Science.gov

Sample records for cells reproduces gene

  1. Electric Pulse Stimulation of Cultured Murine Muscle Cells Reproduces Gene Expression Changes of Trained Mouse Muscle

    PubMed Central

    Burch, Nathalie; Arnold, Anne-Sophie; Item, Flurin; Summermatter, Serge; Brochmann Santana Santos, Gesa; Christe, Martine; Boutellier, Urs; Toigo, Marco; Handschin, Christoph

    2010-01-01

    Adequate levels of physical activity are at the center of a healthy lifestyle. However, the molecular mechanisms that mediate the beneficial effects of exercise remain enigmatic. This gap in knowledge is caused by the lack of an amenable experimental model system. Therefore, we optimized electric pulse stimulation of muscle cells to closely recapitulate the plastic changes in gene expression observed in a trained skeletal muscle. The exact experimental conditions were established using the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) as a marker for an endurance-trained muscle fiber. We subsequently compared the changes in the relative expression of metabolic and myofibrillar genes in the muscle cell system with those observed in mouse muscle in vivo following either an acute or repeated bouts of treadmill exercise. Importantly, in electrically stimulated C2C12 mouse muscle cells, the qualitative transcriptional adaptations were almost identical to those in trained muscle, but differ from the acute effects of exercise on muscle gene expression. In addition, significant alterations in the expression of myofibrillar proteins indicate that this stimulation could be used to modulate the fiber-type of muscle cells in culture. Our data thus describe an experimental cell culture model for the study of at least some of the transcriptional aspects of skeletal muscle adaptation to physical activity. This system will be useful for the study of the molecular mechanisms that regulate exercise adaptation in muscle. PMID:20532042

  2. Quartz-Seq: a highly reproducible and sensitive single-cell RNA sequencing method, reveals non-genetic gene-expression heterogeneity.

    PubMed

    Sasagawa, Yohei; Nikaido, Itoshi; Hayashi, Tetsutaro; Danno, Hiroki; Uno, Kenichiro D; Imai, Takeshi; Ueda, Hiroki R

    2013-04-17

    Development of a highly reproducible and sensitive single-cell RNA sequencing (RNA-seq) method would facilitate the understanding of the biological roles and underlying mechanisms of non-genetic cellular heterogeneity. In this study, we report a novel single-cell RNA-seq method called Quartz-Seq that has a simpler protocol and higher reproducibility and sensitivity than existing methods. We show that single-cell Quartz-Seq can quantitatively detect various kinds of non-genetic cellular heterogeneity, and can detect different cell types and different cell-cycle phases of a single cell type. Moreover, this method can comprehensively reveal gene-expression heterogeneity between single cells of the same cell type in the same cell-cycle phase.

  3. Intra- and inter-laboratory reproducibility and accuracy of the LuSens assay: A reporter gene-cell line to detect keratinocyte activation by skin sensitizers.

    PubMed

    Ramirez, Tzutzuy; Stein, Nadine; Aumann, Alexandra; Remus, Tina; Edwards, Amber; Norman, Kimberly G; Ryan, Cindy; Bader, Jackie E; Fehr, Markus; Burleson, Florence; Foertsch, Leslie; Wang, Xiaohong; Gerberick, Frank; Beilstein, Paul; Hoffmann, Sebastian; Mehling, Annette; van Ravenzwaay, Bennard; Landsiedel, Robert

    2016-04-01

    Several non-animal methods are now available to address the key events leading to skin sensitization as defined by the adverse outcome pathway. The KeratinoSens assay addresses the cellular event of keratinocyte activation and is a method accepted under OECD TG 442D. In this study, the results of an inter-laboratory evaluation of the "me-too" LuSens assay, a bioassay that uses a human keratinocyte cell line harboring a reporter gene construct composed of the rat antioxidant response element (ARE) of the NADPH:quinone oxidoreductase 1 gene and the luciferase gene, are described. Earlier in-house validation with 74 substances showed an accuracy of 82% in comparison to human data. When used in a battery of non-animal methods, even higher predictivity is achieved. To meet European validation criteria, a multicenter study was conducted in 5 laboratories. The study was divided into two phases, to assess 1) transferability of the method, and 2) reproducibility and accuracy. Phase I was performed by testing 8 non-coded test substances; the results showed a good transferability to naïve laboratories even without on-site training. Phase II was performed with 20 coded test substances (performance standards recommended by OECD, 2015). In this phase, the intra- and inter-laboratory reproducibility as well as accuracy of the method was evaluated. The data demonstrate a remarkable reproducibility of 100% and an accuracy of over 80% in identifying skin sensitizers, indicating a good concordance with in vivo data. These results demonstrate good transferability, reliability and accuracy of the method thereby achieving the standards necessary for use in a regulatory setting to detect skin sensitizers.

  4. Empowering Multi-Cohort Gene Expression Analysis to Increase Reproducibility

    PubMed Central

    Haynes, Winston A; Vallania, Francesco; Liu, Charles; Bongen, Erika; Tomczak, Aurelie; Andres-Terrè, Marta; Lofgren, Shane; Tam, Andrew; Deisseroth, Cole A; Li, Matthew D; Sweeney, Timothy E

    2016-01-01

    A major contributor to the scientific reproducibility crisis has been that the results from homogeneous, single-center studies do not generalize to heterogeneous, real world populations. Multi-cohort gene expression analysis has helped to increase reproducibility by aggregating data from diverse populations into a single analysis. To make the multi-cohort analysis process more feasible, we have assembled an analysis pipeline which implements rigorously studied meta-analysis best practices. We have compiled and made publicly available the results of our own multi-cohort gene expression analysis of 103 diseases, spanning 615 studies and 36,915 samples, through a novel and interactive web application. As a result, we have made both the process of and the results from multi-cohort gene expression analysis more approachable for non-technical users. PMID:27896970

  5. Efficient and reproducible mammalian cell bioprocesses without probes and controllers?

    PubMed

    Tissot, Stéphanie; Oberbek, Agata; Reclari, Martino; Dreyer, Matthieu; Hacker, David L; Baldi, Lucia; Farhat, Mohamed; Wurm, Florian M

    2011-07-01

    Bioprocesses for recombinant protein production with mammalian cells are typically controlled for several physicochemical parameters including the pH and dissolved oxygen concentration (DO) of the culture medium. Here we studied whether these controls are necessary for efficient and reproducible bioprocesses in an orbitally shaken bioreactor (OSR). Mixing, gas transfer, and volumetric power consumption (P(V)) were determined in both a 5-L OSR and a 3-L stirred-tank bioreactor (STR). The two cultivation systems had a similar mixing intensity, but the STR had a lower volumetric mass transfer coefficient of oxygen (k(L)a) and a higher P(V) than the OSR. Recombinant CHO cell lines expressing either tumor necrosis factor receptor as an Fc fusion protein (TNFR:Fc) or an anti-RhesusD monoclonal antibody were cultivated in the two systems. The 5-L OSR was operated in an incubator shaker with 5% CO(2) in the gas environment but without pH and DO control whereas the STR was operated with or without pH and DO control. Higher cell densities and recombinant protein titers were obtained in the OSR as compared to both the controlled and the non-controlled STRs. To test the reproducibility of a bioprocess in a non-controlled OSR, the two CHO cell lines were each cultivated in parallel in six 5-L OSRs. Similar cell densities, cell viabilities, and recombinant protein titers along with similar pH and DO profiles were achieved in each group of replicates. Our study demonstrated that bioprocesses can be performed in OSRs without pH or DO control in a highly reproducible manner, at least at the scale of operation studied here.

  6. Identifying reproducible cancer-associated highly expressed genes with important functional significances using multiple datasets

    PubMed Central

    Huang, Haiyan; Li, Xiangyu; Guo, You; Zhang, Yuncong; Deng, Xusheng; Chen, Lufei; Zhang, Jiahui; Guo, Zheng; Ao, Lu

    2016-01-01

    Identifying differentially expressed (DE) genes between cancer and normal tissues is of basic importance for studying cancer mechanisms. However, current methods, such as the commonly used Significance Analysis of Microarrays (SAM), are biased to genes with low expression levels. Recently, we proposed an algorithm, named the pairwise difference (PD) algorithm, to identify highly expressed DE genes based on reproducibility evaluation of top-ranked expression differences between paired technical replicates of cells under two experimental conditions. In this study, we extended the application of the algorithm to the identification of DE genes between two types of tissue samples (biological replicates) based on several independent datasets or sub-datasets of a dataset, by constructing multiple paired average gene expression profiles for the two types of samples. Using multiple datasets for lung and esophageal cancers, we demonstrated that PD could identify many DE genes highly expressed in both cancer and normal tissues that tended to be missed by the commonly used SAM. These highly expressed DE genes, including many housekeeping genes, were significantly enriched in many conservative pathways, such as ribosome, proteasome, phagosome and TNF signaling pathways with important functional significances in oncogenesis. PMID:27796338

  7. Planar heterojunction perovskite solar cells with superior reproducibility

    PubMed Central

    Jeon, Ye-Jin; Lee, Sehyun; Kang, Rira; Kim, Jueng-Eun; Yeo, Jun-Seok; Lee, Seung-Hoon; Kim, Seok-Soon; Yun, Jin-Mun; Kim, Dong-Yu

    2014-01-01

    Perovskite solar cells (PeSCs) have been considered one of the competitive next generation power sources. To date, light-to-electric conversion efficiencies have rapidly increased to over 10%, and further improvements are expected. However, the poor device reproducibility of PeSCs ascribed to their inhomogeneously covered film morphology has hindered their practical application. Here, we demonstrate high-performance PeSCs with superior reproducibility by introducing small amounts of N-cyclohexyl-2-pyrrolidone (CHP) as a morphology controller into N,N-dimethylformamide (DMF). As a result, highly homogeneous film morphology, similar to that achieved by vacuum-deposition methods, as well as a high PCE of 10% and an extremely small performance deviation within 0.14% were achieved. This study represents a method for realizing efficient and reproducible planar heterojunction (PHJ) PeSCs through morphology control, taking a major step forward in the low-cost and rapid production of PeSCs by solving one of the biggest problems of PHJ perovskite photovoltaic technology through a facile method. PMID:25377945

  8. Planar heterojunction perovskite solar cells with superior reproducibility

    NASA Astrophysics Data System (ADS)

    Jeon, Ye-Jin; Lee, Sehyun; Kang, Rira; Kim, Jueng-Eun; Yeo, Jun-Seok; Lee, Seung-Hoon; Kim, Seok-Soon; Yun, Jin-Mun; Kim, Dong-Yu

    2014-11-01

    Perovskite solar cells (PeSCs) have been considered one of the competitive next generation power sources. To date, light-to-electric conversion efficiencies have rapidly increased to over 10%, and further improvements are expected. However, the poor device reproducibility of PeSCs ascribed to their inhomogeneously covered film morphology has hindered their practical application. Here, we demonstrate high-performance PeSCs with superior reproducibility by introducing small amounts of N-cyclohexyl-2-pyrrolidone (CHP) as a morphology controller into N,N-dimethylformamide (DMF). As a result, highly homogeneous film morphology, similar to that achieved by vacuum-deposition methods, as well as a high PCE of 10% and an extremely small performance deviation within 0.14% were achieved. This study represents a method for realizing efficient and reproducible planar heterojunction (PHJ) PeSCs through morphology control, taking a major step forward in the low-cost and rapid production of PeSCs by solving one of the biggest problems of PHJ perovskite photovoltaic technology through a facile method.

  9. Planar heterojunction perovskite solar cells with superior reproducibility.

    PubMed

    Jeon, Ye-Jin; Lee, Sehyun; Kang, Rira; Kim, Jueng-Eun; Yeo, Jun-Seok; Lee, Seung-Hoon; Kim, Seok-Soon; Yun, Jin-Mun; Kim, Dong-Yu

    2014-11-07

    Perovskite solar cells (PeSCs) have been considered one of the competitive next generation power sources. To date, light-to-electric conversion efficiencies have rapidly increased to over 10%, and further improvements are expected. However, the poor device reproducibility of PeSCs ascribed to their inhomogeneously covered film morphology has hindered their practical application. Here, we demonstrate high-performance PeSCs with superior reproducibility by introducing small amounts of N-cyclohexyl-2-pyrrolidone (CHP) as a morphology controller into N,N-dimethylformamide (DMF). As a result, highly homogeneous film morphology, similar to that achieved by vacuum-deposition methods, as well as a high PCE of 10% and an extremely small performance deviation within 0.14% were achieved. This study represents a method for realizing efficient and reproducible planar heterojunction (PHJ) PeSCs through morphology control, taking a major step forward in the low-cost and rapid production of PeSCs by solving one of the biggest problems of PHJ perovskite photovoltaic technology through a facile method.

  10. Evolutionary adaptation after crippling cell polarization follows reproducible trajectories

    PubMed Central

    Laan, Liedewij; Koschwanez, John H; Murray, Andrew W

    2015-01-01

    Cells are organized by functional modules, which typically contain components whose removal severely compromises the module's function. Despite their importance, these components are not absolutely conserved between parts of the tree of life, suggesting that cells can evolve to perform the same biological functions with different proteins. We evolved Saccharomyces cerevisiae for 1000 generations without the important polarity gene BEM1. Initially the bem1∆ lineages rapidly increase in fitness and then slowly reach >90% of the fitness of their BEM1 ancestors at the end of the evolution. Sequencing their genomes and monitoring polarization reveals a common evolutionary trajectory, with a fixed sequence of adaptive mutations, each improving cell polarization by inactivating proteins. Our results show that organisms can be evolutionarily robust to physiologically destructive perturbations and suggest that recovery by gene inactivation can lead to rapid divergence in the parts list for cell biologically important functions. DOI: http://dx.doi.org/10.7554/eLife.09638.001 PMID:26426479

  11. Methods to increase reproducibility in differential gene expression via meta-analysis

    PubMed Central

    Sweeney, Timothy E.; Haynes, Winston A.; Vallania, Francesco; Ioannidis, John P.; Khatri, Purvesh

    2017-01-01

    Findings from clinical and biological studies are often not reproducible when tested in independent cohorts. Due to the testing of a large number of hypotheses and relatively small sample sizes, results from whole-genome expression studies in particular are often not reproducible. Compared to single-study analysis, gene expression meta-analysis can improve reproducibility by integrating data from multiple studies. However, there are multiple choices in designing and carrying out a meta-analysis. Yet, clear guidelines on best practices are scarce. Here, we hypothesized that studying subsets of very large meta-analyses would allow for systematic identification of best practices to improve reproducibility. We therefore constructed three very large gene expression meta-analyses from clinical samples, and then examined meta-analyses of subsets of the datasets (all combinations of datasets with up to N/2 samples and K/2 datasets) compared to a ‘silver standard’ of differentially expressed genes found in the entire cohort. We tested three random-effects meta-analysis models using this procedure. We showed relatively greater reproducibility with more-stringent effect size thresholds with relaxed significance thresholds; relatively lower reproducibility when imposing extraneous constraints on residual heterogeneity; and an underestimation of actual false positive rate by Benjamini–Hochberg correction. In addition, multivariate regression showed that the accuracy of a meta-analysis increased significantly with more included datasets even when controlling for sample size. PMID:27634930

  12. Factors Affecting Polymer Electrolyte Fuel Cells Performance and Reproducibility

    SciTech Connect

    Moller-Holst S.

    1998-11-01

    Development of fuel cells is often based on small-scale laboratory studies. Due to limited time and budgets, a minimum number of cells are usually prepared and tested, thus, conclusions about improved performance are often drawn from studies of a few cells. Generally, statistics showing the significance of an effect are seldom reported. In this work a simple PEM fuel cell electrode optimization experiment is used as an example to illustrate the importance of statistical evaluation of factors affecting cell performance. The use of fractional factorial design of experiments to reduce the number of cells that have to be studied is also addressed.

  13. A Reproducible Immunopotency Assay to Measure Mesenchymal Stromal Cell Mediated T cell Suppression

    PubMed Central

    Bloom, Debra D.; Centanni, John M.; Bhatia, Neehar; Emler, Carol A.; Drier, Diana; Leverson, Glen E.; McKenna, David H.; Gee, Adrian P.; Lindblad, Robert; Hei, Derek J.; Hematti, Peiman

    2014-01-01

    Background The T cell suppressive property of bone marrow derived mesenchymal stromal cells (MSCs) has been considered a major mode of action and basis for their utilization in a number of human clinical trials. However, there is no well-established reproducible assay to measure MSC-mediated T cell suppression. Methods At the University of Wisconsin-Madison Production Assistance for Cellular Therapy (PACT) Center we developed an in vitro quality control T cell suppression immunopotency assay (IPA) which utilizes anti-CD3 and anti-CD28 antibodies to stimulate T cell proliferation. We measured MSC-induced suppression of CD4+ T cell proliferation at various effector to target cell ratios using defined peripheral blood mononuclear cells and in parallel compared to a reference standard MSC product. We calculated an IPA value for suppression of CD4+ T cells for each MSC product. Results Eleven MSC products generated at three independent PACT centers were evaluated for cell surface phenotypic markers and T cell suppressive properties. Flow cytometry results demonstrated typical MSC cell surface marker profiles. There was significant variability in the level of suppression of T cell proliferation with IPA values ranging from 27% to 88%. However, MSC suppression did not correlate with HLA-DR expression. Discussion We have developed a reproducible immunopotency assay to measure allogeneic MSC-mediated suppression of CD4+ T cells. Additional studies may be warranted to determine how these in vitro assay results may correlate with other immunomodulatory properties of MSCs, in addition to evaluating the ability of this assay to predict in vivo efficacy. PMID:25455739

  14. Characterization of Adipogenic Chemicals in Three Different Cell Culture Systems: Implications for Reproducibility Based on Cell Source and Handling.

    PubMed

    Kassotis, Christopher D; Masse, Lauren; Kim, Stephanie; Schlezinger, Jennifer J; Webster, Thomas F; Stapleton, Heather M

    2017-02-08

    The potential for chemical exposures to exacerbate the development and/or prevalence of metabolic disorders, such as obesity, is currently of great societal concern. Various in vitro assays are available to assess adipocyte differentiation, though little work has been done to standardize protocols and compare models effectively. This study compares several adipogenic cell culture systems under a variety of conditions to assess variability in responses. Two sources of 3T3-L1 preadipocytes as well as OP9 preadipocytes were assessed for cell proliferation and triglyceride accumulation following different induction periods and using various tissue culture plates. Both cell line and cell source had a significant impact on potencies and efficacies of adipogenic chemicals. Gene expression analyses suggested that differential expression of nuclear receptors involved in adipogenesis underlie the differences between OP9 and 3T3-L1 cells; however, there were also differences based on 3T3-L1 cell source. Induction period modulated potency and efficacy of response depending on cell line and test chemical, and large variations were observed in triglyceride accumulation and cell proliferation between brands of tissue culture plates. Our results suggest that the selection of a cell system and differentiation protocol significantly impacts the detection of adipogenic chemicals, and therefore, influences reproducibility of these studies.

  15. Characterization of Adipogenic Chemicals in Three Different Cell Culture Systems: Implications for Reproducibility Based on Cell Source and Handling

    PubMed Central

    Kassotis, Christopher D.; Masse, Lauren; Kim, Stephanie; Schlezinger, Jennifer J.; Webster, Thomas F.; Stapleton, Heather M.

    2017-01-01

    The potential for chemical exposures to exacerbate the development and/or prevalence of metabolic disorders, such as obesity, is currently of great societal concern. Various in vitro assays are available to assess adipocyte differentiation, though little work has been done to standardize protocols and compare models effectively. This study compares several adipogenic cell culture systems under a variety of conditions to assess variability in responses. Two sources of 3T3-L1 preadipocytes as well as OP9 preadipocytes were assessed for cell proliferation and triglyceride accumulation following different induction periods and using various tissue culture plates. Both cell line and cell source had a significant impact on potencies and efficacies of adipogenic chemicals. Gene expression analyses suggested that differential expression of nuclear receptors involved in adipogenesis underlie the differences between OP9 and 3T3-L1 cells; however, there were also differences based on 3T3-L1 cell source. Induction period modulated potency and efficacy of response depending on cell line and test chemical, and large variations were observed in triglyceride accumulation and cell proliferation between brands of tissue culture plates. Our results suggest that the selection of a cell system and differentiation protocol significantly impacts the detection of adipogenic chemicals, and therefore, influences reproducibility of these studies. PMID:28176856

  16. Reproducibility-optimized test statistic for ranking genes in microarray studies.

    PubMed

    Elo, Laura L; Filén, Sanna; Lahesmaa, Riitta; Aittokallio, Tero

    2008-01-01

    A principal goal of microarray studies is to identify the genes showing differential expression under distinct conditions. In such studies, the selection of an optimal test statistic is a crucial challenge, which depends on the type and amount of data under analysis. While previous studies on simulated or spike-in datasets do not provide practical guidance on how to choose the best method for a given real dataset, we introduce an enhanced reproducibility-optimization procedure, which enables the selection of a suitable gene- anking statistic directly from the data. In comparison with existing ranking methods, the reproducibilityoptimized statistic shows good performance consistently under various simulated conditions and on Affymetrix spike-in dataset. Further, the feasibility of the novel statistic is confirmed in a practical research setting using data from an in-house cDNA microarray study of asthma-related gene expression changes. These results suggest that the procedure facilitates the selection of an appropriate test statistic for a given dataset without relying on a priori assumptions, which may bias the findings and their interpretation. Moreover, the general reproducibilityoptimization procedure is not limited to detecting differential expression only but could be extended to a wide range of other applications as well.

  17. Confronting the missing epistasis problem: on the reproducibility of gene-gene interactions.

    PubMed

    Murk, William; Bracken, Michael B; DeWan, Andrew T

    2015-08-01

    Epistasis (gene-gene interaction) is thought to play an integral role in the genetic basis of complex traits, and a significant amount of research has been invested into identifying this phenomenon in human disease. However, the overall success of empirical studies of epistasis in humans is unclear, as such studies are rarely systematically evaluated. Here, we have selected asthma as an example of a well-studied, complex human disease, and provide a critical analysis and replication attempt of nearly all prior reports of epistasis for this disease. Of 191 previously reported interactions, we find that 39.8% were not originally identified using an explicit test for interaction and thus may not have been true epistatic effects to begin with. Moreover, directions of effect were not described for 46.1% of the interactions, which prevents their rigorous replication. In the original studies, attempts at replication were made for 15.2% of the interactions, and 7.3% were actually replicated. In the current study, we were able to evaluate 85.9% of the interactions using a large asthma dataset from the GABRIEL Consortium. None of these interactions could be replicated based on strict criteria. However, we found nominally significant (p < 0.05) evidence in support of 23.8% of the evaluated interactions. Although many reports of epistasis are not robustly supported in the published literature, our results suggest that at least some of these reports may have been true-positive examples of epistasis. In general, improvements in empirical studies of epistasis are called for, in order to better understand the importance of this phenomenon in human disease.

  18. Safe, efficient, and reproducible gene therapy of the brain in the dog models of Sanfilippo and Hurler syndromes.

    PubMed

    Ellinwood, N Matthew; Ausseil, Jérôme; Desmaris, Nathalie; Bigou, Stéphanie; Liu, Song; Jens, Jackie K; Snella, Elizabeth M; Mohammed, Eman E A; Thomson, Christopher B; Raoul, Sylvie; Joussemet, Béatrice; Roux, Françoise; Chérel, Yan; Lajat, Yaouen; Piraud, Monique; Benchaouir, Rachid; Hermening, Stephan; Petry, Harald; Froissart, Roseline; Tardieu, Marc; Ciron, Carine; Moullier, Philippe; Parkes, Jennifer; Kline, Karen L; Maire, Irène; Vanier, Marie-Thérèse; Heard, Jean-Michel; Colle, Marie-Anne

    2011-02-01

    Recent trials in patients with neurodegenerative diseases documented the safety of gene therapy based on adeno-associated virus (AAV) vectors deposited into the brain. Inborn errors of the metabolism are the most frequent causes of neurodegeneration in pre-adulthood. In Sanfilippo syndrome, a lysosomal storage disease in which heparan sulfate oligosaccharides accumulate, the onset of clinical manifestation is before 5 years. Studies in the mouse model showed that gene therapy providing the missing enzyme α-N-acetyl-glucosaminidase to brain cells prevents neurodegeneration and improves behavior. We now document safety and efficacy in affected dogs. Animals received eight deposits of a serotype 5 AAV vector, including vector prepared in insect Sf9 cells. As shown previously in dogs with the closely related Hurler syndrome, immunosuppression was necessary to prevent neuroinflammation and elimination of transduced cells. In immunosuppressed dogs, vector was efficiently delivered throughout the brain, induced α-N-acetyl-glucosaminidase production, cleared stored compounds and storage lesions. The suitability of the procedure for clinical application was further assessed in Hurler dogs, providing information on reproducibility, tolerance, appropriate vector type and dosage, and optimal age for treatment in a total number of 25 treated dogs. Results strongly support projects of human trials aimed at assessing this treatment in Sanfilippo syndrome.

  19. Cell and gene therapy.

    PubMed

    Rao, Rajesh C; Zacks, David N

    2014-01-01

    Replacement or repair of a dysfunctional gene combined with promoting cell survival is a two-pronged approach that addresses an unmet need in the therapy of retinal degenerative diseases. In this chapter, we discuss various strategies toward achieving both goals: transplantation of wild-type cells to replace degenerating cells and to rescue gene function, sequential gene and cell therapy, and in vivo reprogramming of rods to cones. These approaches highlight cutting-edge advances in cell and gene therapy, and cellular lineage conversion in order to devise new therapies for various retinal degenerative diseases.

