Yuki; Najima, Yuho; Takagi, Shinsuke; Suzuki, Nahoko; Suzuki, Emi; Kubo, Masanori; Mimuro, Jun; Kashiwakura, Yuji; Madoiwa, Seiji; Sakata, Yoichi; Perry, Anthony C F; Ishikawa, Fumihiko; Onishi, Akira

    2012-06-14

    A porcine model of severe combined immunodeficiency (SCID) promises to facilitate human cancer studies, the humanization of tissue for xenotransplantation, and the evaluation of stem cells for clinical therapy, but SCID pigs have not been described. We report here the generation and preliminary evaluation of a porcine SCID model. Fibroblasts containing a targeted disruption of the X-linked interleukin-2 receptor gamma chain gene, Il2rg, were used as donors to generate cloned pigs by serial nuclear transfer. Germline transmission of the Il2rg deletion produced healthy Il2rg(+/-) females, while Il2rg(-/Y) males were athymic and exhibited markedly impaired immunoglobulin and T and NK cell production, robustly recapitulating human SCID. Following allogeneic bone marrow transplantation, donor cells stably integrated in Il2rg(-/Y) heterozygotes and reconstituted the Il2rg(-/Y) lymphoid lineage. The SCID pigs described here represent a step toward the comprehensive evaluation of preclinical cellular regenerative strategies. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Identification of Multiple Cryptococcal Fungicidal Drug Targets by Combined Gene Dosing and Drug Affinity Responsive Target Stability Screening

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    Yoon-Dong Park

    2016-08-01

    Full Text Available Cryptococcus neoformans is a pathogenic fungus that is responsible for up to half a million cases of meningitis globally, especially in immunocompromised individuals. Common fungistatic drugs, such as fluconazole, are less toxic for patients but have low efficacy for initial therapy of the disease. Effective therapy against the disease is provided by the fungicidal drug amphotericin B; however, due to its high toxicity and the difficulty in administering its intravenous formulation, it is imperative to find new therapies targeting the fungus. The antiparasitic drug bithionol has been recently identified as having potent fungicidal activity. In this study, we used a combined gene dosing and drug affinity responsive target stability (GD-DARTS screen as well as protein modeling to identify a common drug binding site of bithionol within multiple NAD-dependent dehydrogenase drug targets. This combination genetic and proteomic method thus provides a powerful method for identifying novel fungicidal drug targets for further development.

  3. Vorinostat in combination with bortezomib in patients with advanced malignancies directly alters transcription of target genes.

    Science.gov (United States)

    Kolesar, Jill M; Traynor, Anne M; Holen, Kyle D; Hoang, Tien; Seo, Songwon; Kim, Kyungmann; Alberti, Dona; Espinoza-Delgado, Igor; Wright, John J; Wilding, George; Bailey, Howard H; Schelman, William R

    2013-09-01

    Vorinostat is a small molecule inhibitor of class I and II histone deacetylase enzymes which alters the expression of target genes including the cell cycle gene p21, leading to cell cycle arrest and apoptosis. Patients enrolled in a phase I trial were treated with vorinostat alone on day 1 and vorinostat and bortezomib in combination on day 9. Paired biopsies were obtained in eleven subjects. Blood samples were obtained on days 1 and 9 of cycle 1 prior to dosing and 2 and 6 h post-dosing in all 60 subjects. Gene expression of p21, HSP70, AKT, Nur77, ERB1, and ERB2 was evaluated in peripheral blood mononuclear cells and tissue samples. Chromatin immunoprecipitation of p21, HSP70, and Nur77 was also performed in biopsy samples. In peripheral blood mononuclear cells, Nur77 was significantly and consistently decreased 2 h after vorinostat administration on both days 1 and 9, median ratio of gene expression relative to baseline of 0.69 with interquartile range 0.49-1.04 (p vorinostat and bortezomib. p21, a downstream target of Nur77, was significantly decreased on day 9, 2 and 6 h after administration of vorinostat and bortezomib, 0.67 (0.41-1.03) (p vorinostat in tissue biopsies in most patients. Vorinostat inhibits Nur77 expression, which in turn may decrease p21 and AKT expression in PBMCs. The influence of vorinostat on target gene expression in tumor tissue was variable; however, most patients demonstrated interaction of acetylated H3 with Nur77, HSP70, and p21 which provides evidence of interaction with the transcriptionally active acetylated H3.

  4. Targeted gene delivery in tumor xenografts by the combination of ultrasound-targeted microbubble destruction and polyethylenimine to inhibit survivin gene expression and induce apoptosis

    Directory of Open Access Journals (Sweden)

    Qiu Ri-Xiang

    2010-11-01

    Full Text Available Abstract Background Noninvasive and tissue-specific technologies of gene transfection would be valuable in clinical gene therapy. This present study was designed to determine whether it could enhance gene transfection in vivo by the combination of ultrasound-targeted microbubble destruction (UTMD with polyethylenimine (PEI in tumor xenografts, and illuminate the effects of gene silencing and apoptosis induction with short hairpin RNA (shRNA interference therapy targeting human survivin by this novel technique. Methods Two different expression vectors (pCMV-LUC and pSIREN were incubated with PEI to prepare cationic complexes (PEI/DNA and confirmed by the gel retardation assay. Human cervical carcinoma (Hela tumors were planted subcutaneously in both flanks of nude mice. Tumor-bearing mice were administered by tail vein with PBS, plasmid, plasmid and SonoVue microbubble, PEI/DNA and SonoVue microbubble. One tumor was exposed to ultrasound irradiation, while the other served as control. The feasibility of targeted delivery and tissue specificity facilitated by UTMD and PEI were investigated. Moreover, immunohistochemistry analyses about gene silencing and apoptosis induction were detected. Results Electrophoresis experiment revealed that PEI could condense DNA efficiently. The application of UTMD significantly increases the tissue transfection. Both expression vectors showed that gene expressions were present in all sections of tumors that received ultrasound exposure but not in control tumors. More importantly, the increases in transgene expression were related to UTMD with the presence of PEI significantly. Silencing of the survivin gene could induce apoptosis effectively by downregulating survivin and bcl-2 expression, also cause up-regulation of bax and caspase-3 expression. Conclusions This noninvasive, novel combination of UTMD with PEI could enhance targeted gene delivery and gene expression in tumor xenografts at intravenous administration

  5. The study of irradiation combined with targeted suicide gene therapy for prostate cancer xenografts

    International Nuclear Information System (INIS)

    Lu Xueguan; Milas, L.

    2007-01-01

    Objective: To study whether RGD-4C AAVP HSV-TK/GCV, one of suicide gene therapy targeting to Integrin αv, can enhance radiotherapeutic effect for DU145 prostate cancer xenografts or not. Methods: When the diameter of tumor in 48 nude mice bearing DU145 prostate cancer in the right leg attained 6.0 mm (5.8-6.3 mm), the mice were entered into the experiment. There were 6 experimental groups (8 mice per group), including the control, radiotherapy only (RT), RGD-4C AAVP HSV-TK/GCV only (Targeted, RGD-4C), AAVP HSV-TK/GCV (Non-targeted, non RGD-4C ), radiotherapy plus RGD- 4C AAVP HSV-TK/GCV(XRT + RGD-4C) and radiotherapy plus AAVP HSV-TK/GCV group (XRT + Non RGD-4C). The effect of treatment was assessed by tumor growth delay ( the time required when tumor grew from 6.0 mm to 12.0 mm) and tumor cure. Results: Five mice died during the treatment course. There were 6 mice without tumor after treatment, including 1 in RT group, 1 in RGD-4C group, 1 in non RGD-4C group and 3 in XRT + RGD-4C group, respectively. For tumor growth delay analysis in 37 mice, the absolute growth delay (AGD) for RGD-4C, non RGD-4C and RT group was 24.4 ± 9.0, 22.6±11.3 and 28.3 ±5.5 days, respectively. When RGD-4C AAVP HSV-TK/GCV or AAVP HSV-TK/GCV combined with radiotherapy, their AGD was 64.7±23.8 and 35.4±9.6 days, and nominal growth delay (NGD) was 40.3 ± 23.8 and 12.8 ± 9.6 days, respectively. The enhancement factor of RGD-4C AAVP HSV-TK/GCV and AAVP HSV-TK/GCV for radiotherapy were 1.42 and 0.45. Conclusion: RGD-4C AAVP HSV-TK/GCV can enhance radiotherapeutic effect for DU145 prostate cancer xenografts. Further study is needed. (authors)

  6. Combined anti-tumor therapeutic effect of targeted gene, hyperthermia, radionuclide brachytherapy in breast carcinoma

    International Nuclear Information System (INIS)

    Chen Daozhen; Tang Qiusha; Xiang Jingying; Xu Fei; Zhang Li; Wang Junfeng

    2011-01-01

    Objective: To investigate the antitumor therapeutic effect of combined therapy of magnetic induction heating by nano-magnetic particles, herpes simplex virus thymidine kinase gene (HSV-tk suicide gene) and internal radiation in mice bearing MCF-7 breast carcinoma. Methods: The transfection reagents, plasmids heat shock protein-HSV-tk (pHSP-HSV-tk), ferroso-ferric oxide nano-magnetic fluid flow and 188 Re-ganciclovir-bovine serum albumin-nanopaticles (GCV-BSA-NP) were prepared. The heating experiments in vivo were carried out using ferroso-ferric oxide nano-magnetic fluid flow. Sixty mice tumor models bearing MCF-7 breast carcinoma were established and randomly divided into six groups. Group A was the control group, B was gene transfection therapy group, C was hyperthermia group, D was gene transfection therapy combined with radionuclide brachytherapy group, E was gene therapy combined with hyperthermia group, and F was gene therapy, hyperthermia combined with radionuclide brachytherapy group. The tumor growth, tumor mass and histopathological changes were evaluated. The expression of HSV-tk in the groups of B, D, E and F was detected by RT-PCR. Poisson distribution and one-way analysis of variance (ANOVA) were used for statistical analysis by SPSS 10.0 software. Results: In the animal heating experiments, the temperature of tumor increased up to 39.6 degree C, 43.2 degree C, and 48.1 degree C quickly with different injected doses (2, 4 and 6 mg respectively) of nano-magnetic particles and maintained for 40 min. The temperature of tumor tissue reduced to 36.8 degree C, 37.5 degree C and 37.8 degree C in 10 min when alternating magnetic field (AMF) stopped. The tumor mass in Groups C ((452.50±30.29) mg), D ((240.98±35.32)mg), E((231.87±27.41) mg) and F ((141.55±23.78) mg) were much lower than that in Group A ((719.12±22.65) mg) (F=800.07, P<0.01), with the most significant treatment effect in Group F.The tumor mass in Group B((684.05±24.02) mg) was higher than

  7. Evaluation of hyaluronic acid-combined ternary complexes for serum-resistant and targeted gene delivery system.

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    Hong, Woong-Gil; Jeong, Gyeong-Won; Nah, Jae-Woon

    2018-04-19

    Branched polyethylenimine (bPEI) was well known as high transfection agent, which has many amine group. However, utilization of bPEI was limited due to high toxicity. To solve these problems, bPEI was introduced to low molecular weight water-soluble chitosan (LMWSC) with coupling agent. In addition, hyaluronic acid (HA), one of natural anion polymer, was introduced to binary complex of pDNA/bPEI-grafted LMWSC (LMPEI) to target the specific cancer cell and impart the serum resistant. Ternary complexes of pDNA/LMPEI/HA were prepared by electrostatic charge interaction and their binding affinity and DNase protection assay were conducted by gel retardation assay. Particle size of ternary complexes showed that had each 482 ± 245.4 (pDNA/LMPEI2%/HA, 1:16:1, w/w/w) and 410 ± 78.5 nm (pDNA/LMPEI4%/HA, 1:16:2, w/w/w). Moreover, to demonstrate serum-resistant effect of ternary complexes, particle size of them was measured according to incubated time (0-10 h) under serum condition. Transfection assay of ternary complexes showed that their transfection efficiency in CD44-receptor overexpressed HCT116 cell was higher than CD44-receptor negative CT26 cell. Additionally, intracellular uptake of ternary complexes with propidium iodide (PI)-labeled pDNA was observed to confirm targeting effect and cellular internalization by fluorescence microscopy. These results suggest that ternary complexes are superb gene carrier with excellent serum-resistant and high gene transfection. Copyright © 2017. Published by Elsevier B.V.

  8. The drug target genes show higher evolutionary conservation than non-target genes.

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    Lv, Wenhua; Xu, Yongdeng; Guo, Yiying; Yu, Ziqi; Feng, Guanglong; Liu, Panpan; Luan, Meiwei; Zhu, Hongjie; Liu, Guiyou; Zhang, Mingming; Lv, Hongchao; Duan, Lian; Shang, Zhenwei; Li, Jin; Jiang, Yongshuai; Zhang, Ruijie

    2016-01-26

    Although evidence indicates that drug target genes share some common evolutionary features, there have been few studies analyzing evolutionary features of drug targets from an overall level. Therefore, we conducted an analysis which aimed to investigate the evolutionary characteristics of drug target genes. We compared the evolutionary conservation between human drug target genes and non-target genes by combining both the evolutionary features and network topological properties in human protein-protein interaction network. The evolution rate, conservation score and the percentage of orthologous genes of 21 species were included in our study. Meanwhile, four topological features including the average shortest path length, betweenness centrality, clustering coefficient and degree were considered for comparison analysis. Then we got four results as following: compared with non-drug target genes, 1) drug target genes had lower evolutionary rates; 2) drug target genes had higher conservation scores; 3) drug target genes had higher percentages of orthologous genes and 4) drug target genes had a tighter network structure including higher degrees, betweenness centrality, clustering coefficients and lower average shortest path lengths. These results demonstrate that drug target genes are more evolutionarily conserved than non-drug target genes. We hope that our study will provide valuable information for other researchers who are interested in evolutionary conservation of drug targets.

  9. Tumor targeted gene therapy

    International Nuclear Information System (INIS)

    Kang, Joo Hyun

    2006-01-01

    Knowledge of molecular mechanisms governing malignant transformation brings new opportunities for therapeutic intervention against cancer using novel approaches. One of them is gene therapy based on the transfer of genetic material to an organism with the aim of correcting a disease. The application of gene therapy to the cancer treatment had led to the development of new experimental approaches such as suicidal gene therapy, inhibition of oncogenes and restoration of tumor-suppressor genes. Suicidal gene therapy is based on the expression in tumor cells of a gene encoding an enzyme that converts a prodrug into a toxic product. Representative suicidal genes are Herpes simplex virus type 1 thymidine kinase (HSV1-tk) and cytosine deaminase (CD). Especially, physicians and scientists of nuclear medicine field take an interest in suicidal gene therapy because they can monitor the location and magnitude, and duration of expression of HSV1-tk and CD by PET scanner

  10. Targeting Aflatoxin Biosynthetic Genes.

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    Srour, Ali Y; Fakhoury, Ahmad M; Brown, Robert L

    2017-01-01

    Chemical detoxification and physical destruction of aflatoxins in foods and feed commodities are mostly unattainable in a way that preserves the edibility of the food. Therefore, preventing mycotoxins in general and aflatoxins in particular from entering the food chain is a better approach. This requires early detection of the aflatoxin-causing organisms. Detection and quantification of aflatoxin-producing fungi has always been a challenge, especially within species of Aspergillus and Penicillium. Culture-based methods require a high level of expertise and a list of sophisticated equipment. Furthermore, even for a trained taxonomist, species that are identical in morphology, physiology, and nutritional aspects can be challenging to classify. Fungal taxonomy has changed over the past few decades; more species are being reclassified, and new species are being described due to advances in sequencing and genome assembly. These developments make the use of PCR-based approaches practical, rapid, and more reliable for the identification of fungi to the species level. This chapter presents a variety of protocols to detect and quantify aflatoxin-producing fungi using mycotoxin biosynthesis pathway genes.

  11. Gene transfer technology and genetic radioisotope targeting therapy

    International Nuclear Information System (INIS)

    Wang Jiaqiong; Wang Zizheng

    2004-01-01

    With deeper cognition about mechanisms of disease at the cellular and molecular level, gene therapy has become one of the most important research fields in medical molecular biology at present. Gene transfer technology plays an important role during the course of gene therapy, and further improvement should be made about vectors carrying target gene sequences. Also, gene survey is needed during gene therapy, and gene imaging is the most effective method. The combination of gene therapy and targeted radiotherapy, that is, 'Genetic Radioisotope Targeting Therapy', will be a novel approach to tumor gene therapy

  12. Single agent- and combination treatment with two targeted suicide gene therapy systems is effective in chemoresistant small cell lung cancer cells

    DEFF Research Database (Denmark)

    Michaelsen, Signe R; Christensen, Camilla L; Sehested, Maxwell

    2012-01-01

    Transcriptional targeted suicide gene (SG) therapy driven by the insulinoma-associated 1 (INSM1) promoter makes it possible to target suicide toxin production and cytotoxicity exclusively to small cell lung cancer (SCLC) cells and tumors. It remains to be determined whether acquired chemoresistance......, as observed in the majority of SCLC patients, desensitizes SCLC cells to INSM1 promoter-driven SG therapy....

  13. Combination of targeting gene-viro therapy with recombinant Fowl-pox viruses with HN and VP3 genes on mouse osteosarcoma.

    Science.gov (United States)

    Zhang, Z-Y; Wang, L-Q; Fu, C-F; Li, X; Cui, Z-L; Zhang, J-Y; Xue, S-H; Sun, N; Xu, F

    2013-03-01

    Osteosarcoma is an aggressive cancerous neoplasm arising from primitive transformed cells of mesenchymal origin that exhibit osteoblastic differentiation and produce malignant osteoid. With the rapid development of tumor molecular biology, gene and viral therapy, a highly promising strategy for the treatment, has shown some therapeutic effects. To study the strategy of cooperative cancer gene therapy, previously, we explored the antitumor effects of recombinant Fowl-pox viruses (FPVs) with both HN (hemagglutinin-neuramidinase) and VP3 genes on mouse osteosarcoma. We constructed vFV-HN, vFV-VP3 and vFV-HN-VP3 inserting CAV VP3 gene, NDV HN gene into fowlpox virus. S180 osteosarcoma were transfected with Recombinant Fowl-pox viruses (FPVs). These cell lines stably expressing tagged proteins were selected by culturing in medium containing puromycin (2 µg/ml) and confirmed by immunoblotting and immunostaining. S180 osteosarcoma model with BALB/c mice and nude mice were established and the vFPV viruses as control, vFV-HN, vFV-VP3, vFV-HN-VP3 were injected into the tumor directly. The rate of tumor growth, tumor suppression and the sialic acid levels in serum were examined and the tumor tissues were analyzed by the method of immunohistochemistry. Flow cytometric analysis was performed using a FACSCalibur flow cytometer. A total of 100,000 events were analyzed for each sample and the experiment was repeated at least twice. Our data indicated that vFV-HN, vFV-VP3 and vFV-HN-VP3 all had growth inhibition effects, the inhibition rate of vFV-HN-VP3 group was 51.7%, which was higher than that of vFV-HN, vFV-VP3 group and control group (p genes into mouse osteosarcoma cancer cells can cause cell a specificity anti-tumor immune activity, suppress tumor growth, and increase the survival rate of the tumor within host.

  14. Enhanced nigrostriatal neuron-specific, long-term expression by using neural-specific promoters in combination with targeted gene transfer by modified helper virus-free HSV-1 vector particles

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    Kong Lingxin

    2008-04-01

    Full Text Available Abstract Background Direct gene transfer into neurons has potential for developing gene therapy treatments for specific neurological conditions, and for elucidating neuronal physiology. Due to the complex cellular composition of specific brain areas, neuronal type-specific recombinant gene expression is required for many potential applications of neuronal gene transfer. One approach is to target gene transfer to a specific type of neuron. We developed modified Herpes Simplex Virus (HSV-1 particles that contain chimeric glycoprotein C (gC – glial cell line-derived neurotrophic factor (GDNF or brain-derived neurotrophic factor (BDNF proteins. HSV-1 vector particles containing either gC – GDNF or gC – BDNF target gene transfer to nigrostriatal neurons, which contain specific receptors for GDNF or BDNF. A second approach to achieve neuronal type-specific expression is to use a cell type-specific promoter, and we have used the tyrosine hydroxylase (TH promoter to restrict expression to catecholaminergic neurons or a modified neurofilament heavy gene promoter to restrict expression to neurons, and both of these promoters support long-term expression from HSV-1 vectors. To both improve nigrostriatal-neuron specific expression, and to establish that targeted gene transfer can be followed by long-term expression, we performed targeted gene transfer with vectors that support long-term, neuronal-specific expression. Results Helper virus-free HSV-1 vector packaging was performed using either gC – GDNF or gC – BDNF and vectors that contain either the TH promoter or the modified neurofilament heavy gene promoter. Vector stocks were injected into the midbrain proximal to the substantia nigra, and the rats were sacrificed at either 4 days or 1 month after gene transfer. Immunofluorescent costaining was performed to detect both recombinant gene products and nigrostriatal neurons. The combination of targeted gene transfer with neuronal

  15. rAAV9 combined with renal vein injection is optimal for kidney-targeted gene delivery: conclusion of a comparative study.

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    Rocca, C J; Ur, S N; Harrison, F; Cherqui, S

    2014-06-01

    Effective gene therapy strategies for the treatment of kidney disorders remain elusive. We report an optimized kidney-targeted gene delivery strategy using recombinant adeno-associated virus (rAAV) administered via retrograde renal vein injection in mice. Renal vein injection of rAAV consistently resulted in superior kidney transduction compared with tail vein injection using as little as half the tail vein dose. We compared rAAV5, 6, 8 and 9, containing either green fluorescent protein (GFP) or luciferase reporter genes driven by the Cytomegalovirus promoter. We demonstrated that although rAAV6 and 8 injected via renal vein transduced the kidney, transgene expression was mainly restricted to the medulla. Transgene expression was systematically low after rAAV5 injection, attributed to T-cell immune response, which could be overcome by transient immunosuppression. However, rAAV9 was the only serotype that permitted high-transduction efficiency of both the cortex and medulla. Moreover, both the glomeruli and tubules were targeted, with a higher efficiency within the glomeruli. To improve the specificity of kidney-targeted gene delivery with rAAV9, we used the parathyroid hormone receptor 'kidney-specific' promoter. We obtained a more efficient transgene expression within the kidney, and a significant reduction in other tissues. Our work represents the first comprehensive and clinically relevant study for kidney gene delivery.

  16. Gene Therapy Targeting HIV Entry

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    Chuka Didigu

    2014-03-01

    Full Text Available Despite the unquestionable success of antiretroviral therapy (ART in the treatment of HIV infection, the cost, need for daily adherence, and HIV-associated morbidities that persist despite ART all underscore the need to develop a cure for HIV. The cure achieved following an allogeneic hematopoietic stem cell transplant (HSCT using HIV-resistant cells, and more recently, the report of short-term but sustained, ART-free control of HIV replication following allogeneic HSCT, using HIV susceptible cells, have served to both reignite interest in HIV cure research, and suggest potential mechanisms for a cure. In this review, we highlight some of the obstacles facing HIV cure research today, and explore the roles of gene therapy targeting HIV entry, and allogeneic stem cell transplantation in the development of strategies to cure HIV infection.

  17. Gene therapy and radionuclides targeting therapy in mammary carcinoma

    International Nuclear Information System (INIS)

    Song Jinhua

    2003-01-01

    Breast carcinoma's gene therapy is a hotspot in study of the tumor's therapy in the recent years. Currently the major therapy methods that in the experimentative and primary clinical application phases include immunological gene therapy, multidrug resistance gene therapy, antisense oligonucleotide therapy and suicide gene therapy. The gene targeting brachytherapy, which is combined with gene therapy and radiotherapy has enhanced the killer effects of the suicide gene and nuclide in tumor cells. That has break a new path in tumor's gene therapy. The further study in this field will step up it's space to the clinical application

  18. Targeted integration of genes in Xenopus tropicalis

    DEFF Research Database (Denmark)

    Shi, Zhaoying; Tian, Dandan; Xin, Huhu

    2017-01-01

    With the successful establishment of both targeted gene disruption and integration methods in the true diploid frog Xenopus tropicalis, this excellent vertebrate genetic model now is making a unique contribution to modelling human diseases. Here, we summarize our efforts on establishing homologous...... recombination-mediated targeted integration in Xenopus tropicalis, the usefulness, and limitation of targeted integration via the homology-independent strategy, and future directions on how to further improve targeted gene integration in Xenopus tropicalis....

  19. Suicide genes or p53 gene and p53 target genes as targets for cancer gene therapy by ionizing radiation

    International Nuclear Information System (INIS)

    Liu Bing; Chinese Academy of Sciences, Beijing; Zhang Hong

    2005-01-01

    Radiotherapy has some disadvantages due to the severe side-effect on the normal tissues at a curative dose of ionizing radiation (IR). Similarly, as a new developing approach, gene therapy also has some disadvantages, such as lack of specificity for tumors, limited expression of therapeutic gene, potential biological risk. To certain extent, above problems would be solved by the suicide genes or p53 gene and its target genes therapies targeted by ionizing radiation. This strategy not only makes up the disadvantage from radiotherapy or gene therapy alone, but also promotes success rate on the base of lower dose. By present, there have been several vectors measuring up to be reaching clinical trials. This review focused on the development of the cancer gene therapy through suicide genes or p53 and its target genes mediated by IR. (authors)

  20. Rational Combinations of Targeted Agents in AML

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    Prithviraj Bose

    2015-04-01

    Full Text Available Despite modest improvements in survival over the last several decades, the treatment of AML continues to present a formidable challenge. Most patients are elderly, and these individuals, as well as those with secondary, therapy-related, or relapsed/refractory AML, are particularly difficult to treat, owing to both aggressive disease biology and the high toxicity of current chemotherapeutic regimens. It has become increasingly apparent in recent years that coordinated interruption of cooperative survival signaling pathways in malignant cells is necessary for optimal therapeutic results. The modest efficacy of monotherapy with both cytotoxic and targeted agents in AML testifies to this. As the complex biology of AML continues to be elucidated, many “synthetic lethal” strategies involving rational combinations of targeted agents have been developed. Unfortunately, relatively few of these have been tested clinically, although there is growing interest in this area. In this article, the preclinical and, where available, clinical data on some of the most promising rational combinations of targeted agents in AML are summarized. While new molecules should continue to be combined with conventional genotoxic drugs of proven efficacy, there is perhaps a need to rethink traditional philosophies of clinical trial development and regulatory approval with a focus on mechanism-based, synergistic strategies.

  1. Targeted Gene Therapy for Breast Cancer

    Science.gov (United States)

    1999-08-01

    or immunotoxin therapy, natural vector-host tropisms must be altered. Recent improvements in monoclonal antibody (mAb) engineering have expanded the...endocytosis. To achieve targeted gene therapy or immunotoxin therapy, natural vector-host tropisms must be altered. Recent improvements in monoclonal...trafficking of monoclonal antibody- antigen to an endolysosomal pathway is important. After altering targeting specificities, prokaryotic and plant

  2. Large scale RNAi screen in Tribolium reveals novel target genes for pest control and the proteasome as prime target.

    Science.gov (United States)

    Ulrich, Julia; Dao, Van Anh; Majumdar, Upalparna; Schmitt-Engel, Christian; Schwirz, Jonas; Schultheis, Dorothea; Ströhlein, Nadi; Troelenberg, Nicole; Grossmann, Daniela; Richter, Tobias; Dönitz, Jürgen; Gerischer, Lizzy; Leboulle, Gérard; Vilcinskas, Andreas; Stanke, Mario; Bucher, Gregor

    2015-09-03

    Insect pest control is challenged by insecticide resistance and negative impact on ecology and health. One promising pest specific alternative is the generation of transgenic plants, which express double stranded RNAs targeting essential genes of a pest species. Upon feeding, the dsRNA induces gene silencing in the pest resulting in its death. However, the identification of efficient RNAi target genes remains a major challenge as genomic tools and breeding capacity is limited in most pest insects impeding whole-animal-high-throughput-screening. We use the red flour beetle Tribolium castaneum as a screening platform in order to identify the most efficient RNAi target genes. From about 5,000 randomly screened genes of the iBeetle RNAi screen we identify 11 novel and highly efficient RNAi targets. Our data allowed us to determine GO term combinations that are predictive for efficient RNAi target genes with proteasomal genes being most predictive. Finally, we show that RNAi target genes do not appear to act synergistically and that protein sequence conservation does not correlate with the number of potential off target sites. Our results will aid the identification of RNAi target genes in many pest species by providing a manageable number of excellent candidate genes to be tested and the proteasome as prime target. Further, the identified GO term combinations will help to identify efficient target genes from organ specific transcriptomes. Our off target analysis is relevant for the sequence selection used in transgenic plants.

  3. TargetMine, an integrated data warehouse for candidate gene prioritisation and target discovery.

    Directory of Open Access Journals (Sweden)

    Yi-An Chen

    Full Text Available Prioritising candidate genes for further experimental characterisation is a non-trivial challenge in drug discovery and biomedical research in general. An integrated approach that combines results from multiple data types is best suited for optimal target selection. We developed TargetMine, a data warehouse for efficient target prioritisation. TargetMine utilises the InterMine framework, with new data models such as protein-DNA interactions integrated in a novel way. It enables complicated searches that are difficult to perform with existing tools and it also offers integration of custom annotations and in-house experimental data. We proposed an objective protocol for target prioritisation using TargetMine and set up a benchmarking procedure to evaluate its performance. The results show that the protocol can identify known disease-associated genes with high precision and coverage. A demonstration version of TargetMine is available at http://targetmine.nibio.go.jp/.

  4. Specific genetic modifications of domestic animals by gene targeting and animal cloning.

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    Wang, Bin; Zhou, Jiangfeng

    2003-11-13

    The technology of gene targeting through homologous recombination has been extremely useful for elucidating gene functions in mice. The application of this technology was thought impossible in the large livestock species until the successful creation of the first mammalian clone "Dolly" the sheep. The combination of the technologies for gene targeting of somatic cells with those of animal cloning made it possible to introduce specific genetic mutations into domestic animals. In this review, the principles of gene targeting in somatic cells and the challenges of nuclear transfer using gene-targeted cells are discussed. The relevance of gene targeting in domestic animals for applications in bio-medicine and agriculture are also examined.

  5. Improved Gene Targeting through Cell Cycle Synchronization.

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    Vasiliki Tsakraklides

    Full Text Available Gene targeting is a challenge in organisms where non-homologous end-joining is the predominant form of recombination. We show that cell division cycle synchronization can be applied to significantly increase the rate of homologous recombination during transformation. Using hydroxyurea-mediated cell cycle arrest, we obtained improved gene targeting rates in Yarrowia lipolytica, Arxula adeninivorans, Saccharomyces cerevisiae, Kluyveromyces lactis and Pichia pastoris demonstrating the broad applicability of the method. Hydroxyurea treatment enriches for S-phase cells that are active in homologous recombination and enables previously unattainable genomic modifications.

  6. Targeting Herpetic Keratitis by Gene Therapy

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    Hossein Mostafa Elbadawy

    2012-01-01

    Full Text Available Ocular gene therapy is rapidly becoming a reality. By November 2012, approximately 28 clinical trials were approved to assess novel gene therapy agents. Viral infections such as herpetic keratitis caused by herpes simplex virus 1 (HSV-1 can cause serious complications that may lead to blindness. Recurrence of the disease is likely and cornea transplantation, therefore, might not be the ideal therapeutic solution. This paper will focus on the current situation of ocular gene therapy research against herpetic keratitis, including the use of viral and nonviral vectors, routes of delivery of therapeutic genes, new techniques, and key research strategies. Whereas the correction of inherited diseases was the initial goal of the field of gene therapy, here we discuss transgene expression, gene replacement, silencing, or clipping. Gene therapy of herpetic keratitis previously reported in the literature is screened emphasizing candidate gene therapy targets. Commonly adopted strategies are discussed to assess the relative advantages of the protective therapy using antiviral drugs and the common gene therapy against long-term HSV-1 ocular infections signs, inflammation and neovascularization. Successful gene therapy can provide innovative physiological and pharmaceutical solutions against herpetic keratitis.

  7. Molecular targeting of gene therapy and radiotherapy

    International Nuclear Information System (INIS)

    Weichselbaum, R.R.; Kufe, D.W.; Advani, S.J.; Roizman, B.

    2001-01-01

    The full promise of gene therapy has been limited by the lack of specificity of vectors for tumor tissue as well as the lack of antitumor efficacy of transgenes encoded by gene delivery systems. In this paper we review our studies investigating two modifications of gene therapy combined with radiotherapy. The first investigations described include studies of radiation inducible gene therapy. In this paradigm, radio-inducible DNA sequences from the CarG elements of the Egr-1 promoter are cloned upstream of a cDNA encoding TNFa. The therapeutic gene (TNFa) is induced by radiation within the tumor microenvironment. In the second paradigm, genetically engineered herpes simplex virus (HSV-1) is induced by ionizing radiation to proliferate within the tumor volume. These modifications of radiotherapy and gene therapy may enhance the efficacy of both treatments

  8. Identification and targeting of genes in atherosclerosis

    NARCIS (Netherlands)

    van Capelleveen, J.C.

    2017-01-01

    The aim of the studies described in this thesis was to assess the relevance of variations in known and novel genes for CVD associated endpoints in order to ultimately identify therapeutic targets for the treatment of atherosclerosis. The type of research is truly translational; it nourishes on the

  9. TF Target Mapper: A BLAST search tool for the identification of Transcription Factor target genes

    Directory of Open Access Journals (Sweden)

    van der Spek Peter

    2006-03-01

    Full Text Available Abstract Background In the current era of high throughput genomics a major challenge is the genome-wide identification of target genes for specific transcription factors. Chromatin immunoprecipitation (ChIP allows the isolation of in vivo binding sites of transcription factors and provides a powerful tool for examining gene regulation. Crosslinked chromatin is immunoprecipitated with antibodies against specific transcription factors, thus enriching for sequences bound in vivo by these factors in the immunoprecipitated DNA. Cloning and sequencing the immunoprecipitated sequences allows identification of transcription factor target genes. Routinely, thousands of such sequenced clones are used in BLAST searches to map their exact location in the genome and the genes located in the vicinity. These genes represent potential targets of the transcription factor of interest. Such bioinformatics analysis is very laborious if performed manually and for this reason there is a need for developing bioinformatic tools to automate and facilitate it. Results In order to facilitate this analysis we generated TF Target Mapper (Transcription Factor Target Mapper. TF Target Mapper is a BLAST search tool allowing rapid extraction of annotated information on genes around each hit. It combines sequence cleaning/filtering, pattern searching and BLAST searches with extraction of information on genes located around each BLAST hit and comparisons of the output list of genes or gene ontology IDs with user-implemented lists. We successfully applied and tested TF Target Mapper to analyse sequences bound in vivo by the transcription factor GATA-1. We show that TF Target Mapper efficiently extracted information on genes around ChIPed sequences, thus identifying known (e.g. α-globin and ζ-globin and potentially novel GATA-1 gene targets. Conclusion TF Target Mapper is a very efficient BLAST search tool that allows the rapid extraction of annotated information on the genes

  10. Genetic nanomedicine: gene delivery by targeted lipoplexes.

    Science.gov (United States)

    Düzgüneş, Nejat; de Ilarduya, Conchita Tros

    2012-01-01

    Cationic liposome-DNA complexes (lipoplexes) are used for the delivery of plasmid DNA to cultured cells and various tissues in vivo. In this chapter, we describe the preparation and evaluation of plain and targeted lipoplexes, using targeting ligands, including epidermal growth factor and transferrin. Ligand-associated lipoplexes may be used to target DNA or other nucleic acid drugs to specific cells, particularly cancer cells that overexpress the receptors for the ligands. We provide examples of the enhancement of gene expression mediated by epidermal growth factor in murine and human oral squamous cell carcinoma cells, and human hepatoblastoma and rat colon adenocarcinoma cells. We also summarize the studies on the use of transferrin-lipoplexes for enhancing gene delivery to cervical carcinoma, murine colon carcinoma, and African green monkey kidney cells. We outline two animal models in which transferrin-lipoplexes have been used for antitumor therapy by delivering either the gene encoding interleukin-12 or a suicide gene: a CT26 murine colon carcinoma, and a syngeneic, orthotopic murine oral squamous cell carcinoma. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. A two-cassette reporter system for assessing target gene translation and target gene product inclusion body formation

    DEFF Research Database (Denmark)

    2016-01-01

    The present invention relates to a dual cassette reporter system capable of assessing target gene translation and target gene product folding. The present invention further relates to vectors and host cells comprising the dual cassette reporter system. In addition the invention relates to the use...... of the dual cassette reporter system for assessing target gene translation and target gene product folding....

  12. Gene recognition by combination of several gene-finding programs.

    Science.gov (United States)

    Murakami, K; Takagi, T

    1998-01-01

    A number of programs have been developed to predict the eukaryotic gene structures in DNA sequences. However, gene finding is still a challenging problem. We have explored the effectiveness when the results of several gene-finding programs were re-analyzed and combined. We studied several methods with four programs (FEXH, GeneParser3, GEN-SCAN and GRAIL2). By HIGHEST-policy combination method or BOUNDARY method, approximate correlation (AC) improved by 3-5% in comparison with the best single gene-finding program. From another viewpoint, OR-based combination of the four programs is the most reliable to know whether a candidate exon overlaps with the real exon or not, although it is less sensitive than GENSCAN for exon-intron boundaries. Our methods can easily be extended to combine other programs. We have developed a server program (Shirokane System) and a client program (GeneScope) to use the methods. GeneScope is available through a WWW site (http://gf.genome.ad.jp/). (katsu,takagi)@ims.u-tokyo.ac.jp

  13. Targeting Conserved Genes in Alternaria Species.

    Science.gov (United States)

    Pavón, Miguel Ángel; López-Calleja, Inés María; González, Isabel; Martín, Rosario; García, Teresa

    2017-01-01

    Real-time polymerase chain reaction (PCR) is a molecular biology technique based on the detection of the fluorescence produced by a reporter molecule, which increases as the reaction proceeds proportionally to the accumulation of the PCR product within each amplification cycle. The fluorescent reporter molecules include dyes that bind to the double-stranded DNA (i.e., SYBR ® Green) or sequence-specific probes (i.e., Molecular Beacons or TaqMan ® Probes). Real-time PCR provides a tool for accurate and sensitive quantification of target fungal DNA. Here, we describe a TaqMan real-time PCR method for specific detection and quantification of Alternaria spp. The method uses Alternaria-specific primers and probe, targeting the internal transcribed spacer regions ITS1 and ITS2 of the rRNA gene, and a positive amplification control based on 18S rRNA gene.

  14. Inhibition of nuclear factor-κB and target genes during combined therapy with proteasome inhibitor bortezomib and reirradiation in patients with recurrent head-and-neck squamous cell carcinoma

    International Nuclear Information System (INIS)

    Van Waes, Carter; Chang, Angela A.; Lebowitz, Peter F.; Druzgal, Colleen H.; Chen, Zhong; Elsayed, Yusri A.; Sunwoo, John B.; Rudy, Susan; Morris, John C.; Mitchell, James B.; Camphausen, Kevin; Gius, David; Adams, Julian; Sausville, Edward A.; Conley, Barbara A.

    2005-01-01

    Purpose: To examine the effects the proteasome inhibitor bortezomib (VELCADE) on transcription factor nuclear factor-κB (NF-κB) and target genes and the feasibility of combination therapy with reirradiation in patients with recurrent head-and-neck squamous cell carcinoma (HNSCC). Methods and Materials: The tolerability and response to bortezomib 0.6 mg/m 2 and 0.9 mg/m 2 given twice weekly concurrent with daily reirradiation to 50-70 Gy was explored. Blood proteasome inhibition and NF-κB-modulated cytokines and factors were measured. Proteasome inhibition, nuclear localization of NF-κB phospho-p65, apoptosis, and expression of NF-κB-modulated mRNAs were compared in serial biopsies from accessible tumors. Results: The maximally tolerated dose was exceeded, and study was limited to 7 and 2 patients, respectively, given bortezomib 0.6 mg/m 2 and 0.9 mg/m 2 /dose with reirradiation. Grade 3 hypotension and hyponatremia were dose limiting. Mucositis was Grade 3 or less and was delayed. The mean blood proteasome inhibition at 1, 24, and 48 h after 0.6 mg/m 2 was 32%, 16%, and 7% and after 0.9 mg/m 2 was 56%, 26%, and 14%, respectively. Differences in proteasome and NF-κB activity, apoptosis, and expression of NF-κB-modulated cell cycle, apoptosis, and angiogenesis factor mRNAs were detected in 2 patients with minor tumor reductions and in serum NF-κB-modulated cytokines in 1 patient with a major tumor reduction. Conclusions: In combination with reirradiation, the maximally tolerated dose of bortezomib was exceeded at a dose of 0.6 mg/m 2 and the threshold of proteasome inhibition. Although this regimen with reirradiation is not feasible, bortezomib induced detectable differences in NF-κB localization, apoptosis, and NF-κB-modulated genes and cytokines in tumor and serum in association with tumor reduction, indicating that other schedules of bortezomib combined with primary radiotherapy or reirradiation may merit future investigation

  15. Engineering liposomal nanoparticles for targeted gene therapy.

    Science.gov (United States)

    Zylberberg, C; Gaskill, K; Pasley, S; Matosevic, S

    2017-08-01

    Recent mechanistic studies have attempted to deepen our understanding of the process by which liposome-mediated delivery of genetic material occurs. Understanding the interactions between lipid nanoparticles and cells is still largely elusive. Liposome-mediated delivery of genetic material faces systemic obstacles alongside entry into the cell, endosomal escape, lysosomal degradation and nuclear uptake. Rational design approaches for targeted delivery have been developed to reduce off-target effects and enhance transfection. These strategies, which have included the modification of lipid nanoparticles with target-specific ligands to enhance intracellular uptake, have shown significant promise at the proof-of-concept stage. Control of physical and chemical specifications of liposome composition, which includes lipid-to-DNA charge, size, presence of ester bonds, chain length and nature of ligand complexation, is integral to the performance of targeted liposomes as genetic delivery agents. Clinical advances are expected to rely on such systems in the therapeutic application of liposome nanoparticle-based gene therapy. Here, we discuss the latest breakthroughs in the development of targeted liposome-based agents for the delivery of genetic material, paying particular attention to new ligand and cationic lipid design as well as recent in vivo advances.

  16. Hyb-Seq: combining target enrichment and genome skimming for plant phylogenomics

    Science.gov (United States)

    Kevin Weitemier; Shannon C.K. Straub; Richard C. Cronn; Mark Fishbein; Roswitha Schmickl; Angela McDonnell; Aaron. Liston

    2014-01-01

    • Premise of the study: Hyb-Seq, the combination of target enrichment and genome skimming, allows simultaneous data collection for low-copy nuclear genes and high-copy genomic targets for plant systematics and evolution studies. • Methods and Results: Genome and transcriptome assemblies for milkweed ( Asclepias syriaca ) were used to design enrichment probes for 3385...

  17. Targeting the human lysozyme gene on bovine αs1- casein gene ...

    African Journals Online (AJOL)

    Targeting an exogenous gene into a favorable gene locus and for expression under endogenous regulators is an ideal method in mammary gland bioreactor research. For this purpose, a gene targeting vector was constructed to targeting the human lysozyme gene on bovine αs1-casein gene locus. In this case, the ...

  18. Targeting the human lysozyme gene on bovine αs1- casein gene ...

    African Journals Online (AJOL)

    ajl yemi

    2011-11-28

    Nov 28, 2011 ... Targeting an exogenous gene into a favorable gene locus and for expression under endogenous regulators is an ideal method in mammary gland bioreactor research. For this purpose, a gene targeting vector was constructed to targeting the human lysozyme gene on bovine αs1-casein gene locus. In this.

  19. Magnetic nanoparticles for targeted therapeutic gene delivery and magnetic-inducing heating on hepatoma

    International Nuclear Information System (INIS)

    Yuan, Chenyan; Zhang, Jia; Li, Hongbo; Zhang, Hao; Wang, Ling; Zhang, Dongsheng; An, Yanli

    2014-01-01

    Gene therapy holds great promise for treating cancers, but their clinical applications are being hampered due to uncontrolled gene delivery and expression. To develop a targeted, safe and efficient tumor therapy system, we constructed a tissue-specific suicide gene delivery system by using magnetic nanoparticles (MNPs) as carriers for the combination of gene therapy and hyperthermia on hepatoma. The suicide gene was hepatoma-targeted and hypoxia-enhanced, and the MNPs possessed the ability to elevate temperature to the effective range for tumor hyperthermia as imposed on an alternating magnetic field (AMF). The tumoricidal effects of targeted gene therapy associated with hyperthermia were evaluated in vitro and in vivo. The experiment demonstrated that hyperthermia combined with a targeted gene therapy system proffer an effective tool for tumor therapy with high selectivity and the synergistic effect of hepatoma suppression. (paper)

  20. Immuno-Oncology-The Translational Runway for Gene Therapy: Gene Therapeutics to Address Multiple Immune Targets.

    Science.gov (United States)

    Weß, Ludger; Schnieders, Frank

    2017-12-01

    Cancer therapy is once again experiencing a paradigm shift. This shift is based on extensive clinical experience demonstrating that cancer cannot be successfully fought by addressing only single targets or pathways. Even the combination of several neo-antigens in cancer vaccines is not sufficient for successful, lasting tumor eradication. The focus has therefore shifted to the immune system's role in cancer and the striking abilities of cancer cells to manipulate and/or deactivate the immune system. Researchers and pharma companies have started to target the processes and cells known to support immune surveillance and the elimination of tumor cells. Immune processes, however, require novel concepts beyond the traditional "single-target-single drug" paradigm and need parallel targeting of diverse cells and mechanisms. This review gives a perspective on the role of gene therapy technologies in the evolving immuno-oncology space and identifies gene therapy as a major driver in the development and regulation of effective cancer immunotherapy. Present challenges and breakthroughs ranging from chimeric antigen receptor T-cell therapy, gene-modified oncolytic viruses, combination cancer vaccines, to RNA therapeutics are spotlighted. Gene therapy is recognized as the most prominent technology enabling effective immuno-oncology strategies.

  1. HIV-1 CCR5 gene therapy will fail unless it is combined with a suicide gene.

    Science.gov (United States)

    Pandit, Aridaman; de Boer, Rob J

    2015-12-17

    Highly active antiretroviral therapy (ART) has successfully turned Human immunodeficiency virus type 1 (HIV-1) from a deadly pathogen into a manageable chronic infection. ART is a lifelong therapy which is both expensive and toxic, and HIV can become resistant to it. An alternative to lifelong ART is gene therapy that targets the CCR5 co-receptor and creates a population of genetically modified host cells that are less susceptible to viral infection. With generic mathematical models we show that gene therapy that only targets the CCR5 co-receptor fails to suppress HIV-1 (which is in agreement with current data). We predict that the same gene therapy can be markedly improved if it is combined with a suicide gene that is only expressed upon HIV-1 infection.

  2. Specific genetic modifications of domestic animals by gene targeting and animal cloning

    Directory of Open Access Journals (Sweden)

    Zhou Jiangfeng

    2003-11-01

    Full Text Available Abstract The technology of gene targeting through homologous recombination has been extremely useful for elucidating gene functions in mice. The application of this technology was thought impossible in the large livestock species until the successful creation of the first mammalian clone "Dolly" the sheep. The combination of the technologies for gene targeting of somatic cells with those of animal cloning made it possible to introduce specific genetic mutations into domestic animals. In this review, the principles of gene targeting in somatic cells and the challenges of nuclear transfer using gene-targeted cells are discussed. The relevance of gene targeting in domestic animals for applications in bio-medicine and agriculture are also examined.

  3. Cancer therapeutic target genes identified on chromosome 20q

    Directory of Open Access Journals (Sweden)

    Editorial Office

    2016-08-01

    , Snijders and Mao described that and “when the selection pressure is removed, amplifications are not maintained and eventually disappear. Thus, amplifications focus on those genes that are important for tumor development,” they said. Their analysis showed that, as tumorous cells progress toward malignancy, the DNA copy number plays a major role in the mechanism of increased expression levels for the 18-gene signature on chromosome 20q. “Strong associations between the DNA copy number and gene expression were observed in the majority of tumor types,” the researchers said. “For example, the RAE1 expression was found to be significantly associated with DNA copy number in 20 tumor types,” the study reported. “Elevated DNA copy numbers of MMP9 and SULF2 were associated with increased gene expressions in only two and seven tumor types, respectively,” it added. With their integrated multi-omics analysis of genes on chromosome 20q, Snijders and Mao believed that the 18-gene signature could become new molecular targets for cancer therapy. “Gene ontology analysis revealed significant enrichment of cell cycle and mitosis-related biological processes in our 18-gene, suggesting that a cluster of functionally related genes localize to chromosome 20q,” they said. The identification of good targets such as theirs is a critical step for the development of targeted therapies for cancer treatment, according to the researchers. Microarray and next generation sequencing technologies have become invaluable tools in cataloging genomic abnormalities in human cancers and identifying new potential therapeutic targets, in addition to the availability of large cancer genomic data sets which allows for unbiased approaches to identify genes that are important in tumor progression, the research study noted. “Here, we aggregated available cancer databases to identify cancer driver genes across tumor types by combining gene transcript and DNA copy number across chromosome 20q to

  4. Chromosomal position effects on AAV-mediated gene targeting.

    Science.gov (United States)

    Cornea, Anda M; Russell, David W

    2010-06-01

    The effects of chromosomal position and neighboring genomic elements on gene targeting in human cells remain largely unexplored. To study these, we used a shuttle vector system in which murine leukemia virus (MLV)-based proviral targets present at different chromosomal locations and containing mutations in the neomycin phosphotransferase (neo) gene were corrected by adeno-associated virus (AAV)-mediated gene targeting. Sixteen identical target loci present in HT-1080 human sarcoma cells were all successfully corrected by gene targeting. The gene targeting frequencies varied by as much as 10-fold, and there was a clear bias for correction of one of the targets in clones containing two target sites. The targeting frequency at each site was correlated to the proximity and density of various genomic elements, and we found a significant association of higher targeting frequencies at loci near a subset of dinucleotide microsatellite repeats (r = -0.55, P targeting frequencies at the target loci (r = 0.52, P targeting frequencies. Our results indicate that certain chromosomal positions are preferred sites for gene targeting in human cells.

  5. Predicting targeted drug combinations based on Pareto optimal patterns of coexpression network connectivity.

    Science.gov (United States)

    Penrod, Nadia M; Greene, Casey S; Moore, Jason H

    2014-01-01

    Molecularly targeted drugs promise a safer and more effective treatment modality than conventional chemotherapy for cancer patients. However, tumors are dynamic systems that readily adapt to these agents activating alternative survival pathways as they evolve resistant phenotypes. Combination therapies can overcome resistance but finding the optimal combinations efficiently presents a formidable challenge. Here we introduce a new paradigm for the design of combination therapy treatment strategies that exploits the tumor adaptive process to identify context-dependent essential genes as druggable targets. We have developed a framework to mine high-throughput transcriptomic data, based on differential coexpression and Pareto optimization, to investigate drug-induced tumor adaptation. We use this approach to identify tumor-essential genes as druggable candidates. We apply our method to a set of ER(+) breast tumor samples, collected before (n = 58) and after (n = 60) neoadjuvant treatment with the aromatase inhibitor letrozole, to prioritize genes as targets for combination therapy with letrozole treatment. We validate letrozole-induced tumor adaptation through coexpression and pathway analyses in an independent data set (n = 18). We find pervasive differential coexpression between the untreated and letrozole-treated tumor samples as evidence of letrozole-induced tumor adaptation. Based on patterns of coexpression, we identify ten genes as potential candidates for combination therapy with letrozole including EPCAM, a letrozole-induced essential gene and a target to which drugs have already been developed as cancer therapeutics. Through replication, we validate six letrozole-induced coexpression relationships and confirm the epithelial-to-mesenchymal transition as a process that is upregulated in the residual tumor samples following letrozole treatment. To derive the greatest benefit from molecularly targeted drugs it is critical to design combination

  6. The mechanism of gene targeting in human somatic cells.

    Directory of Open Access Journals (Sweden)

    Yinan Kan

    2014-04-01

    Full Text Available Gene targeting in human somatic cells is of importance because it can be used to either delineate the loss-of-function phenotype of a gene or correct a mutated gene back to wild-type. Both of these outcomes require a form of DNA double-strand break (DSB repair known as homologous recombination (HR. The mechanism of HR leading to gene targeting, however, is not well understood in human cells. Here, we demonstrate that a two-end, ends-out HR intermediate is valid for human gene targeting. Furthermore, the resolution step of this intermediate occurs via the classic DSB repair model of HR while synthesis-dependent strand annealing and Holliday Junction dissolution are, at best, minor pathways. Moreover, and in contrast to other systems, the positions of Holliday Junction resolution are evenly distributed along the homology arms of the targeting vector. Most unexpectedly, we demonstrate that when a meganuclease is used to introduce a chromosomal DSB to augment gene targeting, the mechanism of gene targeting is inverted to an ends-in process. Finally, we demonstrate that the anti-recombination activity of mismatch repair is a significant impediment to gene targeting. These observations significantly advance our understanding of HR and gene targeting in human cells.

  7. Transcriptionally targeted gene therapy to detect and treat cancer

    OpenAIRE

    Wu, Lily; Johnson, Mai; Sato, Makoto

    2003-01-01

    The greatest challenge in cancer treatment is to achieve the highest levels of specificity and efficacy. Cancer gene therapy could be designed specifically to express therapeutic genes to induce cancer cell destruction. Cancer-specific promoters are useful tools to accomplish targeted expression; however, high levels of gene expression are needed to achieve therapeutic efficacy. Incorporating an imaging reporter gene in tandem with the therapeutic gene will allow tangible proof of principle t...

  8. Chemotherapy and molecular target therapy combined with radiation therapy

    International Nuclear Information System (INIS)

    Akimoto, Tetsuo

    2012-01-01

    Combined chemotherapy and radiation therapy has been established as standard treatment approach for locally advanced head and neck cancer, esophageal cancer and so on through randomized clinical trials. However, radiation-related morbidity such as acute toxicity also increased as treatment intensity has increased. In underlining mechanism for enhancement of normal tissue reaction in chemo-radiation therapy, chemotherapy enhanced radiosensitivity of normal tissues in addition to cancer cells. Molecular target-based drugs combined with radiation therapy have been expected as promising approach that makes it possible to achieve cancer-specific enhancement of radiosensitivity, and clinical trials using combined modalities have been performed to evaluate the feasibility and efficacy of this approach. In order to obtain maximum radiotherapeutic gain, a detailed understanding of the mechanism underlying the interaction between radiation and Molecular target-based drugs is indispensable. Among molecular target-based drugs, inhibitors targeting epidermal growth factor receptor (EGFR) and its signal transduction pathways have been vigorously investigated, and mechanisms regarding the radiosensitizing effect have been getting clear. In addition, the results of randomized clinical trials demonstrated that radiation therapy combined with cetuximab resulted in improvement of overall and disease-specific survival rate compared with radiation therapy in locally advanced head and neck cancer. In this review, clinical usefulness of chemo-radiation therapy and potential molecular targets for potentiation of radiation-induced cell killing are summarized. (author)

  9. Gene therapy of cancer and development of therapeutic target gene

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Chang Min; Kwon, Hee Chung

    1998-04-01

    We applied HSV-tk/GCV strategy to orthotopic rat hepatoma model and showed anticancer effects of hepatoma. The increased expression of Lac Z gene after adenovirus-mediated gene delivery throughout hepatic artery was thought that is increased the possibility of gene therapy for curing hepatoma. With the construction of kGLP-laboratory, it is possible to produce a good quantity and quality of adenovirus in lage-scale production and purification of adenovirus vector. Also, the analysis of hepatoma related genes by PCR-LOH could be used for the diagnosis of patients and the development of therapeutic gene.

  10. Gene therapy of cancer and development of therapeutic target gene

    International Nuclear Information System (INIS)

    Kim, Chang Min; Kwon, Hee Chung

    1998-04-01

    We applied HSV-tk/GCV strategy to orthotopic rat hepatoma model and showed anticancer effects of hepatoma. The increased expression of Lac Z gene after adenovirus-mediated gene delivery throughout hepatic artery was thought that is increased the possibility of gene therapy for curing hepatoma. With the construction of kGLP-laboratory, it is possible to produce a good quantity and quality of adenovirus in lage-scale production and purification of adenovirus vector. Also, the analysis of hepatoma related genes by PCR-LOH could be used for the diagnosis of patients and the development of therapeutic gene

  11. The combination of checkpoint immunotherapy and targeted therapy in cancer.

    Science.gov (United States)

    Karachaliou, Niki; Gonzalez-Cao, Maria; Sosa, Aaron; Berenguer, Jordi; Bracht, Jillian Wilhelmina Paulina; Ito, Masaoki; Rosell, Rafael

    2017-10-01

    The therapeutic possibilities for patients with metastatic melanoma have changed due to the development of targeted therapies that inhibit oncogenic signaling pathways as well as immune modulating therapies that unleash the patient antitumor immunity. These therapeutic changes have impressively increased the median overall survival of the patients. Considering the dramatic but transient responses that occur with targeted therapies for a subgroup of patients and the durable responses that can be achieved with immunotherapy in a subset of patients, a lot of effort is ongoing for the clinical development of combinations of these two therapeutic approaches. Herein we discuss the existing preclinical and clinical data for the combination of targeted therapies and immunotherapy focusing mainly on melanoma and non-small cell lung cancer (NSCLC).

  12. Novel, Synergistic Antifungal Combinations that Target Translation Fidelity

    Science.gov (United States)

    Moreno-Martinez, Elena; Vallieres, Cindy; Holland, Sara L.; Avery, Simon V.

    2015-01-01

    There is an unmet need for new antifungal or fungicide treatments, as resistance to existing treatments grows. Combination treatments help to combat resistance. Here we develop a novel, effective target for combination antifungal therapy. Different aminoglycoside antibiotics combined with different sulphate-transport inhibitors produced strong, synergistic growth-inhibition of several fungi. Combinations decreased the respective MICs by ≥8-fold. Synergy was suppressed in yeast mutants resistant to effects of sulphate-mimetics (like chromate or molybdate) on sulphate transport. By different mechanisms, aminoglycosides and inhibition of sulphate transport cause errors in mRNA translation. The mistranslation rate was stimulated up to 10-fold when the agents were used in combination, consistent with this being the mode of synergistic action. A range of undesirable fungi were susceptible to synergistic inhibition by the combinations, including the human pathogens Candida albicans, C. glabrata and Cryptococcus neoformans, the food spoilage organism Zygosaccharomyces bailii and the phytopathogens Rhizoctonia solani and Zymoseptoria tritici. There was some specificity as certain fungi were unaffected. There was no synergy against bacterial or mammalian cells. The results indicate that translation fidelity is a promising new target for combinatorial treatment of undesirable fungi, the combinations requiring substantially decreased doses of active components compared to each agent alone. PMID:26573415

  13. Targeted Gene Therapy of Cancer: Second Amendment toward Holistic Therapy.

    Science.gov (United States)

    Barar, Jaleh; Omidi, Yadollah

    2013-01-01

    It seems solid tumors are developing smart organs with specialized cells creating specified bio-territory, the so called "tumor microenvironment (TME)", in which there is reciprocal crosstalk among cancer cells, immune system cells and stromal cells. TME as an intricate milieu also consists of cancer stem cells (CSCs) that can resist against chemotherapies. In solid tumors, metabolism and vascularization appears to be aberrant and tumor interstitial fluid (TIF) functions as physiologic barrier. Thus, chemotherapy, immunotherapy and gene therapy often fail to provide cogent clinical outcomes. It looms that it is the time to accept the fact that initiation of cancer could be generation of another form of life that involves a cluster of thousands of genes, while we have failed to observe all aspects of it. Hence, the current treatment modalities need to be re-visited to cover all key aspects of disease using combination therapy based on the condition of patients. Perhaps personalized cluster of genes need to be simultaneously targeted.

  14. Targeted Gene Therapy of Cancer: Second Amendment toward Holistic Therapy

    Directory of Open Access Journals (Sweden)

    Jaleh Barar

    2013-02-01

    Full Text Available It seems solid tumors are developing smart organs with specialized cells creating specified bio-territory, the so called “tumor microenvironment (TME”, in which there is reciprocal crosstalk among cancer cells, immune system cells and stromal cells. TME as an intricate milieu also consists of cancer stem cells (CSCs that can resist against chemotherapies. In solid tumors, metabolism and vascularization appears to be aberrant and tumor interstitial fluid (TIF functions as physiologic barrier. Thus, chemotherapy, immunotherapy and gene therapy often fail to provide cogent clinical outcomes. It looms that it is the time to accept the fact that initiation of cancer could be generation of another form of life that involves a cluster of thousands of genes, while we have failed to observe all aspects of it. Hence, the current treatment modalities need to be re-visited to cover all key aspects of disease using combination therapy based on the condition of patients. Perhaps personalized cluster of genes need to be simultaneously targeted.

  15. A new electrospray method for targeted gene delivery.

    Science.gov (United States)

    Boehringer, Stephan; Ruzgys, Paulius; Tamò, Luca; Šatkauskas, Saulius; Geiser, Thomas; Gazdhar, Amiq; Hradetzky, David

    2018-03-05

    A challenge for gene therapy is absence of safe and efficient local delivery of therapeutic genetic material. An efficient and reproducible physical method of electrospray for localized and targeted gene delivery is presented. Electrospray works on the principle of coulombs repulsion, under influence of electric field the liquid carrying genetic material is dispersed into micro droplets and is accelerated towards the targeted tissue, acting as a counter electrode. The accelerated droplets penetrate the targeted cells thus facilitating the transfer of genetic material into the cell. The work described here presents the principle of electrospray for gene delivery, the basic instrument design, and the various optimized parameters to enhance gene transfer in vitro. We estimate a transfection efficiency of up to 60% was achieved. We describe an efficient gene transfer method and a potential electrospray-mediated gene transfer mechanism.

  16. Characterisation of genome-wide PLZF/RARA target genes.

    Directory of Open Access Journals (Sweden)

    Salvatore Spicuglia

    Full Text Available The PLZF/RARA fusion protein generated by the t(11;17(q23;q21 translocation in acute promyelocytic leukaemia (APL is believed to act as an oncogenic transcriptional regulator recruiting epigenetic factors to genes important for its transforming potential. However, molecular mechanisms associated with PLZF/RARA-dependent leukaemogenesis still remain unclear.We searched for specific PLZF/RARA target genes by ChIP-on-chip in the haematopoietic cell line U937 conditionally expressing PLZF/RARA. By comparing bound regions found in U937 cells expressing endogenous PLZF with PLZF/RARA-induced U937 cells, we isolated specific PLZF/RARA target gene promoters. We next analysed gene expression profiles of our identified target genes in PLZF/RARA APL patients and analysed DNA sequences and epigenetic modification at PLZF/RARA binding sites. We identify 413 specific PLZF/RARA target genes including a number encoding transcription factors involved in the regulation of haematopoiesis. Among these genes, 22 were significantly down regulated in primary PLZF/RARA APL cells. In addition, repressed PLZF/RARA target genes were associated with increased levels of H3K27me3 and decreased levels of H3K9K14ac. Finally, sequence analysis of PLZF/RARA bound sequences reveals the presence of both consensus and degenerated RAREs as well as enrichment for tissue-specific transcription factor motifs, highlighting the complexity of targeting fusion protein to chromatin. Our study suggests that PLZF/RARA directly targets genes important for haematopoietic development and supports the notion that PLZF/RARA acts mainly as an epigenetic regulator of its direct target genes.

  17. Gene therapy for meningioma: improved gene delivery with targeted adenoviruses

    NARCIS (Netherlands)

    Dirven, Clemens M. F.; Grill, Jacques; Lamfers, Martine L. M.; van der Valk, Paul; Leonhart, Angelique M.; van Beusechem, Victor W.; Haisma, Hidde J.; Pinedo, Herbert M.; Curiel, David T.; Vandertop, W. Peter; Gerritsen, Winald R.

    2002-01-01

    OBJECT: Due to their surgical inaccessibility or aggressive behavior, some meningiomas cannot be cured with current treatment strategies. Gene therapy is an emerging strategy for the treatment of brain tumors, which the authors investigated to determine whether adenoviruses could be used for gene

  18. Gene therapy for meningioma : improved gene delivery with targeted adenoviruses

    NARCIS (Netherlands)

    Dirven, CMF; Grill, J; Lamfers, MLM; Van der Valk, P; Leonhart, AM; Van Beusechem, VW; Haisma, HJ; Pinedo, HM; Curiel, DT; Vandertop, WP; Gerritsen, WR

    Object. Due to their surgical inaccessibility or aggressive behavior, some meningiomas cannot be cured with current treatment strategies. Gene therapy is an emerging strategy for the treatment of brain tumors, which the authors investigated to determine whether adenoviruses could be used for gene

  19. TBC2target: A Resource of Predicted Target Genes of Tea Bioactive Compounds.

    Science.gov (United States)

    Zhang, Shihua; Zhang, Liang; Wang, Yijun; Yang, Jian; Liao, Mingzhi; Bi, Shoudong; Xie, Zhongwen; Ho, Chi-Tang; Wan, Xiaochun

    2018-01-01

    Tea is one of the most popular non-alcoholic beverages consumed worldwide. Numerous bioactive constituents of tea were confirmed to possess healthy benefits via the mechanisms of regulating gene expressions or protein activities. However, a complete interacting profile between tea bioactive compounds (TBCs) and their target genes is lacking, which put an obstacle in the study of healthy function of tea. To fill this gap, we developed a database of target genes of TBCs (TBC2target, http://camellia.ahau.edu.cn/TBC2target) based on a pharmacophore mapping approach. In TBC2target, 6,226 interactions between 240 TBCs and 673 target genes were documented. TBC2target contains detailed information about each interacting entry, such as TBC, CAS number, PubChem CID, source of compound (e.g., green, black), compound type, target gene(s) of TBC, gene symbol, gene ID, ENSEMBL ID, PDB ID, TBC bioactivity and the reference. Using the TBC-target associations, we constructed a bipartite network and provided users the global network and local sub-network visualization and topological analyses. The entire database is free for online browsing, searching and downloading. In addition, TBC2target provides a BLAST search function to facilitate use of the database. The particular strengths of TBC2target are the inclusion of the comprehensive TBC-target interactions, and the capacity to visualize and analyze the interacting networks, which may help uncovering the beneficial effects of tea on human health as a central resource in tea health community.

  20. TBC2target: A Resource of Predicted Target Genes of Tea Bioactive Compounds

    Directory of Open Access Journals (Sweden)

    Shihua Zhang

    2018-02-01

    Full Text Available Tea is one of the most popular non-alcoholic beverages consumed worldwide. Numerous bioactive constituents of tea were confirmed to possess healthy benefits via the mechanisms of regulating gene expressions or protein activities. However, a complete interacting profile between tea bioactive compounds (TBCs and their target genes is lacking, which put an obstacle in the study of healthy function of tea. To fill this gap, we developed a database of target genes of TBCs (TBC2target, http://camellia.ahau.edu.cn/TBC2target based on a pharmacophore mapping approach. In TBC2target, 6,226 interactions between 240 TBCs and 673 target genes were documented. TBC2target contains detailed information about each interacting entry, such as TBC, CAS number, PubChem CID, source of compound (e.g., green, black, compound type, target gene(s of TBC, gene symbol, gene ID, ENSEMBL ID, PDB ID, TBC bioactivity and the reference. Using the TBC-target associations, we constructed a bipartite network and provided users the global network and local sub-network visualization and topological analyses. The entire database is free for online browsing, searching and downloading. In addition, TBC2target provides a BLAST search function to facilitate use of the database. The particular strengths of TBC2target are the inclusion of the comprehensive TBC-target interactions, and the capacity to visualize and analyze the interacting networks, which may help uncovering the beneficial effects of tea on human health as a central resource in tea health community.

  1. Targeting a Novel Vector for Breast Cancer Gene Therapy

    National Research Council Canada - National Science Library

    Bzik, David

    2002-01-01

    .... The primary purpose and scope of this IDEA award project is to experimentally examine approaches to safely target the Toxoplasma gondii parasite gene therapy vector to breast cancer tissue using...

  2. Gene-Specific Demethylation as Targeted Therapy in MDS

    Science.gov (United States)

    2017-07-01

    AWARD NUMBER: W81XWH-15-1-0161 TITLE: Gene-Specific Demethylation as Targeted Therapy in MDS PRINCIPAL INVESTIGATOR: Daniel G. Tenen, M.D...15JUN2016-14JUN2017 4. TITLE AND SUBTITLE Gene-Specific Demethylation as Targeted Therapy in MDS 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM... magnetic beads capture all newly synthesized transcripts. To compare the transcriptional profiles under these conditions with our previous results, we

  3. Targeted gene deletion in Zygosaccharomyces bailii.

    Science.gov (United States)

    Mollapour, M; Piper, P

    2001-01-30

    Yeasts of the genus Zygosaccharomyces are notable agents of large-scale food spoilage. Despite the economic importance of these organisms, little is known about the stress adaptations whereby they adapt to many of the more severe conditions of food preservation. In this study it was shown that genes of Z. bailii, a yeast notable for its high resistances to food preservatives and ethanol, can be isolated by complementation of the corresponding mutant strains of Saccharomyces cerevisiae. It was also discovered that the acquisition by S. cerevisiae of a single small Z. bailii gene (ZbYME2) was sufficient for the former yeast to acquire the ability to degrade two major food preservatives, benzoic acid and sorbic acid. Using DNA cassettes containing dominant selectable markers and methods originally developed for performing gene deletions in S. cerevisiae, the two copies of ZbYME2 in the Z. bailii genome were sequentially deleted. The resulting Zbyme2/Zbyme2 homozygous deletant strain had lost any ability to utilize benzoate as sole carbon source and was more sensitive to weak acid preservatives during growth on glucose. Thus, ZbYME2, probably the nuclear gene for a mitochondrial mono-oxygenase function, is essential for Z. bailii to degrade food preservatives. This ability to catabolize weak acid preservatives is a significant factor contributing to the preservative resistance of Z. bailii under aerobic conditions. This study is the first to demonstrate that it is possible to delete in Z. bailii genes that are suspected as being important for growth of this organism in preserved foods and beverages. With the construction of further mutant of Z. bailii strains, a clearer picture should emerge of how this yeast adapts to the conditions of food preservation. This information will, in turn, allow the design of new preservation strategies. GenBank Accession Nos: ZbURA3 (AF279259), ZbTIM9 (AF279260), ZbYME2 (AF279261), ZbTRP1 (AF279262), ZbHHT1(AF296170). Copyright 2000 John

  4. Inducement of radionuclides targeting therapy by gene transfection

    International Nuclear Information System (INIS)

    Luo Quanyong

    2001-01-01

    The author presents an overview of gene transfection methods to genetically induce tumor cells to express enhanced levels of cell surface antigens and receptors to intake radiolabeled antibody and peptide targeting and thus increase their therapeutic effect in radiotherapy. The current research include inducement of radioimmunotherapy through CEA gene transfection, inducement of iodine-131 therapy by sodium iodide symporter gene transfection and inducement of MIBG therapy by noradrenaline transporter gene transfection. These studies raise the prospect that gene-therapy techniques could be used to enable the treatment of a wide range of tumors with radiopharmaceuticals of established clinical acceptability

  5. Epigenetic Editing: targeted rewriting of epigenetic marks to modulate expression of selected target genes.

    NARCIS (Netherlands)

    de Groote, M.L.; Verschure, P.J.; Rots, M.G.

    2012-01-01

    Despite significant advances made in epigenetic research in recent decades, many questions remain unresolved, especially concerning cause and consequence of epigenetic marks with respect to gene expression modulation (GEM). Technologies allowing the targeting of epigenetic enzymes to predetermined

  6. Epigenetic Editing : targeted rewriting of epigenetic marks to modulate expression of selected target genes

    NARCIS (Netherlands)

    de Groote, Marloes L.; Verschure, Pernette J.; Rots, Marianne G.

    2012-01-01

    Despite significant advances made in epigenetic research in recent decades, many questions remain unresolved, especially concerning cause and consequence of epigenetic marks with respect to gene expression modulation (GEM). Technologies allowing the targeting of epigenetic enzymes to predetermined

  7. Epigenetic Modification of the Repair Donor Regulates Targeted Gene Correction

    Directory of Open Access Journals (Sweden)

    Olivier Humbert

    2012-01-01

    Full Text Available Optimizing design of vectors is critical to effective gene therapy. In targeted gene correction (TGC, cleavage of chromosomal DNA near a mutation stimulates homology-directed repair of a target gene using a donor provided in trans. We have systematically addressed epigenetic parameters of donor design, using a flow-based assay to quantify correction frequencies and expression levels of a green fluorescent protein (GFP reporter gene in a human cell line. We show that active transcription of the donor increased correction frequency by threefold, establishing that a proximal promoter enhances donor use. Conversely, CpG methylation of the donor diminished correction frequency and reduced expression of the repaired gene. However, bisulfite sequencing of the target revealed no transfer of methylation marks during repair with a methylated donor. Treatment with histone deacetylase (HDAC inhibitors can partially compensate for epigenetic inactivation, suggesting a role for class I and II HDACs in regulation of donor use. These results establish that epigenetic status of a trans-donor determines both the efficiency and outcome of gene correction, and identify and clarify parameters that should guide donor design for targeted gene therapy.

  8. Combining Targeted Agents With Modern Radiotherapy in Soft Tissue Sarcomas

    Science.gov (United States)

    Wong, Philip; Houghton, Peter; Kirsch, David G.; Finkelstein, Steven E.; Monjazeb, Arta M.; Xu-Welliver, Meng; Dicker, Adam P.; Ahmed, Mansoor; Vikram, Bhadrasain; Teicher, Beverly A.; Coleman, C. Norman; Machtay, Mitchell; Curran, Walter J.