  20. Reproducibility of detecting silent cerebral infarcts in pediatric sickle cell anemia.

    PubMed

    Liem, Robert I; Liu, Jingxia; Gordon, Mae O; Vendt, Bruce A; McKinstry, Robert C; Kraut, Michael A; Strouse, John J; Ball, William S; DeBaun, Michael R

    2014-12-01

    Detecting silent cerebral infarcts on magnetic resonance images (MRIs) in children with sickle cell anemia is challenging, yet reproducibility of readings has not been examined in this population. We evaluated consensus rating, inter-, and intra-grader agreement associated with detecting silent cerebral infarct on screening MRI in the Silent Infarct Transfusion Trial. Three neuroradiologists provided consensus decisions for 1073 MRIs. A random sample of 53 scans was reanalyzed in blinded fashion. Agreement between first and second consensus ratings was substantial (κ = 0.70, P < .0001), as was overall intergrader agreement (κ = 0.76, P < .0001). In the test-retest sample, intragrader agreement ranged from κ of 0.57 to 0.76. Consensus decisions were more concordant when MRIs contained more than one larger lesions. Routine use of MRI to screen for silent cerebral infarcts in the research setting is reproducible in sickle cell anemia and agreement among neuroradiologists is sufficient.

  1. Reproducible copy number variation patterns among single circulating tumor cells of lung cancer patients

    PubMed Central

    Ni, Xiaohui; Zhuo, Minglei; Su, Zhe; Duan, Jianchun; Gao, Yan; Wang, Zhijie; Zong, Chenghang; Bai, Hua; Chapman, Alec R.; Zhao, Jun; Xu, Liya; An, Tongtong; Ma, Qi; Wang, Yuyan; Wu, Meina; Sun, Yu; Wang, Shuhang; Li, Zhenxiang; Yang, Xiaodan; Yong, Jun; Su, Xiao-Dong; Lu, Youyong; Bai, Fan; Xie, X. Sunney; Wang, Jie

    2013-01-01

    Circulating tumor cells (CTCs) enter peripheral blood from primary tumors and seed metastases. The genome sequencing of CTCs could offer noninvasive prognosis or even diagnosis, but has been hampered by low single-cell genome coverage of scarce CTCs. Here, we report the use of the recently developed multiple annealing and looping-based amplification cycles for whole-genome amplification of single CTCs from lung cancer patients. We observed characteristic cancer-associated single-nucleotide variations and insertions/deletions in exomes of CTCs. These mutations provided information needed for individualized therapy, such as drug resistance and phenotypic transition, but were heterogeneous from cell to cell. In contrast, every CTC from an individual patient, regardless of the cancer subtypes, exhibited reproducible copy number variation (CNV) patterns, similar to those of the metastatic tumor of the same patient. Interestingly, different patients with the same lung cancer adenocarcinoma (ADC) shared similar CNV patterns in their CTCs. Even more interestingly, patients of small-cell lung cancer have CNV patterns distinctly different from those of ADC patients. Our finding suggests that CNVs at certain genomic loci are selected for the metastasis of cancer. The reproducibility of cancer-specific CNVs offers potential for CTC-based cancer diagnostics. PMID:24324171

  2. Improved reproducibility of unit-cell parameters in macromolecular cryocrystallography by limiting dehydration during crystal mounting.

    PubMed

    Farley, Christopher; Burks, Geoffry; Siegert, Thomas; Juers, Douglas H

    2014-08-01

    In macromolecular cryocrystallography unit-cell parameters can have low reproducibility, limiting the effectiveness of combining data sets from multiple crystals and inhibiting the development of defined repeatable cooling protocols. Here, potential sources of unit-cell variation are investigated and crystal dehydration during loop-mounting is found to be an important factor. The amount of water lost by the unit cell depends on the crystal size, the loop size, the ambient relative humidity and the transfer distance to the cooling medium. To limit water loss during crystal mounting, a threefold strategy has been implemented. Firstly, crystal manipulations are performed in a humid environment similar to the humidity of the crystal-growth or soaking solution. Secondly, the looped crystal is transferred to a vial containing a small amount of the crystal soaking solution. Upon loop transfer, the vial is sealed, which allows transport of the crystal at its equilibrated humidity. Thirdly, the crystal loop is directly mounted from the vial into the cold gas stream. This strategy minimizes the exposure of the crystal to relatively low humidity ambient air, improves the reproducibility of low-temperature unit-cell parameters and offers some new approaches to crystal handling and cryoprotection.

  3. Consecutive Morphology Controlling Operations for Highly Reproducible Mesostructured Perovskite Solar Cells.

    PubMed

    Wu, Yongzhen; Chen, Wei; Yue, Youfeng; Liu, Jian; Bi, Enbing; Yang, Xudong; Islam, Ashraful; Han, Liyuan

    2015-09-23

    Perovskite solar cells have shown high photovoltaic performance but suffer from low reproducibility, which is mainly caused by low uniformity of the active perovskite layer in the devices. The nonuniform perovskites further limit the fabrication of large size solar cells. In this work, we control the morphology of CH3NH3PbI3 on a mesoporous TiO2 substrate by employing consecutive antisolvent dripping and solvent-vapor fumigation during spin coating of the precursor solution. The solvent-vapor treatment is found to enhance the perovskite pore filling and increase the uniformity of CH3NH3PbI3 in the porous scaffold layer but slightly decrease the uniformity of the perovskite capping layer. An additional antisolvent dripping is employed to recover the uniform perovskite capping layer. Such consecutive morphology controlling operations lead to highly uniform perovskite in both porous and capping layers. By using the optimized perovskite deposition procedure, the reproducibility of mesostructured solar cells was greatly improved such that a total of 40 devices showed an average efficiency of 15.3% with a very small standard deviation of 0.32. Moreover, a high efficiency of 14.9% was achieved on a large-size cell with a working area of 1.02 cm(2).

  4. A rat tail temporary static compression model reproduces different stages of intervertebral disc degeneration with decreased notochordal cell phenotype.

    PubMed

    Hirata, Hiroaki; Yurube, Takashi; Kakutani, Kenichiro; Maeno, Koichiro; Takada, Toru; Yamamoto, Junya; Kurakawa, Takuto; Akisue, Toshihiro; Kuroda, Ryosuke; Kurosaka, Masahiro; Nishida, Kotaro

    2014-03-01

    The intervertebral disc nucleus pulposus (NP) has two phenotypically distinct cell types-notochordal cells (NCs) and non-notochordal chondrocyte-like cells. In human discs, NCs are lost during adolescence, which is also when discs begin to show degenerative signs. However, little evidence exists regarding the link between NC disappearance and the pathogenesis of disc degeneration. To clarify this, a rat tail disc degeneration model induced by static compression at 1.3 MPa for 0, 1, or 7 days was designed and assessed for up to 56 postoperative days. Radiography, MRI, and histomorphology showed degenerative disc findings in response to the compression period. Immunofluorescence displayed that the number of DAPI-positive NP cells decreased with compression; particularly, the decrease was notable in larger, vacuolated, cytokeratin-8- and galectin-3-co-positive cells, identified as NCs. The proportion of TUNEL-positive cells, which predominantly comprised non-NCs, increased with compression. Quantitative PCR demonstrated isolated mRNA up-regulation of ADAMTS-5 in the 1-day loaded group and MMP-3 in the 7-day loaded group. Aggrecan-1 and collagen type 2α-1 mRNA levels were down-regulated in both groups. This rat tail temporary static compression model, which exhibits decreased NC phenotype, increased apoptotic cell death, and imbalanced catabolic and anabolic gene expression, reproduces different stages of intervertebral disc degeneration.

  5. Distinct Antigen Delivery Systems Induce Dendritic Cells' Divergent Transcriptional Response: New Insights from a Comparative and Reproducible Computational Analysis.

    PubMed

    Costa, Valerio; Righelli, Dario; Russo, Francesco; De Berardinis, Piergiuseppe; Angelini, Claudia; D'Apice, Luciana

    2017-02-25

    Vaccination is the most successful and cost-effective method to prevent infectious diseases. However, many vaccine antigens have poor in vivo immunogenic potential and need adjuvants to enhance immune response. The application of systems biology to immunity and vaccinology has yielded crucial insights about how vaccines and adjuvants work. We have previously characterized two safe and powerful delivery systems derived from non-pathogenic prokaryotic organisms: E2 and fd filamentous bacteriophage systems. They elicit an in vivo immune response inducing CD8+ T-cell responses, even in absence of adjuvants or stimuli for dendritic cells' maturation. Nonetheless, a systematic and comparative analysis of the complex gene expression network underlying such activation is missing. Therefore, we compared the transcriptomes of ex vivo isolated bone marrow-derived dendritic cells exposed to these antigen delivery systems. Significant differences emerged, especially for genes involved in innate immunity, co-stimulation, and cytokine production. Results indicate that E2 drives polarization toward the Th2 phenotype, mainly mediated by Irf4, Ccl17, and Ccr4 over-expression. Conversely, fd-scαDEC-205 triggers Th1 T cells' polarization through the induction of Il12b, Il12rb, Il6, and other molecules involved in its signal transduction. The data analysis was performed using RNASeqGUI, hence, addressing the increasing need of transparency and reproducibility of computational analysis.

  6. Incurred sample reproducibility and stability assessment in a cell-based drug concentration assay.

    PubMed

    White, Joleen T; Crossman, Mary; Subramanyam, Meena

    2015-01-01

    Joleen White is Principal Scientist in Translational Sciences at Biogen Idec. Throughout her career, she has applied her background in biophysical protein chemistry to pharmaceutical development in therapeutic indications with significant unmet medical need. In her current role, she supports method development and regulated bioanalysis of biomarkers, biopharmaceuticals, and immunogenicity in biological samples from nonclinical and clinical studies. Her experience with measuring macromolecules includes enzymes, monoclonal antibodies, Fc fusions, oligonucleotides, PEGylated proteins, and other novel protein constructs. She has supported studies from discovery through all phases of development including GLP nonclinical, clinical, and post-marketing commitments. Incurred samplereproducibility is one aspect of in-study validation, with white papers outlining expectations for chromatographic assays and immunoassays. This manuscript outlines an approach for performing incurred sample reproducibility for a bioequivalence study using a cell-based assay, with the complication of time elapsed between original and repeat assays. The incurred sample reproducibility passed the pre-established acceptance criteria of 45% for at least 2/3 of the samples: 174/216 samples (80.6%). Data trends between the two crossover arms were qualitatively similar. The passed incurred sample reproducibility and stability further supports the validity of the original study conclusion that the two manufacturing processes were bioequivalent. This illustrates one approach to extrapolating industry and regulatory recommendations for situations outside current guidance.

  7. In vitro treatment of HepG2 cells with saturated fatty acids reproduces mitochondrial dysfunction found in nonalcoholic steatohepatitis.

    PubMed

    García-Ruiz, Inmaculada; Solís-Muñoz, Pablo; Fernández-Moreira, Daniel; Muñoz-Yagüe, Teresa; Solís-Herruzo, José A

    2015-02-01

    Activity of the oxidative phosphorylation system (OXPHOS) is decreased in humans and mice with nonalcoholic steatohepatitis. Nitro-oxidative stress seems to be involved in its pathogenesis. The aim of this study was to determine whether fatty acids are implicated in the pathogenesis of this mitochondrial defect. In HepG2 cells, we analyzed the effect of saturated (palmitic and stearic acids) and monounsaturated (oleic acid) fatty acids on: OXPHOS activity; levels of protein expression of OXPHOS complexes and their subunits; gene expression and half-life of OXPHOS complexes; nitro-oxidative stress; and NADPH oxidase gene expression and activity. We also studied the effects of inhibiting or silencing NADPH oxidase on the palmitic-acid-induced nitro-oxidative stress and subsequent OXPHOS inhibition. Exposure of cultured HepG2 cells to saturated fatty acids resulted in a significant decrease in the OXPHOS activity. This effect was prevented in the presence of a mimic of manganese superoxide dismutase. Palmitic acid reduced the amount of both fully-assembled OXPHOS complexes and of complex subunits. This reduction was due mainly to an accelerated degradation of these subunits, which was associated with a 3-tyrosine nitration of mitochondrial proteins. Pretreatment of cells with uric acid, an antiperoxynitrite agent, prevented protein degradation induced by palmitic acid. A reduced gene expression also contributed to decrease mitochondrial DNA (mtDNA)-encoded subunits. Saturated fatty acids induced oxidative stress and caused mtDNA oxidative damage. This effect was prevented by inhibiting NADPH oxidase. These acids activated NADPH oxidase gene expression and increased NADPH oxidase activity. Silencing this oxidase abrogated totally the inhibitory effect of palmitic acid on OXPHOS complex activity. We conclude that saturated fatty acids caused nitro-oxidative stress, reduced OXPHOS complex half-life and activity, and decreased gene expression of mtDNA-encoded subunits

  8. Improving the reproducibility of the MCF-7 cell proliferation assay for the detection of xenoestrogens.

    PubMed

    Payne, J; Jones, C; Lakhani, S; Kortenkamp, A

    2000-03-29

    The MCF-7 cell proliferation assay is potentially a simple and highly reproducible tool for the identification of estrogenic compounds. However, its widespread use has been complicated by the lack of a standardised protocol, resulting in considerable inter-laboratory variability. We have explored the sources of variability both in relation to cell lines and test regimens and report on optimised procedures for the identification of estrogenic agents. Two supposedly identical MCF-7 parent cell lines (designated UCL and SOP), and the BUS subline were cultured according to an existing protocol, and responses to 17-estradiol (E2) assessed. Despite yielding almost identical EC50 values, the proliferative response varied widely between cell lines from 0.98-fold over controls (UCL) to 8.9-fold (BUS) indicating major differences between them. The underlying causes may be genetic, and to assess this we used comparative genomic hybridisation (CGH), a technique which allows the detection of DNA sequence copy number changes on a genome-wide scale. Although numerous similarities existed between the different cell lines, the least oestrogen-responsive line (MCF-7/UCL) exhibited the greatest number of cytogenetic changes, many of which were not seen in MCF-7/SOP cells. We suggest that care must be taken, therefore, when choosing a cell line for MCF-7 cell-based experiments. Selecting the MCF-7/SOP line for further work, we carried out a thorough and systematic optimisation of the MCF-7 cell proliferation assay, finding that a 72-h period in oestrogen-free medium before treatment strongly influenced the cells response to E2. With 1 nM E2, proliferation increased from 1.5-fold to 6.5-fold relative to vehicle-treated controls, a response similar to that seen with MCF-7/BUS cells in the E-SCREEN protocol devised by Soto et al. With parent MCF-7 cells, other laboratories have reported only 4.5-fold increases as maximal. Here we present evidence that the choice of cell line and culture

  9. Device Engineering Towards Improved Tin Sulfide Solar Cell Performance and Performance Reproducibility

    SciTech Connect

    Steinmann, Vera; Chakraborty, Rupak; Rekemeyer, Paul; Siol, Sebastian; Martinot, Loic; Polizzotti, Alex; Yang, Chuanxi; Hartman, Katy; Gradecak, Silvija; Zakutayev, Andriy; Gordon, Roy G.; Buonassisi, Tonio

    2016-11-21

    As novel absorber materials are developed and screened for their photovoltaic (PV) properties, the challenge remains to rapidly test promising candidates in high-performing PV devices. There is a need to engineer new compatible device architectures, including the development of novel transparent conductive oxides and buffer layers. Here, we consider the two approaches of a substrate-style and a superstrate-style device architecture for novel thin-film solar cells. We use tin sulfide as a test absorber material. Upon device engineering, we demonstrate new approaches to improve device performance and performance reproducibility.

  10. Determinants, reproducibility, and seasonal variation of bacterial cell wall components and viable counts in house dust.

    PubMed

    Leppänen, H K; Täubel, M; Roponen, M; Vepsäläinen, A; Rantakokko, P; Pekkanen, J; Nevalainen, A; von Mutius, E; Hyvärinen, A

    2015-06-01

    The objectives of this study were (i) to assess the determinants that affect concentrations of the bacterial cell wall components 3-hydroxy fatty acids (3-OH FAs) and muramic acid and of total viable bacteria and actinomycetes in house dust; and (ii) to examine the seasonal variation and reproducibility of these bacterial cell wall components in house dust. A number of lifestyle and environmental factors, mostly not consistent for different bacterial measures but commonly including the type of dwelling and farming (number of livestock), explained up to 37% of the variation of the bacterial concentrations in 212 homes in Eastern Finland. The reproducibility of 3-OH FAs and muramic acid measurements in house dust were studied in five urban homes and were found to be generally high (ICC 74-84%). Temporal variation observed in repeated sampling of the same home throughout a year was more pronounced for 3-OH FAs determinations (ICC 22%) than for muramic acid (ICC 55-66%). We conclude that determinants vary largely for different types of bacterial measurements in house dust; the measured parameters represent different aspects of the bacterial content indoors. More than one sample is needed to describe bacterial concentrations in house dust in the home environment due to large temporal variation.

  11. Safe and Reproducible Preparation of Functional Dendritic Cells for Immunotherapy in Glioblastoma Patients

    PubMed Central

    Lisini, Daniela; Pogliani, Simona; Dossena, Marta; Bersano, Anna; Pellegatta, Serena; Parati, Eugenio; Finocchiaro, Gaetano; Frigerio, Simona

    2015-01-01

    Cell therapy based on dendritic cells (DCs) pulsed with tumor lysate is a promising approach in addition to conventional therapy for the treatment of patients with glioblastoma (GB). The success of this approach strongly depends on the ability to generate high-quality, functionally mature DCs (mDCs), with a high level of standardization and in compliance with Good Manufacturing Practices. In the cell factory of the Carlo Besta Foundation, two phase I clinical trials on immunotherapy with tumor lysate-loaded DCs as treatment for GB are ongoing. From 2010 to 2014, 54 patients were enrolled in the studies and 54 batches of DCs were prepared. We retrospectively analyzed the results of the quality control tests carried out on each produced batch, evaluating yield of mDCs and their quality in terms of microbiological safety and immunological efficacy. The number of mDCs obtained allowed the treatment of all the enrolled patients. All 54 batches were sterile, conformed to acceptable endotoxin levels, and were free of Mycoplasma species and adventitious viruses. During culture, cells maintained a high percentage of viability (87%–98%), and all batches showed high viability after thawing (mean ± SD: 94.6% ± 2.9%). Phenotype evaluation of mDCs showed an evident upregulation of markers typical of DC maturation; mixed lymphocyte reaction tests for the functional evaluation of DCs demonstrated that all batches were able to induce lymphocyte responses. These results demonstrated that our protocol for DC preparation is highly reproducible and permits generation of large numbers of safe and functional DCs for in vivo use in immunotherapy approaches. Significance Cell therapy based on antigen-pulsed dendritic cells (DCs) is a promising approach for the treatment of glioblastoma patients. The success of this approach strongly depends on the ability to generate high-quality, functional DCs with a high level of standardization, ensuring reproducibility, efficacy, and safety of the

  12. High-throughput miniaturized bioreactors for cell culture process development: reproducibility, scalability, and control.

    PubMed

    Rameez, Shahid; Mostafa, Sigma S; Miller, Christopher; Shukla, Abhinav A

    2014-01-01

    Decreasing the timeframe for cell culture process development has been a key goal toward accelerating biopharmaceutical development. Advanced Microscale Bioreactors (ambr™) is an automated micro-bioreactor system with miniature single-use bioreactors with a 10-15 mL working volume controlled by an automated workstation. This system was compared to conventional bioreactor systems in terms of its performance for the production of a monoclonal antibody in a recombinant Chinese Hamster Ovary cell line. The miniaturized bioreactor system was found to produce cell culture profiles that matched across scales to 3 L, 15 L, and 200 L stirred tank bioreactors. The processes used in this article involve complex feed formulations, perturbations, and strict process control within the design space, which are in-line with processes used for commercial scale manufacturing of biopharmaceuticals. Changes to important process parameters in ambr™ resulted in predictable cell growth, viability and titer changes, which were in good agreement to data from the conventional larger scale bioreactors. ambr™ was found to successfully reproduce variations in temperature, dissolved oxygen (DO), and pH conditions similar to the larger bioreactor systems. Additionally, the miniature bioreactors were found to react well to perturbations in pH and DO through adjustments to the Proportional and Integral control loop. The data presented here demonstrates the utility of the ambr™ system as a high throughput system for cell culture process development.

  13. A Stable and Reproducible Human Blood-Brain Barrier Model Derived from Hematopoietic Stem Cells

    PubMed Central

    Sevin, Emmanuel; Almeida, Catarina; Culot, Maxime; Dehouck, Lucie; Coisne, Caroline; Engelhardt, Britta; Dehouck, Marie-Pierre; Ferreira, Lino

    2014-01-01

    The human blood brain barrier (BBB) is a selective barrier formed by human brain endothelial cells (hBECs), which is important to ensure adequate neuronal function and protect the central nervous system (CNS) from disease. The development of human in vitro BBB models is thus of utmost importance for drug discovery programs related to CNS diseases. Here, we describe a method to generate a human BBB model using cord blood-derived hematopoietic stem cells. The cells were initially differentiated into ECs followed by the induction of BBB properties by co-culture with pericytes. The brain-like endothelial cells (BLECs) express tight junctions and transporters typically observed in brain endothelium and maintain expression of most in vivo BBB properties for at least 20 days. The model is very reproducible since it can be generated from stem cells isolated from different donors and in different laboratories, and could be used to predict CNS distribution of compounds in human. Finally, we provide evidence that Wnt/β-catenin signaling pathway mediates in part the BBB inductive properties of pericytes. PMID:24936790

  14. Reproducibility of Detecting Silent Cerebral Infarcts in Pediatric Sickle Cell Anemia

    PubMed Central

    Liem, Robert I.; Liu, Jingxia; Gordon, Mae O.; Vendt, Bruce A.; McKinstry, Robert C.; Kraut, Michael A.; Strouse, John J.; Ball, William S.; DeBaun, Michael R.

    2014-01-01

    Detecting silent cerebral infarcts (SCI) on MRI in children with sickle cell anemia (SCA) is challenging, yet reproducibility of readings has not been examined in this population. We evaluated consensus rating, inter- and intra-grader agreement associated with detecting SCI on screening MRI in the Silent Infarct Transfusion (SIT) Trial. Three neuroradiologists provided consensus decisions for 1,073 MRIs. A random sample of 53 scans was re-analyzed in blinded fashion. Agreement between first and second consensus ratings was substantial (κ = 0.70, p < 0.0001), as was overall inter-grader agreement (κ = 0.76, p < 0.0001). In the test-retest sample, intra-grader agreement ranged from κ of 0.57 to 0.76. Consensus decisions were more concordant when MRIs contained more than one lesion and lesions were larger. We conclude that the routine use of MRI to screen for SCI in the research setting is reproducible in SCA and agreement among neuroradiologists is sufficient. PMID:24309240

  15. Fabrication of highly reproducible polymer solar cells using ultrasonic substrate vibration posttreatment

    NASA Astrophysics Data System (ADS)

    Xie, Yu; Zabihi, Fatemeh; Eslamian, Morteza

    2016-10-01

    Organic solar cells are usually nonreproducible due to the presence of defects in the structure of their constituting thin films. To minimize the density of pinholes and defects in PEDOT:PSS, which is the hole transporting layer of a standard polymer solar cell, i.e., glass/ITO/PEDOT:PSS/P3HT:PCBM/Al, and to reduce scattering in device performance, wet spun-on PEDOT:PSS films are subjected to imposed ultrasonic substrate vibration posttreatment (SVPT). The imposed vibration improves the mixing and homogeneity of the wet spun-on films, and consequently the nanostructure of the ensuing thin solid films. For instance, our results show that by using the SVPT, which is a mechanical, single-step and low-cost process, the average power conversion efficiency of 14 identical cells increases by 25% and the standard deviation decreases by 22% indicating that the device photovoltaic performance becomes more consistent and significantly improved. This eliminates several tedious and expensive chemical and thermal treatments currently performed to improve the cell reproducibility.

  16. In Vitro Adult Astrocytes Are Derived from Mature Cells and Reproduce In Vivo Redox Profile.

    PubMed

    Souza, Débora Guerini; Bellaver, Bruna; Terra, Silvia Resende; Guma, Fatima Costa Rodrigues; Souza, Diogo Onofre; Quincozes-Santos, André

    2017-04-04

    Astrocytes are versatile cells involved in synaptic information processing, energy metabolism, redox homeostasis, inflammatory response and structural support of the brain. Recently, we established a routine protocol of cultured astrocytes derived from adult and aged Wistar rats, which present several different responses compared to newborn astrocytes, commonly used to characterize the role of the astrocytes in the central nervous system. Previous studies hypothesized that astrocyte cultures prepared from adult animals derive from immature precursors present in the adult tissue throughout life. Since our group has already demonstrated that the glial functionality of adult astrocytes differs from newborn cultures, the aim of this study was to confirm that our in vitro astrocytes were derived from mature cells. Therefore, we evaluated cytoskeleton proteins, such as glial fibrillary acidic protein and vimentin, as well as Sox10, an essential marker of immature glial cells, in ex vivo tissue and in in vitro astrocytes from the same animals (1, 90 and 180 days old). In addition, we examined the mitochondrial functionality and the cellular redox homeostasis. Our results suggest that adult and aged astrocytes are derived from mature cells and that changes in mitochondrial parameters in ex vivo tissue were reproduced in in vitro astrocytes. This article is protected by copyright. All rights reserved.

  17. Reproducibility blues.

    PubMed

    Pulverer, Bernd

    2015-11-12

    Research findings advance science only if they are significant, reliable and reproducible. Scientists and journals must publish robust data in a way that renders it optimally reproducible. Reproducibility has to be incentivized and supported by the research infrastructure but without dampening innovation.

  18. AGA: Interactive pipeline for reproducible gene expression and DNA methylation data analyses.

    PubMed

    Considine, Michael; Parker, Hilary; Wei, Yingying; Xia, Xaio; Cope, Leslie; Ochs, Michael; Fertig, Elana

    2015-01-01

    Automated Genomics Analysis (AGA) is an interactive program to analyze high-throughput genomic data sets on a variety of platforms. An easy to use, point and click, guided pipeline is implemented to combine, define, and compare datasets, and customize their outputs. In contrast to other automated programs, AGA enables flexible selection of sample groups for comparison from complex sample annotations. Batch correction techniques are also included to further enable the combination of datasets from diverse studies in this comparison. AGA also allows users to save plots, tables and data, and log files containing key portions of the R script run for reproducible analyses. The link between the interface and R supports collaborative research, enabling advanced R users to extend preliminary analyses generated from bioinformatics novices.

  19. AGA: Interactive pipeline for reproducible gene expression and DNA methylation data analyses

    PubMed Central

    Considine, Michael; Parker, Hilary; Wei, Yingying; Xia, Xaio; Cope, Leslie; Ochs, Michael; Fertig, Elana

    2015-01-01

    Automated Genomics Analysis (AGA) is an interactive program to analyze high-throughput genomic data sets on a variety of platforms. An easy to use, point and click, guided pipeline is implemented to combine, define, and compare datasets, and customize their outputs. In contrast to other automated programs, AGA enables flexible selection of sample groups for comparison from complex sample annotations. Batch correction techniques are also included to further enable the combination of datasets from diverse studies in this comparison. AGA also allows users to save plots, tables and data, and log files containing key portions of the R script run for reproducible analyses. The link between the interface and R supports collaborative research, enabling advanced R users to extend preliminary analyses generated from bioinformatics novices. PMID:26535111

  20. Commonly dysregulated genes in murine APL cells

    PubMed Central

    Yuan, Wenlin; Payton, Jacqueline E.; Holt, Matthew S.; Link, Daniel C.; Watson, Mark A.; DiPersio, John F.; Ley, Timothy J.

    2007-01-01

    To identify genes that are commonly dysregulated in a murine model of acute promyelocytic leukemia (APL), we first defined gene expression patterns during normal murine myeloid development; serial gene expression profiling studies were performed with primary murine hematopoietic progenitors that were induced to undergo myeloid maturation in vitro with G-CSF. Many genes were reproducibly expressed in restricted developmental “windows,” suggesting a structured hierarchy of expression that is relevant for the induction of developmental fates and/or differentiated cell functions. We compared the normal myeloid developmental transcriptome with that of APL cells derived from mice expressing PML-RARα under control of the murine cathepsin G locus. While many promyelocyte-specific genes were highly expressed in all APL samples, 116 genes were reproducibly dysregulated in many independent APL samples, including Fos, Jun, Egr1, Tnf, and Vcam1. However, this set of commonly dysregulated genes was expressed normally in preleukemic, early myeloid cells from the same mouse model, suggesting that dysregulation occurs as a “downstream” event during disease progression. These studies suggest that the genetic events that lead to APL progression may converge on common pathways that are important for leukemia pathogenesis. PMID:17008535

  1. Generation of a murine hepatic angiosarcoma cell line and reproducible mouse tumor model.

    PubMed

    Rothweiler, Sonja; Dill, Michael T; Terracciano, Luigi; Makowska, Zuzanna; Quagliata, Luca; Hlushchuk, Ruslan; Djonov, Valentin; Heim, Markus H; Semela, David

    2015-03-01

    Hepatic angiosarcoma (AS) is a rare and highly aggressive tumor of endothelial origin with dismal prognosis. Studies of the molecular biology of AS and treatment options are limited as animal models are rare. We have previously shown that inducible knockout of Notch1 in mice leads to spontaneous formation of hepatic AS. The aims of this study were to: (1) establish and characterize a cell line derived from this murine AS, (2) identify molecular pathways involved in the pathogenesis and potential therapeutic targets, and (3) generate a tumor transplantation model. AS cells retained specific endothelial properties such as tube formation activity, as well as expression of CD31 and Von Willebrand factor. However, electron microscopy analysis revealed signs of dedifferentiation with loss of fenestrae and loss of contact inhibition. Microarray and pathway analysis showed substantial changes in gene expression and revealed activation of the Myc pathway. Exposing the AS cells to sorafenib reduced migration, filopodia dynamics, and cell proliferation but did not induce apoptosis. In addition, sorafenib suppressed ERK phosphorylation and expression of cyclin D2. Injection of AS cells into NOD/SCID mice resulted in formation of undifferentiated tumors, confirming the tumorigenic potential of these cells. In summary, we established and characterized a murine model of spontaneous AS formation and hepatic AS cell lines as a useful in vitro tool. Our data demonstrate antitumor activity of sorafenib in AS cells with potent inhibition of migration, filopodia formation, and cell proliferation, supporting further evaluation of sorafenib as a novel treatment strategy. In addition, AS cell transplantation provides a subcutaneous tumor model useful for in vivo preclinical drug testing.

  2. Human genome-specific real-time PCR method for sensitive detection and reproducible quantitation of human cells in mice.

    PubMed

    Song, Pengyue; Xie, Zhenhua; Guo, Ling; Wang, Chengmei; Xie, Weidong; Wu, Yaojiong

    2012-12-01

    Xenotransplantation of human cells into immunodeficiency mice has been frequently used to study stem cells in tissue repair and regeneration and cancer cell metastasis. However, a sensitive and reproducible method to quantify cell engraftment lacks. Here, we developed a Real-Time PCR-based method which facilitated consistent detection and quantification of small amounts of human cells distributed in mouse organs after infusion. The principle of the method was to directly detect a humans-specific sequence in the human-murine genomic DNA mixture. In a mouse myocardial infarction model, the Real-Time PCR-based method consistently determined the amounts of human mesenchymal stem cells (hMSCs) engrafted into the heart and other organs 7 days after infusion of as little as 2.5 × 10(5) cells, indicating a high sensitivity, and the amounts of hMSCs detected in mice highly correlated to the numbers of hMSCs transplanted. Importantly, different from previous PCR-based methods, our method produced highly consistent and reproducible results. The reliability of the method was further proven by parallel analyses of DiI-labeled hMSCs in tissue sections and in single cell suspensions of mice. Our data show that the present human genomic DNA-specific primers-based Real-Time PCR method is sensitive and highly reproducible in determining the amount of xenotransplanted human cells in murine tissues.

  3. Dynamic Contrast-enhanced MR Imaging in Renal Cell Carcinoma: Reproducibility of Histogram Analysis on Pharmacokinetic Parameters

    PubMed Central

    Wang, Hai-yi; Su, Zi-hua; Xu, Xiao; Sun, Zhi-peng; Duan, Fei-xue; Song, Yuan-yuan; Li, Lu; Wang, Ying-wei; Ma, Xin; Guo, Ai-tao; Ma, Lin; Ye, Hui-yi

    2016-01-01

    Pharmacokinetic parameters derived from dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) have been increasingly used to evaluate the permeability of tumor vessel. Histogram metrics are a recognized promising method of quantitative MR imaging that has been recently introduced in analysis of DCE-MRI pharmacokinetic parameters in oncology due to tumor heterogeneity. In this study, 21 patients with renal cell carcinoma (RCC) underwent paired DCE-MRI studies on a 3.0 T MR system. Extended Tofts model and population-based arterial input function were used to calculate kinetic parameters of RCC tumors. Mean value and histogram metrics (Mode, Skewness and Kurtosis) of each pharmacokinetic parameter were generated automatically using ImageJ software. Intra- and inter-observer reproducibility and scan–rescan reproducibility were evaluated using intra-class correlation coefficients (ICCs) and coefficient of variation (CoV). Our results demonstrated that the histogram method (Mode, Skewness and Kurtosis) was not superior to the conventional Mean value method in reproducibility evaluation on DCE-MRI pharmacokinetic parameters (K trans & Ve) in renal cell carcinoma, especially for Skewness and Kurtosis which showed lower intra-, inter-observer and scan-rescan reproducibility than Mean value. Our findings suggest that additional studies are necessary before wide incorporation of histogram metrics in quantitative analysis of DCE-MRI pharmacokinetic parameters. PMID:27380733

  4. Molecular Interactions of the Min Protein System Reproduce Spatiotemporal Patterning in Growing and Dividing Escherichia coli Cells

    PubMed Central

    Walsh, James C.; Angstmann, Christopher N.; Duggin, Iain G.; Curmi, Paul M. G.

    2015-01-01

    Oscillations of the Min protein system are involved in the correct midcell placement of the divisome during Escherichia coli cell division. Based on molecular interactions of the Min system, we formulated a mathematical model that reproduces Min patterning during cell growth and division. Specifically, the increase in the residence time of MinD attached to the membrane as its own concentration increases, is accounted for by dimerisation of membrane-bound MinD and its interaction with MinE. Simulation of this system generates unparalleled correlation between the waveshape of experimental and theoretical MinD distributions, suggesting that the dominant interactions of the physical system have been successfully incorporated into the model. For cells where MinD is fully-labelled with GFP, the model reproduces the stationary localization of MinD-GFP for short cells, followed by oscillations from pole to pole in larger cells, and the transition to the symmetric distribution during cell filamentation. Cells containing a secondary, GFP-labelled MinD display a contrasting pattern. The model is able to account for these differences, including temporary midcell localization just prior to division, by increasing the rate constant controlling MinD ATPase and heterotetramer dissociation. For both experimental conditions, the model can explain how cell division results in an equal distribution of MinD and MinE in the two daughter cells, and accounts for the temperature dependence of the period of Min oscillations. Thus, we show that while other interactions may be present, they are not needed to reproduce the main characteristics of the Min system in vivo. PMID:26018614

  5. Neurons Derived from Induced Pluripotent Stem Cells of Patients with Down Syndrome Reproduce Early Stages of Alzheimer's Disease Type Pathology in vitro.

    PubMed

    Dashinimaev, Erdem B; Artyuhov, Alexander S; Bolshakov, Alexey P; Vorotelyak, Ekaterina A; Vasiliev, Andrey V

    2017-01-01

    People with Down syndrome (DS) are at high risk of developing pathology similar to Alzheimer's disease (AD). Modeling of this pathology in vitro may be useful for studying this phenomenon. In this study, we analyzed three different cultures of neural cells carrying trisomy of chromosome 21, which were generated by directed differentiation from induced pluripotent stem cells (iPS cells). We report here that in vitro generated DS neural cells have abnormal metabolism of amyloid-β (Aβ) manifested by increased secretion and accumulation of Aβ granules of Aβ42 pathological isoform with upregulated expression of the APP gene. Additionally, we found increased expression levels of genes that are considered to be associated with AD (BACE2, RCAN1, ETS2, TMED10), as compared to healthy controls. Thus, the neural cells generated from induced pluripotent stem cells with DS reproduce initial cellular signs of AD-type pathology and can be useful tools for modeling and studying this variant of AD in vitro.

  6. A multiple-selenization process for enhanced reproducibility of Cu2ZnSn(S,Se)4 solar cells

    NASA Astrophysics Data System (ADS)

    Neuwirth, Markus; Zhou, Huijuan; Schnabel, Thomas; Ahlswede, Erik; Kalt, Heinz; Hetterich, Michael

    2016-12-01

    A multiple-selenization process for wet-chemically fabricated kesterite-type Cu2ZnSn(S,Se)4 solar cells is reported that significantly improves the overall sample quality of the absorber layer and especially the reproducibility of device characteristics. Conversion efficiencies of up to 7.2% are obtained. With this method, the absorber forms a very compact, hole- and crack-free layer and avoids the formation of multilayer or trilayer structures. Mainly, the series resistance and therefore the short-circuit current can be stabilized, which leads to lower fluctuation of the energy conversion efficiency of the solar cells on the same sample.

  7. A Reproducible Method for Isolation and In Vitro Culture of Functional Human Lymphoid Stromal Cells from Tonsils

    PubMed Central

    Bar-Ephraim, Yotam E.; Konijn, Tanja; Gönültas, Mehmet; Mebius, Reina E.

    2016-01-01

    The stromal compartment of secondary lymphoid organs is classicaly known for providing a mechanical scaffold for the complex interactions between hematopoietic cells during immune activation as well as for providing a niche which is favorable for survival of lymphocytes. In recent years, it became increasingly clear that these cells also play an active role during such a response. Currently, knowledge of the interactions between human lymphoid stroma and hematopoietic cells is still lacking and most insight is based on murine systems. Although methods to isolate stromal cells from tonsils have been reported, data on stability in culture, characterization, and functional properties are lacking. Here, we describe a reproducible and easy method for isolation and in vitro culture of functional human lymphoid stromal cells from palatine tonsils. The cells isolated express markers and characteristics of T cell zone fibroblastic reticular cells (FRCs) and react to inflammatory stimuli by upregulating inflammatory cytokines and chemokines as well as adhesion molecules, as previously described for mouse lymphoid stroma. Also, cultured tonsil stromal cells support survival of human innate lymphoid cells, showing that these stromal cells can function as bone fide FRCs, providing a favorable microenvironment for hematopoietic cells. PMID:27907202

  8. Modeling stochastic cell dynamics with adhesion anisotropy quantitatively reproduces convergent extension

    NASA Astrophysics Data System (ADS)

    Firman, Taylor; Ghosh, Kingshuk; Blankenship, J. Todd; Loerke, Dinah

    2014-03-01

    Epithelial cells in Drosophila embryos intercalate together during germ-band extension in order to elongate the entire embryo along the anterior-posterior axis, a process more broadly known as convergent extension. In silico simulation of hexagonal cell matrices provides an inexpensive way to test the validity of possible mechanisms governing convergent extension of epithelial tissues. Our proposed system is node-based as opposed to pixel-based, storing data only for the node points defining the idealized polygons representing individual cells. This brings simulation times down from days to hours. Using Monte Carlo simulation techniques, the energy function used takes into account cell volume and membrane conservation as well as adhesion between surrounding cells. Our model takes a passive adhesion approach by assuming planar polarized distributions of adhesiveness within the cell. This leads to convergent extension using only Brownian motion. This adhesion-based model also allows us to add in a level of heterogeneity, where cell polarizations don't align perfectly along the dorsal-ventral axis due to a mistake in cellular machinery. This results in longer monopolar adhesions along interfaces, leading to slower interface contraction and complex cell behaviors.

  9. Prognostic Value and Reproducibility of Pretreatment CT Texture Features in Stage III Non-Small Cell Lung Cancer

    SciTech Connect

    Fried, David V.; Tucker, Susan L.; Zhou, Shouhao; Liao, Zhongxing; Mawlawi, Osama; Ibbott, Geoffrey; Court, Laurence E.

    2014-11-15

    Purpose: To determine whether pretreatment CT texture features can improve patient risk stratification beyond conventional prognostic factors (CPFs) in stage III non-small cell lung cancer (NSCLC). Methods and Materials: We retrospectively reviewed 91 cases with stage III NSCLC treated with definitive chemoradiation therapy. All patients underwent pretreatment diagnostic contrast enhanced computed tomography (CE-CT) followed by 4-dimensional CT (4D-CT) for treatment simulation. We used the average-CT and expiratory (T50-CT) images from the 4D-CT along with the CE-CT for texture extraction. Histogram, gradient, co-occurrence, gray tone difference, and filtration-based techniques were used for texture feature extraction. Penalized Cox regression implementing cross-validation was used for covariate selection and modeling. Models incorporating texture features from the 33 image types and CPFs were compared to those with models incorporating CPFs alone for overall survival (OS), local-regional control (LRC), and freedom from distant metastases (FFDM). Predictive Kaplan-Meier curves were generated using leave-one-out cross-validation. Patients were stratified based on whether their predicted outcome was above or below the median. Reproducibility of texture features was evaluated using test-retest scans from independent patients and quantified using concordance correlation coefficients (CCC). We compared models incorporating the reproducibility seen on test-retest scans to our original models and determined the classification reproducibility. Results: Models incorporating both texture features and CPFs demonstrated a significant improvement in risk stratification compared to models using CPFs alone for OS (P=.046), LRC (P=.01), and FFDM (P=.005). The average CCCs were 0.89, 0.91, and 0.67 for texture features extracted from the average-CT, T50-CT, and CE-CT, respectively. Incorporating reproducibility within our models yielded 80.4% (±3.7% SD), 78.3% (±4.0% SD), and 78

  10. Instrumental improvements and sample preparations that enable reproducible, reliable acquisition of mass spectra from whole bacterial cells

    PubMed Central

    Alusta, Pierre; Buzatu, Dan; Williams, Anna; Cooper, Willie-Mae; Tarasenko, Olga; Dorey, R Cameron; Hall, Reggie; Parker, W Ryan; Wilkes, Jon G

    2015-01-01

    Rationale Rapid sub-species characterization of pathogens is required for timely responses in outbreak situations. Pyrolysis mass spectrometry (PyMS) has the potential to be used for this purpose. Methods However, in order to make PyMS practical for traceback applications, certain improvements related to spectrum reproducibility and data acquisition speed were required. The main objectives of this study were to facilitate fast detection (<30 min to analyze 6 samples, including preparation) and sub-species-level bacterial characterization based on pattern recognition of mass spectral fingerprints acquired from whole cells volatilized and ionized at atmospheric pressure. An AccuTOF DART mass spectrometer was re-engineered to permit ionization of low-volatility bacteria by means of Plasma Jet Ionization (PJI), in which an electric discharge, and, by extension, a plasma beam, impinges on sample cells. Results Instrumental improvements and spectral acquisition methodology are described. Performance of the re-engineered system was assessed using a small challenge set comprised of assorted bacterial isolates differing in identity by varying amounts. In general, the spectral patterns obtained allowed differentiation of all samples tested, including those of the same genus and species but different serotypes. Conclusions Fluctuations of ±15% in bacterial cell concentrations did not substantially compromise replicate spectra reproducibility. © 2015 National Center for Toxicological Research. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd. PMID:26443394

  11. Continuous Flow Polymer Synthesis toward Reproducible Large-Scale Production for Efficient Bulk Heterojunction Organic Solar Cells.

    PubMed

    Pirotte, Geert; Kesters, Jurgen; Verstappen, Pieter; Govaerts, Sanne; Manca, Jean; Lutsen, Laurence; Vanderzande, Dirk; Maes, Wouter

    2015-10-12

    Organic photovoltaics (OPV) have attracted great interest as a solar cell technology with appealing mechanical, aesthetical, and economies-of-scale features. To drive OPV toward economic viability, low-cost, large-scale module production has to be realized in combination with increased top-quality material availability and minimal batch-to-batch variation. To this extent, continuous flow chemistry can serve as a powerful tool. In this contribution, a flow protocol is optimized for the high performance benzodithiophene-thienopyrroledione copolymer PBDTTPD and the material quality is probed through systematic solar-cell evaluation. A stepwise approach is adopted to turn the batch process into a reproducible and scalable continuous flow procedure. Solar cell devices fabricated using the obtained polymer batches deliver an average power conversion efficiency of 7.2 %. Upon incorporation of an ionic polythiophene-based cathodic interlayer, the photovoltaic performance could be enhanced to a maximum efficiency of 9.1 %.

  12. Kidney specific protein-positive cells derived from embryonic stem cells reproduce tubular structures in vitro and differentiate into renal tubular cells.

    PubMed

    Morizane, Ryuji; Monkawa, Toshiaki; Fujii, Shizuka; Yamaguchi, Shintaro; Homma, Koichiro; Matsuzaki, Yumi; Okano, Hideyuki; Itoh, Hiroshi

    2014-01-01

    Embryonic stem cells and induced pluripotent stem cells have the ability to differentiate into various organs and tissues, and are regarded as new tools for the elucidation of disease mechanisms as well as sources for regenerative therapies. However, a method of inducing organ-specific cells from pluripotent stem cells is urgently needed. Although many scientists have been developing methods to induce various organ-specific cells from pluripotent stem cells, renal lineage cells have yet to be induced in vitro because of the complexity of kidney structures and the diversity of kidney-component cells. Here, we describe a method of inducing renal tubular cells from mouse embryonic stem cells via the cell purification of kidney specific protein (KSP)-positive cells using an anti-KSP antibody. The global gene expression profiles of KSP-positive cells derived from ES cells exhibited characteristics similar to those of cells in the developing kidney, and KSP-positive cells had the capacity to form tubular structures resembling renal tubular cells when grown in a 3D culture in Matrigel. Moreover, our results indicated that KSP-positive cells acquired the characteristics of each segment of renal tubular cells through tubular formation when stimulated with Wnt4. This method is an important step toward kidney disease research using pluripotent stem cells, and the development of kidney regeneration therapies.

  13. Improvement of Charge Collection and Performance Reproducibility in Inverted Organic Solar Cells by Suppression of ZnO Subgap States.

    PubMed

    Wu, Bo; Wu, Zhenghui; Yang, Qingyi; Zhu, Furong; Ng, Tsz-Wai; Lee, Chun-Sing; Cheung, Sin-Hang; So, Shu-Kong

    2016-06-15

    Organic solar cells (OSCs) with inverted structure usually exhibit higher power conversion efficiency (PCE) and are more stable than corresponding devices with regular configuration. Indium tin oxide (ITO) surface is often modified with solution-processed low work function metal oxides, such as ZnO, serving as the transparent cathode. However, the defect-induced subgap states in the ZnO interlayer hamper the efficient charge collection and the performance reproducibility of the OSCs. In this work, we demonstrate that suppression of the ZnO subgap states by modification of its surface with an ultrathin Al layer significantly improves the charge extraction and performance reproducibility, achieving PCE of 8.0%, which is ∼15% higher than that of a structurally identical control cell made with a pristine ZnO interlayer. Light intensity-dependent current density-voltage characteristic, photothermal deflection spectroscopy, and X-ray photoelectron spectroscopy measurements point out the enhancement of charge collection efficiency at the organic/cathode interface, due to the suppression of the subgap states in the ZnO interlayer.

  14. Cell assemblies for reproducible multi-anvil experiments (the COMPRES assemblies)

    SciTech Connect

    Leinenweber, Kurt D.; Tyburczy, James A.; Sharp, Thomas G.; Soignard, Emmanuel; Diedrich, Tamara; Petuskey, William B.; Wang, Yanbin; Mosenfelder, Jed L.

    2012-01-31

    The multi-anvil high-pressure technique is an important tool in high-pressure mineralogy and petrology, as well as in chemical synthesis, allowing the treatment of large (millimeter-size) samples of minerals, rocks, and other materials at pressures of a few GPa to over 25 GPa and simultaneous uniform temperatures up to 2500 C and higher. A series of cell assemblies specially designed and implemented for interlaboratory use are described here. In terms of the size of the pressure medium and the anvil truncation size, the five sizes of assemblies developed here are an 8/3, 10/5, 14/8, 18/12, and 25/15 assembly. As of this writing, these assemblies are in widespread use at many laboratories. The details of design, construction, and materials developed or used for the assemblies are presented here.

  15. Stem cell directed gene therapy.

    PubMed

    Engel, B C; Kohn, D B

    1999-05-01

    A potential therapeutic approach to HIV-1 infection is the genetic modification of cells of a patient to make them resistant to HIV-1. Hematopoietic stem cells are an attractive target for gene therapy of AIDS because of their ability to generate a broad repertoire of mature T lymphocytes, as well as the monocytic cells (macrophages, dendritic cells and microglia) which are also involved in HIV-1 pathogenesis. A number of synthetic "anti-HIV-1 genes" have been developed which inhibit HIV-1 replication. However, current methods for gene transfer into human hematopoietic stem cells, using retroviral vectors derived from the Moloney murine leukemia virus, have been minimally effective. Clinical trials performed to date in which hematopoietic cells from HIV-1-positive patients have been transduced with retroviral vectors and then reinfused have produced low to undetectable levels of gene-containing peripheral blood leukocytes. New vector delivery systems, such as lentiviral vectors, need to be developed to ensure efficient gene transfer and persistent transgene expression to provide life-long resistance to the cells targeted by HIV-1.

  16. Effects of Thermochemical Treatment on CuSbS 2 Photovoltaic Absorber Quality and Solar Cell Reproducibility

    SciTech Connect

    de Souza Lucas, Francisco Willian; Welch, Adam W.; Baranowski, Lauryn L.; Dippo, Patricia C.; Hempel, Hannes; Unold, Thomas; Eichberger, Rainer; Blank, Beatrix; Rau, Uwe; Mascaro, Lucia H.; Zakutayev, Andriy

    2016-08-25

    CuSbS2 is a promising nontoxic and earth-abundant photovoltaic absorber that is chemically simpler than the widely studied Cu2ZnSnS4. However, CuSbS2 photovoltaic (PV) devices currently have relatively low efficiency and poor reproducibility, often due to suboptimal material quality and insufficient optoelectronic properties. To address these issues, here we develop a thermochemical treatment (TT) for CuSbS2 thin films, which consists of annealing in Sb2S3 vapor followed by a selective KOH surface chemical etch. The annealed CuSbS2 films show improved structural quality and optoelectronic properties, such as stronger band-edge photoluminescence and longer photoexcited carrier lifetime. These improvements also lead to more reproducible CuSbS2 PV devices, with performance currently limited by a large cliff-type interface band offset with CdS contact. Overall, these results point to the potential avenues to further increase the performance of CuSbS2 thin film solar cell, and the findings can be transferred to other thin film photovoltaic technologies.