    2014-01-01

    Improved understanding of soft-tissue sarcoma (STS) biology has led to better distinction and subtyping of these diseases with the hope of exploiting the molecular characteristics of each subtype to develop appropriately targeted treatment regimens. In the care of patients with extremity STS, adjunctive radiation therapy (RT) is used to facilitate limb and function, preserving surgeries while maintaining five-year local control above 85%. In contrast, for STS originating from nonextremity anatomical sites, the rate of local recurrence is much higher (five-year local control is approximately 50%) and a major cause of death and morbidity in these patients. Incorporating novel technological advancements to administer accurate RT in combination with novel radiosensitizing agents could potentially improve local control and overall survival. RT efficacy in STS can be increased by modulating biological pathways such as angiogenesis, cell cycle regulation, cell survival signaling, and cancer-host immune interactions. Previous experiences, advancements, ongoing research, and current clinical trials combining RT with agents modulating one or more of the above pathways are reviewed. The standard clinical management of patients with STS with pretreatment biopsy, neoadjuvant treatment, and primary surgery provides an opportune disease model for interrogating translational hypotheses. The purpose of this review is to outline a strategic vision for clinical translation of preclinical findings and to identify appropriate targeted agents to combine with radiotherapy in the treatment of STS from different sites and/or different histology subtypes. PMID:25326640

  9. A proposal for combining mapping, localization and target recognition

    Science.gov (United States)

    Grönwall, Christina; Hendeby, Gustaf; Sinivaara, Kristian

    2015-10-01

    Simultaneous localization and mapping (SLAM) is a well-known positioning approach in GPS-denied environments such as urban canyons and inside buildings. Autonomous/aided target detection and recognition (ATR) is commonly used in military application to detect threats and targets in outdoor environments. This papers present approaches to combine SLAM with ATR in ways that compensate for the drawbacks in each method. The methods use physical objects that are recognizable by ATR as unambiguous features in SLAM, while SLAM provides the ATR with better position estimates. Landmarks in the form of 3D point features based on normal aligned radial features (NARF) are used in conjunction with identified objects and 3D object models that replace landmarks when possible. This leads to a more compact map representation with fewer landmarks, which partly compensates for the introduced cost of the ATR. We analyze three approaches to combine SLAM and 3D-data; point-point matching ignoring NARF features, point-point matching using the set of points that are selected by NARF feature analysis, and matching of NARF features using nearest neighbor analysis. The first two approaches are is similar to the common iterative closest point (ICP). We propose an algorithm that combines EKF-SLAM and ATR based on rectangle estimation. The intended application is to improve the positioning of a first responder moving through an indoor environment, where the map offers localization and simultaneously helps locate people, furniture and potentially dangerous objects such as gas canisters.

  10. Targeted radionuclide therapy in combined-modality regimens.

    Science.gov (United States)

    Gill, Martin R; Falzone, Nadia; Du, Yong; Vallis, Katherine A

    2017-07-01

    Targeted radionuclide therapy (TRT) is a branch of cancer medicine concerned with the use of radioisotopes, radiolabelled molecules, nanoparticles, or microparticles that either naturally accumulate in or are designed to target tumours. TRT combines the specificity of molecular and sometimes physical targeting with the potent cytotoxicity of ionising radiation. Targeting vectors for TRT include antibodies, antibody fragments, proteins, peptides, and small molecules. The diversity of available carrier molecules, together with the large panel of suitable radioisotopes with unique physicochemical properties, allows vector-radionuclide pairings to be matched to the molecular, pathological, and physical characteristics of a tumour. Some pairings are designed for dual therapeutic and diagnostic applications. Use of TRT is increasing with the adoption into practice of radium-223 dichloride for the treatment of bone metastases and with the ongoing clinical development of, among others, 177 Lu-dodecanetetraacetic acid tyrosine-3-octreotate (DOTATATE) for the treatment of neuroendocrine tumours and 90 Y-microspheres for the treatment of hepatic tumours. The increasing use of TRT raises the question of how best to integrate TRT into multimodality protocols. Achievements in this area and the future prospects of TRT are evaluated in this Review. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Cancer Nanomedicine: From Targeted Delivery to Combination Therapy

    Science.gov (United States)

    Xu, Xiaoyang; Ho, William; Zhang, Xueqing; Bertrand, Nicolas; Farokhzad, Omid

    2015-01-01

    The advent of nanomedicine marks an unparalleled opportunity to advance the treatment of a variety of diseases, including cancer. The unique properties of nanoparticles, such as large surface-to volume ratio, small size, the ability to encapsulate a variety of drugs, and tunable surface chemistry, gives them many advantages over their bulk counterparts. This includes multivalent surface modification with targeting ligands, efficient navigation of the complex in vivo environment, increased intracellular trafficking, and sustained release of drug payload. These advantages make nanoparticles a mode of treatment potentially superior to conventional cancer therapies. This article highlights the most recent developments in cancer treatment using nanoparticles as drug-delivery vehicles, including promising opportunities in targeted and combination therapy. PMID:25656384

  12. Cancer gene therapy targeting angiogenesis: An updated Review

    Science.gov (United States)

    Liu, Ching-Chiu; Shen, Zan; Kung, Hsiang-Fu; Lin, Marie CM

    2006-01-01

    Since the relationship between angiogenesis and tumor growth was established by Folkman in 1971, scientists have made efforts exploring the possibilities in treating cancer by targeting angiogenesis. Inhibition of angiogenesis growth factors and administration of angiogenesis inhibitors are the basics of anti-angiogenesis therapy. Transfer of anti-angiogenesis genes has received attention recently not only because of the advancement of recombinant vectors, but also because of the localized and sustained expression of therapeutic gene product inside the tumor after gene transfer. This review provides the up-to-date information about the strategies and the vectors studied in the field of anti-angiogenesis cancer gene therapy. PMID:17109514

  13. Targeting angiogenesis-dependent calcified neoplasms using combined polymer therapeutics.

    Directory of Open Access Journals (Sweden)

    Ehud Segal

    Full Text Available There is an immense clinical need for novel therapeutics for the treatment of angiogenesis-dependent calcified neoplasms such as osteosarcomas and bone metastases. We developed a new therapeutic strategy to target bone metastases and calcified neoplasms using combined polymer-bound angiogenesis inhibitors. Using an advanced "living polymerization" technique, the reversible addition-fragmentation chain transfer (RAFT, we conjugated the aminobisphosphonate alendronate (ALN, and the potent anti-angiogenic agent TNP-470 with N-(2-hydroxypropylmethacrylamide (HPMA copolymer through a Glycine-Glycine-Proline-Norleucine linker, cleaved by cathepsin K, a cysteine protease overexpressed at resorption sites in bone tissues. In this approach, dual targeting is achieved. Passive accumulation is possible due to the increase in molecular weight following polymer conjugation of the drugs, thus extravasating from the tumor leaky vessels and not from normal healthy vessels. Active targeting to the calcified tissues is achieved by ALN's affinity to bone mineral.The anti-angiogenic and antitumor potency of HPMA copolymer-ALN-TNP-470 conjugate was evaluated both in vitro and in vivo. We show that free and conjugated ALN-TNP-470 have synergistic anti-angiogenic and antitumor activity by inhibiting proliferation, migration and capillary-like tube formation of endothelial and human osteosarcoma cells in vitro. Evaluation of anti-angiogenic, antitumor activity and body distribution of HPMA copolymer-ALN-TNP-470 conjugate was performed on severe combined immunodeficiency (SCID male mice inoculated with mCherry-labeled MG-63-Ras human osteosarcoma and by modified Miles permeability assay. Our targeted bi-specific conjugate reduced VEGF-induced vascular hyperpermeability by 92% and remarkably inhibited osteosarcoma growth in mice by 96%.This is the first report to describe a new concept of a narrowly-dispersed combined polymer therapeutic designed to target both tumor and

  14. Positive-negative-selection-mediated gene targeting in rice

    Directory of Open Access Journals (Sweden)

    Zenpei eShimatani

    2015-01-01

    Full Text Available Gene targeting (GT refers to the designed modification of genomic sequence(s through homologous recombination (HR. GT is a powerful tool both for the study of gene function and for molecular breeding. However, in transformation of higher plants, non-homologous end joining (NHEJ occurs overwhelmingly in somatic cells, masking HR-mediated GT. Positive-negative selection (PNS is an approach for finding HR-mediated GT events because it can eliminate NHEJ effectively by expression of a negative-selection marker gene. In rice—a major crop worldwide—reproducible PNS-mediated GT of endogenous genes has now been successfully achieved. The procedure is based on strong PNS using diphtheria toxin A-fragment as a negative marker, and has succeeded in the directed modification of several endogenous rice genes in various ways. In addition to gene knock-outs and knock-ins, a nucleotide substitution in a target gene was also achieved recently. This review presents a summary of the development of the rice PNS system, highlighting its advantages. Different types of gene modification and gene editing aimed at developing new plant breeding technology (NPBT based on PNS are discussed.

  15. Gene-carried hepatoma targeting complex induced high gene transfection efficiency with low toxicity and significant antitumor activity

    Directory of Open Access Journals (Sweden)

    Zhao QQ

    2012-06-01

    Full Text Available Qing-Qing Zhao,1,2 Yu-Lan Hu,1 Yang Zhou,3 Ni Li,1 Min Han,1 Gu-Ping Tang,4 Feng Qiu,2 Yasuhiko Tabata,5 Jian-Qing Gao,11Institute of Pharmaceutics, Zhejiang University, Hangzhou, China; 2Department of Pharmacy, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China; 3Institute of Biochemistry, Iowa State University, Ames, IA, USA; 4Institute of Chemical Biology and Pharmaceutical Chemistry, Zhejiang University, Hangzhou, China; 5Institute for Frontier Medical Sciences, Kyoto University, Kyoto, JapanBackground: The success of gene transfection is largely dependent on the development of a vehicle or vector that can efficiently deliver a gene to cells with minimal toxicity.Methods: A liver cancer-targeted specific peptide (FQHPSF sequence was successfully synthesized and linked with chitosan-linked polyethylenimine (CP to form a new targeted gene delivery vector called CPT (CP/peptide. The structure of CPT was confirmed by 1H nuclear magnetic resonance spectroscopy and ultraviolet spectrophotometry. The particle size of CPT/DNA complexes was measured using laser diffraction spectrometry and the cytotoxicity of the copolymer was evaluated by methylthiazol tetrazolium method. The transfection efficiency evaluation of the CP copolymer was performed using luciferase activity assay. Cellular internalization of the CP/DNA complex was observed under confocal laser scanning microscopy. The targeting specificity of the polymer coupled to peptide was measured by competitive inhibition transfection study. The liver targeting specificity of the CPT copolymer in vivo was demonstrated by combining the copolymer with a therapeutic gene, interleukin-12, and assessed by its abilities in suppressing the growth of ascites tumor in mouse model.Results: The results showed that the liver cancer-targeted specific peptide was successfully synthesized and linked with CP to form a new targeted gene delivery vector called CPT. The composition of CPT

  16. Combining orthogonal polarization for elongated target detection with GPR

    International Nuclear Information System (INIS)

    Lualdi, Maurizio; Lombardi, Federico

    2014-01-01

    For an accurate imaging of ground penetrating radar data the polarization characteristics of the propagating electromagnetic (EM) wavefield and wave amplitude variations with antenna pattern orientation must be taken into account. For objects that show some directionality feature and cylindrical shape any misalignment between transmitter and target can strongly modify the polarization state of the backscattered wavefield, thus conditioning the detection capability of the system. Hints on the depolarization can be used to design the optimal GPR antenna survey to avoid omissions and pitfalls during data processing. This research addresses the issue of elongated target detection through a multi azimuth (or multi polarization) approach based on the combination of mutually orthogonal GPR data. Results from the analysis of the formal scattering problem demonstrate how this strategy can reach a scalar formulation of the scattering matrix and achieve a rotational invariant quantity. The effectiveness of the algorithm is then evaluated with a detailed field example showing results closely proximal to those obtained under the optimal alignment condition: detection is significantly improved and the risk of target missing is reduced. (paper)

  17. Pancreatic Cancer Gene Therapy: From Molecular Targets to Delivery Systems

    Energy Technology Data Exchange (ETDEWEB)

    Fillat, Cristina, E-mail: cristina.fillat@crg.es; Jose, Anabel; Ros, Xavier Bofill-De; Mato-Berciano, Ana; Maliandi, Maria Victoria; Sobrevals, Luciano [Programa Gens i Malaltia, Centre de Regulació Genòmica-CRG, UPF, Parc de Recerca Biomedica de Barcelona-PRBB and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Barcelona (Spain)

    2011-01-18

    The continuous identification of molecular changes deregulating critical pathways in pancreatic tumor cells provides us with a large number of novel candidates to engineer gene-targeted approaches for pancreatic cancer treatment. Targets—both protein coding and non-coding—are being exploited in gene therapy to influence the deregulated pathways to facilitate cytotoxicity, enhance the immune response or sensitize to current treatments. Delivery vehicles based on viral or non-viral systems as well as cellular vectors with tumor homing characteristics are a critical part of the design of gene therapy strategies. The different behavior of tumoral versus non-tumoral cells inspires vector engineering with the generation of tumor selective products that can prevent potential toxic-associated effects. In the current review, a detailed analysis of the different targets, the delivery vectors, the preclinical approaches and a descriptive update on the conducted clinical trials are presented. Moreover, future possibilities in pancreatic cancer treatment by gene therapy strategies are discussed.

  18. Folate receptor-targeted liposomes enhanced the antitumor potency of imatinib through the combination of active targeting and molecular targeting

    Science.gov (United States)

    Ye, Peng; Zhang, Wendian; Yang, Tan; Lu, Yao; Lu, Miao; Gai, Yongkang; Ma, Xiang; Xiang, Guangya

    2014-01-01

    liposomes may constitute a promising strategy in cervical cancer therapy through the combination of active targeting and molecular targeting. PMID:24855354

  19. Optimizing the design of oligonucleotides for homology directed gene targeting.

    Science.gov (United States)

    Miné-Hattab, Judith; Fleury, Geneviève; Prevost, Chantal; Dutreix, Marie; Viovy, Jean-Louis

    2011-04-05

    Gene targeting depends on the ability of cells to use homologous recombination to integrate exogenous DNA into their own genome. A robust mechanistic model of homologous recombination is necessary to fully exploit gene targeting for therapeutic benefit. In this work, our recently developed numerical simulation model for homology search is employed to develop rules for the design of oligonucleotides used in gene targeting. A Metropolis Monte-Carlo algorithm is used to predict the pairing dynamics of an oligonucleotide with the target double-stranded DNA. The model calculates the base-alignment between a long, target double-stranded DNA and a probe nucleoprotein filament comprised of homologous recombination proteins (Rad51 or RecA) polymerized on a single strand DNA. In this study, we considered different sizes of oligonucleotides containing 1 or 3 base heterologies with the target; different positions on the probe were tested to investigate the effect of the mismatch position on the pairing dynamics and stability. We show that the optimal design is a compromise between the mean time to reach a perfect alignment between the two molecules and the stability of the complex. A single heterology can be placed anywhere without significantly affecting the stability of the triplex. In the case of three consecutive heterologies, our modeling recommends using long oligonucleotides (at least 35 bases) in which the heterologous sequences are positioned at an intermediate position. Oligonucleotides should not contain more than 10% consecutive heterologies to guarantee a stable pairing with the target dsDNA. Theoretical modeling cannot replace experiments, but we believe that our model can considerably accelerate optimization of oligonucleotides for gene therapy by predicting their pairing dynamics with the target dsDNA.

  20. Optimizing the design of oligonucleotides for homology directed gene targeting.

    Directory of Open Access Journals (Sweden)

    Judith Miné-Hattab

    Full Text Available BACKGROUND: Gene targeting depends on the ability of cells to use homologous recombination to integrate exogenous DNA into their own genome. A robust mechanistic model of homologous recombination is necessary to fully exploit gene targeting for therapeutic benefit. METHODOLOGY/PRINCIPAL FINDINGS: In this work, our recently developed numerical simulation model for homology search is employed to develop rules for the design of oligonucleotides used in gene targeting. A Metropolis Monte-Carlo algorithm is used to predict the pairing dynamics of an oligonucleotide with the target double-stranded DNA. The model calculates the base-alignment between a long, target double-stranded DNA and a probe nucleoprotein filament comprised of homologous recombination proteins (Rad51 or RecA polymerized on a single strand DNA. In this study, we considered different sizes of oligonucleotides containing 1 or 3 base heterologies with the target; different positions on the probe were tested to investigate the effect of the mismatch position on the pairing dynamics and stability. We show that the optimal design is a compromise between the mean time to reach a perfect alignment between the two molecules and the stability of the complex. CONCLUSION AND SIGNIFICANCE: A single heterology can be placed anywhere without significantly affecting the stability of the triplex. In the case of three consecutive heterologies, our modeling recommends using long oligonucleotides (at least 35 bases in which the heterologous sequences are positioned at an intermediate position. Oligonucleotides should not contain more than 10% consecutive heterologies to guarantee a stable pairing with the target dsDNA. Theoretical modeling cannot replace experiments, but we believe that our model can considerably accelerate optimization of oligonucleotides for gene therapy by predicting their pairing dynamics with the target dsDNA.

  1. Advances in sarcoma gene mutations and therapeutic targets.

    Science.gov (United States)

    Gao, Peng; Seebacher, Nicole A; Hornicek, Francis; Guo, Zheng; Duan, Zhenfeng

    2018-01-01

    Sarcomas are rare and complex malignancies that have been associated with a poor prognostic outcome. Over the last few decades, traditional treatment with surgery and/or chemotherapy has not significantly improved outcomes for most types of sarcomas. In recent years, there have been significant advances in the understanding of specific gene mutations that are important in driving the pathogenesis and progression of sarcomas. Identification of these new gene mutations, using next-generation sequencing and advanced molecular techniques, has revealed a range of potential therapeutic targets. This, in turn, may lead to the development of novel agents targeted to different sarcoma subtypes. In this review, we highlight the advances made in identifying sarcoma gene mutations, including those of p53, RB, PI3K and IDH genes, as well as novel therapeutic strategies aimed at utilizing these mutant genes. In addition, we discuss a number of preclinical studies and ongoing early clinical trials in sarcoma targeting therapies, as well as gene editing technology, which may provide a better choice for sarcoma patient management. Published by Elsevier Ltd.

  2. Hyb-Seq: Combining Target Enrichment and Genome Skimming for Plant Phylogenomics

    Directory of Open Access Journals (Sweden)

    Kevin Weitemier

    2014-08-01

    Full Text Available Premise of the study: Hyb-Seq, the combination of target enrichment and genome skimming, allows simultaneous data collection for low-copy nuclear genes and high-copy genomic targets for plant systematics and evolution studies. Methods and Results: Genome and transcriptome assemblies for milkweed (Asclepias syriaca were used to design enrichment probes for 3385 exons from 768 genes (>1.6 Mbp followed by Illumina sequencing of enriched libraries. Hyb-Seq of 12 individuals (10 Asclepias species and two related genera resulted in at least partial assembly of 92.6% of exons and 99.7% of genes and an average assembly length >2 Mbp. Importantly, complete plastomes and nuclear ribosomal DNA cistrons were assembled using off-target reads. Phylogenomic analyses demonstrated signal conflict between genomes. Conclusions: The Hyb-Seq approach enables targeted sequencing of thousands of low-copy nuclear exons and flanking regions, as well as genome skimming of high-copy repeats and organellar genomes, to efficiently produce genome-scale data sets for phylogenomics.

  3. Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer.

    Science.gov (United States)

    Zhou, Xiaoqing; Xin, Jige; Fan, Nana; Zou, Qingjian; Huang, Jiao; Ouyang, Zhen; Zhao, Yu; Zhao, Bentian; Liu, Zhaoming; Lai, Sisi; Yi, Xiaoling; Guo, Lin; Esteban, Miguel A; Zeng, Yangzhi; Yang, Huaqiang; Lai, Liangxue

    2015-03-01

    The domestic pig has been widely used as an important large animal model. Precise and efficient genetic modification in pig provides a great promise in biomedical research. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has been successfully used to produce many gene-targeted animals. However, these animals have been generated by co-injection of Cas9 mRNA and single-guide RNA (sgRNA) into one-cell stage embryos, which mostly resulted in mosaicism of the modification. One or two rounds of further breeding should be performed to obtain homozygotes with identical genotype and phenotype. To address this issue, gene-targeted somatic cells can be used as donor for somatic cell nuclear transfer (SCNT) to produce gene-targeted animals with single and identical mutations. In this study, we applied Cas9/sgRNAs to effectively direct gene editing in porcine fetal fibroblasts and then mutant cell colonies were used as donor to generate homozygous gene-targeted pigs through single round of SCNT. As a result, we successfully obtained 15 tyrosinase (TYR) biallelic mutant pigs and 20 PARK2 and PINK1 double-gene knockout (KO) pigs. They were all homozygous and no off-target mutagenesis was detected by comprehensive analysis. TYR (-/-) pigs showed typical albinism and the expression of parkin and PINK1 were depleted in PARK2 (-/-)/PINK1 (-/-) pigs. The results demonstrated that single- or double-gene targeted pigs can be effectively achieved by using the CRISPR/Cas9 system combined with SCNT without mosaic mutation and detectable off-target effects. This gene-editing system provides an efficient, rapid, and less costly manner to generate genetically modified pigs or other large animals.

  4. Identification of target genes of transcription factor activator protein 2 gamma in breast cancer cells

    International Nuclear Information System (INIS)

    Ailan, He; Shuanglin, Xiang; Xiangwen, Xiao; Daolong, Ren; Lu, Gan; Xiaofeng, Ding; Xi, Qiao; Xingwang, Hu; Rushi, Liu; Jian, Zhang

    2009-01-01

    Activator protein 2 gamma (AP-2γ) is a member of the transcription factor activator protein-2 (AP-2) family, which is developmentally regulated and plays a role in human neoplasia. AP-2γ has been found to be overexpressed in most breast cancers, and have a dual role to inhibit tumor initiation and promote tumor progression afterwards during mammary tumorigensis. To identify the gene targets that mediate its effects, we performed chromatin immunoprecipitation (ChIP) to isolate AP-2γ binding sites on genomic DNA from human breast cancer cell line MDA-MB-453. 20 novel DNA fragments proximal to potential AP-2γ targets were obtained. They are categorized into functional groups of carcinogenesis, metabolism and others. A combination of sequence analysis, reporter gene assays, quantitative real-time PCR, electrophoretic gel mobility shift assays and immunoblot analysis further confirmed the four AP-2γ target genes in carcinogenesis group: ErbB2, CDH2, HPSE and IGSF11. Our results were consistent with the previous reports that ErbB2 was the target gene of AP-2γ. Decreased expression and overexpression of AP-2γ in human breast cancer cells significantly altered the expression of these four genes, indicating that AP-2γ directly regulates them. This suggested that AP-2γ can coordinate the expression of a network of genes, involving in carcinogenesis, especially in breast cancer. They could serve as therapeutic targets against breast cancers in the future

  5. Targeted Gene Delivery to Macrophages by Biodegradable Star-Shaped Polymers.

    Science.gov (United States)

    Zhang, Yajie; Wang, Yafeng; Zhang, Chi; Wang, Jin; Pan, Dejing; Liu, Jianghuai; Feng, Fude

    2016-02-17

    In this report, two biodegradable star-shaped polyasparamide derivatives and four analogues modified with either mannose or folic acid moiety for preferential targeting of a difficult-to-transfect immune cell type, i.e., macrophage, have been synthesized. Each of the prepared star polymers complexes with plasmid DNA to form nanosized particles featuring a core-shell-like morphology. Mannose or folate functionalized star polymers can greatly improve the transfection performance on a macrophage cell line RAW 264.7. As a result, a combination of targeting ligand modification and topological structures of gene carriers is a promising strategy for immune cells-based gene therapy.

  6. The Human Kinome Targeted by FDA Approved Multi-Target Drugs and Combination Products: A Comparative Study from the Drug-Target Interaction Network Perspective.

    Science.gov (United States)

    Li, Ying Hong; Wang, Pan Pan; Li, Xiao Xu; Yu, Chun Yan; Yang, Hong; Zhou, Jin; Xue, Wei Wei; Tan, Jun; Zhu, Feng

    2016-01-01

    The human kinome is one of the most productive classes of drug target, and there is emerging necessity for treating complex diseases by means of polypharmacology (multi-target drugs and combination products). However, the advantages of the multi-target drugs and the combination products are still under debate. A comparative analysis between FDA approved multi-target drugs and combination products, targeting the human kinome, was conducted by mapping targets onto the phylogenetic tree of the human kinome. The approach of network medicine illustrating the drug-target interactions was applied to identify popular targets of multi-target drugs and combination products. As identified, the multi-target drugs tended to inhibit target pairs in the human kinome, especially the receptor tyrosine kinase family, while the combination products were able to against targets of distant homology relationship. This finding asked for choosing the combination products as a better solution for designing drugs aiming at targets of distant homology relationship. Moreover, sub-networks of drug-target interactions in specific disease were generated, and mechanisms shared by multi-target drugs and combination products were identified. In conclusion, this study performed an analysis between approved multi-target drugs and combination products against the human kinome, which could assist the discovery of next generation polypharmacology.

  7. Yeast-based assay identifies novel Shh/Gli target genes in vertebrate development

    Directory of Open Access Journals (Sweden)

    Milla Luis A

    2012-01-01

    Full Text Available Abstract Background The increasing number of developmental events and molecular mechanisms associated with the Hedgehog (Hh pathway from Drosophila to vertebrates, suggest that gene regulation is crucial for diverse cellular responses, including target genes not yet described. Although several high-throughput, genome-wide approaches have yielded information at the genomic, transcriptional and proteomic levels, the specificity of Gli binding sites related to direct target gene activation still remain elusive. This study aims to identify novel putative targets of Gli transcription factors through a protein-DNA binding assay using yeast, and validating a subset of targets both in-vitro and in-vivo. Testing in different Hh/Gli gain- and loss-of-function scenarios we here identified known (e.g., ptc1 and novel Hh-regulated genes in zebrafish embryos. Results The combined yeast-based screening and MEME/MAST analysis were able to predict Gli transcription factor binding sites, and position mapping of these sequences upstream or in the first intron of promoters served to identify new putative target genes of Gli regulation. These candidates were validated by qPCR in combination with either the pharmacological Hh/Gli antagonist cyc or the agonist pur in Hh-responsive C3H10T1/2 cells. We also used small-hairpin RNAs against Gli proteins to evaluate targets and confirm specific Gli regulation their expression. Taking advantage of mutants that have been identified affecting different components of the Hh/Gli signaling system in the zebrafish model, we further analyzed specific novel candidates. Studying Hh function with pharmacological inhibition or activation complemented these genetic loss-of-function approaches. We provide evidence that in zebrafish embryos, Hh signaling regulates sfrp2, neo1, and c-myc expression in-vivo. Conclusion A recently described yeast-based screening allowed us to identify new Hh/Gli target genes, functionally important in

  8. Novel Data Fusion Method and Exploration of Multiple Information Sources for Transcription Factor Target Gene Prediction

    Science.gov (United States)

    Dai, Xiaofeng; Yli-Harja, Olli; Lähdesmäki, Harri

    2010-12-01

    Background. Revealing protein-DNA interactions is a key problem in understanding transcriptional regulation at mechanistic level. Computational methods have an important role in predicting transcription factor target gene genomewide. Multiple data fusion provides a natural way to improve transcription factor target gene predictions because sequence specificities alone are not sufficient to accurately predict transcription factor binding sites. Methods. Here we develop a new data fusion method to combine multiple genome-level data sources and study the extent to which DNA duplex stability and nucleosome positioning information, either alone or in combination with other data sources, can improve the prediction of transcription factor target gene. Results. Results on a carefully constructed test set of verified binding sites in mouse genome demonstrate that our new multiple data fusion method can reduce false positive rates, and that DNA duplex stability and nucleosome occupation data can improve the accuracy of transcription factor target gene predictions, especially when combined with other genome-level data sources. Cross-validation and other randomization tests confirm the predictive performance of our method. Our results also show that nonredundant data sources provide the most efficient data fusion.

  9. Revealing targeted therapy for human cancer by gene module maps

    NARCIS (Netherlands)

    Wong, David J.; Nuyten, Dimitry S. A.; Regev, Aviv; Lin, Meihong; Adler, Adam S.; Segal, Eran; van de Vijver, Marc J.; Chang, Howard Y.

    2008-01-01

    A major goal of cancer research is to match specific therapies to molecular targets in cancer. Genome-scale expression profiling has identified new subtypes of cancer based on consistent patterns of variation in gene expression, leading to improved prognostic predictions. However, how these new

  10. E2F target genes: unraveling the biology

    DEFF Research Database (Denmark)

    Bracken, Adrian P; Ciro, Marco; Cocito, Andrea

    2004-01-01

    The E2F transcription factors are downstream effectors of the retinoblastoma protein (pRB) pathway and are required for the timely regulation of numerous genes essential for DNA replication and cell cycle progression. Several laboratories have used genome-wide approaches to discover novel target...

  11. Evolutionary dynamics of cancer in response to targeted combination therapy

    Science.gov (United States)

    Bozic, Ivana; Reiter, Johannes G; Allen, Benjamin; Antal, Tibor; Chatterjee, Krishnendu; Shah, Preya; Moon, Yo Sup; Yaqubie, Amin; Kelly, Nicole; Le, Dung T; Lipson, Evan J; Chapman, Paul B; Diaz, Luis A; Vogelstein, Bert; Nowak, Martin A

    2013-01-01

    In solid tumors, targeted treatments can lead to dramatic regressions, but responses are often short-lived because resistant cancer cells arise. The major strategy proposed for overcoming resistance is combination therapy. We present a mathematical model describing the evolutionary dynamics of lesions in response to treatment. We first studied 20 melanoma patients receiving vemurafenib. We then applied our model to an independent set of pancreatic, colorectal, and melanoma cancer patients with metastatic disease. We find that dual therapy results in long-term disease control for most patients, if there are no single mutations that cause cross-resistance to both drugs; in patients with large disease burden, triple therapy is needed. We also find that simultaneous therapy with two drugs is much more effective than sequential therapy. Our results provide realistic expectations for the efficacy of new drug combinations and inform the design of trials for new cancer therapeutics. DOI: http://dx.doi.org/10.7554/eLife.00747.001 PMID:23805382

  12. Identification of Plagl1/Zac1 binding sites and target genes establishes its role in the regulation of extracellular matrix genes and the imprinted gene network.

    Science.gov (United States)

    Varrault, Annie; Dantec, Christelle; Le Digarcher, Anne; Chotard, Laëtitia; Bilanges, Benoit; Parrinello, Hugues; Dubois, Emeric; Rialle, Stéphanie; Severac, Dany; Bouschet, Tristan; Journot, Laurent

    2017-10-13

    PLAGL1/ZAC1 undergoes parental genomic imprinting, is paternally expressed, and is a member of the imprinted gene network (IGN). It encodes a zinc finger transcription factor with anti-proliferative activity and is a candidate tumor suppressor gene on 6q24 whose expression is frequently lost in various neoplasms. Conversely, gain of PLAGL1 function is responsible for transient neonatal diabetes mellitus, a rare genetic disease that results from defective pancreas development. In the present work, we showed that Plagl1 up-regulation was not associated with DNA damage-induced cell cycle arrest. It was rather associated with physiological cell cycle exit that occurred with contact inhibition, growth factor withdrawal, or cell differentiation. To gain insights into Plagl1 mechanism of action, we identified Plagl1 target genes by combining chromatin immunoprecipitation and genome-wide transcriptomics in transfected cell lines. Plagl1-elicited gene regulation correlated with multiple binding to the proximal promoter region through a GC-rich motif. Plagl1 target genes included numerous genes involved in signaling, cell adhesion, and extracellular matrix composition, including collagens. Plagl1 targets also included 22% of the 409 genes that make up the IGN. Altogether, this work identified Plagl1 as a transcription factor that coordinated the regulation of a subset of IGN genes and controlled extracellular matrix composition. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. W::Neo: a novel dual-selection marker for high efficiency gene targeting in Drosophila.

    Science.gov (United States)

    Zhou, Wenke; Huang, Juan; Watson, Annie M; Hong, Yang

    2012-01-01

    We have recently developed a so-called genomic engineering approach that allows for directed, efficient and versatile modifications of Drosophila genome by combining the homologous recombination (HR)-based gene targeting with site-specific DNA integration. In genomic engineering and several similar approaches, a "founder" knock-out line must be generated first through HR-based gene targeting, which can still be a potentially time and resource intensive process. To significantly improve the efficiency and success rate of HR-based gene targeting in Drosophila, we have generated a new dual-selection marker termed W::Neo, which is a direct fusion between proteins of eye color marker White (W) and neomycin resistance (Neo). In HR-based gene targeting experiments, mutants carrying W::Neo as the selection marker can be enriched as much as fifty times by taking advantage of the antibiotic selection in Drosophila larvae. We have successfully carried out three independent gene targeting experiments using the W::Neo to generate genomic engineering founder knock-out lines in Drosophila.

  14. Identification of novel androgen receptor target genes in prostate cancer

    Directory of Open Access Journals (Sweden)

    Gerald William L

    2007-06-01

    Full Text Available Abstract Background The androgen receptor (AR plays critical roles in both androgen-dependent and castrate-resistant prostate cancer (PCa. However, little is known about AR target genes that mediate the receptor's roles in disease progression. Results Using Chromatin Immunoprecipitation (ChIP Display, we discovered 19 novel loci occupied by the AR in castrate resistant C4-2B PCa cells. Only four of the 19 AR-occupied regions were within 10-kb 5'-flanking regulatory sequences. Three were located up to 4-kb 3' of the nearest gene, eight were intragenic and four were in gene deserts. Whereas the AR occupied the same loci in C4-2B (castrate resistant and LNCaP (androgen-dependent PCa cells, differences between the two cell lines were observed in the response of nearby genes to androgens. Among the genes strongly stimulated by DHT in C4-2B cells – D-dopachrome tautomerase (DDT, Protein kinase C delta (PRKCD, Glutathione S- transferase theta 2 (GSTT2, Transient receptor potential cation channel subfamily V member 3 (TRPV3, and Pyrroline-5-carboxylate reductase 1 (PYCR1 – most were less strongly or hardly stimulated in LNCaP cells. Another AR target gene, ornithine aminotransferase (OAT, was AR-stimulated in a ligand-independent manner, since it was repressed by AR siRNA knockdown, but not stimulated by DHT. We also present evidence for in vivo AR-mediated regulation of several genes identified by ChIP Display. For example, PRKCD and PYCR1, which may contribute to PCa cell growth and survival, are expressed in PCa biopsies from primary tumors before and after ablation and in metastatic lesions in a manner consistent with AR-mediated stimulation. Conclusion AR genomic occupancy is similar between LNCaP and C4-2B cells and is not biased towards 5' gene flanking sequences. The AR transcriptionally regulates less than half the genes nearby AR-occupied regions, usually but not always, in a ligand-dependent manner. Most are stimulated and a few are

  15. Recurrent Fusion Genes in Leukemia: An Attractive Target for Diagnosis and Treatment.

    Science.gov (United States)

    Wang, Yuhui; Wu, Nan; Liu, Duo; Jin, Yan

    2017-10-01

    Since the first fusion gene was discovered decades ago, a considerable number of fusion genes have been detected in leukemia. The majority of them are generated through chromosomal rearrangement or abnormal transcription. With the development of techniques, high-throughput sequencing method makes it possible to detect fusion genes systematically in multiple human cancers. Owing to their biological significance and tumor-specific expression, some of the fusion genes are attractive diagnostic tools and therapeutic targets. Tyrosine kinase inhibitors (TKI) targeting BCR-ABL1 fusions have been widely used to treat CML. The combination of ATRA and ATO targeting PML-RARA fusions has proven to be effective in acute promyelocytic leukemia (APL). Moreover, therapy with high dose cytarabine (HDAC) has significantly improved the prognosis of core binding factor (CBF) acute myeloid leukemia (AML) patients. Therefore, studies on fusion genes may benefit patients with leukemia by providing more diagnostic markers and therapies in the future. The presented review focuses on the history of fusion genes, mechanisms of formation, and treatments against specific fusion genes in leukemia.

  16. TF Target Mapper: a BLAST search tool for the identification of Transcription Factor target genes.

    NARCIS (Netherlands)

    S. Horsman (Sebastiaan); M.J. Moorhouse (Michael); V. de Jager (Victor); P.J. van der Spek (Peter); F.G. Grosveld (Frank); J. Strouboulis (John); E. Katsantoni (Eleni)

    2006-01-01

    textabstractBACKGROUND: In the current era of high throughput genomics a major challenge is the genome-wide identification of target genes for specific transcription factors. Chromatin immunoprecipitation (ChIP) allows the isolation of in vivo binding sites of transcription factors and provides a

  17. STAT3 Target Genes Relevant to Human Cancers

    International Nuclear Information System (INIS)

    Carpenter, Richard L.; Lo, Hui-Wen

    2014-01-01

    Since its discovery, the STAT3 transcription factor has been extensively studied for its function as a transcriptional regulator and its role as a mediator of development, normal physiology, and pathology of many diseases, including cancers. These efforts have uncovered an array of genes that can be positively and negatively regulated by STAT3, alone and in cooperation with other transcription factors. Through regulating gene expression, STAT3 has been demonstrated to play a pivotal role in many cellular processes including oncogenesis, tumor growth and progression, and stemness. Interestingly, recent studies suggest that STAT3 may behave as a tumor suppressor by activating expression of genes known to inhibit tumorigenesis. Additional evidence suggested that STAT3 may elicit opposing effects depending on cellular context and tumor types. These mixed results signify the need for a deeper understanding of STAT3, including its upstream regulators, parallel transcription co-regulators, and downstream target genes. To help facilitate fulfilling this unmet need, this review will be primarily focused on STAT3 downstream target genes that have been validated to associate with tumorigenesis and/or malignant biology of human cancers

  18. Multilevel Regulation of Bacterial Gene Expression with the Combined STAR and Antisense RNA System.

    Science.gov (United States)

    Lee, Young Je; Kim, Soo-Jung; Moon, Tae Seok

    2018-02-16

    Synthetic small RNA regulators have emerged as a versatile tool to predictably control bacterial gene expression. Owing to their simple design principles, small size, and highly orthogonal behavior, these engineered genetic parts have been incorporated into genetic circuits. However, efforts to achieve more sophisticated cellular functions using RNA regulators have been hindered by our limited ability to integrate different RNA regulators into complex circuits. Here, we present a combined RNA regulatory system in Escherichia coli that uses small transcription activating RNA (STAR) and antisense RNA (asRNA) to activate or deactivate target gene expression in a programmable manner. Specifically, we demonstrated that the activated target output by the STAR system can be deactivated by expressing two different types of asRNAs: one binds to and sequesters the STAR regulator, affecting the transcription process, while the other binds to the target mRNA, affecting the translation process. We improved deactivation efficiencies (up to 96%) by optimizing each type of asRNA and then integrating the two optimized asRNAs into a single circuit. Furthermore, we demonstrated that the combined STAR and asRNA system can control gene expression in a reversible way and can regulate expression of a gene in the genome. Lastly, we constructed and simultaneously tested two A AND NOT B logic gates in the same cell to show sophisticated multigene regulation by the combined system. Our approach establishes a methodology for integrating multiple RNA regulators to rationally control multiple genes.

  19. RFMirTarget: predicting human microRNA target genes with a random forest classifier.

    Directory of Open Access Journals (Sweden)

    Mariana R Mendoza

    Full Text Available MicroRNAs are key regulators of eukaryotic gene expression whose fundamental role has already been identified in many cell pathways. The correct identification of miRNAs targets is still a major challenge in bioinformatics and has motivated the development of several computational methods to overcome inherent limitations of experimental analysis. Indeed, the best results reported so far in terms of specificity and sensitivity are associated to machine learning-based methods for microRNA-target prediction. Following this trend, in the current paper we discuss and explore a microRNA-target prediction method based on a random forest classifier, namely RFMirTarget. Despite its well-known robustness regarding general classifying tasks, to the best of our knowledge, random forest have not been deeply explored for the specific context of predicting microRNAs targets. Our framework first analyzes alignments between candidate microRNA-target pairs and extracts a set of structural, thermodynamics, alignment, seed and position-based features, upon which classification is performed. Experiments have shown that RFMirTarget outperforms several well-known classifiers with statistical significance, and that its performance is not impaired by the class imbalance problem or features correlation. Moreover, comparing it against other algorithms for microRNA target prediction using independent test data sets from TarBase and starBase, we observe a very promising performance, with higher sensitivity in relation to other methods. Finally, tests performed with RFMirTarget show the benefits of feature selection even for a classifier with embedded feature importance analysis, and the consistency between relevant features identified and important biological properties for effective microRNA-target gene alignment.

  20. Demonstration of the effective performance of a combined enrichment/real-time PCR method targeting the prfA gene of Listeria monocytogenes by testing fresh naturally contaminated acid curd cheese.

    Science.gov (United States)

    Rossmanith, P; Mester, P; Wagner, M; Schoder, D

    2010-10-01

    A rapid real-time PCR-based method for the detection of Listeria monocytogenes was applied to the examination of 44 Quargel cheese samples from a recent outbreak in Austria and compared to the standard method according to ISO-16140. The combined enrichment/real-time PCR method amplifying the prfA locus was performed according to [Rossmanith et al.(2006) Res Microbiol, 157, 763-771]. Qualitative and quantitative examination of the samples was performed according to the standard method ISO-11290. Comparison of the combined enrichment/real-time PCR method with ISO-11290 resulted in 100% relative accuracy, 100% relative sensitivity and 100% relative specificity. A previously published study describing the validation of the method, including samples after storage at -80 degrees C, resulted in lower performance values. In contrast, the samples were stored at +4 degrees C in this study. The results of this study indicate an effect of storage, thus masking the true performance of the method. The results of this study are discussed together with the previously published data to demonstrate the excellent qualities of this rapid (fresh specimens stored at +4 degrees C.

  1. Genome-wide ChIP-seq profiling of PPARγ/RXR target sites and gene program during adipogenesis

    DEFF Research Database (Denmark)

    Nielsen, Ronni; Pedersen, Thomas Åskov; Hagenbeek, Dik

    directly a large number of genes involved in lipid metabolism. Using ChIP combined with deep sequencing we have generated a genome-wide map of PPARγ-RXR binding to chromatin as well as the activation of associated target genes during differentiation of murine 3T3-L1 adipocytes. Our analysis shows...

  2. Reproducible gene targeting in recalcitrant Escherichia coli isolates

    Directory of Open Access Journals (Sweden)

    De Greve Henri

    2011-06-01

    Full Text Available Abstract Background A number of allele replacement methods can be used to mutate bacterial genes. For instance, the Red recombinase system of phage Lambda has been used very efficiently to inactivate chromosomal genes in E. coli K-12, through recombination between regions of homology. However, this method does not work reproducibly in some clinical E. coli isolates. Findings The procedure was modified by using longer homologous regions (85 bp and 500-600 bp, to inactivate genes in the uropathogenic E. coli strain UTI89. An lrhA regulator mutant, and deletions of the lac operon as well as the complete type 1 fimbrial gene cluster, were obtained reproducibly. The modified method is also functional in other recalcitrant E. coli, like the avian pathogenic E. coli strain APEC1. The lrhA regulator and lac operon deletion mutants of APEC1 were successfully constructed in the same way as the UTI89 mutants. In other avian pathogenic E. coli strains (APEC3E, APEC11A and APEC16A it was very difficult or impossible to construct these mutants, with the original Red recombinase-based method, with a Red recombinase-based method using longer (85 bp homologous regions or with our modified protocol, using 500 - 600 bp homologous regions. Conclusions The method using 500-600 bp homologous regions can be used reliably in some clinical isolates, to delete single genes or entire operons by homologous recombination. However, it does not invariably show a greater efficiency in obtaining mutants, when compared to the original Red-mediated gene targeting method or to the gene targeting method with 85 bp homologous regions. Therefore the length of the homology regions is not the only limiting factor for the construction of mutants in these recalcitrant strains.

  3. Stimulation of IgY responses in gene gun immunized laying hens by combined administration of vector DNA coding for the target antigen Botulinum toxin A1 and for avian cytokine adjuvants.

    Science.gov (United States)

    Niederstadt, Lars; Hohn, Oliver; Dorner, Brigitte G; Schade, Rüdiger; Bannert, Norbert

    2012-08-31

    DNA immunization is a convenient and effective way of inducing a specific antibody response. In mammals, co-administration of vectors encoding immunostimulatory cytokines can enhance the humoral response resulting in elevated antibody titers. We therefore set out to investigate the effect using avian interleukin 1β (IL-1β) and avian interleukin 6 (IL-6) as genetic adjuvants when immunizing laying hens. A BoNT A1 holotoxoid DNA immunogen carrying two inactivating mutations was evaluated for its ability to induce a specific and sustained IgY antibody response. Both the holotoxoid and the cytokine sequences were codon-optimized. In vitro, the proteins were efficiently expressed in transfected HEK 293T cells and the cytokines were secreted into the culture supernatants. Whereas eggs from hens immunized via gene gun using a prime boost strategy showed no differences in their total IgY content, the specific αBoNT A1 response was slightly elevated up to 1.4× by the IL-1β adjuvant vector and increased by 3.8× by the IL-6 vector. Finally, although hens receiving the IL-1β adjuvant had laying capacities above the average, hens receiving the IL-6 adjuvant experienced laying problems. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Inheritable Silencing of Endogenous Genes by Hit-and-Run Targeted Epigenetic Editing.

    Science.gov (United States)

    Amabile, Angelo; Migliara, Alessandro; Capasso, Paola; Biffi, Mauro; Cittaro, Davide; Naldini, Luigi; Lombardo, Angelo

    2016-09-22

    Gene silencing is instrumental to interrogate gene function and holds promise for therapeutic applications. Here, we repurpose the endogenous retroviruses' silencing machinery of embryonic stem cells to stably silence three highly expressed genes in somatic cells by epigenetics. This was achieved by transiently expressing combinations of engineered transcriptional repressors that bind to and synergize at the target locus to instruct repressive histone marks and de novo DNA methylation, thus ensuring long-term memory of the repressive epigenetic state. Silencing was highly specific, as shown by genome-wide analyses, sharply confined to the targeted locus without spreading to nearby genes, resistant to activation induced by cytokine stimulation, and relieved only by targeted DNA demethylation. We demonstrate the portability of this technology by multiplex gene silencing, adopting different DNA binding platforms and interrogating thousands of genomic loci in different cell types, including primary T lymphocytes. Targeted epigenome editing might have broad application in research and medicine. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  5. Experimentally validated positive target genes for human from ...

    Indian Academy of Sciences (India)

    Ravish

    Experimentally validated positive target genes for human from miRecords databse. AGTR1. BACE1. BCL2. BCL6. C21orf33. C6orf134. CDK6. CDKN2A. CYP1B1. DNMT3B. E2F3. E2F6. ERBB2. ERBB3. ESR1. FBXW11. Fmr1. GJA1. HCN2. HCN4. HMGA2. HOXA11. HOXB8. HOXC8. HOXD10. HOXD8. HSPA1A. HSPD1.

  6. Mannan-Modified PLGA Nanoparticles for Targeted Gene Delivery

    Directory of Open Access Journals (Sweden)

    Fansheng Kong

    2012-01-01

    Full Text Available The studies of targeted gene delivery nanocarriers have gained increasing attention during the past decades. In this study, mannan modified DNA loaded bioadhesive PLGA nanoparticles (MAN-DNA-NPs were investigated for targeted gene delivery to the Kupffer cells (KCs. Bioadhesive PLGA nanoparticles were prepared and subsequently bound with pEGFP. Following the coupling of the mannan-based PE-grafted ligands (MAN-PE with the DNA-NPs, the MAN-DNA-NPs were delivered intravenously to rats. The transfection efficiency was determined from the isolated KCs and flow cytometry was applied for the quantitation of gene expression after 48 h post transfection. The size of the MAN-DNA-NPs was found to be around 190 nm and the Zeta potential was determined to be −15.46mV. The pEGFP binding capacity of MAN-DNA-NPs was (88.9±5.8% and the in vitro release profiles of the MAN-DNA-NPs follow the Higuchi model. When compared with non-modified DNA-NPs and Lipofectamine 2000-DNA, MAN-DNA-NPs produced the highest gene expressions, especially in vivo. The in vivo data from flow cytometry analysis showed that MAN-DNA-NPs displayed a remarkably higher transfection efficiency (39% than non-modified DNA-NPs (25% and Lipofectamine 2000-DNA (23% in KCs. The results illustrate that MAN-DNA-NPs have the ability to target liver KCs and could function as promising active targeting drug delivery vectors.

  7. Normal Collagen and Bone Production by Gene-targeted Human Osteogenesis Imperfecta iPSCs

    Science.gov (United States)

    Deyle, David R; Khan, Iram F; Ren, Gaoying; Wang, Pei-Rong; Kho, Jordan; Schwarze, Ulrike; Russell, David W

    2012-01-01

    Osteogenesis imperfecta (OI) is caused by dominant mutations in the type I collagen genes. In principle, the skeletal abnormalities of OI could be treated by transplantation of patient-specific, bone-forming cells that no longer express the mutant gene. Here, we develop this approach by isolating mesenchymal cells from OI patients, inactivating their mutant collagen genes by adeno-associated virus (AAV)-mediated gene targeting, and deriving induced pluripotent stem cells (iPSCs) that were expanded and differentiated into mesenchymal stem cells (iMSCs). Gene-targeted iMSCs produced normal collagen and formed bone in vivo, but were less senescent and proliferated more than bone-derived MSCs. To generate iPSCs that would be more appropriate for clinical use, the reprogramming and selectable marker transgenes were removed by Cre recombinase. These results demonstrate that the combination of gene targeting and iPSC derivation can be used to produce potentially therapeutic cells from patients with genetic disease. PMID:22031238

  8. Specifically targeted gene therapy for small-cell lung cancer

    DEFF Research Database (Denmark)

    Christensen, C.L.; Zandi, R.; Gjetting, T.

    2009-01-01

    Small-cell lung cancer (SCLC) is a highly malignant disease with poor prognosis. Hence, there is great demand for new therapies that can replace or supplement the current available treatment regimes. Gene therapy constitutes a promising strategy and relies on the principle of introducing exogenous....... This review describes and discusses the current status of the application of gene therapy in relation to SCLC Udgivelsesdato: 2009/4...... DNA into malignant cells causing them to die. Since SCLC is a highly disseminated malignancy, the gene therapeutic agent must be administered systemically, obligating a high level of targeting of tumor tissue and the use of delivery vehicles designed for systemic circulation of the therapeutic DNA...

  9. Pancreatic Cancer Gene Therapy: From Molecular Targets to Delivery Systems

    Directory of Open Access Journals (Sweden)

    Maria Victoria Maliandi

    2011-01-01

    Full Text Available The continuous identification of molecular changes deregulating critical pathways in pancreatic tumor cells provides us with a large number of novel candidates to engineer gene-targeted approaches for pancreatic cancer treatment. Targets—both protein coding and non-coding—are being exploited in gene therapy to influence the deregulated pathways to facilitate cytotoxicity, enhance the immune response or sensitize to current treatments. Delivery vehicles based on viral or non-viral systems as well as cellular vectors with tumor homing characteristics are a critical part of the design of gene therapy strategies. The different behavior of tumoral versus non-tumoral cells inspires vector engineering with the generation of tumor selective products that can prevent potential toxic-associated effects. In the current review, a detailed analysis of the different targets, the delivery vectors, the preclinical approaches and a descriptive update on the conducted clinical trials are presented. Moreover, future possibilities in pancreatic cancer treatment by gene therapy strategies are discussed.

  10. RNAi-Based Identification of Gene-Specific Nuclear Cofactor Networks Regulating Interleukin-1 Target Genes

    Directory of Open Access Journals (Sweden)

    Johanna Meier-Soelch

    2018-04-01

    Full Text Available The potent proinflammatory cytokine interleukin (IL-1 triggers gene expression through the NF-κB signaling pathway. Here, we investigated the cofactor requirements of strongly regulated IL-1 target genes whose expression is impaired in p65 NF-κB-deficient murine embryonic fibroblasts. By two independent small-hairpin (shRNA screens, we examined 170 genes annotated to encode nuclear cofactors for their role in Cxcl2 mRNA expression and identified 22 factors that modulated basal or IL-1-inducible Cxcl2 levels. The functions of 16 of these factors were validated for Cxcl2 and further analyzed for their role in regulation of 10 additional IL-1 target genes by RT-qPCR. These data reveal that each inducible gene has its own (quantitative requirement of cofactors to maintain basal levels and to respond to IL-1. Twelve factors (Epc1, H2afz, Kdm2b, Kdm6a, Mbd3, Mta2, Phf21a, Ruvbl1, Sin3b, Suv420h1, Taf1, and Ube3a have not been previously implicated in inflammatory cytokine functions. Bioinformatics analysis indicates that they are components of complex nuclear protein networks that regulate chromatin functions and gene transcription. Collectively, these data suggest that downstream from the essential NF-κB signal each cytokine-inducible target gene has further subtle requirements for individual sets of nuclear cofactors that shape its transcriptional activation profile.

  11. Construction of RNAi lentiviral vector targeting mouse Islet-1 gene

    Directory of Open Access Journals (Sweden)

    Shen-shen ZHI

    2011-02-01

    Full Text Available Objective To construct and select RNAi lentiviral vectors that can silence mouse Islet-1 gene effectively.Methods Three groups of RNAi-target of mouse Islet-1 gene were designed,and corresponding shRNA oligo(sh1,sh2 and sh3 were synthesized,and then they were respectively inserted to the PLVTHM vector that had been digested by endonuclease.Agarose gel electrophoresis and sequencing were used to select and indentify the positive clones.The positive clones were extracted and then mixed with E.coli to amplify positive clones.The amplified clones were then infected into 293T along with the other 3 helper plasmids to produce lentiviral vector.After the construction of the lentiviral vector,plaque formation test was performed to determine the titer of lentiviral vector.The lentiviral vectors were then infected into C3H10T1/2 cells.The transfect efficiency of the lentiviral vectors was determined with flow cytometry with detection of green fluorescent protein(GFP.Q-PCR was employed to detect the RNAi efficiency of the lentiviral vectors.Results Agarose gel electrophoresis analysis showed that the clones with right gene at the target size were successfully established;gene sequencing showed that the right DNA fragments had been inserted;plaque formation test showed that the titer of the virus solution was 3.87×108TU/ml;the transfect efficiency of the lentiviral vector infected into C3H10T1/2 cells was 90.36%.All the 3 groups of shRNA targets(sh1,sh2 and sh3 showed an inhibitory effect on Islet-1 gene,and the sh1 showed the highest inhibitory effect(76.8%,as compared with that of normal cells(P < 0.05.Conclusion The RNAi lentiviral vector that can effectively silence the mouse Islet-1 gene has been constructed successfully,which may lay a foundation for further investigation of Islet-1 gene.

  12. A targeted ultrasound contrast agent carrying gene and cell-penetrating peptide: preparation and gene transfection in vitro.

    Science.gov (United States)

    Ren, Jianli; Zhang, Ping; Tian, Ju; Zhou, Zhiyi; Liu, Xingzhao; Wang, Dong; Wang, Zhigang

    2014-09-01

    Targeted and high efficient gene delivery is a main issue in gene treatment. Taking advantage of ischemic memory target P-selectin and our previous study-synergistic effects of ultrasound-targeted microbubble destruction (UTMD) and TAT peptide on gene transfection, which were characterized by targeted aggregation and high efficient gene transfection, we set up a 'smart' gene delivery system-targeted ultrasound contrast agent (UCA) carrying gene and cell-permeable peptides (CPP). Such UCA had a strong binding force with DNA which was protected from being hydrolysed by nuclease. Moreover, synergistic effects of UTMD and TAT peptide increased gene transfection. Specifically, the UCA were reacted with an ischemic memory target P-selectin overexpressed by ischemic issues (including ischemic heart disease) and loaded with gene and CPP, which enabled targeted localization and gene delivery to ischemic cells overexpressing P-selectin. We demonstrated their targeting affinity for hypoxia human umbilical vein endothelial cell (HUVEC) and gene transfection in vitro. The results of confocal laser scanning microscopy (CLSM) showed that gene and CPP were distributed on the shell of UCA. Red fluorescence was observed on the surface of targeted UCA using immunofluorescent microscopy, which demonstrated that the antibody was successfully connected to the UCA. The targeted UCA was specifically and tightly binded to hypoxia HUVEC, while there were no or little non-targeted UCA binding around hypoxia HUVEC. 24h after transfection, gene transfection efficiency detected by FCM was higher in targeted group than non-targeted group. Overall, the targeted UCA carrying gene and CPP was prepared successfully. It had a strong target binding capacity to hypoxia HUVEC and high efficient gene transfection, which maybe provide a novel strategy for gene therapy. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Efficient production of multi-modified pigs for xenotransplantation by 'combineering', gene stacking and gene editing.

    Science.gov (United States)

    Fischer, Konrad; Kraner-Scheiber, Simone; Petersen, Björn; Rieblinger, Beate; Buermann, Anna; Flisikowska, Tatiana; Flisikowski, Krzysztof; Christan, Susanne; Edlinger, Marlene; Baars, Wiebke; Kurome, Mayuko; Zakhartchenko, Valeri; Kessler, Barbara; Plotzki, Elena; Szczerbal, Izabela; Switonski, Marek; Denner, Joachim; Wolf, Eckhard; Schwinzer, Reinhard; Niemann, Heiner; Kind, Alexander; Schnieke, Angelika

    2016-06-29

    Xenotransplantation from pigs could alleviate the shortage of human tissues and organs for transplantation. Means have been identified to overcome hyperacute rejection and acute vascular rejection mechanisms mounted by the recipient. The challenge is to combine multiple genetic modifications to enable normal animal breeding and meet the demand for transplants. We used two methods to colocate xenoprotective transgenes at one locus, sequential targeted transgene placement - 'gene stacking', and cointegration of multiple engineered large vectors - 'combineering', to generate pigs carrying modifications considered necessary to inhibit short to mid-term xenograft rejection. Pigs were generated by serial nuclear transfer and analysed at intermediate stages. Human complement inhibitors CD46, CD55 and CD59 were abundantly expressed in all tissues examined, human HO1 and human A20 were widely expressed. ZFN or CRISPR/Cas9 mediated homozygous GGTA1 and CMAH knockout abolished α-Gal and Neu5Gc epitopes. Cells from multi-transgenic piglets showed complete protection against human complement-mediated lysis, even before GGTA1 knockout. Blockade of endothelial activation reduced TNFα-induced E-selectin expression, IFNγ-induced MHC class-II upregulation and TNFα/cycloheximide caspase induction. Microbial analysis found no PERV-C, PCMV or 13 other infectious agents. These animals are a major advance towards clinical porcine xenotransplantation and demonstrate that livestock engineering has come of age.

  14. Identification of apoptosis-related PLZF target genes

    International Nuclear Information System (INIS)

    Bernardo, Maria Victoria; Yelo, Estefania; Gimeno, Lourdes; Campillo, Jose Antonio; Parrado, Antonio

    2007-01-01

    The PLZF gene encodes a BTB/POZ-zinc finger-type transcription factor, involved in physiological development, proliferation, differentiation, and apoptosis. In this paper, we investigate proliferation, survival, and gene expression regulation in stable clones from the human haematopoietic K562, DG75, and Jurkat cell lines with inducible expression of PLZF. In Jurkat cells, but not in K562 and DG75 cells, PLZF induced growth suppression and apoptosis in a cell density-dependent manner. Deletion of the BTB/POZ domain of PLZF abrogated growth suppression and apoptosis. PLZF was expressed with a nuclear speckled pattern distinctively in the full-length PLZF-expressing Jurkat clones, suggesting that the nuclear speckled localization is required for PLZF-induced apoptosis. By microarray analysis, we identified that the apoptosis-inducer TP53INP1, ID1, and ID3 genes were upregulated, and the apoptosis-inhibitor TERT gene was downregulated. The identification of apoptosis-related PLZF target genes may have biological and clinical relevance in cancer typified by altered PLZF expression

  15. COMBINING ABILITY AND GENE ACTION ESTIMATES FOR SOME ...

    African Journals Online (AJOL)

    A. Hannachi

    1 sept. 2017 ... Département d'Agronomie, Fac. Sci. Nat. 4. Département d'Ecologie et Biologie Végétale,. Received: 22 January 2017 / Accepted: 19. ABSTRACT. Combining ability and gene action. 6 half diallel crosses. Results revealed th ability mean squares were significant for a additive and nonadditive components ...

  16. Haplotype combination of the bovine PCSK1 gene sequence ...

    Indian Academy of Sciences (India)

    Haplotype combination of the bovine PCSK1 gene sequence variants and association with growth traits in Jiaxian cattle. JIAJIE SUN1,2, LIMIN SHAN2, CHUNLEI ZHANG2 and HONG CHEN1∗. 1College of Animal Science and Technology, Northwest A&F University, Shaanxi Key Laboratory of Molecular Biology.

  17. Domain combination of the vertebrate-like TLR gene family ...

    Indian Academy of Sciences (India)

    Domain combination of the vertebrate-like TLR gene family: implications for their origin and evolution. Baojun Wu, Tianxiao Huan, Jing Gong, Pin Zhou and Zengliang Bai. J. Genet. 90, 401–408. Figure 1. Unrooted NJ phylogenetic tree of P-Toll proteins from selected phyla based on TIR domains. The putative V-TIR protein.

  18. Anti-EGFR immunonanoparticles containing IL12 and salmosin genes for targeted cancer gene therapy.

    Science.gov (United States)

    Kim, Jung Seok; Kang, Seong Jae; Jeong, Hwa Yeon; Kim, Min Woo; Park, Sang Il; Lee, Yeon Kyung; Kim, Hong Sung; Kim, Keun Sik; Park, Yong Serk

    2016-09-01

    Tumor-directed gene delivery is of major interest in the field of cancer gene therapy. Varied functionalizations of non-viral vectors have been suggested to enhance tumor targetability. In the present study, we prepared two different types of anti-EGF receptor (EGFR) immunonanoparticles containing pDNA, neutrally charged liposomes and cationic lipoplexes, for tumor-directed transfection of cancer therapeutic genes. Even though both anti-EGFR immunonanoparticles had a high binding affinity to the EGFR-positive cancer cells, the anti-EGFR immunolipoplex formulation exhibited approximately 100-fold higher transfection to the target cells than anti-EGFR immunoliposomes. The lipoplex formulation also showed a higher transfection to SK-OV-3 tumor xenografts in mice. Thus, IL12 and/or salmosin genes were loaded in the anti-EGFR immunolipoplexes and intravenously administered to mice carrying SK-OV-3 tumors. Co-transfection of IL12 and salmosin genes using anti-EGFR immunolipoplexes significantly reduced tumor growth and pulmonary metastasis. Furthermore, combinatorial treatment with doxorubicin synergistically inhibited tumor growth. These results suggest that anti-EGFR immunolipoplexes containing pDNA encoding therapeutic genes could be utilized as a gene-transfer modality for cancer gene therapy.

  19. Target Marketing and Direct Mail: A Smart Campaign Combination.

    Science.gov (United States)

    Brostoff, Mark J.

    1994-01-01

    Market segmentation is a marketing strategy that helps identify and classify a camp's product or service and determine the needs of a targeted market for the purpose of allocating marketing resources. Offers strategies for defining a target market and discusses the benefits of direct mail, deriving a mailing list, and suggestions for using a…

  20. Combining target enrichment with barcode multiplexing for high throughput SNP discovery

    Directory of Open Access Journals (Sweden)

    Lunke Sebastian

    2010-11-01

    Full Text Available Abstract Background The primary goal of genetic linkage analysis is to identify genes affecting a phenotypic trait. After localisation of the linkage region, efficient genetic dissection of the disease linked loci requires that functional variants are identified across the loci. These functional variations are difficult to detect due to extent of genetic diversity and, to date, incomplete cataloguing of the large number of variants present both within and between populations. Massively parallel sequencing platforms offer unprecedented capacity for variant discovery, however the number of samples analysed are still limited by cost per sample. Some progress has been made in reducing the cost of resequencing using either multiplexing methodologies or through the utilisation of targeted enrichment technologies which provide the ability to resequence genomic areas of interest rather that full genome sequencing. Results We developed a method that combines current multiplexing methodologies with a solution-based target enrichment method to further reduce the cost of resequencing where region-specific sequencing is required. Our multiplex/enrichment strategy produced high quality data with nominal reduction of sequencing depth. We undertook a genotyping study and were successful in the discovery of novel SNP alleles in all samples at uniplex, duplex and pentaplex levels. Conclusion Our work describes the successful combination of a targeted enrichment method and index barcode multiplexing to reduce costs, time and labour associated with processing large sample sets. Furthermore, we have shown that the sequencing depth obtained is adequate for credible SNP genotyping analysis at uniplex, duplex and pentaplex levels.

  1. Evaluating Transcription Factor Activity Changes by Scoring Unexplained Target Genes in Expression Data.

    Directory of Open Access Journals (Sweden)

    Evi Berchtold

    Full Text Available Several methods predict activity changes of transcription factors (TFs from a given regulatory network and measured expression data. But available gene regulatory networks are incomplete and contain many condition-dependent regulations that are not relevant for the specific expression measurement. It is not known which combination of active TFs is needed to cause a change in the expression of a target gene. A method to systematically evaluate the inferred activity changes is missing. We present such an evaluation strategy that indicates for how many target genes the observed expression changes can be explained by a given set of active TFs. To overcome the problem that the exact combination of active TFs needed to activate a gene is typically not known, we assume a gene to be explained if there exists any combination for which the predicted active TFs can possibly explain the observed change of the gene. We introduce the i-score (inconsistency score, which quantifies how many genes could not be explained by the set of activity changes of TFs. We observe that, even for these minimal requirements, published methods yield many unexplained target genes, i.e. large i-scores. This holds for all methods and all expression datasets we evaluated. We provide new optimization methods to calculate the best possible (minimal i-score given the network and measured expression data. The evaluation of this optimized i-score on a large data compendium yields many unexplained target genes for almost every case. This indicates that currently available regulatory networks are still far from being complete. Both the presented Act-SAT and Act-A* methods produce optimal sets of TF activity changes, which can be used to investigate the difficult interplay of expression and network data. A web server and a command line tool to calculate our i-score and to find the active TFs associated with the minimal i-score is available from https://services.bio.ifi.lmu.de/i-score.

  2. Gene therapy to target ER stress in brain diseases.

    Science.gov (United States)

    Valenzuela, Vicente; Martínez, Gabriela; Duran-Aniotz, Claudia; Hetz, Claudio

    2016-10-01

    Gene therapy based on the use of Adeno-associated viruses (AAVs) is emerging as a safe and stable strategy to target molecular pathways involved in a variety of brain diseases. Endoplasmic reticulum (ER) stress is proposed as a transversal feature of most animal models and clinical samples from patients affected with neurodegenerative diseases. Manipulation of the unfolded protein response (UPR), a major homeostatic reaction under ER stress conditions, had proved beneficial in diverse models of neurodegeneration. Although increasing number of drugs are available to target ER stress, the use of small molecules to treat chronic brain diseases is challenging because of poor blood brain barrier permeability and undesirable side effects due to the role of the UPR in the physiology of peripheral organs. Gene therapy is currently considered a possible future alternative to circumvent these problems by the delivery of therapeutic agents to selective regions and cell types of the nervous system. Here we discuss current efforts to design gene therapy strategies to alleviate ER stress on a disease context. This article is part of a Special Issue entitled SI:ER stress. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Targeting EGFR with photodynamic therapy in combination with Erbitux enhances in vivo bladder tumor response

    Directory of Open Access Journals (Sweden)

    Soo Khee

    2009-11-01

    Full Text Available Abstract Background Photodynamic therapy (PDT is a promising cancer treatment modality that involves the interaction of the photosensitizer, molecular oxygen and light of specific wavelength to destroy tumor cells. Treatment induced hypoxia is one of the main side effects of PDT and efforts are underway to optimize PDT protocols for improved efficacy. The aim of this study was to investigate the anti-tumor effects of PDT plus Erbitux, an angiogenesis inhibitor that targets epidermal growth factor receptor (EGFR, on human bladder cancer model. Tumor-bearing nude mice were assigned to four groups that included control, PDT, Erbitux and PDT plus Erbitux and tumor volume was charted over 90-day period. Results Our results demonstrate that combination of Erbitux with PDT strongly inhibits tumor growth in the bladder tumor xenograft model when compared to the other groups. Downregulation of EGFR was detected using immunohistochemistry, immunofluorescence and western blotting. Increased apoptosis was associated with tumor inhibition in the combination therapy group. In addition, we identified the dephosphorylation of ErbB4 at tyrosine 1284 site to play a major role in tumor inhibition. Also, at the RNA level downregulation of EGFR target genes cyclin D1 and c-myc was observed in tumors treated with PDT plus Erbitux. Conclusion The combination therapy of PDT and Erbitux effectively inhibits tumor growth and is a promising therapeutic approach in the treatment of bladder tumors.

  4. Combining Gene Signatures Improves Prediction of Breast Cancer Survival

    Science.gov (United States)

    Zhao, Xi; Naume, Bjørn; Langerød, Anita; Frigessi, Arnoldo; Kristensen, Vessela N.; Børresen-Dale, Anne-Lise; Lingjærde, Ole Christian

    2011-01-01

    Background Several gene sets for prediction of breast cancer survival have been derived from whole-genome mRNA expression profiles. Here, we develop a statistical framework to explore whether combination of the information from such sets may improve prediction of recurrence and breast cancer specific death in early-stage breast cancers. Microarray data from two clinically similar cohorts of breast cancer patients are used as training (n = 123) and test set (n = 81), respectively. Gene sets from eleven previously published gene signatures are included in the study. Principal Findings To investigate the relationship between breast cancer survival and gene expression on a particular gene set, a Cox proportional hazards model is applied using partial likelihood regression with an L2 penalty to avoid overfitting and using cross-validation to determine the penalty weight. The fitted models are applied to an independent test set to obtain a predicted risk for each individual and each gene set. Hierarchical clustering of the test individuals on the basis of the vector of predicted risks results in two clusters with distinct clinical characteristics in terms of the distribution of molecular subtypes, ER, PR status, TP53 mutation status and histological grade category, and associated with significantly different survival probabilities (recurrence: p = 0.005; breast cancer death: p = 0.014). Finally, principal components analysis of the gene signatures is used to derive combined predictors used to fit a new Cox model. This model classifies test individuals into two risk groups with distinct survival characteristics (recurrence: p = 0.003; breast cancer death: p = 0.001). The latter classifier outperforms all the individual gene signatures, as well as Cox models based on traditional clinical parameters and the Adjuvant! Online for survival prediction. Conclusion Combining the predictive strength of multiple gene signatures improves prediction of breast

  5. Combining gene signatures improves prediction of breast cancer survival.

    Directory of Open Access Journals (Sweden)

    Xi Zhao

    Full Text Available BACKGROUND: Several gene sets for prediction of breast cancer survival have been derived from whole-genome mRNA expression profiles. Here, we develop a statistical framework to explore whether combination of the information from such sets may improve prediction of recurrence and breast cancer specific death in early-stage breast cancers. Microarray data from two clinically similar cohorts of breast cancer patients are used as training (n = 123 and test set (n = 81, respectively. Gene sets from eleven previously published gene signatures are included in the study. PRINCIPAL FINDINGS: To investigate the relationship between breast cancer survival and gene expression on a particular gene set, a Cox proportional hazards model is applied using partial likelihood regression with an L2 penalty to avoid overfitting and using cross-validation to determine the penalty weight. The fitted models are applied to an independent test set to obtain a predicted risk for each individual and each gene set. Hierarchical clustering of the test individuals on the basis of the vector of predicted risks results in two clusters with distinct clinical characteristics in terms of the distribution of molecular subtypes, ER, PR status, TP53 mutation status and histological grade category, and associated with significantly different survival probabilities (recurrence: p = 0.005; breast cancer death: p = 0.014. Finally, principal components analysis of the gene signatures is used to derive combined predictors used to fit a new Cox model. This model classifies test individuals into two risk groups with distinct survival characteristics (recurrence: p = 0.003; breast cancer death: p = 0.001. The latter classifier outperforms all the individual gene signatures, as well as Cox models based on traditional clinical parameters and the Adjuvant! Online for survival prediction. CONCLUSION: Combining the predictive strength of multiple gene signatures improves

  6. Modeling and Targeting MYC Genes in Childhood Brain Tumors

    Science.gov (United States)

    Hutter, Sonja; Bolin, Sara; Weishaupt, Holger; Swartling, Fredrik J.

    2017-01-01

    Brain tumors are the second most common group of childhood cancers, accounting for about 20%–25% of all pediatric tumors. Deregulated expression of the MYC family of transcription factors, particularly c-MYC and MYCN genes, has been found in many of these neoplasms, and their expression levels are often correlated with poor prognosis. Elevated c-MYC/MYCN initiates and drives tumorigenesis in many in vivo model systems of pediatric brain tumors. Therefore, inhibition of their oncogenic function is an attractive therapeutic target. In this review, we explore the roles of MYC oncoproteins and their molecular targets during the formation, maintenance, and recurrence of childhood brain tumors. We also briefly summarize recent progress in the development of therapeutic approaches for pharmacological inhibition of MYC activity in these tumors. PMID:28333115

  7. Global Identification of EVI1 Target Genes in Acute Myeloid Leukemia.

    Directory of Open Access Journals (Sweden)

    Carolyn Glass

    Full Text Available The ecotropic virus integration site 1 (EVI1 transcription factor is associated with human myeloid malignancy of poor prognosis and is overexpressed in 8-10% of adult AML and strikingly up to 27% of pediatric MLL-rearranged leukemias. For the first time, we report comprehensive genomewide EVI1 binding and whole transcriptome gene deregulation in leukemic cells using a combination of ChIP-Seq and RNA-Seq expression profiling. We found disruption of terminal myeloid differentiation and cell cycle regulation to be prominent in EVI-induced leukemogenesis. Specifically, we identified EVI1 directly binds to and downregulates the master myeloid differentiation gene Cebpe and several of its downstream gene targets critical for terminal myeloid differentiation. We also found EVI1 binds to and downregulates Serpinb2 as well as numerous genes involved in the Jak-Stat signaling pathway. Finally, we identified decreased expression of several ATP-dependent P2X purinoreceptors genes involved in apoptosis mechanisms. These findings provide a foundation for future study of potential therapeutic gene targets for EVI1-induced leukemia.

  8. Advances in ultrasound-targeted microbubble-mediated gene therapy for liver fibrosis

    Directory of Open Access Journals (Sweden)

    Cuiyuan Huang

    2017-07-01

    Full Text Available Hepatic fibrosis develops as a wound-healing scar in response to acute and chronic liver inflammation and can lead to cirrhosis in patients with chronic hepatitis B and C. The condition arises due to increased synthesis and reduced degradation of extracellular matrix (ECM and is a common pathological sequela of chronic liver disease. Excessive deposition of ECM in the liver causes liver dysfunction, ascites, and eventually upper gastrointestinal bleeding as well as a series of complications. However, fibrosis can be reversed before developing into cirrhosis and has thus been the subject of extensive researches particularly at the gene level. Currently, therapeutic genes are imported into the damaged liver to delay or prevent the development of liver fibrosis by regulating the expression of exogenous genes. One technique of gene delivery uses ultrasound targeting of microbubbles combined with therapeutic genes where the time and intensity of the ultrasound can control the release process. Ultrasound irradiation of microbubbles in the vicinity of cells changes the permeability of the cell membrane by its cavitation effect and enhances gene transfection. In this paper, recent progress in the field is reviewed with emphasis on the following aspects: the types of ultrasound microbubbles, the construction of an ultrasound-mediated gene delivery system, the mechanism of ultrasound microbubble–mediated gene transfer and the application of ultrasound microbubbles in the treatment of liver fibrosis.

  9. Improvement of Hydrodynamics-Based Gene Transfer of Nonviral DNA Targeted to Murine Hepatocytes

    Directory of Open Access Journals (Sweden)

    Shingo Nakamura

    2013-01-01

    Full Text Available The liver is an important organ for supporting the life of an individual. Gene transfer toward this organ has been attempted in many laboratories to date; however, there have been few reports on improved liver-targeted gene delivery by using a nonviral vector. In this study, we examined the effect of various types of gene delivery carriers on enhancing the uptake and gene expression of exogenous DNA in murine hepatocytes when a hydrodynamics-based gene delivery (HGD is performed via tail-vein injection. Mice were singly injected with a large amount of phosphate-buffered saline containing reporter plasmid DNA and/or with a gene delivery carrier. One day after the gene delivery, the animals' livers were dissected and subjected to biochemical, histochemical, and molecular biological analyses. The strongest signal from the reporter plasmid DNA was observed when the DNA was mixed with a polyethylenimine- (PEI- based reagent. Coinjection with pCRTEIL (a loxP-floxed reporter construct and pTR/NCre (a liver-specific Cre expression vector resulted in the liver-specific recombination of pCRTEIL. The combination of PEI with HGD would thus be a valuable tool for liver-specific manipulation to examine the function of a gene of interest in the liver and for creating liver disease models.

  10. Efficient in planta gene targeting in tomato using geminiviral replicons and the CRISPR/Cas9 system.

    Science.gov (United States)

    Dahan-Meir, Tal; Filler-Hayut, Shdema; Melamed-Bessudo, Cathy; Bocobza, Samuel; Czosnek, Henryk; Aharoni, Asaph; Levy, Avraham A

    2018-04-18

    Current breeding relies mostly on random mutagenesis and recombination to generate novel genetic variation. However, targeted genome editing is becoming an increasingly important tool for precise plant breeding. Using the CRISPR-Cas system combined with the bean yellow dwarf virus rolling circle replicon we optimized a method for targeted mutagenesis and gene replacement in tomato. The carotenoid isomerase (CRTISO) and phytoene synthase 1 (PSY1) genes from the carotenoid biosynthesis pathway were chosen as targets due to their easily detectable change of phenotype. We took advantage of the geminiviral replicon amplification as a mean to provide a large amount of donor template for the repair of a CRISPR-Cas-induced DNA double strand break (DSB) in the target gene, via homologous recombination. Mutagenesis experiments, performed in the Micro-Tom variety achieved precise modification of the CRTISO and PSY1 loci at an efficiency of up to 90%. In the gene targeting experiments, our target was a fast-neutron-induced crtiso allele that contained a 281bp deletion. This deletion was repaired with the wildtype sequence through homologous recombination between the CRISPR-Cas-induced DSB in the crtiso target and the amplified donor in 25% of the plants transformed. This shows that efficient gene targeting can be achieved in the absence of selection markers or reporters using a single and modular construct that is adaptable to other tomato targets and other crops. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  11. Synthetic Lethal Screen Identifies NF-κB as a Target for Combination Therapy with Topotecan for patients with Neuroblastoma

    International Nuclear Information System (INIS)

    Tsang, Patricia S; Cheuk, Adam T; Chen, Qing-Rong; Song, Young K; Badgett, Thomas C; Wei, Jun S; Khan, Javed

    2012-01-01

    Despite aggressive multimodal treatments the overall survival of patients with high-risk neuroblastoma remains poor. The aim of this study was to identify novel combination chemotherapy to improve survival rate in patients with high-risk neuroblastoma. We took a synthetic lethal approach using a siRNA library targeting 418 apoptosis-related genes and identified genes and pathways whose inhibition synergized with topotecan. Microarray analyses of cells treated with topotecan were performed to identify if the same genes or pathways were altered by the drug. An inhibitor of this pathway was used in combination with topotecan to confirm synergism by in vitro and in vivo studies. We found that there were nine genes whose suppression synergized with topotecan to enhance cell death, and the NF-κB signaling pathway was significantly enriched. Microarray analysis of cells treated with topotecan revealed a significant enrichment of NF-κB target genes among the differentially altered genes, suggesting that NF-κB pathway was activated in the treated cells. Combination of topotecan and known NF-κB inhibitors (NSC 676914 or bortezomib) significantly reduced cell growth and induced caspase 3 activity in vitro. Furthermore, in a neuroblastoma xenograft mouse model, combined treatment of topotecan and bortezomib significantly delayed tumor formation compared to single-drug treatments. Synthetic lethal screening provides a rational approach for selecting drugs for use in combination therapy and warrants clinical evaluation of the efficacy of the combination of topotecan and bortezomib or other NF-κB inhibitors in patients with high risk neuroblastoma

  12. Synthetic Lethal Screen Identifies NF-κB as a Target for Combination Therapy with Topotecan for patients with Neuroblastoma

    Directory of Open Access Journals (Sweden)

    Tsang Patricia S

    2012-03-01

    Full Text Available Abstract Background Despite aggressive multimodal treatments the overall survival of patients with high-risk neuroblastoma remains poor. The aim of this study was to identify novel combination chemotherapy to improve survival rate in patients with high-risk neuroblastoma. Methods We took a synthetic lethal approach using a siRNA library targeting 418 apoptosis-related genes and identified genes and pathways whose inhibition synergized with topotecan. Microarray analyses of cells treated with topotecan were performed to identify if the same genes or pathways were altered by the drug. An inhibitor of this pathway was used in combination with topotecan to confirm synergism by in vitro and in vivo studies. Results We found that there were nine genes whose suppression synergized with topotecan to enhance cell death, and the NF-κB signaling pathway was significantly enriched. Microarray analysis of cells treated with topotecan revealed a significant enrichment of NF-κB target genes among the differentially altered genes, suggesting that NF-κB pathway was activated in the treated cells. Combination of topotecan and known NF-κB inhibitors (NSC 676914 or bortezomib significantly reduced cell growth and induced caspase 3 activity in vitro. Furthermore, in a neuroblastoma xenograft mouse model, combined treatment of topotecan and bortezomib significantly delayed tumor formation compared to single-drug treatments. Conclusions Synthetic lethal screening provides a rational approach for selecting drugs for use in combination therapy and warrants clinical evaluation of the efficacy of the combination of topotecan and bortezomib or other NF-κB inhibitors in patients with high risk neuroblastoma.

  13. Combining many interaction networks to predict gene function and analyze gene lists.

    Science.gov (United States)

    Mostafavi, Sara; Morris, Quaid

    2012-05-01

    In this article, we review how interaction networks can be used alone or in combination in an automated fashion to provide insight into gene and protein function. We describe the concept of a "gene-recommender system" that can be applied to any large collection of interaction networks to make predictions about gene or protein function based on a query list of proteins that share a function of interest. We discuss these systems in general and focus on one specific system, GeneMANIA, that has unique features and uses different algorithms from the majority of other systems. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Combined effects of thrombosis pathway gene variants predict cardiovascular events.

    Directory of Open Access Journals (Sweden)

    Kirsi Auro

    2007-07-01

    Full Text Available The genetic background of complex diseases is proposed to consist of several low-penetrance risk loci. Addressing this complexity likely requires both large sample size and simultaneous analysis of different predisposing variants. We investigated the role of four thrombosis genes: coagulation factor V (F5, intercellular adhesion molecule 1 (ICAM1, protein C (PROC, and thrombomodulin (THBD in cardiovascular diseases. Single allelic gene variants and their pair-wise combinations were analyzed in two independently sampled population cohorts from Finland. From among 14,140 FINRISK participants (FINRISK-92, n = 5,999 and FINRISK-97, n = 8,141, we selected for genotyping a sample of 2,222, including 528 incident cardiovascular disease (CVD cases and random subcohorts totaling 786. To cover all known common haplotypes (>10%, 54 single nucleotide polymorphisms (SNPs were genotyped. Classification-tree analysis identified 11 SNPs that were further analyzed in Cox's proportional hazard model as single variants and pair-wise combinations. Multiple testing was controlled by use of two independent cohorts and with false-discovery rate. Several CVD risk variants were identified: In women, the combination of F5 rs7542281 x THBD rs1042580, together with three single F5 SNPs, was associated with CVD events. Among men, PROC rs1041296, when combined with either ICAM1 rs5030341 or F5 rs2269648, was associated with total mortality. As a single variant, PROC rs1401296, together with the F5 Leiden mutation, was associated with ischemic stroke events. Our strategy to combine the classification-tree analysis with more traditional genetic models was successful in identifying SNPs-acting either in combination or as single variants--predisposing to CVD, and produced consistent results in two independent cohorts. These results suggest that variants in these four thrombosis genes contribute to arterial cardiovascular events at population level.

  15. Expression of target and reference genes in Daphnia magna exposed to ibuprofen.

    Science.gov (United States)

    Heckmann, Lars-Henrik; Connon, Richard; Hutchinson, Thomas H; Maund, Steve J; Sibly, Richard M; Callaghan, Amanda

    2006-07-07

    Transcriptomic techniques are now being applied in ecotoxicology and toxicology to measure the impact of stressors and develop understanding of mechanisms of toxicity. Microarray technology in particular offers the potential to measure thousands of gene responses simultaneously. However, it is important that microarrays responses should be validated, at least initially, using real-time quantitative polymerase chain reaction (QPCR). The accurate measurement of target gene expression requires normalisation to an invariant internal control e.g., total RNA or reference genes. Reference genes are preferable, as they control for variation inherent in the cDNA synthesis and PCR. However, reference gene expression can vary between tissues and experimental conditions, which makes it crucial to validate them prior to application. We evaluated 10 candidate reference genes for QPCR in Daphnia magna following a 24 h exposure to the non-steroidal anti-inflammatory drug (NSAID) ibuprofen (IB) at 0, 20, 40 and 80 mg IB l-1. Six of the 10 candidates appeared suitable for use as reference genes. As a robust approach, we used a combination normalisation factor (NF), calculated using the geNorm application, based on the geometric mean of three selected reference genes: glyceraldehyde-3-phosphate dehydrogenase, ubiquitin conjugating enzyme and actin. The effects of normalisation are illustrated using as target gene leukotriene B4 12-hydroxydehydrogenase (Ltb4dh), which was up-regulated following 24 h exposure to 63-81 mg IB l-1. As anticipated, use of the NF clarified the response of Ltb4dh in daphnids exposed to sublethal levels of ibuprofen. Our findings emphasise the importance in toxicogenomics of finding and applying invariant internal QPCR control(s) relevant to the study conditions.

  16. Expression of target and reference genes in Daphnia magna exposed to ibuprofen

    Directory of Open Access Journals (Sweden)

    Sibly Richard M

    2006-07-01

    Full Text Available Abstract Background Transcriptomic techniques are now being applied in ecotoxicology and toxicology to measure the impact of stressors and develop understanding of mechanisms of toxicity. Microarray technology in particular offers the potential to measure thousands of gene responses simultaneously. However, it is important that microarrays responses should be validated, at least initially, using real-time quantitative polymerase chain reaction (QPCR. The accurate measurement of target gene expression requires normalisation to an invariant internal control e.g., total RNA or reference genes. Reference genes are preferable, as they control for variation inherent in the cDNA synthesis and PCR. However, reference gene expression can vary between tissues and experimental conditions, which makes it crucial to validate them prior to application. Results We evaluated 10 candidate reference genes for QPCR in Daphnia magna following a 24 h exposure to the non-steroidal anti-inflammatory drug (NSAID ibuprofen (IB at 0, 20, 40 and 80 mg IB l-1. Six of the 10 candidates appeared suitable for use as reference genes. As a robust approach, we used a combination normalisation factor (NF, calculated using the geNorm application, based on the geometric mean of three selected reference genes: glyceraldehyde-3-phosphate dehydrogenase, ubiquitin conjugating enzyme and actin. The effects of normalisation are illustrated using as target gene leukotriene B4 12-hydroxydehydrogenase (Ltb4dh, which was up-regulated following 24 h exposure to 63–81 mg IB l-1. Conclusions As anticipated, use of the NF clarified the response of Ltb4dh in daphnids exposed to sublethal levels of ibuprofen. Our findings emphasise the importance in toxicogenomics of finding and applying invariant internal QPCR control(s relevant to the study conditions.

  17. Gene effects and combining abilities for oil content in sunflower

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    Jocković Milan

    2014-01-01

    Full Text Available Considering the worldwide importance of sunflower oil, objective of this study was to evaluate gene effects and combining abilities of six sunflower open pollinated varieties. Varieties were crossed according to incomplete diallel method and produced fifteen F1 progenies. Comparing the mean values of F1 progenies to parents mean in most cases superdominance was expressed as a mode of inheritance. Nonetheless, dominance of better parent and partial dominance of better parent were also recorded as a mode of inheritance. GCA/SCA ratio indicated greater importance of non-additive genetic component in oil content expression. The genetic variance analysis showed that dominant component was more important and dominant genes prevailed compared to recessive genes for oil content in sunflower.

  18. AAV-Mediated Gene Targeting Is Significantly Enhanced by Transient Inhibition of Nonhomologous End Joining or the Proteasome In Vivo

    Science.gov (United States)

    Paulk, Nicole K.; Loza, Laura Marquez; Finegold, Milton J.

    2012-01-01

    Abstract Recombinant adeno-associated virus (rAAV) vectors have clear potential for use in gene targeting but low correction efficiencies remain the primary drawback. One approach to enhancing efficiency is a block of undesired repair pathways like nonhomologous end joining (NHEJ) to promote the use of homologous recombination. The natural product vanillin acts as a potent inhibitor of NHEJ by inhibiting DNA-dependent protein kinase (DNA-PK). Using a homology containing rAAV vector, we previously demonstrated in vivo gene repair frequencies of up to 0.1% in a model of liver disease hereditary tyrosinemia type I. To increase targeting frequencies, we administered vanillin in combination with rAAV. Gene targeting frequencies increased up to 10-fold over AAV alone, approaching 1%. Fah−/−Ku70−/− double knockout mice also had increased gene repair frequencies, genetically confirming the beneficial effects of blocking NHEJ. A second strategy, transient proteasomal inhibition, also increased gene-targeting frequencies but was not additive to NHEJ inhibition. This study establishes the benefit of transient NHEJ inhibition with vanillin, or proteasome blockage with bortezomib, for increasing hepatic gene targeting with rAAV. Functional metabolic correction of a clinically relevant disease model was demonstrated and provided evidence for the feasibility of gene targeting as a therapeutic strategy. PMID:22486314

  19. Systematic targeted integration to study Albumin gene control elements.

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    Sanchari Bhattacharyya

    Full Text Available To study transcriptional regulation by distant enhancers, we devised a system of easily modified reporter plasmids for integration into single-copy targeting cassettes in clones of HuH7, a human hepatocellular carcinoma. The plasmid constructs tested transcriptional function of a 35-kb region that contained the rat albumin gene and its upstream flanking region. Expression of integrants was analyzed in two orientations, and compared to transient expression of non-integrated plasmids. Enhancers were studied in their natural positions relative to the promoter and localized by deletion. All constructs were also analyzed by transient transfection assays. In addition to the known albumin gene enhancer (E1 at -10 kb, we demonstrated two new enhancers, E2 at -13, and E4 at +1.2 kb. All three enhancers functioned in both transient assays and integrated constructs. However, chromosomal integration demonstrated several differences from transient expression. For example, analysis of E2 showed that enhancer function within the chromosome required a larger gene region than in transient assays. Another conserved region, E3 at -0.7 kb, functioned as an enhancer in transient assays but inhibited the function of E1 and E2 when chromosomally integrated. The enhancers did not show additive or synergistic behavior,an effect consistent with competition for the promoter or inhibitory interactions among enhancers. Growth arrest by serum starvation strongly stimulated the function of some integrated enhancers, consistent with the expected disruption of enhancer-promoter looping during the cell cycle.

  20. AAC as a Potential Target Gene to Control Verticillium dahliae

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    Xiaofeng Su

    2017-01-01

    Full Text Available Verticillium dahliae invades the roots of host plants and causes vascular wilt, which seriously diminishes the yield of cotton and other important crops. The protein AAC (ADP, ATP carrier is responsible for transferring ATP from the mitochondria into the cytoplasm. When V. dahliae protoplasts were transformed with short interfering RNAs (siRNAs targeting the VdAAC gene, fungal growth and sporulation were significantly inhibited. To further confirm a role for VdAAC in fungal development, we generated knockout mutants (ΔVdACC. Compared with wild-type V. dahliae (Vd wt, ΔVdAAC was impaired in germination and virulence; these impairments were rescued in the complementary strains (ΔVdAAC-C. Moreover, when an RNAi construct of VdAAC under the control of the 35S promoter was used to transform Nicotiana benthamiana, the expression of VdAAC was downregulated in the transgenic seedlings, and they had elevated resistance against V. dahliae. The results of this study suggest that VdAAC contributes to fungal development, virulence and is a promising candidate gene to control V. dahliae. In addition, RNAi is a highly efficient way to silence fungal genes and provides a novel strategy to improve disease resistance in plants.

  1. Combining Oncolytic Virotherapy with p53 Tumor Suppressor Gene Therapy

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    Christian Bressy

    2017-06-01

    Full Text Available Oncolytic virus (OV therapy utilizes replication-competent viruses to kill cancer cells, leaving non-malignant cells unharmed. With the first U.S. Food and Drug Administration-approved OV, dozens of clinical trials ongoing, and an abundance of translational research in the field, OV therapy is poised to be one of the leading treatments for cancer. A number of recombinant OVs expressing a transgene for p53 (TP53 or another p53 family member (TP63 or TP73 were engineered with the goal of generating more potent OVs that function synergistically with host immunity and/or other therapies to reduce or eliminate tumor burden. Such transgenes have proven effective at improving OV therapies, and basic research has shown mechanisms of p53-mediated enhancement of OV therapy, provided optimized p53 transgenes, explored drug-OV combinational treatments, and challenged canonical roles for p53 in virus-host interactions and tumor suppression. This review summarizes studies combining p53 gene therapy with replication-competent OV therapy, reviews preclinical and clinical studies with replication-deficient gene therapy vectors expressing p53 transgene, examines how wild-type p53 and p53 modifications affect OV replication and anti-tumor effects of OV therapy, and explores future directions for rational design of OV therapy combined with p53 gene therapy.

  2. Supervised classification of combined copy number and gene expression data

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    Riccadonna S.

    2007-12-01

    Full Text Available In this paper we apply a predictive profiling method to genome copy number aberrations (CNA in combination with gene expression and clinical data to identify molecular patterns of cancer pathophysiology. Predictive models and optimal feature lists for the platforms are developed by a complete validation SVM-based machine learning system. Ranked list of genome CNA sites (assessed by comparative genomic hybridization arrays – aCGH and of differentially expressed genes (assessed by microarray profiling with Affy HG-U133A chips are computed and combined on a breast cancer dataset for the discrimination of Luminal/ ER+ (Lum/ER+ and Basal-like/ER- classes. Different encodings are developed and applied to the CNA data, and predictive variable selection is discussed. We analyze the combination of profiling information between the platforms, also considering the pathophysiological data. A specific subset of patients is identified that has a different response to classification by chromosomal gains and losses and by differentially expressed genes, corroborating the idea that genomic CNA can represent an independent source for tumor classification.

  3. DNMTi/HDACi combined epigenetic targeted treatment induces reprogramming of myeloma cells in the direction of normal plasma cells.

    Science.gov (United States)

    Bruyer, Angelique; Maes, Ken; Herviou, Laurie; Kassambara, Alboukadel; Seckinger, Anja; Cartron, Guillaume; Rème, Thierry; Robert, Nicolas; Requirand, Guilhem; Boireau, Stéphanie; Müller-Tidow, Carsten; Veyrune, Jean-Luc; Vincent, Laure; Bouhya, Salahedine; Goldschmidt, Hartmut; Vanderkerken, Karin; Hose, Dirk; Klein, Bernard; De Bruyne, Elke; Moreaux, Jerome

    2018-03-02

    Multiple myeloma (MM) is the second most common hematologic malignancy. Aberrant epigenetic modifications have been reported in MM and could be promising therapeutic targets. As response rates are overall limited but deep responses occur, it is important to identify those patients who could indeed benefit from epigenetic-targeted therapy. Since HDACi and DNMTi combination have potential therapeutic value in MM, we aimed to build a GEP-based score that could be useful to design future epigenetic-targeted combination trials. In addition, we investigated the changes in GEP upon HDACi/DNMTi treatment. We report a new gene expression-based score to predict MM cell sensitivity to the combination of DNMTi/HDACi. A high Combo score in MM patients identified a group with a worse overall survival but a higher sensitivity of their MM cells to DNMTi/HDACi therapy compared to a low Combo score. In addition, treatment with DNMTi/HDACi downregulated IRF4 and MYC expression and appeared to induce a mature BMPC plasma cell gene expression profile in myeloma cell lines. In conclusion, we developed a score for the prediction of primary MM cell sensitivity to DNMTi/HDACi and found that this combination could be beneficial in high-risk patients by targeting proliferation and inducing maturation.

  4. Targeted Editing of Myostatin Gene in Sheep by Transcription Activator-like Effector Nucleases

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    Xinxia Zhao

    2016-03-01

    Full Text Available Myostatin (MSTN is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Gene knockout of MSTN can result in increasing muscle mass in sheep. The objectives were to investigate whether myostatin gene can be edited in sheep by transcription activator-like effector nucleases (TALENs in tandem with single-stranded DNA oligonucleotides (ssODNs. We designed a pair of TALENs to target a highly conserved sequence in the coding region of the sheep MSTN gene. The activity of the TALENs was verified by using luciferase single-strand annealing reporter assay in HEK 293T cell line. Co-transfection of TALENs and ssODNs oligonucleotides induced precise gene editing of myostatin gene in sheep primary fibroblasts. MSTN gene-edited cells were successfully used as nuclear donors for generating cloned embryos. TALENs combined with ssDNA oligonucleotides provide a useful approach for precise gene modification in livestock animals.

  5. A rapid high-resolution melting method for differentiation of Leishmania species targeting lack gene.

    Science.gov (United States)

    Kuang, Ziwei; Zhang, Chunying; Pang, Huasheng; Ma, Ying

    2018-02-01

    The aim of this research is to verify that if lack gene can be used for differentiation of Leishmania under HRM assay. Two specific primers were designed targeting polymorphic sites on the lack gene sequence. DNA from promastigotes of six species of Leishmania based on reference strains were tested following a HRM protocol. We also tested ten Chinese isolates in blind to validate our method. Combined with amplicon of the two primers, the six reference strains can be easily discriminated without the effect of initial concentration of DNA templates. Ten Chinese isolates detected by our HRM method resulted in full accord with the standard identification results in previous study. HRM is a rapid and reproducible method to discriminate different Leishmania species and lack gene is a potential novel biological characteristic for easy differentiation of Leishmania isolates in China. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Rice and Bean Targets for Biofortification Combined with High Carotenoid Content Crops Regulate Transcriptional Mechanisms Increasing Iron Bioavailability

    OpenAIRE

    Dias, Desirrê; Moreira, Maria de Castro; Gomes, Mariana; Toledo, Renata Lopes; Nutti, Marilia; Sant’Ana, Helena Pinheiro; Martino, Hércia

    2015-01-01

    Iron deficiency affects thousands of people worldwide. Biofortification of staple food crops aims to support the reduction of this deficiency. This study evaluates the effect of combinations of common beans and rice, targets for biofortification, with high carotenoid content crops on the iron bioavailability, protein gene expression, and antioxidant effect. Iron bioavailability was measured by the depletion/repletion method. Seven groups were tested (n = 7): Pontal bean (PB); rice + Pontal be...

  7. Identification of epigenetically altered genes and potential gene targets in melanoma using bioinformatic methods

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    Duan HH

    2017-12-01

    Full Text Available Honghao Duan, Ke Jiang, Dengke Wei, Lijun Zhang, Deliang Cheng, Min Lv, Yuben Xu, Aimin He Department of Hand Surgery, Honghui Hospital, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi, People’s Republic of China Abstract: This study aimed to analyze epigenetically and genetically altered genes in melanoma to get a better understanding of the molecular circuitry of melanoma and identify potential gene targets for the treatment of melanoma. The microarray data of GSE31879, including mRNA expression profiles (seven melanoma and four melanocyte samples and DNA methylation profiles (seven melanoma and five melanocyte samples, were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs and differentially methylated positions (DMPs were screened using the linear models for microarray data (limma package in melanoma compared with melanocyte samples. Gene ontology (GO and pathway enrichment analysis of the DEGs were carried out using the Database for Annotation, Visualization, and Integrated Discovery. Moreover, differentially methylated genes (DMGs were identified, and a transcriptional regulatory network was constructed using the University of California Santa Cruz genome browser database. A total of 1,215 DEGs (199 upregulated and 1,016 downregulated and 14,094 DMPs (10,450 upregulated and 3,644 downregulated were identified in melanoma compared with melanocyte samples. Additionally, the upregulated and downregulated DEGs were significantly associated with different GO terms and pathways, such as pigment cell differentiation, biosynthesis, and metabolism. Furthermore, the transcriptional regulatory network showed that DMGs such as Aristaless-related homeobox (ARX, damage-specific DNA binding protein 2 (DDB2, and myelin basic protein (MBP had higher node degrees. Our results showed that several methylated genes (ARX, DDB2, and MBP may be involved in melanoma progression. Keywords: melanoma, DNA

  8. Preparation and characterization of magnetic gene vectors for targeting gene delivery

    Science.gov (United States)

    Zheng, S. W.; Liu, G.; Hong, R. Y.; Li, H. Z.; Li, Y. G.; Wei, D. G.

    2012-10-01

    The PEI-CMD-MNPs were successfully prepared by the surface modification of magnetic Fe3O4 nanoparticles with carboxymethyl dextran (CMD) and polyethyleneimine (PEI). The PEI-CMD-MNPs polyplexes exhibited a typical superparamagnetic behavior and were well stable over the entire range of pH and NaCl concentration. These PEI-CMD-MNPs were used as magnetic gene vectors for targeting gene delivery. The prepared MNPs at different surface modification stages were characterized using Fourier transform infrared (FT-IR), thermogravimetric analysis (TGA), field emissions canning electron microscopy (FE-SEM), powder X-ray diffraction (XRD) and dynamic laser light scattering (DLS) analysis. The magnetic properties were studied by vibrating sample magnetometer (VSM). To evaluate the performance of the magnetic nanoparticles as gene transfer vector, the PEI-CMD-MNPs were used to delivery green fluorescent protein (GFP) gene into BHK21 cells. The expression of GFP gene was detected by fluorescence microscope. DNA-PEI-CMD-MNPs polyplexes absorbed by the cells were also monitored by Magnetic resonance imaging (MRI). The transfection efficiency and gene expression efficiency of that transfected with a magnet were much higher than that of standard transfection.

  9. Hypoxia targeted bifunctional suicide gene expression enhances radiotherapy in vitro and in vivo

    International Nuclear Information System (INIS)

    Sun, Xiaorong; Xing, Ligang; Deng, Xuelong; Hsiao, Hung Tsung; Manami, Akiko; Koutcher, Jason A.; Clifton Ling, C.; Li, Gloria C.

    2012-01-01

    Purpose: To investigate whether hypoxia targeted bifunctional suicide gene expression-cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) with 5-FC treatments can enhance radiotherapy. Materials and methods: Stable transfectants of R3327-AT cells were established which express a triple-fusion-gene: CD, UPRT and monomoric DsRed (mDsRed) controlled by a hypoxia inducible promoter. Hypoxia-induced expression/function of CDUPRTmDsRed was verified by western blot, flow cytometry, fluorescent microscopy, and cytotoxicity assay of 5-FU and 5-FC. Tumor-bearing mice were treated with 5-FC and local radiation. Tumor volume was monitored and compared with those treated with 5-FC or radiation alone. In addition, the CDUPRTmDsRed distribution in hypoxic regions of tumor sections was visualized with fluorescent microscopy. Results: Hypoxic induction of CDUPRTmDsRed protein correlated with increased sensitivity to 5-FC and 5-FU. Significant radiosensitization effects were detected after 5-FC treatments under hypoxic conditions. In the tumor xenografts, the distribution of CDUPRTmDsRed expression visualized with fluorescence microscopy was co-localized with the hypoxia marker pimonidazole positive staining cells. Furthermore, administration of 5-FC to mice in combination with local irradiation resulted in significant tumor regression, as in comparison with 5-FC or radiation treatments alone. Conclusions: Our data suggest that the hypoxia-inducible CDUPRT/5-FC gene therapy strategy has the ability to specifically target hypoxic cancer cells and significantly improve the tumor control in combination with radiotherapy.

  10. Therapeutic effect of photodynamic therapy combined with targeted delivery of silencing vascular endothelial growth factor (Conference Presentation)

    Science.gov (United States)

    Hsu, Yih-Chih

    2016-03-01

    Photodynamic therapy is a novel therapeutic modality to treat cancer by using a photosensitizer which is activated by a light source to produce reactive oxygen species and mediates tumours oxygen-independent hypoxic conditions. Vascular endothelial growth factor (VEGF) is one of the primary factors that affect tumor angiogenesis. Another emerging treatment to cure cancer is the use of interference RNA to silence a specific mRNA sequence. Such treatment requires a delivery system such as liposomes. The nanoparticle size measured was about 30 nm. Cellular uptake study was performed to verify that the nanoparticles have a sigma receptor mediated pathway. Non-targeted LCP NPs did not show significant difference with or without haloperidol but has a lower intensity as than targeted LCP NPs. These results confirm that LCP NPs have a receptor mediated pathway. Cell viability was found to decrease at 25 nM of transfected VEGF siRNA. Combined therapy of PDT and VEGF siRNA showed significant response as compared with PDT and gene therapy alone. In vivo toxicity assay with mice treated with targeted LCP NPs containing control siRNA or VEGF siRNA and non-targeted LCP NPs containing VEGF siRNA did not show any significant difference with the PBS injected group which suggests that there is no toxicity with the dose. It suggests that PDT combined with targeted gene therapy has a potential mean to achieve better therapeutic outcome.

  11. Targeting Gene-Viro-Therapy with AFP driving Apoptin gene shows potent antitumor effect in hepatocarcinoma

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    Zhang Kang-Jian

    2012-02-01

    Full Text Available Abstract Background Gene therapy and viral therapy are used for cancer therapy for many years, but the results are less than satisfactory. Our aim was to construct a new recombinant adenovirus which is more efficient to kill hepatocarcinoma cells but more safe to normal cells. Methods By using the Cancer Targeting Gene-Viro-Therapy strategy, Apoptin, a promising cancer therapeutic gene was inserted into the double-regulated oncolytic adenovirus AD55 in which E1A gene was driven by alpha fetoprotein promoter along with a 55 kDa deletion in E1B gene to form AD55-Apoptin. The anti-tumor effects and safety were examined by western blotting, virus yield assay, real time polymerase chain reaction, 3-(4,5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide assay, Hoechst33342 staining, Fluorescence-activated cell sorting, xenograft tumor model, Immunohistochemical assay, liver function analysis and Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling assay. Results The recombinant virus AD55-Apoptin has more significant antitumor effect for hepatocelluar carcinoma cell lines (in vitro than that of AD55 and even ONYX-015 but no or little impair on normal cell lines. Furthermore, it also shows an obvious in vivo antitumor effect on the Huh-7 liver carcinoma xenograft in nude mice with bigger beginning tumor volume till about 425 mm3 but has no any damage on the function of liver. The induction of apoptosis is involved in AD55-Apoptin induced antitumor effects. Conclusion The AD55-Apoptin can be a potential anti-hepatoma agent with remarkable antitumor efficacy as well as higher safety in cancer targeting gene-viro-therapy system.

  12. Preparation and characterization of magnetic gene vectors for targeting gene delivery

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, S.W.; Liu, G. [College of Chemistry, Chemical Engineering and Materials Science and Key Laboratory of Organic Synthesis of Jiangsu Province, Soochow University, SIP, Suzhou 215123 (China); Hong, R.Y., E-mail: rhong@suda.edu.cn [College of Chemistry, Chemical Engineering and Materials Science and Key Laboratory of Organic Synthesis of Jiangsu Province, Soochow University, SIP, Suzhou 215123 (China); State Key Laboratory of Multi-phase Complex Systems, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100080 (China); Li, H.Z. [State Key Laboratory of Multi-phase Complex Systems, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100080 (China); Li, Y.G., E-mail: ilguoliang@sohu.com [Department of radiology, the First Affiliated Hospital of Soochow University, Suzhou 215007 (China); Wei, D.G., E-mail: dougwei@deas.harvard.edu [Center for Nanoscale Systems, School of Engineering and Applied Science, Harvard University, 11 Oxford Street, Cambridge, MA 02139 (United States)

    2012-10-15

    Highlights: Black-Right-Pointing-Pointer PEI is ideal candidate polymer for the design of gene delivery systems. Black-Right-Pointing-Pointer PEI-CMD-MNPs exhibited a typical superparamagnetic behavior. Black-Right-Pointing-Pointer PEI-CMD-MNPs were well stable over the entire range of pH and NaCl concentration. Black-Right-Pointing-Pointer DNA-PEI-CMD-MNPs transfected cells by a magnet have higher transfection efficiency and gene expression efficiency. - Abstract: The PEI-CMD-MNPs were successfully prepared by the surface modification of magnetic Fe{sub 3}O{sub 4} nanoparticles with carboxymethyl dextran (CMD) and polyethyleneimine (PEI). The PEI-CMD-MNPs polyplexes exhibited a typical superparamagnetic behavior and were well stable over the entire range of pH and NaCl concentration. These PEI-CMD-MNPs were used as magnetic gene vectors for targeting gene delivery. The prepared MNPs at different surface modification stages were characterized using Fourier transform infrared (FT-IR), thermogravimetric analysis (TGA), field emissions canning electron microscopy (FE-SEM), powder X-ray diffraction (XRD) and dynamic laser light scattering (DLS) analysis. The magnetic properties were studied by vibrating sample magnetometer (VSM). To evaluate the performance of the magnetic nanoparticles as gene transfer vector, the PEI-CMD-MNPs were used to delivery green fluorescent protein (GFP) gene into BHK21 cells. The expression of GFP gene was detected by fluorescence microscope. DNA-PEI-CMD-MNPs polyplexes absorbed by the cells were also monitored by Magnetic resonance imaging (MRI). The transfection efficiency and gene expression efficiency of that transfected with a magnet were much higher than that of standard transfection.

  13. Hypoxia-targeted suicidal gene therapy system enhances antitumor effects of radiotherapy

    International Nuclear Information System (INIS)

    Liu Junye; Guo Yao; Guo Guozhen

    2006-01-01

    Objective: To explore the effects of hypoxia-targeted suicidal gene therapy system combined with radiotherapy on pancreatic cancer. Methods: The recombinant adenovirus Ad-5HRE/hCMVmp-BCD was constructed by DNA recombinant technique. Western blot was used to detect hypoxia-induced expression of bacterial cytosine deaminase (BCD). Cell growth inhibition assay was used to determine the sensitivity of human pancreatic cancer cells MIA-PACA2 to 5-fluorocytosine (5-FC). Tumor xenograft growth delay assays was used to evaluate the effects of Ad-5HRE/hCMVmp-BCD/5-FC combined with radiotherapy on pancreatic cancer. Results: Western blot analysis demonstrated that hypoxia-induced BCD protein expression was achieved in MIA-PACA2 cells infected with Ad-5HRE/hCMVmp-BCD. With hypoxia treatment, the sensitivity of MIA-PACA2 cells infected with Ad-5HRE/hCMVmp-BCD to 5-FC significantly increased. Administration of either Ad-5HRE/hCMVmp-BCD/5-FC or radiotherapy could inhibit the growth of MIA-PACA2 xenografts in nude mice. Moreover, combination of Ad-5HRE/hCMVmp-BCD/5-FC could significantly enhance suppressing effects of radiotherapy on MIA-PACA2 xenografts. Conclusion: Hypoxia-targeted suicidal gene therapy system Ad-5HRE/hCMVmp-BCD/5-FC could enhance antitumor effects of radiotherapy on pancreatic cancer and can be used as a powerful adjunct to conventional radiotherapy. (authors)

  14. Id-1 gene and gene products as therapeutic targets for treatment of breast cancer and other types of carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Desprez, Pierre-Yves; Campisi, Judith

    2014-08-19

    A method for treatment of breast cancer and other types of cancer. The method comprises targeting and modulating Id-1 gene expression, if any, for the Id-1 gene, or gene products in breast or other epithelial cancers in a patient by delivering products that modulate Id-1 gene expression. When expressed, Id-1 gene is a prognostic indicator that cancer cells are invasive and metastatic.

  15. Targeted Exome Sequencing of Deafness Genes After Failure of Auditory Phenotype-Driven Candidate Gene Screening.

    Science.gov (United States)

    Kim, Bong Jik; Kim, Ah Reum; Park, Gibeom; Park, Woong Yang; Chang, Sun O; Oh, Seung-Ha; Choi, Byung Yoon

    2015-07-01

    To demonstrate the efficacy and advantages of targeted exome sequencing (TES) of known deafness genes in cases with failed or misleading auditory phenotype-driven candidate gene screening. Prospective cohort survey. Otolaryngology department of a tertiary referral hospital. Six hearing-impaired probands with seemingly non-syndromic features from six deaf families were enrolled in this study after failure of genetic diagnosis using auditory phenotype-driven candidate gene screening. TES of known deafness genes was performed in the six probands, and a final causative variant was pursued using subsequent filtering steps. Potential causative variants determined using TES were confirmed by previously introduced filtering steps. We detected causative variants in three (50%) of six families, and these variants were in the COCH, PAX3, and GJB2 genes. Additionally, we also recapitulated the recent finding from other report arguing for the non-pathogenic potential of MYO1A variant. TES of a deafness panel provides a comprehensive genetic screening tool that can be implemented without being misled by the audiogram configuration information and can complement incomplete clinical physical examinations. In addition, the secondary incidental finding obtained by TES contributes useful information regarding the deafness field.

  16. A model of gene-gene and gene-environment interactions and its implications for targeting environmental interventions by genotype.

    Science.gov (United States)

    Wallace, Helen M

    2006-10-09

    The potential public health benefits of targeting environmental interventions by genotype depend on the environmental and genetic contributions to the variance of common diseases, and the magnitude of any gene-environment interaction. In the absence of prior knowledge of all risk factors, twin, family and environmental data may help to define the potential limits of these benefits in a given population. However, a general methodology to analyze twin data is required because of the potential importance of gene-gene interactions (epistasis), gene-environment interactions, and conditions that break the 'equal environments' assumption for monozygotic and dizygotic twins. A new model for gene-gene and gene-environment interactions is developed that abandons the assumptions of the classical twin study, including Fisher's (1918) assumption that genes act as risk factors for common traits in a manner necessarily dominated by an additive polygenic term. Provided there are no confounders, the model can be used to implement a top-down approach to quantifying the potential utility of genetic prediction and prevention, using twin, family and environmental data. The results describe a solution space for each disease or trait, which may or may not include the classical twin study result. Each point in the solution space corresponds to a different model of genotypic risk and gene-environment interaction. The results show that the potential for reducing the incidence of common diseases using environmental interventions targeted by genotype may be limited, except in special cases. The model also confirms that the importance of an individual's genotype in determining their risk of complex diseases tends to be exaggerated by the classical twin studies method, owing to the 'equal environments' assumption and the assumption of no gene-environment interaction. In addition, if phenotypes are genetically robust, because of epistasis, a largely environmental explanation for shared sibling

  17. Identification and validation of reference genes for quantification of target gene expression with quantitative real-time PCR for tall fescue under four abiotic stresses.

    Directory of Open Access Journals (Sweden)

    Zhimin Yang

    Full Text Available Tall fescue (Festuca arundinacea Schreb. is widely utilized as a major forage and turfgrass species in the temperate regions of the world and is a valuable plant material for studying molecular mechanisms of grass stress tolerance due to its superior drought and heat tolerance among cool-season species. Selection of suitable reference genes for quantification of target gene expression is important for the discovery of molecular mechanisms underlying improved growth traits and stress tolerance. The stability of nine potential reference genes (ACT, TUB, EF1a, GAPDH, SAND, CACS, F-box, PEPKR1 and TIP41 was evaluated using four programs, GeNorm, NormFinder, BestKeeper, and RefFinder. The combinations of SAND and TUB or TIP41 and TUB were most stably expressed in salt-treated roots or leaves. The combinations of GAPDH with TIP41 or TUB were stable in roots and leaves under drought stress. TIP41 and PEPKR1 exhibited stable expression in cold-treated roots, and the combination of F-box, TIP41 and TUB was also stable in cold-treated leaves. CACS and TUB were the two most stable reference genes in heat-stressed roots. TIP41 combined with TUB and ACT was stably expressed in heat-stressed leaves. Finally, quantitative real-time polymerase chain reaction (qRT-PCR assays of the target gene FaWRKY1 using the identified most stable reference genes confirmed the reliability of selected reference genes. The selection of suitable reference genes in tall fescue will allow for more accurate identification of stress-tolerance genes and molecular mechanisms conferring stress tolerance in this stress-tolerant species.

  18. Combining gene expression data from different generations of oligonucleotide arrays

    Directory of Open Access Journals (Sweden)

    Kong Sek

    2004-10-01

    Full Text Available Abstract Background One of the important challenges in microarray analysis is to take full advantage of previously accumulated data, both from one's own laboratory and from public repositories. Through a comparative analysis on a variety of datasets, a more comprehensive view of the underlying mechanism or structure can be obtained. However, as we discover in this work, continual changes in genomic sequence annotations and probe design criteria make it difficult to compare gene expression data even from different generations of the same microarray platform. Results We first describe the extent of discordance between the results derived from two generations of Affymetrix oligonucleotide arrays, as revealed in cluster analysis and in identification of differentially expressed genes. We then propose a method for increasing comparability. The dataset we use consists of a set of 14 human muscle biopsy samples from patients with inflammatory myopathies that were hybridized on both HG-U95Av2 and HG-U133A human arrays. We find that the use of the probe set matching table for comparative analysis provided by Affymetrix produces better results than matching by UniGene or LocusLink identifiers but still remains inadequate. Rescaling of expression values for each gene across samples and data filtering by expression values enhance comparability but only for few specific analyses. As a generic method for improving comparability, we select a subset of probes with overlapping sequence segments in the two array types and recalculate expression values based only on the selected probes. We show that this filtering of probes significantly improves the comparability while retaining a sufficient number of probe sets for further analysis. Conclusions Compatibility between high-density oligonucleotide arrays is significantly affected by probe-level sequence information. With a careful filtering of the probes based on their sequence overlaps, data from different

  19. A model of gene-gene and gene-environment interactions and its implications for targeting environmental interventions by genotype

    OpenAIRE

    Wallace Helen M

    2006-01-01

    Abstract Background The potential public health benefits of targeting environmental interventions by genotype depend on the environmental and genetic contributions to the variance of common diseases, and the magnitude of any gene-environment interaction. In the absence of prior knowledge of all risk factors, twin, family and environmental data may help to define the potential limits of these benefits in a given population. However, a general methodology to analyze twin data is required becaus...

  20. Gene targeting, genome editing: from Dolly to editors.

    Science.gov (United States)

    Tan, Wenfang; Proudfoot, Chris; Lillico, Simon G; Whitelaw, C Bruce A

    2016-06-01

    One of the most powerful strategies to investigate biology we have as scientists, is the ability to transfer genetic material in a controlled and deliberate manner between organisms. When applied to livestock, applications worthy of commercial venture can be devised. Although initial methods used to generate transgenic livestock resulted in random transgene insertion, the development of SCNT technology enabled homologous recombination gene targeting strategies to be used in livestock. Much has been accomplished using this approach. However, now we have the ability to change a specific base in the genome without leaving any other DNA mark, with no need for a transgene. With the advent of the genome editors this is now possible and like other significant technological leaps, the result is an even greater diversity of possible applications. Indeed, in merely 5 years, these 'molecular scissors' have enabled the production of more than 300 differently edited pigs, cattle, sheep and goats. The advent of genome editors has brought genetic engineering of livestock to a position where industry, the public and politicians are all eager to see real use of genetically engineered livestock to address societal needs. Since the first transgenic livestock reported just over three decades ago the field of livestock biotechnology has come a long way-but the most exciting period is just starting.

  1. Landscape of Targeted Anti-Cancer Drug Synergies in Melanoma Identifies a Novel BRAF-VEGFR/PDGFR Combination Treatment.

    Directory of Open Access Journals (Sweden)

    Adam A Friedman

    Full Text Available A newer generation of anti-cancer drugs targeting underlying somatic genetic driver events have resulted in high single-agent or single-pathway response rates in selected patients, but few patients achieve complete responses and a sizeable fraction of patients relapse within a year. Thus, there is a pressing need for identification of combinations of targeted agents which induce more complete responses and prevent disease progression. We describe the results of a combination screen of an unprecedented scale in mammalian cells performed using a collection of targeted, clinically tractable agents across a large panel of melanoma cell lines. We find that even the most synergistic drug pairs are effective only in a discrete number of cell lines, underlying a strong context dependency for synergy, with strong, widespread synergies often corresponding to non-specific or off-target drug effects such as multidrug resistance protein 1 (MDR1 transporter inhibition. We identified drugs sensitizing cell lines that are BRAFV600E mutant but intrinsically resistant to BRAF inhibitor PLX4720, including the vascular endothelial growth factor receptor/kinase insert domain receptor (VEGFR/KDR and platelet derived growth factor receptor (PDGFR family inhibitor cediranib. The combination of cediranib and PLX4720 induced apoptosis in vitro and tumor regression in animal models. This synergistic interaction is likely due to engagement of multiple receptor tyrosine kinases (RTKs, demonstrating the potential of drug- rather than gene-specific combination discovery approaches. Patients with elevated biopsy KDR expression showed decreased progression free survival in trials of mitogen-activated protein kinase (MAPK kinase pathway inhibitors. Thus, high-throughput unbiased screening of targeted drug combinations, with appropriate library selection and mechanistic follow-up, can yield clinically-actionable drug combinations.

  2. Genome-wide gene expression dataset used to identify potential therapeutic targets in androgenetic alopecia

    Directory of Open Access Journals (Sweden)

    R. Dey-Rao

    2017-08-01

    Full Text Available The microarray dataset attached to this report is related to the research article with the title: “A genomic approach to susceptibility and pathogenesis leads to identifying potential novel therapeutic targets in androgenetic alopecia” (Dey-Rao and Sinha, 2017 [1]. Male-pattern hair loss that is induced by androgens (testosterone in genetically predisposed individuals is known as androgenetic alopecia (AGA. The raw dataset is being made publicly available to enable critical and/or extended analyses. Our related research paper utilizes the attached raw dataset, for genome-wide gene-expression associated investigations. Combined with several in silico bioinformatics-based analyses we were able to delineate five strategic molecular elements as potential novel targets towards future AGA-therapy.

  3. Co-factors necessary for PPAR mediated transactivation of endogenous target genes

    DEFF Research Database (Denmark)

    Grøntved, Lars; Nielsen, Ronni; Stunnenberg, Henk

    of endogenous target gene in different cell types are elusive. To mutually compare the ability of the PPAR subtypes to activate endogenous target genes in a given cell, PPARa, PPARb/d and PPARg2 were HA tagged and rapidly, equally and synchronously expressed using adenoviral delivery. Within a few hours after...... subtype specific activation of target genes. Accumulating evidence suggests that transcriptional co-factors can function as master regulators for nuclear receptors and impose promoter selectivity. To study co-factor necessity for PPAR mediated transactivation of endogenous target genes, specific co...

  4. Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors

    Directory of Open Access Journals (Sweden)

    Andrew H. Baker

    2010-10-01

    Full Text Available Achieving high efficiency, targeted gene delivery with adenoviral vectors is a long-standing goal in the field of clinical gene therapy. To achieve this, platform vectors must combine efficient retargeting strategies with detargeting modifications to ablate native receptor binding (i.e. CAR/integrins/heparan sulfate proteoglycans and “bridging” interactions. “Bridging” interactions refer to coagulation factor binding, namely coagulation factor X (FX, which bridges hepatocyte transduction in vivo through engagement with surface expressed heparan sulfate proteoglycans (HSPGs. These interactions can contribute to the off-target sequestration of Ad5 in the liver and its characteristic dose-limiting hepatotoxicity, thereby significantly limiting the in vivo targeting efficiency and clinical potential of Ad5-based therapeutics. To date, various approaches to retargeting adenoviruses (Ad have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon, pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping with non-human Ad species or with capsid proteins derived from other viral families, hexon hypervariable region (HVR substitutions and adapter-based conjugation/crosslinking of scFv, growth factors or monoclonal antibodies directed against surface-expressed target antigens. In order to maximize retargeting, strategies which permit detargeting from undesirable interactions between the Ad capsid and components of the circulatory system (e.g. coagulation factors, erythrocytes, pre-existing neutralizing antibodies, can be employed simultaneously. Detargeting can be achieved by genetic ablation of native receptor-binding determinants, ablation of “bridging interactions” such as those which occur between the hexon of Ad5 and coagulation factor X (FX, or alternatively, through the use of polymer

  5. Fine and Predictable Tuning of TALEN Gene Editing Targeting for Improved T Cell Adoptive Immunotherapy.

    Science.gov (United States)

    Gautron, Anne-Sophie; Juillerat, Alexandre; Guyot, Valérie; Filhol, Jean-Marie; Dessez, Emilie; Duclert, Aymeric; Duchateau, Philippe; Poirot, Laurent

    2017-12-15

    Using a TALEN-mediated gene-editing approach, we have previously described a process for the large-scale manufacturing of "off-the-shelf" CAR T cells from third-party donor T cells by disrupting the gene encoding TCRα constant chain (TRAC). Taking advantage of a previously described strategy to control TALEN targeting based on the exclusion capacities of non-conventional RVDs, we have developed highly efficient and specific nucleases targeting a key T cell immune checkpoint, PD-1, to improve engineered CAR T cells' functionalities. Here, we demonstrate that this approach allows combined TRAC and PDCD1 TALEN processing at the desired locus while eliminating low-frequency off-site processing. Thus, by replacing few RVDs, we provide here an easy and rapid redesign of optimal TALEN combinations. We anticipate that this method can greatly benefit multiplex editing, which is of key importance especially for therapeutic applications where high editing efficiencies need to be associated with maximal specificity and safety. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Fine and Predictable Tuning of TALEN Gene Editing Targeting for Improved T Cell Adoptive Immunotherapy

    Directory of Open Access Journals (Sweden)

    Anne-Sophie Gautron

    2017-12-01

    Full Text Available Using a TALEN-mediated gene-editing approach, we have previously described a process for the large-scale manufacturing of “off-the-shelf” CAR T cells from third-party donor T cells by disrupting the gene encoding TCRα constant chain (TRAC. Taking advantage of a previously described strategy to control TALEN targeting based on the exclusion capacities of non-conventional RVDs, we have developed highly efficient and specific nucleases targeting a key T cell immune checkpoint, PD-1, to improve engineered CAR T cells’ functionalities. Here, we demonstrate that this approach allows combined TRAC and PDCD1 TALEN processing at the desired locus while eliminating low-frequency off-site processing. Thus, by replacing few RVDs, we provide here an easy and rapid redesign of optimal TALEN combinations. We anticipate that this method can greatly benefit multiplex editing, which is of key importance especially for therapeutic applications where high editing efficiencies need to be associated with maximal specificity and safety.

  7. Mapping of Wnt-Frizzled interactions by multiplex CRISPR targeting of receptor gene families.

    Science.gov (United States)

    Voloshanenko, Oksana; Gmach, Philipp; Winter, Jan; Kranz, Dominique; Boutros, Michael

    2017-11-01

    Signaling pathway modules are often encoded by several closely related paralogous genes that can have redundant roles and are therefore difficult to analyze by loss-of-function analysis. A typical example is the Wnt signaling pathway, which in mammals is mediated by 19 Wnt ligands that can bind to 10 Frizzled (FZD) receptors. Although significant progress in understanding Wnt-FZD receptor interactions has been made in recent years, tools to generate systematic interaction maps have been largely lacking. Here we generated cell lines with multiplex mutant alleles of FZD1 , FZD2 , and FZD7 and demonstrate that these cells are unresponsive to canonical Wnt ligands. Subsequently, we performed genetic rescue experiments with combinations of FZDs and canonical Wnts to create a functional ligand-receptor interaction map. These experiments showed that whereas several Wnt ligands, such as Wnt3a, induce signaling through a broad spectrum of FZD receptors, others, such as Wnt8a, act through a restricted set of FZD genes. Together, our results map functional interactions of FZDs and 10 Wnt ligands and demonstrate how multiplex targeting by clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 can be used to systematically elucidate the functions of multigene families.-Voloshanenko, O., Gmach, P., Winter, J., Kranz, D., Boutros, M. Mapping of Wnt-Frizzled interactions by multiplex CRISPR targeting of receptor gene families. © The Author(s).

  8. Targeted gene delivery in the cricket brain, using in vivo electroporation.

    Science.gov (United States)

    Matsumoto, Chihiro Sato; Shidara, Hisashi; Matsuda, Koji; Nakamura, Taro; Mito, Taro; Matsumoto, Yukihisa; Oka, Kotaro; Ogawa, Hiroto

    2013-12-01

    The cricket (Gryllus bimaculatus) is a hemimetabolous insect that is emerging as a model organism for the study of neural and molecular mechanisms of behavioral traits. However, research strategies have been limited by a lack of genetic manipulation techniques that target the nervous system of the cricket. The development of a new method for efficient gene delivery into cricket brains, using in vivo electroporation, is described here. Plasmid DNA, which contained an enhanced green fluorescent protein (eGFP) gene, under the control of a G. bimaculatus actin (Gb'-act) promoter, was injected into adult cricket brains. Injection was followed by electroporation at a sufficient voltage. Expression of eGFP was observed within the brain tissue. Localized gene expression, targeted to specific regions of the brain, was also achieved using a combination of local DNA injection and fine arrangement of the electroporation electrodes. Further studies using this technique will lead to a better understanding of the neural and molecular mechanisms that underlie cricket behaviors. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. HAND2 Target Gene Regulatory Networks Control Atrioventricular Canal and Cardiac Valve Development.

    Science.gov (United States)

    Laurent, Frédéric; Girdziusaite, Ausra; Gamart, Julie; Barozzi, Iros; Osterwalder, Marco; Akiyama, Jennifer A; Lincoln, Joy; Lopez-Rios, Javier; Visel, Axel; Zuniga, Aimée; Zeller, Rolf

    2017-05-23

    The HAND2 transcriptional regulator controls cardiac development, and we uncover additional essential functions in the endothelial to mesenchymal transition (EMT) underlying cardiac cushion development in the atrioventricular canal (AVC). In Hand2-deficient mouse embryos, the EMT underlying AVC cardiac cushion formation is disrupted, and we combined ChIP-seq of embryonic hearts with transcriptome analysis of wild-type and mutants AVCs to identify the functionally relevant HAND2 target genes. The HAND2 target gene regulatory network (GRN) includes most genes with known functions in EMT processes and AVC cardiac cushion formation. One of these is Snai1, an EMT master regulator whose expression is lost from Hand2-deficient AVCs. Re-expression of Snai1 in mutant AVC explants partially restores this EMT and mesenchymal cell migration. Furthermore, the HAND2-interacting enhancers in the Snai1 genomic landscape are active in embryonic hearts and other Snai1-expressing tissues. These results show that HAND2 directly regulates the molecular cascades initiating AVC cardiac valve development. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  10. Advancing cancer drug discovery towards more agile development of targeted combination therapies.

    Science.gov (United States)

    Carragher, Neil O; Unciti-Broceta, Asier; Cameron, David A

    2012-01-01

    Current drug-discovery strategies are typically 'target-centric' and are based upon high-throughput screening of large chemical libraries against nominated targets and a selection of lead compounds with optimized 'on-target' potency and selectivity profiles. However, high attrition of targeted agents in clinical development suggest that combinations of targeted agents will be most effective in treating solid tumors if the biological networks that permit cancer cells to subvert monotherapies are identified and retargeted. Conventional drug-discovery and development strategies are suboptimal for the rational design and development of novel drug combinations. In this article, we highlight a series of emerging technologies supporting a less reductionist, more agile, drug-discovery and development approach for the rational design, validation, prioritization and clinical development of novel drug combinations.

  11. Homologous gene targeting of a carotenoids biosynthetic gene in Rhodosporidium toruloides by Agrobacterium-mediated transformation.

    Science.gov (United States)

    Sun, Wenyi; Yang, Xiaobing; Wang, Xueying; Lin, Xinping; Wang, Yanan; Zhang, Sufang; Luan, Yushi; Zhao, Zongbao K

    2017-07-01

    To target a carotenoid biosynthetic gene in the oleaginous yeast Rhodosporidium toruloides by using the Agrobacterium-mediated transformation (AMT) method. The RHTO_04602 locus of R. toruloides NP11, previously assigned to code the carotenoid biosynthetic gene CRTI, was amplified from genomic DNA and cloned into the binary plasmid pZPK-mcs, resulting in pZPK-CRT. A HYG-expression cassette was inserted into the CRTI sequence of pZPK-CRT by utilizing the restriction-free clone strategy. The resulted plasmid was used to transform R. toruloides cells according to the AMT method, leading to a few white transformants. Sequencing analysis of those transformants confirmed homologous recombination and insertional inactivation of CRTI. When the white variants were transformed with a CRTI-expression cassette, cells became red and produced carotenoids as did the wild-type strain NP11. Successful homologous targeting of the CrtI locus confirmed the function of RHTO_04602 in carotenoids biosynthesis in R. toruloides. It provided valuable information for metabolic engineering of this non-model yeast species.

  12. Transcriptome Analysis of Targeted Mouse Mutations Reveals the Topography of Local Changes in Gene Expression.

    Directory of Open Access Journals (Sweden)

    David B West

    2016-02-01

    Full Text Available The unintended consequences of gene targeting in mouse models have not been thoroughly studied and a more systematic analysis is needed to understand the frequency and characteristics of off-target effects. Using RNA-seq, we evaluated targeted and neighboring gene expression in tissues from 44 homozygous mutants compared with C57BL/6N control mice. Two allele types were evaluated: 15 targeted trap mutations (TRAP; and 29 deletion alleles (DEL, usually a deletion between the translational start and the 3' UTR. Both targeting strategies insert a bacterial beta-galactosidase reporter (LacZ and a neomycin resistance selection cassette. Evaluating transcription of genes in +/- 500 kb of flanking DNA around the targeted gene, we found up-regulated genes more frequently around DEL compared with TRAP alleles, however the frequency of alleles with local down-regulated genes flanking DEL and TRAP targets was similar. Down-regulated genes around both DEL and TRAP targets were found at a higher frequency than expected from a genome-wide survey. However, only around DEL targets were up-regulated genes found with a significantly higher frequency compared with genome-wide sampling. Transcriptome analysis confirms targeting in 97% of DEL alleles, but in only 47% of TRAP alleles probably due to non-functional splice variants, and some splicing around the gene trap. Local effects on gene expression are likely due to a number of factors including compensatory regulation, loss or disruption of intragenic regulatory elements, the exogenous promoter in the neo selection cassette, removal of insulating DNA in the DEL mutants, and local silencing due to disruption of normal chromatin organization or presence of exogenous DNA. An understanding of local position effects is important for understanding and interpreting any phenotype attributed to targeted gene mutations, or to spontaneous indels.

  13. Control of target gene specificity during metamorphosis by the steroid response gene E93

    Science.gov (United States)

    Mou, Xiaochun; Duncan, Dianne M.; Baehrecke, Eric H.; Duncan, Ian

    2012-01-01

    Hormonal control of sexual maturation is a common feature in animal development. A particularly dramatic example is the metamorphosis of insects, in which pulses of the steroid hormone ecdysone drive the wholesale transformation of the larva into an adult. The mechanisms responsible for this transformation are not well understood. Work in Drosophila indicates that the larval and adult forms are patterned by the same underlying sets of developmental regulators, but it is not understood how the same regulators pattern two distinct forms. Recent studies indicate that this ability is facilitated by a global change in the responsiveness of target genes during metamorphosis. Here we show that this shift is controlled in part by the ecdysone-induced transcription factor E93. Although long considered a dedicated regulator of larval cell death, we find that E93 is expressed widely in adult cells at the pupal stage and is required for many patterning processes at this time. To understand the role of E93 in adult patterning, we focused on a simple E93-dependent process, the induction of the Dll gene within bract cells of the pupal leg by EGF receptor signaling. In this system, we show that E93 functions to cause Dll to become responsive to EGF receptor signaling. We demonstrate that E93 is both necessary and sufficient for directing this switch. E93 likely controls the responsiveness of many other target genes because it is required broadly for patterning during metamorphosis. The wide conservation of E93 orthologs suggests that similar mechanisms control life-cycle transitions in other organisms, including vertebrates. PMID:22308414

  14. Control of target gene specificity during metamorphosis by the steroid response gene E93.

    Science.gov (United States)

    Mou, Xiaochun; Duncan, Dianne M; Baehrecke, Eric H; Duncan, Ian

    2012-02-21

    Hormonal control of sexual maturation is a common feature in animal development. A particularly dramatic example is the metamorphosis of insects, in which pulses of the steroid hormone ecdysone drive the wholesale transformation of the larva into an adult. The mechanisms responsible for this transformation are not well understood. Work in Drosophila indicates that the larval and adult forms are patterned by the same underlying sets of developmental regulators, but it is not understood how the same regulators pattern two distinct forms. Recent studies indicate that this ability is facilitated by a global change in the responsiveness of target genes during metamorphosis. Here we show that this shift is controlled in part by the ecdysone-induced transcription factor E93. Although long considered a dedicated regulator of larval cell death, we find that E93 is expressed widely in adult cells at the pupal stage and is required for many patterning processes at this time. To understand the role of E93 in adult patterning, we focused on a simple E93-dependent process, the induction of the Dll gene within bract cells of the pupal leg by EGF receptor signaling. In this system, we show that E93 functions to cause Dll to become responsive to EGF receptor signaling. We demonstrate that E93 is both necessary and sufficient for directing this switch. E93 likely controls the responsiveness of many other target genes because it is required broadly for patterning during metamorphosis. The wide conservation of E93 orthologs suggests that similar mechanisms control life-cycle transitions in other organisms, including vertebrates.

  15. Identification of the human ApoAV gene as a novel RORα target gene

    International Nuclear Information System (INIS)

    Lind, Ulrika; Nilsson, Tina; McPheat, Jane; Stroemstedt, Per-Erik; Bamberg, Krister; Balendran, Clare; Kang, Daiwu

    2005-01-01

    Retinoic acid receptor-related orphan receptor-α (RORα) (NR1F1) is an orphan nuclear receptor with a potential role in metabolism. Previous studies have shown that RORα regulates transcription of the murine Apolipoprotein AI gene and human Apolipoprotein CIII genes. In the present study, we present evidence that RORα also induces transcription of the human Apolipoprotein AV gene, a recently identified apolipoprotein associated with triglyceride levels. Adenovirus-mediated overexpression of RORα increased the endogenous expression of ApoAV in HepG2 cells and RORα also enhanced the activity of an ApoAV promoter construct in transiently transfected HepG2 cells. Deletion and mutation studies identified three AGGTCA motifs in the ApoAV promoter that mediate RORα transactivation, one of which overlaps with a previously identified binding site for PPARα. Together, these results suggest a novel mechanism whereby RORα modulates lipid metabolism and implies RORα as a potential target for the treatment of dyslipidemia and atherosclerosis

  16. Using PCR to Target Misconceptions about Gene Expression

    Directory of Open Access Journals (Sweden)

    Leslie K. Wright

    2013-02-01

    Full Text Available We present a PCR-based laboratory exercise that can be used with first- or second-year biology students to help overcome common misconceptions about gene expression. Biology students typically do not have a clear understanding of the difference between genes (DNA and gene expression (mRNA/protein and often believe that genes exist in an organism or cell only when they are expressed. This laboratory exercise allows students to carry out a PCR-based experiment designed to challenge their misunderstanding of the difference between genes and gene expression. Students first transform E. coli with an inducible GFP gene containing plasmid and observe induced and un-induced colonies. The following exercise creates cognitive dissonance when actual PCR results contradict their initial (incorrect predictions of the presence of the GFP gene in transformed cells. Field testing of this laboratory exercise resulted in learning gains on both knowledge and application questions on concepts related to genes and gene expression.

  17. Gene targeting in human-induced pluripotent stem cells with adenoviral vectors.

    Science.gov (United States)

    Mitani, Kohnosuke

    2014-01-01

    Helper-dependent adenoviral vector (HDAdV), which is also called gutless AdV, has been used to deliver donor DNA for gene targeting in human pluripotent stem cells. Surprisingly, the targeting efficacies, both per chromosomal integration (drug-resistant colony) and per treated cells, are much higher than those by standard electroporation and equivalent to those by utilizing artificial nucleases, such as TAL effector nucleases (Aizawa et al., Mol Ther 20:424-431, 2012; Suzuki, Proc Natl Acad Sci U S A 105:13781-13786, 2008). Importantly, gene targeting with HDAdVs was equally efficient in transcriptionally inactive loci in human ES/iPS cells. Therefore, multiple gene-targeted clones can be obtained from human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) cultured in one 100-mm dish. For virus-mediated gene targeting, it is not required to introduce artificial double-strand breaks. By using electroporation for gene targeting, target cells should be expanded to 10⁶-10⁷ cells. In contrast, as an advantage of virus-mediated method, DNA delivery efficiency is high even in a smaller number of cells, resulting in minimizing the number of passages/cell divisions before performing gene targeting. The characteristics suggest that HDAdV-mediated gene targeting has potential advantages for manipulation of chromosomes of pluripotent stem cells for therapeutic applications.

  18. A Genome-Wide Map of AAV-Mediated Human Gene Targeting

    OpenAIRE

    Deyle, David R.; Hansen, R. Scott; Cornea, Anda M.; Li, Li B.; Burt, Amber A.; Alexander, Ian E.; Sandstrom, Richard S.; Stamatoyannopoulos, John A.; Wei, Chia-Lin; Russell, David W.

    2014-01-01

    To determine which genomic features promote homologous recombination, we created a genome-wide map of gene targeting sites. An adeno-associated virus vector was used to target identical loci introduced as transcriptionally active retroviral vector proviruses. A comparison of ~2,000 targeted and untargeted sites showed that targeting occurred throughout the human genome and was not influenced by the presence of nearby CpG islands, sequence repeats, or DNase I hypersensitive sites. Targeted sit...

  19. Genome-wide analysis of murine renal distal convoluted tubular cells for the target genes of mineralocorticoid receptor

    Energy Technology Data Exchange (ETDEWEB)

    Ueda, Kohei [Department of Nephrology and Endocrinology, The University of Tokyo, Tokyo (Japan); Fujiki, Katsunori; Shirahige, Katsuhiko [Research Center for Epigenetic Disease, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo (Japan); Gomez-Sanchez, Celso E. [Endocrine Section, G.V. (Sonny) Montgomery VA Medical Center, MS (United States); Endocrinology, University of Mississippi Medical Center, MS (United States); Fujita, Toshiro [Division of Clinical Epigenetics, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo (Japan); Nangaku, Masaomi [Department of Nephrology and Endocrinology, The University of Tokyo, Tokyo (Japan); Nagase, Miki, E-mail: mnagase-tky@umin.ac.jp [Department of Nephrology and Endocrinology, The University of Tokyo, Tokyo (Japan); Department of Anatomy and Life Structure, School of Medicine Juntendo University, Tokyo (Japan)

    2014-02-28

    Highlights: • We define a target gene of MR as that with MR-binding to the adjacent region of DNA. • We use ChIP-seq analysis in combination with microarray. • We, for the first time, explore the genome-wide binding profile of MR. • We reveal 5 genes as the direct target genes of MR in the renal epithelial cell-line. - Abstract: Background and objective: Mineralocorticoid receptor (MR) is a member of nuclear receptor family proteins and contributes to fluid homeostasis in the kidney. Although aldosterone-MR pathway induces several gene expressions in the kidney, it is often unclear whether the gene expressions are accompanied by direct regulations of MR through its binding to the regulatory region of each gene. The purpose of this study is to identify the direct target genes of MR in a murine distal convoluted tubular epithelial cell-line (mDCT). Methods: We analyzed the DNA samples of mDCT cells overexpressing 3xFLAG-hMR after treatment with 10{sup −7} M aldosterone for 1 h by chromatin immunoprecipitation with deep-sequence (ChIP-seq) and mRNA of the cell-line with treatment of 10{sup −7} M aldosterone for 3 h by microarray. Results: 3xFLAG-hMR overexpressed in mDCT cells accumulated in the nucleus in response to 10{sup −9} M aldosterone. Twenty-five genes were indicated as the candidate target genes of MR by ChIP-seq and microarray analyses. Five genes, Sgk1, Fkbp5, Rasl12, Tns1 and Tsc22d3 (Gilz), were validated as the direct target genes of MR by quantitative RT-qPCR and ChIP-qPCR. MR binding regions adjacent to Ctgf and Serpine1 were also validated. Conclusions: We, for the first time, captured the genome-wide distribution of MR in mDCT cells and, furthermore, identified five MR target genes in the cell-line. These results will contribute to further studies on the mechanisms of kidney diseases.

  20. GeneChip microarrays facilitate identification of Protease Nexin-1 as a target gene of the Prx2 (S8) homeoprotein.

    Science.gov (United States)

    Scott, Karen K; Norris, Russell A; Potter, S Steven; Norrington, David W; Baybo, Mary Ann; Hicklin, David M; Kern, Michael J

    2003-02-01

    The paired-related homeobox genes, Prx1 and Prx2, are important for normal skeletal and cardiovascular development as well as adult vascular remodeling. The identification and characterization of Prx downstream targets is crucial to understanding their function in normal developmental processes and congenital malformations. To identify Prx2 regulated genes, stably transfected NIH3T3 clones expressing Prx2 sense or antisense transcripts were generated. Expression profiles initially were established for two of the clones using Affymetrix GeneChip arrays. Over 6,400 genes were screened by the microarray approach, and approximately 500 genes differed in expression by a factor of two or more. Fifteen genes were chosen for further analysis. RT-PCR of the two transfectants used in the GeneChip analysis demonstrated that five out of the 15 genes were differentially expressed. However, after screening additional stable transfectant clones only one of the 15 genes, Protease Nexin-1 (PN-1), was differentially expressed. Subsequent Northern blot, RT-PCR, and further GeneChip analysis of additional stable transfectants confirmed that PN-1 expression is increased at least fivefold when Prx2 is overexpressed. It was demonstrated that Prx2 directly regulates PN-1 because (1) Prx2 binds to a cis element in the PN-1 promoter in vitro, and (2) Prx2 regulates the PN-1 promoter in transient transfection assays. The GeneChip analysis generated a prioritized list of other potential targets. The utility and limitations of cell culture models combined with microarray analysis for elucidating complex regulatory cascades are discussed.

  1. Rice and Bean Targets for Biofortification Combined with High Carotenoid Content Crops Regulate Transcriptional Mechanisms Increasing Iron Bioavailability

    Directory of Open Access Journals (Sweden)

    Desirrê Morais Dias

    2015-11-01

    Full Text Available Iron deficiency affects thousands of people worldwide. Biofortification of staple food crops aims to support the reduction of this deficiency. This study evaluates the effect of combinations of common beans and rice, targets for biofortification, with high carotenoid content crops on the iron bioavailability, protein gene expression, and antioxidant effect. Iron bioavailability was measured by the depletion/repletion method. Seven groups were tested (n = 7: Pontal bean (PB; rice + Pontal bean (R + BP; Pontal bean + sweet potato (PB + SP; Pontal bean + pumpkin (PB + P; Pontal bean + rice + sweet potato (PB + R + P; Pontal bean + rice + sweet potato (PB + R + SP; positive control (Ferrous Sulfate. The evaluations included: hemoglobin gain, hemoglobin regeneration efficiency (HRE, gene expression of divalente metal transporter 1 (DMT-1, duodenal citocromo B (DcytB, ferroportin, hephaestin, transferrin and ferritin and total plasma antioxidant capacity (TAC. The test groups, except the PB, showed higher HRE (p < 0.05 than the control. Gene expression of DMT-1, DcytB and ferroportin increased (p < 0.05 in the groups fed with high content carotenoid crops (sweet potato or pumpkin. The PB group presented lower (p < 0.05 TAC than the other groups. The combination of rice and common beans, and those with high carotenoid content crops increased protein gene expression, increasing the iron bioavailability and antioxidant capacity.