  17. Can radiomics features be reproducibly measured from CBCT images for patients with non-small cell lung cancer?

    SciTech Connect

    Fave, Xenia Fried, David; Mackin, Dennis; Yang, Jinzhong; Zhang, Joy; Balter, Peter; Followill, David; Gomez, Daniel; Kyle Jones, A.; Stingo, Francesco; Fontenot, Jonas; Court, Laurence

    2015-12-15

    Purpose: Increasing evidence suggests radiomics features extracted from computed tomography (CT) images may be useful in prognostic models for patients with nonsmall cell lung cancer (NSCLC). This study was designed to determine whether such features can be reproducibly obtained from cone-beam CT (CBCT) images taken using medical Linac onboard-imaging systems in order to track them through treatment. Methods: Test-retest CBCT images of ten patients previously enrolled in a clinical trial were retrospectively obtained and used to determine the concordance correlation coefficient (CCC) for 68 different texture features. The volume dependence of each feature was also measured using the Spearman rank correlation coefficient. Features with a high reproducibility (CCC > 0.9) that were not due to volume dependence in the patient test-retest set were further examined for their sensitivity to differences in imaging protocol, level of scatter, and amount of motion by using two phantoms. The first phantom was a texture phantom composed of rectangular cartridges to represent different textures. Features were measured from two cartridges, shredded rubber and dense cork, in this study. The texture phantom was scanned with 19 different CBCT imagers to establish the features’ interscanner variability. The effect of scatter on these features was studied by surrounding the same texture phantom with scattering material (rice and solid water). The effect of respiratory motion on these features was studied using a dynamic-motion thoracic phantom and a specially designed tumor texture insert of the shredded rubber material. The differences between scans acquired with different Linacs and protocols, varying amounts of scatter, and with different levels of motion were compared to the mean intrapatient difference from the test-retest image set. Results: Of the original 68 features, 37 had a CCC >0.9 that was not due to volume dependence. When the Linac manufacturer and imaging protocol

  18. Can radiomics features be reproducibly measured from CBCT images for patients with non-small cell lung cancer?

    PubMed Central

    Fave, Xenia; Mackin, Dennis; Zhang, Joy; Fried, David; Balter, Peter; Followill, David; Gomez, Daniel; Kyle Jones, A.; Stingo, Francesco; Fontenot, Jonas; Court, Laurence

    2015-01-01

    Purpose: Increasing evidence suggests radiomics features extracted from computed tomography (CT) images may be useful in prognostic models for patients with nonsmall cell lung cancer (NSCLC). This study was designed to determine whether such features can be reproducibly obtained from cone-beam CT (CBCT) images taken using medical Linac onboard-imaging systems in order to track them through treatment. Methods: Test-retest CBCT images of ten patients previously enrolled in a clinical trial were retrospectively obtained and used to determine the concordance correlation coefficient (CCC) for 68 different texture features. The volume dependence of each feature was also measured using the Spearman rank correlation coefficient. Features with a high reproducibility (CCC > 0.9) that were not due to volume dependence in the patient test-retest set were further examined for their sensitivity to differences in imaging protocol, level of scatter, and amount of motion by using two phantoms. The first phantom was a texture phantom composed of rectangular cartridges to represent different textures. Features were measured from two cartridges, shredded rubber and dense cork, in this study. The texture phantom was scanned with 19 different CBCT imagers to establish the features’ interscanner variability. The effect of scatter on these features was studied by surrounding the same texture phantom with scattering material (rice and solid water). The effect of respiratory motion on these features was studied using a dynamic-motion thoracic phantom and a specially designed tumor texture insert of the shredded rubber material. The differences between scans acquired with different Linacs and protocols, varying amounts of scatter, and with different levels of motion were compared to the mean intrapatient difference from the test-retest image set. Results: Of the original 68 features, 37 had a CCC >0.9 that was not due to volume dependence. When the Linac manufacturer and imaging protocol

  19. Minimum information about tolerogenic antigen-presenting cells (MITAP): a first step towards reproducibility and standardisation of cellular therapies.

    PubMed

    Lord, Phillip; Spiering, Rachel; Aguillon, Juan C; Anderson, Amy E; Appel, Silke; Benitez-Ribas, Daniel; Ten Brinke, Anja; Broere, Femke; Cools, Nathalie; Cuturi, Maria Cristina; Diboll, Julie; Geissler, Edward K; Giannoukakis, Nick; Gregori, Silvia; van Ham, S Marieke; Lattimer, Staci; Marshall, Lindsay; Harry, Rachel A; Hutchinson, James A; Isaacs, John D; Joosten, Irma; van Kooten, Cees; Lopez Diaz de Cerio, Ascension; Nikolic, Tatjana; Oral, Haluk Barbaros; Sofronic-Milosavljevic, Ljiljana; Ritter, Thomas; Riquelme, Paloma; Thomson, Angus W; Trucco, Massimo; Vives-Pi, Marta; Martinez-Caceres, Eva M; Hilkens, Catharien M U

    2016-01-01

    Cellular therapies with tolerogenic antigen-presenting cells (tolAPC) show great promise for the treatment of autoimmune diseases and for the prevention of destructive immune responses after transplantation. The methodologies for generating tolAPC vary greatly between different laboratories, making it difficult to compare data from different studies; thus constituting a major hurdle for the development of standardised tolAPC therapeutic products. Here we describe an initiative by members of the tolAPC field to generate a minimum information model for tolAPC (MITAP), providing a reporting framework that will make differences and similarities between tolAPC products transparent. In this way, MITAP constitutes a first but important step towards the production of standardised and reproducible tolAPC for clinical application.

  20. Minimum information about tolerogenic antigen-presenting cells (MITAP): a first step towards reproducibility and standardisation of cellular therapies

    PubMed Central

    Spiering, Rachel; Aguillon, Juan C.; Anderson, Amy E.; Appel, Silke; Benitez-Ribas, Daniel; ten Brinke, Anja; Broere, Femke; Cools, Nathalie; Cuturi, Maria Cristina; Diboll, Julie; Geissler, Edward K.; Giannoukakis, Nick; Gregori, Silvia; van Ham, S. Marieke; Lattimer, Staci; Marshall, Lindsay; Harry, Rachel A.; Hutchinson, James A.; Isaacs, John D.; Joosten, Irma; van Kooten, Cees; Lopez Diaz de Cerio, Ascension; Nikolic, Tatjana; Oral, Haluk Barbaros; Sofronic-Milosavljevic, Ljiljana; Ritter, Thomas; Riquelme, Paloma; Thomson, Angus W.; Trucco, Massimo; Vives-Pi, Marta; Martinez-Caceres, Eva M.

    2016-01-01

    Cellular therapies with tolerogenic antigen-presenting cells (tolAPC) show great promise for the treatment of autoimmune diseases and for the prevention of destructive immune responses after transplantation. The methodologies for generating tolAPC vary greatly between different laboratories, making it difficult to compare data from different studies; thus constituting a major hurdle for the development of standardised tolAPC therapeutic products. Here we describe an initiative by members of the tolAPC field to generate a minimum information model for tolAPC (MITAP), providing a reporting framework that will make differences and similarities between tolAPC products transparent. In this way, MITAP constitutes a first but important step towards the production of standardised and reproducible tolAPC for clinical application. PMID:27635311

  1. The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements

    EPA Science Inventory

    Over the last decade, the introduction of microarray technology has had a profound impact on gene expression research. The publication of studies with dissimilar or altogether contradictory results, obtained using different microarray platforms to analyze identical RNA samples, ...

  2. Improved Reproducibility for Perovskite Solar Cells with 1 cm(2) Active Area by a Modified Two-Step Process.

    PubMed

    Shen, Heping; Wu, Yiliang; Peng, Jun; Duong, The; Fu, Xiao; Barugkin, Chog; White, Thomas P; Weber, Klaus; Catchpole, Kylie R

    2017-02-22

    With rapid progress in recent years, organohalide perovskite solar cells (PSC) are promising candidates for a new generation of highly efficient thin-film photovoltaic technologies, for which up-scaling is an essential step toward commercialization. In this work, we propose a modified two-step method to deposit the CH3NH3PbI3 (MAPbI3) perovskite film that improves the uniformity, photovoltaic performance, and repeatability of large-area perovskite solar cells. This method is based on the commonly used two-step method, with one additional process involving treating the perovskite film with concentrated methylammonium iodide (MAI) solution. This additional treatment is proved to be helpful for tailoring the residual PbI2 level to an optimal range that is favorable for both optical absorption and inhibition of recombination. Scanning electron microscopy and photoluminescence image analysis further reveal that, compared to the standard two-step and one-step methods, this method is very robust for achieving uniform and pinhole-free large-area films. This is validated by the photovoltaic performance of the prototype devices with an active area of 1 cm(2), where we achieved the champion efficiency of ∼14.5% and an average efficiency of ∼13.5%, with excellent reproducibility.

  3. Clock Genes in Glia Cells

    PubMed Central

    Chi-Castañeda, Donají

    2016-01-01

    Circadian rhythms are periodic patterns in biological processes that allow the organisms to anticipate changes in the environment. These rhythms are driven by the suprachiasmatic nucleus (SCN), the master circadian clock in vertebrates. At a molecular level, circadian rhythms are regulated by the so-called clock genes, which oscillate in a periodic manner. The protein products of clock genes are transcription factors that control their own and other genes’ transcription, collectively known as “clock-controlled genes.” Several brain regions other than the SCN express circadian rhythms of clock genes, including the amygdala, the olfactory bulb, the retina, and the cerebellum. Glia cells in these structures are expected to participate in rhythmicity. However, only certain types of glia cells may be called “glial clocks,” since they express PER-based circadian oscillators, which depend of the SCN for their synchronization. This contribution summarizes the current information about clock genes in glia cells, their plausible role as oscillators and their medical implications. PMID:27666286

  4. Reproducibility of the EGFR immunohistochemistry scores for tumor samples from patients with advanced non-small cell lung cancer

    PubMed Central

    Avilés-Salas, Alejandro; Muñiz-Hernández, Saé; Maldonado-Martínez, Héctor Aquiles; Chanona-Vilchis, José G.; Ramírez-Tirado, Laura-Alejandra; HernáNdez-Pedro, Norma; Dorantes-Heredia, Rita; RuíZ-Morales, José Manuel; Motola-Kuba, Daniel; Arrieta, Oscar

    2017-01-01

    Epidermal growth factor receptor (EGFR) is overexpressed in >60% of non-small cell lung cancer (NSCLC) cases. In combination with radiotherapy or chemotherapy, first-line treatments with antibodies against EGFR, including cetuximab and necitumumab, have demonstrated benefits by increasing overall survival (OS), particularly in patients who overexpress EGFR. The present study evaluated the interobserver agreement among three senior pathologists, who were blinded to the clinical outcomes and assessed tumor samples from 85 patients with NSCLC using the H-score method. EGFR immunohistochemistry was performed using a qualitative immunohistochemical kit. The reported (mean ± standard deviation) H-scores from each pathologist were 111±102, 127±103 and 128.53±104.03. The patients with average H-scores ≥1, ≥100, ≥200 and between 250–300 were 85.9, 54.1, 28.2 and 12.9, respectively. Patients who had an average H-score >100 had a shorter OS time compared with those with lower scores. Furthermore, patients with EGFR mutations who were treated with EGFR-tyrosine kinase inhibitors (TKIs) and had an average H-score >100 had a longer OS time compared with those with an average H-score <100. The interobserver concordance for the total H-scores were 0.982, 0.980 and 0.988, and for a positive H-score ≥200, the interobserver concordance was 0.773, 0.710 and 0.675, respectively. The determination of EGFR expression by the H-score method is highly reproducible among pathologists and is a prognostic factor associated with a poor OS in all patients. Additionally, the results of the present study suggest that patients with EGFR mutations that are treated with EGFR-TKIs and present with a high H-score have a longer OS time. PMID:28356978

  5. American Society of Gene & Cell Therapy

    MedlinePlus

    ... Join ASGCT! Job Bank Donate Media The American Society of Gene & Cell Therapy The American Society of Gene & Cell Therapy is the primary professional membership organization for gene and cell therapy. The Society's members are scientists, physicians, patient advocates, and other ...

  6. From single-cell to cell-pool transcriptomes: stochasticity in gene expression and RNA splicing.

    PubMed

    Marinov, Georgi K; Williams, Brian A; McCue, Ken; Schroth, Gary P; Gertz, Jason; Myers, Richard M; Wold, Barbara J

    2014-03-01

    Single-cell RNA-seq mammalian transcriptome studies are at an early stage in uncovering cell-to-cell variation in gene expression, transcript processing and editing, and regulatory module activity. Despite great progress recently, substantial challenges remain, including discriminating biological variation from technical noise. Here we apply the SMART-seq single-cell RNA-seq protocol to study the reference lymphoblastoid cell line GM12878. By using spike-in quantification standards, we estimate the absolute number of RNA molecules per cell for each gene and find significant variation in total mRNA content: between 50,000 and 300,000 transcripts per cell. We directly measure technical stochasticity by a pool/split design and find that there are significant differences in expression between individual cells, over and above technical variation. Specific gene coexpression modules were preferentially expressed in subsets of individual cells, including one enriched for mRNA processing and splicing factors. We assess cell-to-cell variation in alternative splicing and allelic bias and report evidence of significant differences in splice site usage that exceed splice variation in the pool/split comparison. Finally, we show that transcriptomes from small pools of 30-100 cells approach the information content and reproducibility of contemporary RNA-seq from large amounts of input material. Together, our results define an experimental and computational path forward for analyzing gene expression in rare cell types and cell states.

  7. Hand1-Luc embryonic stem cell test (Hand1-Luc EST): a novel rapid and highly reproducible in vitro test for embryotoxicity by measuring cytotoxicity and differentiation toxicity using engineered mouse ES cells.

    PubMed

    Le Coz, Florian; Suzuki, Noriyuki; Nagahori, Hirohisa; Omori, Takashi; Saito, Koichi

    2015-04-01

    The embryonic stem cell test (EST) is a promising alternative method for evaluating embryotoxicity of test chemicals by measuring cytotoxicity and differentiation toxicity using mouse ES cells. Differentiation toxicity is analyzed by microscopically counting the beating of embryonic bodies after 10 days of culture. However, improvements are necessary to reduce the laborious manipulations involved and the time required to obtain results. We have previously reported the successful stable transfection of ES cells (ES-D3) with the heart and neural crest derivatives expressed transcript 1 (Hand1) gene and the establishment of a 96-well multi-plate-based new EST with luciferase reporter assay 6 days after treatment with test chemicals. Now, we propose an even more rapid and easier EST, named Hand1-Luc EST. We established another cell line to monitor the Hand1 gene expression via a luciferase reporter gene. By mRNA analysis and luciferase assay, we examined in detail the luciferase activity during cell differentiation, which allowed us to reduce the time of measurement from day 6 to day 5 (120 hr). Furthermore, the protocol was improved, with, among others, the measurement of cytotoxicity and differentiation toxicity taking place in the same 96-well round bottom plate instead of two different plates. With the positive control, 5-fluorouracil (5-FU), and 9 test chemicals, data with high reproducibility and very low variation (CV < 50%) in the relevant endpoints were obtained. This study shows that the Hand1-Luc EST could provide an accurate and sensitive short-term test for prediction of embryotoxicants by measuring cytotoxicity and differentiation toxicity from the same sample.

  8. The Need for Reproducibility

    SciTech Connect

    Robey, Robert W.

    2016-06-27

    The purpose of this presentation is to consider issues of reproducibility, specifically it determines whether bitwise reproducible computation is possible, if computational research in DOE improves its publication process, and if reproducible results can be achieved apart from the peer review process?

  9. Highly reproducible SERS detection in sequential injection analysis: real time preparation and application of photo-reduced silver substrate in a moving flow-cell.

    PubMed

    El-Zahry, Marwa R; Genner, Andreas; Refaat, Ibrahim H; Mohamed, Horria A; Lendl, Bernhard

    2013-11-15

    This paper reports an improved way for performing highly reproducible surface enhanced Raman scattering of different analytes using an automated flow system. The method uses a confocal Raman microscope to prepare SERS active silver spots on the window of a flow cell by photo-reduction of silver nitrate in the presence of citrate. Placement of the flow cell on an automated x and y stages of the Raman microscope allows to prepare a fresh spot for every new measurement. This procedure thus efficiently avoids any carry over effects which might result from adsorption of the analyte on the SERS active material and enables highly reproducible SERS measurements. For reproducible liquid handling the used sequential injection analysis system as well as the Raman microscope was operated by the flexible LabVIEW based software ATLAS developed in our group. Quantitative aspects were investigated using Cu(PAR)2 as a model analyte. Concentration down to 5×10(-6) M provided clear SERS spectra, a linear concentration dependence of the SERS intensities at 1333 cm(-1) was obtained from 5×10(-5) to 1×10(-3) with a correlation coefficient r=0.999. The coefficient of variation of the method Vxo was found to be 5.6% and the calculated limit of detection 1.7×10(-5) M. The results demonstrate the potential of SERS spectroscopy to be used as a molecular specific detector in aqueous flow systems.

  10. Intra- and Inter-visit Reproducibility of Ganglion Cell – Inner Plexiform Layer Measurements Using Handheld Optical Coherence Tomography in Children with Optic Pathway Gliomas

    PubMed Central

    Avery, Robert A.; Cnaan, Avital; Schuman, Joel S.; Chen, Chieh-Li; Glaug, Natalie C.; Packer, Roger J.; Quinn, Graham E.; Ishikawa, Hiroshi

    2014-01-01

    Purpose To determine the intra- and inter-visit reproducibility of ganglion cell-inner plexiform layer thickness measures using handheld optical coherence tomography (OCT) in sedated children with optic pathway gliomas and/or Neurofibromatosis type 1 (NF1). Design Prospective longitudinal cohort study Methods Children with sporadic optic pathway gliomas and/or NF1 who had ≥ 2 volumes acquired over the macula using handheld OCT during sedation for a clinically indicated MRI were eligible for the intra-visit cohort. Children with repeat handheld OCT imaging within 6 months were eligible for the inter-visit cohort. Total retinal thickness and ganglion cell-inner plexiform layer thickness were measured using custom designed automated segmentation software. Reproducibility was compared across average and anatomic quadrant by calculating the coefficient of variation (CV) and intraclass correlation coefficient (ICC). Results Forty-two subjects (median age 5.4 years, range 0.8–12.7 years) contributed 45 eyes to the intra-visit cohort. Thirty-one subject eyes had normal vision and 14 had abnormal vision (decreased visual acuity and/or visual field). Average and quadrant ganglion cell-inner plexiform layer measures demonstrated CVs ≤ 4.5% with excellent ICCs (> .935). The superior quadrant CV differed between subjects with (4.4%) and without (2.1%) vision loss (P < 0.05). Twenty-five subject eyes were eligible for the inter-visit cohort, demonstrating CVs from 1.6% to 5.2%. Inter-visit ICCs were excellent (.955 – .995). Discussion Handheld OCT imaging in sedated children with optic pathway gliomas produces highly reproducible measures of ganglion cell-inner plexiform layer thickness. PMID:25068639

  11. Patenting human genes and stem cells.

    PubMed

    Martin-Rendon, Enca; Blake, Derek J

    2007-01-01

    Cell lines and genetically modified single cell organisms have been considered patentable subjects for the last two decades. However, despite the technical patentability of genes and stem cell lines, social and legal controversy concerning their 'ownership' has surrounded stem cell research in recent years. Some granted patents on stem cells with extremely broad claims are casting a shadow over the commercialization of these cells as therapeutics. However, in spite of those early patents, the number of patent applications related to stem cells is growing exponentially. Both embryonic and adult stem cells have the ability to differentiate into several cell lineages in an organism as a result of specific genetic programs that direct their commitment and cell fate. Genes that control the pluripotency of stem cells have been recently identified and the genetic manipulation of these cells is becoming more efficient with the advance of new technologies. This review summarizes some of the recent published patents on pluripotency genes, gene transfer into stem cells and genetic reprogramming and takes the hematopoietic and embryonic stem cell as model systems.

  12. Predicting skin sensitization potential and inter-laboratory reproducibility of a human Cell Line Activation Test (h-CLAT) in the European Cosmetics Association (COLIPA) ring trials.

    PubMed

    Sakaguchi, Hitoshi; Ryan, Cindy; Ovigne, Jean-Marc; Schroeder, Klaus R; Ashikaga, Takao

    2010-09-01

    Regulatory policies in Europe prohibited the testing of cosmetic ingredients in animals for a number of toxicological endpoints. Currently no validated non-animal test methods exist for skin sensitization. Evaluation of changes in cell surface marker expression in dendritic cell (DC)-surrogate cell lines represents one non-animal approach. The human Cell Line Activation Test (h-CLAT) examines the level of CD86 and CD54 expression on the surface of THP-1 cells, a human monocytic leukemia cell line, following 24h of chemical exposure. To examine protocol transferability, between-lab reproducibility, and predictive capacity, the h-CLAT has been evaluated by five independent laboratories in several ring trials (RTs) coordinated by the European Cosmetics Association (COLIPA). The results of the first and second RTs demonstrated that the protocol was transferable and basically had good between-lab reproducibility and predictivity, but there were some false negative data. To improve performance, protocol and prediction model were modified. Using the modified prediction model in the first and second RT, accuracy was improved. However, about 15% of the outcomes were not correctly identified, which exposes some of the limitations of the assay. For the chemicals evaluated, the limitation may due to chemical being a weak allergen or having low solubility (ex. alpha-hexylcinnamaldehyde). The third RT evaluated the modified prediction model and satisfactory results were obtained. From the RT data, the feasibility of utilizing cell lines as surrogate DC in development of in vitro skin sensitization methods shows promise. The data also support initiating formal pre-validation of the h-CLAT in order to fully understand the capabilities and limitations of the assay.

  13. Reproducible Construction of Surface Tension-Mediated Honeycomb Concave Microwell Arrays for Engineering of 3D Microtissues with Minimal Cell Loss

    PubMed Central

    Lee, GeonHui; Lee, JaeSeo; Oh, HyunJik; Lee, SangHoon

    2016-01-01

    The creation of engineered 3D microtissues has attracted prodigious interest because of the fact that this microtissue structure is able to mimic in vivo environments. Such microtissues can be applied extensively in the fields of regenerative medicine and tissue engineering, as well as in drug and toxicity screening. Here, we develop a novel method of fabricating a large number of dense honeycomb concave microwells via surface tension-mediated self-construction. More specifically, in order to control the curvature and shape of the concavity in a precise and reproducible manner, a custom-made jig system was designed and fabricated. By applying a pre-set force using the jig system, the shape of the honeycomb concave well was precisely and uniformly controlled, despite the fact that wells were densely packed. The thin wall between the honeycomb wells enables the minimization of cell loss during the cell-seeding process. To evaluate the performance of the honeycomb microwell array, rat hepatocytes were seeded, and spheroids were successfully formed with uniform shape and size. Liver-specific functions such as albumin secretion and cytochrome P450 were subsequently analyzed. The proposed method of fabricating honeycomb concave wells is cost-effective, simple, and reproducible. The honeycomb well array can produce multiple spheroids with minimal cell loss, and can lead to significant contributions in tissue engineering and organ regeneration. PMID:27513567

  14. Reproducible Construction of Surface Tension-Mediated Honeycomb Concave Microwell Arrays for Engineering of 3D Microtissues with Minimal Cell Loss.

    PubMed

    Lee, GeonHui; Lee, JaeSeo; Oh, HyunJik; Lee, SangHoon

    2016-01-01

    The creation of engineered 3D microtissues has attracted prodigious interest because of the fact that this microtissue structure is able to mimic in vivo environments. Such microtissues can be applied extensively in the fields of regenerative medicine and tissue engineering, as well as in drug and toxicity screening. Here, we develop a novel method of fabricating a large number of dense honeycomb concave microwells via surface tension-mediated self-construction. More specifically, in order to control the curvature and shape of the concavity in a precise and reproducible manner, a custom-made jig system was designed and fabricated. By applying a pre-set force using the jig system, the shape of the honeycomb concave well was precisely and uniformly controlled, despite the fact that wells were densely packed. The thin wall between the honeycomb wells enables the minimization of cell loss during the cell-seeding process. To evaluate the performance of the honeycomb microwell array, rat hepatocytes were seeded, and spheroids were successfully formed with uniform shape and size. Liver-specific functions such as albumin secretion and cytochrome P450 were subsequently analyzed. The proposed method of fabricating honeycomb concave wells is cost-effective, simple, and reproducible. The honeycomb well array can produce multiple spheroids with minimal cell loss, and can lead to significant contributions in tissue engineering and organ regeneration.

  15. Gene sensitizes cancer cells to chemotherapy drugs

    Cancer.gov

    NCI scientists have found that a gene, Schlafen-11 (SLFN11), sensitizes cells to substances known to cause irreparable damage to DNA.  As part of their study, the researchers used a repository of 60 cell types to identify predictors of cancer cell respons

  16. Hematopoietic Stem Cell Expansion and Gene Therapy

    PubMed Central

    Watts, Korashon Lynn; Adair, Jennifer; Kiem, Hans-Peter

    2012-01-01

    Hematopoietic stem cell (HSC) gene therapy remains a highly attractive treatment option for many disorders including hematologic conditions, immunodeficiencies including HIV/AIDS, and other genetic disorders like lysosomal storage diseases, among others. In this review, we discuss the successes, side effects, and limitations of current gene therapy protocols. In addition, we describe the opportunities presented by implementing ex vivo expansion of gene-modified HSCs, as well as summarize the most promising ex vivo expansion techniques currently available. We conclude by discussing how some of the current limitations of HSC gene therapy could be overcome by combining novel HSC expansion strategies with gene therapy. PMID:21999373

  17. Expression of bacterial genes in plant cells.

    PubMed Central

    Fraley, R T; Rogers, S G; Horsch, R B; Sanders, P R; Flick, J S; Adams, S P; Bittner, M L; Brand, L A; Fink, C L; Fry, J S; Galluppi, G R; Goldberg, S B; Hoffmann, N L; Woo, S C

    1983-01-01

    Chimeric bacterial genes conferring resistance to aminoglycoside antibiotics have been inserted into the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid and introduced into plant cells by in vitro transformation techniques. The chimeric genes contain the nopaline synthase 5' and 3' regulatory regions joined to the genes for neomycin phosphotransferase type I or type II. The chimeric genes were cloned into an intermediate vector, pMON120, and inserted into pTiB6S3 by recombination and then introduced into petunia and tobacco cells by cocultivating A. tumefaciens cells with protoplast-derived cells. Southern hybridization was used to confirm the presence of the chimeric genes in the transformed plant tissues. Expression of the chimeric genes was determined by the ability of the transformed cells to proliferate on medium containing normally inhibitory levels of kanamycin (50 micrograms/ml) or other aminoglycoside antibiotics. Plant cells transformed by wild-type pTiB6S3 or derivatives carrying the bacterial neomycin phosphotransferase genes with their own promoters failed to grow under these conditions. The significance of these results for plant genetic engineering is discussed. Images PMID:6308651

  18. Pluripotent Stem Cells and Gene Therapy

    PubMed Central

    Simara, Pavel; Motl, Jason A.; Kaufman, Dan S.

    2013-01-01

    Human pluripotent stem cells represent an accessible cell source for novel cell-based clinical research and therapies. With the realization of induced pluripotent stem cells (iPSCs), it is possible to produce almost any desired cell type from any patient's cells. Current developments in gene modification methods have opened the possibility for creating genetically corrected human iPSCs for certain genetic diseases that could be used later in autologous transplantation. Promising preclinical studies have demonstrated correction of disease-causing mutations in a number of hematological, neuronal and muscular disorders. This review aims to summarize these recent advances with a focus on iPSC generation techniques, as well as gene modification methods. We will then further discuss some of the main obstacles remaining to be overcome before successful application of human pluripotent stem cell-based therapy arrives in the clinic and what the future of stem cell research may look like. PMID:23353080

  19. Keggin-Type PMo11V as a P-type Dopant for Enhancing the Efficiency and Reproducibility of Perovskite Solar Cells.

    PubMed

    Dong, Guohua; Xia, Debin; Yang, Yulin; Shenga, Li; Ye, Tengling; Fan, Ruiqing

    2017-01-25

    The conventional perovskite solar cells (PSCs) with 2,2',7,7'-tetrakis(N,N-dimethoxyphenylamine)-9,9'-spirobifluorene (spiro-OMeTAD) as a hole transporting material commonly suffer from poor stability and reproducibility mainly due to the process of placing the devices in air and illumination for oxidizing the spiro-OMeTAD. Herein, Keggin-type polyoxometalates (POMs)-phosphovanadomolybdate (H4PMo11V·nH2O, denoted as PMo11V) is for the first time employed as a p-type dopant for promoting the oxidation of spiro-OMeTAD. Thereby, without illumination and air, the conductivity and hole extraction efficiency of the PMo11V doped spiro-OMeTAD with assistance of lithium bis(trifluoromethanesulfonyl)imide (Li-TFSI) and 4-tert-butylpyridine (TBP) can be dramatically enhanced. On the basis of this strategy, the corresponding PSCs exhibit substantially improved photovoltaic performance and good reproducibility. The best performing device yields a power conversion efficiency (PCE) of 14.05%. This work indicates a great potential of polyoxometalates for further applications in solar cells and other optoelectronics devices.

  20. Gene and Cell Therapy for Heart Failure

    PubMed Central

    2009-01-01

    Abstract Cardiac gene and cell therapy have both entered clinical trials aimed at ameliorating ventricular dysfunction in patients with chronic congestive heart failure. The transduction of myocardial cells with viral constructs encoding a specific cardiomyocyte Ca2+ pump in the sarcoplasmic reticulum (SR), SRCa2+-ATPase has been shown to correct deficient Ca2+ handling in cardiomyocytes and improvements in contractility in preclinical studies, thus leading to the first clinical trial of gene therapy for heart failure. In cell therapy, it is not clear whether beneficial effects are cell-type specific and how improvements in contractility are brought about. Despite these uncertainties, a number of clinical trials are under way, supported by safety and efficacy data from trials of cell therapy in the setting of myocardial infarction. Safety concerns for gene therapy center on inflammatory and immune responses triggered by viral constructs, and for cell therapy with myoblast cells, the major concern is increased incidence of ventricular arrhythmia after cell transplantation. Principles and mechanisms of action of gene and cell therapy for heart failure are discussed, together with the potential influence of reactive oxygen species on the efficacy of these treatments and the status of myocardial-delivery techniques for viral constructs and cells. Antioxid. Redox Signal. 11, 2025–2042. PMID:19416058

  1. A robust and reproducible animal serum-free culture method for clinical-grade bone marrow-derived mesenchymal stromal cells.

    PubMed

    Laitinen, Anita; Oja, Sofia; Kilpinen, Lotta; Kaartinen, Tanja; Möller, Johanna; Laitinen, Saara; Korhonen, Matti; Nystedt, Johanna

    2016-08-01

    Efficient xenofree expansion methods to replace fetal bovine serum (FBS)-based culture methods are strongly encouraged by the regulators and are needed to facilitate the adoption of mesenchymal stromal cell (MSC)-based therapies. In the current study we established a clinically-compliant and reproducible animal serum-free culture protocol for bone marrow-(BM-) MSCs based on an optimized platelet-derived supplement. Our study compared two different platelet-derived supplements, platelet lysate PL1 versus PL2, produced by two different methods and lysed with different amounts of freeze-thaw cycles. Our study also explored the effect of a low oxygen concentration on BM-MSCs. FBS-supplemented BM-MSC culture served as control. Growth kinetics, differentiation and immunomodulatory potential, morphology, karyotype and immunophenotype was analysed. Growth kinetics in long-term culture was also studied. Based on the initial results, we chose to further process develop the PL1-supplemented culture protocol at 20 % oxygen. The results from 11 individual BM-MSC batches expanded in the chosen condition were consistent, yielding 6.60 × 10(9) ± 4.74 × 10(9) cells from only 20 ml of bone marrow. The cells suppressed T-cell proliferation, displayed normal karyotype and typical MSC differentiation potential and phenotype. The BM-MSCs were, however, consistently HLA-DR positive when cultured in platelet lysate (7.5-66.1 %). We additionally show that culture media antibiotics and sterile filtration of the platelet lysate can be successfully omitted. We present a robust and reproducible clinically-compliant culture method for BM-MSCs based on platelet lysate, which enables high quantities of HLA-DR positive MSCs at a low passage number (p2) and suitable for clinical use.

  2. Introduction of genes into living cells

    NASA Astrophysics Data System (ADS)

    Nishimura, E.; Nagai, A.; Tomimasu, T.; Kina, T.; Fujimoto, S.; Katsura, Y.

    1998-02-01

    One of our main subjects is an application of the FEL to gene therapy of genetic diseases, immunodeficiency syndromes and cancer. In this study, using the FEL, we tried to establish a model system for introducing genes into the stem cells from which all blood cells are derived. Our aim is to specifically mark the stem cells with monoclonal antibodies which are conjugated with efficient FEL absorbers. Cells are then irradiated with FEL at a wavelength corresponding to the absorption energy of the absorber. We speculate that the gap formation of cell membrane will occur, caused by the thermal shock due to the absorption of the FEL energy. As an animal model for gene therapy, we tried to transfer the RAG-2 genes into hematopoietic stem cells from RAG-2 deficient mice, which have severe immunodeficiency because of the lack of RAG-2 gene required for lymphocyte development. As the results by this construct, the infant lymphocytes (T and B cells) could be observed in the thymus of the RAG-2 deficient mice 2 weeks post-operative.

  3. Reproducibility of parameter learning with missing observations in naive Wnt Bayesian network trained on colorectal cancer samples and doxycycline-treated cell lines.

    PubMed

    Sinha, Shriprakash

    2015-07-01

    In this manuscript the reproducibility of parameter learning with missing observations in a naive Bayesian network and its effect on the prediction results for Wnt signaling activation in colorectal cancer is tested. The training of the network is carried out separately on doxycycline-treated LS174T cell lines (GSE18560) as well as normal and adenoma samples (GSE8671). A computational framework to test the reproducibility of the parameters is designed in order check the veracity of the prediction results. Detailed experimental analysis suggests that the prediction results are accurate and reproducible with negligible deviations. Anomalies in estimated parameters are accounted for due to the representation issues of the Bayesian network model. High prediction accuracies are reported for normal (N) and colon-related adenomas (AD), colorectal cancer (CRC), carcinomas (C), adenocarcinomas (ADC) and replication error colorectal cancer (RER CRC) test samples. Test samples from inflammatory bowel diseases (IBD) do not fare well in the prediction test. Also, an interesting case regarding hypothesis testing came up while proving the statistical significance of the different design setups of the Bayesian network model. It was found that hypothesis testing may not be the correct way to check the significance between design setups, especially when the structure of the model is the same, given that the model is trained on a single piece of test data. The significance test does have value when the datasets are independent. Finally, in comparison to the biologically inspired models, the naive Bayesian model may give accurate results, but this accuracy comes at the cost of a loss of crucial biological knowledge which might help reveal hidden relations among intra/extracellular factors affecting the Wnt pathway.

  4. An innovative stand-alone bioreactor for the highly reproducible transfer of cyclic mechanical stretch to stem cells cultured in a 3D scaffold.

    PubMed

    Govoni, Marco; Lotti, Fabrizio; Biagiotti, Luigi; Lannocca, Maurizio; Pasquinelli, Gianandrea; Valente, Sabrina; Muscari, Claudio; Bonafè, Francesca; Caldarera, Claudio M; Guarnieri, Carlo; Cavalcanti, Silvio; Giordano, Emanuele

    2014-10-01

    Much evidence in the literature demonstrates the effect of cyclic mechanical stretch in maintaining, or addressing, a muscle phenotype. Such results were obtained using several technical approaches, useful for the experimental collection of proofs of principle but probably unsuitable for application in clinical regenerative medicine. Here we aimed to design a reliable innovative bioreactor, acting as a stand-alone cell culture incubator, easy to operate and effective in addressing mesenchymal stem cells (MSCs) seeded onto a 3D bioreabsorbable scaffold, towards a muscle phenotype via the transfer of a controlled and highly-reproducible cyclic deformation. Electron microscopy, immunohistochemistry and biochemical analysis of the obtained pseudotissue constructs showed that cells 'trained' over 1 week: (a) displayed multilayer organization and invaded the 3D mesh of the scaffold; and (b) expressed typical markers of muscle cells. This effect was due only to physical stimulation of the cells, without the need of any other chemical or genetic manipulation. This device is thus proposed as a prototypal instrument to obtain pseudotissue constructs to test in cardiovascular regenerative medicine, using good manufacturing procedures.

  5. How did bacterial ancestors reproduce? Lessons from L-form cells and giant lipid vesicles: multiplication similarities between lipid vesicles and L-form bacteria.

    PubMed

    Briers, Yves; Walde, Peter; Schuppler, Markus; Loessner, Martin J

    2012-12-01

    In possible scenarios on the origin of life, protocells represent the precursors of the first living cells. To study such hypothetical protocells, giant vesicles are being widely used as a simple model. Lipid vesicles can undergo complex morphological changes enabling self-reproduction such as growth, fission, and extra- and intravesicular budding. These properties of vesicular systems may in some way reflect the mechanism of reproduction used by protocells. Moreover, remarkable similarities exist between the morphological changes observed in giant vesicles and bacterial L-form cells, which represent bacteria that have lost their rigid cell wall, but retain the ability to reproduce. L-forms feature a dismantled cellular structure and are unable to carry out classical binary fission. We propose that the striking similarities in morphological transitions of L-forms and giant lipid vesicles may provide insights into primitive reproductive mechanisms and contribute to a better understanding of the origin and evolution of mechanisms of cell reproduction. Editor's suggested further reading in BioEssays Synthesizing artificial cells from giant unilamellar vesicles: State-of-the art in the development of microfluidic technology Abstract.

  6. A reproducible method for the isolation and expansion of ovine mesenchymal stromal cells from bone marrow for use in regenerative medicine preclinical studies.

    PubMed

    Caminal, Marta; Vélez, Roberto; Rabanal, Rosa Maria; Vivas, Daniel; Batlle-Morera, Laura; Aguirre, Màrius; Barquinero, Jordi; García, Joan; Vives, Joaquim

    2016-11-18

    The use of multipotent mesenchymal stromal cells (MSCs) as candidate medicines for treating a variety of pathologies is based on their qualities as either progenitors for the regeneration of damaged tissue or producers of a number of molecules with pharmacological properties. Preclinical product development programmes include the use of well characterized cell populations for proof of efficacy and safety studies before testing in humans. In the field of orthopaedics, an increasing number of translational studies use sheep as an in vivo test system because of the similarities with humans in size and musculoskeletal architecture. However, robust and reproducible methods for the isolation, expansion, manipulation and characterization of ovine MSCs have not yet been standardised. The present study describes a method for isolation and expansion of fibroblastic-like, adherent ovine MSCs that express CD44, CD90, CD140a, CD105 and CD166, and display trilineage differentiation potential. The 3-week bioprocess proposed here typically yielded cell densities of 1.4 × 10(4) MSCs/cm(2) at passage 2, with an expansion factor of 37.8 and approximately eight cumulative population doublings. The osteogenic potential of MSCs derived following this methodology was further evaluated in vivo in a translational model of osteonecrosis of the femoral head, in which the persistence of grafted cells in the host tissue and their lineage commitment into osteoblasts and osteocytes was demonstrated by tracking enhanced green fluorescent protein-labelled cells. Copyright © 2016 John Wiley & Sons, Ltd.

  7. Heat induces gene amplification in cancer cells

    SciTech Connect

    Yan, Bin; Ouyang, Ruoyun; Huang, Chenghui; Liu, Franklin; Neill, Daniel; Li, Chuanyuan; Dewhirst, Mark

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer This study discovered that heat exposure (hyperthermia) results in gene amplification in cancer cells. Black-Right-Pointing-Pointer Hyperthermia induces DNA double strand breaks. Black-Right-Pointing-Pointer DNA double strand breaks are considered to be required for the initiation of gene amplification. Black-Right-Pointing-Pointer The underlying mechanism of heat-induced gene amplification is generation of DNA double strand breaks. -- Abstract: Background: Hyperthermia plays an important role in cancer therapy. However, as with radiation, it can cause DNA damage and therefore genetic instability. We studied whether hyperthermia can induce gene amplification in cancer cells and explored potential underlying molecular mechanisms. Materials and methods: (1) Hyperthermia: HCT116 colon cancer cells received water-submerged heating treatment at 42 or 44 Degree-Sign C for 30 min; (2) gene amplification assay using N-(phosphoacetyl)-L-aspartate (PALA) selection of cabamyl-P-synthetase, aspartate transcarbarmylase, dihydro-orotase (cad) gene amplified cells; (3) southern blotting for confirmation of increased cad gene copies in PALA-resistant cells; (4) {gamma}H2AX immunostaining to detect {gamma}H2AX foci as an indication for DNA double strand breaks. Results: (1) Heat exposure at 42 or 44 Degree-Sign C for 30 min induces gene amplification. The frequency of cad gene amplification increased by 2.8 and 6.5 folds respectively; (2) heat exposure at both 42 and 44 Degree-Sign C for 30 min induces DNA double strand breaks in HCT116 cells as shown by {gamma}H2AX immunostaining. Conclusion: This study shows that heat exposure can induce gene amplification in cancer cells, likely through the generation of DNA double strand breaks, which are believed to be required for the initiation of gene amplification. This process may be promoted by heat when cellular proteins that are responsible for checkpoints, DNA replication, DNA repair and

  8. Novel Cell and Gene Therapies for HIV

    PubMed Central

    Hoxie, James A.; June, Carl H.

    2012-01-01

    Highly active antiretroviral therapy dramatically improves survival in HIV-infected patients. However, persistence of HIV in reservoirs has necessitated lifelong treatment that can be complicated by cumulative toxicities, incomplete immune restoration, and the emergence of drug-resistant escape mutants. Cell and gene therapies offer the promise of preventing progressive HIV infection by interfering with HIV replication in the absence of chronic antiviral therapy. Individuals homozygous for a deletion in the CCR5 gene (CCR5Δ32) are largely resistant to infection from R5-topic HIV-1 strains, which are most commonly transmitted. A recent report that an HIV-infected patient with relapsed acute myelogenous leukemia was effectively cured from HIV infection after transplantation of hematopoietic stem/progenitor cells (HSC) from a CCR5Δ32 homozygous donor has generated renewed interest in developing treatment strategies that target viral reservoirs and generate HIV resistance in a patient’s own cells. Although the development of cell-based and gene transfer therapies has been slow, progress in a number of areas is evident. Advances in the fields of gene-targeting strategies, T-cell-based approaches, and HSCs have been encouraging, and a series of ongoing and planned trials to establish proof of concept for strategies that could lead to successful cell and gene therapies for HIV are under way. The eventual goal of these studies is to eliminate latent viral reservoirs and the need for lifelong antiretroviral therapy. PMID:23028130

  9. Improvement of CH₃NH₃PbI₃ Formation for Efficient and Better Reproducible Mesoscopic Perovskite Solar Cells.

    PubMed

    Jiang, Changyun; Lim, Siew Lay; Goh, Wei Peng; Wei, Feng Xia; Zhang, Jie

    2015-11-11

    High-performance perovskite solar cells (PSCs) are obtained through optimization of the formation of CH3NH3PbI3 nanocrystals on mesoporous TiO2 film, using a two-step sequential deposition process by first spin-coating a PbI2 film and then submerging it into CH3NH3I solution for perovskite conversion (PbI2 + CH3NH3I → CH3NH3PbI3). It is found that the PbI2 morphology from different film formation process (thermal drying, solvent extraction, and as-deposited) has a profound effect on the CH3NH3PbI3 active layer formation and its nanocrystalline composition. The residual PbI2 in the active layer contributes to substantial photocurrent losses, thus resulting in low and inconsistent PSC performances. The PbI2 film dried by solvent extraction shows enhanced CH3NH3PbI3 conversion as the loosely packed disk-like PbI2 crystals allow better CH3NH3I penetration and reaction in comparison to the multicrystal aggregates that are commonly obtained in the thermally dried PbI2 film. The as-deposited PbI2 wet film, without any further drying, exhibits complete conversion to CH3NH3PbI3 in MAI solution. The resulting PSCs reveal high power conversion efficiency of 15.60% with a batch-to-batch consistency of 14.60 ± 0.55%, whereas a lower efficiency of 13.80% with a poorer consistency of 11.20 ± 3.10% are obtained from the PSCs using thermally dried PbI2 films.

  10. Dynamics of single-cell gene expression

    PubMed Central

    Longo, Diane; Hasty, Jeff

    2006-01-01

    Cellular behavior has traditionally been investigated by utilizing bulk-scale methods that measure average values for a population of cells. Such population-wide studies mask the behavior of individual cells and are often insufficient for characterizing biological processes in which cellular heterogeneity plays a key role. A unifying theme of many recent studies has been a focus on the development and utilization of single-cell experimental techniques that are capable of probing key biological phenomena in individual living cells. Recently, novel information about gene expression dynamics has been obtained from single-cell experiments that draw upon the unique capabilities of fluorescent reporter proteins. PMID:17130866

  11. Correlation of immunohistochemical staining p63 and TTF-1 with EGFR and K-ras mutational spectrum and diagnostic reproducibility in non small cell lung carcinoma.

    PubMed

    Thunnissen, Erik; Boers, Evan; Heideman, Daniëlle A M; Grünberg, Katrien; Kuik, Dirk J; Noorduin, Arnold; van Oosterhout, Matthijs; Pronk, Divera; Seldenrijk, Cees; Sietsma, Hannie; Smit, Egbert F; van Suylen, Robertjan; von der Thusen, Jan; Vrugt, Bart; Wiersma, Anne; Witte, Birgit I; den Bakker, Michael

    2012-12-01

    For treatment purposes, distinction between squamous cell carcinoma and adenocarcinoma is important. The aim of this study is to examine the diagnostic accuracy on lung cancer small biopsies for the distinction between adenocarcinoma and squamous cell carcinoma and relate these to immunohistochemical and KRAS and EGFR mutation analysis. An interobserver study was performed on 110 prospectively collected biopsies obtained by bronchoscopy or transthoracic needle biopsy of patients with non-small cell lung cancer. The diagnosis was correlated with immunohistochemical (IHC) analysis for markers of adeno- (TTF1 and/or mucin positivity) and squamous cell differentiation (P63 and CK5/6) as well as KRAS and EGFR mutation analysis. Eleven observers independently read H&E-stained slides of 110 cases, resulting in a kappa value of 0.55 ± 0.10. The diagnosis non-small cell lung cancer not otherwise specified was given on average on 29.5 % of the biopsies. A high concordance was observed between hematoxylin-eosin-based consensus diagnosis (≥8/11 readings concordant) and IHC markers. In all cases with EGFR (n = 1) and KRAS (n = 20) mutations, adenodifferentiation as determined by IHC was present and p63 staining was absent. In 2 of 25 cases with a consensus diagnosis of squamous cell carcinoma, additional stainings favored adenodifferentation, and a KRAS mutation was present. P63 is most useful for distinction between EGFR/KRAS mutation positive and negative patients. In the diagnostic work-up of non-small cell lung carcinoma the limited reproducibility on small biopsies is optimized with immunohistochemical analysis, resulting in reliable delineation for predictive analysis.

  12. Expression of bacterial superantigen genes in mice induces localized mononuclear cell inflammatory responses.

    PubMed Central

    Dow, S W; Potter, T A

    1997-01-01

    Bacterial superantigens are potent T cell activators, and superantigen proteins have been injected into mice and other animals to study T cell responses in vivo. When superantigen proteins are injected, however, the T cell stimulatory effects cannot be confined to specific tissues. Therefore, to target superantigen expression to specific tissues, we used gene transfer techniques to express bacterial superantigen genes in mammalian cells in vitro and in tissues in vivo. Murine, human, and canine cells transfected with superantigen genes in vitro all produced superantigen proteins both intracellularly and extracellularly, as assessed by bioassay, immunocytochemistry, and antigen ELISA. Superantigens produced by transfected eukaryotic cells retained their biologic specificity for T cell receptor binding. Intramuscular injection of superantigen plasmid DNA in vivo induced an intense intramuscular mononuclear cell infiltrate, an effect that could not be reproduced by intramuscular injection of superantigen protein. Intradermal and intravenous injection of superantigen DNA induced cutaneous and intrapulmonary mononuclear cell inflammatory responses, respectively. Thus, superantigen genes can be expressed by mammalian cells in vivo. Superantigen gene therapy represents a novel method of targeting localized T cell inflammatory reactions, with potential application to treatment of cancer and certain infectious diseases. PMID:9169491

  13. Diverse marrow stromal cells protect CLL cells from spontaneous and drug-induced apoptosis: development of a reliable and reproducible system to assess stromal cell adhesion-mediated drug resistance.

    PubMed

    Kurtova, Antonina V; Balakrishnan, Kumudha; Chen, Rong; Ding, Wei; Schnabl, Susanne; Quiroga, Maite P; Sivina, Mariela; Wierda, William G; Estrov, Zeev; Keating, Michael J; Shehata, Medhat; Jäger, Ulrich; Gandhi, Varsha; Kay, Neil E; Plunkett, William; Burger, Jan A

    2009-11-12

    Marrow stromal cells (MSCs) provide important survival and drug resistance signals to chronic lymphocytic leukemia (CLL) cells, but current models to analyze CLL-MSC interactions are heterogeneous. Therefore, we tested different human and murine MSC lines and primary human MSCs for their ability to protect CLL cells from spontaneous and drug-induced apoptosis. Our results show that both human and murine MSCs are equally effective in protecting CLL cells from fludarabine-induced apoptosis. This protective effect was sustained over a wide range of CLL-MSC ratios (5:1 to 100:1), and the levels of protection were reproducible in 4 different laboratories. Human and murine MSCs also protected CLL cells from dexamethasone- and cyclophosphamide-induced apoptosis. This protection required cell-cell contact and was virtually absent when CLL cells were separated from the MSCs by micropore filters. Furthermore, MSCs maintained Mcl-1 and protected CLL cells from spontaneous and fludarabine-induced Mcl-1 and PARP cleavage. Collectively, these studies define common denominators for CLL cocultures with MSCs. They also provide a reliable, validated tool for future investigations into the mechanism of MSC-CLL cross talk and for drug testing in a more relevant fashion than the commonly used suspension cultures.