  2. Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi

    DEFF Research Database (Denmark)

    Frandsen, Rasmus John Normand; Andersson, Jens A.; Kristensen, Matilde Bylov

    2008-01-01

    technique that allows single step cloning of the two required homologous recombination sequences into different sites of a recipient vector. The advantages are: A simple experimental design, free choice of target sequence, few procedures and user convenience. The vectors are intented for Agrobacterium...... with an average efficiency of 84% for gene replacement and 80% for targeted overexpression. Conclusion: The new vectors designed for USER Friendly cloning provided a fast reliable method to construct vectors for targeted gene manipulations in fungi....

  3. Detection of Balamuthia mandrillaris DNA by real-time PCR targeting the RNase P gene

    Directory of Open Access Journals (Sweden)

    Lewin Astrid

    2008-12-01

    Full Text Available Abstract Background The free-living amoeba Balamuthia mandrillaris may cause fatal encephalitis both in immunocompromised and in – apparently – immunocompetent humans and other mammalian species. Rapid, specific, sensitive, and reliable detection requiring little pathogen-specific expertise is an absolute prerequisite for a successful therapy and a welcome tool for both experimental and epidemiological research. Results A real-time polymerase chain reaction assay using TaqMan® probes (real-time PCR was established specifically targeting the RNase P gene of B. mandrillaris amoebae. The assay detected at least 2 (down to 0.5 genomes of B. mandrillaris grown in axenic culture. It did not react with DNA from closely related Acanthamoeba (3 species, nor with DNA from Toxoplasma gondii, Leishmania major, Pneumocystis murina, Mycobacterium bovis (BCG, human brain, various mouse organs, or from human and murine cell lines. The assay efficiently detected B. mandrillaris DNA in spiked cell cultures, spiked murine organ homogenates, B. mandrillaris-infected mice, and CNS tissue-DNA preparations from 2 patients with proven cerebral balamuthiasis. This novel primer set was successfully combined with a published set that targets the B. mandrillaris 18S rRNA gene in a duplex real-time PCR assay to ensure maximum specificity and as a precaution against false negative results. Conclusion A real-time PCR assay for B. mandrillaris amoebae is presented, that is highly specific, sensitive, and reliable and thus suited both for diagnosis and for research.

  4. Genome-wide identification of Bcl11b gene targets reveals role in brain-derived neurotrophic factor signaling.

    Directory of Open Access Journals (Sweden)

    Bin Tang

    Full Text Available B-cell leukemia/lymphoma 11B (Bcl11b is a transcription factor showing predominant expression in the striatum. To date, there are no known gene targets of Bcl11b in the nervous system. Here, we define targets for Bcl11b in striatal cells by performing chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq in combination with genome-wide expression profiling. Transcriptome-wide analysis revealed that 694 genes were significantly altered in striatal cells over-expressing Bcl11b, including genes showing striatal-enriched expression similar to Bcl11b. ChIP-seq analysis demonstrated that Bcl11b bound a mixture of coding and non-coding sequences that were within 10 kb of the transcription start site of an annotated gene. Integrating all ChIP-seq hits with the microarray expression data, 248 direct targets of Bcl11b were identified. Functional analysis on the integrated gene target list identified several zinc-finger encoding genes as Bcl11b targets, and further revealed a significant association of Bcl11b to brain-derived neurotrophic factor/neurotrophin signaling. Analysis of ChIP-seq binding regions revealed significant consensus DNA binding motifs for Bcl11b. These data implicate Bcl11b as a novel regulator of the BDNF signaling pathway, which is disrupted in many neurological disorders. Specific targeting of the Bcl11b-DNA interaction could represent a novel therapeutic approach to lowering BDNF signaling specifically in striatal cells.

  5. Combined analysis of fourteen nuclear genes refines the Ursidae phylogeny.

    Science.gov (United States)

    Pagès, Marie; Calvignac, Sébastien; Klein, Catherine; Paris, Mathilde; Hughes, Sandrine; Hänni, Catherine

    2008-04-01

    Despite numerous studies, questions remain about the evolutionary history of Ursidae and additional independent genetic markers were needed to elucidate these ambiguities. For this purpose, we sequenced ten nuclear genes for all the eight extant bear species. By combining these new sequences with those of four other recently published nuclear markers, we provide new insights into the phylogenetic relationships of the Ursidae family members. The hypothesis that the giant panda was the first species to diverge among ursids is definitively confirmed and the precise branching order within the Ursus genus is clarified for the first time. Moreover, our analyses indicate that the American and the Asiatic black bears do not cluster as sister taxa, as had been previously hypothesised. Sun and sloth bears clearly appear as the most basal ursine species but uncertainties about their exact relationships remain. Since our larger dataset did not enable us to clarify this last question, identifying rare genomic changes in bear genomes could be a promising solution for further studies.

  6. Combinational Spinal GAD65 Gene Delivery and Systemic GABA-Mimetic Treatment for Modulation of Spasticity

    Science.gov (United States)

    Kakinohana, Osamu; Hefferan, Michael P.; Miyanohara, Atsushi; Nejime, Tetsuya; Marsala, Silvia; Juhas, Stefan; Juhasova, Jana; Motlik, Jan; Kucharova, Karolina; Strnadel, Jan; Platoshyn, Oleksandr; Lazar, Peter; Galik, Jan; Vinay, Laurent; Marsala, Martin

    2012-01-01

    Background Loss of GABA-mediated pre-synaptic inhibition after spinal injury plays a key role in the progressive increase in spinal reflexes and the appearance of spasticity. Clinical studies show that the use of baclofen (GABAB receptor agonist), while effective in modulating spasticity is associated with major side effects such as general sedation and progressive tolerance development. The goal of the present study was to assess if a combined therapy composed of spinal segment-specific upregulation of GAD65 (glutamate decarboxylase) gene once combined with systemic treatment with tiagabine (GABA uptake inhibitor) will lead to an antispasticity effect and whether such an effect will only be present in GAD65 gene over-expressing spinal segments. Methods/Principal Findings Adult Sprague-Dawley (SD) rats were exposed to transient spinal ischemia (10 min) to induce muscle spasticity. Animals then received lumbar injection of HIV1-CMV-GAD65 lentivirus (LVs) targeting ventral α-motoneuronal pools. At 2–3 weeks after lentivirus delivery animals were treated systemically with tiagabine (4, 10, 20 or 40 mg/kg or vehicle) and the degree of spasticity response measured. In a separate experiment the expression of GAD65 gene after spinal parenchymal delivery of GAD65-lentivirus in naive minipigs was studied. Spastic SD rats receiving spinal injections of the GAD65 gene and treated with systemic tiagabine showed potent and tiagabine-dose-dependent alleviation of spasticity. Neither treatment alone (i.e., GAD65-LVs injection only or tiagabine treatment only) had any significant antispasticity effect nor had any detectable side effect. Measured antispasticity effect correlated with increase in spinal parenchymal GABA synthesis and was restricted to spinal segments overexpressing GAD65 gene. Conclusions/Significance These data show that treatment with orally bioavailable GABA-mimetic drugs if combined with spinal-segment-specific GAD65 gene overexpression can represent a novel

  7. Genome-wide analysis of PDX1 target genes in human pancreatic progenitors

    Directory of Open Access Journals (Sweden)

    Xianming Wang

    2018-03-01

    Full Text Available Objective: Homozygous loss-of-function mutations in the gene coding for the homeobox transcription factor (TF PDX1 leads to pancreatic agenesis, whereas heterozygous mutations can cause Maturity-Onset Diabetes of the Young 4 (MODY4. Although the function of Pdx1 is well studied in pre-clinical models during insulin-producing β-cell development and homeostasis, it remains elusive how this TF controls human pancreas development by regulating a downstream transcriptional program. Also, comparative studies of PDX1 binding patterns in pancreatic progenitors and adult β-cells have not been conducted so far. Furthermore, many studies reported the association between single nucleotide polymorphisms (SNPs and T2DM, and it has been shown that islet enhancers are enriched in T2DM-associated SNPs. Whether regions, harboring T2DM-associated SNPs are PDX1 bound and active at the pancreatic progenitor stage has not been reported so far. Methods: In this study, we have generated a novel induced pluripotent stem cell (iPSC line that efficiently differentiates into human pancreatic progenitors (PPs. Furthermore, PDX1 and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq was used to identify PDX1 transcriptional targets and active enhancer and promoter regions. To address potential differences in the function of PDX1 during development and adulthood, we compared PDX1 binding profiles from PPs and adult islets. Moreover, combining ChIP-seq and GWAS meta-analysis data we identified T2DM-associated SNPs in PDX1 binding sites and active chromatin regions. Results: ChIP-seq for PDX1 revealed a total of 8088 PDX1-bound regions that map to 5664 genes in iPSC-derived PPs. The PDX1 target regions include important pancreatic TFs, such as PDX1 itself, RFX6, HNF1B, and MEIS1, which were activated during the differentiation process as revealed by the active chromatin mark H3K27ac and mRNA expression profiling, suggesting that auto-regulatory feedback regulation

  8. Rice and Bean Targets for Biofortification Combined with High Carotenoid Content Crops Regulate Transcriptional Mechanisms Increasing Iron Bioavailability

    Science.gov (United States)

    Dias, Desirrê Morais; de Castro Moreira, Maria Eliza; Gomes, Mariana Juste Contin; Lopes Toledo, Renata Celi; Nutti, Marilia Regini; Pinheiro Sant’Ana, Helena Maria; Martino, Hércia Stampini Duarte

    2015-01-01

    Iron deficiency affects thousands of people worldwide. Biofortification of staple food crops aims to support the reduction of this deficiency. This study evaluates the effect of combinations of common beans and rice, targets for biofortification, with high carotenoid content crops on the iron bioavailability, protein gene expression, and antioxidant effect. Iron bioavailability was measured by the depletion/repletion method. Seven groups were tested (n = 7): Pontal bean (PB); rice + Pontal bean (R + BP); Pontal bean + sweet potato (PB + SP); Pontal bean + pumpkin (PB + P); Pontal bean + rice + sweet potato (PB + R + P); Pontal bean + rice + sweet potato (PB + R + SP); positive control (Ferrous Sulfate). The evaluations included: hemoglobin gain, hemoglobin regeneration efficiency (HRE), gene expression of divalente metal transporter 1 (DMT-1), duodenal citocromo B (DcytB), ferroportin, hephaestin, transferrin and ferritin and total plasma antioxidant capacity (TAC). The test groups, except the PB, showed higher HRE (p bioavailability and antioxidant capacity. PMID:26610564

  9. Predicting human miRNA target genes using a novel evolutionary methodology

    KAUST Repository

    Aigli, Korfiati

    2012-01-01

    The discovery of miRNAs had great impacts on traditional biology. Typically, miRNAs have the potential to bind to the 3\\'untraslated region (UTR) of their mRNA target genes for cleavage or translational repression. The experimental identification of their targets has many drawbacks including cost, time and low specificity and these are the reasons why many computational approaches have been developed so far. However, existing computational approaches do not include any advanced feature selection technique and they are facing problems concerning their classification performance and their interpretability. In the present paper, we propose a novel hybrid methodology which combines genetic algorithms and support vector machines in order to locate the optimal feature subset while achieving high classification performance. The proposed methodology was compared with two of the most promising existing methodologies in the problem of predicting human miRNA targets. Our approach outperforms existing methodologies in terms of classification performances while selecting a much smaller feature subset. © 2012 Springer-Verlag.

  10. Identification of MicroRNAs and target genes involvement in hepatocellular carcinoma with microarray data.

    Science.gov (United States)

    Wang, Dadong; Tan, Jingwang; Xu, Yong; Tan, Xianglong; Han, Mingming; Tu, Yuliang; Zhu, Ziman; Zen, Jianping; Dou, Chunqing; Cai, Shouwang

    2015-01-01

    The aim of the study is to identify the differentially expressed microRNAs (miRNAs) between hepatocellular carcinoma (HCC) samples and controls and provide new diagnostic potential miRNAs for HCC. The miRNAs expression profile data GSE20077 included 7 HCC samples, 1 HeLa sample and 3 controls. Differentially expressed miRNAs (DE-miRNAs) were identified by t-test and wilcox test. The miRNA with significantly differential expression was chosen for further analysis. Target genes for this miRNA were selected using TargetScan and miRbase database. STRING software was applied to construct the target genes interaction network and topology analysis was carried out to identify the hub gene in the network. And we identified the mechanism for affecting miRNA function. A total of 54 differentially expressed miRNAs were identified, in which there were 13 miRNAs published to be related to HCC. The differentially expressed hsa-miR-106b was chosen for further analysis and PTPRT (Receptor-type tyrosine-protein phosphatase T) was its potential target gene. The target genes interaction network was constructed among 33 genes, in which PTPRT was the hub gene. We got the conclusion that the differentially expressed hsa-miR-106b may play an important role in the development of HCC by regulating the expression of its potential target gene PT-PRT.

  11. Identification of target genes of synovial sarcoma-associated fusion oncoprotein using human pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Hayakawa, Kazuo [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Ikeya, Makoto [Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Fukuta, Makoto [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Woltjen, Knut [Department of Reprogramming Sciences, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Tamaki, Sakura; Takahara, Naoko; Kato, Tomohisa; Sato, Shingo [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Otsuka, Takanobu [Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Toguchida, Junya, E-mail: togjun@frontier.kyoto-u.ac.jp [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, Kyoto (Japan)

    2013-03-22

    Highlights: ► We tried to identify targets of synovial sarcoma (SS)-associated SYT–SSX fusion gene. ► We established pluripotent stem cell (PSC) lines with inducible SYT–SSX gene. ► SYT–SSX responsive genes were identified by the induction of SYT–SSX in PSC. ► SS-related genes were selected from database by in silico analyses. ► 51 genes were finally identified among SS-related genes as targets of SYT–SSX in PSC. -- Abstract: Synovial sarcoma (SS) is a malignant soft tissue tumor harboring chromosomal translocation t(X; 18)(p11.2; q11.2), which produces SS-specific fusion gene, SYT–SSX. Although precise function of SYT–SSX remains to be investigated, accumulating evidences suggest its role in gene regulation via epigenetic mechanisms, and the product of SYT–SSX target genes may serve as biomarkers of SS. Lack of knowledge about the cell-of-origin of SS, however, has placed obstacle in the way of target identification. Here we report a novel approach to identify SYT–SSX2 target genes using human pluripotent stem cells (hPSCs) containing a doxycycline-inducible SYT–SSX2 gene. SYT–SSX2 was efficiently induced both at mRNA and protein levels within three hours after doxycycline administration, while no morphological change of hPSCs was observed until 24 h. Serial microarray analyses identified genes of which the expression level changed more than twofold within 24 h. Surprisingly, the majority (297/312, 95.2%) were up-regulated genes and a result inconsistent with the current concept of SYT–SSX as a transcriptional repressor. Comparing these genes with SS-related genes which were selected by a series of in silico analyses, 49 and 2 genes were finally identified as candidates of up- and down-regulated target of SYT–SSX, respectively. Association of these genes with SYT–SSX in SS cells was confirmed by knockdown experiments. Expression profiles of SS-related genes in hPSCs and human mesenchymal stem cells (hMSCs) were strikingly

  12. Prediction of Effective Drug Combinations by Chemical Interaction, Protein Interaction and Target Enrichment of KEGG Pathways

    Directory of Open Access Journals (Sweden)

    Lei Chen

    2013-01-01

    Full Text Available Drug combinatorial therapy could be more effective in treating some complex diseases than single agents due to better efficacy and reduced side effects. Although some drug combinations are being used, their underlying molecular mechanisms are still poorly understood. Therefore, it is of great interest to deduce a novel drug combination by their molecular mechanisms in a robust and rigorous way. This paper attempts to predict effective drug combinations by a combined consideration of: (1 chemical interaction between drugs, (2 protein interactions between drugs’ targets, and (3 target enrichment of KEGG pathways. A benchmark dataset was constructed, consisting of 121 confirmed effective combinations and 605 random combinations. Each drug combination was represented by 465 features derived from the aforementioned three properties. Some feature selection techniques, including Minimum Redundancy Maximum Relevance and Incremental Feature Selection, were adopted to extract the key features. Random forest model was built with its performance evaluated by 5-fold cross-validation. As a result, 55 key features providing the best prediction result were selected. These important features may help to gain insights into the mechanisms of drug combinations, and the proposed prediction model could become a useful tool for screening possible drug combinations.

  13. Prediction of Effective Drug Combinations by Chemical Interaction, Protein Interaction and Target Enrichment of KEGG Pathways

    Science.gov (United States)

    Chen, Lei; Zheng, Ming-Yue; Zhang, Jian; Feng, Kai-Yan; Cai, Yu-Dong

    2013-01-01

    Drug combinatorial therapy could be more effective in treating some complex diseases than single agents due to better efficacy and reduced side effects. Although some drug combinations are being used, their underlying molecular mechanisms are still poorly understood. Therefore, it is of great interest to deduce a novel drug combination by their molecular mechanisms in a robust and rigorous way. This paper attempts to predict effective drug combinations by a combined consideration of: (1) chemical interaction between drugs, (2) protein interactions between drugs' targets, and (3) target enrichment of KEGG pathways. A benchmark dataset was constructed, consisting of 121 confirmed effective combinations and 605 random combinations. Each drug combination was represented by 465 features derived from the aforementioned three properties. Some feature selection techniques, including Minimum Redundancy Maximum Relevance and Incremental Feature Selection, were adopted to extract the key features. Random forest model was built with its performance evaluated by 5-fold cross-validation. As a result, 55 key features providing the best prediction result were selected. These important features may help to gain insights into the mechanisms of drug combinations, and the proposed prediction model could become a useful tool for screening possible drug combinations. PMID:24083237

  14. Identification of potential target genes of ROR-alpha in THP1 and HUVEC cell lines

    International Nuclear Information System (INIS)

    Gulec, Cagri; Coban, Neslihan; Ozsait-Selcuk, Bilge; Sirma-Ekmekci, Sema; Yildirim, Ozlem; Erginel-Unaltuna, Nihan

    2017-01-01

    ROR-alpha is a nuclear receptor, activity of which can be modulated by natural or synthetic ligands. Due to its possible involvement in, and potential therapeutic target for atherosclerosis, we aimed to identify ROR-alpha target genes in monocytic and endothelial cell lines. We performed chromatin immunoprecipitation (ChIP) followed by tiling array (ChIP-on-chip) for ROR-alpha in monocytic cell line THP1 and endothelial cell line HUVEC. Following bioinformatic analysis of the array data, we tested four candidate genes in terms of dependence of their expression level on ligand-mediated ROR-alpha activity, and two of them in terms of promoter occupancy by ROR-alpha. Bioinformatic analyses of ChIP-on-chip data suggested that ROR-alpha binds to genomic regions near the transcription start site (TSS) of more than 3000 genes in THP1 and HUVEC. Potential ROR-alpha target genes in both cell types seem to be involved mainly in membrane receptor activity, signal transduction and ion transport. While SPP1 and IKBKA were shown to be direct target genes of ROR-alpha in THP1 monocytes, inflammation related gene HMOX1 and heat shock protein gene HSPA8 were shown to be potential target genes of ROR-alpha. Our results suggest that ROR-alpha may regulate signaling receptor activity, and transmembrane transport activity through its potential target genes. ROR-alpha seems also to play role in cellular sensitivity to environmental substances like arsenite and chloroprene. Although, the expression analyses have shown that synthetic ROR-alpha ligands can modulate some of potential ROR-alpha target genes, functional significance of ligand-dependent modulation of gene expression needs to be confirmed with further analyses.

  15. Identification of potential target genes of ROR-alpha in THP1 and HUVEC cell lines.

    Science.gov (United States)

    Gulec, Cagri; Coban, Neslihan; Ozsait-Selcuk, Bilge; Sirma-Ekmekci, Sema; Yildirim, Ozlem; Erginel-Unaltuna, Nihan

    2017-04-01

    ROR-alpha is a nuclear receptor, activity of which can be modulated by natural or synthetic ligands. Due to its possible involvement in, and potential therapeutic target for atherosclerosis, we aimed to identify ROR-alpha target genes in monocytic and endothelial cell lines. We performed chromatin immunoprecipitation (ChIP) followed by tiling array (ChIP-on-chip) for ROR-alpha in monocytic cell line THP1 and endothelial cell line HUVEC. Following bioinformatic analysis of the array data, we tested four candidate genes in terms of dependence of their expression level on ligand-mediated ROR-alpha activity, and two of them in terms of promoter occupancy by ROR-alpha. Bioinformatic analyses of ChIP-on-chip data suggested that ROR-alpha binds to genomic regions near the transcription start site (TSS) of more than 3000 genes in THP1 and HUVEC. Potential ROR-alpha target genes in both cell types seem to be involved mainly in membrane receptor activity, signal transduction and ion transport. While SPP1 and IKBKA were shown to be direct target genes of ROR-alpha in THP1 monocytes, inflammation related gene HMOX1 and heat shock protein gene HSPA8 were shown to be potential target genes of ROR-alpha. Our results suggest that ROR-alpha may regulate signaling receptor activity, and transmembrane transport activity through its potential target genes. ROR-alpha seems also to play role in cellular sensitivity to environmental substances like arsenite and chloroprene. Although, the expression analyses have shown that synthetic ROR-alpha ligands can modulate some of potential ROR-alpha target genes, functional significance of ligand-dependent modulation of gene expression needs to be confirmed with further analyses. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Identification of potential target genes of ROR-alpha in THP1 and HUVEC cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Gulec, Cagri, E-mail: cagri.gulec@gmail.com; Coban, Neslihan, E-mail: neslic@istanbul.edu.tr; Ozsait-Selcuk, Bilge, E-mail: ozsaitb@istanbul.edu.tr; Sirma-Ekmekci, Sema, E-mail: semasirma@gmail.com; Yildirim, Ozlem, E-mail: ozlm-yildirim@hotmail.com; Erginel-Unaltuna, Nihan, E-mail: nihanerginel@yahoo.com

    2017-04-01

    ROR-alpha is a nuclear receptor, activity of which can be modulated by natural or synthetic ligands. Due to its possible involvement in, and potential therapeutic target for atherosclerosis, we aimed to identify ROR-alpha target genes in monocytic and endothelial cell lines. We performed chromatin immunoprecipitation (ChIP) followed by tiling array (ChIP-on-chip) for ROR-alpha in monocytic cell line THP1 and endothelial cell line HUVEC. Following bioinformatic analysis of the array data, we tested four candidate genes in terms of dependence of their expression level on ligand-mediated ROR-alpha activity, and two of them in terms of promoter occupancy by ROR-alpha. Bioinformatic analyses of ChIP-on-chip data suggested that ROR-alpha binds to genomic regions near the transcription start site (TSS) of more than 3000 genes in THP1 and HUVEC. Potential ROR-alpha target genes in both cell types seem to be involved mainly in membrane receptor activity, signal transduction and ion transport. While SPP1 and IKBKA were shown to be direct target genes of ROR-alpha in THP1 monocytes, inflammation related gene HMOX1 and heat shock protein gene HSPA8 were shown to be potential target genes of ROR-alpha. Our results suggest that ROR-alpha may regulate signaling receptor activity, and transmembrane transport activity through its potential target genes. ROR-alpha seems also to play role in cellular sensitivity to environmental substances like arsenite and chloroprene. Although, the expression analyses have shown that synthetic ROR-alpha ligands can modulate some of potential ROR-alpha target genes, functional significance of ligand-dependent modulation of gene expression needs to be confirmed with further analyses.

  17. Identification of downstream metastasis-associated target genes regulated by LSD1 in colon cancer cells.

    Science.gov (United States)

    Chen, Jiang; Ding, Jie; Wang, Ziwei; Zhu, Jian; Wang, Xuejian; Du, Jiyi

    2017-03-21

    This study aims to identify downstream target genes regulated by lysine-specific demethylase 1 (LSD1) in colon cancer cells and investigate the molecular mechanisms of LSD1 influencing invasion and metastasis of colon cancer. We obtained the expression changes of downstream target genes regulated by small-interfering RNA-LSD1 and LSD1-overexpression via gene expression profiling in two human colon cancer cell lines. An Affymetrix Human Transcriptome Array 2.0 was used to identify differentially expressed genes (DEGs). We screened out LSD1-target gene associated with proliferation, metastasis, and invasion from DEGs via Gene Ontology and Pathway Studio. Subsequently, four key genes (CABYR, FOXF2, TLE4, and CDH1) were computationally predicted as metastasis-related LSD1-target genes. ChIp-PCR was applied after RT-PCR and Western blot validations to detect the occupancy of LSD1-target gene promoter-bound LSD1. A total of 3633 DEGs were significantly upregulated, and 4642 DEGs were downregulated in LSD1-silenced SW620 cells. A total of 4047 DEGs and 4240 DEGs were upregulated and downregulated in LSD1-overexpressed HT-29 cells, respectively. RT-PCR and Western blot validated the microarray analysis results. ChIP assay results demonstrated that LSD1 might be negative regulators for target genes CABYR and CDH1. The expression level of LSD1 is negatively correlated with mono- and dimethylation of histone H3 lysine4(H3K4) at LSD1- target gene promoter region. No significant mono-methylation and dimethylation of H3 lysine9 methylation was detected at the promoter region of CABYR and CDH1. LSD1- depletion contributed to the upregulation of CABYR and CDH1 through enhancing the dimethylation of H3K4 at the LSD1-target genes promoter. LSD1- overexpression mediated the downregulation of CABYR and CDH1expression through decreasing the mono- and dimethylation of H3K4 at LSD1-target gene promoter in colon cancer cells. CABYR and CDH1 might be potential LSD1-target genes in colon

  18. Stable gene replacement in barley by targeted double-strand break induction.

    Science.gov (United States)

    Watanabe, Koichi; Breier, Ulrike; Hensel, Götz; Kumlehn, Jochen; Schubert, Ingo; Reiss, Bernd

    2016-03-01

    Gene targeting is becoming an important tool for precision genome engineering in plants. During gene replacement, a variant of gene targeting, transformed DNA integrates into the genome by homologous recombination (HR) to replace resident sequences. We have analysed gene targeting in barley (Hordeum vulgare) using a model system based on double-strand break (DSB) induction by the meganuclease I-SceI and a transgenic, artificial target locus. In the plants we obtained, the donor construct was inserted at the target locus by homology-directed DNA integration in at least two transformants obtained in a single experiment and was stably inherited as a single Mendelian trait. Both events were produced by one-sided integration. Our data suggest that gene replacement can be achieved in barley with a frequency suitable for routine application. The use of a codon-optimized nuclease and co-transfer of the nuclease gene together with the donor construct are probably the components important for efficient gene targeting. Such an approach, employing the recently developed synthetic nucleases/nickases that allow DSB induction at almost any sequence of a genome of interest, sets the stage for precision genome engineering as a routine tool even for important crops such as barley. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  19. Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi

    Directory of Open Access Journals (Sweden)

    Kristensen Matilde B

    2008-08-01

    Full Text Available Abstract Background The rapid increase in whole genome fungal sequence information allows large scale functional analyses of target genes. Efficient transformation methods to obtain site-directed gene replacement, targeted over-expression by promoter replacement, in-frame epitope tagging or fusion of coding sequences with fluorescent markers such as GFP are essential for this process. Construction of vectors for these experiments depends on the directional cloning of two homologous recombination sequences on each side of a selection marker gene. Results Here, we present a USER Friendly cloning based technique that allows single step cloning of the two required homologous recombination sequences into different sites of a recipient vector. The advantages are: A simple experimental design, free choice of target sequence, few procedures and user convenience. The vectors are intented for Agrobacterium tumefaciens and protoplast based transformation technologies. The system has been tested by the construction of vectors for targeted replacement of 17 genes and overexpression of 12 genes in Fusarium graminearum. The results show that four fragment vectors can be constructed in a single cloning step with an average efficiency of 84% for gene replacement and 80% for targeted overexpression. Conclusion The new vectors designed for USER Friendly cloning provided a fast reliable method to construct vectors for targeted gene manipulations in fungi.

  20. Gene targeting using homologous recombination in embryonic stem cells: The future for behavior genetics?

    Directory of Open Access Journals (Sweden)

    Robert eGerlai

    2016-04-01

    Full Text Available Gene targeting with homologous recombination in embryonic stem cells created a revolution in the analysis of the function of genes in behavioral brain research. The technology allowed unprecedented precision with which one could manipulate genes and study the effect of this manipulation on the central nervous system. With gene targeting, the uncertainty inherent in psychopharmacology regarding whether a particular compound would act only through a specific target was removed. Thus, gene targeting became highly popular. However, with this popularity came the realization that like other methods, gene targeting also suffered from some technical and principal problems. For example, two decades ago, issues about compensatory changes and about genetic linkage were raised. Since then, the technology developed, and its utility has been better delineated. This review will discuss the pros and cons of the technique along with these advancements from the perspective of the neuroscientist user. It will also compare and contrast methods that may represent novel alternatives to the homologous recombination based gene targeting approach, including the TALEN and the CRISPR/Cas9 systems. The goal of the review is not to provide detailed recipes, but to attempt to present a short summary of these approaches a behavioral geneticist or neuroscientist may consider for the analysis of brain function and behavior.

  1. Gene targeting in embryonic stem cells, II: conditional technologies

    Science.gov (United States)

    Genome modification via transgenesis has allowed researchers to link genotype and phenotype as an alternative approach to the characterization of random mutations through evolution. The synergy of technologies from the fields of embryonic stem (ES) cells, gene knockouts, and protein-mediated recombi...

  2. Specifically targeted gene therapy for small-cell lung cancer

    DEFF Research Database (Denmark)

    Christensen, C.L.; Zandi, R.; Gjetting, T.

    2009-01-01

    Small-cell lung cancer (SCLC) is a highly malignant disease with poor prognosis. Hence, there is great demand for new therapies that can replace or supplement the current available treatment regimes. Gene therapy constitutes a promising strategy and relies on the principle of introducing exogenous...

  3. Targeting the ABCB4 gene to control cholesterol homeostasis

    NARCIS (Netherlands)

    Oude Elferink, Ronald Pj; Beuers, Ulrich

    2011-01-01

    INTRODUCTION: Multidrug resistance 3 (MDR3) P-glycoprotein is a lipid floppase that is encoded by the ATP-binding cassette sub-family B member 4 (ABCB4) gene and plays a crucial role in proper bile formation by transporting phosphatidylcholine across the canalicular plasma membrane of the hepatocyte

  4. Haplotype combination of the caprine PC1 gene sequence variants ...

    Indian Academy of Sciences (India)

    Mutations in PC1 gene are related to obesity-related traits which may influence the performance of animals, and therefore it has been the focus of this study. In this study, polymorphisms of the caprine PC1 gene were detected in. 407 individuals from three breeds by PCR-SSCP and DNA sequencing methods. The results ...

  5. Gene expression profiling reveals novel TGFβ targets in adult lung fibroblasts

    Directory of Open Access Journals (Sweden)

    Pearson Jeremy D

    2004-11-01

    Full Text Available Abstract Background Transforming growth factor beta (TGFβ, a multifunctional cytokine, plays a crucial role in the accumulation of extracellular matrix components in lung fibrosis, where lung fibroblasts are considered to play a major role. Even though the effects of TGFβ on the gene expression of several proteins have been investigated in several lung fibroblast cell lines, the global pattern of response to this cytokine in adult lung fibroblasts is still unknown. Methods We used Affymetrix oligonucleotide microarrays U95v2, containing approximately 12,000 human genes, to study the transcriptional profile in response to a four hour treatment with TGFβ in control lung fibroblasts and in fibroblasts from patients with idiopathic and scleroderma-associated pulmonary fibrosis. A combination of the Affymetrix change algorithm (Microarray Suite 5 and of analysis of variance models was used to identify TGFβ-regulated genes. Additional criteria were an average up- or down- regulation of at least two fold. Results Exposure of fibroblasts to TGFβ had a profound impact on gene expression, resulting in regulation of 129 transcripts. We focused on genes not previously found to be regulated by TGFβ in lung fibroblasts or other cell types, including nuclear co-repressor 2, SMAD specific E3 ubiquitin protein ligase 2 (SMURF2, bone morphogenetic protein 4, and angiotensin II receptor type 1 (AGTR1, and confirmed the microarray results by real time-PCR. Western Blotting confirmed induction at the protein level of AGTR1, the most highly induced gene in both control and fibrotic lung fibroblasts among genes encoding for signal transduction molecules. Upregulation of AGTR1 occurred through the MKK1/MKK2 signalling pathway. Immunohistochemical staining showed AGTR1 expression by lung fibroblasts in fibroblastic foci within biopsies of idiopathic pulmonary fibrosis. Conclusions This study identifies several novel TGFβ targets in lung fibroblasts, and confirms

  6. International Test Comparisons: Reviewing Translation Error in Different Source Language-Target Language Combinations

    Science.gov (United States)

    Zhao, Xueyu; Solano-Flores, Guillermo; Qian, Ming

    2018-01-01

    This article addresses test translation review in international test comparisons. We investigated the applicability of the theory of test translation error--a theory of the multidimensionality and inevitability of test translation error--across source language-target language combinations in the translation of PISA (Programme of International…

  7. On combining spectral and spatial information of hyperspectral image for camouflaged target detecting

    Science.gov (United States)

    Hua, Wenshen; Liu, Xun; Yang, Jia

    2013-12-01

    Detecting enemy's targets and being undetectable play increasingly important roles in modern warfare. Hyperspectral images can provide large spectral range and high spectral resolution, which are invaluable in discriminating between camouflaged targets and backgrounds. As supervised classification requires prior knowledge which cannot be acquired easily, unsupervised classification usually is adopted to process hyperspectral images to detect camouflaged target. But one of its drawbacks—low detecting accuracy confines its application for camouflaged target detecting. Most research on the processing of hyperspectral image tends to focus exclusively on spectral domain and ignores spatial domain. However current hyperspectral image provides high spatial resolution which contains useful information for camouflaged target detecting. A new method combining spectral and spatial information is proposed to increase the detecting accuracy using unsupervised classification. The method has two steps. In the first step, a traditional unsupervised classifier (i.e. K-MEANS, ISODATA) is adopted to classify the hyperspectral image to acquire basic classifications or clusters. During the second step, a 3×3 model and spectral angle mapping are utilized to test the spatial character of the hyperspectral image. The spatial character is defined as spatial homogeneity and calculated by spectral angle mapping. Theory analysis and experiment shows the method is reasonable and efficient. Camouflaged targets are extracted from the background and different camouflaged targets are also recognized. And the proposed algorithm outperforms K-MEANS in terms of detecting accuracy, robustness and edge's distinction. This paper demonstrates the new method is meaningful to camouflaged targets detecting.

  8. Stripy Ftz target genes are coordinately regulated by Ftz-F1.

    Science.gov (United States)

    Hou, Hui Ying; Heffer, Alison; Anderson, W Ray; Liu, Jingnan; Bowler, Timothy; Pick, Leslie

    2009-11-15

    During development, cascades of regulatory genes act in a hierarchical fashion to subdivide the embryo into increasingly specified body regions. This has been best characterized in Drosophila, where genes encoding regulatory transcription factors form a network to direct the development of the basic segmented body plan. The pair-rule genes are pivotal in this process as they are responsible for the first subdivision of the embryo into repeated metameric units. The Drosophila pair-rule gene fushi tarazu (ftz) is a derived Hox gene expressed in and required for the development of alternate parasegments. Previous studies suggested that Ftz achieves its distinct regulatory specificity as a segmentation protein by interacting with a ubiquitously expressed cofactor, the nuclear receptor Ftz-F1. However, the downstream target genes regulated by Ftz and other pair-rule genes to direct segment formation are not known. In this study, we selected candidate Ftz targets by virtue of their early expression in Ftz-like stripes. This identified two new Ftz target genes, drumstick (drm) and no ocelli (noc), and confirmed that Ftz regulates a serotonin receptor (5-HT2). These are the earliest Ftz targets identified to date and all are coordinately regulated by Ftz-F1. Engrailed (En), the best-characterized Ftz/Ftz-F1 downstream target, is not an intermediate in regulation. The drm genomic region harbors two separate seven-stripe enhancers, identified by virtue of predicted Ftz-F1 binding sites, and these sites are necessary for stripe expression in vivo. We propose that pair-rule genes, exemplified by Ftz/Ftz-F1, promote segmentation by acting at different hierarchical levels, regulating first, other segmentation genes; second, other regulatory genes that in turn control specific cellular processes such as tissue differentiation; and, third, 'segmentation realizator genes' that are directly involved in morphogenesis.

  9. Combination of immunotherapy with targeted therapies in advanced non-small cell lung cancer (NSCLC).

    Science.gov (United States)

    Moya-Horno, Irene; Viteri, Santiago; Karachaliou, Niki; Rosell, Rafael

    2018-01-01

    Treatment for advanced non-small cell lung cancer (NSCLC) has been significantly improved in recent years with the incorporation of drugs targeting antiangiogenesis and more specifically genomic alterations such as the EGFR mutations and ALK translocations. However, most patients invariably progress and die. The emergence of immune checkpoint inhibitors targeting the pathways involved in tumor-induced immunosuppression have redefined the management of the disease, achieving significant long-lasting responses with manageable safety profiles, regardless of histology. Still, response rates with immunotherapy are deemed suboptimal. Current efforts are focusing on new potential combination strategies with synergistic antitumor activity, using immune checkpoint blockade as a partner for targeted agents. Herein we discuss the available data on the combined use of immunotherapy, including PD-1/PD-L1 and CTLA-4 inhibitors, with EGFR and ALK inhibitors and comment on the current status of immunotherapy plus antiangiogenic drugs for molecularly unselected advanced NSCLC.

  10. In vitro therapeutic effect of PDT combined with VEGF-A gene therapy

    Science.gov (United States)

    Lecaros, Rumwald Leo G.; Huang, Leaf; Hsu, Yih-Chih

    2014-02-01

    Vascular endothelial growth factor A (VEGF-A), commonly known as VEGF, is one of the primary factors that affect tumor angiogenesis. It was found to be expressed in cancer cell lines including oral squamous cell carcinoma. Photodynamic therapy (PDT) is a novel therapeutic modality to treat cancer by using a photosensitizer which is activated by a light source to produce reactive oxygen species and mediates oxygen-independent hypoxic conditions to tumor. Another emerging treatment to cure cancer is the use of interference RNA (e.g. siRNA) to silence a specific mRNA sequence. VEGF-A was found to be expressed in oral squamous cell carcinoma and overexpressed after 24 hour post-PDT by Western blot analysis. Cell viability was found to decrease at 25 nM of transfected VEGF-A siRNA. In vitro combined therapy of PDT and VEGF-A siRNA showed better response as compared with PDT and gene therapy alone. The results suggest that PDT combined with targeted gene therapy has a potential mean to achieve better therapeutic outcome.

  11. Expression of RNA-interference/antisense transgenes by the cognate promoters of target genes is a better gene-silencing strategy to study gene functions in rice.

    Science.gov (United States)

    Li, Jing; Jiang, Dagang; Zhou, Hai; Li, Feng; Yang, Jiawei; Hong, Laifa; Fu, Xiao; Li, Zhibin; Liu, Zhenlan; Li, Jianming; Zhuang, Chuxiong

    2011-03-03

    Antisense and RNA interference (RNAi)-mediated gene silencing systems are powerful reverse genetic methods for studying gene function. Most RNAi and antisense experiments used constitutive promoters to drive the expression of RNAi/antisense transgenes; however, several reports showed that constitutive promoters were not expressed in all cell types in cereal plants, suggesting that the constitutive promoter systems are not effective for silencing gene expression in certain tissues/organs. To develop an alternative method that complements the constitutive promoter systems, we constructed RNAi and/or antisense transgenes for four rice genes using a constitutive promoter or a cognate promoter of a selected rice target gene and generated many independent transgenic lines. Genetic, molecular, and phenotypic analyses of these RNAi/antisense transgenic rice plants, in comparison to previously-reported transgenic lines that silenced similar genes, revealed that expression of the cognate promoter-driven RNAi/antisense transgenes resulted in novel growth/developmental defects that were not observed in transgenic lines expressing constitutive promoter-driven gene-silencing transgenes of the same target genes. Our results strongly suggested that expression of RNAi/antisense transgenes by cognate promoters of target genes is a better gene-silencing approach to discovery gene function in rice.

  12. UP Finder: A COBRA toolbox extension for identifying gene overexpression strategies for targeted overproduction

    Directory of Open Access Journals (Sweden)

    Xi Wang

    2017-12-01

    Full Text Available Overexpression of key genes is a basic strategy for overproducing target products via metabolic engineering. Traditionally, identifying those key genes/pathways largely relies on the knowledge of biochemistry and bioinformatics. In this study, a modeling tool named UP Finder was developed to facilitate the rapid identification of gene overexpression strategies. It was based on the COBRA toolbox under MATLAB environment. All the key gene/pathway targets are identified in one click after simply loading a Systems Biology Markup Language model and specifying a metabolite as the targeted product. The outputs are also quantitatively ranked to show the preference for determining overexpression strategies in pathway design. Analysis examples for overproducing lycopene precursor in Escherichia coli and fatty acyl-ACP in the cyanobacterium Synechocystis sp. PCC 6803 by the UP Finder showed high degree of agreement with the reported key genes in the literatures.

  13. Target gene approaches: Gene expression in Daphnia magna exposed to predator-borne kairomones or to microcystin-producing and microcystin-free Microcystis aeruginosa

    Directory of Open Access Journals (Sweden)

    Courts Cornelius

    2009-11-01

    Full Text Available Abstract Background Two major biological stressors of freshwater zooplankton of the genus Daphnia are predation and fluctuations in food quality. Here we use kairomones released from a planktivorous fish (Leucaspius delineatus and from an invertebrate predator (larvae of Chaoborus flavicans to simulate predation pressure; a microcystin-producing culture of the cyanobacterium Microcystis aeruginosa and a microcystin-deficient mutant are used to investigate effects of low food quality. Real-time quantitative polymerase chain reaction (QPCR allows quantification of the impact of biotic stressors on differential gene activity. The draft genome sequence for Daphnia pulex facilitates the use of candidate genes by precisely identifying orthologs to functionally characterized genes in other model species. This information is obtained by constructing phylogenetic trees of candidate genes with the knowledge that the Daphnia genome is composed of many expanded gene families. Results We evaluated seven candidate reference genes for QPCR in Daphnia magna after exposure to kairomones. As a robust approach, a combination normalisation factor (NF was calculated based on the geometric mean of three of these seven reference genes: glyceraldehyde-3-phosphate dehydrogenase, TATA-box binding protein and succinate dehydrogenase. Using this NF, expression of the target genes actin and alpha-tubulin were revealed to be unchanged in the presence of the tested kairomones. The presence of fish kairomone up-regulated one gene (cyclophilin involved in the folding of proteins, whereas Chaoborus kairomone down-regulated the same gene. We evaluated the same set of candidate reference genes for QPCR in Daphnia magna after exposure to a microcystin-producing and a microcystin-free strain of the cyanobacterium Microcystis aeruginosa. The NF was calculated based on the reference genes 18S ribosomal RNA, alpha-tubulin and TATA-box binding protein. We found glyceraldehyde-3

  14. Multi-targeted priming for genome-wide gene expression assays.

    Science.gov (United States)

    Adomas, Aleksandra B; Lopez-Giraldez, Francesc; Clark, Travis A; Wang, Zheng; Townsend, Jeffrey P

    2010-08-17

    Complementary approaches to assaying global gene expression are needed to assess gene expression in regions that are poorly assayed by current methodologies. A key component of nearly all gene expression assays is the reverse transcription of transcribed sequences that has traditionally been performed by priming the poly-A tails on many of the transcribed genes in eukaryotes with oligo-dT, or by priming RNA indiscriminately with random hexamers. We designed an algorithm to find common sequence motifs that were present within most protein-coding genes of Saccharomyces cerevisiae and of Neurospora crassa, but that were not present within their ribosomal RNA or transfer RNA genes. We then experimentally tested whether degenerately priming these motifs with multi-targeted primers improved the accuracy and completeness of transcriptomic assays. We discovered two multi-targeted primers that would prime a preponderance of genes in the genomes of Saccharomyces cerevisiae and Neurospora crassa while avoiding priming ribosomal RNA or transfer RNA. Examining the response of Saccharomyces cerevisiae to nitrogen deficiency and profiling Neurospora crassa early sexual development, we demonstrated that using multi-targeted primers in reverse transcription led to superior performance of microarray profiling and next-generation RNA tag sequencing. Priming with multi-targeted primers in addition to oligo-dT resulted in higher sensitivity, a larger number of well-measured genes and greater power to detect differences in gene expression. Our results provide the most complete and detailed expression profiles of the yeast nitrogen starvation response and N. crassa early sexual development to date. Furthermore, our multi-targeting priming methodology for genome-wide gene expression assays provides selective targeting of multiple sequences and counter-selection against undesirable sequences, facilitating a more complete and precise assay of the transcribed sequences within the genome.

  15. Multi-targeted priming for genome-wide gene expression assays

    Directory of Open Access Journals (Sweden)

    Adomas Aleksandra B

    2010-08-01

    Full Text Available Abstract Background Complementary approaches to assaying global gene expression are needed to assess gene expression in regions that are poorly assayed by current methodologies. A key component of nearly all gene expression assays is the reverse transcription of transcribed sequences that has traditionally been performed by priming the poly-A tails on many of the transcribed genes in eukaryotes with oligo-dT, or by priming RNA indiscriminately with random hexamers. We designed an algorithm to find common sequence motifs that were present within most protein-coding genes of Saccharomyces cerevisiae and of Neurospora crassa, but that were not present within their ribosomal RNA or transfer RNA genes. We then experimentally tested whether degenerately priming these motifs with multi-targeted primers improved the accuracy and completeness of transcriptomic assays. Results We discovered two multi-targeted primers that would prime a preponderance of genes in the genomes of Saccharomyces cerevisiae and Neurospora crassa while avoiding priming ribosomal RNA or transfer RNA. Examining the response of Saccharomyces cerevisiae to nitrogen deficiency and profiling Neurospora crassa early sexual development, we demonstrated that using multi-targeted primers in reverse transcription led to superior performance of microarray profiling and next-generation RNA tag sequencing. Priming with multi-targeted primers in addition to oligo-dT resulted in higher sensitivity, a larger number of well-measured genes and greater power to detect differences in gene expression. Conclusions Our results provide the most complete and detailed expression profiles of the yeast nitrogen starvation response and N. crassa early sexual development to date. Furthermore, our multi-targeting priming methodology for genome-wide gene expression assays provides selective targeting of multiple sequences and counter-selection against undesirable sequences, facilitating a more complete and

  16. Combination of Supervised and Unsupervised Approaches for miRNA Target Prediction.

    Science.gov (United States)

    Sedaghat, Nafiseh; Fathy, Mahmood; Modarressi, Mohammad Hossein; Shojaie, Ali

    2017-07-13

    MicroRNAs (miRNAs) are short non-coding RNAs which target mRNAs by binding to them and regulating their expression. Involvement of miRNAs has been discovered in many diseases, so it is fruitful to investigate the miRNAs and their targets to develop new therapeutic ways by designing anti-miRNA oligonucleotides. There are various computational methods to predict the target genes, however, their precisions are not good enough. In this paper, we apply a two-step approach to refine the results of sequence-based prediction algorithms. The first step is based on consensus learning according to our previous research, then the second step includes utilizing Support Vector Machine (SVM) classifier in unary and binary modes to identify validated interactions based on binding features as well as network features of genes in constructed gene regulatory network. Experimental results on two real data sets from TCGA database show the binary SVM classification significantly increases the precision of finding validated interactions.

  17. Radar correlated imaging for extended target by the combination of negative exponential restraint and total variation

    Science.gov (United States)

    Qian, Tingting; Wang, Lianlian; Lu, Guanghua

    2017-07-01

    Radar correlated imaging (RCI) introduces the optical correlated imaging technology to traditional microwave imaging, which has raised widespread concern recently. Conventional RCI methods neglect the structural information of complex extended target, which makes the quality of recovery result not really perfect, thus a novel combination of negative exponential restraint and total variation (NER-TV) algorithm for extended target imaging is proposed in this paper. The sparsity is measured by a sequential order one negative exponential function, then the 2D total variation technique is introduced to design a novel optimization problem for extended target imaging. And the proven alternating direction method of multipliers is applied to solve the new problem. Experimental results show that the proposed algorithm could realize high resolution imaging efficiently for extended target.

  18. Application of gene targeting to designed mutation breeding of high-tryptophan rice.

    Science.gov (United States)

    Saika, Hiroaki; Oikawa, Akira; Matsuda, Fumio; Onodera, Haruko; Saito, Kazuki; Toki, Seiichi

    2011-07-01

    Site-directed mutagenesis via gene targeting (GT) based on homologous recombination is the ultimate mutation breeding technology because it enables useful information acquired from structural- and computational-based protein engineering to be applied directly to molecular breeding, including metabolic engineering, of crops. Here, we employed this rationale to introduce precise mutations in OASA2--an α-subunit of anthranilate synthase that is a key enzyme of tryptophan (Trp) biosynthesis in rice (Oryza sativa)--via GT, with subsequent selection of GT cells using a Trp analog. The expression level of OASA2 in plants homozygous and heterozygous for modified OASA2 was similar to that of nontransformants, suggesting that OASA2 transcription in GT plants was controlled in the same manner as endogenous OASA2, and that GT could lead to a lower risk of gene silencing than in conventional overexpression approaches. Moreover, we showed that enzymatic properties deduced from protein engineering or in vitro analysis could be reproduced in GT plants as evidenced by Trp accumulation levels. Interestingly, mature seeds of homozygous GT plants accumulated Trp levels 230-fold higher than in nontransformants without any apparent morphological or developmental changes. Thus, we have succeeded in producing a novel rice plant of great potential nutritional benefit for both man and livestock that could not have been selected using conventional mutagenesis approaches. Our results demonstrate the effectiveness of directed crop improvement by combining precision mutagenesis via GT with a knowledge of protein engineering.

  19. Genome-wide identification of structural variants in genes encoding drug targets

    DEFF Research Database (Denmark)

    Rasmussen, Henrik Berg; Dahmcke, Christina Mackeprang

    2012-01-01

    The objective of the present study was to identify structural variants of drug target-encoding genes on a genome-wide scale. We also aimed at identifying drugs that are potentially amenable for individualization of treatments based on knowledge about structural variation in the genes encoding...

  20. RYP1 gene as a target for molecular diagnosis of histoplasmosis.

    Science.gov (United States)

    Brilhante, Raimunda Sâmia Nogueira; Guedes, Glaucia Morgana de Melo; Riello, Giovanna Barbosa; Ribeiro, Joyce Fonteles; Alencar, Lucas Pereira; Bandeira, Silviane Praciano; Castelo-Branco, Débora Souza Collares Maia; Oliveira, Jonathas Sales; Freire, Janaína Maria Maia; Mesquita, Jacó Ricarte Lima de; Camargo, Zoilo Pires de; Cordeiro, Rossana de Aguiar; Rocha, Marcos Fábio Gadelha; Sidrim, José Júlio Costa

    2016-11-01

    This study analyzed the RYP1 gene as a target for the molecular diagnosis of histoplasmosis. This assay detected fungal DNA in 13/13 blood samples from HIV/AIDS-patients with histoplasmosis. Therefore, the detection of RYP1 gene in whole blood sample is a quick and sensitive test to diagnose histoplasmosis. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Computational design and application of endogenous promoters for transcriptionally targeted gene therapy for rheumatoid arthritis.

    NARCIS (Netherlands)

    Geurts, J.; Joosten, L.A.B.; Takahashi, N.; Arntz, O.J.; Gluck, A.; Bennink, M.B.; Berg, W.B. van den; Loo, F.A.J. van de

    2009-01-01

    The promoter regions of genes that are differentially regulated in the synovial membrane during the course of rheumatoid arthritis (RA) represent attractive candidates for application in transcriptionally targeted gene therapy. In this study, we applied an unbiased computational approach to define

  2. PDGF-receptor beta-targeted adenovirus redirects gene transfer from hepatocytes to activated stellate cells

    NARCIS (Netherlands)

    Schoemaker, Marieke H.; Rots, Marianne G.; Beljaars, Leonie; Ypma, Arjen Y.; Jansen, Peter L. M.; Poelstra, Klaas; Moshage, Albert; Haisma, Hidde J.

    2008-01-01

    Chronic liver damage may lead to liver fibrosis. In this process, hepatic activated stellate cells are the key players. Thus, activated stellate cells are attractive targets for antifibrotic gene therapy. Recombinant, adenovirus is a promising vehicle for delivering therapeutic genes to liver cells.

  3. Combination therapy targeting the tumor microenvironment is effective in a model of human ocular melanoma

    Directory of Open Access Journals (Sweden)

    Schafer Peter H

    2007-07-01

    Full Text Available Abstract Background Ocular melanoma is the leading intraocular malignancy. There is no effective treatment for metastatic ocular melanoma. We sought a treatment targeting the tumor microenvironment as well as the tumor cells. Methods Migration of HUVEC cells, the ability of HUVEC cells to form tubes, and proliferative capacity of a human ocular melanoma cell line were tested in the presence of lenalidomide and sorafenib alone and in combination. The compounds were also tested in a rat aortic ring assay and were tested in a highly aggressive human ocular melanoma xenograft model. Results Lenalidomide and Sorafenib inhibit HUVEC ability to migrate and form tubes and when used in combination the inhibition is increased. The agents alone and in combination inhibit outgrowth in the rat aortic ring model. The combination of the agents improved the inhibition over either single agent. In a xenograft model, combination therapy inhibited tumor growth over inhibition by single agent alone in a significant fashion (p Conclusion Lenalidomide and sorafenib are effective at targeting endothelial cells, inhibiting growth of ocular melanoma cells and can inhibit growth of tumors in a xenograft model as well as inhibit development of metastases. Combining these agents works in an additive to synergistic way to inhibit the growth of tumors and development of metastases.

  4. Application of an Efficient Gene Targeting System Linking Secondary Metabolites to their Biosynthetic Genes in Aspergillus terreus

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Chun-Jun; Knox, Benjamin P.; Sanchez, James F.; Chiang, Yi-Ming; Bruno, Kenneth S.; Wang, Clay C.

    2013-07-19

    Nonribosomal peptides (NRPs) are natural products biosynthesized by NRP synthetases. A kusA-, pyrG- mutant strain of Aspergillusterreus NIH 2624 was developed that greatly facilitated the gene targeting efficiency in this organism. Application of this tool allowed us to link four major types of NRP related secondary metabolites to their responsible genes in A. terreus. In addition, an NRP related melanin synthetase was also identified in this species.

  5. Global gene expression analysis reveals reduced abundance of putative microRNA targets in human prostate tumours

    Directory of Open Access Journals (Sweden)

    Xie Yi

    2009-02-01

    Full Text Available Abstract Background Recently, microRNAs (miRNAs have taken centre stage in the field of human molecular oncology. Several studies have shown that miRNA profiling analyses offer new possibilities in cancer classification, diagnosis and prognosis. However, the function of miRNAs that are dysregulated in tumours remains largely a mystery. Global analysis of miRNA-target gene expression has helped illuminate the role of miRNAs in developmental gene expression programs, but such an approach has not been reported in cancer transcriptomics. Results In this study, we globally analysed the expression patterns of miRNA target genes in prostate cancer by using several public microarray datasets. Intriguingly, we found that, in contrast to global mRNA transcript levels, putative miRNA targets showed a reduced abundance in prostate tumours relative to benign prostate tissue. Additionally, the down-regulation of these miRNA targets positively correlated with the number of types of miRNA target-sites in the 3' untranslated regions of these targets. Further investigation revealed that the globally low expression was mainly driven by the targets of 36 specific miRNAs that were reported to be up-regulated in prostate cancer by a miRNA expression profiling study. We also found that the transcript levels of miRNA targets were lower in androgen-independent prostate cancer than in androgen-dependent prostate cancer. Moreover, when the global analysis was extended to four other cancers, significant differences in transcript levels between miRNA targets and total mRNA backgrounds were found. Conclusion Global gene expression analysis, along with further investigation, suggests that miRNA targets have a significantly reduced transcript abundance in prostate cancer, when compared with the combined pool of all mRNAs. The abnormal expression pattern of miRNA targets in human cancer could be a common feature of the human cancer transcriptome. Our study may help to shed new

  6. Combining biological gene expression signatures in predicting outcome in breast cancer: An alternative to supervised classification

    NARCIS (Netherlands)

    Nuyten, Dimitry S. A.; Hastie, Trevor; Chi, Jen-Tsan Ashley; Chang, Howard Y.; van de Vijver, Marc J.

    2008-01-01

    INTRODUCTION: Gene expression profiling has been extensively used to predict outcome in breast cancer patients. We have previously reported on biological hypothesis-driven analysis of gene expression profiling data and we wished to extend this approach through the combinations of various gene

  7. Chemical Genetic Screens Identify Kinase Inhibitor Combinations that Target Anti-Apoptotic Proteins for Cancer Therapy.

    Science.gov (United States)

    Contreras, Jacob I; Robb, Caroline M; King, Hannah M; Baxter, Jared; Crawford, Ayrianne J; Kour, Smit; Kizhake, Smitha; Sonawane, Yogesh A; Rana, Sandeep; Hollingsworth, Michael A; Luo, Xu; Natarajan, Amarnath

    2018-04-05

    The study presented here provides a framework for the discovery of unique inhibitor combinations that target the apoptosis network for cancer therapy. A pair of doxycycline (Dox)-inducible cell lines that specifically report on the ability of an inhibitor to induce apoptosis by targeting either the Mcl-1 arm or the Bcl-2/Bcl-xL/Bcl-w arm were used. Cell-based assays were optimized for high throughput screening (HTS) with caspase 3/7 as a read out. HTS with a 355-member kinase inhibitor library and the panel of Dox-inducible cell lines revealed that cyclin dependent kinase (CDK) inhibitors induced apoptosis by targeting the Mcl-1 arm, whereas PI3K inhibitors induced apoptosis by targeting the Bcl-2/Bcl-xL/Bcl-w arm. Validation studies identified unique combinations that synergistically inhibited growth and induced apoptosis in a panel of cancer cell lines. Since these inhibitors have been or are currently in clinical trials as single agents, the combinations can be rapidly translated to the clinics.

  8. Towards prostate cancer gene therapy: Development of a chlorotoxin-targeted nanovector for toxic (melittin) gene delivery.

    Science.gov (United States)

    Tarokh, Zahra; Naderi-Manesh, Hossein; Nazari, Mahboobeh

    2017-03-01

    Prostate cancer is the second leading cause of death due to cancer in men. Owing to shortcomings in the current treatments, other therapies are being considered. Toxic gene delivery is one of the most effective methods for cancer therapy. Cationic polymers are able to form stable nanoparticles via interaction with nucleic acids electrostatically. Branched polyethylenimine that contains amine groups has notable buffering capacity and the ability to escape from endosome through the proton sponge effect. However, the cytotoxicity of this polymer is high, and modification is one of the applicable strategies to overcome this problem. In this study, PEI was targeted with chlorotoxin (CTX) via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) cross-linker. CTX can bind specifically to matrix metalloproteinase-2 that is overexpressed in certain cancers. Melittin as the major component of bee venom has been reported to have anti-cancer activity. This was thus selected to deliver to PC3 cell line. Flow cytometry analysis revealed that transfection efficiency of targeted nanoparticles is significantly higher compared to non-targeted nanoparticles. Targeted nanoparticles carrying the melittin gene also decreased cell viability of PC3 cells significantly while no toxic effects were observed on NIH3T3 cell line. Therefore, CTX-targeted nanoparticles carrying the melittin gene could serve as an appropriate gene delivery system for prostate and other MMP-2 positive cancer cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Targeted cancer gene therapy : the flexibility of adenoviral gene therapy vectors

    NARCIS (Netherlands)

    Rots, MG; Curiel, DT; Gerritsen, WR; Haisma, HJ

    2003-01-01

    Recombinant adenoviral vectors are promising reagents for therapeutic interventions in humans, including gene therapy for biologically complex diseases like cancer and cardiovascular diseases. In this regard, the major advantage of adenoviral vectors is their superior in vivo gene transfer

  10. Biallelic targeting of expressed genes in mouse embryonic stem cells using the Cas9 system

    NARCIS (Netherlands)

    Zhang, Yu; Vanoli, Fabio; LaRocque, Jeannine R.; Krawczyk, Przemek M.; Jasin, Maria

    2014-01-01

    Gene targeting - homologous recombination between transfected DNA and a chromosomal locus - is greatly stimulated by a DNA break in the target locus. Recently, the RNA-guided Cas9 endonuclease, involved in bacterial adaptive immunity, has been modified to function in mammalian cells. Unlike other

  11. Manipulating the in vivo immune response by targeted gene knockdown.

    Science.gov (United States)

    Lieberman, Judy

    2015-08-01

    Aptamers, nucleic acids selected for high affinity binding to proteins, can be used to activate or antagonize immune mediators or receptors in a location and cell-type specific manner and to enhance antigen presentation. They can also be linked to other molecules (other aptamers, siRNAs or miRNAs, proteins, toxins) to produce multifunctional compounds for targeted immune modulation in vivo. Aptamer-siRNA chimeras (AsiCs) that induce efficient cell-specific knockdown in immune cells in vitro and in vivo can be used as an immunological research tool or potentially as an immunomodulating therapeutic. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Expression of androgen receptor target genes in skeletal muscle

    Directory of Open Access Journals (Sweden)

    Kesha Rana

    2014-10-01

    Full Text Available We aimed to determine the mechanisms of the anabolic actions of androgens in skeletal muscle by investigating potential androgen receptor (AR-regulated genes in in vitro and in vivo models. The expression of the myogenic regulatory factor myogenin was significantly decreased in skeletal muscle from testosterone-treated orchidectomized male mice compared to control orchidectomized males, and was increased in muscle from male AR knockout mice that lacked DNA binding activity (ARΔZF2 versus wildtype mice, demonstrating that myogenin is repressed by the androgen/AR pathway. The ubiquitin ligase Fbxo32 was repressed by 12 h dihydrotestosterone treatment in human skeletal muscle cell myoblasts, and c-Myc expression was decreased in testosterone-treated orchidectomized male muscle compared to control orchidectomized male muscle, and increased in AR∆ZF2 muscle. The expression of a group of genes that regulate the transition from myoblast proliferation to differentiation, Tceal7 , p57 Kip2, Igf2 and calcineurin Aa, was increased in AR∆ZF2 muscle, and the expression of all but p57 Kip2 was also decreased in testosterone-treated orchidectomized male muscle compared to control orchidectomized male muscle. We conclude that in males, androgens act via the AR in part to promote peak muscle mass by maintaining myoblasts in the proliferative state and delaying the transition to differentiation during muscle growth and development, and by suppressing ubiquitin ligase-mediated atrophy pathways to preserve muscle mass in adult muscle.

  13. The combination of novel targeted molecular agents and radiation in the treatment of pediatric gliomas

    Directory of Open Access Journals (Sweden)

    Tina eDasgupta

    2013-05-01

    Full Text Available Brain tumors are the most common solid pediatric malignancy. For high-grade, recurrent or refractory pediatric brain tumors, radiation therapy (XRT is an integral treatment modality. In the era of personalized cancer therapy, molecularly targeted agents have been designed to inhibit pathways critical to tumorigenesis. Our evolving knowledge of genetic aberrations in low-grade gliomas is being exploited with targeted inhibitors. These agents are also being combined with XRT to increase their efficacy. In this review, we discuss novel agents targeting three different pathways in low-grade gliomas, and their potential combination with XRT. B-Raf is a kinase in the Ras/Raf/MAPK kinase pathway, which is integral to cellular division, survival and metabolism. In low-grade pediatric gliomas, point mutations in BRAF (BRAF V600E or a BRAF fusion mutation (KIAA1549:BRAF causes overactivation of the MEK/MAPK pathway. Pre-clinical data shows cooperation between XRT and tagrgeted inhibitors of BRAF V600E, and MEK and mTOR inhibitors in the gliomas with the BRAF fusion. A second important signaling cascade in pediatric glioma pathogenesis is the PI3 kinase (PI3K/mTOR pathway. Dual PI3K/mTOR inhibitors are poised to enter studies of pediatric tumors. Finally, many brain tumors express potent stimulators of angiogenesis. Several inhibitors of immunomodulators are currently being evaluated in in clinical trials for the treatment of recurrent or refractory pediatric central nervous system (CNS tumors. In summary, combinations of these targeted inhibitors with radiation are currently under investigation in both translational bench research and early clinical trials. We summarize the molecular rationale for, and the pre-clinical data supporting the combinations of these targeted agents with other anti-cancer agents and XRT in pediatric gliomas. Parallels are drawn to adult gliomas, and the molecular mechanisms underlying the efficacy of these agents is discussed

  14. Simple and Efficient Targeting of Multiple Genes Through CRISPR-Cas9 in Physcomitrella patens

    Directory of Open Access Journals (Sweden)

    Mauricio Lopez-Obando

    2016-11-01

    Full Text Available Powerful genome editing technologies are needed for efficient gene function analysis. The CRISPR-Cas9 system has been adapted as an efficient gene-knock-out technology in a variety of species. However, in a number of situations, knocking out or modifying a single gene is not sufficient; this is particularly true for genes belonging to a common family, or for genes showing redundant functions. Like many plants, the model organism Physcomitrella patens has experienced multiple events of polyploidization during evolution that has resulted in a number of families of duplicated genes. Here, we report a robust CRISPR-Cas9 system, based on the codelivery of a CAS9 expressing cassette, multiple sgRNA vectors, and a cassette for transient transformation selection, for gene knock-out in multiple gene families. We demonstrate that CRISPR-Cas9-mediated targeting of five different genes allows the selection of a quintuple mutant, and all possible subcombinations of mutants, in one experiment, with no mutations detected in potential off-target sequences. Furthermore, we confirmed the observation that the presence of repeats in the vicinity of the cutting region favors deletion due to the alternative end joining pathway, for which induced frameshift mutations can be potentially predicted. Because the number of multiple gene families in Physcomitrella is substantial, this tool opens new perspectives to study the role of expanded gene families in the colonization of land by plants.

  15. Prioritization of EGFR/IGF-IR/VEGFR2 combination targeted therapies utilizing cancer models.

    Science.gov (United States)

    Tonra, James R; Corcoran, Erik; Deevi, Dhanvanthri S; Steiner, Philipp; Kearney, Jessica; Li, Huiling; Ludwig, Dale L; Zhu, Zhenping; Witte, Larry; Surguladze, David; Hicklin, Daniel J

    2009-06-01

    Rational strategies utilizing anticancer efficacy and biological principles are needed for the prioritization of specific combination targeted therapy approaches for clinical development, from among the many with experimental support. Antibodies targeting epidermal growth factor receptor (EGFR) (cetuximab), insulin-like growth factor-1 receptor (IGF-IR) (IMC-A12) or vascular endothelial growth factor receptor 2 (VEGFR2) (DC101), were dosed alone or in combination, in 11 human tumor xenograft models established in mice. Efficacy readouts included the tumor burden and incidence of metastasis, as well as tumor active hypoxia inducible factor-1 (HIF-1), human VEGF and blood vessel density. Cetuximab and DC101 contributed potent and non-overlapping benefits to the combination approach. Moreover, DC101 prevented escape from IMC-A12 + cetuximab in a colorectal cancer model and cetuximab prevented escape from DC101 therapy in a pancreatic cancer model. Targeting VEGFR2 + EGFR was prioritized over other treatment strategies utilizing EGFR, IGF-IR and VEGFR2 antibodies. The criteria that proved to be valuable were a non-overlapping spectrum of anticancer activity and the prevention of resistance to another therapy in the combination.

  16. High efficiency of BRCA1 knockout using rAAV-mediated gene targeting: developing a pig model for breast cancer.

    Science.gov (United States)

    Luo, Yonglun; Li, Juan; Liu, Ying; Lin, Lin; Du, Yutao; Li, Shengting; Yang, Huanming; Vajta, Gábor; Callesen, Henrik; Bolund, Lars; Sørensen, Charlotte Brandt

    2011-10-01

    Germline inactivating mutations of the breast cancer associated gene 1 (BRCA1) predispose to breast cancer and account for most cases of familiar breast and/or ovarian cancer. The pig is an excellent model for medical research as well as testing of new methods and drugs for disease prevention and treatment. We have generated cloned BRCA1 knockout (KO) Yucatan miniature piglets by targeting exon 11 using recombinant adeno-associated virus (rAAV)-mediated gene targeting and somatic cell nuclear transfer by Handmade Cloning (HMC). We found a very high targeting rate of rAAV-mediated BRCA1 KO. Approximately 35% of the selected cells were BRCA1 targeted. One BRCA1 KO cell clone (5D1), identified by PCR and Southern blot, was used as nuclear donor for HMC. Reconstructed embryos were transferred to three recipient sows which gave birth to 8 piglets in total. Genotyping identified seven piglets as BRCA1 heterozygotes (BRCA1(+/∆11)), and one as wild type. The BRCA1 expression was decreased at the mRNA level in BRCA1(+/∆11) fibroblasts. However, all BRCA1(+/∆11) piglets died within 18 days after birth. The causes of perinatal mortality remain unclear. Possible explanations may include a combination of the BRCA1 haploinsufficiency, problems of epigenetic reprogramming, presence of the marker gene, single cell clone effects, and/or the special genetic background of the minipigs.

  17. Targeting fungal genes by diced siRNAs: a rapid tool to decipher gene function in Aspergillus nidulans.

    Science.gov (United States)

    Kalleda, Natarajaswamy; Naorem, Aruna; Manchikatla, Rajam V

    2013-01-01

    Gene silencing triggered by chemically synthesized small interfering RNAs (siRNAs) has become a powerful tool for deciphering gene function in many eukaryotes. However, prediction and validation of a single siRNA duplex specific to a target gene is often ineffective. RNA interference (RNAi) with synthetic siRNA suffers from lower silencing efficacy, off-target effects and is cost-intensive, especially for functional genomic studies. With the explosion of fungal genomic information, there is an increasing need to analyze gene function in a rapid manner. Therefore, studies were performed in order to investigate the efficacy of gene silencing induced by RNase III-diced-siRNAs (d-siRNA) in model filamentous fungus, Aspergillus nidulans. Stable expression of heterologous reporter gene in A. nidulans eases the examination of a new RNAi-induction route. Hence, we have optimized Agrobacterium tumefaciens-mediated transformation (AMT) of A. nidulans for stable expression of sGFP gene. This study demonstrates that the reporter GFP gene stably introduced into A. nidulans can be effectively silenced by treatment of GFP-d-siRNAs. We have shown the down-regulation of two endogenous genes, AnrasA and AnrasB of A. nidulans by d-siRNAs. We have also elucidated the function of an uncharacterized Ras homolog, rasB gene, which was found to be involved in hyphal growth and development. Further, silencing potency of d-siRNA was higher as compared to synthetic siRNA duplex, targeting AnrasA. Silencing was shown to be sequence-specific, since expression profiles of other closely related Ras family genes in d-siRNA treated AnrasA and AnrasB silenced lines exhibited no change in gene expression. We have developed and applied a fast, specific and efficient gene silencing approach for elucidating gene function in A. nidulans using d-siRNAs. We have also optimized an efficient AMT in A. nidulans, which is useful for stable integration of transgenes.

  18. Sequence-Specific Targeting of Bacterial Resistance Genes Increases Antibiotic Efficacy.

    Science.gov (United States)

    Ayhan, Dilay Hazal; Tamer, Yusuf Talha; Akbar, Mohammed; Bailey, Stacey M; Wong, Michael; Daly, Seth M; Greenberg, David E; Toprak, Erdal

    2016-09-01

    The lack of effective and well-tolerated therapies against antibiotic-resistant bacteria is a global public health problem leading to prolonged treatment and increased mortality. To improve the efficacy of existing antibiotic compounds, we introduce a new method for strategically inducing antibiotic hypersensitivity in pathogenic bacteria. Following the systematic verification that the AcrAB-TolC efflux system is one of the major determinants of the intrinsic antibiotic resistance levels in Escherichia coli, we have developed a short antisense oligomer designed to inhibit the expression of acrA and increase antibiotic susceptibility in E. coli. By employing this strategy, we can inhibit E. coli growth using 2- to 40-fold lower antibiotic doses, depending on the antibiotic compound utilized. The sensitizing effect of the antisense oligomer is highly specific to the targeted gene's sequence, which is conserved in several bacterial genera, and the oligomer does not have any detectable toxicity against human cells. Finally, we demonstrate that antisense oligomers improve the efficacy of antibiotic combinations, allowing the combined use of even antagonistic antibiotic pairs that are typically not favored due to their reduced activities.

  19. Gold Nanoparticle Approach to the Selective Delivery of Gene Silencing in Cancer—The Case for Combined Delivery?

    Directory of Open Access Journals (Sweden)

    Rita Mendes

    2017-03-01

    Full Text Available Gene therapy arises as a great promise for cancer therapeutics due to its potential to silence genes involved in tumor development. In fact, there are some pivotal gene drivers that suffer critical alterations leading to cell transformation and ultimately to tumor growth. In this vein, gene silencing has been proposed as an active tool to selectively silence these molecular triggers of cancer, thus improving treatment. However, naked nucleic acid (DNA/RNA sequences are reported to have a short lifetime in the body, promptly degraded by circulating enzymes, which in turn speed up elimination and decrease the therapeutic potential of these drugs. The use of nanoparticles for the effective delivery of these silencers to the specific target locations has allowed researchers to overcome this issue. Particularly, gold nanoparticles (AuNPs have been used as attractive vehicles for the target-specific delivery of gene-silencing moieties, alone or in combination with other drugs. We shall discuss current trends in AuNP-based delivery of gene-silencing tools, considering the promising road ahead without overlooking existing concerns for their translation to clinics.

  20. A Combination of CRISPR/Cas9 and Standardized RNAi as a Versatile Platform for the Characterization of Gene Function

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    Sebastian Wissel

    2016-08-01

    Full Text Available Traditional loss-of-function studies in Drosophila suffer from a number of shortcomings, including off-target effects in the case of RNA interference (RNAi or the stochastic nature of mosaic clonal analysis. Here, we describe minimal in vivo GFP interference (miGFPi as a versatile strategy to characterize gene function and to conduct highly stringent, cell type-specific loss-of-function experiments in Drosophila. miGFPi combines CRISPR/Cas9-mediated tagging of genes at their endogenous locus with an immunotag and an exogenous 21 nucleotide RNAi effector sequence with the use of a single reagent, highly validated RNAi line targeting this sequence. We demonstrate the utility and time effectiveness of this method by characterizing the function of the Polymerase I (Pol I-associated transcription factor Tif-1a, and the previously uncharacterized gene MESR4, in the Drosophila female germline stem cell lineage. In addition, we show that miGFPi serves as a powerful technique to functionally characterize individual isoforms of a gene. We exemplify this aspect of miGFPi by studying isoform-specific loss-of-function phenotypes of the longitudinals lacking (lola gene in neural stem cells. Altogether, the miGFPi strategy constitutes a generalized loss-of-function approach that is amenable to the study of the function of all genes in the genome in a stringent and highly time effective manner.

  1. Bioinformatic methods for finding differentially expressed genes in cDNA libraries, applied to the identification of tumour vascular targets.

    Science.gov (United States)

    Herbert, John M J; Stekel, Dov J; Mura, Manuela; Sychev, Michail; Bicknell, Roy

    2011-01-01

    The aim of this method is to guide a bench scientist to maximise cDNA library analyses to predict biologically relevant genes to pursue in the laboratory. Many groups have successfully utilised cDNA libraries to discover novel and/or differentially expressed genes in pathologies of interest. This is despite the high cost of cDNA library production using the Sanger method of sequencing, which produces modest numbers of expressed sequences compared to the total transcriptome. Both public and propriety cDNA libraries can be utilised in this way, and combining biologically relevant data can reveal biologically interesting genes. Pivotal to the quality of target identification are the selection of biologically relevant libraries, the accuracy of Expressed Sequence Tag to gene assignment, and the statistics used. The key steps, methods, and tools used to this end will be described using vascular targeting as an example. With the advent of next-generation sequencing, these or similar methods can be applied to find novel genes with this new source of data.

  2. Targeting New Candidate Genes by Small Molecules Approaching Neurodegenerative Diseases

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    Hueng-Chuen Fan

    2015-12-01

    Full Text Available Neurodegenerative diseases (NDs are among the most feared of the disorders that afflict humankind for the lack of specific diagnostic tests and effective treatments. Understanding the molecular, cellular, biochemical changes of NDs may hold therapeutic promise against debilitating central nerve system (CNS disorders. In the present review, we summarized the clinical presentations and biology backgrounds of NDs, including Parkinson’s disease (PD, Huntington’s disease (HD, and Alzheimer’s disease (AD and explored the role of molecular mechanisms, including dys-regulation of epigenetic control mechanisms, Ataxia-telangiectasia-mutated protein kinase (ATM, and neuroinflammation in the pathogenesis of NDs. Targeting these mechanisms may hold therapeutic promise against these devastating diseases.

  3. The MSX1 homeoprotein recruits G9a methyltransferase to repressed target genes in myoblast cells.

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    Jingqiang Wang

    Full Text Available Although the significance of lysine modifications of core histones for regulating gene expression is widely appreciated, the mechanisms by which these modifications are incorporated at specific regulatory elements during cellular differentiation remains largely unknown. In our previous studies, we have shown that in developing myoblasts the Msx1 homeoprotein represses gene expression by influencing the modification status of chromatin at its target genes. We now show that genomic binding by Msx1 promotes enrichment of the H3K9me2 mark on repressed target genes via recruitment of G9a histone methyltransferase, the enzyme responsible for catalyzing this histone mark. Interaction of Msx1 with G9a is mediated via the homeodomain and is required for transcriptional repression and regulation of cellular differentiation, as well as enrichment of the H3K9me2 mark in proximity to Msx1 binding sites on repressed target genes in myoblast cells as well as the developing limb. We propose that regulation of chromatin status by Msx1 recruitment of G9a and other histone modifying enzymes to regulatory regions of target genes represents an important means of regulating the gene expression during development.

  4. Combining Results from Distinct MicroRNA Target Prediction Tools Enhances the Performance of Analyses.

    Science.gov (United States)

    Oliveira, Arthur C; Bovolenta, Luiz A; Nachtigall, Pedro G; Herkenhoff, Marcos E; Lemke, Ney; Pinhal, Danillo

    2017-01-01

    Target prediction is generally the first step toward recognition of bona fide microRNA (miRNA)-target interactions in living cells. Several target prediction tools are now available, which use distinct criteria and stringency to provide the best set of candidate targets for a single miRNA or a subset of miRNAs. However, there are many false-negative predictions, and consensus about the optimum strategy to select and use the output information provided by the target prediction tools is lacking. We compared the performance of four tools cited in literature-TargetScan (TS), miRanda-mirSVR (MR), Pita, and RNA22 (R22), and we determined the most effective approach for analyzing target prediction data (individual, union, or intersection). For this purpose, we calculated the sensitivity, specificity, precision, and correlation of these approaches using 10 miRNAs (miR-1-3p, miR-17-5p, miR-21-5p, miR-24-3p, miR-29a-3p, miR-34a-5p, miR-124-3p, miR-125b-5p, miR-145-5p, and miR-155-5p) and 1,400 genes (700 validated and 700 non-validated) as targets of these miRNAs. The four tools provided a subset of high-quality predictions and returned few false-positive predictions; however, they could not identify several known true targets. We demonstrate that union of TS/MR and TS/MR/R22 enhanced the quality of in silico prediction analysis of miRNA targets. We conclude that the union rather than the intersection of the aforementioned tools is the best strategy for maximizing performance while minimizing the loss of time and resources in subsequent in vivo and in vitro experiments for functional validation of miRNA-target interactions.

  5. Haplotype combination of the caprine PC1 gene sequence variants ...

    Indian Academy of Sciences (India)

    Proprotein convertase 1 (PC1) is an endopeptidase involved in proteolytic processing of peptide hormone precursors in granules of the regulated secretory pathway of endocrine cells. Mutations in PC1 gene are related to obesity-related traits which may influence the performance of animals, and therefore it has been the ...

  6. Detection of miR-33 Expression and the Verification of Its Target Genes in the Fatty Liver of Geese

    Directory of Open Access Journals (Sweden)

    Yun Zheng

    2015-06-01

    CROT is highest and reaches 70%; (3 The seed sequence (5′ 2–8 bases is the acting site of miR-33. The two predicted target sites of CROT are the target sites of miR-33. Moreover, the predicted target site of HADHB and NPC1 is the target site of miR-33. Conclusions/significance: (1 After 19 days of overfeeding, the expression level of miR-33 increases significantly in the livers of geese; (2 CROT, HADHB and NPC1 are the target genes of miR-33 in geese. These genes determine the combined target site.

  7. Combination of chemotherapy and cancer stem cell targeting agents: Preclinical and clinical studies.

    Science.gov (United States)

    Li, Yanyan; Atkinson, Katharine; Zhang, Tao

    2017-06-28

    The cancer stem cell model claims that the initiation, maintenance, and growth of a tumor are driven by a small population of cancer cells termed cancer stem cells. Cancer stem cells possess a variety of phenotypes associated with therapeutic resistance and often cause recurrence of the diseases. Several strategies have been investigated to target cancer stem cells in a variety of cancers, such as blocking one or more self-renewal signaling pathways, reducing the expression of drug efflux and ATP-binding cassette efflux transporters, modulating epigenetic aberrations, and promoting cancer stem cell differentiation. A number of cell and animal studies strongly support the potential benefits of combining chemotherapeutic drugs with cancer stem cell targeting agents. Clinical trials are still underway to address the pharmacokinetics, safety, and efficacy of combination treatment. This mini-review provides an updated discussion of these preclinical and clinical studies. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Enhancing potency of siRNA targeting fusion genes by optimization outside of target sequence.

    Science.gov (United States)

    Gavrilov, Kseniya; Seo, Young-Eun; Tietjen, Gregory T; Cui, Jiajia; Cheng, Christopher J; Saltzman, W Mark

    2015-12-01

    Canonical siRNA design algorithms have become remarkably effective at predicting favorable binding regions within a target mRNA, but in some cases (e.g., a fusion junction site) region choice is restricted. In these instances, alternative approaches are necessary to obtain a highly potent silencing molecule. Here we focus on strategies for rational optimization of two siRNAs that target the junction sites of fusion oncogenes BCR-ABL and TMPRSS2-ERG. We demonstrate that modifying the termini of these siRNAs with a terminal G-U wobble pair or a carefully selected pair of terminal asymmetry-enhancing mismatches can result in an increase in potency at low doses. Importantly, we observed that improvements in silencing at the mRNA level do not necessarily translate to reductions in protein level and/or cell death. Decline in protein level is also heavily influenced by targeted protein half-life, and delivery vehicle toxicity can confound measures of cell death due to silencing. Therefore, for BCR-ABL, which has a long protein half-life that is difficult to overcome using siRNA, we also developed a nontoxic transfection vector: poly(lactic-coglycolic acid) nanoparticles that release siRNA over many days. We show that this system can achieve effective killing of leukemic cells. These findings provide insights into the implications of siRNA sequence for potency and suggest strategies for the design of more effective therapeutic siRNA molecules. Furthermore, this work points to the importance of integrating studies of siRNA design and delivery, while heeding and addressing potential limitations such as restricted targetable mRNA regions, long protein half-lives, and nonspecific toxicities.

  9. A combination of trastuzumab and BAG-1 inhibition synergistically targets HER2 positive breast cancer cells.

    Science.gov (United States)

    Papadakis, Emmanouil; Robson, Natalia; Yeomans, Alison; Bailey, Sarah; Laversin, Stephanie; Beers, Stephen; Sayan, A Emre; Ashton-Key, Margaret; Schwaiger, Stefan; Stuppner, Hermann; Troppmair, Jakob; Packham, Graham; Cutress, Ramsey

    2016-04-05

    Treatment of HER2+ breast cancer with trastuzumab is effective and combination anti-HER2 therapies have demonstrated benefit over monotherapy in the neoadjuvant and metastatic settings. This study investigated the therapeutic potential of targeting the BAG-1 protein co-chaperone in trastuzumab-responsive or -resistant cells. In the METABRIC dataset, BAG-1 mRNA was significantly elevated in HER2+ breast tumors and predicted overall survival in a multivariate analysis (HR = 0.81; p = 0.022). In a breast cell line panel, BAG-1 protein was increased in HER2+ cells and was required for optimal growth as shown by siRNA knockdown. Overexpression of BAG-1S in HER2+ SKBR3 cells blocked growth inhibition by trastuzumab, whereas overexpression of a mutant BAG-1S protein (BAG-1S H3AB), defective in binding HSC70, potentiated the effect of trastuzumab. Injection of a Tet-On SKBR3 clone, induced to overexpress myc-BAG-1S into the mammary fat pads of immunocompromised mice, resulted in 2-fold larger tumors compared to uninduced controls. Induction of myc-BAG-1S expression in two Tet-On SKBR3 clones attenuated growth inhibition by trastuzumab in vitro. Targeting endogenous BAG-1 by siRNA enhanced growth inhibition of SKBR3 and BT474 cells by trastuzumab, while BAG-1 protein-protein interaction inhibitor (Thio-S or Thio-2) plus trastuzumab combination treatment synergistically attenuated growth. In BT474 cells this reduced protein synthesis, caused G1/S cell cycle arrest and targeted the ERK and AKT signaling pathways. In a SKBR3 subpopulation with acquired resistance to trastuzumab BAG-1 targeting remained effective and either Thio-2 or BAG-1 siRNA reduced growth more compared to trastuzumab-responsive parental cells. In summary, targeting BAG-1 function in combination with anti-HER2 therapy might prove beneficial.

  10. Rice and Bean Targets for Biofortification Combined with High Carotenoid Content Crops Regulate Transcriptional Mechanisms Increasing Iron Bioavailability.

    Science.gov (United States)

    Dias, Desirrê Morais; de Castro Moreira, Maria Eliza; Gomes, Mariana Juste Contin; Lopes Toledo, Renata Celi; Nutti, Marilia Regini; Pinheiro Sant'Ana, Helena Maria; Martino, Hércia Stampini Duarte

    2015-11-23

    Iron deficiency affects thousands of people worldwide. Biofortification of staple food crops aims to support the reduction of this deficiency. This study evaluates the effect of combinations of common beans and rice, targets for biofortification, with high carotenoid content crops on the iron bioavailability, protein gene expression, and antioxidant effect. Iron bioavailability was measured by the depletion/repletion method. Seven groups were tested (n = 7): Pontal bean (PB); rice + Pontal bean (R + BP); Pontal bean + sweet potato (PB + SP); Pontal bean + pumpkin (PB + P); Pontal bean + rice + sweet potato (PB + R + P); Pontal bean + rice + sweet potato (PB + R + SP); positive control (Ferrous Sulfate). The evaluations included: hemoglobin gain, hemoglobin regeneration efficiency (HRE), gene expression of divalente metal transporter 1 (DMT-1), duodenal citocromo B (DcytB), ferroportin, hephaestin, transferrin and ferritin and total plasma antioxidant capacity (TAC). The test groups, except the PB, showed higher HRE (p common beans, and those with high carotenoid content crops increased protein gene expression, increasing the iron bioavailability and antioxidant capacity.

  11. Nano-siRNA Particles and Combination Therapies for Ovarian Tumor Targeting

    Science.gov (United States)

    2015-08-01

    reaction parameters. Treating the multi-RNAi-MS particles with different polycations, such as poly-L- lysine (PLL) and polyethylene imine (PEI), yielded...perspective and guidance in pre-clinical studies of specific combination drug/siRNA sets for targeting OC, and in the use of appropriate animal models...an amino-acid linker spacer exhibit potent antagonism that suppress the ErbB3-NRG dimerization and reduce the downstream signaling pathways of ErbB3

  12. Efficient vaccine against pandemic influenza: combining DNA vaccination and targeted delivery to MHC class II molecules.

    Science.gov (United States)

    Grødeland, Gunnveig; Bogen, Bjarne

    2015-06-01

    There are two major limitations to vaccine preparedness in the event of devastating influenza pandemics: the time needed to generate a vaccine and rapid generation of sufficient amounts. DNA vaccination could represent a solution to these problems, but efficacy needs to be enhanced. In a separate line of research, it has been established that targeting of vaccine molecules to antigen-presenting cells enhances immune responses. We have combined the two principles by constructing DNA vaccines that encode bivalent fusion proteins; these target hemagglutinin to MHC class II molecules on antigen-presenting cells. Such DNA vaccines rapidly induce hemagglutinin-specific antibodies and T cell responses in immunized mice. Responses are long-lasting and protect mice against challenge with influenza virus. In a pandemic situation, targeted DNA vaccines could be produced and tested within a month. The novel DNA vaccines could represent a solution to pandemic preparedness in the advent of novel influenza pandemics.

  13. Potential benefits of combining cytosine deaminase/5-fluorocytosine gene therapy and irradiation for prostate cancer. Experimental study

    Energy Technology Data Exchange (ETDEWEB)

    Kato, Hiroaki; Koshida, Kiyoshi; Yokoyama, Kunihiko; Mizokami, Atsushi; Namiki, Mikio [Kanazawa Univ. (Japan). School of Medicine

    2002-10-01

    The purpose of this study was to investigate the potential of combining cytosine deaminase/5-fluorocytosine (CD/5-FC) gene therapy and radiation therapy (either external beam radiation or radioimmunotherapy [RIT]), for the treatment of prostate cancer. Tumor xenografts of CD-transduced LNCaP cells grown in the testes of severe combined immunodeficiency (SCID) mice were used to evaluate antitumor effect. The mice were injected intraperitoneally with 500 mg/kg of 5-FC, or with 5, 15 or 30 mg/kg of 5-fluorouracil (5-FU), for 9 days. The tumors were treated with fractionated radiation at a dose of 1 or 3 Gy/day for 3 days, or I-131 labelled anti-prostate specific antigen (anti-PSA) monoclonal antibody (mAb) administration at a subtherapeutic dose of 20 or 80 {mu}Ci. Intratumoral and serum concentrations of 5-FU were measured using high performance liquid chromatography. Mice treated with CD/5-FC gene therapy presented a significant tumor growth inhibition comparable to that obtained with 15 mg/kg, 5-FU systemic administration without marked weight loss. Treatment with CD/5-FC gene therapy resulted in higher tumor but lower serum concentrations of 5-FU than treatment with systemic 5-FU chemotherapy. An additive antitumor effect was obtained when CD/5-FC therapy was combined with 1 Gy irradiation, which by itself did not produce a significant antitumor effect. However, the efficacy of CD/5-FC therapy was not enhanced when combined with RIT, probably due to poor accumulation of the mAb as the tumor/blood ratio never exceeded 1. These findings indicate that CD/5-FC gene therapy for prostate cancer may function with enhanced antitumor effect when combined with external beam radiation. However, combining CD/5-FC gene therapy and RIT using an anti-PSA mAb may not be effective because of insufficient accumulation of the mAb at the target tumors. (author)

  14. Potential benefits of combining cytosine deaminase/5-fluorocytosine gene therapy and irradiation for prostate cancer. Experimental study

    International Nuclear Information System (INIS)

    Kato, Hiroaki; Koshida, Kiyoshi; Yokoyama, Kunihiko; Mizokami, Atsushi; Namiki, Mikio

    2002-01-01

    The purpose of this study was to investigate the potential of combining cytosine deaminase/5-fluorocytosine (CD/5-FC) gene therapy and radiation therapy (either external beam radiation or radioimmunotherapy [RIT]), for the treatment of prostate cancer. Tumor xenografts of CD-transduced LNCaP cells grown in the testes of severe combined immunodeficiency (SCID) mice were used to evaluate antitumor effect. The mice were injected intraperitoneally with 500 mg/kg of 5-FC, or with 5, 15 or 30 mg/kg of 5-fluorouracil (5-FU), for 9 days. The tumors were treated with fractionated radiation at a dose of 1 or 3 Gy/day for 3 days, or I-131 labelled anti-prostate specific antigen (anti-PSA) monoclonal antibody (mAb) administration at a subtherapeutic dose of 20 or 80 μCi. Intratumoral and serum concentrations of 5-FU were measured using high performance liquid chromatography. Mice treated with CD/5-FC gene therapy presented a significant tumor growth inhibition comparable to that obtained with 15 mg/kg, 5-FU systemic administration without marked weight loss. Treatment with CD/5-FC gene therapy resulted in higher tumor but lower serum concentrations of 5-FU than treatment with systemic 5-FU chemotherapy. An additive antitumor effect was obtained when CD/5-FC therapy was combined with 1 Gy irradiation, which by itself did not produce a significant antitumor effect. However, the efficacy of CD/5-FC therapy was not enhanced when combined with RIT, probably due to poor accumulation of the mAb as the tumor/blood ratio never exceeded 1. These findings indicate that CD/5-FC gene therapy for prostate cancer may function with enhanced antitumor effect when combined with external beam radiation. However, combining CD/5-FC gene therapy and RIT using an anti-PSA mAb may not be effective because of insufficient accumulation of the mAb at the target tumors. (author)

  15. A genome-wide screen for genetic variants that modify the recruitment of REST to its target genes.

    Directory of Open Access Journals (Sweden)

    Rory Johnson

    Full Text Available Increasing numbers of human diseases are being linked to genetic variants, but our understanding of the mechanistic links leading from DNA sequence to disease phenotype is limited. The majority of disease-causing nucleotide variants fall within the non-protein-coding portion of the genome, making it likely that they act by altering gene regulatory sequences. We hypothesised that SNPs within the binding sites of the transcriptional repressor REST alter the degree of repression of target genes. Given that changes in the effective concentration of REST contribute to several pathologies-various cancers, Huntington's disease, cardiac hypertrophy, vascular smooth muscle proliferation-these SNPs should alter disease-susceptibility in carriers. We devised a strategy to identify SNPs that affect the recruitment of REST to target genes through the alteration of its DNA recognition element, the RE1. A multi-step screen combining genetic, genomic, and experimental filters yielded 56 polymorphic RE1 sequences with robust and statistically significant differences of affinity between alleles. These SNPs have a considerable effect on the the functional recruitment of REST to DNA in a range of in vitro, reporter gene, and in vivo analyses. Furthermore, we observe allele-specific biases in deeply sequenced chromatin immunoprecipitation data, consistent with predicted differenes in RE1 affinity. Amongst the targets of polymorphic RE1 elements are important disease genes including NPPA, PTPRT, and CDH4. Thus, considerable genetic variation exists in the DNA motifs that connect gene regulatory networks. Recently available ChIP-seq data allow the annotation of human genetic polymorphisms with regulatory information to generate prior hypotheses about their disease-causing mechanism.

  16. A Genome-Wide Screen for Genetic Variants That Modify the Recruitment of REST to Its Target Genes

    Science.gov (United States)

    Johnson, Rory; Richter, Nadine; Bogu, Gireesh K.; Bhinge, Akshay; Teng, Siaw Wei; Choo, Siew Hua; Andrieux, Lise O.; de Benedictis, Cinzia; Jauch, Ralf; Stanton, Lawrence W.

    2012-01-01

    Increasing numbers of human diseases are being linked to genetic variants, but our understanding of the mechanistic links leading from DNA sequence to disease phenotype is limited. The majority of disease-causing nucleotide variants fall within the non-protein-coding portion of the genome, making it likely that they act by altering gene regulatory sequences. We hypothesised that SNPs within the binding sites of the transcriptional repressor REST alter the degree of repression of target genes. Given that changes in the effective concentration of REST contribute to several pathologies—various cancers, Huntington's disease, cardiac hypertrophy, vascular smooth muscle proliferation—these SNPs should alter disease-susceptibility in carriers. We devised a strategy to identify SNPs that affect the recruitment of REST to target genes through the alteration of its DNA recognition element, the RE1. A multi-step screen combining genetic, genomic, and experimental filters yielded 56 polymorphic RE1 sequences with robust and statistically significant differences of affinity between alleles. These SNPs have a considerable effect on the the functional recruitment of REST to DNA in a range of in vitro, reporter gene, and in vivo analyses. Furthermore, we observe allele-specific biases in deeply sequenced chromatin immunoprecipitation data, consistent with predicted differenes in RE1 affinity. Amongst the targets of polymorphic RE1 elements are important disease genes including NPPA, PTPRT, and CDH4. Thus, considerable genetic variation exists in the DNA motifs that connect gene regulatory networks. Recently available ChIP–seq data allow the annotation of human genetic polymorphisms with regulatory information to generate prior hypotheses about their disease-causing mechanism. PMID:22496669

  17. An analysis of possible off target effects following CAS9/CRISPR targeted deletions of neuropeptide gene enhancers from the mouse genome.

    Science.gov (United States)

    Hay, Elizabeth Anne; Khalaf, Abdulla Razak; Marini, Pietro; Brown, Andrew; Heath, Karyn; Sheppard, Darrin; MacKenzie, Alasdair

    2017-08-01

    We have successfully used comparative genomics to identify putative regulatory elements within the human genome that contribute to the tissue specific expression of neuropeptides such as galanin and receptors such as CB1. However, a previous inability to rapidly delete these elements from the mouse genome has prevented optimal assessment of their function in-vivo. This has been solved using CAS9/CRISPR genome editing technology which uses a bacterial endonuclease called CAS9 that, in combination with specifically designed guide RNA (gRNA) molecules, cuts specific regions of the mouse genome. However, reports of "off target" effects, whereby the CAS9 endonuclease is able to cut sites other than those targeted, limits the appeal of this technology. We used cytoplasmic microinjection of gRNA and CAS9 mRNA into 1-cell mouse embryos to rapidly generate enhancer knockout mouse lines. The current study describes our analysis of the genomes of these enhancer knockout lines to detect possible off-target effects. Bioinformatic analysis was used to identify the most likely putative off-target sites and to design PCR primers that would amplify these sequences from genomic DNA of founder enhancer deletion mouse lines. Amplified DNA was then sequenced and blasted against the mouse genome sequence to detect off-target effects. Using this approach we were unable to detect any evidence of off-target effects in the genomes of three founder lines using any of the four gRNAs used in the analysis. This study suggests that the problem of off-target effects in transgenic mice have been exaggerated and that CAS9/CRISPR represents a highly effective and accurate method of deleting putative neuropeptide gene enhancer sequences from the mouse genome. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  18. Tumor-targeted inhibition by a novel strategy - mimoretrovirus expressing siRNA targeting the Pokemon gene.

    Science.gov (United States)

    Tian, Zhiqiang; Wang, Huaizhi; Jia, Zhengcai; Shi, Jinglei; Tang, Jun; Mao, Liwei; Liu, Hongli; Deng, Yijing; He, Yangdong; Ruan, Zhihua; Li, Jintao; Wu, Yuzhang; Ni, Bing

    2010-12-01

    Pokemon gene has crucial but versatile functions in cell differentiation, proliferation and tumorigenesis. It is a master regulator of the ARF-HDM2-p53 and Rb-E2F pathways. The facts that the expression of Pokemon is essential for tumor formation and many kinds of tumors over-express the Pokemon gene make it an attractive target for therapeutic intervention for cancer treatment. In this study, we used an RNAi strategy to silence the Pokemon gene in a cervical cancer model. To address the issues involving tumor specific delivery and durable expression of siRNA, we applied the Arg-Gly-Asp (RGD) peptide ligand and polylysine (K(18)) fusion peptide to encapsulate a recombinant retrovirus plasmid expressing a siRNA targeting the Pokemon gene and produced the 'mimoretrovirus'. At charge ratio 2.0 of fusion peptide/plasmid, the mimoretrovirus formed stable and homogenous nanoparticles, and provided complete DNase I protection and complete gel retardation. This nanoparticle inhibited SiHa cell proliferation and invasion, while it promoted SiHa cell apoptosis. The binding of the nanoparticle to SiHa cells was mediated via the RGD-integrin α(v)β(3) interaction, as evidenced by the finding that unconjugated RGD peptide inhibited this binding significantly. This tumor-targeting mimoretrovirus exhibited excellent anti-tumor capacity in vivo in a nude mouse model. Moreover, the mimoretrovirus inhibited tumor growth with a much higher efficiency than recombinant retrovirus expressing siRNA or the K(18)/P4 nanoparticle lacking the RGD peptide. Results suggest that the RNAi/RGD-based mimoretrovirus developed in this study represents a novel anti-tumor strategy that may be applicable to most research involving cancer therapy and, thus, has promising potential as a cervical cancer treatment.

  19. Molecular landscape of acquired resistance to targeted therapy combinations in BRAF mutant colorectal cancer

    Science.gov (United States)

    Oddo, Daniele; Sennott, Erin M.; Barault, Ludovic; Valtorta, Emanuele; Arena, Sabrina; Cassingena, Andrea; Filiciotto, Genny; Marzolla, Giulia; Elez, Elena; van Geel, Robin M.J.M.; Bartolini, Alice; Crisafulli, Giovanni; Boscaro, Valentina; Godfrey, Jason T.; Buscarino, Michela; Cancelliere, Carlotta; Linnebacher, Michael; Corti, Giorgio; Truini, Mauro; Siravegna, Giulia; Grasselli, Julieta; Gallicchio, Margherita; Bernards, René; Schellens, Jan H.M.; Tabernero, Josep; Engelman, Jeffrey A.; Sartore-Bianchi, Andrea; Bardelli, Alberto; Siena, Salvatore; Corcoran, Ryan B.; Di Nicolantonio, Federica

    2016-01-01

    Summary Although recent clinical trials of BRAF inhibitor combinations have demonstrated improved efficacy in BRAF mutant colorectal cancer, emergence of acquired resistance limits clinical benefit. Here, we undertook a comprehensive effort to define mechanisms underlying drug resistance with the goal of guiding development of therapeutic strategies to overcome this limitation. We generated a broad panel of BRAF mutant resistant cell line models across seven different clinically-relevant drug combinations. Combinatorial drug treatments were able to abrogate ERK1/2 phosphorylation in parental sensitive cells, but not in their resistant counterparts, indicating that resistant cells escaped drug treatments through one or more mechanisms leading to biochemical reactivation of the MAPK signaling pathway. Genotyping of resistant cells identified gene amplification of EGFR, KRAS and mutant BRAF, as well as acquired mutations in KRAS, EGFR, and MAP2K1. These mechanisms were clinically relevant, as we identified emergence of a KRAS G12C mutation and increase of mutant BRAF V600E allele frequency in the circulating tumor DNA of a patient at relapse from combined treatment with BRAF and MEK inhibitors. In order to identify therapeutic combinations capable of overcoming drug resistance, we performed a systematic assessment of candidate therapies across the panel of resistant cell lines. Independent of the molecular alteration acquired upon drug pressure, most resistant cells retained sensitivity to vertical MAPK pathway suppression when combinations of ERK, BRAF, and EGFR inhibitors were applied. These therapeutic combinations represent promising strategies for future clinical trials in BRAF mutant colorectal cancer. PMID:27312529

  20. Synergistic effects of ultrasound-targeted microbubble destruction and TAT peptide on gene transfection: an experimental study in vitro and in vivo.

    Science.gov (United States)

    Zhou, Zhiyi; Zhang, Ping; Ren, Jianli; Ran, Haitao; Zheng, Yuanyi; Li, Pan; Zhang, Qunxia; Zhang, Maohui; Wang, Zhigang

    2013-09-28

    Cell-permeable peptides (CPPs) and ultrasound-targeted microbubble destruction (UTMD) have tremendous potential for gene delivery. However, their applications are limited due to nonspecificity of CPPs and low transfection efficiency of UTMD. Here, we developed a 'smart' gene delivery system by encapsulating TAT peptide (TATp) and hepatocyte growth factor (HGF) gene within lipid microbubbles, in which TATp was protected from being enzymatically cleaved and HGF gene was protected from degradation. This new strategy had synergistic effects of UTMD and TATp on gene transfection. We investigated the efficacy and safety of HGF gene transfection mediated by the combination of UTMD and TATp in vitro and in vivo. The results from MTT assay and flow cytometry analyses indicated that the combination of UTMD and TATp could enhance HGF gene expression in HUVECs without any significant side effect on cell viability. In rat myocardial infarction models, we demonstrated that the protein and mRNA expressions of HGF in myocardium caused by the combination of UTMD and TATp were the highest. Histopathological findings demonstrated that the combination of UTMD and TATp enhanced myocardial microvasculature and ameliorated myocardial fibrosis. In conclusion, the combination of UTMD and TATp might be a safe and efficient technique for gene delivery. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Novel Hematopoietic Target Genes in the NRF2-Mediated Transcriptional Pathway

    Directory of Open Access Journals (Sweden)

    Michelle R. Campbell

    2013-01-01

    Full Text Available Nuclear factor- (erythroid-derived 2 like 2 (NFE2L2, NRF2 is a key transcriptional activator of the antioxidant response pathway and is closely related to erythroid transcription factor NFE2. Under oxidative stress, NRF2 heterodimerizes with small Maf proteins and binds cis-acting enhancer sequences found near oxidative stress response genes. Using the dietary isothiocyanate sulforaphane (SFN to activate NRF2, chromatin immunoprecipitation sequencing (ChIP-seq identified several hundred novel NRF2-mediated targets beyond its role in oxidative stress. Activated NRF2 bound the antioxidant response element (ARE in promoters of several known and novel target genes involved in iron homeostasis and heme metabolism, including known targets FTL and FTH1, as well as novel binding in the globin locus control region. Five novel NRF2 target genes were chosen for followup: AMBP, ABCB6, FECH, HRG-1 (SLC48A1, and TBXAS1. SFN-induced gene expression in erythroid K562 and lymphoid cells were compared for each target gene. NRF2 silencing showed reduced expression in lymphoid, lung, and hepatic cells. Furthermore, stable knockdown of NRF2 negative regulator KEAP1 in K562 cells resulted in increased NQO1, AMBP, and TBXAS1 expression. NFE2 binding sites in K562 cells revealed similar binding profiles as lymphoid NRF2 sites in all potential NRF2 candidates supporting a role for NRF2 in heme metabolism and erythropoiesis.

  2. Combining vascular and cellular targeting regimens enhances the efficacy of photodynamic therapy

    International Nuclear Information System (INIS)

    Chen Bin; Pogue, Brian W.; Hoopes, P. Jack; Hasan, Tayyaba

    2005-01-01

    Purpose: Photodynamic therapy (PDT) can be designed to target either tumor vasculature or tumor cells by varying the drug-light interval. Photodynamic therapy treatments with different drug-light intervals can be combined to increase tumor response by targeting both tumor vasculature and tumor cells. The sequence of photosensitizer and light delivery can influence the effect of combined treatments. Methods and materials: The R3327-MatLyLu rat prostate tumor model was used in this study. Photosensitizer verteporfin distribution was quantified by fluorescence microscopy. Tumor blood flow changes were monitored by laser-Doppler system and tumor hypoxia was quantified by the immunohistochemical staining for the hypoxic marker EF5. The therapeutic effects of PDT treatments were evaluated by the histologic examination and tumor regrowth assay. Results: Fluorescence microscopic studies indicated that tumor localization of verteporfin changed from predominantly within the tumor vasculature at 15 min after injection, to being throughout the tumor parenchyma at 3 h after injection. Light treatment (50 J/cm 2 ) at 15 min after verteporfin injection (0.25 mg/kg, i.v.) induced significant tumor vascular damage, as manifested by tumor blood flow reduction and increase in the tumor hypoxic fraction. In contrast, the vascular effect observed after the same light dose (50 J/cm 2 ) delivered 3 h after administration of verteporfin (1 mg/kg, i.v.) was an initial acute decrease in blood flow, followed by recovery to the level of control. The EF5 staining revealed no significant increase in hypoxic fraction at 1 h after PDT using 3 h drug-light interval. The combination of 3-h interval PDT and 15-min interval PDT was more effective in inhibiting tumor growth than each individual PDT treatment. However, it was found that the combined treatment with the sequence of 3-h interval PDT before 15-min interval PDT led to a superior antitumor effect than the other combinative PDT treatments

  3. Integrated analysis of the molecular action of Vorinostat identifies epi-sensitised targets for combination therapy.

    Science.gov (United States)

    Hay, Jodie F; Lappin, Katrina; Liberante, Fabio; Kettyle, Laura M; Matchett, Kyle B; Thompson, Alexander; Mills, Ken I

    2017-09-15

    Several histone deacetylase inhibitors including Vorinostat have received FDA approval for the treatment of haematological malignancies. However, data from these trials indicate that Vorinostat has limited efficacy as a monotherapy, prompting the need for rational design of combination therapies. A number of epi-sensitised pathways, including sonic hedgehog (SHH), were identified in AML cells by integration of global patterns of histone H3 lysine 9 (H3K9) acetylation with transcriptomic analysis following Vorinostat-treatment. Direct targeting of the SHH pathway with SANT-1, following Vorinostat induced epi-sensitisation, resulted in synergistic cell death of AML cells. In addition, xenograft studies demonstrated that combination therapy induced a marked reduction in leukemic burden compared to control or single agents. Together, the data supports epi-sensitisation as a potential component of the strategy for the rational development of combination therapies in AML.

  4. Gene Therapy for RAG-deficient Severe Combined Immunodeficiency

    NARCIS (Netherlands)

    K. Pike (Karin)

    2007-01-01

    textabstractSevere combined immunodeficiency (SCID) is a rare class of primary, inherited, immunodeficiency causing infants to suffer from persistent diarrhea, opportunistic infections and a failure to thrive. RAG proteins play a crucial role in the initiation of V(D)J recombination of

  5. Concordant and discordant regulation of target genes by miR-31 and its isoforms.

    Directory of Open Access Journals (Sweden)

    Yu-Tzu Chan

    Full Text Available It has been shown that imprecise cleavage of a primary or precursor RNA by Drosha or Dicer, respectively, may yield a group of microRNA (miRNA variants designated as "isomiR". Variations in the relative abundance of isoforms for a given miRNA among different species and different cell types beg the question whether these isomiRs might regulate target genes differentially. We compared the capacity of three miR-31 isoforms (miR-31-H, miR-31-P, and miR-31-M, which differ only slightly in their 5'- and/or 3'-end sequences, to regulate several known targets and a predicted target, Dicer. Notably, we found isomiR-31s displayed concordant and discordant regulation of 6 known target genes. Furthermore, we validated a predicted target gene, Dicer, to be a novel target of miR-31 but only miR-31-P could directly repress Dicer expression in both MCF-7 breast cancer cells and A549 lung cancer cells, resulting in their enhanced sensitivity to cisplatin, a known attribute of Dicer knockdown. This was further supported by reporter assay using full length 3'-untranslated region (UTR of Dicer. Our findings not only revealed Dicer to be a direct target of miR-31, but also demonstrated that isomiRs displayed similar and disparate regulation of target genes in cell-based systems. Coupled with the variations in the distribution of isomiRs among different cells or conditions, our findings support the possibility of fine-tuning gene expression by miRNAs.

  6. Identification of target genes of synovial sarcoma-associated fusion oncoprotein using human pluripotent stem cells.

    Science.gov (United States)

    Hayakawa, Kazuo; Ikeya, Makoto; Fukuta, Makoto; Woltjen, Knut; Tamaki, Sakura; Takahara, Naoko; Kato, Tomohisa; Sato, Shingo; Otsuka, Takanobu; Toguchida, Junya

    2013-03-22

    Synovial sarcoma (SS) is a malignant soft tissue tumor harboring chromosomal translocation t(X; 18)(p11.2; q11.2), which produces SS-specific fusion gene, SYT-SSX. Although precise function of SYT-SSX remains to be investigated, accumulating evidences suggest its role in gene regulation via epigenetic mechanisms, and the product of SYT-SSX target genes may serve as biomarkers of SS. Lack of knowledge about the cell-of-origin of SS, however, has placed obstacle in the way of target identification. Here we report a novel approach to identify SYT-SSX2 target genes using human pluripotent stem cells (hPSCs) containing a doxycycline-inducible SYT-SSX2 gene. SYT-SSX2 was efficiently induced both at mRNA and protein levels within three hours after doxycycline administration, while no morphological change of hPSCs was observed until 24h. Serial microarray analyses identified genes of which the expression level changed more than twofold within 24h. Surprisingly, the majority (297/312, 95.2%) were up-regulated genes and a result inconsistent with the current concept of SYT-SSX as a transcriptional repressor. Comparing these genes with SS-related genes which were selected by a series of in silico analyses, 49 and 2 genes were finally identified as candidates of up- and down-regulated target of SYT-SSX, respectively. Association of these genes with SYT-SSX in SS cells was confirmed by knockdown experiments. Expression profiles of SS-related genes in hPSCs and human mesenchymal stem cells (hMSCs) were strikingly different in response to the induction of SYT-SSX, and more than half of SYT-SSX target genes in hPSCs were not induced in hMSCs. These results suggest the importance of cellular context for correct understanding of SYT-SSX function, and demonstrated how our new system will help to overcome this issue. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Integrated microarray and ChIP analysis identifies multiple Foxa2 dependent target genes in the notochord.

    Science.gov (United States)

    Tamplin, Owen J; Cox, Brian J; Rossant, Janet

    2011-12-15

    The node and notochord are key tissues required for patterning of the vertebrate body plan. Understanding the gene regulatory network that drives their formation and function is therefore important. Foxa2 is a key transcription factor at the top of this genetic hierarchy and finding its targets will help us to better understand node and notochord development. We performed an extensive microarray-based gene expression screen using sorted embryonic notochord cells to identify early notochord-enriched genes. We validated their specificity to the node and notochord by whole mount in situ hybridization. This provides the largest available resource of notochord-expressed genes, and therefore candidate Foxa2 target genes in the notochord. Using existing Foxa2 ChIP-seq data from adult liver, we were able to identify a set of genes expressed in the notochord that had associated regions of Foxa2-bound chromatin. Given that Foxa2 is a pioneer transcription factor, we reasoned that these sites might represent notochord-specific enhancers. Candidate Foxa2-bound regions were tested for notochord specific enhancer function in a zebrafish reporter assay and 7 novel notochord enhancers were identified. Importantly, sequence conservation or predictive models could not have readily identified these regions. Mutation of putative Foxa2 binding elements in two of these novel enhancers abrogated reporter expression and confirmed their Foxa2 dependence. The combination of highly specific gene expression profiling and genome-wide ChIP analysis is a powerful means of understanding developmental pathways, even for small cell populations such as the notochord. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. Hyaluronic acid-functionalized polymeric nanoparticles for colon cancer-targeted combination chemotherapy

    Science.gov (United States)

    Xiao, Bo; Han, Moon Kwon; Viennois, Emilie; Wang, Lixin; Zhang, Mingzhen; Si, Xiaoying; Merlin, Didier

    2015-10-01

    Nanoparticle (NP)-based combination chemotherapy has been proposed as an effective strategy for achieving synergistic effects and targeted drug delivery for colon cancer therapy. Here, we fabricated a series of hyaluronic acid (HA)-functionalized camptothecin (CPT)/curcumin (CUR)-loaded polymeric NPs (HA-CPT/CUR-NPs) with various weight ratios of CPT to CUR (1 : 1, 2 : 1 and 4 : 1). The resultant spherical HA-CPT/CUR-NPs had a desirable particle size (around 289 nm), relative narrow size distribution, and slightly negative zeta potential. These NPs exhibited a simultaneous sustained release profile for both drugs throughout the time frame examined. Subsequent cellular uptake experiments demonstrated that the introduction of HA to the NP surface endowed NPs with colon cancer-targeting capability and markedly increased cellular uptake efficiency compared with chitosan-coated NPs. Importantly, the combined delivery of CPT and CUR in one HA-functionalized NP exerted strong synergistic effects. HA-CPT/CUR-NP (1 : 1) showed the highest antitumor activity among the three HA-CPT/CUR-NPs, resulting in an extremely low combination index. Collectively, our findings indicate that this HA-CPT/CUR-NP can be exploited as an efficient formulation for colon cancer-targeted combination chemotherapy.Nanoparticle (NP)-based combination chemotherapy has been proposed as an effective strategy for achieving synergistic effects and targeted drug delivery for colon cancer therapy. Here, we fabricated a series of hyaluronic acid (HA)-functionalized camptothecin (CPT)/curcumin (CUR)-loaded polymeric NPs (HA-CPT/CUR-NPs) with various weight ratios of CPT to CUR (1 : 1, 2 : 1 and 4 : 1). The resultant spherical HA-CPT/CUR-NPs had a desirable particle size (around 289 nm), relative narrow size distribution, and slightly negative zeta potential. These NPs exhibited a simultaneous sustained release profile for both drugs throughout the time frame examined. Subsequent cellular uptake experiments

  9. In silico prediction of novel therapeutic targets using gene-disease association data.

    Science.gov (United States)

    Ferrero, Enrico; Dunham, Ian; Sanseau, Philippe

    2017-08-29

    Target identification and validation is a pressing challenge in the pharmaceutical industry, with many of the programmes that fail for efficacy reasons showing poor association between the drug target and the disease. Computational prediction of successful targets could have a considerable impact on attrition rates in the drug discovery pipeline by significantly reducing the initial search space. Here, we explore whether gene-disease association data from the Open Targets platform is sufficient to predict therapeutic targets that are actively being pursued by pharmaceutical companies or are already on the market. To test our hypothesis, we train four different classifiers (a random forest, a support vector machine, a neural network and a gradient boosting machine) on partially labelled data and evaluate their performance using nested cross-validation and testing on an independent set. We then select the best performing model and use it to make predictions on more than 15,000 genes. Finally, we validate our predictions by mining the scientific literature for proposed therapeutic targets. We observe that the data types with the best predictive power are animal models showing a disease-relevant phenotype, differential expression in diseased tissue and genetic association with the disease under investigation. On a test set, the neural network classifier achieves over 71% accuracy with an AUC of 0.76 when predicting therapeutic targets in a semi-supervised learning setting. We use this model to gain insights into current and failed programmes and to predict 1431 novel targets, of which a highly significant proportion has been independently proposed in the literature. Our in silico approach shows that data linking genes and diseases is sufficient to predict novel therapeutic targets effectively and confirms that this type of evidence is essential for formulating or strengthening hypotheses in the target discovery process. Ultimately, more rapid and automated target

  10. Microbial population index and community structure in saline-alkaline soil using gene targeted metagenomics.

    Science.gov (United States)

    Keshri, Jitendra; Mishra, Avinash; Jha, Bhavanath

    2013-03-30

    Population indices of bacteria and archaea were investigated from saline-alkaline soil and a possible microbe-environment pattern was established using gene targeted metagenomics. Clone libraries were constructed using 16S rRNA and functional gene(s) involved in carbon fixation (cbbL), nitrogen fixation (nifH), ammonia oxidation (amoA) and sulfur metabolism (apsA). Molecular phylogeny revealed the dominance of Actinobacteria, Firmicutes and Proteobacteria along with archaeal members of Halobacteraceae. The library consisted of novel bacterial (20%) and archaeal (38%) genera showing ≤95% similarity to previously retrieved sequences. Phylogenetic analysis indicated ability of inhabitant to survive in stress condition. The 16S rRNA gene libraries contained novel gene sequences and were distantly homologous with cultured bacteria. Functional gene libraries were found unique and most of the clones were distantly related to Proteobacteria, while clones of nifH gene library also showed homology with Cyanobacteria and Firmicutes. Quantitative real-time PCR exhibited that bacterial abundance was two orders of magnitude higher than archaeal. The gene(s) quantification indicated the size of the functional guilds harboring relevant key genes. The study provides insights on microbial ecology and different metabolic interactions occurring in saline-alkaline soil, possessing phylogenetically diverse groups of bacteria and archaea, which may be explored further for gene cataloging and metabolic profiling. Copyright © 2012 Elsevier GmbH. All rights reserved.

  11. Significant impact of miRNA-target gene networks on genetics of human complex traits.

    Science.gov (United States)

    Okada, Yukinori; Muramatsu, Tomoki; Suita, Naomasa; Kanai, Masahiro; Kawakami, Eiryo; Iotchkova, Valentina; Soranzo, Nicole; Inazawa, Johji; Tanaka, Toshihiro

    2016-03-01

    The impact of microRNA (miRNA) on the genetics of human complex traits, especially in the context of miRNA-target gene networks, has not been fully assessed. Here, we developed a novel analytical method, MIGWAS, to comprehensively evaluate enrichment of genome-wide association study (GWAS) signals in miRNA-target gene networks. We applied the method to the GWAS results of the 18 human complex traits from >1.75 million subjects, and identified significant enrichment in rheumatoid arthritis (RA), kidney function, and adult height (P impact of miRNA-target gene networks on the genetics of human complex traits, and provided resources which should contribute to drug discovery and nucleic acid medicine.

  12. Amplification of distant estrogen response elements deregulates target genes associated with tamoxifen resistance in breast cancer.

    Science.gov (United States)

    Hsu, Pei-Yin; Hsu, Hang-Kai; Lan, Xun; Juan, Liran; Yan, Pearlly S; Labanowska, Jadwiga; Heerema, Nyla; Hsiao, Tzu-Hung; Chiu, Yu-Chiao; Chen, Yidong; Liu, Yunlong; Li, Lang; Li, Rong; Thompson, Ian M; Nephew, Kenneth P; Sharp, Zelton D; Kirma, Nameer B; Jin, Victor X; Huang, Tim H-M

    2013-08-12

    A causal role of gene amplification in tumorigenesis is well known, whereas amplification of DNA regulatory elements as an oncogenic driver remains unclear. In this study, we integrated next-generation sequencing approaches to map distant estrogen response elements (DEREs) that remotely control the transcription of target genes through chromatin proximity. Two densely mapped DERE regions located on chromosomes 17q23 and 20q13 were frequently amplified in estrogen receptor-α-positive luminal breast cancer. These aberrantly amplified DEREs deregulated target gene expression potentially linked to cancer development and tamoxifen resistance. Progressive accumulation of DERE copies was observed in normal breast progenitor cells chronically exposed to estrogenic chemicals. These findings may extend to other DNA regulatory elements, the amplification of which can profoundly alter target transcriptome during tumorigenesis. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. Generation of B cell-deficient pigs by highly efficient CRISPR/Cas9-mediated gene targeting.

    Science.gov (United States)

    Chen, Fengjiao; Wang, Ying; Yuan, Yilin; Zhang, Wei; Ren, Zijian; Jin, Yong; Liu, Xiaorui; Xiong, Qiang; Chen, Qin; Zhang, Manling; Li, Xiaokang; Zhao, Lihua; Li, Ze; Wu, Zhaoqiang; Zhang, Yanfei; Hu, Feifei; Huang, Juan; Li, Rongfeng; Dai, Yifan

    2015-08-20

    Generating B cell-deficient mutant is the first step to produce human antibody repertoires in large animal models. In this study, we applied the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system to target the JH region of the pig IgM heavy chain gene which is crucial for B cell development and differentiation. Transfection of IgM-targeting Cas9 plasmid in primary porcine fetal fibroblasts (PFFs) enabled inducing gene knock out (KO) in up to 53.3% of colonies analyzed, a quarter of which harbored biallelic modification, which was much higher than that of the traditional homologous recombination (HR). With the aid of somatic cell nuclear transfer (SCNT) technology, three piglets with the biallelic IgM heavy chain gene mutation were produced. The piglets showed no antibody-producing B cells which indicated that the biallelic mutation of the IgM heavy chain gene effectively knocked out the function of the IgM and resulted in a B cell-deficient phenotype. Our study suggests that the CRISPR/Cas9 system combined with SCNT technology is an efficient genome-editing approach in pigs. Copyright © 2015 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

  14. JAK2/IDH-mutant–driven myeloproliferative neoplasm is sensitive to combined targeted inhibition

    Science.gov (United States)

    McKenney, Anna Sophia; Somasundara, Amritha Varshini Hanasoge; Spitzer, Barbara; Intlekofer, Andrew M.; Ahn, Jihae; Shank, Kaitlyn; Rapaport, Franck T.; Patel, Minal A.; Papalexi, Efthymia; Shih, Alan H.; Chiu, April; Freinkman, Elizaveta; Akbay, Esra A.; Steadman, Mya; Nagaraja, Raj; Yen, Katharine; Teruya-Feldstein, Julie; Wong, Kwok-Kin; Rampal, Raajit; Thompson, Craig B.

    2018-01-01

    Patients with myeloproliferative neoplasms (MPNs) frequently progress to bone marrow failure or acute myeloid leukemia (AML), and mutations in epigenetic regulators such as the metabolic enzyme isocitrate dehydrogenase (IDH) are associated with poor outcomes. Here, we showed that combined expression of Jak2V617F and mutant IDH1R132H or Idh2R140Q induces MPN progression, alters stem/progenitor cell function, and impairs differentiation in mice. Jak2V617F Idh2R140Q–mutant MPNs were sensitive to small-molecule inhibition of IDH. Combined inhibition of JAK2 and IDH2 normalized the stem and progenitor cell compartments in the murine model and reduced disease burden to a greater extent than was seen with JAK inhibition alone. In addition, combined JAK2 and IDH2 inhibitor treatment also reversed aberrant gene expression in MPN stem cells and reversed the metabolite perturbations induced by concurrent JAK2 and IDH2 mutations. Combined JAK2 and IDH2 inhibitor therapy also showed cooperative efficacy in cells from MPN patients with both JAK2mut and IDH2mut mutations. Taken together, these data suggest that combined JAK and IDH inhibition may offer a therapeutic advantage in this high-risk MPN subtype. PMID:29355841

  15. Tyrosine kinase receptor inhibitor-targeted combined chemotherapy for metastatic bladder cancer

    Directory of Open Access Journals (Sweden)

    Chia-Lun Wu

    2012-04-01

    increased subG1 in cell cycle was seen in the epirubicin and sunitinib combination treatment group. The activation of apoptosis pathway was confirmed by increased cleaved caspase-3 and cleaved PARP in MBT-2 cells. In tail vein tumor inoculation C3H mice model, epirubicin alone and sunitinib combination therapy decreased tumor growth in lungs with marginal effect. Sunitinib and epirubicin combination had shown a synergistic cytotoxic effect and inhibited cell migration ability in MBT-2 cells. The combination can induce cell cycle arrest at G2/M phase and increase subG1 cells. Metastatic animal study also showed that sunitinib combined with epirubicin has a marginal effect on inhibition of tumor growth of lungs. The tyrosine kinase receptor inhibitor-targeted combined chemotherapy regimen may provide as a new treatment modality for advanced bladder cancer in the future.

  16. Human disease MiRNA inference by combining target information based on heterogeneous manifolds.

    Science.gov (United States)

    Ding, Pingjian; Luo, Jiawei; Liang, Cheng; Xiao, Qiu; Cao, Buwen

    2018-04-01

    The emergence of network medicine has provided great insight into the identification of disease-related molecules, which could help with the development of personalized medicine. However, the state-of-the-art methods could neither simultaneously consider target information and the known miRNA-disease associations nor effectively explore novel gene-disease associations as a by-product during the process of inferring disease-related miRNAs. Computational methods incorporating multiple sources of information offer more opportunities to infer disease-related molecules, including miRNAs and genes in heterogeneous networks at a system level. In this study, we developed a novel algorithm, named inference of Disease-related MiRNAs based on Heterogeneous Manifold (DMHM), to accurately and efficiently identify miRNA-disease associations by integrating multi-omics data. Graph-based regularization was utilized to obtain a smooth function on the data manifold, which constitutes the main principle of DMHM. The novelty of this framework lies in the relatedness between diseases and miRNAs, which are measured via heterogeneous manifolds on heterogeneous networks integrating target information. To demonstrate the effectiveness of DMHM, we conducted comprehensive experiments based on HMDD datasets and compared DMHM with six state-of-the-art methods. Experimental results indicated that DMHM significantly outperformed the other six methods under fivefold cross validation and de novo prediction tests. Case studies have further confirmed the practical usefulness of DMHM. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. Identification of miRNAs and their target genes in developing soybean seeds by deep sequencing

    Directory of Open Access Journals (Sweden)

    Chen Shou-Yi

    2011-01-01

    Full Text Available Abstract Background MicroRNAs (miRNAs regulate gene expression by mediating gene silencing at transcriptional and post-transcriptional levels in higher plants. miRNAs and related target genes have been widely studied in model plants such as Arabidopsis and rice; however, the number of identified miRNAs in soybean (Glycine max is limited, and global identification of the related miRNA targets has not been reported in previous research. Results In our study, a small RNA library and a degradome library were constructed from developing soybean seeds for deep sequencing. We identified 26 new miRNAs in soybean by bioinformatic analysis and further confirmed their expression by stem-loop RT-PCR. The miRNA star sequences of 38 known miRNAs and 8 new miRNAs were also discovered, providing additional evidence for the existence of miRNAs. Through degradome sequencing, 145 and 25 genes were identified as targets of annotated miRNAs and new miRNAs, respectively. GO analysis indicated that many of the identified miRNA targets may function in soybean seed development. Additionally, a soybean homolog of Arabidopsis SUPPRESSOR OF GENE SLIENCING 3 (AtSGS3 was detected as a target of the newly identified miRNA Soy_25, suggesting the presence of feedback control of miRNA biogenesis. Conclusions We have identified large numbers of miRNAs and their related target genes through deep sequencing of a small RNA library and a degradome library. Our study provides more information about the regulatory network of miRNAs in soybean and advances our understanding of miRNA functions during seed development.

  18. Drosophila switch gene Sex-lethal can bypass its switch-gene target transformer to regulate aspects of female behavior

    Science.gov (United States)

    Evans, Daniel S.; Cline, Thomas W.

    2013-01-01

    The switch gene Sex-lethal (Sxl) was thought to elicit all aspects of Drosophila female somatic differentiation other than size dimorphism by controlling only the switch gene transformer (tra). Here we show instead that Sxl controls an aspect of female sexual behavior by acting on a target other than or in addition to tra. We inferred the existence of this unknown Sxl target from the observation that a constitutively feminizing tra transgene that restores fertility to tra− females failed to restore fertility to Sxl-mutant females that were adult viable but functionally tra−. The sterility of these mutant females was caused by an ovulation failure. Because tra expression is not sufficient to render these Sxl-mutant females fertile, we refer to this pathway as the tra-insufficient feminization (TIF) branch of the sex-determination regulatory pathway. Using a transgene that conditionally expresses two Sxl feminizing isoforms, we find that the TIF branch is required developmentally for neurons that also sex-specifically express fruitless, a tra gene target controlling sexual behavior. Thus, in a subset of fruitless neurons, targets of the TIF and tra pathways appear to collaborate to control ovulation. In most insects, Sxl has no sex-specific functions, and tra, rather than Sxl, is both the target of the primary sex signal and the gene that maintains the female developmental commitment via positive autoregulation. The TIF pathway may represent an ancestral female-specific function acquired by Sxl in an early evolutionary step toward its becoming the regulator of tra in Drosophila. PMID:24191002

  19. Targeted sequencing of venom genes from cone snail genomes improves understanding of conotoxin molecular evolution.

    Science.gov (United States)

    Phuong, Mark A; Mahardika, Gusti N

    2018-03-05

    To expand our capacity to discover venom sequences from the genomes of venomous organisms, we applied targeted sequencing techniques to selectively recover venom gene superfamilies and non-toxin loci from the genomes of 32 cone snail species (family, Conidae), a diverse group of marine gastropods that capture their prey using a cocktail of neurotoxic peptides (conotoxins). We were able to successfully recover conotoxin gene superfamilies across all species with high confidence (> 100X coverage) and used these data to provide new insights into conotoxin evolution. First, we found that conotoxin gene superfamilies are composed of 1-6 exons and are typically short in length (mean = ∼85bp). Second, we expanded our understanding of the following genetic features of conotoxin evolution: (a) positive selection, where exons coding the mature toxin region were often three times more divergent than their adjacent noncoding regions, (b) expression regulation, with comparisons to transcriptome data showing that cone snails only express a fraction of the genes available in their genome (24%-63%), and (c) extensive gene turnover, where Conidae species varied from 120-859 conotoxin gene copies. Finally, using comparative phylogenetic methods, we found that while diet specificity did not predict patterns of conotoxin evolution, dietary breadth was positively correlated with total conotoxin gene diversity. Overall, the targeted sequencing technique demonstrated here has the potential to radically increase the pace at which venom gene families are sequenced and studied, reshaping our ability to understand the impact of genetic changes on ecologically relevant phenotypes and subsequent diversification.

  20. SVMRFE based approach for prediction of most discriminatory gene target for type II diabetes

    Directory of Open Access Journals (Sweden)

    Atul Kumar

    2017-06-01

    Full Text Available Type II diabetes is a chronic condition that affects the way our body metabolizes sugar. The body's important source of fuel is now becoming a chronic disease all over the world. It is now very necessary to identify the new potential targets for the drugs which not only control the disease but also can treat it. Support vector machines are the classifier which has a potential to make a classification of the discriminatory genes and non-discriminatory genes. SVMRFE a modification of SVM ranks the genes based on their discriminatory power and eliminate the genes which are not involved in causing the disease. A gene regulatory network has been formed with the top ranked coding genes to identify their role in causing diabetes. To further validate the results pathway study was performed to identify the involvement of the coding genes in type II diabetes. The genes obtained from this study showed a significant involvement in causing the disease, which may be used as a potential drug target.

  1. Targeted delivery of genes to endothelial cells and cell- and gene-based therapy in pulmonary vascular diseases.

    Science.gov (United States)

    Suen, Colin M; Mei, Shirley H J; Kugathasan, Lakshmi; Stewart, Duncan J

    2013-10-01

    Pulmonary arterial hypertension (PAH) is a devastating disease that, despite significant advances in medical therapies over the last several decades, continues to have an extremely poor prognosis. Gene therapy is a method to deliver therapeutic genes to replace defective or mutant genes or supplement existing cellular processes to modify disease. Over the last few decades, several viral and nonviral methods of gene therapy have been developed for preclinical PAH studies with varying degrees of efficacy. However, these gene delivery methods face challenges of immunogenicity, low transduction rates, and nonspecific targeting which have limited their translation to clinical studies. More recently, the emergence of regenerative approaches using stem and progenitor cells such as endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) have offered a new approach to gene therapy. Cell-based gene therapy is an approach that augments the therapeutic potential of EPCs and MSCs and may deliver on the promise of reversal of established PAH. These new regenerative approaches have shown tremendous potential in preclinical studies; however, large, rigorously designed clinical studies will be necessary to evaluate clinical efficacy and safety. © 2013 American Physiological Society. Compr Physiol 3:1749-1779, 2013.

  2. Integrative Analysis of CRISPR/Cas9 Target Sites in the Human HBB Gene

    Directory of Open Access Journals (Sweden)

    Yumei Luo

    2015-01-01

    Full Text Available Recently, the clustered regularly interspaced short palindromic repeats (CRISPR system has emerged as a powerful customizable artificial nuclease to facilitate precise genetic correction for tissue regeneration and isogenic disease modeling. However, previous studies reported substantial off-target activities of CRISPR system in human cells, and the enormous putative off-target sites are labor-intensive to be validated experimentally, thus motivating bioinformatics methods for rational design of CRISPR system and prediction of its potential off-target effects. Here, we describe an integrative analytical process to identify specific CRISPR target sites in the human β-globin gene (HBB and predict their off-target effects. Our method includes off-target analysis in both coding and noncoding regions, which was neglected by previous studies. It was found that the CRISPR target sites in the introns have fewer off-target sites in the coding regions than those in the exons. Remarkably, target sites containing certain transcriptional factor motif have enriched binding sites of relevant transcriptional factor in their off-target sets. We also found that the intron sites have fewer SNPs, which leads to less variation of CRISPR efficiency in different individuals during clinical applications. Our studies provide a standard analytical procedure to select specific CRISPR targets for genetic correction.

  3. A PCA3 gene-based transcriptional amplification system targeting primary prostate cancer

    OpenAIRE

    Neveu, Bertrand; Jain, Pallavi; T?tu, Bernard; Wu, Lily; Fradet, Yves; Pouliot, Fr?d?ric

    2015-01-01

    Targeting specifically primary prostate cancer (PCa) cells for immune therapy, gene therapy or molecular imaging is of high importance. The PCA3 long non-coding RNA is a unique PCa biomarker and oncogene that has been widely studied. This gene has been mainly exploited as an accurate diagnostic urine biomarker for PCa detection. In this study, the PCA3 promoter was introduced into a new transcriptional amplification system named the 3-Step Transcriptional Amplification System (PCA3-3STA) and ...

  4. In Vivo Targeted Gene Transfer by Direct Irradiation with Nanosecond Pulsed Laser

    Science.gov (United States)

    Ogura, Makoto; Sato, Shunichi; Ashida, Hiroshi; Obara, Minoru

    2004-10-01

    We demonstrated in vivo targeted gene transfer to rat skin by direct irradiation with nanosecond laser pulses without major side effects. Expressions of enhanced green fluorescent protein (EGFP) were observed only in the area irradiated with laser pulses; in the skin, epidermal cells were selectively transfected. Unlike other physical methods, this method enables noncontact gene transfer. Moreover, the laser intensity required in this method is as low as 20 MW/cm2, and thus fiber-based beam delivery is possible.

  5. Disabled-2 is a FOXP3 target gene required for regulatory T cell function

    OpenAIRE

    Jain, N; Nguyen, H; Friedline, RH; Malhotra, N; Brehm, M; Koyonagi, M; Bix, M; Cooper, JA; Chambers, CA; Kang, J

    2009-01-01

    FOXP3 expressing regulatory T cells are vital for maintaining peripheral T cell tolerance and homeostasis. The mechanisms by which FOXP3 target genes orchestrate context-dependent Treg cell function are largely unknown. Here we show that in mouse peripheral lymphocytes, the Drosophila Disabled-2 (Dab2) homolog, a gene that is involved in enhancing TGFβ responses, is exclusively expressed in FOXP3+ regulatory T cells. Dab2 is a direct target of FOXP3 and regulatory T cells lacking DAB2 are fun...

  6. Cooperative interactions between CBP and TORC2 confer selectivity to CREB target gene expression

    DEFF Research Database (Denmark)

    Ravnskjær, Kim; Kester, Henri; Liu, Yi

    2007-01-01

    , but have minimal effects on CRE-dependent transcription. Here, we show that the latent cytoplasmic coactivator TORC2 mediates target gene activation in response to cAMP signaling by associating with CBP/p300 and increasing its recruitment to a subset of CREB target genes. TORC2 is not activated in response...... to stress signals, however; and in its absence, P-CREB is unable to stimulate CRE-dependent transcription, due to a block in CBP recruitment. The effect of TORC2 on CBP/p300 promoter occupancy appears pivotal because a gain of function mutant CREB polypeptide with increased affinity for CBP restored CRE...

  7. Enteropeptidase: a gene associated with a starvation human phenotype and a novel target for obesity treatment.

    Directory of Open Access Journals (Sweden)

    Sandrine Braud

    Full Text Available BACKGROUND: Obesity research focuses essentially on gene targets associated with the obese phenotype. None of these targets have yet provided a viable drug therapy. Focusing instead on genes that are involved in energy absorption and that are associated with a "human starvation phenotype", we have identified enteropeptidase (EP, a gene associated with congenital enteropeptidase deficiency, as a novel target for obesity treatment. The advantages of this target are that the gene is expressed exclusively in the brush border of the intestine; it is peripheral and not redundant. METHODOLOGY/PRINCIPAL FINDINGS: Potent and selective EP inhibitors were designed around a boroarginine or borolysine motif. Oral administration of these compounds to mice restricted the bioavailability of dietary energy, and in a long-term treatment it significantly diminished the rate of increase in body weight, despite ad libitum food intake. No adverse reactions of the type seen with lipase inhibitors, such as diarrhea or steatorrhea, were observed. This validates EP as a novel, druggable target for obesity treatment. CONCLUSIONS: In vivo testing of novel boroarginine or borolysine-based EP inhibitors validates a novel approach to the treatment of obesity.

  8. A meta analysis of pancreatic microarray datasets yields new targets as cancer genes and biomarkers.

    Directory of Open Access Journals (Sweden)

    Nalin C W Goonesekere

    Full Text Available The lack of specific symptoms at early tumor stages, together with a high biological aggressiveness of the tumor contribute to the high mortality rate for pancreatic cancer (PC, which has a five year survival rate of less than 5%. Improved screening for earlier diagnosis, through the detection of diagnostic and prognostic biomarkers provides the best hope of increasing the rate of curatively resectable carcinomas. Though many serum markers have been reported to be elevated in patients with PC, so far, most of these markers have not been implemented into clinical routine due to low sensitivity or specificity. In this study, we have identified genes that are significantly upregulated in PC, through a meta-analysis of large number of microarray datasets. We demonstrate that the biological functions ascribed to these genes are clearly associated with PC and metastasis, and that that these genes exhibit a strong link to pathways involved with inflammation and the immune response. This investigation has yielded new targets for cancer genes, and potential biomarkers for pancreatic cancer. The candidate list of cancer genes includes protein kinase genes, new members of gene families currently associated with PC, as well as genes not previously linked to PC. In this study, we are also able to move towards developing a signature for hypomethylated genes, which could be useful for early detection of PC. We also show that the significantly upregulated 800+ genes in our analysis can serve as an enriched pool for tissue and serum protein biomarkers in pancreatic cancer.

  9. Detection of targeted GFP-Hox gene fusions during mouse embryogenesis.

    Science.gov (United States)

    Godwin, A R; Stadler, H S; Nakamura, K; Capecchi, M R

    1998-10-27

    The ability to use a vital cell marker to study mouse embryogenesis will open new avenues of experimental research. Recently, the use of transgenic mice, containing multiple copies of the jellyfish gene encoding the green fluorescent protein (GFP), has begun to realize this potential. Here, we show that the fluorescent signals produced by single-copy, targeted GFP in-frame fusions with two different murine Hox genes, Hoxa1 and Hoxc13, are readily detectable by using confocal microscopy. Since Hoxa1 is expressed early and Hoxc13 is expressed late in mouse embryogenesis, this study shows that single-copy GFP gene fusions can be used through most of mouse embryogenesis. Previously, targeted lacZ gene fusions have been very useful for analyzing mouse mutants. Use of GFP gene fusions extends the benefits of targeted lacZ gene fusions by providing the additional utility of a vital marker. Our analysis of the Hoxc13(GFPneo) embryos reveals GFP expression in each of the sites expected from analysis of Hoxc13(lacZneo) embryos. Similarly, Hoxa1(GFPneo) expression was detected in all of the sites predicted from RNA in situ analysis. GFP expression in the foregut pocket of Hoxa1(GFPneo) embryos suggests a role for Hoxa1 in foregut-mediated differentiation of the cardiogenic mesoderm.

  10. Targeted disruption of Ataxia-telangiectasia mutated gene in miniature pigs by somatic cell nuclear transfer

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Young June; Ahn, Kwang Sung; Kim, Minjeong; Kim, Min Ju; Park, Sang-Min; Ryu, Junghyun; Ahn, Jin Seop; Heo, Soon Young; Kang, Jee Hyun; Choi, You Jung [Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan (Korea, Republic of); Choi, Seong-Jun [Institute of Tissue Regeneration Engineering, Dankook University, Cheonan (Korea, Republic of); Shim, Hosup, E-mail: shim@dku.edu [Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan (Korea, Republic of); Institute of Tissue Regeneration Engineering, Dankook University, Cheonan (Korea, Republic of); Department of Physiology, Dankook University School of Medicine, Cheonan (Korea, Republic of)

    2014-10-03

    Highlights: • ATM gene-targeted pigs were produced by somatic cell nuclear transfer. • A novel large animal model for ataxia telangiectasia was developed. • The new model may provide an alternative to the mouse model. - Abstract: Ataxia telangiectasia (A-T) is a recessive autosomal disorder associated with pleiotropic phenotypes, including progressive cerebellar degeneration, gonad atrophy, and growth retardation. Even though A-T is known to be caused by the mutations in the Ataxia telangiectasia mutated (ATM) gene, the correlation between abnormal cellular physiology caused by ATM mutations and the multiple symptoms of A-T disease has not been clearly determined. None of the existing ATM mouse models properly reflects the extent to which neurological degeneration occurs in human. In an attempt to provide a large animal model for A-T, we produced gene-targeted pigs with mutations in the ATM gene by somatic cell nuclear transfer. The disrupted allele in the ATM gene of cloned piglets was confirmed via PCR and Southern blot analysis. The ATM gene-targeted pigs generated in the present study may provide an alternative to the current mouse model for the study of mechanisms underlying A-T disorder and for the development of new therapies.

  11. Functional characterization of endogenous siRNA target genes in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Heikkinen Liisa

    2008-06-01

    Full Text Available Abstract Background Small interfering RNA (siRNA molecules mediate sequence specific silencing in RNA interference (RNAi, a gene regulatory phenomenon observed in almost all organisms. Large scale sequencing of small RNA libraries obtained from C. elegans has revealed that a broad spectrum of siRNAs is endogenously transcribed from genomic sequences. The biological role and molecular diversity of C. elegans endogenous siRNA (endo-siRNA molecules, nonetheless, remain poorly understood. In order to gain insight into their biological function, we annotated two large libraries of endo-siRNA sequences, identified their cognate targets, and performed gene ontology analysis to identify enriched functional categories. Results Systematic trends in categorization of target genes according to the specific length of siRNA sequences were observed: 18- to 22-mer siRNAs were associated with genes required for embryonic development; 23-mers were associated uniquely with post-embryonic development; 24–26-mers were associated with phosphorus metabolism or protein modification. Moreover, we observe that some argonaute related genes associate with siRNAs with multiple reads. Sequence frequency graphs suggest that different lengths of siRNAs share similarities in overall sequence structure: the 5' end begins with G, while the body predominates with U and C. Conclusion These results suggest that the lengths of endogenous siRNA molecules are consequential to their biological functions since the gene ontology categories for their cognate mRNA targets vary depending upon their lengths.

  12. Combining immunotherapy with oncogene-targeted therapy: a new road for melanoma treatment

    Directory of Open Access Journals (Sweden)

    Mariana eAris

    2015-02-01

    Full Text Available Cutaneous melanoma arises from the malignant transformation of skin melanocytes; its incidence and mortality have been increasing steadily over the last fifty-years, now representing 3% of total tumors. Once melanoma metastasizes, prognosis is somber and therapeutic options are limited. However, the discovery of prevalent BRAF mutations in at least 50% of melanoma tumors led to development of BRAF inhibitors, and other drugs targeting the MAPK pathway including MEK inhibitors, are changing this reality. These recently approved treatments for metastatic melanoma have made a significant impact on patient survival; though the results are shadowed by the appearance of drug-resistance. Combination therapies provide a rational strategy to potentiate efficacy and potentially overcome resistance. Undoubtedly, the last decade has also born an renaissance of immunotherapy, and encouraging advances in metastatic melanoma treatment are illuminating the road. Immune checkpoint blockades, such as CTLA-4 antagonist-antibodies, and multiple cancer vaccines are now invaluable arms of anti-tumor therapy. Recent work has brought to light the delicate relationship between tumor biology and the immune system. Host immunity contributes to the antitumor activity of oncogene-targeted inhibitors within a complex network of cytokines and chemokines. Therefore, combining immunotherapy with oncogene-targeted drugs may be the key to melanoma control. Here we review ongoing clinical studies of combination therapies using both oncogene inhibitors and immunotherapeutic strategies in melanoma patients. We will revisit the preclinical evidence that tested sequential and concurrent schemes in suitable animal models and formed the basis for the current trials. Finally, we will discuss potential future directions of the field.