  14. Gene trapping in embryonic stem cells.

    PubMed

    Stanford, William L; Epp, Trevor; Reid, Tammy; Rossant, Janet

    2006-01-01

    Gene trapping in embryonic stem cells (ESCs) generates random, sequence-tagged insertional mutations, which can often report the gene expression pattern of the mutated gene. This mutagenesis strategy has often been coupled to expression or function-based assays in gene discovery screens. The availability of the mouse genome sequence has shifted gene trapping from a gene discovery platform to a high-throughput mutagenesis platform. At present, a concerted worldwide effort is underway to develop a library of loss-of-function mutations in all mouse genes. The International Gene Trap Consortium (IGTC) is leading the way by making a first pass of the genome by random mutagenesis before a high-throughput gene targeting program takes over. In this chapter, we provide a methods guidebook to exploring and using the IGTC resource, explain the different kinds of vectors and insertions that reside in the different libraries, and provide advice and methods for investigators to design novel expression-based "cottage industry" screens.

  15. CIRCADIAN CLOCK AND CELL CYCLE GENE EXPRESSION

    PubMed Central

    Metz, Richard P.; Qu, Xiaoyu; Laffin, Brian; Earnest, David; Porter, Weston W.

    2009-01-01

    Mouse mammary epithelial cells (HC-11) and mammary tissues were analyzed for developmental changes in circadian clock, cellular proliferation and differentiation marker genes. Expression of the clock genes, Per1 and Bmal1, were elevated in differentiated HC-11 cells whereas Per2 mRNA levels were higher in undifferentiated cells. This differentiation-dependent profile of clock gene expression was consistent with that observed in mouse mammary glands as Per1 and Bmal1 mRNA levels were elevated in late pregnant and lactating mammary tissues, while Per2 expression was higher in proliferating virgin and early pregnant glands. In both HC-11 cells and mammary glands, elevated Per2 expression was positively correlated with c-Myc and Cyclin D1 mRNA levels while Per1 and Bmal1 expression changed in conjunction with ß-casein mRNA levels. Interestingly, developmental stage had differential effects on rhythms of clock gene expression in the mammary gland. These data suggest that circadian clock genes may play a role in mouse mammary gland development and differentiation. PMID:16261617

  16. Diverse marrow stromal cells protect CLL cells from spontaneous and drug-induced apoptosis: development of a reliable and reproducible system to assess stromal cell adhesion-mediated drug resistance

    PubMed Central

    Kurtova, Antonina V.; Balakrishnan, Kumudha; Chen, Rong; Ding, Wei; Schnabl, Susanne; Quiroga, Maite P.; Sivina, Mariela; Wierda, William G.; Estrov, Zeev; Keating, Michael J.; Shehata, Medhat; Jäger, Ulrich; Gandhi, Varsha; Kay, Neil E.; Plunkett, William

    2009-01-01

    Marrow stromal cells (MSCs) provide important survival and drug resistance signals to chronic lymphocytic leukemia (CLL) cells, but current models to analyze CLL–MSC interactions are heterogeneous. Therefore, we tested different human and murine MSC lines and primary human MSCs for their ability to protect CLL cells from spontaneous and drug-induced apoptosis. Our results show that both human and murine MSCs are equally effective in protecting CLL cells from fludarabine-induced apoptosis. This protective effect was sustained over a wide range of CLL–MSC ratios (5:1 to 100:1), and the levels of protection were reproducible in 4 different laboratories. Human and murine MSCs also protected CLL cells from dexamethasone- and cyclophosphamide-induced apoptosis. This protection required cell–cell contact and was virtually absent when CLL cells were separated from the MSCs by micropore filters. Furthermore, MSCs maintained Mcl-1 and protected CLL cells from spontaneous and fludarabine-induced Mcl-1 and PARP cleavage. Collectively, these studies define common denominators for CLL cocultures with MSCs. They also provide a reliable, validated tool for future investigations into the mechanism of MSC–CLL cross talk and for drug testing in a more relevant fashion than the commonly used suspension cultures. PMID:19762485

  17. Cell Cycle and Cell Size Dependent Gene Expression Reveals Distinct Subpopulations at Single-Cell Level

    PubMed Central

    Dolatabadi, Soheila; Candia, Julián; Akrap, Nina; Vannas, Christoffer; Tesan Tomic, Tajana; Losert, Wolfgang; Landberg, Göran; Åman, Pierre; Ståhlberg, Anders

    2017-01-01

    Cell proliferation includes a series of events that is tightly regulated by several checkpoints and layers of control mechanisms. Most studies have been performed on large cell populations, but detailed understanding of cell dynamics and heterogeneity requires single-cell analysis. Here, we used quantitative real-time PCR, profiling the expression of 93 genes in single-cells from three different cell lines. Individual unsynchronized cells from three different cell lines were collected in different cell cycle phases (G0/G1 – S – G2/M) with variable cell sizes. We found that the total transcript level per cell and the expression of most individual genes correlated with progression through the cell cycle, but not with cell size. By applying the random forests algorithm, a supervised machine learning approach, we show how a multi-gene signature that classifies individual cells into their correct cell cycle phase and cell size can be generated. To identify the most predictive genes we used a variable selection strategy. Detailed analysis of cell cycle predictive genes allowed us to define subpopulations with distinct gene expression profiles and to calculate a cell cycle index that illustrates the transition of cells between cell cycle phases. In conclusion, we provide useful experimental approaches and bioinformatics to identify informative and predictive genes at the single-cell level, which opens up new means to describe and understand cell proliferation and subpopulation dynamics. PMID:28179914

  18. Modifying the Chemical Structure of a Porphyrin Small Molecule with Benzothiophene Groups for the Reproducible Fabrication of High Performance Solar Cells.

    PubMed

    Liang, Tianxiang; Xiao, Liangang; Gao, Ke; Xu, Wenzhan; Peng, Xiaobin; Cao, Yong

    2017-03-01

    A porphyrin-based molecule DPPEZnP-BzTBO with bulky benzothiophene groups was designed and synthesized as an electron donor material for bulk heterojunction (BHJ) solar cells. The optimized devices under thermal annealing (TA) and then chloroform solvent vapor anneanling (SVA) for 80 s exhibited an outstanding power conversion efficiencie (PCE) of 9.08%. Contrasted with the smaller thienyl substituted analogues we reported previously, DPPEZnP-BzTBO-based BHJ solar cells exhibited a higher open circuit voltage due to the lower highest occupied molecular orbital energy level. The TA post-treatment of the active layers induced the formation of more crystallized components, and the subsequent SVA provided a driving force for the domain growth, resulting in more obvious phase segregation between the donor and the acceptor in nanoscale. Furthermore, the PCEs kept above 95% upon the further SVA treatment within the time range of 60 to 95 s probably because the bulky benzothiophene groups retard the too quick change of crystallinity, providing a wide processing window for the reproducible device fabrication.

  19. Reproducibility in Chemical Research.

    PubMed

    Bergman, Robert G; Danheiser, Rick L

    2016-10-04

    "… To what extent is reproducibility a significant issue in chemical research? How can problems involving irreproducibility be minimized? … Researchers should be aware of the dangers of unconscious investigator bias, all papers should provide adequate experimental detail, and Reviewers have a responsibility to carefully examine papers for adequacy of experimental detail and support for the conclusions …" Read more in the Editorial by Robert G. Bergman and Rick L. Danheiser.

  20. Gene expression profiles in irradiated cancer cells

    NASA Astrophysics Data System (ADS)

    Minafra, L.; Bravatà, V.; Russo, G.; Ripamonti, M.; Gilardi, M. C.

    2013-07-01

    Knowledge of the molecular and genetic mechanisms underlying cellular response to radiation may provide new avenues to develop innovative predictive tests of radiosensitivity of tumours and normal tissues and to improve individual therapy. Nowadays very few studies describe molecular changes induced by hadrontherapy treatments, therefore this field has to be explored and clarified. High-throughput methodologies, such as DNA microarray, allow us to analyse mRNA expression of thousands of genes simultaneously in order to discover new genes and pathways as targets of response to hadrontherapy. Our aim is to elucidate the molecular networks involved in the sensitivity/resistance of cancer cell lines subjected to hadrontherapy treatments with a genomewide approach by using cDNA microarray technology to identify gene expression profiles and candidate genes responsible of differential cellular responses.

  1. Gene expression profiles in irradiated cancer cells

    SciTech Connect

    Minafra, L.; Bravatà, V.; Russo, G.; Ripamonti, M.; Gilardi, M. C.

    2013-07-26

    Knowledge of the molecular and genetic mechanisms underlying cellular response to radiation may provide new avenues to develop innovative predictive tests of radiosensitivity of tumours and normal tissues and to improve individual therapy. Nowadays very few studies describe molecular changes induced by hadrontherapy treatments, therefore this field has to be explored and clarified. High-throughput methodologies, such as DNA microarray, allow us to analyse mRNA expression of thousands of genes simultaneously in order to discover new genes and pathways as targets of response to hadrontherapy. Our aim is to elucidate the molecular networks involved in the sensitivity/resistance of cancer cell lines subjected to hadrontherapy treatments with a genomewide approach by using cDNA microarray technology to identify gene expression profiles and candidate genes responsible of differential cellular responses.

  2. Cell cycle gene expression under clinorotation

    NASA Astrophysics Data System (ADS)

    Artemenko, Olga

    2016-07-01

    Cyclins and cyclin-dependent kinase (CDK) are main regulators of the cell cycle of eukaryotes. It's assumes a significant change of their level in cells under microgravity conditions and by other physical factors actions. The clinorotation use enables to determine the influence of gravity on simulated events in the cell during the cell cycle - exit from the state of quiet stage and promotion presynthetic phase (G1) and DNA synthesis phase (S) of the cell cycle. For the clinorotation effect study on cell proliferation activity is the necessary studies of molecular mechanisms of cell cycle regulation and development of plants under altered gravity condition. The activity of cyclin D, which is responsible for the events of the cell cycle in presynthetic phase can be controlled by the action of endogenous as well as exogenous factors, but clinorotation is one of the factors that influence on genes expression that regulate the cell cycle.These data can be used as a model for further research of cyclin - CDK complex for study of molecular mechanisms regulation of growth and proliferation. In this investigation we tried to summarize and analyze known literature and own data we obtained relatively the main regulators of the cell cycle in altered gravity condition.

  3. Epigenetic landscapes explain partially reprogrammed cells and identify key reprogramming gene

    NASA Astrophysics Data System (ADS)

    Lang, Alex; Li, Hu; Collins, James; Mehta, Pankaj

    2013-03-01

    A common metaphor for describing development is a rugged epigenetic landscape where cell fates are represented as attracting valleys resulting from a complex regulatory network. Here, we introduce a framework for explicitly constructing epigenetic landscapes that combines genomic data with techniques from physics, specifically Hopfield neural networks. Each cell fate is a dynamic attractor, yet cells can change fate in response to external signals. Our model suggests that partially reprogrammed cells (cells found in reprogramming experiments but not in vivo) are a natural consequence of high-dimensional landscapes and predicts that partially reprogrammed cells should be hybrids that coexpress genes from multiple cell fates. We verify this prediction by reanalyzing existing data sets. Our model reproduces known reprogramming protocols and identifies candidate transcription factors for reprogramming to novel cell fates, suggesting epigenetic landscapes are a powerful paradigm for understanding cellular identity.

  4. Reproducible research in palaeomagnetism

    NASA Astrophysics Data System (ADS)

    Lurcock, Pontus; Florindo, Fabio

    2015-04-01

    The reproducibility of research findings is attracting increasing attention across all scientific disciplines. In palaeomagnetism as elsewhere, computer-based analysis techniques are becoming more commonplace, complex, and diverse. Analyses can often be difficult to reproduce from scratch, both for the original researchers and for others seeking to build on the work. We present a palaeomagnetic plotting and analysis program designed to make reproducibility easier. Part of the problem is the divide between interactive and scripted (batch) analysis programs. An interactive desktop program with a graphical interface is a powerful tool for exploring data and iteratively refining analyses, but usually cannot operate without human interaction. This makes it impossible to re-run an analysis automatically, or to integrate it into a larger automated scientific workflow - for example, a script to generate figures and tables for a paper. In some cases the parameters of the analysis process itself are not saved explicitly, making it hard to repeat or improve the analysis even with human interaction. Conversely, non-interactive batch tools can be controlled by pre-written scripts and configuration files, allowing an analysis to be 'replayed' automatically from the raw data. However, this advantage comes at the expense of exploratory capability: iteratively improving an analysis entails a time-consuming cycle of editing scripts, running them, and viewing the output. Batch tools also tend to require more computer expertise from their users. PuffinPlot is a palaeomagnetic plotting and analysis program which aims to bridge this gap. First released in 2012, it offers both an interactive, user-friendly desktop interface and a batch scripting interface, both making use of the same core library of palaeomagnetic functions. We present new improvements to the program that help to integrate the interactive and batch approaches, allowing an analysis to be interactively explored and refined

  5. Novel Analysis of Immune Cells from Nasal Microbiopsy Demonstrates Reliable, Reproducible Data for Immune Populations, and Superior Cytokine Detection Compared to Nasal Wash

    PubMed Central

    Rylance, Jamie; Adler, Hugh; Carniel, Beatriz F.; Collins, Andrea; Gritzfeld, Jenna F.; Hancock, Carole; Hill, Helen; Reiné, Jesus; Seddon, Alexandra; Solórzano, Carla; Sunny, Syba; Trimble, Ashleigh; Wright, Angela D.; Zaidi, Seher; Gordon, Stephen B.; Ferreira, Daniela M.

    2017-01-01

    The morbidity and mortality related to respiratory tract diseases is enormous, with hundreds of millions of individuals afflicted and four million people dying each year. Understanding the immunological processes in the mucosa that govern outcome following pathogenic encounter could lead to novel therapies. There is a need to study responses at mucosal surfaces in humans for two reasons: (i) Immunological findings in mice, or other animals, often fail to translate to humans. (ii) Compartmentalization of the immune system dictates a need to study sites where pathogens reside. In this manuscript, we describe two novel non-invasive nasal mucosal microsampling techniques and their use for measuring immunological parameters: 1) using nasal curettes to collect cells from the inferior turbinate and; 2) absorptive matrices to collect nasal lining fluid. Both techniques were well tolerated and yielded reproducible and robust data. We demonstrated differences in immune populations and activation state in nasal mucosa compared to blood as well as compared to nasopharyngeal lumen in healthy adults. We also found superior cytokine detection with absorptive matrices compared to nasal wash. These techniques are promising new tools that will facilitate studies of the immunological signatures underlying susceptibility and resistance to respiratory infections. PMID:28107457

  6. Use of a Novel Embryonic Mammary Stem Cell Gene Signature to Improve Human Breast Cancer Diagnostics and Therapeutic Decision Making

    DTIC Science & Technology

    2013-10-01

    most recent funding period, our bioinformatic analysis identified subsets of fMaSC signature genes that are coordinately expressed in archived human...adapted a new microfluidics -based, single-cell capture and library preparation system to improve reproducibility in the generation of gene expression...fraction, a two-class Significance Analysis of Microarrays (SAM) [7, 8] was performed within each dataset comparing each given FACS population

  7. Patent Law's Reproducibility Paradox.

    PubMed

    Sherkow, Jacob S

    2017-01-01

    Clinical research faces a reproducibility crisis. Many recent clinical and preclinical studies appear to be irreproducible--their results cannot be verified by outside researchers. This is problematic for not only scientific reasons but also legal ones: patents grounded in irreproducible research appear to fail their constitutional bargain of property rights in exchange for working disclosures of inventions. The culprit is likely patent law’s doctrine of enablement. Although the doctrine requires patents to enable others to make and use their claimed inventions, current difficulties in applying the doctrine hamper or even actively dissuade reproducible data in patents. This Article assesses the difficulties in reconciling these basic goals of scientific research and patent law. More concretely, it provides several examples of irreproducibility in patents on blockbuster drugs--Prempro, Xigris, Plavix, and Avastin--and discusses some of the social costs of the misalignment between good clinical practice and patent doctrine. Ultimately, this analysis illuminates several current debates concerning innovation policy. It strongly suggests that a proper conception of enablement should take into account after-arising evidence. It also sheds light on the true purpose--and limits--of patent disclosure. And lastly, it untangles the doctrines of enablement and utility.

  8. Opening Reproducible Research

    NASA Astrophysics Data System (ADS)

    Nüst, Daniel; Konkol, Markus; Pebesma, Edzer; Kray, Christian; Klötgen, Stephanie; Schutzeichel, Marc; Lorenz, Jörg; Przibytzin, Holger; Kussmann, Dirk

    2016-04-01

    Open access is not only a form of publishing such that research papers become available to the large public free of charge, it also refers to a trend in science that the act of doing research becomes more open and transparent. When science transforms to open access we not only mean access to papers, research data being collected, or data being generated, but also access to the data used and the procedures carried out in the research paper. Increasingly, scientific results are generated by numerical manipulation of data that were already collected, and may involve simulation experiments that are completely carried out computationally. Reproducibility of research findings, the ability to repeat experimental procedures and confirm previously found results, is at the heart of the scientific method (Pebesma, Nüst and Bivand, 2012). As opposed to the collection of experimental data in labs or nature, computational experiments lend themselves very well for reproduction. Some of the reasons why scientists do not publish data and computational procedures that allow reproduction will be hard to change, e.g. privacy concerns in the data, fear for embarrassment or of losing a competitive advantage. Others reasons however involve technical aspects, and include the lack of standard procedures to publish such information and the lack of benefits after publishing them. We aim to resolve these two technical aspects. We propose a system that supports the evolution of scientific publications from static papers into dynamic, executable research documents. The DFG-funded experimental project Opening Reproducible Research (ORR) aims for the main aspects of open access, by improving the exchange of, by facilitating productive access to, and by simplifying reuse of research results that are published over the Internet. Central to the project is a new form for creating and providing research results, the executable research compendium (ERC), which not only enables third parties to

  9. Epigenetic Landscapes Explain Partially Reprogrammed Cells and Identify Key Reprogramming Genes

    PubMed Central

    Lang, Alex H.; Li, Hu; Collins, James J.; Mehta, Pankaj

    2014-01-01

    A common metaphor for describing development is a rugged “epigenetic landscape” where cell fates are represented as attracting valleys resulting from a complex regulatory network. Here, we introduce a framework for explicitly constructing epigenetic landscapes that combines genomic data with techniques from spin-glass physics. Each cell fate is a dynamic attractor, yet cells can change fate in response to external signals. Our model suggests that partially reprogrammed cells are a natural consequence of high-dimensional landscapes, and predicts that partially reprogrammed cells should be hybrids that co-express genes from multiple cell fates. We verify this prediction by reanalyzing existing datasets. Our model reproduces known reprogramming protocols and identifies candidate transcription factors for reprogramming to novel cell fates, suggesting epigenetic landscapes are a powerful paradigm for understanding cellular identity. PMID:25122086

  10. Antioxidant gene therapy against neuronal cell death

    PubMed Central

    Navarro-Yepes, Juliana; Zavala-Flores, Laura; Annadurai, Anandhan; Wang, Fang; Skotak, Maciej; Chandra, Namas; Li, Ming; Pappa, Aglaia; Martinez-Fong, Daniel; Razo, Luz Maria Del; Quintanilla-Vega, Betzabet; Franco, Rodrigo

    2014-01-01

    Oxidative stress is a common hallmark of neuronal cell death associated with neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, as well as brain stroke/ischemia and traumatic brain injury. Increased accumulation of reactive species of both oxygen (ROS) and nitrogen (RNS) has been implicated in mitochondrial dysfunction, energy impairment, alterations in metal homeostasis and accumulation of aggregated proteins observed in neurodegenerative disorders, which lead to the activation/modulation of cell death mechanisms that include apoptotic, necrotic and autophagic pathways. Thus, the design of novel antioxidant strategies to selectively target oxidative stress and redox imbalance might represent important therapeutic approaches against neurological disorders. This work reviews the evidence demonstrating the ability of genetically encoded antioxidant systems to selectively counteract neuronal cell loss in neurodegenerative diseases and ischemic brain damage. Because gene therapy approaches to treat inherited and acquired disorders offer many unique advantages over conventional therapeutic approaches, we discussed basic research/clinical evidence and the potential of virus-mediated gene delivery techniques for antioxidant gene therapy. PMID:24333264

  11. Ankylosing Spondylitis: From Cells to Genes

    PubMed Central

    Zambrano-Zaragoza, José Francisco; Agraz-Cibrian, Juan Manuel; González-Reyes, Christian; Durán-Avelar, Ma. de Jesús; Vibanco-Pérez, Norberto

    2013-01-01

    Ankylosing spondylitis (AS) is a chronic inflammatory disease of unknown etiology, though it is considered an autoimmune disease. HLA-B27 is the risk factor most often associated with AS, and although the mechanism of involvement is unclear, the subtypes and other features of the relationship between HLA-B27 and AS have been studied for years. Additionally, the key role of IL-17 and Th17 cells in autoimmunity and inflammation suggests that the latter and the cytokines involved in their generation could play a role in the pathogenesis of this disease. Recent studies have described the sources of IL-17 and IL-23, as well as the characterization of Th17 cells in autoimmune diseases. Other cells, such as NK and regulatory T cells, have been implicated in autoimmunity and have been evaluated to ascertain their possible role in AS. Moreover, several polymorphisms, mutations and deletions in the regulatory proteins, protein-coding regions, and promoter regions of different genes involved in immune responses have been discovered and evaluated for possible genetic linkages to AS. In this review, we analyze the features of HLA-B27 and the suggested mechanisms of its involvement in AS while also focusing on the characterization of the immune response and the identification of genes associated with AS. PMID:23970995

  12. Highly reproducible, efficient hysteresis-less CH3NH3PbI3-xClx planar hybrid solar cells without requiring heat-treatment

    NASA Astrophysics Data System (ADS)

    Heo, Jin Hyuck; Im, Sang Hyuk

    2016-01-01

    CH3NH3PbI3-xClx(MAPbI3-xClx) mixed halide perovskite powder with uniform composition was synthesized via simple solution chemistry, which demonstrates highly reproducible, efficient planar type MAPbI3-xClx mixed halide perovskite solar cells. Pure MAPbI3-xClx mixed halide perovskite powder was synthesized by reacting a 3 : 1 molar ratio of MAI : PbCl2 powder mixture in isopropanol (IPA) solution for 30 min at 60 °C with subsequent repeated centrifugation and washing in IPA. IPA functions as both the reaction medium for the formation of MAPbI3-xClx mixed halide and a selective remover of unreacted MAI and MACl byproducts. Accordingly, we could deposit a pinhole-free dense MAPbI3-xClx mixed halide perovskite film on a TiO2/FTO substrate through a simple one step spin-coating of pure MAPbI3-xClx mixed halide perovskite powder in DMF solution with HI additive, without further long heat-treatment processes. The deposited MAPbI3-xClx mixed halide perovskite film revealed uniform composition throughout the entire area, and the ratio of Cl to I + Cl and I + Cl to Pb was constant at ~0.03 and ~1/3, respectively. On the other hand, the conventional MAPbI3-xClx mixed halide perovskite film prepared by the long heat-treatment process had non-uniform composition because the ratio of Cl to I + Cl fluctuated greatly from 0 to 7.2. The average efficiency of planar type MAPbI3-xClx mixed halide perovskite solar cells was 18.65% +/- 0.30% and the champion cell had 1.11 V Voc, 22.1 mA cm-2Jsc, 77% F.F., and 18.9% η for forward scan conditions and 1.11 V Voc, 22.1 mA cm-2Jsc, 78% F.F., and 19.1% η for reverse scan conditions. Although the thickness of the MAPbI3-xClx mixed halide perovskite layer varied from ~500 nm to ~900 nm, the efficiency was within the range of 18.3%-19.0%.CH3NH3PbI3-xClx(MAPbI3-xClx) mixed halide perovskite powder with uniform composition was synthesized via simple solution chemistry, which demonstrates highly reproducible, efficient planar type MAPbI3-x

  13. Neural stem cell gene therapy ameliorates pathology and function in a mouse model of globoid cell leukodystrophy.

    PubMed

    Neri, Margherita; Ricca, Alessandra; di Girolamo, Ilaria; Alcala'-Franco, Beatriz; Cavazzin, Chiara; Orlacchio, Aldo; Martino, Sabata; Naldini, Luigi; Gritti, Angela

    2011-10-01

    Murine neural stem cells (mNSCs), either naive or genetically modified to express supranormal levels of β-galactocerebrosidase (GALC), were transplanted into the brain of Twitcher mice, a murine model of globoid cell leukodystrophy, a severe sphingolipidosis. Cells engrafted long-term into the host cytoarchitecture, producing functional GALC. Levels of enzyme activity in brain and spinal cord tissues were enhanced when GALC-overexpressing NSC were used. Enzymatic correction correlated with reduced tissue storage, decreased activation of astroglia and microglia, delayed onset of symptoms, and longer lifespan. Mechanisms underlying the therapeutic effect of mNSC included widespread enzyme distribution, cross-correction of host cells, anti-inflammatory activity, and neuroprotection. Similar cell engraftment and metabolic correction were reproduced using human NSC. Thus, NSC gene therapy rapidly reconstitutes sustained and long-lasting enzyme activity in central nervous system tissues. Combining this approach with treatments targeting the systemic disease associated with leukodystrophies may provide significant therapeutic benefit.

  14. The stem-cell profile of ovarian surface epithelium is reproduced in the oviductal fimbriae, with increased stem-cell marker density in distal parts of the fimbriae.

    PubMed

    Auersperg, Nelly

    2013-09-01

    High-grade serous ovarian carcinomas are the most common and most lethal ovarian cancers, but their histologic origin is still controversial. Current evidence suggests that they may originate in the ovarian surface epithelium (OSE) and/or epithelium of oviductal fimbriae (FE). To further investigate this question we compared the stem-cell profiles of these epithelia. Formalin-fixed sections of normal FE (N=21) and ovaries (N=21) were stained immunohistochemically for the stem-cell markers NANOG, SFRP1, LHX9, ALDH1A1, and ALDH1A2. All markers were detected in both OSE and FE. A total of 75% to 100% of surface OSE expressed all markers except ALDH1A1, which occurred in about 25% of cells. Among epithelial inclusion cysts with flat-to-cuboidal epithelium, resembling OSE, ALDH1A1 was significantly increased, whereas SFRP1 was reduced compared with surface OSE, suggesting an increased trend towards malignant transformation. Similarly, among cysts lined by columnar cells resembling FE, SFRP1 expression was low, whereas ALDH1A1 approached 100% of the cysts. FE exhibited considerable variation between and within specimens. In about half of the samples, SFRP1 and NANOG were detected in ≤25% FE. The most widespread markers were ALDH1A1 and ALDH1A2. The highest proportion of all markers occurred in the distal parts of the FE, the site of the putative ovarian cancer precursors. Marker expression in tubal ampullae was low or absent except for ALDH1A1 and ALDH1A2. The results provide an explanation for the characteristic distal location of fimbrial high-grade serous ovarian carcinoma precursor lesions, and indicate that both OSE and FE have the capacity to undergo neoplastic transformation.

  15. Investigation of deregulated genes of Notch signaling pathway in human T cell acute lymphoblastic leukemia cell lines and clinical samples.

    PubMed

    Paryan, Mahdi; Mohammadi-Yeganeh, Samira; Samiee, Siamak Mirab; Soleimani, Masoud; Arefian, Ehsan; Azadmanesh, Keyhan; Poopak, Behzad; Mostafavi, Ehsan; Karimipoor, Morteza; Mahdian, Reza

    2013-10-01

    In diagnostic research challenges, quantitative real-time PCR (QPCR) has been widely utilized in gene expression analysis because of its sensitivity, accuracy, reproducibility, and most importantly, quantitativeness. Real-time PCR base kits are wildly applicable in cancer signaling pathways, especially in cancer investigations. T-cell acute lymphoblastic leukemia (T-ALL) is a type of leukemia that is more common in older children and teenagers. Deregulation of the Notch signaling pathway promotes proliferation and inhibits apoptosis of the lymphoblastic T cells. The aim of this study was to investigate the effect of Notch signaling activation on the expression of target genes using real-time QPCR and further use this method in clinical examination after validation. Two T-ALL cell lines, Jurkat and Molt-4, were used as models for activation of the Notch signaling via over-expression of the Notch1 intracellular domain. Expression analysis was performed for six downstream target genes (NCSTN, APH1, PSEN1, ADAM17, NOTCH1 and C-MYC) which play critical roles in the Notch signaling pathway. The results showed significant difference in the expression of target genes in the deregulated Notch signaling pathway. These results were also verified in 12 clinical samples bearing over-expression of the Notch signaling pathway. Identification of such downstream Notch target genes, which have not been studied inclusively, provides insights into the mechanisms of the Notch function in T cell leukemia, and may help identify novel diagnoses and therapeutic targets in acute lymphoblastic leukemia.

  16. Ex Vivo Generated Natural Killer Cells Acquire Typical Natural Killer Receptors and Display a Cytotoxic Gene Expression Profile Similar to Peripheral Blood Natural Killer Cells

    PubMed Central

    Lehmann, Dorit; Spanholtz, Jan; Osl, Markus; Tordoir, Marleen; Lipnik, Karoline; Bilban, Martin; Schlechta, Bernhard; Dolstra, Harry

    2012-01-01

    Ex vivo differentiation systems of natural killer (NK) cells from CD34+ hematopoietic stem cells are of potential importance for adjuvant immunotherapy of cancer. Here, we analyzed ex vivo differentiation of NK cells from cord blood-derived CD34+ stem cells by gene expression profiling, real-time RT-PCR, flow cytometry, and functional analysis. Additionally, we compared the identified characteristics to peripheral blood (PB) CD56bright and CD56dim NK cells. The data show sequential expression of CD56 and the CD94 and NKG2 receptor chains during ex vivo NK cell development, resulting finally in the expression of a range of genes with partial characteristics of CD56bright and CD56dim NK cells from PB. Expression of characteristic NK cell receptors and cytotoxic genes was mainly found within the predominant ex vivo generated population of NKG2A+ NK cells, indicating the importance of NKG2A expression during NK cell differentiation and maturation. Furthermore, despite distinct phenotypic characteristics, the detailed analysis of cytolytic genes expressed within the ex vivo differentiated NK cells revealed a pattern close to CD56dim NK cells. In line with this finding, ex vivo generated NK cells displayed potent cytotoxicity. This supports that the ex vivo differentiation system faithfully reproduces major steps of the differentiation of NK cells from their progenitors, constitutes an excellent model to study NK cell differentiation, and is valuable to generate large-scale NK cells appropriate for immunotherapy. PMID:22571679

  17. Ex vivo generated natural killer cells acquire typical natural killer receptors and display a cytotoxic gene expression profile similar to peripheral blood natural killer cells.

    PubMed

    Lehmann, Dorit; Spanholtz, Jan; Osl, Markus; Tordoir, Marleen; Lipnik, Karoline; Bilban, Martin; Schlechta, Bernhard; Dolstra, Harry; Hofer, Erhard

    2012-11-01

    Ex vivo differentiation systems of natural killer (NK) cells from CD34+ hematopoietic stem cells are of potential importance for adjuvant immunotherapy of cancer. Here, we analyzed ex vivo differentiation of NK cells from cord blood-derived CD34+ stem cells by gene expression profiling, real-time RT-PCR, flow cytometry, and functional analysis. Additionally, we compared the identified characteristics to peripheral blood (PB) CD56(bright) and CD56(dim) NK cells. The data show sequential expression of CD56 and the CD94 and NKG2 receptor chains during ex vivo NK cell development, resulting finally in the expression of a range of genes with partial characteristics of CD56(bright) and CD56(dim) NK cells from PB. Expression of characteristic NK cell receptors and cytotoxic genes was mainly found within the predominant ex vivo generated population of NKG2A+ NK cells, indicating the importance of NKG2A expression during NK cell differentiation and maturation. Furthermore, despite distinct phenotypic characteristics, the detailed analysis of cytolytic genes expressed within the ex vivo differentiated NK cells revealed a pattern close to CD56(dim) NK cells. In line with this finding, ex vivo generated NK cells displayed potent cytotoxicity. This supports that the ex vivo differentiation system faithfully reproduces major steps of the differentiation of NK cells from their progenitors, constitutes an excellent model to study NK cell differentiation, and is valuable to generate large-scale NK cells appropriate for immunotherapy.

  18. Gene therapy of primary T cell immunodeficiencies.

    PubMed

    Fischer, Alain; Hacein-Bey-Abina, Salima; Cavazzana-Calvo, Marina

    2013-08-10

    Gene therapy of severe combined immunodeficiencies has been proven to be effective to provide sustained correction of the T cell immunodeficiencies. This has been achieved for 2 forms of SCID, i.e SCID-X1 (γc deficiency) and adenosine deaminase deficiency. Occurrence of gene toxicity generated by integration of first generation retroviral vectors, as observed in the SCID-X1 trials has led to replace these vectors by self inactivated (SIN) retro(or lenti) viruses that may provide equivalent efficacy with a better safety profile. Results of ongoing clinical studies in SCID as well as in other primary immunodeficiencies, such as the Wiskott Aldrich syndrome, will be thus very informative.

  19. IL-1/IL-3 gene therapy of non-small cell lung cancer (NSCLC) in rats using 'cracked' adenoproducer cells.

    PubMed

    Esandi, M C; van Someren, G D; Bout, A; Mulder, A H; van Bekkum, D W; Valerio, D; Noteboom, J L

    1998-06-01

    Cytokine gene therapy was studied in established L42 tumours in syngeneic rats. L42 is a transplantable non-immunogenic non-small cell lung cancer (NSCLC). Genes coding for human interleukin-1 alpha and for rat interleukin-3 beta were transferred by injecting producer cells of recombinant adenovirus vectors into the tumour in attempts to achieve high concentrations of the cytokines inside the tumor without systemic toxicity. Limited tumour growth delay was obtained with viable producer cells. For logistic reasons stocks of pooled frozen producer cells allowed intensive treatment of groups of tumour bearing rats. The cells were lysed by thawing before administration. Ten daily injections of such 'cracked' producer cells induced reproducible tumour responses. These were due to local release of cytokines, not to systemic effects. Growth retardation also occurred in contralateral tumours which were not injected. When rats carrying established tumours were vaccinated with lysates of tumours collected during treatment with 'cracked' producer cells, significant tumour growth retardation was obtained. We speculate that both cytokines, if produced at sufficiently high concentrations in tumours, induce inflammation which in turn initiates an immune response against tumours growing at a distant site. These findings seem to justify further exploration of IL-1 and IL-3 gene transfer for the treatment of cancers.

  20. Gene Expression by Mouse Inner Ear Hair Cells during Development

    PubMed Central

    Scheffer, Déborah I.; Shen, Jun

    2015-01-01

    Hair cells of the inner ear are essential for hearing and balance. As a consequence, pathogenic variants in genes specifically expressed in hair cells often cause hereditary deafness. Hair cells are few in number and not easily isolated from the adjacent supporting cells, so the biochemistry and molecular biology of hair cells can be difficult to study. To study gene expression in hair cells, we developed a protocol for hair cell isolation by FACS. With nearly pure hair cells and surrounding cells, from cochlea and utricle and from E16 to P7, we performed a comprehensive cell type-specific RNA-Seq study of gene expression during mouse inner ear development. Expression profiling revealed new hair cell genes with distinct expression patterns: some are specific for vestibular hair cells, others for cochlear hair cells, and some are expressed just before or after maturation of mechanosensitivity. We found that many of the known hereditary deafness genes are much more highly expressed in hair cells than surrounding cells, suggesting that genes preferentially expressed in hair cells are good candidates for unknown deafness genes. PMID:25904789

  1. Toward stable gene expression in CHO cells

    PubMed Central

    Mariati; Koh, Esther YC; Yeo, Jessna HM; Ho, Steven CL; Yang, Yuansheng

    2014-01-01

    Maintaining high gene expression level during long-term culture is critical when producing therapeutic recombinant proteins using mammalian cells. Transcriptional silencing of promoters, most likely due to epigenetic events such as DNA methylation and histone modifications, is one of the major mechanisms causing production instability. Previous studies demonstrated that the core CpG island element (IE) from the hamster adenine phosphoribosyltransferase gene is effective to prevent DNA methylation. We generated one set of modified human cytomegalovirus (hCMV) promoters by insertion of one or two copies of IE in either forward or reverse orientations into different locations of the hCMV promoter. The modified hCMV with one copy of IE inserted between the hCMV enhancer and core promoter in reverse orientation (MR1) was most effective at enhancing expression stability in CHO cells without comprising expression level when compared with the wild type hCMV. We also found that insertion of IE into a chimeric murine CMV (mCMV) enhancer and human elongation factor-1α core (hEF) promoter in reverse orientation did not enhance expression stability, indicating that the effect of IE on expression stability is possibly promoter specific. PMID:25482237

  2. Aire knockdown in medullary thymic epithelial cells affects Aire protein, deregulates cell adhesion genes and decreases thymocyte interaction.

    PubMed

    Pezzi, Nicole; Assis, Amanda Freire; Cotrim-Sousa, Larissa Cotrim; Lopes, Gabriel Sarti; Mosella, Maritza Salas; Lima, Djalma Sousa; Bombonato-Prado, Karina F; Passos, Geraldo Aleixo

    2016-09-01

    We demonstrate that even a partial reduction of Aire mRNA levels by siRNA-induced Aire knockdown (Aire KD) has important consequences to medullary thymic epithelial cells (mTECs). Aire knockdown is sufficient to reduce Aire protein levels, impair its nuclear location, and cause an imbalance in large-scale gene expression, including genes that encode cell adhesion molecules. These genes drew our attention because adhesion molecules are implicated in the process of mTEC-thymocyte adhesion, which is critical for T cell development and the establishment of central self-tolerance. Accordingly, we consider the following: 1) mTECs contribute to the elimination of self-reactive thymocytes through adhesion; 2) Adhesion molecules play a crucial role during physical contact between these cells; and 3) Aire is an important transcriptional regulator in mTECs. However, its role in controlling mTEC-thymocyte adhesion remains unclear. Because Aire controls adhesion molecule genes, we hypothesized that the disruption of its expression could influence mTEC-thymocyte interaction. To test this hypothesis, we used a murine Aire(+) mTEC cell line as a model system to reproduce mTEC-thymocyte adhesion in vitro. Transcriptome analysis of the mTEC cell line revealed that Aire KD led to the down-modulation of more than 800 genes, including those encoding for proteins involved in cell adhesion, i.e., the extracellular matrix constituent Lama1, the CAM family adhesion molecules Vcam1 and Icam4, and those that encode peripheral tissue antigens. Thymocytes co-cultured with Aire KD mTECs had a significantly reduced capacity to adhere to these cells. This finding is the first direct evidence that Aire also plays a role in controlling mTEC-thymocyte adhesion.

  3. DNA repair genes of mammalian cells

    SciTech Connect

    Thompson, L.H.; Brookman, K.W.; Salazar, E.P.; Fuscoe, J.C.; Weber, C.A.

    1985-09-27

    In the CHO cell line various mutations affecting DNA repair have been obtained. Mutants that belong to five genetic complementation groups for UV sensitivity and resemble the cells from individuals having the cancer-prone genetic disorder xeroderma pigmentosum were previously identified. Each mutant is defective in the incision step of nucleotide excision repair and hypersensitive to bulky DNA lesions. A sixth genetic complementation group for UV sensitivity has now been identified with UV27-1. These UV mutants can be divided into two subgroups; only Groups 2 and 4 are extremely sensitive to mitomycin C and other DNA cross-linking agents. The clear-cut phenotypes of the CHO mutants have allowed us to construct hybrid cells by fusion with human lymphocytes and thereby identify which human chromosomes carry genes that correct the CHO mutations. The first two mutants analyzed, UV20 (excision-repair deficient; UV Group 2) and EM9, which has very high SCE, are both corrected by chromosome 19. 46 refs., 3 figs.

  4. Transient gene expression in epidermal cells of plant leaves by biolistic DNA delivery.

    PubMed

    Ueki, Shoko; Magori, Shimpei; Lacroix, Benoît; Citovsky, Vitaly

    2013-01-01

    Transient gene expression is a useful approach for studying the functions of gene products. In the case of plants, Agrobacterium infiltration is a method of choice for transient introduction of genes for many species. However, this technique does not work efficiently in some species, such as Arabidopsis thaliana. Moreover, the infection of Agrobacterium is known to induce dynamic changes in gene expression patterns in the host plants, possibly affecting the function and localization of the proteins to be tested. These problems can be circumvented by biolistic delivery of the genes of interest. Here, we present an optimized protocol for biolistic delivery of plasmid DNA into epidermal cells of plant leaves, which can be easily performed using the Bio-Rad Helios gene gun system. This protocol allows efficient and reproducible transient expression of diverse genes in Arabidopsis, Nicotiana benthamiana and N. tabacum, and is suitable for studies of the biological function and subcellular localization of the gene products directly in planta. The protocol also can be easily adapted to other species by optimizing the delivery gas pressure.

  5. Reproducibility in a multiprocessor system

    SciTech Connect

    Bellofatto, Ralph A; Chen, Dong; Coteus, Paul W; Eisley, Noel A; Gara, Alan; Gooding, Thomas M; Haring, Rudolf A; Heidelberger, Philip; Kopcsay, Gerard V; Liebsch, Thomas A; Ohmacht, Martin; Reed, Don D; Senger, Robert M; Steinmacher-Burow, Burkhard; Sugawara, Yutaka

    2013-11-26

    Fixing a problem is usually greatly aided if the problem is reproducible. To ensure reproducibility of a multiprocessor system, the following aspects are proposed; a deterministic system start state, a single system clock, phase alignment of clocks in the system, system-wide synchronization events, reproducible execution of system components, deterministic chip interfaces, zero-impact communication with the system, precise stop of the system and a scan of the system state.

  6. cell type–specific gene expression differences in complex tissues

    PubMed Central

    Shen-Orr, Shai S; Tibshirani, Robert; Khatri, Purvesh; Bodian, Dale L; Staedtler, Frank; Perry, Nicholas M; Hastie, Trevor; Sarwal, Minnie M; Davis, Mark M; Butte, Atul J

    2013-01-01

    We describe cell type–specific significance analysis of microarrays (cssam) for analyzing differential gene expression for each cell type in a biological sample from microarray data and relative cell-type frequencies. first, we validated cssam with predesigned mixtures and then applied it to whole-blood gene expression datasets from stable post-transplant kidney transplant recipients and those experiencing acute transplant rejection, which revealed hundreds of differentially expressed genes that were otherwise undetectable. PMID:20208531

  7. Cell dynamics and gene expression control in tissue homeostasis and development.

    PubMed

    Rué, Pau; Martinez Arias, Alfonso

    2015-02-25

    During tissue and organ development and maintenance, the dynamic regulation of cellular proliferation and differentiation allows cells to build highly elaborate structures. The development of the vertebrate retina or the maintenance of adult intestinal crypts, for instance, involves the arrangement of newly created cells with different phenotypes, the proportions of which need to be tightly controlled. While some of the basic principles underlying these processes developing and maintaining these organs are known, much remains to be learnt from how cells encode the necessary information and use it to attain those complex but reproducible arrangements. Here, we review the current knowledge on the principles underlying cell population dynamics during tissue development and homeostasis. In particular, we discuss how stochastic fate assignment, cell division, feedback control and cellular transition states interact during organ and tissue development and maintenance in multicellular organisms. We propose a framework, involving the existence of a transition state in which cells are more susceptible to signals that can affect their gene expression state and influence their cell fate decisions. This framework, which also applies to systems much more amenable to quantitative analysis like differentiating embryonic stem cells, links gene expression programmes with cell population dynamics.

  8. Changes in expression of genes involved in apoptosis in activated human T-cells in response to modeled microgravity

    NASA Astrophysics Data System (ADS)

    Ward, Nancy E.; Pellis, Neal R.; Risin, Diana; Risin, Semyon A.; Liu, Wenbin

    2006-09-01

    Space flights result in remarkable effects on various physiological systems, including a decline in cellular immune functions. Previous studies have shown that exposure to microgravity, both true and modeled, can cause significant changes in numerous lymphocyte functions. The purpose of this study was to search for microgravity-sensitive genes, and specifically for apoptotic genes influenced by the microgravity environment and other genes related to immune response. The experiments were performed on anti-CD3 and IL-2 activated human T cells. To model microgravity conditions we have utilized the NASA rotating wall vessel bioreactor. Control lymphocytes were cultured in static 1g conditions. To assess gene expression we used DNA microarray chip technology. We had shown that multiple genes (approximately 3-8% of tested genes) respond to microgravity conditions by 1.5 and more fold change in expression. There is a significant variability in the response. However, a certain reproducible pattern in gene response could be identified. Among the genes showing reproducible changes in expression in modeled microgravity, several genes involved in apoptosis as well as in immune response were identified. These are IL-7 receptor, Granzyme B, Beta-3-endonexin, Apo2 ligand and STAT1. Possible functional consequences of these changes are discussed.

  9. Cotransformation and gene targeting in mouse embryonic stem cells.

    PubMed Central

    Reid, L H; Shesely, E G; Kim, H S; Smithies, O

    1991-01-01

    We have investigated cotransformation in mammalian cells and its potential for identifying cells that have been modified by gene targeting. Selectable genes on separate DNA fragments were simultaneously introduced into cells by coelectroporation. When the introduced fragments were scored for random integration, 75% of the transformed cells integrated both fragments within the genome of the same cell. When one of the cointroduced fragments was scored for integration at a specific locus by gene targeting, only 4% of the targeted cells cointegrated the second fragment. Apparently, cells that have been modified by gene targeting with one DNA fragment rarely incorporate a second DNA fragment. Despite this limitation, we were able to use the cotransformation protocol to identify targeted cells by screening populations of colonies that had been transformed with a cointroduced selectable gene. When hypoxanthine phosphoribosyltransferase (hprt) targeting DNA was coelectroporated with a selectable neomycin phosphotransferase (neo) gene into embryonic stem (ES) cells, hprt-targeted colonies were isolated from the population of neo transformants at a frequency of 1 per 70 G418-resistant colonies. In parallel experiments with the same targeting construct, hprt-targeted cells were found at a frequency of 1 per 5,500 nonselected colonies. Thus, an 80-fold enrichment for targeted cells was observed within the population of colonies transformed with the cointroduced DNA compared with the population of nonselected colonies. This enrichment for targeted cells after cotransformation should be useful in the isolation of colonies that contain targeted but nonselectable gene alterations. Images PMID:1850104

  10. Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control.

    PubMed

    Grant, Gavin D; Gamsby, Joshua; Martyanov, Viktor; Brooks, Lionel; George, Lacy K; Mahoney, J Matthew; Loros, Jennifer J; Dunlap, Jay C; Whitfield, Michael L

    2012-08-01

    We developed a system to monitor periodic luciferase activity from cell cycle-regulated promoters in synchronous cells. Reporters were driven by a minimal human E2F1 promoter with peak expression in G1/S or a basal promoter with six Forkhead DNA-binding sites with peak expression at G2/M. After cell cycle synchronization, luciferase activity was measured in live cells at 10-min intervals across three to four synchronous cell cycles, allowing unprecedented resolution of cell cycle-regulated gene expression. We used this assay to screen Forkhead transcription factors for control of periodic gene expression. We confirmed a role for FOXM1 and identified two novel cell cycle regulators, FOXJ3 and FOXK1. Knockdown of FOXJ3 and FOXK1 eliminated cell cycle-dependent oscillations and resulted in decreased cell proliferation rates. Analysis of genes regulated by FOXJ3 and FOXK1 showed that FOXJ3 may regulate a network of zinc finger proteins and that FOXK1 binds to the promoter and regulates DHFR, TYMS, GSDMD, and the E2F binding partner TFDP1. Chromatin immunoprecipitation followed by high-throughput sequencing analysis identified 4329 genomic loci bound by FOXK1, 83% of which contained a FOXK1-binding motif. We verified that a subset of these loci are activated by wild-type FOXK1 but not by a FOXK1 (H355A) DNA-binding mutant.

  11. Identification of Stem Cells in a Novel Human Mammary Epithelial Culture (HMEC) System that Reproducibly Demonstrates Ductal Organotypic Architecture in 3 Weeks

    DTIC Science & Technology

    2005-10-01

    maintaining HME stem cells in vitro. Clinically this study on stem cells will allow many laboratories to test the long held hypothesis that some...for CD24, a very marked difference between these cells and the BRL cell lines tested . In addition a very different profile for CD90 and CD81 was...different conditions were tested for 2 of these cell lines but cloning is achievable for all. 17 Fig. 12. Single cell pick up was physically

  12. Identification of Stem Cells in a Novel Human Mammary Epithelial Culture (HMEC) System that Reproducibly Demonstrates Ductal Organotypic Architecture in 3 Weeks

    DTIC Science & Technology

    2006-10-01

    maintaining HME stem cells in vitro. Clinically this study on stem cells will allow many laboratories to test the long held hypothesis that some...for CD24, a very marked difference between these cells and the BRL cell lines tested . In addition a very different profile for CD90 and CD81 was...different conditions were tested for 2 of these cell lines but cloning is achievable for all. 17 Fig. 12. Single cell pick up was physically

  13. Cesium-containing triple cation perovskite solar cells: improved stability, reproducibility and high efficiency† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ee03874j Click here for additional data file.

    PubMed Central

    Matsui, Taisuke; Seo, Ji-Youn; Domanski, Konrad; Correa-Baena, Juan-Pablo; Nazeeruddin, Mohammad Khaja; Zakeeruddin, Shaik M.; Tress, Wolfgang; Abate, Antonio; Hagfeldt, Anders; Grätzel, Michael

    2016-01-01

    Today's best perovskite solar cells use a mixture of formamidinium and methylammonium as the monovalent cations. With the addition of inorganic cesium, the resulting triple cation perovskite compositions are thermally more stable, contain less phase impurities and are less sensitive to processing conditions. This enables more reproducible device performances to reach a stabilized power output of 21.1% and ∼18% after 250 hours under operational conditions. These properties are key for the industrialization of perovskite photovoltaics. PMID:27478500

  14. Gene Therapy: a Breakthrough for Sickle Cell Anemia?

    MedlinePlus

    ... html Gene Therapy: A Breakthrough for Sickle Cell Anemia? But treatment has only been given to one ... to treat, or even potentially cure, sickle cell anemia. The findings come from just one patient, a ...