  13. Targeted Chemo-Photodynamic Combination Platform Based on the DOX Prodrug Nanoparticles for Enhanced Cancer Therapy.

    Science.gov (United States)

    Zhang, Yumin; Huang, Fan; Ren, Chunhua; Yang, Lijun; Liu, Jianfeng; Cheng, Zhen; Chu, Liping; Liu, Jinjian

    2017-04-19

    Chemo-photodynamic combination therapy has been received widespread attention in cancer treatment due to its excellent characteristics, such as reducing the adverse side effects of chemo-drugs and improving the therapeutic effects for various cancers. In this study, RGD and DOX was conjugated to PEG by thiol-ene addition and Schiff's base reaction, respectively, to prepare the targeted and pH-sensitive antitumor prodrug nanoparticles (RGD-PEG-DOX NPs, RGD-NPs). Subsequently, the photosensitizer chlorin e6 (Ce6) was encapsulated into RGD-NPs, thus obtaining a simple and efficient chemo-photodynamic combination platform (RGD-PEG-DOX/Ce6 NPs, RGD-NPs/Ce6). This nanoparticle possessed high drug loading property of both the chemo-drug and photosensitizer and could simultaneously release them under the mild acidic microenvironment of cancer cells, which was expected to realize the synchronization therapy of chemotherapy and photodynamic therapy (PDT). Compared with free DOX and Ce6, RGD-NPs/Ce6 could significantly improve the cellular uptake capacities of DOX and Ce6, resulting in the increased contents of ROS in cancer cells and effective cytotoxicity for tumor cells (MDA-MB-231 cells and MCF-7 cells) upon a laser radiation. The in vivo experiment showed that RGD-NPs/Ce6 displayed superior tumor targeting, accumulation, and retention ability than the other groups (free DOX, free Ce6 and NPs/Ce6), and thus significantly enhancing the antitumor effect in vivo with a laser radiation. In addition, the cardiotoxicity induced by DOX was thoroughly wiped out after being loaded and delivered by the nanoparticles according to the pathological analysis. Therefore, the targeted chemo-photodynamic combination therapeutic platform may be a promising candidate for enhanced cancer therapy.

  14. Generation of Gene-Edited Chrysanthemum morifolium Using Multicopy Transgenes as Targets and Markers.

    Science.gov (United States)

    Kishi-Kaboshi, Mitsuko; Aida, Ryutaro; Sasaki, Katsutomo

    2017-02-01

    The most widely used gene editing technology-the CRISPR/Cas9 system-employs a bacterial monomeric DNA endonuclease known as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) and single-guide RNA (sgRNA) that directs Cas9 to a complementary target DNA. However, introducing mutations into higher polyploid plant species, especially for species without genome information, has been difficult. Chrysanthemum morifolium (chrysanthemum) is one of the most important ornamental plants, but it is a hexaploid with a large genome; moreover, it lacks whole-genome information. These characteristics hinder genome editing in chrysanthemum. In the present study, we attempted to perform gene editing using the CRISPR/Cas9 system to introduce mutations into chrysanthemum. We constructed transgenic chrysanthemum plants expressing the yellowish-green fluorescent protein gene from Chiridius poppei (CpYGFP) and targeted CpYGFP for gene editing. We compared the activity of a Cauliflower mosaic virus (CaMV) 35S promoter and parsley ubiquitin promoter in chrysanthemum calli and chose the parsley ubiquitin promoter to drive Cas9. We selected two sgRNAs to target different positions in the CpYGFP gene and obtained transgenic calli containing mutated CpYGFP genes (CRISPR-CpYGFP-chrysanthemum). A DNA sequencing analysis and fluorescence observations indicated that cells containing the mutated CpYGFP gene grew independently of cells containing the original CpYGFP gene in one callus. We finally obtained the CRISPR-CpYGFP-chrysanthemum shoot containing a mutation in the CpYGFP sequence. This is the first report of gene editing using the CRISPR/Cas9 system in chrysanthemum and sheds light on chrysanthemum genome editing. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  15. In silico identification and characterization of microRNAs and their putative target genes in Solanaceae plants.

    Science.gov (United States)

    Kim, Hyun-Jin; Baek, Kwang-Hyun; Lee, Bong-Woo; Choi, Doil; Hur, Cheol-Goo

    2011-02-01

    MicroRNAs (miRNAs) are a class of small, single-stranded, noncoding RNAs ranging from 19 to 25 nucleotides. The miRNA control various cellular functions by negatively regulating gene expression at the post-transcriptional level. The miRNA regulation over their target genes has a central role in regulating plant growth and development; however, only a few reports have been published on the function of miRNAs in the family Solanaceae. We identified Solanaceae miRNAs and their target genes by analyzing expressed sequence tag (EST) data from five different Solanaceae species. A comprehensive bioinformatic analysis of EST data of Solanaceae species revealed the presence of at least 11 miRNAs and 54 target genes in pepper (Capsicum annuum L.), 22 miRNAs and 221 target genes in potato (Solanum tuberosum L.), 12 miRNAs and 417 target genes in tomato (Solanum lycopersicum L.), 46 miRNAs and 60 target genes in tobacco (Nicotiana tabacum L.), and 7 miRNAs and 28 target genes in Nicotiana benthamiana. The identified Solanaceae miRNAs and their target genes were deposited in the SolmiRNA database, which is freely available for academic research only at http://genepool.kribb.re.kr/SolmiRNA. Our data indicate that the Solanaceae family has both conserved and specific miRNAs and that their target genes may play important roles in growth and development of Solanaceae plants.

  16. Modern dose-finding designs for cancer phase I trials drug combinations and molecularly targeted agents

    CERN Document Server

    Hirakawa, Akihiro; Daimon, Takashi; Matsui, Shigeyuki

    2018-01-01

    This book deals with advanced methods for adaptive phase I dose-finding clinical trials for combination of two agents and molecularly targeted agents (MTAs) in oncology. It provides not only methodological aspects of the dose-finding methods, but also software implementations and practical considerations in applying these complex methods to real cancer clinical trials. Thus, the book aims to furnish researchers in biostatistics and statistical science with a good summary of recent developments of adaptive dose-finding methods as well as providing practitioners in biostatistics and clinical investigators with advanced materials for designing, conducting, monitoring, and analyzing adaptive dose-finding trials. The topics in the book are mainly related to cancer clinical trials, but many of those topics are potentially applicable or can be extended to trials for other diseases. The focus is mainly on model-based dose-finding methods for two kinds of phase I trials. One is clinical trials with combinations of tw...

  17. Construction of a mouse model of factor VIII deficiency by gene targeting

    Energy Technology Data Exchange (ETDEWEB)

    Bi, L.; Lawler, A.; Gearhart, J. [Univ. of Pennsylvania School of Medicine, Philadelphia, PA (United States)] [and others

    1994-09-01

    To develop a small animal model of hemophilia A for gene therapy experiments, we set out to construct a mouse model for factor VIII deficiency by gene targeting. First, we screened a mouse liver cDNA library using a human FVIII cDNA probe. We cloned a 2.6 Kb partial mouse factor VIII cDNA which extends from 800 base pairs of the 3{prime} end of exon 14 to the 5{prime} end of exon 26. A mouse genomic library made from strain 129 was then screened to obtain genomic fragments covering the exons desired for homologous recombination. Two genomic clones were obtained, and one covering exon 15 through 22 was used for gene targeting. To make gene targeting constructs, a 5.8 Kb genomic DNA fragment covering exons 15 to 19 of the mouse FVIII gene was subcloned, and the neo expression cassette was inserted into exons 16 and 17 separately by different strategies. These two constructs were named MFVIIIC-16 and MFVIIIC-17. The constructs were linearized and transfected into strain 129 mouse ES cells by electroporation. Factor VIII gene-knockout ES cell lines were selected by G-418 and screened by genomic Southern blots. Eight exon 16 targeted cell lines and five exon 17 targeted cell lines were obtained. Three cell lines from each construct were injected into blastocysts and surgically transferred into foster mothers. Multiple chimeric mice with 70-90% hair color derived from the ES-cell genotype were seen with both constructs. Germ line transmission of the ES-cell genotype has been obtained for the MFVIIIC-16 construct, and multiple hemophilia A carrier females have been identified. Factor VIII-deficient males will be conceived soon.

  18. The effects of transfection reagent polyethyleneimine (PEI) and non-targeting control siRNAs on global gene expression in human aortic smooth muscle cells.

    Science.gov (United States)

    Raof, Nurazhani A; Rajamani, Deepa; Chu, Hsun-Chieh; Gurav, Aniket; Johnson, Joel M; LoGerfo, Frank W; Pradhan-Nabzdyk, Leena; Bhasin, Manoj

    2016-01-05

    RNA interference (RNAi) is a powerful platform utilized to target transcription of specific genes and downregulate the protein product. To achieve effective silencing, RNAi is usually applied to cells or tissue with a transfection reagent to enhance entry into cells. A commonly used control is the same transfection reagent plus a "noncoding RNAi". However, this does not control for the genomic response to the transfection reagent alone or in combination with the noncoding RNAi. These control effects while not directly targeting the gene in question may influence expression of other genes that in turn alter expression of the target. The current study was prompted by our work focused on prevention of vascular bypass graft failure and our experience with gene silencing in human aortic smooth muscle cells (HAoSMCs) where we suspected that off target effects through this mechanism might be substantial. We have used Next Generation Sequencing (NGS) technology and bioinformatics analysis to examine the genomic response of HAoSMCs to the transfection reagent alone (polyethyleneimine (PEI)) or in combination with commercially obtained control small interfering RNA (siRNAs) (Dharmacon and Invitrogen). Compared to untreated cells, global gene expression of HAoSMcs after transfection either with PEI or in combination with control siRNAs displayed significant alterations in gene transcriptome after 24 h. HAoSMCs transfected by PEI alone revealed alterations of 213 genes mainly involved in inflammatory and immune responses. HAoSMCs transfected by PEI complexed with siRNA from either Dharmacon or Invitrogen showed substantial gene variation of 113 and 85 genes respectively. Transfection of cells with only PEI or with PEI and control siRNAs resulted in identification of 20 set of overlapping altered genes. Further, systems biology analysis revealed key master regulators in cells transfected with control siRNAs including the cytokine, Interleukin (IL)-1, transcription factor GATA

  19. Inducing gene expression by targeting promoter sequences using small activating RNAs

    Directory of Open Access Journals (Sweden)

    Ji Wang

    2015-02-01

    Full Text Available Vector-based systems comprised of exogenous nucleic acid sequences remain the standard for ectopic expression of a particular gene. Such systems offer robust overexpression, but have inherent drawbacks such the tedious process of construction, excluding sequences (e.g. introns and untranslated regions important for gene function and potential insertional mutagenesis of host genome associated with the use of viral vectors. We and others have recently reported that short double-stranded RNAs (dsRNAs can induce endogenous gene expression by targeting promoter sequences in a phenomenon referred to as RNA activation (RNAa and such dsRNAs are termed small activating RNAs (saRNAs. To date, RNAa has been successfully utilized to induce the expression of different genes such as tumor suppressor genes. Here, we describe a detailed protocol for target selection and dsRNA design with associated experiments to facilitate RNAa in cultured cells. This technique may be applied to selectively activate endogenous gene expression for studying gene function, interrogating molecular pathways and reprogramming cell fate.

  20. Targeted sequencing of cancer-related genes in colorectal cancer using next-generation sequencing.

    Directory of Open Access Journals (Sweden)

    Sae-Won Han

    Full Text Available Recent advance in sequencing technology has enabled comprehensive profiling of genetic alterations in cancer. We have established a targeted sequencing platform using next-generation sequencing (NGS technology for clinical use, which can provide mutation and copy number variation data. NGS was performed with paired-end library enriched with exons of 183 cancer-related genes. Normal and tumor tissue pairs of 60 colorectal adenocarcinomas were used to test feasibility. Somatic mutation and copy number alteration were analyzed. A total of 526 somatic non-synonymous sequence variations were found in 113 genes. Among these, 278 single nucleotide variations were 232 different somatic point mutations. 216 SNV were 79 known single nucleotide polymorphisms in the dbSNP. 32 indels were 28 different indel mutations. Median number of mutated gene per tumor was 4 (range 0-23. Copy number gain (>X2 fold was found in 65 genes in 40 patients, whereas copy number loss (genes in 39 patients. The most frequently altered genes (mutation and/or copy number alteration were APC in 35 patients (58%, TP53 in 34 (57%, and KRAS in 24 (40%. Altered gene list revealed ErbB signaling pathway as the most commonly involved pathway (25 patients, 42%. Targeted sequencing platform using NGS technology is feasible for clinical use and provides comprehensive genetic alteration data.

  1. Identification of the Drosophila skpA gene as a novel target of the transcription factor DREF

    International Nuclear Information System (INIS)

    Dang Thi Phuong Thao; Ida, Hiroyuki; Yoshida, Hideki; Yamaguchi, Masamitsu

    2006-01-01

    SKPa is component of a Drosophila SCF complex that functions in combination with the ubiquitin-conjugating enzyme UbcD1. skpA null mutation results in centrosome overduplication, unusual chromatin condensation, defective endoreduplication and cell-cycle progression. While the molecular mechanisms that regulate expression of the skpA gene are poorly understood, the DNA replication-related element (DRE) and the DRE-binding factor (DREF) play important roles in regulating proliferation-related genes in Drosophila and DRE (5'-TATCGATA) and DRE-like (5'-CATCGATT) sequences were here found to be involved in skpA promoter activity. Thus both luciferase transient expression assays in cultured Drosophila S2 cells using skpA promoter-luciferase fusion plasmids and anti-lacZ immunostaining of various tissues from transgenic third instar larvae carrying the skpA promoter-lacZ fusion genes provided supportive evidence. Furthermore, anti-SKPa immunostaining of eye imaginal discs from flies overexpressing DREF showed ectopic expression of protein in the region posterior to the morphogenetic furrow where DREF is overexpressed. Knockdown of DREF in some tissues where SKPa distribution is well known almost completely abrogated the skpA gene expression. These findings, taken together, indicate that the Drosophila skpA gene is a novel target of the transcription factor DREF

  2. Analysis of the siRNA-Mediated Gene Silencing Process Targeting Three Homologous Genes Controlling Soybean Seed Oil Quality.

    Directory of Open Access Journals (Sweden)

    Sha Lu

    Full Text Available In the past decade, RNA silencing has gained significant attention because of its success in genomic scale research and also in the genetic improvement of crop plants. However, little is known about the molecular basis of siRNA processing in association with its target transcript. To reveal this process for improving hpRNA-mediated gene silencing in crop plants, the soybean GmFAD3 gene family was chosen as a test model. We analyzed RNAi mutant soybean lines in which three members of the GmFAD3 gene family were silenced. The silencing levels of FAD3A, FAD3B and FAD3C were correlated with the degrees of sequence homology between the inverted repeat of hpRNA and the GmFAD3 transcripts in the RNAi lines. Strikingly, transgenes in two of the three RNAi lines were heavily methylated, leading to a dramatic reduction of hpRNA-derived siRNAs. Small RNAs corresponding to the loop portion of the hairpin transcript were detected while much lower levels of siRNAs were found outside of the target region. siRNAs generated from the 318-bp inverted repeat were found to be diced much more frequently at stem sequences close to the loop and associated with the inferred cleavage sites on the target transcripts, manifesting "hot spots". The top candidate hpRNA-derived siRNA share certain sequence features with mature miRNA. This is the first comprehensive and detailed study revealing the siRNA-mediated gene silencing mechanism in crop plants using gene family GmFAD3 as a test model.

  3. Analysis of the siRNA-Mediated Gene Silencing Process Targeting Three Homologous Genes Controlling Soybean Seed Oil Quality.

    Science.gov (United States)

    Lu, Sha; Yin, Xiaoyan; Spollen, William; Zhang, Ning; Xu, Dong; Schoelz, James; Bilyeu, Kristin; Zhang, Zhanyuan J

    2015-01-01

    In the past decade, RNA silencing has gained significant attention because of its success in genomic scale research and also in the genetic improvement of crop plants. However, little is known about the molecular basis of siRNA processing in association with its target transcript. To reveal this process for improving hpRNA-mediated gene silencing in crop plants, the soybean GmFAD3 gene family was chosen as a test model. We analyzed RNAi mutant soybean lines in which three members of the GmFAD3 gene family were silenced. The silencing levels of FAD3A, FAD3B and FAD3C were correlated with the degrees of sequence homology between the inverted repeat of hpRNA and the GmFAD3 transcripts in the RNAi lines. Strikingly, transgenes in two of the three RNAi lines were heavily methylated, leading to a dramatic reduction of hpRNA-derived siRNAs. Small RNAs corresponding to the loop portion of the hairpin transcript were detected while much lower levels of siRNAs were found outside of the target region. siRNAs generated from the 318-bp inverted repeat were found to be diced much more frequently at stem sequences close to the loop and associated with the inferred cleavage sites on the target transcripts, manifesting "hot spots". The top candidate hpRNA-derived siRNA share certain sequence features with mature miRNA. This is the first comprehensive and detailed study revealing the siRNA-mediated gene silencing mechanism in crop plants using gene family GmFAD3 as a test model.

  4. Targeting the NF-κB Pathway as a Combination Therapy for Advanced Thyroid Cancer.

    Directory of Open Access Journals (Sweden)

    Nikita Pozdeyev

    Full Text Available NF-κB signaling plays an important role in tumor cell proliferation, cell survival, angiogenesis, invasion, metastasis and drug/radiation resistance. Combination therapy involving NF-κB pathway inhibition is an attractive strategy for the treatment of advanced forms of thyroid cancer. This study was designed to test the efficacy of NF-κB pathway inhibition in combination with cytotoxic chemotherapy, using docetaxel and ionizing radiation in in vitro models of thyroid cancer. We found that while both docetaxel and ionizing radiation activated NF-κB signaling in thyroid cancer cells, there was no synergistic effect on cell proliferation and/or programmed cell death with either genetic (transduction of a dominant negative mutant form of IκBα or pharmacologic (proteasome inhibitor bortezomib and IKKβ inhibitor GO-Y030 inhibition of the NF-κB pathway in thyroid cancer cell lines BCPAP, 8505C, THJ16T and SW1736. Docetaxel plus bortezomib synergistically decreased in vitro invasion of 8505C cells, but not in the other cell lines. Screening of a panel of clinically relevant targeted therapies for synergy with genetic NF-κB inhibition in a proliferation/cytotoxicity assay identified the histone deacetylase (HDAC inhibitor suberoylanilide hydroxamic acid (SAHA as a potential candidate. However, the synergistic effect was confirmed only in the BCPAP cells. These results indicate that NF-κB inhibitors are unlikely to be beneficial as combination therapy with taxane cytotoxic chemotherapy, external radiation therapy or radioiodine therapy. There may be unique circumstances where NF-κB inhibitors may be considered in combination with docetaxel to reduce tumor invasion or in combination with HDAC inhibitors to reduce tumor growth, but this does not appear to be a combination therapy that could be broadly applied to patients with advanced thyroid cancer. Further research may identify which subsets of patients/tumors may respond to this therapeutic

  5. Mining predicted essential genes of Brugia malayi for nematode drug targets.

    Directory of Open Access Journals (Sweden)

    Sanjay Kumar

    Full Text Available We report results from the first genome-wide application of a rational drug target selection methodology to a metazoan pathogen genome, the completed draft sequence of Brugia malayi, a parasitic nematode responsible for human lymphatic filariasis. More than 1.5 billion people worldwide are at risk of contracting lymphatic filariasis and onchocerciasis, a related filarial disease. Drug treatments for filariasis have not changed significantly in over 20 years, and with the risk of resistance rising, there is an urgent need for the development of new anti-filarial drug therapies. The recent publication of the draft genomic sequence for B. malayi enables a genome-wide search for new drug targets. However, there is no functional genomics data in B. malayi to guide the selection of potential drug targets. To circumvent this problem, we have utilized the free-living model nematode Caenorhabditis elegans as a surrogate for B. malayi. Sequence comparisons between the two genomes allow us to map C. elegans orthologs to B. malayi genes. Using these orthology mappings and by incorporating the extensive genomic and functional genomic data, including genome-wide RNAi screens, that already exist for C. elegans, we identify potentially essential genes in B. malayi. Further incorporation of human host genome sequence data and a custom algorithm for prioritization enables us to collect and rank nearly 600 drug target candidates. Previously identified potential drug targets cluster near the top of our prioritized list, lending credibility to our methodology. Over-represented Gene Ontology terms, predicted InterPro domains, and RNAi phenotypes of C. elegans orthologs associated with the potential target pool are identified. By virtue of the selection procedure, the potential B. malayi drug targets highlight components of key processes in nematode biology such as central metabolism, molting and regulation of gene expression.

  6. Evaluation of nested PCR in detection of Helicobacter pylori targeting a highly conserved gene: HSP60.

    Science.gov (United States)

    Singh, Varsha; Mishra, Shrutkirti; Rao, G R K; Jain, Ashok Kumar; Dixit, V K; Gulati, Anil Kumar; Mahajan, Divya; McClelland, Michael; Nath, Gopal

    2008-02-01

    To comparatively evaluate a new nested set of primers designed for the detection of Helicobacter pylori targeting a highly conserved heat shock protein gene (Hsp60). A total of 60 subjects having peptic ulcer diseases were tested for the detection of H. pylori using rapid urease test (RUT), histology, culture, and polymerase chain reaction (PCR) in their antral biopsy specimens. A newly designed Hsp60 gene-based primer set was evaluated against commonly used PCR primers for detection of H. pylori. Forty-six of the 60 study subjects were found positive for culture isolation and all the 46 culture-positive specimens were also positive with Hsp60 gene PCR. Of the 46 culture-positive specimens, 44 were positive for 16S rRNA gene, ureC gene, RUT, and histology whereas only 29 were positive with ureA gene PCR. Of the 14 culture-negative subjects, 10 were positive with 16S rRNA gene, 4 were positive with ureC (glmM) gene PCR, and 2 were positive with RUT and 1 was positive on histology. This study shows that nested amplification targeting Hsp60 gene is the most sensitive and specific with LR+ and LR- values of proportional, variant and 0, respectively, when compared with the other three PCR methods. Also, HSP60 gene-specific nested protocol was the most appropriate for detection of H. pylori in clinical specimens. This is particularly valuable because it can be used as a noninvasive method for detecting H. pylori infection in young children and also, in follow-up studies with peptic ulcer patients, on samples like feces and saliva.

  7. Regulation of the β-globin gene family expression, useful in the search for new therapeutic targets for hemoglobinopathies

    Directory of Open Access Journals (Sweden)

    Karen G. Scheps

    2016-12-01

    Full Text Available Different hemoglobin isoforms are expressed during the embryonic, fetal and postnatal stages. They are formed by combination of polypeptide chains synthesized from the α- and β-globin gene clusters. Based on the fact that the presence of high hemoglobin F levels is beneficial in both sickle cell disease and severe thalassemic syndromes, a revision of the regulation of the β-globin cluster expression is proposed, especially regarding the genes encoding the y-globin chains (HBG1 and HBG2. In this review we describe the current knowledge about transcription factors and epigenetic regulators involved in the switches of the β-globin cluster. It is expected that the consolidation of knowledge in this field will allow finding new therapeutic targets for the treatment of hemoglobinopathies.

  8. The down regulation of target genes by photo activated DNA nanoscissors.

    Science.gov (United States)

    Tsai, Tsung-Lin; Shieh, Dar-Bin; Yeh, Chen-Sheng; Tzeng, Yonhua; Htet, Khant; Chuang, Kao-Shu; Hwu, Jih Ru; Su, Wu-Chou

    2010-09-01

    An artificial, targeted, light-activated nanoscissor (ATLANS) was developed for precision photonic cleavage of DNA at selectable target sequences. The ATLANS is comprised of nanoparticle core and a monolayer of hydrazone-modified triplex-forming oligonucleotides (TFOs), which recognize and capture the targeted DNA duplex. Upon photo-illumination (lambda = 460 nm), the attached hydrazone scissor specifically cleaves the targeted DNA at a pre-designed nucleotide pair. Electrophoretic mobility shift and co-precipitation assays revealed sequence-specific binding with the short-fragment and long-form plasmid DNA of both TFO and TFO-nanoparticle probes. Upon photo-illumination, ATLANS introduced a precise double-stranded break 12bp downstream the TFO binding sequence and down-regulated the target gene in HeLa cell system. Gold nanoparticles multiplexed the cutting efficiency and potential for simultaneous manipulation of multiple targets, as well as protected DNA from non-specific photo-damage. This photon-mediated DNA manipulation technology will facilitate high spatial and temporal precision in simultaneous silencing at the genome level, and advanced simultaneous manipulation of multiple targeted genes. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  9. Generation of novel resistance genes using mutation and targeted gene editing

    Science.gov (United States)

    Classical breeding for virus resistance is a lengthy process and is restricted by the availability of resistance genes. Precise genome editing is a "dream technology" to improve plants for virus resistance and these tools have opened new and very promising ways to generate virus resistant plants by ...

  10. Multiple functions of CREB-binding protein during postembryonic development: identification of target genes.

    Science.gov (United States)

    Roy, Amit; George, Smitha; Palli, Subba Reddy

    2017-12-29

    Juvenile hormones (JH) and ecdysteroids control postembryonic development in insects. They serve as valuable targets for pest management. Hence, understanding the molecular mechanisms of their action is of crucial importance. CREB-binding protein (CBP) is a universal transcriptional co-regulator. It controls the expression of several genes including those from hormone signaling pathways through co-activation of many transcription factors. However, the role of CBP during postembryonic development in insects is not well understood. Therefore, we have studied the role of CBP in postembryonic development in Tribolium, a model coleopteran insect. CBP is ubiquitously expressed in the red flour beetle, Tribolium castaneum. RNA interference (RNAi) mediated knockdown of CBP resulted in a decrease in JH induction of Kr-h1 gene expression in Tribolium larvae and led to a block in their development. Moreover, the injection of CBP double-stranded RNA (dsRNA) showed lethal phenotypes within 8 days of injection. RNA-seq and subsequent differential gene expression analysis identified CBP target genes in Tribolium. Knockdown of CBP caused a decrease in the expression of 1306 genes coding for transcription factors and other proteins associated with growth and development. Depletion of CBP impaired the expression of several JH response genes (e.g., Kr-h1, Hairy, early trypsin) and ecdysone response genes (EcR, E74, E75, and broad complex). Further, GO enrichment analyses of the downregulated genes showed enrichment in different functions including developmental processes, pigmentation, anatomical structure development, regulation of biological and cellular processes, etc. These data suggest diverse but crucial roles for CBP during postembryonic development in the coleopteran model insect, Tribolium. It can serve as a target for RNAi mediated pest management of this stored product pest.

  11. Gene Delivery Particle Engineering Strategies for Shape-dependent Targeting of Cells and Tissues.

    Science.gov (United States)

    Kozielski, Kristen L; Sitti, Metin

    2017-01-01

    Successful gene delivery requires overcoming both systemic and intracellular obstacles before the nucleic acid cargo can successfully reach its tissue and subcellular target location. Materials & Methods: Non-viral mechanisms to enable targeting while avoiding off-target delivery have arisen via biological, chemical, and physical engineering strategies. Herein we will discuss the physical parameters in particle design that promote tissue- and cell-targeted delivery of genetic cargo. We will discuss systemic concerns, such as circulation, tissue localization, and clearance, as well as cell-scale obstacles, such as cellular uptake and nucleic acid packaging. In particular, we will focus on engineering particle shape and size in order to enhance delivery and promote precise targeting. We will also address methods to program or change particle shape in situ using environmentally triggered cues. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  12. Principles and limitations of computational microRNA gene and target finding

    DEFF Research Database (Denmark)

    Lindow, Morten; Gorodkin, Jan

    2007-01-01

    of sequence data being generated, the need for analysis by computational approaches is obvious. Here, we review the general principles used in computational gene and target finding, and discuss the strengths and weaknesses of the methods. Several methods rely on detection of evolutionary conserved candidates...

  13. A single gene target of an ETS-family transcription factor determines neuronal CO2-chemosensitivity

    DEFF Research Database (Denmark)

    Brandt, Julia P; Aziz-Zaman, Sonya; Juozaityte, Vaida

    2012-01-01

    . We report here a mechanism that endows C. elegans neurons with the ability to detect CO(2). The ETS-5 transcription factor is necessary for the specification of CO(2)-sensing BAG neurons. Expression of a single ETS-5 target gene, gcy-9, which encodes a receptor-type guanylate cyclase, is sufficient...

  14. Nonviral Gene Targeting at rDNA Locus of Human Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Youjin Hu

    2013-01-01

    Full Text Available Background. Genetic modification, such as the addition of exogenous genes to the MSC genome, is crucial to their use as cellular vehicles. Due to the risks associated with viral vectors such as insertional mutagenesis, the safer nonviral vectors have drawn a great deal of attention. Methods. VEGF, bFGF, vitamin C, and insulin-transferrin-selenium-X were supplemented in the MSC culture medium. The cells’ proliferation and survival capacity was measured by MTT, determination of the cumulative number of cells, and a colony-forming efficiency assay. The plasmid pHr2-NL was constructed and nucleofected into MSCs. The recombinants were selected using G418 and characterized using PCR and Southern blotting. Results. BFGF is critical to MSC growth and it acted synergistically with vitamin C, VEGF, and ITS-X, causing the cells to expand significantly. The neomycin gene was targeted to the rDNA locus of human MSCs using a nonviral human ribosomal targeting vector. The recombinant MSCs retained multipotential differentiation capacity, typical levels of hMSC surface marker expression, and a normal karyotype, and none were tumorigenic in nude mice. Conclusions. Exogenous genes can be targeted to the rDNA locus of human MSCs while maintaining the characteristics of MSCs. This is the first nonviral gene targeting of hMSCs.

  15. The effect of COMT gene on the target precision of the athlete movement

    Directory of Open Access Journals (Sweden)

    E. V. Mikhailova

    2014-01-01

    Full Text Available The aim of the study was to find correlation between COMT gene alleles and the target precision of the athlete movement. 68 Russian competing athletes involved in boxing and volleyball, participated in the study. We found interrelation between COMT Met allele and a tall stature in the volleyball players.

  16. Interactome of Radiation-Induced microRNA-Predicted Target Genes

    Directory of Open Access Journals (Sweden)

    Tenzin W. Lhakhang

    2012-01-01

    Full Text Available The microRNAs (miRNAs function as global negative regulators of gene expression and have been associated with a multitude of biological processes. The dysfunction of the microRNAome has been linked to various diseases including cancer. Our laboratory recently reported modulation in the expression of miRNA in a variety of cell types exposed to ionizing radiation (IR. To further understand miRNA role in IR-induced stress pathways, we catalogued a set of common miRNAs modulated in various irradiated cell lines and generated a list of predicted target genes. Using advanced bioinformatics tools we identified cellular pathways where miRNA predicted target genes function. The miRNA-targeted genes were found to play key roles in previously identified IR stress pathways such as cell cycle, p53 pathway, TGF-beta pathway, ubiquitin-mediated proteolysis, focal adhesion pathway, MAPK signaling, thyroid cancer pathway, adherens junction, insulin signaling pathway, oocyte meiosis, regulation of actin cytoskeleton, and renal cell carcinoma pathway. Interestingly, we were able to identify novel targeted pathways that have not been identified in cellular radiation response, such as aldosterone-regulated sodium reabsorption, long-term potentiation, and neutrotrophin signaling pathways. Our analysis indicates that the miRNA interactome in irradiated cells provides a platform for comprehensive modeling of the cellular stress response to IR exposure.

  17. Problem-Solving Test: Conditional Gene Targeting Using the Cre/loxP Recombination System

    Science.gov (United States)

    Szeberényi, József

    2013-01-01

    Terms to be familiar with before you start to solve the test: gene targeting, knock-out mutation, bacteriophage, complementary base-pairing, homologous recombination, deletion, transgenic organisms, promoter, polyadenylation element, transgene, DNA replication, RNA polymerase, Shine-Dalgarno sequence, restriction endonuclease, polymerase chain…

  18. Targeting Neuronal Networks with Combined Drug and Stimulation Paradigms Guided by Neuroimaging to Treat Brain Disorders.

    Science.gov (United States)

    Faingold, Carl L; Blumenfeld, Hal

    2015-10-01

    Improved therapy of brain disorders can be achieved by focusing on neuronal networks, utilizing combined pharmacological and stimulation paradigms guided by neuroimaging. Neuronal networks that mediate normal brain functions, such as hearing, interact with other networks, which is important but commonly neglected. Network interaction changes often underlie brain disorders, including epilepsy. "Conditional multireceptive" (CMR) brain areas (e.g., brainstem reticular formation and amygdala) are critical in mediating neuroplastic changes that facilitate network interactions. CMR neurons receive multiple inputs but exhibit extensive response variability due to milieu and behavioral state changes and are exquisitely sensitive to agents that increase or inhibit GABA-mediated inhibition. Enhanced CMR neuronal responsiveness leads to expression of emergent properties--nonlinear events--resulting from network self-organization. Determining brain disorder mechanisms requires animals that model behaviors and neuroanatomical substrates of human disorders identified by neuroimaging. However, not all sites activated during network operation are requisite for that operation. Other active sites are ancillary, because their blockade does not alter network function. Requisite network sites exhibit emergent properties that are critical targets for pharmacological and stimulation therapies. Improved treatment of brain disorders should involve combined pharmacological and stimulation therapies, guided by neuroimaging, to correct network malfunctions by targeting specific network neurons. © The Author(s) 2015.

  19. Pathophysiologic effects of vascular-targeting agents and the implications for combination with conventional therapies

    DEFF Research Database (Denmark)

    Horsman, Michael Robert; Siemann, D.W.

    2006-01-01

    A functional vascular supply is critical for the continued growth and development of solid tumors. It also plays a major role in metastatic spread of tumor cells. This importance has led to the concept of targeting the vasculature of the tumor as a form of cancer therapy. Two major types of vascu...... on the tumor response to more conventional therapies. This review aims to discuss the pathophysiologic changes induced by VTAs and the implications of these effects on the potential use of VTAs in combined modality therapy....... successful in the clinic they will need to be combined with more conventional therapies. However, by affecting the tumor vascular supply, these VTAs should induce pathophysiologic changes in variables, such as blood flow, pH, and oxygenation. Such changes could have negative or positive influences......A functional vascular supply is critical for the continued growth and development of solid tumors. It also plays a major role in metastatic spread of tumor cells. This importance has led to the concept of targeting the vasculature of the tumor as a form of cancer therapy. Two major types...

  20. EFFICACY OF COMBINED ANTIHYPERTENSIVE THERAPY IN ACHIEVEMENT OF TARGET BLOOD PRESSURE IN DIABETIC PATIENTS

    Directory of Open Access Journals (Sweden)

    O. A. Koshel'skaya

    2012-01-01

    Full Text Available Aim. To evaluate the efficacy of long-term combined antihypertensive therapy (AHT based on renin-angiotensin-aldosterone system (RAAS blockers, indapamide and calcium channel blocker (CCB in hypertensive patients with diabetes mellitus (DM in accordance with target blood pressure (BP <130/80 mm Hg achievement rate, dynamics of 24-hour BP profile, metabolic indices, and local stiffness of the main arteries. Besides, to study the effects of the CCB addition to dual therapy on these parameters. Material and methods. Patients (16 men, 31 women, 57.2±6.6 years old with arterial hypertension degrees 1–3 and mild to moderate DM type 2 were included into the study. The patients were treated with perindopril (5–10 mg/day or valsartan (80–160 mg/day in combination with indapamide SR (1.5 mg/day and amlodipine (5–10 mg/day. Examination included office BP measurement and ambulatory BP monitoring (ABPM, common carotid arteries sonarography , evaluation of serum levels of potassium, creatinine, uric acid, glucose metabolism and lipid profile parameters, calculation of insulin resistance index (HOMA at baseline and after 30–32 weeks of treatment. Results. Target BP was achieved in 86.7% of patients. Evenly reduction of day and night BP without reflex tachycardia and hypotension episodes was observed. Office BP decreased from 149.5±12.0/90.0±8.3 to 125.0±7.6/76.8±4.9 mm Hg (p<0.05 and average daily BP (ABPM decreased to 120.1±10.0/71.7±6.9 mmHg. Three drugs were needed to achieve target BP in baseline systolic BP >150 mm Hg (office or >134 mmHg (ABPM. Marked beneficial effect on the morphological and functional characteristics of the vascular wall and its elastic properties, improvement of glycemic control, tissue insulin sensitivity and lipids profile were found. These effects were associated mainly with amlodipine inclusion into the therapy. Conclusion. The combined AHT based on RAAS blockers, indapamide SR and CCB provides achievement of

  1. EFFICACY OF COMBINED ANTIHYPERTENSIVE THERAPY IN ACHIEVEMENT OF TARGET BLOOD PRESSURE IN DIABETIC PATIENTS

    Directory of Open Access Journals (Sweden)

    O. A. Koshel'skaya

    2015-12-01

    Full Text Available Aim. To evaluate the efficacy of long-term combined antihypertensive therapy (AHT based on renin-angiotensin-aldosterone system (RAAS blockers, indapamide and calcium channel blocker (CCB in hypertensive patients with diabetes mellitus (DM in accordance with target blood pressure (BP <130/80 mm Hg achievement rate, dynamics of 24-hour BP profile, metabolic indices, and local stiffness of the main arteries. Besides, to study the effects of the CCB addition to dual therapy on these parameters. Material and methods. Patients (16 men, 31 women, 57.2±6.6 years old with arterial hypertension degrees 1–3 and mild to moderate DM type 2 were included into the study. The patients were treated with perindopril (5–10 mg/day or valsartan (80–160 mg/day in combination with indapamide SR (1.5 mg/day and amlodipine (5–10 mg/day. Examination included office BP measurement and ambulatory BP monitoring (ABPM, common carotid arteries sonarography , evaluation of serum levels of potassium, creatinine, uric acid, glucose metabolism and lipid profile parameters, calculation of insulin resistance index (HOMA at baseline and after 30–32 weeks of treatment. Results. Target BP was achieved in 86.7% of patients. Evenly reduction of day and night BP without reflex tachycardia and hypotension episodes was observed. Office BP decreased from 149.5±12.0/90.0±8.3 to 125.0±7.6/76.8±4.9 mm Hg (p<0.05 and average daily BP (ABPM decreased to 120.1±10.0/71.7±6.9 mmHg. Three drugs were needed to achieve target BP in baseline systolic BP >150 mm Hg (office or >134 mmHg (ABPM. Marked beneficial effect on the morphological and functional characteristics of the vascular wall and its elastic properties, improvement of glycemic control, tissue insulin sensitivity and lipids profile were found. These effects were associated mainly with amlodipine inclusion into the therapy. Conclusion. The combined AHT based on RAAS blockers, indapamide SR and CCB provides achievement of

  2. Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yongchao [ORNL; Tschaplinski, Timothy J [ORNL; Engle, Nancy L [ORNL; Hamilton, Choo Yieng [ORNL; Rodriguez, Jr., Miguel [ORNL; Liao, James C [ORNL; Schadt, Christopher Warren [ORNL; Guss, Adam M [ORNL; Yang, Yunfeng [ORNL; Graham, David E [ORNL

    2012-01-01

    Background: The model bacterium Clostridium cellulolyticum efficiently hydrolyzes crystalline cellulose and hemicellulose, using cellulosomes to degrade lignocellulosic biomass. Although it imports and ferments both pentose and hexose sugars to produce a mixture of ethanol, acetate, lactate, H2 and CO2, the proportion of ethanol is low, which impedes its use in consolidated bioprocessing for biofuels. Therefore genetic engineering will likely be required to improve the ethanol yield. Random mutagenesis, plasmid transformation, and heterologous expression systems have previously been developed for C. cellulolyticum, but targeted mutagenesis has not been reported for this organism. Results: The first targeted gene inactivation system was developed for C. cellulolyticum, based on a mobile group II intron originating from the Lactococcus lactis L1.LtrB intron. This markerless mutagenesis system was used to disrupt both the paralogous L-lactate dehydrogenase (Ccel_2485; ldh) and L-malate dehydrogenase (Ccel_0137; mdh) genes, distinguishing the overlapping substrate specificities of these enzymes. Both mutations were then combined in a single strain. This double mutant produced 8.5-times more ethanol than wild-type cells growing on crystalline cellulose. Ethanol constituted 93% of the major fermentation products (by molarity), corresponding to a molar ratio of ethanol to organic acids of 15, versus 0.18 in wild-type cells. During growth on acid-pretreated switchgrass, the double mutant also produced four-times as much ethanol as wild-type cells. Detailed metabolomic analyses identified increased flux through the oxidative branch of the mutant s TCA pathway. Conclusions: The efficient intron-based gene inactivation system produced the first gene-targeted mutations in C. cellulolyticum. As a key component of the genetic toolbox for this bacterium, markerless targeted mutagenesis enables functional genomic research in C. cellulolyticum and rapid genetic engineering to

  3. miRNA signatures and transcriptional regulation of their target genes in vitiligo.

    Science.gov (United States)

    Mansuri, Mohmmad Shoab; Singh, Mala; Begum, Rasheedunnisa

    2016-10-01

    miRNAs are small non-coding RNA molecules that post-transcriptionally regulate gene expression. We have earlier reported the skin miRNA expression profiling in patients with non-segmental vitiligo. In the present study, we show the expression of previously identified skin miRNAs signatures in blood and their target genes in whole blood and PBMCs as well as skin micro-environment of vitiligo patients and controls. miRNA expression profiling in whole blood was performed using customized TaqMan ® Low Density Array. We predicted the potential targets of differentially expressed miRNAs and investigated their expression levels in skin, whole blood and PBMCs from patients and controls using Real-time PCR. Our results showed miR-1, miR-184, miR-328, miR-383 and miR-577 hold similar pattern of expression as of skin, suggesting their potent eminence for being putative markers for vitiligo. In silico target prediction revealed miR-1 targets EDN1, G6PD, HSP60, HSP70, SERP1, SIRT1 & TYR; miR-184 targets EZR & LAMP1; miR-328 targets IL1B, POLH & TRPM1; miR-383 targets EDN1 & TYRP1; and miR-577 targets PTPN22 & TYRP1 which were corroborated by our validation study. In conclusion, the present study for the first time provides new insights into the crucial role of miRNA regulated gene network involved in oxidative stress, autoimmunity and ER stress mediated pathogenesis of vitiligo. Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

  4. Combined targeting of Raf and Mek synergistically inhibits tumorigenesis in triple negative breast cancer model systems.

    Science.gov (United States)

    Nagaria, Teddy S; Shi, Changnian; Leduc, Charles; Hoskin, Victoria; Sikdar, Soma; Sangrar, Waheed; Greer, Peter A

    2017-10-06

    Aberrant Ras-MAPK signaling from receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor-2 (HER2), is a hallmark of triple negative breast cancer (TNBC); thus providing rationale for targeting the Ras-MAPK pathway. Components of this EGFR/HER2-Ras-Raf-Mek-Erk pathway were co-targeted in the MDA-MB-231 and MDA-MB-468 human TNBC cell lines, and in vitro effects on signaling and cytotoxicity, as well as in vivo effects on xenograft tumor growth and metastasis were assessed. The dual EGFR/HER2 inhibitor lapatinib (LPN) displayed greater cytotoxic potency and MAPK signaling inhibition than the EGFR inhibitor erlotinib, suggesting both EGFR and HER2 contribute to MAPK signaling in this TNBC model. The Raf inhibitor sorafenib (SFN) or the Mek inhibitor U0126 suppressed MAPK signaling to a greater extent than LPN; which correlated with greater cytotoxic potency of SFN, but not U0126. However, U0126 potentiated the cytotoxic efficacy of LPN and SFN in an additive and synergistic manner, respectively. This in-series Raf-Mek co-targeting synergy was recapitulated in orthotopic mouse xenografts, where SFN and the Mek inhibitor selumitinib (AZD6244) inhibited primary tumor growth and pulmonary metastasis. Raf and Mek co-inhibition exhibits synergy in TNBC models and represent a promising combination therapy for this aggressive breast cancer type.

  5. Multi-target trapping in constrained environments using gene regulatory network-based pattern formation

    Directory of Open Access Journals (Sweden)

    Xingguang Peng

    2016-10-01

    Full Text Available Inspired by the morphogenesis of biological organisms, gene regulatory network-based methods have been used in complex pattern formation of swarm robotic systems. In this article, obstacle information was embedded into the gene regulatory network model to make the robots trap targets with an expected pattern while avoiding obstacles in a distributed manner. Based on the modified gene regulatory network model, an implicit function method was adopted to represent the expected pattern which is easily adjusted by adding extra feature points. Considering environmental constraints (e.g. tunnels or gaps in which robots must adjust their pattern to conduct trapping task, a pattern adaptation strategy was proposed for the pattern modeler to adaptively adjust the expected pattern. Also to trap multiple targets, a splitting pattern adaptation strategy was proposed for diffusively moving targets so that the robots can trap each target separately with split sub-patterns. The proposed model and strategies were verified through a set of simulation with complex environmental constraints and non-consensus movements of targets.

  6. Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

    Science.gov (United States)

    Reddy, Thimma R.; Kelsall, Emma J.; Fevat, Léna M.S.

    2015-01-01

    Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a ‘targeting’ vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors. PMID:25590226

  7. Analysis of Deregulated microRNAs and Their Target Genes in Gastric Cancer.

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    Simonas Juzėnas

    Full Text Available MicroRNAs (miRNAs are widely studied non-coding RNAs that modulate gene expression. MiRNAs are deregulated in different tumors including gastric cancer (GC and have potential diagnostic and prognostic implications. The aim of our study was to determine miRNA profile in GC tissues, followed by evaluation of deregulated miRNAs in plasma of GC patients. Using available databases and bioinformatics methods we also aimed to evaluate potential target genes of confirmed differentially expressed miRNA and validate these findings in GC tissues.The study included 51 GC patients and 51 controls. Initially, we screened miRNA expression profile in 13 tissue samples of GC and 12 normal gastric tissues with TaqMan low density array (TLDA. In the second stage, differentially expressed miRNAs were validated in a replication cohort using qRT-PCR in tissue and plasma samples. Subsequently, we analyzed potential target genes of deregulated miRNAs using bioinformatics approach, determined their expression in GC tissues and performed correlation analysis with targeting miRNAs.Profiling with TLDA revealed 15 deregulated miRNAs in GC tissues compared to normal gastric mucosa. Replication analysis confirmed that miR-148a-3p, miR-204-5p, miR-223-3p and miR-375 were consistently deregulated in GC tissues. Analysis of GC patients' plasma samples showed significant down-regulation of miR-148a-3p, miR-375 and up-regulation of miR-223-3p compared to healthy subjects. Further, using bioinformatic tools we identified targets of replicated miRNAs and performed disease-associated gene enrichment analysis. Ultimately, we evaluated potential target gene BCL2 and DNMT3B expression by qRT-PCR in GC tissue, which correlated with targeting miRNA expression.Our study revealed miRNA profile in GC tissues and showed that miR-148a-3p, miR-223-3p and miR-375 are deregulated in GC plasma samples, but these circulating miRNAs showed relatively weak diagnostic performance as sole biomarkers

  8. Identification of Brassinosteroid Target Genes by Chromatin Immunoprecipitation Followed by High-Throughput Sequencing (ChIP-seq) and RNA-Sequencing.

    Science.gov (United States)

    Nolan, Trevor; Liu, Sanzhen; Guo, Hongqing; Li, Lei; Schnable, Patrick; Yin, Yanhai

    2017-01-01

    Brassinosteroids (BRs) play important roles in many growth and developmental processes. BRs signal to regulate BR-INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 (BES1) and BRASSINAZOLE-RESISTANT1 (BZR1) transcription factors (TFs), which, in turn, regulate several hundreds of transcription factors (termed BES1/BZR1-targeted TFs or BTFs) and thousands of genes to mediate various BR responses. Chromatin Immunoprecipitation followed by high-throughput sequencing (ChIP-seq) with BES1/BZR1 and BTFs is an important approach to identify BR target genes. In combination with RNA-sequencing experiments, these genomic methods have become powerful tools to detect BR target genes and reveal transcriptional networks underlying BR-regulated processes.

  9. Recombinant adeno-associated virus targets passenger gene expression to cones in primate retina

    Science.gov (United States)

    Mancuso, Katherine; Hendrickson, Anita E.; Connor, Thomas B., Jr.; Mauck, Matthew C.; Kinsella, James J.; Hauswirth, William W.; Neitz, Jay; Neitz, Maureen

    2007-05-01

    Recombinant adeno-associated virus (rAAV) is a promising vector for gene therapy of photoreceptor-based diseases. Previous studies have demonstrated that rAAV serotypes 2 and 5 can transduce both rod and cone photoreceptors in rodents and dogs, and it can target rods, but not cones in primates. Here we report that using a human cone-specific enhancer and promoter to regulate expression of a green fluorescent protein (GFP) reporter gene in an rAAV-5 vector successfully targeted expression of the reporter gene to primate cones, and the time course of GFP expression was able to be monitored in a living animal using the RetCam II digital imaging system.

  10. Classification of Breast Cancer Subtypes by combining Gene Expression and DNA Methylation Data

    DEFF Research Database (Denmark)

    List, Markus; Hauschild, Anne-Christin; Tan, Qihua

    2014-01-01

    expression data for hundreds of patients, the challenge is to extract a minimal optimal set of genes with good prognostic properties from a large bulk of genes making a moderate contribution to classification. Several studies have successfully applied machine learning algorithms to solve this so-called gene...... on the transcriptomic, but also on an epigenetic level. We compared so-called random forest derived classification models based on gene expression and methylation data alone, to a model based on the combined features and to a model based on the gold standard PAM50. We obtained bootstrap errors of 10...

  11. Control of African swine fever virus replication by small interfering RNA targeting the A151R and VP72 genes.

    Science.gov (United States)

    Keita, Djénéba; Heath, Livio; Albina, Emmanuel

    2010-01-01

    African swine fever virus (ASFV) is the unique member of the Asfarviridae family and Asfivirus genus. It is an enveloped double-stranded DNA arbovirus that replicates in the cell cytoplasm, similar to poxviruses. There is no vaccine and no treatment available to control this virus. We describe the use of small interfering RNA (siRNA) targeting the A151R and VP72 (B646L) genes to control the ASFV replication in vitro. Results suggest that siRNA targeting the A151R and VP72 genes can reduce both the virus replication and its levels of messenger RNA transcripts. The reduction was up to 4 log(10) copies on the virus titre and up to 3 log(10) copies on virus RNA transcripts levels. The combination of multiple siRNA did not improve the antiviral effect significantly, compared with use of individual siRNAs. The function of the A151R gene product in the virus replication cycle is yet unclear, but is essential. We also demonstrate that it is possible to inhibit, using small interfering RNA, a virus that replicates exclusively in the cell cytoplasm in specific viral factories.

  12. A novel mechanism for p53 to regulate its target gene ECK in signaling apoptosis.

    Science.gov (United States)

    Jin, Y Jenny; Wang, Jianli; Qiao, Changhong; Hei, Tom K; Brandt-Rauf, Paul W; Yin, Yuxin

    2006-10-01

    Transcription factor p53 regulates its target genes through binding to DNA consensus sequence and activating the promoters of its downstream genes. The conventional p53 consensus binding sequence was defined as two copies of the 10-bp motif 5'-PuPuPuC(A/T)(T/A)GPyPyPy-3' with a spacer of 0 to 13 bp, which exists in the regulatory regions of some p53 target genes. However, there is no such p53 consensus sequence in the promoters of a number of p53-responsive genes, suggesting that there might be other mechanisms whereby p53 transactivates the promoters of its target genes. We report here that p53 uses a novel binding mechanism to regulate the transcription of epithelial cell kinase (ECK), a receptor protein-tyrosine kinase implicated in signal transduction. We show that p53 binds to a 10-bp perfect palindromic decanucleotide (GTGACGTCAC) in the ECK promoter, activates the ECK promoter, and increases the transcription of ECK. This palindrome is required for p53-mediated transactivation of the ECK promoter. ECK is highly responsive to oxidative damage that leads to cell death. Ectopic expression of ECK causes spontaneous apoptosis in breast cancer cells. We found that ectopic expression of a mutant ECK fails to induce apoptosis in cancer cells. Our findings show that p53 is a transcriptional regulator of ECK in mediating apoptosis. The discovery of the novel p53-binding motif in the promoter may lead to the identification of a new class of p53 target genes.

  13. Diverse, Biologically Relevant, and Targetable Gene Rearrangements in Triple-Negative Breast Cancer and Other Malignancies.

    Science.gov (United States)

    Shaver, Timothy M; Lehmann, Brian D; Beeler, J Scott; Li, Chung-I; Li, Zhu; Jin, Hailing; Stricker, Thomas P; Shyr, Yu; Pietenpol, Jennifer A

    2016-08-15

    Triple-negative breast cancer (TNBC) and other molecularly heterogeneous malignancies present a significant clinical challenge due to a lack of high-frequency "driver" alterations amenable to therapeutic intervention. These cancers often exhibit genomic instability, resulting in chromosomal rearrangements that affect the structure and expression of protein-coding genes. However, identification of these rearrangements remains technically challenging. Using a newly developed approach that quantitatively predicts gene rearrangements in tumor-derived genetic material, we identified and characterized a novel oncogenic fusion involving the MER proto-oncogene tyrosine kinase (MERTK) and discovered a clinical occurrence and cell line model of the targetable FGFR3-TACC3 fusion in TNBC. Expanding our analysis to other malignancies, we identified a diverse array of novel and known hybrid transcripts, including rearrangements between noncoding regions and clinically relevant genes such as ALK, CSF1R, and CD274/PD-L1 The over 1,000 genetic alterations we identified highlight the importance of considering noncoding gene rearrangement partners, and the targetable gene fusions identified in TNBC demonstrate the need to advance gene fusion detection for molecularly heterogeneous cancers. Cancer Res; 76(16); 4850-60. ©2016 AACR. ©2016 American Association for Cancer Research.

  14. Oligonucleotide-based microarray analysis of retinoic acid target genes in the protochordate, Ciona intestinalis.

    Science.gov (United States)

    Ishibashi, Tomoko; Usami, Takeshi; Fujie, Manabu; Azumi, Kaoru; Satoh, Nori; Fujiwara, Shigeki

    2005-08-01

    Oligonucleotide-based microarray analyses were carried out to identify retinoic acid target genes in embryos of the ascidian Ciona intestinalis. Of 21,938 spots, 50 (corresponding to 43 genes) showed over twofold up-regulation in retinoic acid-treated tail bud embryos. In situ hybridization verified retinoic acid-induced up-regulation of 23 genes. Many of them were expressed in the anterior tail region, where a retinaldehyde dehydrogenase homolog is expressed. Homologs of vertebrate genes involved in neurogenesis and/or neuronal functions (e.g., COUP-TF, Ci-Hox1, and SCO-spondin) were expressed in the central nervous system of Ciona embryos, and activated by retinoic acid. Genes encoding transcription factors (e.g., Ci-lmx1.2, vitamin D receptor, and Hox proteins) and apoptosis-related proteins (e.g., transglutaminase and an apoptosis-inducing factor homolog) were also activated by retinoic acid. Simultaneous treatment of embryos with retinoic acid and puromycin revealed a few direct targets, including genes encoding Ci-Hox1, Ci-Cyp26, and an Rnf126-like ring finger protein. (c) 2005 Wiley-Liss, Inc.

  15. Reprogramming of the ERRα and ERα target gene landscape triggers tamoxifen resistance in breast cancer.

    Science.gov (United States)

    Thewes, Verena; Simon, Ronald; Schroeter, Petra; Schlotter, Magdalena; Anzeneder, Tobias; Büttner, Reinhard; Benes, Vladimir; Sauter, Guido; Burwinkel, Barbara; Nicholson, Robert I; Sinn, Hans-Peter; Schneeweiss, Andreas; Deuschle, Ulrich; Zapatka, Marc; Heck, Stefanie; Lichter, Peter

    2015-02-15

    Endocrine treatment regimens for breast cancer that target the estrogen receptor-α (ERα) are effective, but acquired resistance remains a limiting drawback. One mechanism of acquired resistance that has been hypothesized is functional substitution of the orphan receptor estrogen-related receptor-α (ERRα) for ERα. To examine this hypothesis, we analyzed ERRα and ERα in recurrent tamoxifen-resistant breast tumors and conducted a genome-wide target gene profiling analysis of MCF-7 breast cancer cell populations that were sensitive or resistant to tamoxifen treatment. This analysis uncovered a global redirection in the target genes controlled by ERα, ERRα, and their coactivator AIB1, defining a novel set of target genes in tamoxifen-resistant cells. Beyond differences in the ERα and ERRα target gene repertoires, both factors were engaged in similar pathobiologic processes relevant to acquired resistance. Functional analyses confirmed a requirement for ERRα in tamoxifen- and fulvestrant-resistant MCF-7 cells, with pharmacologic inhibition of ERRα sufficient to partly restore sensitivity to antiestrogens. In clinical specimens (n = 1041), increased expression of ERRα was associated with enhanced proliferation and aggressive disease parameters, including increased levels of p53 in ERα-positive cases. In addition, increased ERRα expression was linked to reduced overall survival in independent tamoxifen-treated patient cohorts. Taken together, our results suggest that ERα and ERRα cooperate to promote endocrine resistance, and they provide a rationale for the exploration of ERRα as a candidate drug target to treat endocrine-resistant breast cancer. ©2015 American Association for Cancer Research.

  16. New target genes of MITF-induced microRNA-211 contribute to melanoma cell invasion.

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    Christiane Margue

    Full Text Available The non-coding microRNAs (miRNA have tissue- and disease-specific expression patterns. They down-regulate target mRNAs, which likely impacts on most fundamental cellular processes. Differential expression patterns of miRNAs are currently being exploited for identification of biomarkers for early disease diagnosis, prediction of progression for melanoma and other cancers and as promising drug targets, since they can easily be inhibited or replaced in a given cellular context. Before successfully manipulating miRNAs in clinical settings, their precise expression levels, endogenous functions and thus their target genes have to be determined. MiR-211, a melanocyte lineage-specific small non-coding miRNA, is located in an intron of TRPM1, a target gene of the microphtalmia-associated transcription factor (MITF. By transcriptionally up-regulating TRPM1, MITF, which is critical for both melanocyte differentiation and survival and for melanoma progression, indirectly drives the expression of miR-211. Expression of this miRNA is often reduced in melanoma samples. Here, we investigated functional roles of miR-211 by identifying and studying new target genes. We show that MITF-correlated miR-211 expression levels are mostly but not always reduced in a panel of 11 melanoma cell lines and in primary and metastatic melanoma compared to normal melanocytes and nevi, respectively. MiR-211 itself only marginally impacted on cell invasion and migration, while perturbation of some new miR-211 target genes, such as AP1S2, SOX11, IGFBP5, and SERINC3 significantly increased invasion. These results and the variable expression levels of miR-211 raise serious doubts on the value of miR-211 as a melanoma tumor-suppressing miRNA and/or as a biomarker for melanoma.

  17. Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases.

    Directory of Open Access Journals (Sweden)

    Tatiana Flisikowska

    Full Text Available Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM(+ and IgG(+ B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ∼1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.

  18. Electrotransfer parameters as a tool for controlled and targeted gene expression in skin

    Directory of Open Access Journals (Sweden)

    Spela Kos

    2016-01-01

    Full Text Available Skin is an attractive target for gene electrotransfer. It consists of different cell types that can be transfected, leading to various responses to gene electrotransfer. We demonstrate that these responses could be controlled by selecting the appropriate electrotransfer parameters. Specifically, the application of low or high electric pulses, applied by multi-electrode array, provided the possibility to control the depth of the transfection in the skin, the duration and the level of gene expression, as well as the local or systemic distribution of the transgene. The influence of electric pulse type was first studied using a plasmid encoding a reporter gene (DsRed. Then, plasmids encoding therapeutic genes (IL-12, shRNA against endoglin, shRNA against melanoma cell adhesion molecule were used, and their effects on wound healing and cutaneous B16F10 melanoma tumors were investigated. The high-voltage pulses resulted in gene expression that was restricted to superficial skin layers and induced a local response. In contrast, the low-voltage electric pulses promoted transfection into the deeper skin layers, resulting in prolonged gene expression and higher transgene production, possibly with systemic distribution. Therefore, in the translation into the clinics, it will be of the utmost importance to adjust the electrotransfer parameters for different therapeutic approaches and specific mode of action of the therapeutic gene.

  19. Camelid Ig V genes reveal significant human homology not seen in therapeutic target genes, providing for a powerful therapeutic antibody platform

    Science.gov (United States)

    Klarenbeek, Alex; Mazouari, Khalil El; Desmyter, Aline; Blanchetot, Christophe; Hultberg, Anna; de Jonge, Natalie; Roovers, Rob C; Cambillau, Christian; Spinelli, Sylvia; Del-Favero, Jurgen; Verrips, Theo; de Haard, Hans J; Achour, Ikbel

    2015-01-01

    Camelid immunoglobulin variable (IGV) regions were found homologous to their human counterparts; however, the germline V repertoires of camelid heavy and light chains are still incomplete and their therapeutic potential is only beginning to be appreciated. We therefore leveraged the publicly available HTG and WGS databases of Lama pacos and Camelus ferus to retrieve the germline repertoire of V genes using human IGV genes as reference. In addition, we amplified IGKV and IGLV genes to uncover the V germline repertoire of Lama glama and sequenced BAC clones covering part of the Lama pacos IGK and IGL loci. Our in silico analysis showed that camelid counterparts of all human IGKV and IGLV families and most IGHV families could be identified, based on canonical structure and sequence homology. Interestingly, this sequence homology seemed largely restricted to the Ig V genes and was far less apparent in other genes: 6 therapeutically relevant target genes differed significantly from their human orthologs. This contributed to efficient immunization of llamas with the human proteins CD70, MET, interleukin (IL)-1β and IL-6, resulting in large panels of functional antibodies. The in silico predicted human-homologous canonical folds of camelid-derived antibodies were confirmed by X-ray crystallography solving the structure of 2 selected camelid anti-CD70 and anti-MET antibodies. These antibodies showed identical fold combinations as found in the corresponding human germline V families, yielding binding site structures closely similar to those occurring in human antibodies. In conclusion, our results indicate that active immunization of camelids can be a powerful therapeutic antibody platform. PMID:26018625

  20. Prediction of novel target genes and pathways involved in irinotecan-resistant colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Precious Takondwa Makondi

    Full Text Available Acquired drug resistance to the chemotherapeutic drug irinotecan (the active metabolite of which is SN-38 is one of the significant obstacles in the treatment of advanced colorectal cancer (CRC. The molecular mechanism or targets mediating irinotecan resistance are still unclear. It is urgent to find the irinotecan response biomarkers to improve CRC patients' therapy.Genetic Omnibus Database GSE42387 which contained the gene expression profiles of parental and irinotecan-resistant HCT-116 cell lines was used. Differentially expressed genes (DEGs between parental and irinotecan-resistant cells, protein-protein interactions (PPIs, gene ontologies (GOs and pathway analysis were performed to identify the overall biological changes. The most common DEGs in the PPIs, GOs and pathways were identified and were validated clinically by their ability to predict overall survival and disease free survival. The gene-gene expression correlation and gene-resistance correlation was also evaluated in CRC patients using The Cancer Genomic Atlas data (TCGA.The 135 DEGs were identified of which 36 were upregulated and 99 were down regulated. After mapping the PPI networks, the GOs and the pathways, nine genes (GNAS, PRKACB, MECOM, PLA2G4C, BMP6, BDNF, DLG4, FGF2 and FGF9 were found to be commonly enriched. Signal transduction was the most significant GO and MAPK pathway was the most significant pathway. The five genes (FGF2, FGF9, PRKACB, MECOM and PLA2G4C in the MAPK pathway were all contained in the signal transduction and the levels of those genes were upregulated. The FGF2, FGF9 and MECOM expression were highly associated with CRC patients' survival rate but not PRKACB and PLA2G4C. In addition, FGF9 was also associated with irinotecan resistance and poor disease free survival. FGF2, FGF9 and PRKACB were positively correlated with each other while MECOM correlated positively with FGF9 and PLA2G4C, and correlated negatively with FGF2 and PRKACB after doing gene-gene

  1. A gene expression signature of acquired chemoresistance to cisplatin and fluorouracil combination chemotherapy in gastric cancer patients.

    Directory of Open Access Journals (Sweden)

    Hark Kyun Kim

    2011-02-01

    Full Text Available We initiated a prospective trial to identify transcriptional alterations associated with acquired chemotherapy resistance from pre- and post-biopsy samples from the same patient and uncover potential molecular pathways involved in treatment failure to help guide therapeutic alternatives.A prospective, high-throughput transcriptional profiling study was performed using endoscopic biopsy samples from 123 metastatic gastric cancer patients prior to cisplatin and fluorouracil (CF combination chemotherapy. 22 patients who initially responded to CF were re-biopsied after they developed resistance to CF. An acquired chemotherapy resistance signature was identified by analyzing the gene expression profiles from the matched pre- and post-CF treated samples. The acquired resistance signature was able to segregate a separate cohort of 101 newly-diagnosed gastric cancer patients according to the time to progression after CF. Hierarchical clustering using a 633-gene acquired resistance signature (feature selection at P<0.01 separated the 101 pretreatment patient samples into two groups with significantly different times to progression (2.5 vs. 4.7 months. This 633-gene signature included the upregulation of AKT1, EIF4B, and RPS6 (mTOR pathway, DNA repair and drug metabolism genes, and was enriched for genes overexpressed in embryonic stem cell signatures. A 72-gene acquired resistance signature (a subset of the 633 gene signature also identified in ES cell-related gene sets was an independent predictor for time to progression (adjusted P = 0.011 and survival (adjusted P = 0.034 of these 101 patients.This signature may offer new insights into identifying new targets and therapies required to overcome the acquired resistance of gastric cancer to CF.

  2. Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions

    DEFF Research Database (Denmark)

    Bracken, Adrian P; Dietrich, Nikolaj; Pasini, Diego

    2006-01-01

    The Polycomb group (PcG) proteins form chromatin-modifying complexes that are essential for embryonic development and stem cell renewal and are commonly deregulated in cancer. Here, we identify their target genes using genome-wide location analysis in human embryonic fibroblasts. We find...... that Polycomb-Repressive Complex 1 (PRC1), PRC2, and tri-methylated histone H3K27 co-occupy >1000 silenced genes with a strong functional bias for embryonic development and cell fate decisions. We functionally identify 40 genes derepressed in human embryonic fibroblasts depleted of the PRC2 components (EZH2...... that PcGs are part of a preprogrammed memory system established during embryogenesis marking certain key genes for repressive signals during subsequent developmental and differentiation processes....

  3. Analysis of a gene panel for targeted sequencing of colorectal cancer samples

    DEFF Research Database (Denmark)

    Jensen, Klaus Højgaard; Izarzugaza, Jose M.G; Sierakowska Juncker, Agnieszka

    2018-01-01

    stratification is necessary for the identification of the subset of patients likely responding to treatment, while sparing others from pernicious treatment. Targeted sequencing approaches may help in this regard, enabling rapid genetic investigation, and at the same time easily applicable in routine diagnosis....... We propose a set of guidelines for the identification, including variant calling and filtering, of somatic mutations driving tumorigenesis in the absence of matched healthy tissue. We also discuss the inclusion criteria for the generation of our gene panel. Furthermore, we evaluate the prognostic...... impact of individual genes, using Cox regression models in the context of overall survival and disease-free survival. These analyses confirmed the role of commonly used biomarkers, and shed light on controversial genes such as CYP2C8. Applying those guidelines, we created a novel gene panel...

  4. Bookmarking target genes in mitosis: a shared epigenetic trait of phenotypic transcription factors and oncogenes?

    Science.gov (United States)

    Zaidi, Sayyed K; Grandy, Rodrigo A; Lopez-Camacho, Cesar; Montecino, Martin; van Wijnen, Andre J; Lian, Jane B; Stein, Janet L; Stein, Gary S

    2014-01-15

    The regulatory information for phenotype, proliferation, and growth of normal and tumor cells must be maintained through genome replication in the S phase and cell division during mitosis. Epigenetic mechanisms that include DNA methylation, posttranslational modifications of histones, selective utilization of histone variants, and inheritable RNA molecules play pivotal roles in maintaining cellular identity through mitotic divisions. Recent studies demonstrate that mitotic occupancy of genes, which are determinants of cell fate, growth, and proliferation, by lineage-restricted transcription factors is a key epigenetic mechanism for retention and transmission of cellular expression memory. Evidence is emerging for the presence of distinct transcriptional regulatory microenvironments in mitotic chromosomes in which the genes bookmarked for reactivation postmitotically reside. Importantly, some oncoproteins are present in mitotic microenvironments where they occupy target genes during mitosis and may contribute to perpetuating the transformed phenotype. We discuss emerging regulatory implications of epigenetically bookmarking genes during mitosis for physiologic control as well as for the onset and progression of cancer.

  5. An update on targeted gene repair in mammalian cells: methods and mechanisms

    Directory of Open Access Journals (Sweden)

    Bolund Lars

    2011-02-01

    Full Text Available Abstract Transfer of full-length genes including regulatory elements has been the preferred gene therapy strategy for clinical applications. However, with significant drawbacks emerging, targeted gene alteration (TGA has recently become a promising alternative to this method. By means of TGA, endogenous DNA repair pathways of the cell are activated leading to specific genetic correction of single-base mutations in the genome. This strategy can be implemented using single-stranded oligodeoxyribonucleotides (ssODNs, small DNA fragments (SDFs, triplex-forming oligonucleotides (TFOs, adeno-associated virus vectors (AAVs and zinc-finger nucleases (ZFNs. Despite difficulties in the use of TGA, including lack of knowledge on the repair mechanisms stimulated by the individual methods, the field holds great promise for the future. The objective of this review is to summarize and evaluate the different methods that exist within this particular area of human gene therapy research.

  6. Adenovirus-mediated IL-12 gene therapy in combination with radiotherapy for murine liver cancer

    International Nuclear Information System (INIS)

    Wei Daoyan; Dai Bingbing; Wang Zhonghe; Chen Shishu

    2001-01-01

    Objective: To investigate the synergistic antitumor effects of adenovirus-mediated IL-12 gene therapy in combination with radiotherapy in mice bearing liver cancer. Methods: Balb/c mice bearing liver cancer received the treatment at day 1 with tumor local irradiation (TLI) of 20 Gy or mask irradiation when tumor size reached 0.6-1.0 cm. Within 1 hour after irradiation, adenovirus containing IL-12 gene or PBS was intra-tumor injected once a week. Forty-eight hours after the second injection, IFN-γ levels in sera and the supernatant of cultured spleen cells were assayed by ELISA, CTL activity of spleen cells was measured by 3 H-TdR release assay, and phenotypes of tumor-infiltrating lymphocytes were analysed by immunohistochemical staining. Results: The growth of tumors in animals treated with a combination of IL-12 gene therapy and TLI was inhibited more significantly than those with either single treatment (P + and CD8 + lymphocyte infiltration and tumor-specific cytolytic activities, and the levels of IFN-γ in sera were higher in IL-12 gene therapy and IL-12 gene therapy combined with TLI groups. Conclusion: These results suggest that IL-12 gene therapy combined with radiotherapy is more effective than both single treatment modalities and can induce specific antitumor immuno-response greatly

  7. A Novel Therapeutic Strategy for the Treatment of Glioma, Combining Chemical and Molecular Targeting of Hsp90α

    International Nuclear Information System (INIS)

    Mehta, Adi; Shervington, Leroy; Munje, Chinmay; Shervington, Amal

    2011-01-01

    Hsp90α's vital role in tumour survival and progression, together with its highly inducible expression profile in gliomas and its absence in normal tissue and cell lines validates it as a therapeutic target for glioma. Hsp90α was downregulated using the post-transcriptional RNAi strategy (sihsp90α) and a post-translational inhibitor, the benzoquinone antibiotic 17-AAG. Glioblastoma U87-MG and normal human astrocyte SVGp12 were treated with sihsp90α, 17-AAG and concurrent sihsp90α/17-AAG (combined treatment). Both Hsp90α gene silencing and the protein inhibitor approaches resulted in a dramatic reduction in cell viability. Results showed that sihsp90α, 17-AAG and a combination of sihsp90α/17-AAG, reduced cell viability by 27%, 75% and 88% (p < 0.001), respectively, after 72 h. hsp90α mRNA copy numbers were downregulated by 65%, 90% and 99% after 72 h treatment with sihsp90α, 17-AAG and sihsp90α/17-AAG, respectively. The relationship between Hsp90α protein expression and its client Akt kinase activity levels were monitored following treatment with sihsp90α, 17-AAG and sihsp90α/17-AAG. Akt kinase activity was downregulated as a direct consequence of Hsp90α inhibition. Both Hsp90α and Akt kinase levels were significantly downregulated after 72 h. Although, 17-AAG when used as a single agent reduces the Hsp90α protein and the Akt kinase levels, the efficacy demonstrated by combinatorial treatment was found to be far more effective. Combination treatment reduced the Hsp90α protein and Akt kinase levels to 4.3% and 43%, respectively, after 72 h. hsp90α mRNA expression detected in SVGp12 was negligible compared to U87-MG, also, the combination treatment did not compromise the normal cell viability. Taking into account the role of Hsp90α in tumour progression and the involvement of Akt kinase in cell signalling and the anti-apoptotic pathways in tumours, this double targets treatment infers a novel therapeutic strategy

  8. Identification of estrogen target genes during zebrafish embryonic development through transcriptomic analysis.

    Directory of Open Access Journals (Sweden)

    Ruixin Hao

    Full Text Available Estrogen signaling is important for vertebrate embryonic development. Here we have used zebrafish (Danio rerio as a vertebrate model to analyze estrogen signaling during development. Zebrafish embryos were exposed to 1 µM 17β-estradiol (E2 or vehicle from 3 hours to 4 days post fertilization (dpf, harvested at 1, 2, 3 and 4 dpf, and subjected to RNA extraction for transcriptome analysis using microarrays. Differentially expressed genes by E2-treatment were analyzed with hierarchical clustering followed by biological process and tissue enrichment analysis. Markedly distinct sets of genes were up and down-regulated by E2 at the four different time points. Among these genes, only the well-known estrogenic marker vtg1 was co-regulated at all time points. Despite this, the biological functional categories targeted by E2 were relatively similar throughout zebrafish development. According to knowledge-based tissue enrichment, estrogen responsive genes were clustered mainly in the liver, pancreas and brain. This was in line with the developmental dynamics of estrogen-target tissues that were visualized using transgenic zebrafish containing estrogen responsive elements driving the expression of GFP (Tg(5xERE:GFP. Finally, the identified embryonic estrogen-responsive genes were compared to already published estrogen-responsive genes identified in male adult zebrafish (Gene Expression Omnibus database. The expressions of a few genes were co-regulated by E2 in both embryonic and adult zebrafish. These could potentially be used as estrogenic biomarkers for exposure to estrogens or estrogenic endocrine disruptors in zebrafish. In conclusion, our data suggests that estrogen effects on early embryonic zebrafish development are stage- and tissue- specific.

  9. MicroRNA-target gene responses to lead-induced stress in cotton (Gossypium hirsutum L.).

    Science.gov (United States)

    He, Qiuling; Zhu, Shuijin; Zhang, Baohong

    2014-09-01

    MicroRNAs (miRNAs) play key roles in plant responses to various metal stresses. To investigate the miRNA-mediated plant response to heavy metals, cotton (Gossypium hirsutum L.), the most important fiber crop in the world, was exposed to different concentrations (0, 25, 50, 100, and 200 µM) of lead (Pb) and then the toxicological effects were investigated. The expression patterns of 16 stress-responsive miRNAs and 10 target genes were monitored in cotton leaves and roots by quantitative real-time PCR (qRT-PCR); of these selected genes, several miRNAs and their target genes are involved in root development. The results show a reciprocal regulation of cotton response to lead stress by miRNAs. The characterization of the miRNAs and the associated target genes in response to lead exposure would help in defining the potential roles of miRNAs in plant adaptation to heavy metal stress and further understanding miRNA regulation in response to abiotic stress.

  10. CRISPR/Cas9-mediated targeted gene mutagenesis in Spodoptera litura.

    Science.gov (United States)

    Bi, Hong-Lun; Xu, Jun; Tan, An-Jiang; Huang, Yong-Ping

    2016-06-01

    Custom-designed nuclease technologies such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) system provide attractive genome editing tools for insect functional genetics. The targeted gene mutagenesis mediated by the CRISPR/Cas9 system has been achieved in several insect orders including Diptera, Lepidoptera and Coleoptera. However, little success has been reported in agricultural pests due to the lack of genomic information and embryonic microinjection techniques in these insect species. Here we report that the CRISPR/Cas9 system induced efficient gene mutagenesis in an important Lepidopteran pest Spodoptera litura. We targeted the S. litura Abdominal-A (Slabd-A) gene which is an important embryonic development gene and plays a significant role in determining the identities of the abdominal segments of insects. Direct injection of Cas9 messenger RNA and Slabd-A-specific single guide RNA (sgRNA) into S. litura embryos successfully induced the typical abd-A deficient phenotype, which shows anomalous segmentation and ectopic pigmentation during the larval stage. A polymerase chain reaction-based analysis revealed that the Cas9/sgRNA complex effectively induced a targeted mutagenesis in S. litura. These results demonstrate that the CRISPR/Cas9 system is a powerful tool for genome manipulation in Lepidopteran pests such as S. litura. © 2016 Institute of Zoology, Chinese Academy of Sciences.

  11. Cooperative interactions between CBP and TORC2 confer selectivity to CREB target gene expression.

    Science.gov (United States)

    Ravnskjaer, Kim; Kester, Henri; Liu, Yi; Zhang, Xinmin; Lee, Dong; Yates, John R; Montminy, Marc

    2007-06-20

    A number of hormones and growth factors stimulate gene expression by promoting the phosphorylation of CREB (P-CREB), thereby enhancing its association with the histone acetylase paralogs p300 and CBP (CBP/p300). Relative to cAMP, stress signals trigger comparable amounts of CREB phosphorylation, but have minimal effects on CRE-dependent transcription. Here, we show that the latent cytoplasmic coactivator TORC2 mediates target gene activation in response to cAMP signaling by associating with CBP/p300 and increasing its recruitment to a subset of CREB target genes. TORC2 is not activated in response to stress signals, however; and in its absence, P-CREB is unable to stimulate CRE-dependent transcription, due to a block in CBP recruitment. The effect of TORC2 on CBP/p300 promoter occupancy appears pivotal because a gain of function mutant CREB polypeptide with increased affinity for CBP restored CRE-mediated transcription in cells exposed to stress signals. Taken together, these results indicate that TORC2 is one of the long sought after cofactors that mediates the differential effects of cAMP and stress pathways on CREB target gene expression.

  12. RBP-Jkappa/SHARP recruits CtIP/CtBP corepressors to silence Notch target genes.

    Science.gov (United States)

    Oswald, Franz; Winkler, Michael; Cao, Ying; Astrahantseff, Kathy; Bourteele, Soizic; Knöchel, Walter; Borggrefe, Tilman

    2005-12-01

    Notch is a transmembrane receptor that determines cell fates and pattern formation in all animal species. After ligand binding, proteolytic cleavage steps occur and the intracellular part of Notch translocates to the nucleus, where it targets the DNA-binding protein RBP-Jkappa/CBF1. In the absence of Notch, RBP-Jkappa represses Notch target genes through the recruitment of a corepressor complex. We and others have identified SHARP as a component of this complex. Here, we functionally demonstrate that the SHARP repression domain is necessary and sufficient to repress transcription and that the absence of this domain causes a dominant negative Notch-like phenotype. We identify the CtIP and CtBP corepressors as novel components of the human RBP-Jkappa/SHARP-corepressor complex and show that CtIP binds directly to the SHARP repression domain. Functionally, CtIP and CtBP augment SHARP-mediated repression. Transcriptional repression of the Notch target gene Hey1 is abolished in CtBP-deficient cells or after the functional knockout of CtBP. Furthermore, the endogenous Hey1 promoter is derepressed in CtBP-deficient cells. We propose that a corepressor complex containing CtIP/CtBP facilitates RBP-Jkappa/SHARP-mediated repression of Notch target genes.

  13. Analysis of bacterial xylose isomerase gene diversity using gene-targeted metagenomics.

    Science.gov (United States)

    Nurdiani, Dini; Ito, Michihiro; Maruyama, Toru; Terahara, Takeshi; Mori, Tetsushi; Ugawa, Shin; Takeyama, Haruko

    2015-08-01

    Bacterial xylose isomerases (XI) are promising resources for efficient biofuel production from xylose in lignocellulosic biomass. Here, we investigated xylose isomerase gene (xylA) diversity in three soil metagenomes differing in plant vegetation and geographical location, using an amplicon pyrosequencing approach and two newly-designed primer sets. A total of 158,555 reads from three metagenomic DNA replicates for each soil sample were classified into 1127 phylotypes, detected in triplicate and defined by 90% amino acid identity. The phylotype coverage was estimated to be within the range of 84.0-92.7%. The xylA gene phylotypes obtained were phylogenetically distributed across the two known xylA groups. They shared 49-100% identities with their closest-related XI sequences in GenBank. Phylotypes demonstrating soil sample were significantly smaller than they were between different soils based on a UniFrac distance analysis, suggesting soil-specific xylA genotypes and taxonomic compositions. The differences among xylA members and their compositions in the soil were strongly correlated with 16S rRNA variation between soil samples, also assessed by amplicon pyrosequencing. This is the first report of xylA diversity in environmental samples assessed by amplicon pyrosequencing. Our data provide information regarding xylA diversity in nature, and can be a basis for the screening of novel xylA genotypes for practical applications. Copyright © 2015. Published by Elsevier B.V.

  14. Identification of novel target genes specifically activated by deregulated E2F in human normal fibroblasts.

    Science.gov (United States)

    Kitamura, Hodaka; Ozono, Eiko; Iwanaga, Ritsuko; Bradford, Andrew P; Okuno, Junko; Shimizu, Emi; Kurayoshi, Kenta; Kugawa, Kazuyuki; Toh, Hiroyuki; Ohtani, Kiyoshi

    2015-09-01

    The transcription factor E2F is the principal target of the tumor suppressor pRB. E2F plays crucial roles not only in cell proliferation by activating growth-related genes but also in tumor suppression by activating pro-apoptotic and growth-suppressive genes. We previously reported that, in human normal fibroblasts, the tumor suppressor genes ARF, p27(Kip1) and TAp73 are activated by deregulated E2F activity induced by forced inactivation of pRB, but not by physiological E2F activity induced by growth stimulation. In contrast, growth-related E2F targets are activated by both E2F activities, underscoring the roles of deregulated E2F in tumor suppression in the context of dysfunctional pRB. In this study, to further understand the roles of deregulated E2F, we explored new targets that are specifically activated by deregulated E2F using DNA microarray. The analysis identified nine novel targets (BIM, RASSF1, PPP1R13B, JMY, MOAP1, RBM38, ABTB1, RBBP4 and RBBP7), many of which are involved in the p53 and RB tumor suppressor pathways. Among these genes, the BIM gene was shown to be activated via atypical E2F-responsive promoter elements and to contribute to E2F1-mediated apoptosis. Our results underscore crucial roles of deregulated E2F in growth suppression to counteract loss of pRB function. © 2015 The Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

  15. Classification of Breast Cancer Subtypes by combining Gene Expression and DNA Methylation Data

    Directory of Open Access Journals (Sweden)

    List Markus

    2014-06-01

    Full Text Available Selecting the most promising treatment strategy for breast cancer crucially depends on determining the correct subtype. In recent years, gene expression profiling has been investigated as an alternative to histochemical methods. Since databases like TCGA provide easy and unrestricted access to gene expression data for hundreds of patients, the challenge is to extract a minimal optimal set of genes with good prognostic properties from a large bulk of genes making a moderate contribution to classification. Several studies have successfully applied machine learning algorithms to solve this so-called gene selection problem. However, more diverse data from other OMICS technologies are available, including methylation. We hypothesize that combining methylation and gene expression data could already lead to a largely improved classification model, since the resulting model will reflect differences not only on the transcriptomic, but also on an epigenetic level. We compared so-called random forest derived classification models based on gene expression and methylation data alone, to a model based on the combined features and to a model based on the gold standard PAM50. We obtained bootstrap errors of 10-20% and classification error of 1-50%, depending on breast cancer subtype and model. The gene expression model was clearly superior to the methylation model, which was also reflected in the combined model, which mainly selected features from gene expression data. However, the methylation model was able to identify unique features not considered as relevant by the gene expression model, which might provide deeper insights into breast cancer subtype differentiation on an epigenetic level.

  16. 5-Fluorocytosine combined with Fcy–hEGF fusion protein targets EGFR-expressing cancer cells

    International Nuclear Information System (INIS)

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

    2012-01-01

    Highlights: ► EGFR-expressing epithelial cancers account for significant portion of cancer deaths. ► EGF–EGFR signaling pathway is validated as an important anticancer drug target. ► EGF and Fcy fusion protein (Fcy–hEGF) can bind to EGFR and convert 5-FC to 5-FU. ► 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 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-expressing cancers.

  17. Combined targeted treatment in early onset epilepsy associated with tuberous sclerosis

    Directory of Open Access Journals (Sweden)

    Romina Moavero

    2016-01-01

    Full Text Available Tuberous sclerosis is associated with epilepsy in up to 85% of cases, and in 2/3, the onset is within the first year of life. An early antiepileptic treatment is crucial to minimize the consequences of epilepsy on cognition and behavior. We present a case report of a child with tuberous sclerosis who presented with infantile spasms at the age of 6 months, immediately treated with vigabatrin. Because of the presence of a subependymal giant cell astrocytoma, he also received everolimus since 18 months of age. We might wonder if an earlier treatment could have produced a better outcome; in fact, despite a targeted combined treatment, he continues to suffer from sporadic focal motor seizures, and at the age of 40 months, he presents severe developmental delay with autism-like behavior.

  18. Huntington's Disease and its therapeutic target genes: a global functional profile based on the HD Research Crossroads database

    Directory of Open Access Journals (Sweden)

    Kalathur Ravi Kiran

    2012-06-01

    Full Text Available Abstract Background Huntington’s disease (HD is a fatal progressive neurodegenerative disorder caused by the expansion of the polyglutamine repeat region in the huntingtin gene. Although the disease is triggered by the mutation of a single gene, intensive research has linked numerous other genes to its pathogenesis. To obtain a systematic overview of these genes, which may serve as therapeutic targets, CHDI Foundation has recently established the HD Research Crossroads database. With currently over 800 cataloged genes, this web-based resource constitutes the most extensive curation of genes relevant to HD. It provides us with an unprecedented opportunity to survey molecular mechanisms involved in HD in a holistic manner. Methods To gain a synoptic view of therapeutic targets for HD, we have carried out a variety of bioinformatical and statistical analyses to scrutinize the functional association of genes curated in the HD Research Crossroads database. In particular, enrichment analyses were performed with respect to Gene Ontology categories, KEGG signaling pathways, and Pfam protein families. For selected processes, we also analyzed differential expression, using published microarray data. Additionally, we generated a candidate set of novel genetic modifiers of HD by combining information from the HD Research Crossroads database with previous genome-wide linkage studies. Results Our analyses led to a comprehensive identification of molecular mechanisms associated with HD. Remarkably, we not only recovered processes and pathways, which have frequently been linked to HD (such as cytotoxicity, apoptosis, and calcium signaling, but also found strong indications for other potentially disease-relevant mechanisms that have been less intensively studied in the context of HD (such as the cell cycle and RNA splicing, as well as Wnt and ErbB signaling. For follow-up studies, we provide a regularly updated compendium of molecular mechanism, that are

  19. Huntington's disease and its therapeutic target genes: a global functional profile based on the HD Research Crossroads database.

    Science.gov (United States)

    Kalathur, Ravi Kiran Reddy; Hernández-Prieto, Miguel A; Futschik, Matthias E

    2012-06-28

    Huntington's disease (HD) is a fatal progressive neurodegenerative disorder caused by the expansion of the polyglutamine repeat region in the huntingtin gene. Although the disease is triggered by the mutation of a single gene, intensive research has linked numerous other genes to its pathogenesis. To obtain a systematic overview of these genes, which may serve as therapeutic targets, CHDI Foundation has recently established the HD Research Crossroads database. With currently over 800 cataloged genes, this web-based resource constitutes the most extensive curation of genes relevant to HD. It provides us with an unprecedented opportunity to survey molecular mechanisms involved in HD in a holistic manner. To gain a synoptic view of therapeutic targets for HD, we have carried out a variety of bioinformatical and statistical analyses to scrutinize the functional association of genes curated in the HD Research Crossroads database. In particular, enrichment analyses were performed with respect to Gene Ontology categories, KEGG signaling pathways, and Pfam protein families. For selected processes, we also analyzed differential expression, using published microarray data. Additionally, we generated a candidate set of novel genetic modifiers of HD by combining information from the HD Research Crossroads database with previous genome-wide linkage studies. Our analyses led to a comprehensive identification of molecular mechanisms associated with HD. Remarkably, we not only recovered processes and pathways, which have frequently been linked to HD (such as cytotoxicity, apoptosis, and calcium signaling), but also found strong indications for other potentially disease-relevant mechanisms that have been less intensively studied in the context of HD (such as the cell cycle and RNA splicing, as well as Wnt and ErbB signaling). For follow-up studies, we provide a regularly updated compendium of molecular mechanism, that are associated with HD, at http://hdtt.sysbiolab.eu Additionally

  20. Safety assessment of liver-targeted hydrodynamic gene delivery in dogs.

    Directory of Open Access Journals (Sweden)

    Kenya Kamimura

    Full Text Available Evidence in support of safety of a gene delivery procedure is essential toward gene therapy. Previous studies using the hydrodynamics-based procedure primarily focus on gene delivery efficiency or gene function analysis in mice. The current study focuses on an assessment of the safety of computer-controlled and liver-targeted hydrodynamic gene delivery in dogs as the first step toward hydrodynamic gene therapy in clinic. We demonstrate that the impacts of the hydrodynamic procedure were limited in the injected region and the influences were transient. Histological examination and the hepatic microcirculation measurement using reflectance spectrophotometry reveal that the liver-specific impact of the procedure involves a transient expansion of the liver sinusoids. No systemic damage or toxicity was observed. Physiological parameters, including electrocardiogram, heart rate, blood pressure, oxygen saturation, and body temperature, remained in normal ranges during and after hydrodynamic injection. Body weight was also examined to assess the long-term effects of the procedure in animals who underwent 3 hydrodynamic injections in 6 weeks with 2-week time interval in between. Serum biochemistry analysis showed a transient increase in liver enzymes and a few cytokines upon injection. These results demonstrate that image-guided, liver-specific hydrodynamic gene delivery is safe.

  1. Herbicide targets and detoxification proteins in sugarcane: from gene assembly to structure modelling.

    Science.gov (United States)

    Lloyd Evans, Dyfed; Joshi, Shailesh Vinay

    2017-07-01

    In a genome context, sugarcane is a classic orphan crop, in that no genome and only very few genes have been assembled. We have devised a novel exome assembly methodology that has allowed us to assemble and characterize 49 genes that serve as herbicide targets, safener interacting proteins, and members of herbicide detoxification pathways within the sugarcane genome. We have structurally modelled the products of each of these genes, as well as determining allelic, genomic, and RNA-Seq based polymorphisms for each gene. This study provides the largest collection of sugarcane structures modelled to date. We demonstrate that sugarcane genes are highly polymorphic, revealing that each genotype is evolving both uniquely and independently. In addition, we present an exome assembly system for orphan crops that can be executed on commodity infrastructure, making exome assembly practical for any group. In terms of knowledge about herbicide modes of action and detoxification, we have advanced sugarcane from a crop where no information about any herbicide-associated gene was available to the situation where sugarcane is now a species with the single largest collection of known and annotated herbicide-associated genes.

  2. Jarid1b targets genes regulating development and is involved in neural differentiation

    DEFF Research Database (Denmark)

    Schmitz, Sandra U; Albert, Mareike; Malatesta, Martina

    2011-01-01

    H3K4 methylation is associated with active transcription and in combination with H3K27me3 thought to keep genes regulating development in a poised state. The contribution of enzymes regulating trimethylation of lysine 4 at histone 3 (H3K4me3) levels to embryonic stem cell (ESC) self-renewal and d...

  3. Methylation of polycomb target genes in intestinal cancer is mediated by inflammation.

    Science.gov (United States)

    Hahn, Maria A; Hahn, Torsten; Lee, Dong-Hyun; Esworthy, R Steven; Kim, Byung-Wook; Riggs, Arthur D; Chu, Fong-Fong; Pfeifer, Gerd P

    2008-12-15

    Epigenetic changes are strongly associated with cancer development. DNA hypermethylation is associated with gene silencing and is often observed in CpG islands. Recently, it was suggested that aberrant CpG island methylation in tumors is directed by Polycomb (PcG) proteins. However, specific mechanisms responsible for methylation of PcG target genes in cancer are not known. Chronic infection and inflammation contribute to up to 25% of all cancers worldwide. Using glutathione peroxidase, Gpx1 and Gpx2, double knockout (Gpx1/2-KO) mice as a model of inflammatory bowel disease predisposing to intestinal cancer, we analyzed genome-wide DNA methylation in the mouse ileum during chronic inflammation, aging, and cancer. We found that inflammation leads to aberrant DNA methylation in PcG target genes, with 70% of the approximately 250 genes methylated in the inflamed tissue being PcG targets in embryonic stem cells and 59% of the methylated genes being marked by H3K27 trimethylation in the ileum of adult wild-type mice. Acquisition of DNA methylation at CpG islands in the ileum of Gpx1/2-KO mice frequently correlates with loss of H3K27 trimethylation at the same loci. Inflammation-associated DNA methylation occurs preferentially in tissue-specific silent genes and, importantly, is much more frequently represented in tumors than is age-dependent DNA methylation. Sixty percent of aberrant methylation found in tumors is also present in the inflamed tissue. In summary, inflammation creates a signature of aberrant DNA methylation, which is observed later in the malignant tissue and is directed by the PcG complex.

  4. Gene silencing by RNA interference in Sarcoptes scabiei: a molecular tool to identify novel therapeutic targets.

    Science.gov (United States)

    Fernando, Deepani D; Marr, Edward J; Zakrzewski, Martha; Reynolds, Simone L; Burgess, Stewart T G; Fischer, Katja

    2017-06-10

    Scabies is one of the most common and widespread parasitic skin infections globally, affecting a large range of mammals including humans, yet the molecular biology of Sarcoptes scabiei is astonishingly understudied. Research has been hampered primarily due to the difficulty of sampling or culturing these obligatory parasitic mites. A further and major impediment to identify and functionally analyse potential therapeutic targets from the recently emerging molecular databases is the lack of appropriate molecular tools. We performed standard BLAST based searches of the existing S. scabiei genome databases using sequences of genes described to be involved in RNA interference in Drosophila and the mite model organism Tetranychus urticae. Experimenting with the S. scabiei mu-class glutathione S-transferase (SsGST-mu1) as a candidate gene we explored the feasibility of gene knockdown in S. scabiei by double-stranded RNA-interference (dsRNAi). We provide here an analysis of the existing S. scabiei draft genomes, confirming the presence of a double stranded RNA (dsRNA) - mediated silencing machinery. We report for the first time experimental gene silencing by RNA interference (RNAi) in S. scabiei. Non-invasive immersion of S. scabiei in dsRNA encoding an S. scabiei glutathione S-transferase mu-class 1 enzyme (SsGST-mu1) resulted in a 35% reduction in the transcription of the target gene compared to controls. A series of experiments identified the optimal conditions allowing systemic experimental RNAi without detrimental side effects on mite viability. This technique can now be used to address the key questions on the fundamental aspects of mite biology and pathogenesis, and to assess the potential therapeutic benefits of silencing S. scabiei target genes.

  5. Targeted gene expression in transgenic Xenopus using the binary Gal4-UAS system.

    Science.gov (United States)

    Hartley, Katharine O; Nutt, Stephen L; Amaya, Enrique

    2002-02-05

    The transgenic technique in Xenopus allows one to misexpress genes in a temporally and spatially controlled manner. However, this system suffers from two experimental limitations. First, the restriction enzyme-mediated integration procedure relies on chromosomal damage, resulting in a percentage of embryos failing to develop normally. Second, every transgenic embryo has unique sites of integration and unique transgene copy number, resulting in variable transgene expression levels and variable phenotypes. For these reasons, we have adapted the Gal4-UAS method for targeted gene expression to Xenopus. This technique relies on the generation of transgenic lines that carry "activator" or "effector" constructs. Activator lines express the yeast transcription factor, Gal4, under the control of a desired promoter, whereas effector lines contain DNA-binding motifs for Gal4-(UAS) linked to the gene of interest. We show that on intercrossing of these lines, the effector gene is transcribed in the temporal and spatial manner of the activator's promoter. Furthermore, we use the Gal4-UAS system to misexpress Xvent-2, a transcriptional target of bone morphogenetic protein 4 (BMP4) signaling during early embryogenesis. Embryos inheriting both the Gal4 activator and Xvent-2 effector transgenes display a consistent microcephalic phenotype. Finally, we exploit this system to characterize the neural and mesodermal defects obtained from early misexpression of Xvent-2. These results emphasize the potential of this system for the controlled analyses of gene function in Xenopus.

  6. Efficient and Heritable Gene Targeting in Tilapia by CRISPR/Cas9

    Science.gov (United States)

    Li, Minghui; Yang, Huihui; Zhao, Jiue; Fang, Lingling; Shi, Hongjuan; Li, Mengru; Sun, Yunlv; Zhang, Xianbo; Jiang, Dongneng; Zhou, Linyan; Wang, Deshou

    2014-01-01

    Studies of gene function in non-model animals have been limited by the approaches available for eliminating gene function. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated) system has recently become a powerful tool for targeted genome editing. Here, we report the use of the CRISPR/Cas9 system to disrupt selected genes, including nanos2, nanos3, dmrt1, and foxl2, with efficiencies as high as 95%. In addition, mutations in dmrt1 and foxl2 induced by CRISPR/Cas9 were efficiently transmitted through the germline to F1. Obvious phenotypes were observed in the G0 generation after mutation of germ cell or somatic cell-specific genes. For example, loss of Nanos2 and Nanos3 in XY and XX fish resulted in germ cell-deficient gonads as demonstrated by GFP labeling and Vasa staining, respectively, while masculinization of somatic cells in both XY and XX gonads was demonstrated by Dmrt1 and Cyp11b2 immunohistochemistry and by up-regulation of serum androgen levels. Our data demonstrate that targeted, heritable gene editing can be achieved in tilapia, providing a convenient and effective approach for generating loss-of-function mutants. Furthermore, our study shows the utility of the CRISPR/Cas9 system for genetic engineering in non-model species like tilapia and potentially in many other teleost species. PMID:24709635

  7. Combining Untargeted and Targeted Proteomic Strategies for Discrimination and Quantification of Cashmere Fibers.

    Science.gov (United States)

    Li, Shanshan; Zhang, Yong; Wang, Jihua; Yang, Yunfei; Miao, Chen; Guo, Yufeng; Zhang, Zhidan; Cao, Qichen; Shui, Wenqing

    2016-01-01

    Cashmere is regarded as a specialty and luxury fiber due to its scarcity and high economic value. For fiber quality assessment, it is technically very challenging to distinguish and quantify the cashmere fiber from yak or wool fibers because of their highly similar physical appearance and substantial protein sequence homology. To address this issue, we propose a workflow combining untargeted and targeted proteomics strategies for selecting, verifying and quantifying biomarkers for cashmere textile authentication. Untargeted proteomic surveys were first applied to identify 174, 157, and 156 proteins from cashmere, wool and yak fibers, respectively. After marker selection at different levels, peptides turned out to afford much higher selectivity than proteins for fiber species discrimination. Subsequently, parallel reaction monitoring (PRM) methods were developed for ten selected peptide markers. The PRM-based targeted analysis of peptide markers enabled accurate determination of fiber species and cashmere percentages in different fiber mixtures. Furthermore, collective use of these peptide makers allowed us to discriminate and quantify cashmere fibers in commercial finished fabrics that have undergone heavy chemical treatments. Cashmere proportion measurement in fabric samples using our proteomic approach was in good agreement with results from traditional light microscopy, yet our method can be more readily standardized to become an objective and robust assay for assessing authenticity of fibers and textiles. We anticipate that the proteomic strategies presented in our study could be further implicated in discovery of quality trait markers for other products containing highly homologous proteomes.

  8. Combining Untargeted and Targeted Proteomic Strategies for Discrimination and Quantification of Cashmere Fibers.

    Directory of Open Access Journals (Sweden)

    Shanshan Li

    Full Text Available Cashmere is regarded as a specialty and luxury fiber due to its scarcity and high economic value. For fiber quality assessment, it is technically very challenging to distinguish and quantify the cashmere fiber from yak or wool fibers because of their highly similar physical appearance and substantial protein sequence homology. To address this issue, we propose a workflow combining untargeted and targeted proteomics strategies for selecting, verifying and quantifying biomarkers for cashmere textile authentication. Untargeted proteomic surveys were first applied to identify 174, 157, and 156 proteins from cashmere, wool and yak fibers, respectively. After marker selection at different levels, peptides turned out to afford much higher selectivity than proteins for fiber species discrimination. Subsequently, parallel reaction monitoring (PRM methods were developed for ten selected peptide markers. The PRM-based targeted analysis of peptide markers enabled accurate determination of fiber species and cashmere percentages in different fiber mixtures. Furthermore, collective use of these peptide makers allowed us to discriminate and quantify cashmere fibers in commercial finished fabrics that have undergone heavy chemical treatments. Cashmere proportion measurement in fabric samples using our proteomic approach was in good agreement with results from traditional light microscopy, yet our method can be more readily standardized to become an objective and robust assay for assessing authenticity of fibers and textiles. We anticipate that the proteomic strategies presented in our study could be further implicated in discovery of quality trait markers for other products containing highly homologous proteomes.

  9. Combinational targeting offsets antigen escape and enhances effector functions of adoptively transferred T cells in glioblastoma.

    Science.gov (United States)

    Hegde, Meenakshi; Corder, Amanda; Chow, Kevin K H; Mukherjee, Malini; Ashoori, Aidin; Kew, Yvonne; Zhang, Yi Jonathan; Baskin, David S; Merchant, Fatima A; Brawley, Vita S; Byrd, Tiara T; Krebs, Simone; Wu, Meng Fen; Liu, Hao; Heslop, Helen E; Gottschalk, Stephen; Gottachalk, Stephen; Yvon, Eric; Ahmed, Nabil

    2013-11-01

    Preclinical and early clinical studies have demonstrated that chimeric antigen receptor (CAR)-redirected T cells are highly promising in cancer therapy. We observed that targeting HER2 in a glioblastoma (GBM) cell line results in the emergence of HER2-null tumor cells that maintain the expression of nontargeted tumor-associated antigens. Combinational targeting of these tumor-associated antigens could therefore offset this escape mechanism. We studied the single-cell coexpression patterns of HER2, IL-13Rα2, and EphA2 in primary GBM samples using multicolor flow cytometry and immunofluorescence, and applied a binomial routine to the permutations of antigen expression and the related odds of complete tumor elimination. This mathematical model demonstrated that cotargeting HER2 and IL-13Rα2 could maximally expand the therapeutic reach of the T cell product in all primary tumors studied. Targeting a third antigen did not predict an added advantage in the tumor cohort studied. We therefore generated bispecific T cell products from healthy donors and from GBM patients by pooling T cells individually expressing HER2 and IL-13Rα2-specific CARs and by making individual T cells to coexpress both molecules. Both HER2/IL-13Rα2-bispecific T cell products offset antigen escape, producing enhanced effector activity in vitro immunoassays (against autologous glioma cells in the case of GBM patient products) and in an orthotopic xenogeneic murine model. Further, T cells coexpressing HER2 and IL-13Rα2-CARs exhibited accentuated yet antigen-dependent downstream signaling and a particularly enhanced antitumor activity.

  10. Three-cohort targeted gene screening reveals a non-synonymous TRKA polymorphism associated with schizophrenia

    DEFF Research Database (Denmark)

    van Schijndel, Jessica E; van Loo, Karen M J; van Zweeden, Martine

    2009-01-01

    Schizophrenia is a complex neurodevelopmental disorder that is thought to be induced by an interaction between predisposing genes and environmental stressors. To identify predisposing genetic factors, we performed a targeted (mostly neurodevelopmental) gene approach involving the screening of 396...... selected non-synonymous single-nucleotide polymorphisms (SNPs) in three independent Caucasian schizophrenia case-control cohorts (USA, Denmark and Norway). A meta-analysis revealed ten non-synonymous SNPs that were nominally associated with schizophrenia, nine of which have not been previously linked...... for schizophrenia....

  11. Dose-dependent regulation of target gene expression and cell proliferation by c-Myc levels

    OpenAIRE

    Schuhmacher, Marino; Eick, Dirk

    2013-01-01

    The proto-oncogene c-myc encodes a basic helix-loop-helix leucine zipper transcription factor (c-Myc). c-Myc plays a crucial role in cell growth and proliferation. Here, we examined how expression of c-Myc target genes and cell proliferation depend on variation of c-Myc protein levels. We show that proliferation rates, the number of cells in S-phase, and cell size increased in a dose-dependent manner in response to increasing c-Myc levels. Likewise, the mRNA levels of c-Myc responsive genes s...

  12. Combining multiple tools outperforms individual methods in gene set enrichment analyses

    Science.gov (United States)

    Ng, Milica; Wilson, Nicholas J.; Sheridan, Julie M.; Huynh, Huy; Wilson, Michael J.

    2017-01-01

    Abstract Motivation: Gene set enrichment (GSE) analysis allows researchers to efficiently extract biological insight from long lists of differentially expressed genes by interrogating them at a systems level. In recent years, there has been a proliferation of GSE analysis methods and hence it has become increasingly difficult for researchers to select an optimal GSE tool based on their particular dataset. Moreover, the majority of GSE analysis methods do not allow researchers to simultaneously compare gene set level results between multiple experimental conditions. Results: The ensemble of genes set enrichment analyses (EGSEA) is a method developed for RNA-sequencing data that combines results from twelve algorithms and calculates collective gene set scores to improve the biological relevance of the highest ranked gene sets. EGSEA’s gene set database contains around 25 000 gene sets from sixteen collections. It has multiple visualization capabilities that allow researchers to view gene sets at various levels of granularity. EGSEA has been tested on simulated data and on a number of human and mouse datasets and, based on biologists’ feedback, consistently outperforms the individual tools that have been combined. Our evaluation demonstrates the superiority of the ensemble approach for GSE analysis, and its utility to effectively and efficiently extrapolate biological functions and potential involvement in disease processes from lists of differentially regulated genes. Availability and Implementation: EGSEA is available as an R package at http://www.bioconductor.org/packages/EGSEA/. The gene sets collections are available in the R package EGSEAdata from http://www.bioconductor.org/packages/EGSEAdata/. Contacts:monther.alhamdoosh@csl.com.au ormritchie@wehi.edu.au Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27694195

  13. Novel noncontiguous duplications identified with a comprehensive mutation analysis in the DMD gene by DMD gene-targeted sequencing.

    Science.gov (United States)

    Xu, Yan; Wang, Huanhuan; Xiao, Bing; Wei, Wei; Liu, Yu; Ye, Hui; Ying, Xiaomin; Chen, Yingwei; Liu, Xiaoqing; Ji, Xing; Sun, Yu

    2018-03-01

    Genomic rearrangements, such as intragenic deletions and duplications, are the most prevalent types of mutation in the DMD gene, and DMD mutations underlie Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). Using multiplex ligation dependent probe amplification (MLPA) and DMD gene-targeted sequencing, we performed a molecular characterization of two cases of complex noncontiguous duplication rearrangements that involved inverted duplications. The breakpoint sequences were analyzed to investigate the mechanisms of the rearrangement. The two cases shared the same duplication events (Dup-nml-Dup/inv), and both involved microhomology and small insertions at the breakpoints. Additionally, in case 1, SNP sequencing results indicated that the de novo duplication mutation arose in the allele that originated from the grandfather. This study has identified a novel type of DMD complex rearrangement and provides insight into the molecular basis of this genomic rearrangement. Copyright © 2017. Published by Elsevier B.V.

  14. Comparative gene expression analysis of Dtg, a novel target gene of Dpp signaling pathway in the early Drosophila melanogaster embryo.

    Science.gov (United States)

    Hodar, Christian; Zuñiga, Alejandro; Pulgar, Rodrigo; Travisany, Dante; Chacon, Carlos; Pino, Michael; Maass, Alejandro; Cambiazo, Verónica

    2014-02-10

    In the early Drosophila melanogaster embryo, Dpp, a secreted molecule that belongs to the TGF-β superfamily of growth factors, activates a set of downstream genes to subdivide the dorsal region into amnioserosa and dorsal epidermis. Here, we examined the expression pattern and transcriptional regulation of Dtg, a new target gene of Dpp signaling pathway that is required for proper amnioserosa differentiation. We showed that the expression of Dtg was controlled by Dpp and characterized a 524-bp enhancer that mediated expression in the dorsal midline, as well as, in the differentiated amnioserosa in transgenic reporter embryos. This enhancer contained a highly conserved region of 48-bp in which bioinformatic predictions and in vitro assays identified three Mad binding motifs. Mutational analysis revealed that these three motifs were necessary for proper expression of a reporter gene in transgenic embryos, suggesting that short and highly conserved genomic sequences may be indicative of functional regulatory regions in D. melanogaster genes. Dtg orthologs were not detected in basal lineages of Dipterans, which unlike D. melanogaster develop two extra-embryonic membranes, amnion and serosa, nevertheless Dtg orthologs were identified in the transcriptome of Musca domestica, in which dorsal ectoderm patterning leads to the formation of a single extra-embryonic membrane. These results suggest that Dtg was recruited as a new component of the network that controls dorsal ectoderm patterning in the lineage leading to higher Cyclorrhaphan flies, such as D. melanogaster and M. domestica. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. The BTB and CNC homology 1 (BACH1) target genes are involved in the oxidative stress response and in control of the cell cycle.

    Science.gov (United States)

    Warnatz, Hans-Jörg; Schmidt, Dominic; Manke, Thomas; Piccini, Ilaria; Sultan, Marc; Borodina, Tatiana; Balzereit, Daniela; Wruck, Wasco; Soldatov, Alexey; Vingron, Martin; Lehrach, Hans; Yaspo, Marie-Laure

    2011-07-01

    The regulation of gene expression in response to environmental signals and metabolic imbalances is a key step in maintaining cellular homeostasis. BTB and CNC homology 1 (BACH1) is a heme-binding transcription factor repressing the transcription from a subset of MAF recognition elements at low intracellular heme levels. Upon heme binding, BACH1 is released from the MAF recognition elements, resulting in increased expression of antioxidant response genes. To systematically address the gene regulatory networks involving BACH1, we combined chromatin immunoprecipitation sequencing analysis of BACH1 target genes in HEK 293 cells with knockdown of BACH1 using three independent types of small interfering RNAs followed by transcriptome profiling using microarrays. The 59 BACH1 target genes identified by chromatin immunoprecipitation sequencing were found highly enriched in genes showing expression changes after BACH1 knockdown, demonstrating the impact of BACH1 repression on transcription. In addition to known and new BACH1 targets involved in heme degradation (HMOX1, FTL, FTH1, ME1, and SLC48A1) and redox regulation (GCLC, GCLM, and SLC7A11), we also discovered BACH1 target genes affecting cell cycle and apoptosis pathways (ITPR2, CALM1, SQSTM1, TFE3, EWSR1, CDK6, BCL2L11, and MAFG) as well as subcellular transport processes (CLSTN1, PSAP, MAPT, and vault RNA). The newly identified impact of BACH1 on genes involved in neurodegenerative processes and proliferation provides an interesting basis for future dissection of BACH1-mediated gene repression in neurodegeneration and virus-induced cancerogenesis.

  16. Radiation targeted TNF gene therapy in TNF-resistant prostate cancer

    International Nuclear Information System (INIS)

    Chung, Theodore D.K.; Mauceri, Helena J.; Hallahan, Dennis E.; Yu, Jenny; Yajnik, Santosh; Weichselbaum, Ralph R.

    1997-01-01

    Purpose/Objectives: Control of gene transcription by ionizing radiation represents a novel method of spatial and temporal regulation of genetic-based medical treatment. We previously reported improved tumor control using this approach in the radioresistant, TNF-sensitive squamous cell carcinoma, SQ-20B. Histopathologic evaluation of treated tumor specimens demonstrated tumor necrosis and vascular obstruction suggesting that the combined TNF and radiation treatment inhibits tumor growth through both direct and indirect mechanisms. The purpose of the present study was to determine the therapeutic potential of this approach in a TNF-resistant prostate cancer xenograft. Materials and Methods: The hormone-refractory human prostate cancer cell line PC-3 was transfected with Ad.Egr-TNF (adenovirus type 5 vector expressing TNFα under the transcriptional regulation of the radiation-inducible Egr-1 promoter) and TNF production was quantified by ELISA. TNF-induced cytotoxicity and radiosensitization were studied using rhTNF and PC-3 in an MTT assay and standard clonogenic survival assays. PC-3 xenografts were established approximately 2 weeks after injecting 2 x 10 7 cells S.C. in the right hind limb of female nude mice. Animals were treated with intratumoral injections of either Ad.Egr-TNF or buffer. Xenografts were irradiated with 5 Gy/fx 4 times per week to a total dose of 40 Gy using a Maxitron 250 x-ray generator (1.88 Gy/min). Results: TNF production in vitro. PC-3 cells infected with Ad.Egr-TNF released >1000 pg/ml TNF. Uninfected cells did not produce detectable levels of TNF. TNF mediated cytotoxicity in vitro. PC-3 cells were highly resistant to rhTNF with 78% cell survival observed at the highest concentration tested (10,000 U/ml). TNF mediated radiosensitization in vitro. rhTNF (1000 U/ml) had no significant effect on clonogenic survival of PC-3 cells following irradiation (D 0 =1.5). Combined Ad.Egr-TNF and 40 Gy in vivo. Combined treatment of PC-3 xenografts

  17. Targeting inhibitor of apoptosis proteins in combination with ErbB antagonists in breast cancer.

    Science.gov (United States)

    Foster, Fiona M; Owens, Thomas W; Tanianis-Hughes, Jolanta; Clarke, Robert B; Brennan, Keith; Bundred, Nigel J; Streuli, Charles H

    2009-01-01

    Inhibitor of apoptosis (IAPs) proteins are a family of proteins that can block apoptosis in normal cells and have been suggested to cause resistance to apoptosis in cancer. Overexpression of oncogenic receptor tyrosine kinases is common in breast cancer; in particular 20% of all cases show elevated Her2. Despite clinical success with the use of targeted therapies, such as Trastuzumab, only up to 35% of Her2-positive patients initially respond. We reasoned that IAP-mediated apoptosis resistance might contribute to this insensitivity to receptor tyrosine kinase therapy, in particular ErbB antagonists. Here we examine the levels of IAPs in breast cancer and evaluate whether targeting IAPs can enhance apoptosis in response to growth factor receptor antagonists and TRAIL. IAP levels were examined in a breast cancer cell line panel and in patient samples. IAPs were inhibited using siRNA or cell permeable mimetics of endogenous inhibitors. Cells were then exposed to TRAIL, Trastuzumab, Lapatinib, or Gefitinib for 48 hours. Examining nuclear morphology and staining for cleaved caspase 3 was used to score apoptosis. Proliferation was examined by Ki67 staining. Four members of the IAP family, Survivin, XIAP, cIAP1 and cIAP2, were all expressed to varying extents in breast cancer cell lines or tumours. MDAMB468, BT474 and BT20 cells all expressed XIAP to varying extents. Depleting the cells of XIAP overcame the intrinsic resistance of BT20 and MDAMB468 cells to TRAIL. Moreover, siRNA-based depletion of XIAP or use of a Smac mimetic to target multiple IAPs increased apoptosis in response to the ErbB antagonists, Trastuzumab, Lapatinib or Gefitinib in Her2-overexpressing BT474 cells, or Gefitinib in EGFR-overexpressing MDAMB468 cells. The novel findings of this study are that multiple IAPs are concomitantly expressed in breast cancers, and that, in combination with clinically relevant Her2 treatments, IAP antagonists promote apoptosis and reduce the cell turnover index of

  18. Identification of Drosophila mitotic genes by combining co-expression analysis and RNA interference.

    Directory of Open Access Journals (Sweden)

    Maria Patrizia Somma

    2008-07-01

    Full Text Available RNAi screens have, to date, identified many genes required for mitotic divisions of Drosophila tissue culture cells. However, the inventory of such genes remains incomplete. We have combined the powers of bioinformatics and RNAi technology to detect novel mitotic genes. We found that Drosophila genes involved in mitosis tend to be transcriptionally co-expressed. We thus constructed a co-expression-based list of 1,000 genes that are highly enriched in mitotic functions, and we performed RNAi for each of these genes. By limiting the number of genes to be examined, we were able to perform a very detailed phenotypic analysis of RNAi cells. We examined dsRNA-treated cells for possible abnormalities in both chromosome structure and spindle organization. This analysis allowed the identification of 142 mitotic genes, which were subdivided into 18 phenoclusters. Seventy of these genes have not previously been associated with mitotic defects; 30 of them are required for spindle assembly and/or chromosome segregation, and 40 are required to prevent spontaneous chromosome breakage. We note that the latter type of genes has never been detected in previous RNAi screens in any system. Finally, we found that RNAi against genes encoding kinetochore components or highly conserved splicing factors results in identical defects in chromosome segregation, highlighting an unanticipated role of splicing factors in centromere function. These findings indicate that our co-expression-based method for the detection of mitotic functions works remarkably well. We can foresee that elaboration of co-expression lists using genes in the same phenocluster will provide many candidate genes for small-scale RNAi screens aimed at completing the inventory of mitotic proteins.

  19. Targeting gene expression to specific cells of kidney tubules in vivo, using adenoviral promoter fragments.

    Science.gov (United States)

    Watanabe, Sumiyo; Ogasawara, Toru; Tamura, Yoshifuru; Saito, Taku; Ikeda, Toshiyuki; Suzuki, Nobuchika; Shimosawa, Tatsuo; Shibata, Shigeru; Chung, Ung-Il; Nangaku, Masaomi; Uchida, Shunya

    2017-01-01

    Although techniques for cell-specific gene expression via viral transfer have advanced, many challenges (e.g., viral vector design, transduction of genes into specific target cells) still remain. We investigated a novel, simple methodology for using adenovirus transfer to target specific cells of the kidney tubules for the expression of exogenous proteins. We selected genes encoding sodium-dependent phosphate transporter type 2a (NPT2a) in the proximal tubule, sodium-potassium-2-chloride cotransporter (NKCC2) in the thick ascending limb of Henle (TALH), and aquaporin 2 (AQP2) in the collecting duct. The promoters of the three genes were linked to a GFP-coding fragment, the final constructs were then incorporated into an adenovirus vector, and this was then used to generate gene-manipulated viruses. After flushing circulating blood, viruses were directly injected into the renal arteries of rats and were allowed to site-specifically expression in tubule cells, and rats were then euthanized to obtain kidney tissues for immunohistochemistry. Double staining with adenovirus-derived EGFP and endogenous proteins were examined to verify orthotopic expression, i.e. "adenovirus driven NPT2a-EGFP and endogenous NHE3 protein", "adenovirus driven NKCC2-EGFP and endogenous NKCC2 protein" and "adenovirus driven AQP2-EGFP and endogenous AQP2 protein". Owing to a lack of finding good working anti-NPT2a antibody, an antibody against a different protein (sodium-hydrogen exchanger 3 or NHE3) that is also specifically expressed in the proximal tubule was used. Kidney structures were well-preserved, and other organ tissues did not show EGFP staining. Our gene transfer method is easier than using genetically engineered animals, and it confers the advantage of allowing the manipulation of gene transfer after birth. This is the first method to successfully target gene expression to specific cells in the kidney tubules. This study may serve as the first step for safe and effective gene

  20. A comparison of Agrobacterium-mediated transformation and protoplast-mediated transformation with CRISPR-Cas9 and bipartite gene targeting substrates, as effective gene targeting tools for Aspergillus carbonarius.

    Science.gov (United States)

    Weyda, István; Yang, Lei; Vang, Jesper; Ahring, Birgitte K; Lübeck, Mette; Lübeck, Peter S

    2017-04-01

    In recent years, versatile genetic tools have been developed and applied to a number of filamentous fungi of industrial importance. However, the existing techniques have limitations when it comes to achieve the desired genetic modifications, especially for efficient gene targeting. In this study, we used Aspergillus carbonarius as a host strain due to its potential as a cell factory, and compared three gene targeting techniques by disrupting the ayg1 gene involved in the biosynthesis of conidial pigment in A. carbonarius. The absence of the ayg1 gene leads to phenotypic change in conidia color, which facilitated the analysis on the gene targeting frequency. The examined transformation techniques included Agrobacterium-mediated transformation (AMT) and protoplast-mediated transformation (PMT). Furthermore, the PMT for the disruption of the ayg1 gene was carried out with bipartite gene targeting fragments and the recently adapted CRISPR-Cas9 system. All three techniques were successful in generating Δayg1 mutants, but showed different efficiencies. The most efficient method for gene targeting was AMT, but further it was shown to be dependent on the choice of Agrobacterium strain. However, there are different advantages and disadvantages of all three gene targeting methods which are discussed, in order to facilitate future approaches for fungal strain improvements. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Targeting synaptic dysfunction in Alzheimer's disease by administering a specific nutrient combination.

    Science.gov (United States)

    van Wijk, Nick; Broersen, Laus M; de Wilde, Martijn C; Hageman, Robert J J; Groenendijk, Martine; Sijben, John W C; Kamphuis, Patrick J G H

    2014-01-01

    Synapse loss and synaptic dysfunction are pathological processes already involved in the early stages of Alzheimer's disease (AD). Synapses consist principally of neuronal membranes, and the neuronal and synaptic losses observed in AD have been linked to the degeneration and altered composition and structure of these membranes. Consequently, synapse loss and membrane-related pathology provide viable targets for intervention in AD. The specific nutrient combination Fortasyn Connect (FC) is designed to ameliorate synapse loss and synaptic dysfunction in AD by addressing distinct nutritional needs believed to be present in these patients. This nutrient combination comprises uridine, docosahexaenoic acid, eicosapentaenoic acid, choline, phospholipids, folic acid, vitamins B12, B6, C, and E, and selenium, and is present in Souvenaid, a medical food intended for use in early AD. It has been hypothesized that FC counteracts synaptic loss and reduces membrane-related pathology in AD by providing nutritional precursors and cofactors that act together to support neuronal membrane formation and function. Preclinical studies formed the basis of this hypothesis which is being validated in a broad clinical study program investigating the potential of this nutrient combination in AD. Memory dysfunction is one key early manifestation in AD and is associated with synapse loss. The clinical studies to date show that the FC-containing medical food improves memory function and preserves functional brain network organization in mild AD compared with controls, supporting the hypothesis that this intervention counteracts synaptic dysfunction. This review provides a comprehensive overview of basic scientific studies that led to the creation of FC and of its effects in various preclinical models.

  2. Cutting edge: Dab2 is a FOXP3 target gene required for regulatory T cell function.

    Science.gov (United States)

    Jain, Nitya; Nguyen, Hai; Friedline, Randall H; Malhotra, Nidhi; Brehm, Michael; Koyanagi, Madoka; Bix, Mark; Cooper, Jonathan A; Chambers, Cynthia A; Kang, Joonsoo

    2009-10-01

    FOXP3-expressing regulatory T (Treg) cells are vital for maintaining peripheral T cell tolerance and homeostasis. The mechanisms by which FOXP3 target genes orchestrate context-dependent Treg cell function are largely unknown. In this study we show that in mouse peripheral lymphocytes the Drosophila Disabled-2 (Dab2) homolog, a gene that is involved in enhancing TGFbeta responses, is exclusively expressed in FOXP3+ regulatory T cells. Dab2 is a direct target of FOXP3, and regulatory T cells lacking DAB2 are functionally impaired in vitro and in vivo. However, not all aspects of Treg cell function are perturbed, and DAB2 appears to be dispensable for Treg cell function in maintaining naive T cell homeostasis.

  3. Disabled-2 is a FOXP3 target gene required for regulatory T cell function

    Science.gov (United States)

    Jain, N; Nguyen, H; Friedline, RH; Malhotra, N; Brehm, M; Koyonagi, M; Bix, M; Cooper, JA; Chambers, CA; Kang, J

    2010-01-01

    FOXP3 expressing regulatory T cells are vital for maintaining peripheral T cell tolerance and homeostasis. The mechanisms by which FOXP3 target genes orchestrate context-dependent Treg cell function are largely unknown. Here we show that in mouse peripheral lymphocytes, the Drosophila Disabled-2 (Dab2) homolog, a gene that is involved in enhancing TGFβ responses, is exclusively expressed in FOXP3+ regulatory T cells. Dab2 is a direct target of FOXP3 and regulatory T cells lacking DAB2 are functionally impaired in vitro and in vivo. However, not all aspects of Treg cell function are perturbed and DAB2 appears dispensable for Treg cell function in maintaining naïve T cell homeostasis. PMID:19767570

  4. The evolution of gene therapy in X-linked severe combined immunodeficiency.

    Science.gov (United States)

    Rans, Tonya S; England, Ronald

    2009-05-01

    To review the evolution of gene therapy in infants with X-linked severe combined immunodeficiency (XL-SCID) and to evaluate the current challenges facing this evolving field. The MEDLINE, OVID, CINAHL, and HealthSTAR databases were searched to identify pertinent articles using the following keywords: gene therapy, XL-SCID, bone marrow transplant, and viral vectors. Journal articles were selected for their relevance to human gene therapy in patients with XL-SCID. Gene therapy with a retrovirus-derived vector has been used to treat 20 patients with XL-SCID internationally. Although most patients derived improvements in T- and B-cell immune numbers and function, severe adverse effects have occurred. After gene therapy, 5 of the 20 patients developed leukemia. This outcome has been associated with insertion of the corrected gene near the T-cell proto-oncogene LMO2. One of the 5 patients subsequently died. Within the past decade, effective improvements in vectorology and cell culture conditions have resulted in clinical success in some infants with SCID and have revived interest after many years of setbacks. However, clinical success and significant adverse events have been reported in patients with XL-SCID who have undergone gene therapy using a retroviral vector. As extensive research into improving safety through vector development and monitoring of gene therapy continues, further progress in gene therapy development can be anticipated.

  5. Proviral HIV-genome-wide and pol-gene specific zinc finger nucleases: usability for targeted HIV gene therapy.

    Science.gov (United States)

    Wayengera, Misaki

    2011-07-22

    Infection with HIV, which culminates in the establishment of a latent proviral reservoir, presents formidable challenges for ultimate cure. Building on the hypothesis that ex-vivo or even in-vivo abolition or disruption of HIV-gene/genome-action by target mutagenesis or excision can irreversibly abrogate HIV's innate fitness to replicate and survive, we previously identified the isoschizomeric bacteria restriction enzymes (REases) AcsI and ApoI as potent cleavers of the HIV-pol gene (11 and 9 times in HIV-1 and 2, respectively). However, both enzymes, along with others found to cleave across the entire HIV-1 genome, slice (SX) at palindromic sequences that are prevalent within the human genome and thereby pose the risk of host genome toxicity. A long-term goal in the field of R-M enzymatic therapeutics has thus been to generate synthetic restriction endonucleases with longer recognition sites limited in specificity to HIV. We aimed (i) to assemble and construct zinc finger arrays and nucleases (ZFN) with either proviral-HIV-pol gene or proviral-HIV-1 whole-genome specificity respectively, and (ii) to advance a model for pre-clinically testing lentiviral vectors (LV) that deliver and transduce either ZFN genotype. First, we computationally generated the consensus sequences of (a) 114 dsDNA-binding zinc finger (Zif) arrays (ZFAs or ZifHIV-pol) and (b) two zinc-finger nucleases (ZFNs) which, unlike the AcsI and ApoI homeodomains, possess specificity to >18 base-pair sequences uniquely present within the HIV-pol gene (ZifHIV-polFN). Another 15 ZFNs targeting >18 bp sequences within the complete HIV-1 proviral genome were constructed (ZifHIV-1FN). Second, a model for constructing lentiviral vectors (LVs) that deliver and transduce a diploid copy of either ZifHIV-polFN or ZifHIV-1FN chimeric genes (termed LV- 2xZifHIV-polFN and LV- 2xZifHIV-1FN, respectively) is proposed. Third, two preclinical models for controlled testing of the safety and efficacy of either of these

  6. Mesenchymal Stem Cell-Based Tumor-Targeted Gene Therapy in Gastrointestinal Cancer

    OpenAIRE

    Bao, Qi; Zhao, Yue; Niess, Hanno; Conrad, Claudius; Schwarz, Bettina; Jauch, Karl-Walter; Huss, Ralf; Nelson, Peter J.; Bruns, Christiane J.

    2012-01-01

    Mesenchymal stem (or stromal) cells (MSCs) are nonhematopoietic progenitor cells that can be obtained from bone marrow aspirates or adipose tissue, expanded and genetically modified in vitro, and then used for cancer therapeutic strategies in vivo. Here, we review available data regarding the application of MSC-based tumor-targeted therapy in gastrointestinal cancer, provide an overview of the general history of MSC-based gene therapy in cancer research, and discuss potential problems associa...

  7. ABCF2, an Nrf2 target gene, contributes to cisplatin resistance in ovarian cancer cells.

    Science.gov (United States)

    Bao, Lingjie; Wu, Jianfa; Dodson, Matthew; Rojo de la Vega, Elisa Montserrat; Ning, Yan; Zhang, Zhenbo; Yao, Ming; Zhang, Donna D; Xu, Congjian; Yi, Xiaofang

    2017-06-01

    Previously, we have demonstrated that NRF2 plays a key role in mediating cisplatin resistance in ovarian cancer. To further explore the mechanism underlying NRF2-dependent cisplatin resistance, we stably overexpressed or knocked down NRF2 in parental and cisplatin-resistant human ovarian cancer cells, respectively. These two pairs of stable cell lines were then subjected to microarray analysis, where we identified 18 putative NRF2 target genes. Among these genes, ABCF2, a cytosolic member of the ABC superfamily of transporters, has previously been reported to contribute to chemoresistance in clear cell ovarian cancer. A detailed analysis on ABCF2 revealed a functional antioxidant response element (ARE) in its promoter region, establishing ABCF2 as an NRF2 target gene. Next, we investigated the contribution of ABCF2 in NRF2-mediated cisplatin resistance using our stable ovarian cancer cell lines. The NRF2-overexpressing cell line, containing high levels of ABCF2, was more resistant to cisplatin-induced apoptosis compared to its control cell line; whereas the NRF2 knockdown cell line with low levels of ABCF2, was more sensitive to cisplatin treatment than its control cell line. Furthermore, transient overexpression of ABCF2 in the parental cells decreased apoptosis and increased cell viability following cisplatin treatment. Conversely, knockdown of ABCF2 using specific siRNA notably increased apoptosis and decreased cell viability in cisplatin-resistant cells treated with cisplatin. This data indicate that the novel NRF2 target gene, ABCF2, plays a critical role in cisplatin resistance in ovarian cancer, and that targeting ABCF2 may be a new strategy to improve chemotherapeutic efficiency. © 2017 Wiley Periodicals, Inc.

  8. Integrating Molecular Imaging Approaches to Monitor Prostate Targeted Suicide and Anti-angiogenic Gene Therapy

    Science.gov (United States)

    2005-02-01

    Bruno , M. D., Korfhagen, T. R., Liu, C., Morrisey, E. E., and Whitsett, J. A. (2000). GATA-6 activates transcription of surfactant protein A. J. Biol...the tail vein (for in- travenous administration), peritoneum, prostate, or forepaw. Before imaging, mice were anesthetized with ketamine–xy- lazine...DOTAP:cholesterol DNA complexes [Iyer et al., 2002]. In- travenous delivery of transferin-targeted DNA-PEI poly- plexes results in specific fluc gene

  9. Efficient gene targeting of the Rosa26 locus in mouse zygotes using TALE nucleases

    Czech Academy of Sciences Publication Activity Database

    Kašpárek, Petr; Krausová, Michaela; Hanečková, Radka; Kříž, Vítězslav; Žbodáková, Olga; Kořínek, Vladimír; Sedláček, Radislav

    2014-01-01

    Roč. 588, č. 21 (2014), 3982-3988 ISSN 0014-5793 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109; GA ČR(CZ) GA14-33952S; GA MŠk(CZ) LM2011032 Institutional support: RVO:68378050 Keywords : Gene targeting * Homologous recombination * Rosa26 locus * Transgenic * Transcription activator-like effector nucleases * Zinc-finger nucleases Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.169, year: 2014

  10. Targeting gene expression to the female larval fat body of transgenic Aedes aegypti mosquitoes.

    Science.gov (United States)

    Totten, D C; Vuong, M; Litvinova, O V; Jinwal, U K; Gulia-Nuss, M; Harrell, R A; Beneš, H

    2013-02-01

    As the fat body is a critical tissue for mosquito development, metamorphosis, immune and reproductive system function, the characterization of regulatory modules targeting gene expression to the female mosquito fat body at distinct life stages is much needed for multiple, varied strategies for controlling vector-borne diseases such as dengue and malaria. The hexameric storage protein, Hexamerin-1.2, of the mosquito Aedes atropalpus is female-specific and uniquely expressed in the fat body of fourth instar larvae and young adults. We have identified in the Hex-1.2 gene, a short regulatory module that directs female-, tissue-, and stage-specific lacZ reporter gene expression using a heterologous promoter in transgenic lines of the dengue vector Aedes aegypti. Male transgenic larvae and pupae of one line expressed no Escherichia coli β-galactosidase or transgene product; in two other lines reporter gene activity was highly female-biased. All transgenic lines expressed the reporter only in the fat body; however, lacZ mRNA levels were no different in males and females at any stage examined, suggesting that the gene regulatory module drives female-specific expression by post-transcriptional regulation in the heterologous mosquito. This regulatory element from the Hex-1.2 gene thus provides a new molecular tool for transgenic mosquito control as well as functional genetic analysis in aedine mosquitoes. © 2012 Royal Entomological Society.

  11. miR-370 suppresses HBV gene expression and replication by targeting nuclear factor IA.

    Science.gov (United States)

    Fan, Hongxia; Lv, Ping; Lv, Jing; Zhao, Xiaopei; Liu, Min; Zhang, Guangling; Tang, Hua

    2017-05-01

    Hepatitis B virus (HBV) infection is a major health problem worldwide. The roles of microRNAs in the regulation of HBV expression are being increasingly recognized. In this study, we found that overexpression of miR-370 suppressed HBV gene expression and replication in Huh7 cells, whereas antisense knockdown of endogenous miR-370 enhanced HBV gene expression and replication in Huh7 cells and HepG2.2.15 cells. Further, we identified the transcription factor nuclear factor IA (NFIA) as a new host target of miR-370. Overexpression and knockdown studies showed that NFIA stimulated HBV gene expression and replication. Importantly, overexpression of NFIA counteracted the effect of miR-370 on HBV gene expression and replication. Further mechanistic studies showed that miR-370 suppressed HBV replication and gene expression by repressing HBV Enhancer I activity, and one of the NFIA binding site in the Enhancer I element was responsible for the repressive effect of miR-370 on HBV Enhancer I activity. Altogether, our results demonstrated that miR-370 suppressed HBV gene expression and replication through repressing NFIA expression, which stimulates HBV replication via direct regulation on HBV Enhancer I activities. Our findings may provide a new antiviral strategy for HBV infection. J. Med. Virol. 89:834-844, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  12. Identification of the Drosophila Mes4 gene as a novel target of the transcription factor DREF

    Energy Technology Data Exchange (ETDEWEB)

    Suyari, Osamu; Ida, Hiroyuki [Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Yoshioka, Yasuhide; Kato, Yasuko; Hashimoto, Reina [Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Venture Laboratory, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Yamaguchi, Masamitsu, E-mail: myamaguc@kit.ac.jp [Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan)

    2009-05-01

    The Mes4 gene has been identified as one of the maternal Dorsal target genes in Drosophila. In the present study, we found a DNA replication-related element (DRE, 5'-TATCGATA) in the Mes4 promoter recognized by the DRE-binding factor (DREF). Luciferase transient expression assays in S2 cells using Mes4 promoter-luciferase fusion plasmids revealed that the DRE sequence is essential for Mes4 promoter activity. Requirement of DRE for Mes4 promoter activity was further confirmed by anti-{beta}-galactosidase antibody-staining of various tissues from transgenic flies carrying Mes4 promoter-lacZ fusion genes. Furthermore, wild type Mes4 promoter activity was decreased by 40% in DREF-depleted S2 cells. These results indicate that DREF positively regulates Mes4 gene expression. Band mobility shift analyses using Kc cell nuclear extracts further indicated that the DRE sequence in the Mes4 promoter is especially important for binding to DREF. Moreover, specific binding of DREF to the involved genomic region could be demonstrated by chromatin immunoprecipitation assays using anti-DREF antibodies. These results, taken together, indicate that the DRE/DREF system activates transcription of the Mes4 gene. In addition, knockdown of the Mes4 gene in wing imaginal discs using the GAL4-UAS system caused an atrophied wing phenotype, suggesting that Mes4 is required for wing morphogenesis.

  13. DNA breaks and chromatin structural changes enhance the transcription of autoimmune regulator target genes.

    Science.gov (United States)

    Guha, Mithu; Saare, Mario; Maslovskaja, Julia; Kisand, Kai; Liiv, Ingrid; Haljasorg, Uku; Tasa, Tõnis; Metspalu, Andres; Milani, Lili; Peterson, Pärt

    2017-04-21

    The autoimmune regulator (AIRE) protein is the key factor in thymic negative selection of autoreactive T cells by promoting the ectopic expression of tissue-specific genes in the thymic medullary epithelium. Mutations in AIRE cause a monogenic autoimmune disease called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. AIRE has been shown to promote DNA breaks via its interaction with topoisomerase 2 (TOP2). In this study, we investigated topoisomerase-induced DNA breaks and chromatin structural alterations in conjunction with AIRE-dependent gene expression. Using RNA sequencing, we found that inhibition of TOP2 religation activity by etoposide in AIRE-expressing cells had a synergistic effect on genes with low expression levels. AIRE-mediated transcription was not only enhanced by TOP2 inhibition but also by the TOP1 inhibitor camptothecin. The transcriptional activation was associated with structural rearrangements in chromatin, notably the accumulation of γH2AX and the exchange of histone H1 with HMGB1 at AIRE target gene promoters. In addition, we found the transcriptional up-regulation to co-occur with the chromatin structural changes within the genomic cluster of carcinoembryonic antigen-like cellular adhesion molecule genes. Overall, our results suggest that the presence of AIRE can trigger molecular events leading to an altered chromatin landscape and the enhanced transcription of low-expressed genes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Genome-Wide Identification of Polycomb Target Genes Reveals a Functional Association of Pho with Scm in Bombyx mori

    OpenAIRE

    Li, Zhiqing; Cheng, Daojun; Mon, Hiroaki; Tatsuke, Tsuneyuki; Zhu, Li; Xu, Jian; Lee, Jae Man; Xia, Qingyou; Kusakabe, Takahiro

    2012-01-01

    Polycomb group (PcG) proteins are evolutionarily conserved chromatin modifiers and act together in three multimeric complexes, Polycomb repressive complex 1 (PRC1), Polycomb repressive complex 2 (PRC2), and Pleiohomeotic repressive complex (PhoRC), to repress transcription of the target genes. Here, we identified Polycomb target genes in Bombyx mori with holocentric centromere using genome-wide expression screening based on the knockdown of BmSCE, BmESC, BmPHO, or BmSCM gene, which represent ...

  15. Combining `OMIC tools and other targeted molecular methods to evaluate iron limitation of diatoms in the Northeast Pacific Ocean.

    Science.gov (United States)

    Chappell, D.; Wallace, J.; Jenkins, B. D.; Powell, K.

    2016-02-01

    Diatoms are an abundant and widespread group of phytoplankton capable of exerting a profound influence on global carbon cycling. It is widely accepted that iron (Fe) controls diatom production in many ocean systems and the Fe stress response has been a focus of research for some time. Assessing the Fe status of natural diatom populations has proven challenging, as diatom species in the same genus may be difficult to distinguish using microscopy and the physiological and molecular responses to Fe stress can vary widely within a genus. A targeted high-throughput sequencing method to characterize the diatom community in field samples has been developed. Additionally, comparative transcriptomics of laboratory experiments with ecologically relevant species have been used to elucidate genes whose expression is tightly coupled to specific limitation scenarios. These two methods have been combined with data mining of the growing database of diatom transcriptomes to develop species-specific markers of physiological status in field populations. Data will be presented on the development and use of these methods to analyze samples collected on field expeditions to the Northeast Pacific Ocean sampling across gradients of Fe.

  16. High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9.

    Science.gov (United States)

    Varshney, Gaurav K; Pei, Wuhong; LaFave, Matthew C; Idol, Jennifer; Xu, Lisha; Gallardo, Viviana; Carrington, Blake; Bishop, Kevin; Jones, MaryPat; Li, Mingyu; Harper, Ursula; Huang, Sunny C; Prakash, Anupam; Chen, Wenbiao; Sood, Raman; Ledin, Johan; Burgess, Shawn M

    2015-07-01

    The use of CRISPR/Cas9 as a genome-editing tool in various model organisms has radically changed targeted mutagenesis. Here, we present a high-throughput targeted mutagenesis pipeline using CRISPR/Cas9 technology in zebrafish that will make possible both saturation mutagenesis of the genome and large-scale phenotyping efforts. We describe a cloning-free single-guide RNA (sgRNA) synthesis, coupled with streamlined mutant identification methods utilizing fluorescent PCR and multiplexed, high-throughput sequencing. We report germline transmission data from 162 loci targeting 83 genes in the zebrafish genome, in which we obtained a 99% success rate for generating mutations and an average germline transmission rate of 28%. We verified 678 unique alleles from 58 genes by high-throughput sequencing. We demonstrate that our method can be used for efficient multiplexed gene targeting. We also demonstrate that phenotyping can be done in the F1 generation by inbreeding two injected founder fish, significantly reducing animal husbandry and time. This study compares germline transmission data from CRISPR/Cas9 with those of TALENs and ZFNs and shows that efficiency of CRISPR/Cas9 is sixfold more efficient than other techniques. We show that the majority of published "rules" for efficient sgRNA design do not effectively predict germline transmission rates in zebrafish, with the exception of a GG or GA dinucleotide genomic match at the 5' end of the sgRNA. Finally, we show that predicted off-target mutagenesis is of low concern for in vivo genetic studies. © 2015 Varshney et al.; Published by Cold Spring Harbor Laboratory Press.

  17. VEGF targeting in mesotheliomas using an interleukin-6 signal inhibitor based on adenovirus gene delivery.

    Science.gov (United States)

    Adachi, Yasuo; Yoshio-Hoshino, Naoko; Aoki, Chieko; Nishimoto, Norihiro

    2010-06-01

    Malignant mesotheliomas reportedly secrete interleukin-6 (IL-6) which augments production of vascular endothelial growth factor (VEGF) from mesothelioma cells. We previously reported the development of a new receptor inhibitor of IL-6 (NRI) by genetically engineering tocilizumab, a humanized anti-IL-6 receptor monoclonal antibody. Since NRI is encoded on a single gene, it is easily applicable to a gene delivery system using virus vehicles. In this study, we report VEGF targeting through NRI expression based on adenovirus-mediated gene delivery in mesothelioma cells. We constructed an NRI expression vector in the context of a tropism-modified adenovirus vector that had enhanced infectivity in mesothelioma cells. This virus effectively induced NRI secretion from mesothelioma cells. This virus infection also reduced the VEGF production in mesothelioma cells. These results indicate that NRI shows potential as an agent in the treatment of mesotheliomas.

  18. Mapping of HNF4alpha target genes in intestinal epithelial cells

    DEFF Research Database (Denmark)

    Boyd, Mette; Bressendorff, Simon; Moller, Jette

    2009-01-01

    not previously been described as being regulated by HNF4alpha. The 1,541 genes contributed significantly to gene ontology (GO) pathways categorized by lipid and amino acid transport and metabolism. An analysis of the homeodomain transcription factor Cdx-2 (CDX2), the disaccharidase trehalase (TREH......ABSTRACT: BACKGROUND: The role of HNF4alpha has been extensively studied in hepatocytes and pancreatic beta-cells, and HNF4alpha is also regarded as key regulator of intestinal epithelial cell differentiation as well. The aim of the present work is to identify novel HNF4alpha target genes...... in the human intestinal epithelial cells in order to elucidate the role of HNF4alpha in the intestinal differentiation progress. METHODS: We have performed a ChIP-chip analysis of the human intestinal cell line Caco-2 in order to make a genome-wide identification of HNF4alpha binding to promoter regions...

  19. Agrobacterium tumefaciens-mediated transformation: an efficient tool for targeted gene disruption in Talaromyces marneffei.

    Science.gov (United States)

    Xiao, Xing; Feng, Jiao; Li, Yu; Chen, Zhiwen; Shi, Minglan; Xi, Liyan; Mylonakis, Eleftherios; Zhang, Junmin

    2017-09-25

    Talaromyces marneffei causes life-threatening infections in immunocompromised hosts. An efficient tool for genetic manipulation of T. marneffei will allow for increased understanding of this thermally dimorphic fungus. Agrobacterium tumefaciens-mediated transformation (ATMT) was optimized for targeted gene disruption in T. marneffei using the plasmid pDHt/acuD::pyrG. Molecular analyses of transformants were performed by PCR, Southern blot and semi-quantitative RT-PCR. A. tumefaciens strain EHA105 was more efficient at transformation than strain AGL-1 in ATMT via solid co-cultivation. An A. tumefaciens:T. marneffei ratio of 1000:1 in an ATMT liquid co-cultivation led to a relatively high transformation efficiency of 90 transformants per 10 6 yeast cells. Using ATMT-mediated knockout mutagenesis, we successfully deleted the acuD gene in T. marneffei. PCR and Southern blot analysis confirmed that acuD was disrupted and that the foreign pyrG gene was integrated into T. marneffei. Semi-quantitative RT-PCR analysis further confirmed that pyrG was expressed normally. These results suggest that ATMT can be a potential platform for targeted gene disruption in T. marneffei and that liquid co-cultivation may provide new opportunities to develop clinical treatments.

  20. NuMA is required for the selective induction of p53 target genes.

    Science.gov (United States)

    Ohata, Hirokazu; Miyazaki, Makoto; Otomo, Ryo; Matsushima-Hibiya, Yuko; Otsubo, Chihiro; Nagase, Takahiro; Arakawa, Hirofumi; Yokota, Jun; Nakagama, Hitoshi; Taya, Yoichi; Enari, Masato

    2013-06-01

    The p53 tumor suppressor protein is a transcription factor controlling various outcomes, such as growth arrest and apoptosis, through the regulation of different sets of target genes. The nuclear mitotic apparatus protein (NuMA) plays important roles in spindle pole organization during mitosis and in chromatin regulation in the nucleus during interphase. Although NuMA has been shown to colocalize with several nuclear proteins, including high-mobility-group proteins I and Y and GAS41, the role of NuMA during interphase remains unclear. Here we report that NuMA binds to p53 to modulate p53-mediated transcription. Acute and partial ablation of NuMA attenuates the induction of the proarrested p21 gene following DNA damage, subsequently causing impaired cell cycle arrest. Interestingly, NuMA knockdown had little effect on the induction of the p53-dependent proapoptotic PUMA gene. Furthermore, NuMA is required for the recruitment of cyclin-dependent kinase 8 (Cdk8), a component of the Mediator complex and a promoter of p53-mediated p21 gene function. These data demonstrate that NuMA is critical for the target selectivity of p53-mediated transcription.

  1. Repression of SRF target genes is critical for Myc-dependent apoptosis of epithelial cells

    Science.gov (United States)

    Wiese, Katrin E; Haikala, Heidi M; von Eyss, Björn; Wolf, Elmar; Esnault, Cyril; Rosenwald, Andreas; Treisman, Richard; Klefström, Juha; Eilers, Martin

    2015-01-01

    Oncogenic levels of Myc expression sensitize cells to multiple apoptotic stimuli, and this protects long-lived organisms from cancer development. How cells discriminate physiological from supraphysiological levels of Myc is largely unknown. Here, we show that induction of apoptosis by Myc in breast epithelial cells requires association of Myc with Miz1. Gene expression and ChIP-Sequencing experiments show that high levels of Myc invade target sites that lack consensus E-boxes in a complex with Miz1 and repress transcription. Myc/Miz1-repressed genes encode proteins involved in cell adhesion and migration and include several integrins. Promoters of repressed genes are enriched for binding sites of the serum-response factor (SRF). Restoring SRF activity antagonizes Myc repression of SRF target genes, attenuates Myc-induced apoptosis, and reverts a Myc-dependent decrease in Akt phosphorylation and activity, a well-characterized suppressor of Myc-induced apoptosis. We propose that high levels of Myc engage Miz1 in repressive DNA binding complexes and suppress an SRF-dependent transcriptional program that supports survival of epithelial cells. PMID:25896507

  2. Interrogating differences in expression of targeted gene sets to predict breast cancer outcome.

    Science.gov (United States)

    Andres, Sarah A; Brock, Guy N; Wittliff, James L

    2013-07-02

    Genomics provides opportunities to develop precise tests for diagnostics, therapy selection and monitoring. From analyses of our studies and those of published results, 32 candidate genes were identified, whose expression appears related to clinical outcome of breast cancer. Expression of these genes was validated by qPCR and correlated with clinical follow-up to identify a gene subset for development of a prognostic test. RNA was isolated from 225 frozen invasive ductal carcinomas,and qRT-PCR was performed. Univariate hazard ratios and 95% confidence intervals for breast cancer mortality and recurrence were calculated for each of the 32 candidate genes. A multivariable gene expression model for predicting each outcome was determined using the LASSO, with 1000 splits of the data into training and testing sets to determine predictive accuracy based on the C-index. Models with gene expression data were compared to models with standard clinical covariates and models with both gene expression and clinical covariates. Univariate analyses revealed over-expression of RABEP1, PGR, NAT1, PTP4A2, SLC39A6, ESR1, EVL, TBC1D9, FUT8, and SCUBE2 were all associated with reduced time to disease-related mortality (HR between 0.8 and 0.91, adjusted p data sets for the gene expression, clinical, and combined models were 0.65, 0.63, and 0.65 for disease mortality and 0.64, 0.63, and 0.66 for disease recurrence, respectively. Molecular signatures consisting of five genes (PGR, GABRP, TBC1D9, SLC39A6 and LRBA) for disease mortality and of six genes (PGR, ESR1, GABRP, TBC1D9, SLC39A6 and LRBA) for disease recurrence were identified. These signatures were as effective as standard clinical parameters in predicting recurrence/mortality, and when combined, offered some improvement relative to clinical information alone for disease recurrence (median difference in C-values of 0.03, 95% CI of -0.08 to 0.13). Collectively, results suggest that these genes form the basis for a clinical

  3. Interrogating differences in expression of targeted gene sets to predict breast cancer outcome

    International Nuclear Information System (INIS)

    Andres, Sarah A; Brock, Guy N; Wittliff, James L

    2013-01-01

    Genomics provides opportunities to develop precise tests for diagnostics, therapy selection and monitoring. From analyses of our studies and those of published results, 32 candidate genes were identified, whose expression appears related to clinical outcome of breast cancer. Expression of these genes was validated by qPCR and correlated with clinical follow-up to identify a gene subset for development of a prognostic test. RNA was isolated from 225 frozen invasive ductal carcinomas,and qRT-PCR was performed. Univariate hazard ratios and 95% confidence intervals for breast cancer mortality and recurrence were calculated for each of the 32 candidate genes. A multivariable gene expression model for predicting each outcome was determined using the LASSO, with 1000 splits of the data into training and testing sets to determine predictive accuracy based on the C-index. Models with gene expression data were compared to models with standard clinical covariates and models with both gene expression and clinical covariates. Univariate analyses revealed over-expression of RABEP1, PGR, NAT1, PTP4A2, SLC39A6, ESR1, EVL, TBC1D9, FUT8, and SCUBE2 were all associated with reduced time to disease-related mortality (HR between 0.8 and 0.91, adjusted p < 0.05), while RABEP1, PGR, SLC39A6, and FUT8 were also associated with reduced recurrence times. Multivariable analyses using the LASSO revealed PGR, ESR1, NAT1, GABRP, TBC1D9, SLC39A6, and LRBA to be the most important predictors for both disease mortality and recurrence. Median C-indexes on test data sets for the gene expression, clinical, and combined models were 0.65, 0.63, and 0.65 for disease mortality and 0.64, 0.63, and 0.66 for disease recurrence, respectively. Molecular signatures consisting of five genes (PGR, GABRP, TBC1D9, SLC39A6 and LRBA) for disease mortality and of six genes (PGR, ESR1, GABRP, TBC1D9, SLC39A6 and LRBA) for disease recurrence were identified. These signatures were as effective as standard clinical

  4. Mitochondrial targeted neuron focused genes in hippocampus of rats with traumatic brain injury.

    Science.gov (United States)

    Sharma, Pushpa; Su, Yan A; Barry, Erin S; Grunberg, Neil E; Lei, Zhang

    2012-09-01

    Mild traumatic brain injury (mTBI) represents a major health problem in civilian populations as well as among the military service members due to (1) lack of effective treatments, and (2) our incomplete understanding about the progression of secondary cell injury cascades resulting in neuronal cell death due to deficient cellular energy metabolism and damaged mitochondria. The aim of this study was to identify and delineate the mitochondrial targeted genes responsible for altered brain energy metabolism in the injured brain. Rats were either grouped into naïve controls or received lateral fluid percussion brain injury (2-2.5 atm) and followed up for 7 days. Rats were either grouped into naïve controls or received lateral fluid percussion brain injury (2-2.5 atm) and followed for 7 days. The severity of brain injury was evaluated by the neurological severity scale-revised (NSS-R) at 3 and 5 days post TBI and immunohistochemical analyses at 7 days post TBI. The expression profiles of mitochondrial-targeted genes across the hippocampus from TBI and naïe rats were also examined by oligo-DNA microarrays. NSS-R scores of TBI rats (5.4 ± 0.5) in comparison to naïe rats (3.9 ± 0.5) and H and E staining of brain sections suggested a mild brain injury. Bioinformatics and systems biology analyses showed 31 dysregulated genes, 10 affected canonical molecular pathways including a number of genes involved in mitochondrial enzymes for oxidative phosphorylation, mitogen-activated protein Kinase (MAP), peroxisome proliferator-activated protein (PPAP), apoptosis signaling, and genes responsible for long-term potentiation of Alzheimer's and Parkinson's diseases. Our results suggest that dysregulated mitochondrial-focused genes in injured brains may have a clinical utility for the development of future therapeutic strategies aimed at the treatment of TBI.

  5. Efficient TALEN-mediated gene targeting of chicken primordial germ cells.

    Science.gov (United States)

    Taylor, Lorna; Carlson, Daniel F; Nandi, Sunil; Sherman, Adrian; Fahrenkrug, Scott C; McGrew, Michael J

    2017-03-01

    In this work we use TALE nucleases (TALENs) to target a reporter construct to the DDX4 ( vasa ) locus in chicken primordial germ cells (PGCs). Vasa is a key germ cell determinant in many animal species and is posited to control avian germ cell formation. We show that TALENs mediate homology-directed repair of the DDX4 locus on the Z sex chromosome at high (8.1%) efficiencies. Large genetic deletions of 30 kb encompassing the entire DDX4 locus were also created using a single TALEN pair. The targeted PGCs were germline competent and were used to produce DDX4 null offspring. In DDX4 knockout chickens, PGCs are initially formed but are lost during meiosis in the developing ovary, leading to adult female sterility. TALEN-mediated gene targeting in avian PGCs is therefore an efficient process. © 2017. Published by The Company of Biologists Ltd.

  6. Genome-scale metabolic models as platforms for identification of novel genes as antimicrobial drug targets.

    Science.gov (United States)

    Mienda, Bashir Sajo; Salihu, Rabiu; Adamu, Aliyu; Idris, Shehu

    2018-03-01

    The growing number of multidrug-resistant pathogenic bacteria is becoming a world leading challenge for the scientific community and for public health. However, advances in high-throughput technologies and whole-genome sequencing of bacterial pathogens make the construction of bacterial genome-scale metabolic models (GEMs) increasingly realistic. The use of GEMs as an alternative platforms will expedite identification of novel unconditionally essential genes and enzymes of target organisms with existing and forthcoming GEMs. This approach will follow the existing protocol for construction of high-quality GEMs, which could ultimately reduce the time, cost and labor-intensive processes involved in identification of novel antimicrobial drug targets in drug discovery pipelines. We discuss the current impact of existing GEMs of selected multidrug-resistant pathogenic bacteria for identification of novel antimicrobial drug targets and the challenges of closing the gap between genome-scale metabolic modeling and conventional experimental trial-and-error approaches in drug discovery pipelines.

  7. DNA demethylases target promoter transposable elements to positively regulate stress responsive genes in Arabidopsis.

    Science.gov (United States)

    Le, Tuan-Ngoc; Schumann, Ulrike; Smith, Neil A; Tiwari, Sameer; Au, Phil Chi Khang; Zhu, Qian-Hao; Taylor, Jennifer M; Kazan, Kemal; Llewellyn, Danny J; Zhang, Ren; Dennis, Elizabeth S; Wang, Ming-Bo

    2014-09-17

    DNA demethylases regulate DNA methylation levels in eukaryotes. Arabidopsis encodes four DNA demethylases, DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), DEMETER-LIKE 2 (DML2), and DML3. While DME is involved in maternal specific gene expression during seed development, the biological function of the remaining DNA demethylases remains unclear. We show that ROS1, DML2, and DML3 play a role in fungal disease resistance in Arabidopsis. A triple DNA demethylase mutant, rdd (ros1 dml2 dml3), shows increased susceptibility to the fungal pathogen Fusarium oxysporum. We identify 348 genes differentially expressed in rdd relative to wild type, and a significant proportion of these genes are downregulated in rdd and have functions in stress response, suggesting that DNA demethylases maintain or positively regulate the expression of stress response genes required for F. oxysporum resistance. The rdd-downregulated stress response genes are enriched for short transposable element sequences in their promoters. Many of these transposable elements and their surrounding sequences show localized DNA methylation changes in rdd, and a general reduction in CHH methylation, suggesting that RNA-directed DNA methylation (RdDM), responsible for CHH methylation, may participate in DNA demethylase-mediated regulation of stress response genes. Many of the rdd-downregulated stress response genes are downregulated in the RdDM mutants nrpd1 and nrpe1, and the RdDM mutants nrpe1 and ago4 show enhanced susceptibility to F. oxysporum infection. Our results suggest that a primary function of DNA demethylases in plants is to regulate the expression of stress response genes by targeting promoter transposable element sequences.

  8. Use of designer nucleases for targeted gene and genome editing in plants.

    Science.gov (United States)

    Weeks, Donald P; Spalding, Martin H; Yang, Bing

    2016-02-01

    The ability to efficiently inactivate or replace genes in model organisms allowed a rapid expansion of our understanding of many of the genetic, biochemical, molecular and cellular mechanisms that support life. With the advent of new techniques for manipulating genes and genomes that are applicable not only to single-celled organisms, but also to more complex organisms such as animals and plants, the speed with which scientists and biotechnologists can expand fundamental knowledge and apply that knowledge to improvements in medicine, industry and agriculture is set to expand in an exponential fashion. At the heart of these advancements will be the use of gene editing tools such as zinc finger nucleases, modified meganucleases, hybrid DNA/RNA oligonucleotides, TAL effector nucleases and modified CRISPR/Cas9. Each of these tools has the ability to precisely target one specific DNA sequence within a genome and (except for DNA/RNA oligonucleotides) to create a double-stranded DNA break. DNA repair to such breaks sometimes leads to gene knockouts or gene replacement by homologous recombination if exogenously supplied homologous DNA fragments are made available. Genome rearrangements are also possible to engineer. Creation and use of such genome rearrangements, gene knockouts and gene replacements by the plant science community is gaining significant momentum. To document some of this progress and to explore the technology's longer term potential, this review highlights present and future uses of designer nucleases to greatly expedite research with model plant systems and to engineer genes and genomes in major and minor crop species for enhanced food production. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  9. Increased Anticancer Efficacy of Intravesical Mitomycin C Therapy when Combined with a PCNA Targeting Peptide

    Directory of Open Access Journals (Sweden)

    Odrun A. Gederaas

    2014-12-01

    Full Text Available Non–muscle-invasive bladder cancers (NMIBCs are tumors confined to the mucosa or the mucosa/submucosa. An important challenge in treatment of NMIBC is both high recurrence and high progression rates. Consequently, more efficacious intravesical treatment regimes are in demand. Inhibition of the cell’s DNA repair systems is a new promising strategy to improve cancer therapy, and proliferating cell nuclear antigen (PCNA is a new promising target. PCNA is an essential scaffold protein in multiple cellular processes including DNA replication and repair. More than 200 proteins, many involved in stress responses, interact with PCNA through the AlkB homologue 2 PCNA-interacting motif (APIM, including several proteins directly or indirectly involved in repair of DNA interstrand crosslinks (ICLs. In this study, we targeted PCNA with a novel peptide drug containing the APIM sequence, ATX-101, to inhibit repair of the DNA damage introduced by the chemotherapeutics. A bladder cancer cell panel and two different orthotopic models of bladder cancer in rats, the AY-27 implantation model and the dietary BBN induction model, were applied. ATX-101 increased the anticancer efficacy of the ICL-inducing drug mitomycin C (MMC, as well as bleomycin and gemcitabine in all bladder cancer cell lines tested. Furthermore, we found that ATX-101 given intravesically in combination with MMC penetrated the bladder wall and further reduced the tumor growth in both the slow growing endogenously induced and the rapidly growing transplanted tumors. These results suggest that ATX-101 has the potential to improve the efficacy of current MMC treatment in NMIBC.

  10. Target genes of myostatin loss-of-function in muscles of late bovine fetuses

    Directory of Open Access Journals (Sweden)

    Hocquette Jean-François

    2007-03-01

    Full Text Available Abstract Background Myostatin, a muscle-specific member of the Transforming Growth Factor beta family, negatively regulates muscle development. Double-muscled (DM cattle have a loss-of-function mutation in their myostatin gene responsible for the hypermuscular phenotype. Thus, these animals are a good model for understanding the mechanisms underpinning muscular hypertrophy. In order to identify individual genes or networks that may be myostatin targets, we looked for genes that were differentially expressed between DM and normal (NM animals (n = 3 per group in the semitendinosus muscle (hypertrophied in DM animals at 260 days of fetal development (when the biochemical differentiation of muscle is intensive. A heterologous microarray (human and murine oligonucleotide sequences of around 6,000 genes expressed in muscle was used. Results Many genes were found to be differentially expressed according to genetic type (some with a more than 5-fold change, and according to the presence of one or two functional myostatin allele(s. They belonged to various functional categories. The genes down-regulated in DM fetuses were mainly those encoding extracellular matrix proteins, slow contractile proteins and ribosomal proteins. The genes up-regulated in DM fetuses were mainly involved in the regulation of transcription, cell cycle/apoptosis, translation or DNA metabolism. These data highlight features indicating that DM muscle is shifted towards a more glycolytic metabolism, and has an altered extracellular matrix composition (e.g. down-regulation of COL1A1 and COL1A2, and up-regulation of COL4A2 and decreased adipocyte differentiation (down-regulation of C1QTNF3. The altered gene expression in the three major muscle compartments (fibers, connective tissue and intramuscular adipose tissue is consistent with the well-known characteristics of DM cattle. In addition, novel potential targets of the myostatin gene were identified (MB, PLN, troponins, ZFHX1B

  11. Strong combined gene-environment effects in anti-cyclic citrullinated peptide-positive rheumatoid arthritis

    DEFF Research Database (Denmark)

    Pedersen, Line Merete Blak; Jacobsen, Søren; Garred, Peter

    2007-01-01

    To study the role of shared epitope (SE) susceptibility genes, alone and in combination with tobacco smoking and other environmental risk factors, for risk of subtypes of rheumatoid arthritis (RA) defined by the presence or absence of serum antibodies against cyclic citrullinated peptides (CCPs)....

  12. The TTSMI database: a catalog of triplex target DNA sites associated with genes and regulatory elements in the human genome.

    Science.gov (United States)

    Jenjaroenpun, Piroon; Chew, Chee Siang; Yong, Tai Pang; Choowongkomon, Kiattawee; Thammasorn, Wimada; Kuznetsov, Vladimir A

    2015-01-01

    A triplex target DNA site (TTS), a stretch of DNA that is composed of polypurines, is able to form a triple-helix (triplex) structure with triplex-forming oligonucleotides (TFOs) and is able to influence the site-specific modulation of gene expression and/or the modification of genomic DNA. The co-localization of a genomic TTS with gene regulatory signals and functional genome structures suggests that TFOs could potentially be exploited in antigene strategies for the therapy of cancers and other genetic diseases. Here, we present the TTS Mapping and Integration (TTSMI; http://ttsmi.bii.a-star.edu.sg) database, which provides a catalog of unique TTS locations in the human genome and tools for analyzing the co-localization of TTSs with genomic regulatory sequences and signals that were identified using next-generation sequencing techniques and/or predicted by computational models. TTSMI was designed as a user-friendly tool that facilitates (i) fast searching/filtering of TTSs using several search terms and criteria associated with sequence stability and specificity, (ii) interactive filtering of TTSs that co-localize with gene regulatory signals and non-B DNA structures, (iii) exploration of dynamic combinations of the biological signals of specific TTSs and (iv) visualization of a TTS simultaneously with diverse annotation tracks via the UCSC genome browser. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Integrated chromatin immunoprecipitation sequencing and microarray analysis identifies FOXA2 target genes in the glands of the mouse uterus.

    Science.gov (United States)

    Filant, Justyna; Lydon, John P; Spencer, Thomas E

    2014-01-01

    Uterine glands and their secretions are indispensable for endometrial function and fertility; however, the mechanisms regulating their development and function are not well understood. Forkhead transcription factor box A2 (FOXA2) is uniquely expressed in the glandular epithelial (GE) cells of the uterus, and conditional deletion of Foxa2 after birth impedes uterine gland development. An integrative approach was used here to define the FOXA2 cistrome in the murine uterus. Genome-wide mapping of FOXA2 binding sites was combined with transcriptomic analyses of isolated GE and Foxa2-deleted uteri. ChIP-Seq analyses found the number of FOXA2 target genes was substantially greater in the adult (8893) than neonatal uterus (1101). In the neonatal uterus, FOXA2-bound and GE-expressed genes (469) were enriched for developmentally related processes, including cell cycle, cell junction, focal adhesion, and WNT signaling. In the adult uterus, FOXA2-bound and GE-expressed genes (3730) were enriched for functional processes, including metabolic pathways, focal adhesion, bacterial invasion of epithelial cells, and WNT signaling. Analysis of the uterine FOXA2 cistrome provides novel insights into mechanisms governing endometrial gland development and function, which are important to understand fundamental aspects of uterine differentiation, regeneration and disease.

  14. Combined ultrasound-targeted microbubble destruction and polyethylenimine-mediated plasmid DNA delivery to the rat retina: enhanced efficiency and accelerated expression.

    Science.gov (United States)

    Li, Hongli; Qian, Jin; Yao, Chunfang; Wan, Caifeng; Li, Fenghua

    2016-04-01

    Gene therapy has potential in the treatment of refractory retinal diseases. It is important to develop an effective delivery system in the retina. The present study aimed to investigate the efficacy and safety of ultrasound (US)-targeted microbubble destruction (UTMD)-mediated polyethylenimine (PEI) to the rat retina. Gene transfer was examined by injecting PEI/plasmid DNA (pDNA) with or without microbubbles (MBs) into the subretinal space of rats that were then exposed to US. We investigated enhanced green fluorescent protein (eGFP) expression on flat fundus oculi and performed quantitative analysis. Hematoxylin and eosin staining was used to observe tissue damage. UTMD significantly enhanced PEI/pDNA transfection efficiency safely by increasing both the transgene expression per cell and the percentage of transfected cells of the retina. PEI/pDNA combined with UTMD significantly increased the number of DNA gene copies and the mRNA level in the retinal pigment epithelium (RPE) and neural retina, respectively, compared to PEI/pDNA alone. The present study demonstrates that enhanced and accelerated pDNA expression can be achieved in the retina/RPE cells in vivo by UTMD physical techniques combined with a PEI chemical vector. Our study provides useful information for further in vivo retinal gene therapy work. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  15. Targeted repression of AXIN2 and MYC gene expression using designer TALEs

    Energy Technology Data Exchange (ETDEWEB)

    Rennoll, Sherri A.; Scott, Samantha A.; Yochum, Gregory S., E-mail: gsy3@psu.edu

    2014-04-18

    Highlights: • We designed TALE–SID fusion proteins to target AXIN2 and MYC. • TALE–SIDs bound the chromosomal AXIN2 and MYC genes and repressed their expression. • TALE–SIDs repress β-catenin{sup S45F}-dependent AXIN2 and MYC transcription. - Abstract: Designer TALEs (dTALEs) are chimeric transcription factors that can be engineered to regulate gene expression in mammalian cells. Whether dTALEs can block gene transcription downstream of signal transduction cascades, however, has yet to be fully explored. Here we tested whether dTALEs can be used to target genes whose expression is controlled by Wnt/β-catenin signaling. TALE DNA binding domains were engineered to recognize sequences adjacent to Wnt responsive enhancer elements (WREs) that control expression of axis inhibition protein 2 (AXIN2) and c-MYC (MYC). These custom DNA binding domains were linked to the mSin3A interaction domain (SID) to generate TALE–SID chimeric repressors. The TALE–SIDs repressed luciferase reporter activity, bound their genomic target sites, and repressed AXIN2 and MYC expression in HEK293 cells. We generated a novel HEK293 cell line to determine whether the TALE–SIDs could function downstream of oncogenic Wnt/β-catenin signaling. Treating these cells with doxycycline and tamoxifen stimulates nuclear accumulation of a stabilized form of β-catenin found in a subset of colorectal cancers. The TALE–SIDs repressed AXIN2 and MYC expression in these cells, which suggests that dTALEs could offer an effective therapeutic strategy for the treatment of colorectal cancer.

  16. High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9.

    Science.gov (United States)

    Gil-Humanes, Javier; Wang, Yanpeng; Liang, Zhen; Shan, Qiwei; Ozuna, Carmen V; Sánchez-León, Susana; Baltes, Nicholas J; Starker, Colby; Barro, Francisco; Gao, Caixia; Voytas, Daniel F

    2017-03-01

    The ability to edit plant genomes through gene targeting (GT) requires efficient methods to deliver both sequence-specific nucleases (SSNs) and repair templates to plant cells. This is typically achieved using Agrobacterium T-DNA, biolistics or by stably integrating nuclease-encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such as Nicotinana tabacum (tobacco) and Solanum lycopersicum (tomato), greater than 10-fold enhancements in GT frequencies have been achieved using DNA virus-based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundant SSNs and repair templates to achieve targeted gene modification. In the present work, we developed a replicon-based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV). In wheat cells, the replicons achieve a 110-fold increase in expression of a reporter gene relative to non-replicating controls. Furthermore, replicons carrying CRISPR/Cas9 nucleases and repair templates achieved GT at an endogenous ubiquitin locus at frequencies 12-fold greater than non-viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these high GT frequencies. We also demonstrate gene-targeted integration by homologous recombination (HR) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with the WDV replicons, multiplexed GT within the same wheat cell can be achieved at frequencies of ~1%. In conclusion, high frequencies of GT using WDV-based DNA replicons will make it possible to edit complex cereal genomes without the need to integrate GT reagents into the genome. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  17. Targeted repression of AXIN2 and MYC gene expression using designer TALEs

    International Nuclear Information System (INIS)

    Rennoll, Sherri A.; Scott, Samantha A.; Yochum, Gregory S.

    2014-01-01

    Highlights: • We designed TALE–SID fusion proteins to target AXIN2 and MYC. • TALE–SIDs bound the chromosomal AXIN2 and MYC genes and repressed their expression. • TALE–SIDs repress β-catenin S45F -dependent AXIN2 and MYC transcription. - Abstract: Designer TALEs (dTALEs) are chimeric transcription factors that can be engineered to regulate gene expression in mammalian cells. Whether dTALEs can block gene transcription downstream of signal transduction cascades, however, has yet to be fully explored. Here we tested whether dTALEs can be used to target genes whose expression is controlled by Wnt/β-catenin signaling. TALE DNA binding domains were engineered to recognize sequences adjacent to Wnt responsive enhancer elements (WREs) that control expression of axis inhibition protein 2 (AXIN2) and c-MYC (MYC). These custom DNA binding domains were linked to the mSin3A interaction domain (SID) to generate TALE–SID chimeric repressors. The TALE–SIDs repressed luciferase reporter activity, bound their genomic target sites, and repressed AXIN2 and MYC expression in HEK293 cells. We generated a novel HEK293 cell line to determine whether the TALE–SIDs could function downstream of oncogenic Wnt/β-catenin signaling. Treating these cells with doxycycline and tamoxifen stimulates nuclear accumulation of a stabilized form of β-catenin found in a subset of colorectal cancers. The TALE–SIDs repressed AXIN2 and MYC expression in these cells, which suggests that dTALEs could offer an effective therapeutic strategy for the treatment of colorectal cancer

  18. Dnmts and Tet target memory-associated genes after appetitive olfactory training in honey bees.

    Science.gov (United States)

    Biergans, Stephanie D; Giovanni Galizia, C; Reinhard, Judith; Claudianos, Charles

    2015-11-04

    DNA methylation and demethylation are epigenetic mechanisms involved in memory formation. In honey bees DNA methyltransferase (Dnmt) function is necessary for long-term memory to be stimulus specific (i.e. to reduce generalization). So far, however, it remains elusive which genes are targeted and what the time-course of DNA methylation is during memory formation. Here, we analyse how DNA methylation affects memory retention, gene expression, and differential methylation in stimulus-specific olfactory long-term memory formation. Out of 30 memory-associated genes investigated here, 9 were upregulated following Dnmt inhibition in trained bees. These included Dnmt3 suggesting a negative feedback loop for DNA methylation. Within these genes also the DNA methylation pattern changed during the first 24 hours after training. Interestingly, this was accompanied by sequential activation of the DNA methylation machinery (i.e. Dnmts and Tet). In sum, memory formation involves a temporally complex epigenetic regulation of memory-associated genes that facilitates stimulus specific long-term memory in the honey bee.

  19. Gene expression levels are a target of recent natural selection in the human genome.

    Science.gov (United States)

    Kudaravalli, Sridhar; Veyrieras, Jean-Baptiste; Stranger, Barbara E; Dermitzakis, Emmanouil T; Pritchard, Jonathan K

    2009-03-01

    Changes in gene expression may represent an important mode of human adaptation. However, to date, there are relatively few known examples in which selection has been shown to act directly on levels or patterns of gene expression. In order to test whether single nucleotide polymorphisms (SNPs) that affect gene expression in cis are frequently targets of positive natural selection in humans, we analyzed genome-wide SNP and expression data from cell lines associated with the International HapMap Project. Using a haplotype-based test for selection that was designed to detect incomplete selective sweeps, we found that SNPs showing signals of selection are more likely than random SNPs to be associated with gene expression levels in cis. This signal is significant in the Yoruba (which is the population that shows the strongest signals of selection overall) and shows a trend in the same direction in the other HapMap populations. Our results argue that selection on gene expression levels is an important type of human adaptation. Finally, our work provides an analytical framework for tackling a more general problem that will become increasingly important: namely, testing whether selection signals overlap significantly with SNPs that are associated with phenotypes of interest.

  20. Potential translational targets revealed by linking mouse grooming behavioral phenotypes to gene expression using public databases.

    Science.gov (United States)

    Roth, Andrew; Kyzar, Evan J; Cachat, Jonathan; Stewart, Adam Michael; Green, Jeremy; Gaikwad, Siddharth; O'Leary, Timothy P; Tabakoff, Boris; Brown, Richard E; Kalueff, Allan V

    2013-01-10

    Rodent self-grooming is an important, evolutionarily conserved behavior, highly sensitive to pharmacological and genetic manipulations. Mice with aberrant grooming phenotypes are currently used to model various human disorders. Therefore, it is critical to understand the biology of grooming behavior, and to assess its translational validity to humans. The present in-silico study used publicly available gene expression and behavioral data obtained from several inbred mouse strains in the open-field, light-dark box, elevated plus- and elevated zero-maze tests. As grooming duration differed between strains, our analysis revealed several candidate genes with significant correlations between gene expression in the brain and grooming duration. The Allen Brain Atlas, STRING, GoMiner and Mouse Genome Informatics databases were used to functionally map and analyze these candidate mouse genes against their human orthologs, assessing the strain ranking of their expression and the regional distribution of expression in the mouse brain. This allowed us to identify an interconnected network of candidate genes (which have expression levels that correlate with grooming behavior), display altered patterns of expression in key brain areas related to grooming, and underlie important functions in the brain. Collectively, our results demonstrate the utility of large-scale, high-throughput data-mining and in-silico modeling for linking genomic and behavioral data, as well as their potential to identify novel neural targets for complex neurobehavioral phenotypes, including grooming. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. Simultaneous Targeting of Multiple Gene Homeologs to Alter Seed Oil Production in Camelina sativa.

    Science.gov (United States)

    Aznar-Moreno, J A; Durrett, T P

    2017-07-01

    The ability to transform Camelina sativa easily with biosynthetic enzymes derived from other plants has made this oil seed crop an ideal platform for the production of unusual lipids valuable for different applications. However, in addition to expressing transgenic enzymes, the suppression of endogenous enzyme activity to reduce competition for common substrates or cofactors is also required to enhance the production of target compounds. As camelina possesses a relatively undifferentiated hexaploid genome, up to three gene homeologs can code for any particular enzymatic activity, complicating efforts to alter endogenous biosynthetic pathways. New genome editing technologies, such as that offered by the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) system, offer the capability to introduce mutations into specifically targeted genomic sites. Here, by using a carefully designed guide RNA identical to all three homeologs, we demonstrate the ability of the CRISPR/Cas genome editing system to introduce mutations in all three CsDGAT1 or CsPDAT1 homeologous genes important for triacylglycerol (TAG) synthesis in developing seeds. Sequence analysis from transgenic T1 plants revealed that each CsDGAT1 or each CsPDAT1 homeolog was altered by multiple mutations, resulting in a genetic mosaic in the plants. Interestingly, seed harvested from both CsDGAT1- and CsPDAT1-targeted lines was often shrunken and wrinkled. Further, lipid analysis revealed that many lines produced seed with reduced oil content and altered fatty acid composition, consistent with the role of the targeted genes in seed oil biosynthesis. The CRISPR/Cas system therefore represents a useful method to alter endogenous biosynthetic pathways efficiently in polyploid species such as camelina. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  2. TIPS Placement via Combined Transjugular and Transhepatic Approach for Cavernous Portal Vein Occlusion: Targeted Approach

    Directory of Open Access Journals (Sweden)

    Natanel Jourabchi

    2013-01-01

    Full Text Available Purpose. We report a novel technique which aided recanalization of an occluded portal vein for transjugular intrahepatic portosystemic shunt (TIPS creation in a patient with symptomatic portal vein thrombosis with cavernous transformation. Some have previously considered cavernous transformation a contraindication to TIPS. Case Presentation. 62-year-old man with chronic pancreatitis, portal vein thrombosis, portal hypertension and recurrent variceal bleeding presents with melena and hematemesis. The patient was severely anemic, hemodynamically unstable, and required emergent portal decompression. Attempts to recanalize the main portal vein using traditional transjugular access were unsuccessful. After percutaneous transhepatic right portal vein access and navigation of a wire through the occluded main portal vein, an angioplasty balloon was inflated at the desired site of shunt takeoff. The balloon was targeted and punctured from the transjugular approach, and a wire was passed into the portal system. TIPS placement then proceeded routinely. Conclusion. Although occlusion of the portal vein increases difficulty of performing TIPS, it should not be considered an absolute contraindication. We have described a method for recanalizing an occluded portal vein using a combined transhepatic and transjugular approach for TIPS. This approach may be useful to relieve portal hypertension in patients who fail endoscopic and/or surgical therapies.

  3. Human Exportin-1 is a Target for Combined Therapy of HIV and AIDS Related Lymphoma

    Directory of Open Access Journals (Sweden)

    Eline Boons

    2015-09-01

    Full Text Available Infection with HIV ultimately leads to advanced immunodeficiency resulting in an increased incidence of cancer. For example primary effusion lymphoma (PEL is an aggressive non-Hodgkin lymphoma with very poor prognosis that typically affects HIV infected individuals in advanced stages of immunodeficiency. Here we report on the dual anti-HIV and anti-PEL effect of targeting a single process common in both diseases. Inhibition of the exportin-1 (XPO1 mediated nuclear transport by clinical stage orally bioavailable small molecule inhibitors (SINE prevented the nuclear export of the late intron-containing HIV RNA species and consequently potently suppressed viral replication. In contrast, in CRISPR-Cas9 genome edited cells expressing mutant C528S XPO1, viral replication was unaffected upon treatment, clearly demonstrating the anti-XPO1 mechanism of action. At the same time, SINE caused the nuclear accumulation of p53 tumor suppressor protein as well as inhibition of NF-κB activity in PEL cells resulting in cell cycle arrest and effective apoptosis induction. In vivo, oral administration arrested PEL tumor growth in engrafted mice. Our findings provide strong rationale for inhibiting XPO1 as an innovative strategy for the combined anti-retroviral and anti-neoplastic treatment of HIV and PEL and offer perspectives for the treatment of other AIDS-associated cancers and potentially other virus-related malignancies.

  4. Combination of arginine deprivation with TRAIL treatment as a targeted-therapy for mesothelioma.

    Science.gov (United States)

    Wangpaichitr, Medhi; Wu, Chunjing; Bigford, Gregory; Theodoropoulos, George; You, Min; Li, Ying Ying; Verona-Santos, Javier; Feun, Lynn G; Nguyen, Dao M; Savaraj, Niramol

    2014-12-01

    In the present study we present data to show that certain tumor cells including malignant pleural mesothelioma (MPM) cells do not express argininosuccinate synthetase (ASS), and thus are unable to synthesize arginine from citrulline. Exposure of these ASS-negative cells to the arginine degrading enzyme, arginine deiminase (ADI-PEG20), for 72 h results in significant increases in cleaved caspase-3. Importantly, this apoptotic signal is further strengthened by the addition of TNF-related apoptosis-inducing ligand (TRAIL). Using flow cytometry, we showed that the combination treatment (ADI-PEG20 at 50 ng/ml and TRAIL at 10 ng/ml) for 24 h resulted in profound cell death with 67% of cells positive for caspase-3 activity, while ADI-PEG20 alone or TRAIL alone resulted in only 10-15% cell death. This positive amplification loop is mediated through the cleavage of proapototic protein "BID". Our work represents a new strategy for treating patients with malignant pleural mesothelioma using targeted molecular therapeutics based on selected tumor markers, thus avoiding the use of potentially cytotoxic chemotherapy. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  5. The combination of suicide gene therapy and radiation enhances the killing of nasopharyngeal carcinoma xenographs

    International Nuclear Information System (INIS)

    Xia Jiahui; Xia Kun; Feng Yong

    2004-01-01

    Nasopharyngeal carcinoma (NPC) is very common in Southern China and Southeast Asian countries. To explore a novel and more effective approach to NPC therapy, a combined strategy of suicide genes and radiation was designed in this study. Five suicide gene expression cassettes, yeast cytosine deaminase (CD), yeast CD/uracil phosphoribosyl-transferase (UPRT), and yeast CDglyTK gene controlled by CMV, and Egr-1 and a synthetic CMV-enhanced Egr-1 promoter (CE) were constructed in an expression vector p11MS. The expression of suicide genes in NPC CNE-2 cells were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. The cytotoxicity of suicide gene therapy and radiation were analyzed by MTT assay. An animal study in which yeast CD/UPRT-expressing CNE-2 tumors in nude mice were treated with 5-fluorocytosine (5-FC) and radiation was also developed. Our results revealed that p11MSCEyCD/UPRT and p11MSCEyCDglyTK are superior over three other constructs in the killing of NPC cells in vitro. We combined suicide gene-expressing tumors, 5-FC treatment, and radiation in vivo and found that the tumors greatly regressed, some disappeared completely in 3 nude mice in the yCD/UPRT group, and a significant difference of tumor volumes was observed between this group and the other four groups (p<0.05). Our results indicated that suicide gene therapy and radiation have a synergic effect on NPC therapy, and the combined strategy of radiogene therapy is of great potential as a substitute for the traditional method, radiation alone, in NPC therapies. (author)

  6. Analysis of essential Arabidopsis nuclear genes encoding plastid-targeted proteins.

    Science.gov (United States)

    Savage, Linda J; Imre, Kathleen M; Hall, David A; Last, Robert L

    2013-01-01

    The Chloroplast 2010 Project (http://www.plastid.msu.edu/) identified and phenotypically characterized homozygous mutants in over three thousand genes, the majority of which encode plastid-targeted proteins. Despite extensive screening by the community, no homozygous mutant alleles were available for several hundred genes, suggesting that these might be enriched for genes of essential function. Attempts were made to generate homozygotes in ~1200 of these lines and 521 of the homozygous viable lines obtained were deposited in the Arabidopsis Biological Resource Center (http://abrc.osu.edu/). Lines that did not yield a homozygote in soil were tested as potentially homozygous lethal due to defects either in seed or seedling development. Mutants were characterized at four stages of development: developing seed, mature seed, at germination, and developing seedlings. To distinguish seed development or seed pigment-defective mutants from seedling development mutants, development of seeds was assayed in siliques from heterozygous plants. Segregating seeds from heterozygous parents were sown on supplemented media in an attempt to rescue homozygous seedlings that could not germinate or survive in soil. Growth of segregating seeds in air and air enriched to 0.3% carbon dioxide was compared to discover mutants potentially impaired in photorespiration or otherwise responsive to CO2 supplementation. Chlorophyll fluorescence measurements identified CO2-responsive mutants with altered photosynthetic parameters. Examples of genes with a viable mutant allele and one or more putative homozygous-lethal alleles were documented. RT-PCR of homozygotes for potentially weak alleles revealed that essential genes may remain undiscovered because of the lack of a true null mutant allele. This work revealed 33 genes with two or more lethal alleles and 73 genes whose essentiality was not confirmed with an independent lethal mutation, although in some cases second leaky alleles were identified.

  7. Common target genes of palatal and gingival fibroblasts for EMD: the microarray approach.

    Science.gov (United States)

    Gruber, R; Stähli, A; Miron, R J; Bosshardt, D D; Sculean, A

    2015-02-01

    Connective tissue grafts are frequently applied, together with Emdogain(®) , for root coverage. However, it is unknown whether fibroblasts from the gingiva and from the palate respond similarly to Emdogain. The aim of this study was therefore to evaluate the effect of Emdogain(®) on fibroblasts from palatal and gingival connective tissue using a genome-wide microarray approach. Human palatal and gingival fibroblasts were exposed to Emdogain(®) and RNA was subjected to microarray analysis followed by gene ontology screening with Database for Annotation, Visualization and Integrated Discovery functional annotation clustering, Kyoto Encyclopedia of Genes and Genomes pathway analysis and the Search Tool for the Retrieval of Interacting Genes/Proteins functional protein association network. Microarray results were confirmed by quantitative RT-PCR analysis. The transcription levels of 106 genes were up-/down-regulated by at least five-fold in both gingival and palatal fibroblasts upon exposure to Emdogain(®) . Gene ontology screening assigned the respective genes into 118 biological processes, six cellular components, eight molecular functions and five pathways. Among the striking patterns observed were the changing expression of ligands targeting the transforming growth factor-beta and gp130 receptor family as well as the transition of mesenchymal epithelial cells. Moreover, Emdogain(®) caused changes in expression of receptors for chemokines, lipids and hormones, and for transcription factors such as SMAD3, peroxisome proliferator-activated receptor gamma and those of the ETS family. The present data suggest that Emdogain(®) causes substantial alterations in gene expression, with similar patterns observed in palatal and gingival fibroblasts. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. Analysis of essential Arabidopsis nuclear genes encoding plastid-targeted proteins.

    Directory of Open Access Journals (Sweden)

    Linda J Savage

    Full Text Available The Chloroplast 2010 Project (http://www.plastid.msu.edu/ identified and phenotypically characterized homozygous mutants in over three thousand genes, the majority of which encode plastid-targeted proteins. Despite extensive screening by the community, no homozygous mutant alleles were available for several hundred genes, suggesting that these might be enriched for genes of essential function. Attempts were made to generate homozygotes in ~1200 of these lines and 521 of the homozygous viable lines obtained were deposited in the Arabidopsis Biological Resource Center (http://abrc.osu.edu/. Lines that did not yield a homozygote in soil were tested as potentially homozygous lethal due to defects either in seed or seedling development. Mutants were characterized at four stages of development: developing seed, mature seed, at germination, and developing seedlings. To distinguish seed development or seed pigment-defective mutants from seedling development mutants, development of seeds was assayed in siliques from heterozygous plants. Segregating seeds from heterozygous parents were sown on supplemented media in an attempt to rescue homozygous seedlings that could not germinate or survive in soil. Growth of segregating seeds in air and air enriched to 0.3% carbon dioxide was compared to discover mutants potentially impaired in photorespiration or otherwise responsive to CO2 supplementation. Chlorophyll fluorescence measurements identified CO2-responsive mutants with altered photosynthetic parameters. Examples of genes with a viable mutant allele and one or more putative homozygous-lethal alleles were documented. RT-PCR of homozygotes for potentially weak alleles revealed that essential genes may remain undiscovered because of the lack of a true null mutant allele. This work revealed 33 genes with two or more lethal alleles and 73 genes whose essentiality was not confirmed with an independent lethal mutation, although in some cases second leaky alleles

  9. Late multiple organ surge in interferon-regulated target genes characterizes staphylococcal enterotoxin B lethality.

    Directory of Open Access Journals (Sweden)

    Gabriela A Ferreyra

    Full Text Available BACKGROUND: Bacterial superantigens are virulence factors that cause toxic shock syndrome. Here, the genome-wide, temporal response of mice to lethal intranasal staphylococcal enterotoxin B (SEB challenge was investigated in six tissues. RESULTS: The earliest responses and largest number of affected genes occurred in peripheral blood mononuclear cells (PBMC, spleen, and lung tissues with the highest content of both T-cells and monocyte/macrophages, the direct cellular targets of SEB. In contrast, the response of liver, kidney, and heart was delayed and involved fewer genes, but revealed a dominant genetic program that was seen in all 6 tissues. Many of the 85 uniquely annotated transcripts participating in this shared genomic response have not been previously linked to SEB. Nine of the 85 genes were subsequently confirmed by RT-PCR in every tissue/organ at 24 h. These 85 transcripts, up-regulated in all tissues, annotated to the interferon (IFN/antiviral-response and included genes belonging to the DNA/RNA sensing system, DNA damage repair, the immunoproteasome, and the ER/metabolic stress-response and apoptosis pathways. Overall, this shared program was identified as a type I and II interferon (IFN-response and the promoters of these genes were highly enriched for IFN regulatory matrices. Several genes whose secreted products induce the IFN pathway were up-regulated at early time points in PBMCs, spleen, and/or lung. Furthermore, IFN regulatory factors including Irf1, Irf7 and Irf8, and Zbp1, a DNA sensor/transcription factor that can directly elicit an IFN innate immune response, participated in this host-wide SEB signature. CONCLUSION: Global gene-expression changes across multiple organs implicated a host-wide IFN-response in SEB-induced death. Therapies aimed at IFN-associated innate immunity may improve outcome in toxic shock syndromes.

  10. Spontaneous autoimmunity in 129 and C57BL/6 mice-implications for autoimmunity described in gene-targeted mice.

    Directory of Open Access Journals (Sweden)

    Anne E Bygrave

    2004-08-01

    Full Text Available Systemic lupus erythematosus (SLE is a multisystem autoimmune disorder in which complex genetic factors play an important role. Several strains of gene-targeted mice have been reported to develop SLE, implicating the null genes in the causation of disease. However, hybrid strains between 129 and C57BL/6 mice, widely used in the generation of gene-targeted mice, develop spontaneous autoimmunity. Furthermore, the genetic background markedly influences the autoimmune phenotype of SLE in gene-targeted mice. This suggests an important role in the expression of autoimmunity of as-yet-uncharacterised background genes originating from these parental mouse strains. Using genome-wide linkage analysis, we identified several susceptibility loci, derived from 129 and C57BL/6 mice, mapped in the lupus-prone hybrid (129 x C57BL/6 model. By creating a C57BL/6 congenic strain carrying a 129-derived Chromosome 1 segment, we found that this 129 interval was sufficient to mediate the loss of tolerance to nuclear antigens, which had previously been attributed to a disrupted gene. These results demonstrate important epistatic modifiers of autoimmunity in 129 and C57BL/6 mouse strains, widely used in gene targeting. These background gene influences may account for some, or even all, of the autoimmune traits described in some gene-targeted models of SLE.

  11. Targeted Gene-Silencing Reveals the Functional Significance of Myocardin Signaling in the Failing Heart

    Science.gov (United States)

    Torrado, Mario; Iglesias, Raquel; Centeno, Alberto; López, Eduardo; Mikhailov, Alexander T.

    2011-01-01

    Background Myocardin (MYOCD), a potent transcriptional coactivator of smooth muscle (SM) and cardiac genes, is upregulated in failing myocardium in animal models and human end-stage heart failure (HF). However, the molecular and functional consequences of myocd upregulation in HF are still unclear. Methodology/Principal Findings The goal of the present study was to investigate if targeted inhibition of upregulated expression of myocd could influence failing heart gene expression and function. To this end, we used the doxorubicin (Dox)-induced diastolic HF (DHF) model in neonatal piglets, in which, as we show, not only myocd but also myocd-dependent SM-marker genes are highly activated in failing left ventricular (LV) myocardium. In this model, intra-myocardial delivery of short-hairpin RNAs, designed to target myocd variants expressed in porcine heart, leads on day 2 post-delivery to: (1) a decrease in the activated expression of myocd and myocd-dependent SM-marker genes in failing myocardium to levels seen in healthy control animals, (2) amelioration of impaired diastolic dysfunction, and (3) higher survival rates of DHF piglets. The posterior restoration of elevated myocd expression (on day 7 post-delivery) led to overexpression of myocd-dependent SM-marker genes in failing LV-myocardium that was associated with a return to altered diastolic function. Conclusions/Significance These data provide the first evidence that a moderate inhibition (e.g., normalization) of the activated MYOCD signaling in the diseased heart may be promising from a therapeutic point of view. PMID:22028870

  12. Targeted gene-silencing reveals the functional significance of myocardin signaling in the failing heart.

    Directory of Open Access Journals (Sweden)

    Mario Torrado

    Full Text Available BACKGROUND: Myocardin (MYOCD, a potent transcriptional coactivator of smooth muscle (SM and cardiac genes, is upregulated in failing myocardium in animal models and human end-stage heart failure (HF. However, the molecular and functional consequences of myocd upregulation in HF are still unclear. METHODOLOGY/PRINCIPAL FINDINGS: The goal of the present study was to investigate if targeted inhibition of upregulated expression of myocd could influence failing heart gene expression and function. To this end, we used the doxorubicin (Dox-induced diastolic HF (DHF model in neonatal piglets, in which, as we show, not only myocd but also myocd-dependent SM-marker genes are highly activated in failing left ventricular (LV myocardium. In this model, intra-myocardial delivery of short-hairpin RNAs, designed to target myocd variants expressed in porcine heart, leads on day 2 post-delivery to: (1 a decrease in the activated expression of myocd and myocd-dependent SM-marker genes in failing myocardium to levels seen in healthy control animals, (2 amelioration of impaired diastolic dysfunction, and (3 higher survival rates of DHF piglets. The posterior restoration of elevated myocd expression (on day 7 post-delivery led to overexpression of myocd-dependent SM-marker genes in failing LV-myocardium that was associated with a return to altered diastolic function. CONCLUSIONS/SIGNIFICANCE: These data provide the first evidence that a moderate inhibition (e.g., normalization of the activated MYOCD signaling in the diseased heart may be promising from a therapeutic point of view.

  13. Constraints on the evolution of a doublesex target gene arising from doublesex’s pleiotropic deployment

    Science.gov (United States)

    Luo, Shengzhan D.; Baker, Bruce S.

    2015-01-01

    “Regulatory evolution,” that is, changes in a gene’s expression pattern through changes at its regulatory sequence, rather than changes at the coding sequence of the gene or changes of the upstream transcription factors, has been increasingly recognized as a pervasive evolution mechanism. Many somatic sexually dimorphic features of Drosophila melanogaster are the results of gene expression regulated by the doublesex (dsx) gene, which encodes sex-specific transcription factors (DSXF in females and DSXM in males). Rapid changes in such sexually dimorphic features are likely a result of changes at the regulatory sequence of the target genes. We focused on the Flavin-containing monooxygenase-2 (Fmo-2) gene, a likely direct dsx target, to elucidate how sexually dimorphic expression and its evolution are brought about. We found that dsx is deployed to regulate the Fmo-2 transcription both in the midgut and in fat body cells of the spermatheca (a female-specific tissue), through a canonical DSX-binding site in the Fmo-2 regulatory sequence. In the melanogaster group, Fmo-2 transcription in the midgut has evolved rapidly, in contrast to the conserved spermathecal transcription. We identified two cis-regulatory modules (CRM-p and CRM-d) that direct sexually monomorphic or dimorphic Fmo-2 transcription, respectively, in the midguts of these species. Changes of Fmo-2 transcription in the midgut from sexually dimorphic to sexually monomorphic in some species are caused by the loss of CRM-d function, but not the loss of the canonical DSX-binding site. Thus, conferring transcriptional regulation on a CRM level allows the regulation to evolve rapidly in one tissue while evading evolutionary constraints posed by other tissues. PMID:25675536

  14. p53 gene targeting by homologous recombination in fish ES cells.

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    Yan Yan

    Full Text Available BACKGROUND: Gene targeting (GT provides a powerful tool for the generation of precise genetic alterations in embryonic stem (ES cells to elucidate gene function and create animal models for human diseases. This technology has, however, been limited to mouse and rat. We have previously established ES cell lines and procedures for gene transfer and selection for homologous recombination (HR events in the fish medaka (Oryzias latipes. METHODOLOGY AND PRINCIPAL FINDINGS: Here we report HR-mediated GT in this organism. We designed a GT vector to disrupt the tumor suppressor gene p53 (also known as tp53. We show that all the three medaka ES cell lines, MES1∼MES3, are highly proficient for HR, as they produced detectable HR without drug selection. Furthermore, the positive-negative selection (PNS procedure enhanced HR by ∼12 folds. Out of 39 PNS-resistant colonies analyzed, 19 (48.7% were positive for GT by PCR genotyping. When 11 of the PCR-positive colonies were further analyzed, 6 (54.5% were found to be bona fide homologous recombinants by Southern blot analysis, sequencing and fluorescent in situ hybridization. This produces a high efficiency of up to 26.6% for p53 GT under PNS conditions. We show that p53 disruption and long-term propagation under drug selection conditions do not compromise the pluripotency, as p53-targeted ES cells retained stable growth, undifferentiated phenotype, pluripotency gene expression profile and differentiation potential in vitro and in vivo. CONCLUSIONS: Our results demonstrate that medaka ES cells are proficient for HR-mediated GT, offering a first model organism of lower vertebrates towards the development of full ES cell-based GT technology.

  15. Rapid and targeted introgression of genes into popular wheat cultivars using marker-assisted background selection.

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    Harpinder S Randhawa

    Full Text Available A marker-assisted background selection (MABS-based gene introgression approach in wheat (Triticum aestivum L. was optimized, where 97% or more of a recurrent parent genome (RPG can be recovered in just two backcross (BC generations. A four-step MABS method was developed based on 'Plabsim' computer simulations and wheat genome structure information. During empirical optimization of this method, double recombinants around the target gene were selected in a step-wise fashion during the two BC cycles followed by selection for recurrent parent genotype on non-carrier chromosomes. The average spacing between carrier chromosome markers was <4 cM. For non-carrier chromosome markers that flanked each of the 48 wheat gene-rich regions, this distance was approximately 12 cM. Employed to introgress seedling stripe rust (Puccinia striiformis f. sp. tritici resistance gene Yr15 into the spring wheat cultivar 'Zak', marker analysis of 2,187 backcross-derived progeny resulted in the recovery of a BC(2F(2ratio3 plant with 97% of the recurrent parent genome. In contrast, only 82% of the recurrent parent genome was recovered in phenotypically selected BC(4F(7 plants developed without MABS. Field evaluation results from 17 locations indicated that the MABS-derived line was either equal or superior to the recurrent parent for the tested agronomic characteristics. Based on these results, MABS is recommended as a strategy for rapidly introgressing a targeted gene into a wheat genotype in just two backcross generations while recovering 97% or more of the recurrent parent genotype.

  16. Nebulisation of receptor-targeted nanocomplexes for gene delivery to the airway epithelium.

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    Maria D I Manunta

    Full Text Available Gene therapy mediated by synthetic vectors may provide opportunities for new treatments for cystic fibrosis (CF via aerosolisation. Vectors for CF must transfect the airway epithelium efficiently and not cause inflammation so they are suitable for repeated dosing. The inhaled aerosol should be deposited in the airways since the cystic fibrosis transmembrane conductance regulator gene (CFTR is expressed predominantly in the epithelium of the submucosal glands and in the surface airway epithelium. The aim of this project was to develop an optimised aerosol delivery approach applicable to treatment of CF lung disease by gene therapy.The vector suspension investigated in this study comprises receptor-targeting peptides, cationic liposomes and plasmid DNA that self-assemble by electrostatic interactions to form a receptor-targeted nanocomplex (RTN of approximately 150 nm with a cationic surface charge of +50 mV. The aerodynamic properties of aerosolised nanocomplexes produced with three different nebulisers were compared by determining aerosol deposition in the different stages of a Next Generation Pharmaceutical Impactor (NGI. We also investigated the yield of intact plasmid DNA by agarose gel electrophoresis and densitometry, and transfection efficacies in vitro and in vivo.RTNs nebulised with the AeroEclipse II BAN were the most effective, compared to other nebulisers tested, for gene delivery both in vitro and in vivo. The biophysical properties of the nanocomplexes were unchanged after nebulisation while the deposition of RTNs suggested a range of aerosol aerodynamic sizes between 5.5 µm-1.4 µm cut off (NGI stages 3-6 compatible with deposition in the central and lower airways.RTNs showed their ability at delivering genes via nebulisation, thus suggesting their potential applications for therapeutic interventions of cystic fibrosis and other respiratory disorders.

  17. Stem cells’ guided gene therapy of cancer: New frontier in personalized and targeted therapy

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    Mavroudi M

    2014-01-01

    Full Text Available Diagnosis and therapy of cancer remain to be the greatest challenges for all physicians working in clinical oncology and molecular medicine. The grim statistics speak for themselves with reports of 1,638,910 men and women diagnosed with cancer and nearly 577,190 patients passed away due to cancer in the USA in 2012. For practicing clinicians, who treat patients suffering from advanced cancers with contemporary systemic therapies, the main challenge is to attain therapeutic efficacy, while minimizing side effects. Unfortunately, all contemporary systemic therapies cause side effects. In treated patients, these side effects may range from nausea to damaged tissues. In cancer survivors, the iatrogenic outcomes of systemic therapies may include genomic mutations and their consequences. Therefore, there is an urgent need for personalized and targeted therapies. Recently, we reviewed the current status of suicide gene therapy for cancer. Herein, we discuss the novel strategy: genetically engineered stem guided gene therapy. Stem cells have the unique potential for self-renewal and differentiation. This potential is the primary reason for introducing them into medicine to regenerate injured or degenerated organs, as well as to rejuvenate aging tissues. Recent advances in genetic engineering and stem cell research have created the foundations for genetic engineering of stem cells as the vectors for delivery of therapeutic transgenes. Specifically in oncology, the stem cells are genetically engineered to deliver the cell suicide inducing genes selectively to the cancer cells. Expression of the transgenes kills the cancer cells, while leaving healthy cells unaffected. Herein, we present various strategies to bioengineer suicide inducing genes and stem cell vectors. Moreover, we review results of the main preclinical studies and clinical trials. However, the main risk for therapeutic use of stem cells is their cancerous transformation. Therefore, we

  18. Explorations of combinational therapy in cancer : targeting the tumor and its microenvironment by combining chemotherapy with chemopreventive approaches

    NARCIS (Netherlands)

    Wijngaarden, Johannes Willem van

    2011-01-01

    One of the most effective anticancer therapy still remains chemotherapy, however, both used as single agent as in combinational regimens, chemotherapy still encounters the problem of therapeutic resistance. Limitations of chemotherapy have led to the exploration of alternative anti-cancer approaches

  19. Effect of suicidal gene combined with irradiation on esophageal carcinoma cell line

    International Nuclear Information System (INIS)

    Pan Jianji; Wang Jiezhong; Zheng Tianrong; Zheng Qiuhong

    2005-01-01

    Objective: As generally known that non-cytotoxic pro-drag can be transformed into cytotoxic drug by suicide gene, this work is to investigate the effect of Coli cytosine deaminase/5-fluorocytosine suicide gene (CD/5-FC) used alone or combined with irradiation in esophageal carcinoma cell line(EC). Methods: CD gene was amplified from Coli DNA genome library with PCR technique, with the eukaryotic vector pcDNA3.1-CD then constructed. ECl09 cells were transfected with pcDNA3.1-CD by liposome method. The cytotoxic effect, bystander effect and radiosensitization effect of CD/5-FC in ECl09 was analyzed. Results: The transfection of CD gene into ECl09 and its transcription was confirmed by RT-PCR method. In vitro, 5-FC showed significantly cytotoxic effect on the EPC cell transfected with CD gene. After adding 5-FC , the survival rate of cultured cell containing 5 % transfect CD gene cell was 41.8 % ± 14.2% while that in the control group was 94.6 ± 4.3 %, (t=3.14, P < 0.05). The survival rate of cultured cell containing 10% transfected CD gene cell was 37.8 ± 4.4% compared to 95.6% ± 5.4% in the control group, (t=9.75, P<0.01). CD/5-FC showed significant radiosen-sitization effect, the survival fraction of CD transfected cell was much lower in 5-FC combined with irradiation, when compared with 5-FC alone and radiotherapy alone group together, (F=11.50, P < 0.01 ). When it was compared with 5-FC alone group and irradiation alone group separately, the difference was also significant( F=4.11, P < 0.05 and F10.53, P < 0.01, respectively). Conclusions: Suicide gene CD/5-FC shows conspicuous by-stander effect and radiosensitization effect. (authors)

  20. Approach of combined cancer gene therapy and radiation: response of promoters to ionizing radiation

    International Nuclear Information System (INIS)

    Anstett, A.

    2005-09-01

    Gene therapy is an emerging cancer treatment modality. We are interested in developing a radiation-inducible gene therapy system to sensitize the tumor vasculature to the effects of ionizing radiation (IR) treatment. An expression system based on irradiation-inducible promoters will drive the expression of anti-tumor genes in the tumor vasculature. Solid tumors are dependent on angio genesis, a process in which new blood vessels are formed from the pre-existing vasculature. Vascular endothelial cells are un transformed and genetically stable, thus avoiding the problem of resistance to the treatments. Vascular endothelial cells may therefore represent a suitable target for this therapeutic gene therapy strategy.The identification of IR-inducible promoters native to endothelial cells was performed by gene expression profiling using cDNA micro array technology. We describe the genes modified by clinically relevant doses of IR. The extension to high doses aimed at studying the effects of total radiation delivery to the tumor. The radio-inductiveness of the genes selected for promoter study was confirmed by RT-PCR. Analysis of the activity of promoters in response to IR was also assessed in a reporter plasmid. We found that authentic promoters cloned onto a plasmid are not suitable for cancer gene therapy due to their low induction after IR. In contrast, synthetic promoters containing repeated sequence-specific binding sites for IR-activated transcription factors such as NF-κB are potential candidates for gene therapy. The activity of five tandemly repeated TGGGGACTTTCCGC elements for NF-κB binding in a luciferase reporter was increased in a dose-dependent manner. Interestingly, the response to fractionated low doses was improved in comparison to the total single dose. Thus, we put present evidence that a synthetic promoter for NF-κB specific binding may have application in the radio-therapeutic treatment of cancer. (author)

  1. Gene identification for risk of relapse in stage I lung adenocarcinoma patients: a combined methodology of gene expression profiling and computational gene network analysis.

    Science.gov (United States)

    Ludovini, Vienna; Bianconi, Fortunato; Siggillino, Annamaria; Piobbico, Danilo; Vannucci, Jacopo; Metro, Giulio; Chiari, Rita; Bellezza, Guido; Puma, Francesco; Della Fazia, Maria Agnese; Servillo, Giuseppe; Crinò, Lucio

    2016-05-24

    Risk assessment and treatment choice remains a challenge in early non-small-cell lung cancer (NSCLC). The aim of this study was to identify novel genes involved in the risk of early relapse (ER) compared to no relapse (NR) in resected lung adenocarcinoma (AD) patients using a combination of high throughput technology and computational analysis. We identified 18 patients (n.13 NR and n.5 ER) with stage I AD. Frozen samples of patients in ER, NR and corresponding normal lung (NL) were subjected to Microarray technology and quantitative-PCR (Q-PCR). A gene network computational analysis was performed to select predictive genes. An independent set of 79 ADs stage I samples was used to validate selected genes by Q-PCR.From microarray analysis we selected 50 genes, using the fold change ratio of ER versus NR. They were validated both in pool and individually in patient samples (ER and NR) by Q-PCR. Fourteen increased and 25 decreased genes showed a concordance between two methods. They were used to perform a computational gene network analysis that identified 4 increased (HOXA10, CLCA2, AKR1B10, FABP3) and 6 decreased (SCGB1A1, PGC, TFF1, PSCA, SPRR1B and PRSS1) genes. Moreover, in an independent dataset of ADs samples, we showed that both high FABP3 expression and low SCGB1A1 expression was associated with a worse disease-free survival (DFS).Our results indicate that it is possible to define, through gene expression and computational analysis, a characteristic gene profiling of patients with an increased risk of relapse that may become a tool for patient selection for adjuvant therapy.

  2. The effect of endostatin gene in combination with radiotherapy on rats with implanted tumor

    International Nuclear Information System (INIS)

    Li Yong; Jin Ning; Yang Haishan; Piao Chunji; Lv Zhe

    2005-01-01

    Objective: To study the combination therapy effect of the radiotherapy with endostatin gene therapy on the rats with implanted tumor. Methods: Immediate Walker-256 cancerous ascetic injection method was used to make a rat tumor-bearing model, then the tumor was treated with saline, endostatin gene, irradiation or endostatin gene plus irradiation. The tumor growth rate and weight were observed, Western blot and RT-PCR were adopted to check the expressions of endostatin mRNA and protein. Results: The expressions of endostatin mRNA and protein were significant in the gene therapy group and the gene plus radiotherapy group, but there was a significant difference between these two groups. As compared with the control group, the tumor growth rate and weight decreased significantly in all the therapy groups (P 0.05). Conclusion: After the pCMV-Endostatin was induced, the expressions of endostatin mRNA and protein was significant in Walker-256 tumor and the tumor growth was inhibited. However, the effect of the endostatin gene plus radiotherapy was obviously better than that of the endostatin gene therapy group or the radiotherapy group for inhibiting tumor growth. (authors)

  3. Prioritization of candidate disease genes by combining topological similarity and semantic similarity.

    Science.gov (United States)

    Liu, Bin; Jin, Min; Zeng, Pan

    2015-10-01

    The identification of gene-phenotype relationships is very important for the treatment of human diseases. Studies have shown that genes causing the same or similar phenotypes tend to interact with each other in a protein-protein interaction (PPI) network. Thus, many identification methods based on the PPI network model have achieved good results. However, in the PPI network, some interactions between the proteins encoded by candidate gene and the proteins encoded by known disease genes are very weak. Therefore, some studies have combined the PPI network with other genomic information and reported good predictive performances. However, we believe that the results could be further improved. In this paper, we propose a new method that uses the semantic similarity between the candidate gene and known disease genes to set the initial probability vector of a random walk with a restart algorithm in a human PPI network. The effectiveness of our method was demonstrated by leave-one-out cross-validation, and the experimental results indicated that our method outperformed other methods. Additionally, our method can predict new causative genes of multifactor diseases, including Parkinson's disease, breast cancer and obesity. The top predictions were good and consistent with the findings in the literature, which further illustrates the effectiveness of our method. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. ARNetMiT R Package: association rules based gene co-expression networks of miRNA targets.

    Science.gov (United States)

    Özgür Cingiz, M; Biricik, G; Diri, B

    2017-03-31

    miRNAs are key regulators that bind to target genes to suppress their gene expression level. The relations between miRNA-target genes enable users to derive co-expressed genes that may be involved in similar biological processes and functions in cells. We hypothesize that target genes of miRNAs are co-expressed, when they are regulated by multiple miRNAs. With the usage of these co-expressed genes, we can theoretically construct co-expression networks (GCNs) related to 152 diseases. In this study, we introduce ARNetMiT that utilize a hash based association rule algorithm in a novel way to infer the GCNs on miRNA-target genes data. We also present R package of ARNetMiT, which infers and visualizes GCNs of diseases that are selected by users. Our approach assumes miRNAs as transactions and target genes as their items. Support and confidence values are used to prune association rules on miRNA-target genes data to construct support based GCNs (sGCNs) along with support and confidence based GCNs (scGCNs). We use overlap analysis and the topological features for the performance analysis of GCNs. We also infer GCNs with popular GNI algorithms for comparison with the GCNs of ARNetMiT. Overlap analysis results show that ARNetMiT outperforms the compared GNI algorithms. We see that using high confidence values in scGCNs increase the ratio of the overlapped gene-gene interactions between the compared methods. According to the evaluation of the topological features of ARNetMiT based GCNs, the degrees of nodes have power-law distribution. The hub genes discovered by ARNetMiT based GCNs are consistent with the literature.

  5. Hairpin RNA Targeting Multiple Viral Genes Confers Strong Resistance to Rice Black-Streaked Dwarf Virus

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    Fangquan Wang

    2016-05-01

    Full Text Available Rice black-streaked dwarf virus (RBSDV belongs to the genus Fijivirus in the family of Reoviridae and causes severe yield loss in rice-producing areas in Asia. RNA silencing, as a natural defence mechanism against plant viruses, has been successfully exploited for engineering virus resistance in plants, including rice. In this study, we generated transgenic rice lines harbouring a hairpin RNA (hpRNA construct targeting four RBSDV genes, S1, S2, S6 and S10, encoding the RNA-dependent RNA polymerase, the putative core protein, the RNA silencing suppressor and the outer capsid protein, respectively. Both field nursery and artificial inoculation assays of three generations of the transgenic lines showed that they had strong resistance to RBSDV infection. The RBSDV resistance in the segregating transgenic populations correlated perfectly with the presence of the hpRNA transgene. Furthermore, the hpRNA transgene was expressed in the highly resistant transgenic lines, giving rise to abundant levels of 21–24 nt small interfering RNA (siRNA. By small RNA deep sequencing, the RBSDV-resistant transgenic lines detected siRNAs from all four viral gene sequences in the hpRNA transgene, indicating that the whole chimeric fusion sequence can be efficiently processed by Dicer into siRNAs. Taken together, our results suggest that long hpRNA targeting multiple viral genes can be used to generate stable and durable virus resistance in rice, as well as other plant species.

  6. Viral Etiology Relationship between Human Papillomavirus and Human Breast Cancer and Target of Gene Therapy.

    Science.gov (United States)

    Yan, Chen; Teng, Zhi Ping; Chen, Yun Xin; Shen, Dan Hua; Li, Jin Tao; Zeng, Yi

    2016-05-01

    To explore the viral etiology of human breast cancer to determine whether there are novel molecular targets for gene therapy of breast cancer and provide evidence for the research of gene therapy and vaccine development for breast cancer. PCR was used to screen HPV16 and HPV18 oncogenes E6 and E7 in the SKBR3 cell line and in 76 paraffin embedded breast cancer tissue samples. RNA interference was used to knock down the expression of HPV18 E6 and E7 in SKBR3 cells, then the changes in the expression of cell-cycle related proteins, cell viability, colony formation, metastasis, and cell cycle progression were determined. HPV18 oncogenes E6 and E7 were amplified and sequenced from the SKBR3 cells. Of the patient samples, 6.58% and 23.68% were tested to be positive for HPV18 E6 and HPV18 E7. In the cell culture models, the knockdown of HPV18 E6 and E7 inhibited the proliferation, metastasis, and cell cycle progression of SKBR3 cell. The knockdown also clearly affected the expression levels of cell cycle related proteins. HPV was a contributor to virus caused human breast cancer, suggesting that the oncogenes in HPV were potential targets for gene therapy of breast cancer. Copyright © 2016 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  7. Properties of a Telomerase-Specific Cre/Lox Switch for Transcriptionally Targeted Cancer Gene Therapy

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    Alan E. Bilsland

    2005-11-01

    Full Text Available Telomerase expression represents a good target for cancer gene therapy. The promoters of the core telomerase catalytic [human telomerase reverse transcriptase (hTERT] and RNA [human telomerase RNA (hTR] subunits show selective activity in cancer cells but not in normal cells. This property can be harnessed to express therapeutic transgenes in a wide range of cancer cells. Unfortunately, weak hTR and hTERT promoter activities in some cancer cells could limit the target cell range. Therefore, strategies to enhance telomerasespecific gene therapy are of interest. We constructed a Cre/Lox reporter switch coupling telomerase promoter specificity with Cytomegalovirus (CMV promoter activity, which is generally considered to be constitutively high. In this approach, a telomerase-specific vector expressing Cre recombinase directs excisive recombination on a second vector, removing a transcriptional blockade to CMV-dependent luciferase expression. We tested switch activation in cell lines over a wide range of telomerase promoter activities. However, Cre/Lox-dependent luciferase expression was not enhanced relative to expression using hTR or hTERT promoters directly. Cell-specific differences between telomerase and CMV promoter activities and incomplete sigmoid switch activation were limiting factors. Notably, CMV activity was not always significantly stronger than telomerase promoter activity. Our conclusions provide a general basis for a more rational design of novel recombinase switches in gene therapy.

  8. Inactivation and inducible oncogenic mutation of p53 in gene targeted pigs.

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    Simon Leuchs

    Full Text Available Mutation of the tumor suppressor p53 plays a major role in human carcinogenesis. Here we describe gene-targeted porcine mesenchymal stem cells (MSCs and live pigs carrying a latent TP53(R167H mutant allele, orthologous to oncogenic human mutant TP53(R175H and mouse Trp53(R172H, that can be activated by Cre recombination. MSCs carrying the latent TP53(R167H mutant allele were analyzed in vitro. Homozygous cells were p53 deficient, and on continued culture exhibited more rapid proliferation, anchorage independent growth, and resistance to the apoptosis-inducing chemotherapeutic drug doxorubicin, all characteristic of cellular transformation. Cre mediated recombination activated the latent TP53(R167H allele as predicted, and in homozygous cells expressed mutant p53-R167H protein at a level ten-fold greater than wild-type MSCs, consistent with the elevated levels found in human cancer cells. Gene targeted MSCs were used for nuclear transfer and fifteen viable piglets were produced carrying the latent TP53(R167H mutant allele in heterozygous form. These animals will allow study of p53 deficiency and expression of mutant p53-R167H to model human germline, or spontaneous somatic p53 mutation. This work represents the first inactivation and mutation of the gatekeeper tumor suppressor gene TP53 in a non-rodent mammal.

  9. Patient mutation in AIRE disrupts P-TEFb binding and target gene transcription

    Science.gov (United States)

    Plemenitaš, Ana; Saksela, Kalle; Peterlin, B. Matija

    2011-01-01

    Autoimmune regulator (AIRE) is a transcription factor that induces the expression of a large subset of otherwise strictly tissue restricted antigens in medullary thymic epithelial cells, thereby enabling their presentation to developing T cells for negative selection. Mutations in AIRE lead to autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a rare monogenetic disease. Although it has been reported that AIRE interacts with proteins involved in nuclear transport, DNA-damage response, chromatin remodeling, transcription and pre-mRNA-splicing, the precise mechanism of AIRE-induced tissue restricted antigen expression has remained elusive. In this study, we investigated an APECED patient mutation that causes the loss of the extreme C-terminus of AIRE and found that this mutant protein is transcriptionaly inactive. When tethered heterologously to DNA, this domain could stimulate transcription and splicing by itself. Moreover, the loss of this C-terminus disrupted inter