  15. Defining the chromatin signature of inducible genes in T cells

    PubMed Central

    2009-01-01

    Background Specific chromatin characteristics, especially the modification status of the core histone proteins, are associated with active and inactive genes. There is growing evidence that genes that respond to environmental or developmental signals may possess distinct chromatin marks. Using a T cell model and both genome-wide and gene-focused approaches, we examined the chromatin characteristics of genes that respond to T cell activation. Results To facilitate comparison of genes with similar basal expression levels, we used expression-profiling data to bin genes according to their basal expression levels. We found that inducible genes in the lower basal expression bins, especially rapidly induced primary response genes, were more likely than their non-responsive counterparts to display the histone modifications of active genes, have RNA polymerase II (Pol II) at their promoters and show evidence of ongoing basal elongation. There was little or no evidence for the presence of active chromatin marks in the absence of promoter Pol II on these inducible genes. In addition, we identified a subgroup of genes with active promoter chromatin marks and promoter Pol II but no evidence of elongation. Following T cell activation, we find little evidence for a major shift in the active chromatin signature around inducible gene promoters but many genes recruit more Pol II and show increased evidence of elongation. Conclusions These results suggest that the majority of inducible genes are primed for activation by having an active chromatin signature and promoter Pol II with or without ongoing elongation. PMID:19807913

  16. Novel radiation response genes identified in gene-trapped MCF10A mammary epithelial cells.

    PubMed

    Malone, Jennifer; Ullrich, Robert

    2007-02-01

    We have used a gene-trapping strategy to screen human mammary epithelial cells for radiation response genes. Relative mRNA expression levels of five candidate genes in MCF10A cells were analyzed, both with and without exposure to radiation. In all five cases, the trapped genes were significantly down-regulated after radiation treatment. Sequence analysis of the fusion transcripts identified the trapped genes: (1) the human androgen receptor, (2) the uncharacterized DREV1 gene, which has known homology to DNA methyltransferases, (3) the human creatine kinase gene, (4) the human eukaryotic translation elongation factor 1 beta 2, and (5) the human ribosomal protein L27. All five genes were down-regulated significantly after treatment with varying doses of ionizing radiation (0.10 to 4.0 Gy) and at varying times (2-30 h after treatment). The genes were also analyzed in human fibroblast and lymphoblastoid cell lines to determine whether the radiation response being observed was cell-type specific. The results verified that the observed radiation response was not a cell-type-specific phenomenon, suggesting that the genes play essential roles in the radiation damage control pathways. This study demonstrates the potential of the gene-trap approach for the identification and functional analysis of novel radiation response genes.

  17. Expression of ets family genes in hematopoietic-cells.

    PubMed

    Romanospica, V; Suzuki, H; Georgiou, P; Chen, S; Ascione, R; Papas, T; Bhat, N

    1994-03-01

    We have examined the expression of the ets family of transcription factors in different types of hematopoietic cells. Our results demonstrate that several members of the ets gene family are expressed differentially in hematopoietic cells. During phorbol ester induced differentiation of HL60 cells, ETS2, PEA3, as well as GABPalpha and GABPbeta mRNAs are coordinately induced. During the activation of T-cells, ETS2 proteins are induced; however, the expression of the ETS1 and ERGB gene products are reduced. These results demonstrate that the regulation of ets family of genes is complex and depends on cell type. This observation leads to the conclusion that the regulation of ets target genes, will be dependent, in part, upon the type of ets genes expressed in each particular cell type.

  18. Glial cell derived neurotrophic factor induces spermatogonial stem cell marker genes in chicken mesenchymal stem cells.

    PubMed

    Boozarpour, Sohrab; Matin, Maryam M; Momeni-Moghaddam, Madjid; Dehghani, Hesam; Mahdavi-Shahri, Naser; Sisakhtnezhad, Sajjad; Heirani-Tabasi, Asieh; Irfan-Maqsood, Muhammad; Bahrami, Ahmad Reza

    2016-06-01

    Mesenchymal stem cells (MSCs) are known with the potential of multi-lineage differentiation. Advances in differentiation technology have also resulted in the conversion of MSCs to other kinds of stem cells. MSCs are considered as a suitable source of cells for biotechnology purposes because they are abundant, easily accessible and well characterized cells. Nowadays small molecules are introduced as novel and efficient factors to differentiate stem cells. In this work, we examined the potential of glial cell derived neurotrophic factor (GDNF) for differentiating chicken MSCs toward spermatogonial stem cells. MSCs were isolated and characterized from chicken and cultured under treatment with all-trans retinoic acid (RA) or glial cell derived neurotrophic factor. Expression analysis of specific genes after 7days of RA treatment, as examined by RT-PCR, proved positive for some germ cell markers such as CVH, STRA8, PLZF and some genes involved in spermatogonial stem cell maintenance like BCL6b and c-KIT. On the other hand, GDNF could additionally induce expression of POU5F1, and NANOG as well as other genes which were induced after RA treatment. These data illustrated that GDNF is relatively more effective in diverting chicken MSCs towards Spermatogonial stem cell -like cells in chickens and suggests GDNF as a new agent to obtain transgenic poultry, nevertheless, exploitability of these cells should be verified by more experiments.

  19. Genes regulating touch cell development in Caenorhabditis elegans.

    PubMed Central

    Du, H; Chalfie, M

    2001-01-01

    To identify genes regulating the development of the six touch receptor neurons, we screened the F(2) progeny of mutated animals expressing an integrated mec-2::gfp transgene that is expressed mainly in these touch cells. From 2638 mutated haploid genomes, we obtained 11 mutations representing 11 genes that affected the production, migration, or outgrowth of the touch cells. Eight of these mutations were in known genes, and 2 defined new genes (mig-21 and vab-15). The mig-21 mutation is the first known to affect the asymmetry of the migrations of Q neuroblasts, the cells that give rise to two of the six touch cells. vab-15 is a msh-like homeobox gene that appears to be needed for the proper production of touch cell precursors, since vab-15 animals lacked the four more posterior touch cells. The remaining touch cells (the ALM cells) were present but mispositioned. A similar touch cell phenotype is produced by mutations in lin-32. A more severe phenotype; i.e., animals often lacked ALM cells, was seen in lin-32 vab-15 double mutants, suggesting that these genes acted redundantly in ALM differentiation. In addition to the touch cell abnormalities, vab-15 animals variably exhibit embryonic or larval lethality, cell degenerations, malformation of the posterior body, uncoordinated movement, and defective egg laying. PMID:11333230

  20. Analysis of mammary specific gene locus regulation in differentiated cells derived by somatic cell fusion

    SciTech Connect

    Robinson, Claire; Kolb, Andreas F.

    2009-02-01

    The transcriptional regulation of a gene is best analysed in the context of its normal chromatin surroundings. However, most somatic cells, in contrast to embryonic stem cells, are refractory to accurate modification by homologous recombination. We show here that it is possible to introduce precise genomic modifications in ES cells and to analyse the phenotypic consequences in differentiated cells by using a combination of gene targeting, site-specific recombination and somatic cell fusion. To provide a proof of principle, we have analysed the regulation of the casein gene locus in mammary gland cells derived from modified murine ES cells by somatic cell fusion. A {beta}-galactosidase reporter gene was inserted in place of the {beta}-casein gene and the modified ES cells, which do not express the reporter gene, were fused with the mouse mammary gland cell line HC11. The resulting cell clones expressed the {beta}-galactosidase gene to a similar extent and with similar hormone responsiveness as the endogenous gene. However, a reporter gene under the control of a minimal {beta}-casein promoter (encompassing the two consensus STAT5 binding sites which mediate the hormone response of the casein genes) was unable to replicate expression levels or hormone responsiveness of the endogenous gene when inserted into the same site of the casein locus. As expected, these results implicate sequences other than the STAT5 sites in the regulation of the {beta}-casein gene.

  1. Baculoviruses deficient in ie1 gene function abrogate viral gene expression in transduced mammalian cells

    SciTech Connect

    Efrose, Rodica; Swevers, Luc; Iatrou, Kostas

    2010-10-25

    One of the newest niches for baculoviruses-based technologies is their use as vectors for mammalian cell transduction and gene therapy applications. However, an outstanding safety issue related to such use is the residual expression of viral genes in infected mammalian cells. Here we show that infectious baculoviruses lacking the major transcriptional regulator, IE1, can be produced in insect host cells stably transformed with IE1 expression constructs lacking targets of homologous recombination that could promote the generation of wt-like revertants. Such ie1-deficient baculoviruses are unable to direct viral gene transcription to any appreciable degree and do not replicate in normal insect host cells. Most importantly, the residual viral gene expression, which occurs in mammalian cells infected with wt baculoviruses is reduced 10 to 100 fold in cells infected with ie1-deficient baculoviruses. Thus, ie1-deficient baculoviruses offer enhanced safety features to baculovirus-based vector systems destined for use in gene therapy applications.

  2. Diverse Gene Expression in Human Regulatory T Cell Subsets Uncovers Connection between Regulatory T Cell Genes and Suppressive Function.

    PubMed

    Hua, Jing; Davis, Scott P; Hill, Jonathan A; Yamagata, Tetsuya

    2015-10-15

    Regulatory T (Treg) cells have a critical role in the control of immunity, and their diverse subpopulations may allow adaptation to different types of immune responses. In this study, we analyzed human Treg cell subpopulations in the peripheral blood by performing genome-wide expression profiling of 40 Treg cell subsets from healthy donors. We found that the human peripheral blood Treg cell population is comprised of five major genomic subgroups, represented by 16 tractable subsets with a particular cell surface phenotype. These subsets possess a range of suppressive function and cytokine secretion and can exert a genomic footprint on target effector T (Teff) cells. Correlation analysis of variability in gene expression in the subsets identified several cell surface molecules associated with Treg suppressive function, and pharmacological interrogation revealed a set of genes having causative effect. The five genomic subgroups of Treg cells imposed a preserved pattern of gene expression on Teff cells, with a varying degree of genes being suppressed or induced. Notably, there was a cluster of genes induced by Treg cells that bolstered an autoinhibitory effect in Teff cells, and this induction appears to be governed by a different set of genes than ones involved in counteracting Teff activation. Our work shows an example of exploiting the diversity within human Treg cell subpopulations to dissect Treg cell biology.

  3. Gene Expression in Single Cells Isolated from the CWR-R1 Prostate Cancer Cell Line and Human Prostate Tissue Based on the Side Population Phenotype

    PubMed Central

    Gangavarapu, Kalyan J; Miller, Austin; Huss, Wendy J

    2016-01-01

    Defining biological signals at the single cell level can identify cancer initiating driver mutations. Techniques to isolate single cells such as microfluidics sorting and magnetic capturing systems have limitations such as: high cost, labor intense, and the requirement of a large number of cells. Therefore, the goal of our current study is to identify a cost and labor effective, reliable, and reproducible technique that allows single cell isolation for analysis to promote regular laboratory use, including standard reverse transcription PCR (RT-PCR). In the current study, we utilized single prostate cells isolated from the CWR-R1 prostate cancer cell line and human prostate clinical specimens, based on the ATP binding cassette (ABC) transporter efflux of dye cycle violet (DCV), side population assay. Expression of four genes: ABCG2; Aldehyde dehydrogenase1A1 (ALDH1A1); androgen receptor (AR); and embryonic stem cell marker, Oct-4, were determined. Results from the current study in the CWR-R1 cell line showed ABCG2 and ALDH1A1 gene expression in 67% of single side population cells and in 17% or 100% of non-side population cells respectively. Studies using single cells isolated from clinical specimens showed that the Oct-4 gene is detected in only 22% of single side population cells and in 78% of single non-side population cells. Whereas, AR gene expression is in 100% single side population and non-side population cells isolated from the same human prostate clinical specimen. These studies show that performing RT-PCR on single cells isolated by FACS can be successfully conducted to determine gene expression in single cells from cell lines and enzymatically digested tissue. While these studies provide a simple yes/no expression readout, the more sensitive quantitative RT-PCR would be able to provide even more information if necessary. PMID:27785389

  4. Contextual sensitivity in scientific reproducibility.

    PubMed

    Van Bavel, Jay J; Mende-Siedlecki, Peter; Brady, William J; Reinero, Diego A

    2016-06-07

    In recent years, scientists have paid increasing attention to reproducibility. For example, the Reproducibility Project, a large-scale replication attempt of 100 studies published in top psychology journals found that only 39% could be unambiguously reproduced. There is a growing consensus among scientists that the lack of reproducibility in psychology and other fields stems from various methodological factors, including low statistical power, researcher's degrees of freedom, and an emphasis on publishing surprising positive results. However, there is a contentious debate about the extent to which failures to reproduce certain results might also reflect contextual differences (often termed "hidden moderators") between the original research and the replication attempt. Although psychologists have found extensive evidence that contextual factors alter behavior, some have argued that context is unlikely to influence the results of direct replications precisely because these studies use the same methods as those used in the original research. To help resolve this debate, we recoded the 100 original studies from the Reproducibility Project on the extent to which the research topic of each study was contextually sensitive. Results suggested that the contextual sensitivity of the research topic was associated with replication success, even after statistically adjusting for several methodological characteristics (e.g., statistical power, effect size). The association between contextual sensitivity and replication success did not differ across psychological subdisciplines. These results suggest that researchers, replicators, and consumers should be mindful of contextual factors that might influence a psychological process. We offer several guidelines for dealing with contextual sensitivity in reproducibility.

  5. Murine somatic cell nuclear transfer using reprogrammed donor cells expressing male germ cell-specific genes.

    PubMed

    Kang, Hoin; Park, Jong Im; Roh, Sangho

    2016-01-01

    In vivo-matured mouse oocytes were enucleated, and a single murine embryonic fibroblast (control or reprogrammed by introducing extracts from murine testis tissue, which showed expression of male germ cell-specific genes) was injected into the cytoplasm of the oocytes. The rate of blastocyst development and expression levels of Oct-4, Eomes and Cdx-2 were not significantly different in both experimental groups. However, the expression levels of Nanog, Sox9 and Glut-1 were significantly increased when reprogrammed cells were used as donor nuclei. Increased expression of Nanog can be supportive of complete reprogramming of somatic cell nuclear transfer murine embryos. The present study suggested that donor cells expressing male germ cell-specific genes can be reconstructed and can develop into embryos with normal high expression of developmentally essential genes.

  6. Transcriptional targeting of tumor endothelial cells for gene therapy

    PubMed Central

    Dong, Zhihong; Nör, Jacques E.

    2009-01-01

    It is well known that angiogenesis plays a critical role in the pathobiology of tumors. Recent clinical trials have shown that inhibition of angiogenesis can be an effective therapeutic strategy for patients with cancer. However, one of the outstanding issues in anti-angiogenic treatment for cancer is the development of toxicities related to off-target effects of drugs. Transcriptional targeting of tumor endothelial cells involves the use of specific promoters for selective expression of therapeutic genes in the endothelial cells lining the blood vessels of tumors. Recently, several genes that are expressed specifically in tumor-associated endothelial cells have been identified and characterized. These discoveries have enhanced the prospectus of transcriptionaly targeting tumor endothelial cells for cancer gene therapy. In this manuscript, we review the promoters, vectors, and therapeutic genes that have been used for transcriptional targeting of tumor endothelial cells, and discuss the prospects of such approaches for cancer gene therapy. PMID:19393703

  7. The companions: regulatory T cells and gene therapy

    PubMed Central

    Eghtesad, Saman; Morel, Penelope A; Clemens, Paula R

    2009-01-01

    Undesired immunological responses to products of therapeutic gene replacement have been obstacles to successful gene therapy. Understanding such responses of the host immune system to achieve immunological tolerance to a transferred gene product is therefore crucial. In this article, we review relevant studies of immunological responses to gene replacement therapy, the role of immunological tolerance mediated by regulatory T cells in down-regulating the unwanted immune responses, and the interrelationship of the two topics. PMID:19368560

  8. ccmGDB: a database for cancer cell metabolism genes.

    PubMed

    Kim, Pora; Cheng, Feixiong; Zhao, Junfei; Zhao, Zhongming

    2016-01-04

    Accumulating evidence has demonstrated that rewiring of metabolism in cells is an important hallmark of cancer. The percentage of patients killed by metabolic disorder has been estimated to be 30% of the advanced-stage cancer patients. Thus, a systematic annotation of cancer cell metabolism genes is imperative. Here, we present ccmGDB (Cancer Cell Metabolism Gene DataBase), a comprehensive annotation database for cell metabolism genes in cancer, available at http://bioinfo.mc.vanderbilt.edu/ccmGDB. We assembled, curated, and integrated genetic, genomic, transcriptomic, proteomic, biological network and functional information for over 2000 cell metabolism genes in more than 30 cancer types. In total, we integrated over 260 000 somatic alterations including non-synonymous mutations, copy number variants and structural variants. We also integrated RNA-Seq data in various primary tumors, gene expression microarray data in over 1000 cancer cell lines and protein expression data. Furthermore, we constructed cancer or tissue type-specific, gene co-expression based protein interaction networks and drug-target interaction networks. Using these systematic annotations, the ccmGDB portal site provides 6 categories: gene summary, phenotypic information, somatic mutations, gene and protein expression, gene co-expression network and drug pharmacological information with a user-friendly interface for browsing and searching. ccmGDB is developed and maintained as a useful resource for the cancer research community.

  9. Single stem cell gene therapy for genetic skin disease.

    PubMed

    Larsimont, Jean-Christophe; Blanpain, Cédric

    2015-04-01

    Stem cell gene therapy followed by transplantation into damaged regions of the skin has been successfully used to treat genetic skin blistering disorder. Usually, many stem cells are virally transduced to obtain a sufficient number of genetically corrected cells required for successful transplantation, as genetic insertion in every stem cell cannot be precisely defined. In this issue of EMBO Molecular Medicine, Droz-Georget Lathion et al developed a new strategy for ex vivo single cell gene therapy that allows extensive genomic and functional characterization of the genetically repaired individual cells before they can be used in clinical settings.

  10. The added value of single-cell gene expression profiling.

    PubMed

    Ståhlberg, Anders; Rusnakova, Vendula; Kubista, Mikael

    2013-03-01

    Cells are the basic unit of life and they have remarkable abilities to respond individually as well as in concert to internal and external stimuli in a specific manner. Studying complex tissues and whole organs requires understanding of cell heterogeneity and responses to stimuli at the single-cell level. In this review, we discuss the potential of single-cell gene expression profiling, focusing on data analysis and biological interpretation. We exemplify several aspects of the added value of single-cell analysis by comparing the same experimental data at both single-cell and cell population level. Data normalization and handling of missing data are two important steps in data analysis that are performed differently at single-cell level compared with cell population level. Furthermore, we discuss how single-cell gene expression data can be viewed and how subpopulations of cells can be identified and characterized.

  11. Providencia stuartii genes activated by cell-to-cell signaling and identification of a gene required for production or activity of an extracellular factor.

    PubMed

    Rather, P N; Ding, X; Baca-DeLancey, R R; Siddiqui, S

    1999-12-01

    By utilizing reporter transposons, five Providencia stuartii genes that are activated by the accumulation of self-produced extracellular signals have been identified. These genes have been designated cma for conditioned medium activated. The presence of conditioned medium from stationary-phase cultures grown in rich media resulted in the premature activation of each gene in cells at early log phase, with activation values ranging from 6- to 26-fold. Preparation of conditioned medium from an M9 salts medium and fractionation by gel filtration chromatography resulted in fractions within the included volume which activated three of the cma fusions. In addition, depending on the reporter fusion, peak activity was found in different fractions. The partially purified factors activated in a dose-dependent manner. Characterization of the factors activating the cma fusions indicated that they were stable to heat, alkali, and acid. Furthermore, for each cma fusion, factor activity was not reproduced by the addition of homoserine lactone, homocysteine thiolactone, pyruvate, Casamino Acids, or alpha-ketoglutarate. The identities of three cma genes have been determined and revealed physiological roles in amino acid biosynthesis and nutrient import. To begin to address the pathways for production of or response to the extracellular factors, we have identified a locus, aarA, that is required for the activation of four cma fusions. The AarA product was required for factor activity in extracellular supernatants, indicating a possible role in biosynthesis or export.

  12. Open Science and Research Reproducibility

    PubMed Central

    Munafò, Marcus

    2016-01-01

    Many scientists, journals and funders are concerned about the low reproducibility of many scientific findings. One approach that may serve to improve the reliability and robustness of research is open science. Here I argue that the process of pre-registering study protocols, sharing study materials and data, and posting preprints of manuscripts may serve to improve quality control procedures at every stage of the research pipeline, and in turn improve the reproducibility of published work. PMID:27350794

  13. A highly sensitive and accurate gene expression analysis by sequencing ("bead-seq") for a single cell.

    PubMed

    Matsunaga, Hiroko; Goto, Mari; Arikawa, Koji; Shirai, Masataka; Tsunoda, Hiroyuki; Huang, Huan; Kambara, Hideki

    2015-02-15

    Analyses of gene expressions in single cells are important for understanding detailed biological phenomena. Here, a highly sensitive and accurate method by sequencing (called "bead-seq") to obtain a whole gene expression profile for a single cell is proposed. A key feature of the method is to use a complementary DNA (cDNA) library on magnetic beads, which enables adding washing steps to remove residual reagents in a sample preparation process. By adding the washing steps, the next steps can be carried out under the optimal conditions without losing cDNAs. Error sources were carefully evaluated to conclude that the first several steps were the key steps. It is demonstrated that bead-seq is superior to the conventional methods for single-cell gene expression analyses in terms of reproducibility, quantitative accuracy, and biases caused during sample preparation and sequencing processes.

  14. Regulation of erythroid cell-specific gene expression during erythropoiesis.

    PubMed Central

    Harrison, P. R.; Plumb, M.; Frampton, J.; Llewellyn, D.; Chester, J.; Chambers, I.; MacLeod, K.; Fleming, J.; O'Prey, J.; Walker, M.

    1988-01-01

    The aim of our group's work over the past few years has been to investigate the molecular mechanisms regulating erythroid cell-specific gene expression during erythroid cell differentiation. In addition to the alpha-globin gene, we have focussed on two non-globin genes of interest encoding the rabbit red cell-specific lipoxygenase (LOX) and the mouse glutathione peroxidase (GSHPX), an important seleno-enzyme responsible for protection against peroxide-damage. Characterisation of the GSHPX gene showed that the seleno-cysteine residue in the active site of the enzyme is encoded by UGA, which usually functions as a translation-termination codon. This novel finding has important implications regarding mRNA sequence context effects affecting codon recognition. The regulation of the GSHPX and red cell LOX genes has been investigated by functional transfection experiments. The 700 bp upstream of the GSHPX promoter seems to function equally well when linked to the bacterial chloramphenicol acetyl transferase (CAT) gene and transfected into mouse erythroid or fibroblast cell lines. However, the presence of tissue-specific DNase I hypersensitive sites (DHSS) in the 3' flanking region of the GSHPX gene suggests that such sites may be important in its regulation in the various cell types in which it is highly expressed, i.e., erythroid cells, liver and kidney. The transcription unit of the RBC LOX gene has also been defined and 5' and 3' flanking regions are being investigated for erythroid-specific regulatory elements: a region upstream of the LOX gene gives increased expression of a linked CAT gene when transfected into mouse erythroid cell lines compared to non-erythroid cell lines.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3151147

  15. Geometry of the Gene Expression Space of Individual Cells

    PubMed Central

    Korem, Yael; Szekely, Pablo; Hart, Yuval; Sheftel, Hila; Hausser, Jean; Mayo, Avi; Rothenberg, Michael E.; Kalisky, Tomer; Alon, Uri

    2015-01-01

    There is a revolution in the ability to analyze gene expression of single cells in a tissue. To understand this data we must comprehend how cells are distributed in a high-dimensional gene expression space. One open question is whether cell types form discrete clusters or whether gene expression forms a continuum of states. If such a continuum exists, what is its geometry? Recent theory on evolutionary trade-offs suggests that cells that need to perform multiple tasks are arranged in a polygon or polyhedron (line, triangle, tetrahedron and so on, generally called polytopes) in gene expression space, whose vertices are the expression profiles optimal for each task. Here, we analyze single-cell data from human and mouse tissues profiled using a variety of single-cell technologies. We fit the data to shapes with different numbers of vertices, compute their statistical significance, and infer their tasks. We find cases in which single cells fill out a continuum of expression states within a polyhedron. This occurs in intestinal progenitor cells, which fill out a tetrahedron in gene expression space. The four vertices of this tetrahedron are each enriched with genes for a specific task related to stemness and early differentiation. A polyhedral continuum of states is also found in spleen dendritic cells, known to perform multiple immune tasks: cells fill out a tetrahedron whose vertices correspond to key tasks related to maturation, pathogen sensing and communication with lymphocytes. A mixture of continuum-like distributions and discrete clusters is found in other cell types, including bone marrow and differentiated intestinal crypt cells. This approach can be used to understand the geometry and biological tasks of a wide range of single-cell datasets. The present results suggest that the concept of cell type may be expanded. In addition to discreet clusters in gene-expression space, we suggest a new possibility: a continuum of states within a polyhedron, in which the

  16. Random Monoallelic Gene Expression Increases upon Embryonic Stem Cell Differentiation

    PubMed Central

    Eckersley-Maslin, Mélanie A.; Thybert, David; Bergmann, Jan H.; Marioni, John C.; Flicek, Paul; Spector, David L.

    2014-01-01

    Summary Random autosomal monoallelic gene expression refers to the transcription of a gene from one of two homologous alleles. We assessed the dynamics of monoallelic expression during development through an allele-specific RNA sequencing screen in clonal populations of hybrid mouse embryonic stem cells (ESCs) and neural progenitor cells (NPCs). We identified 67 and 376 inheritable autosomal random monoallelically expressed genes in ESCs and NPCs respectively, a 5.6-fold increase upon differentiation. While DNA methylation and nuclear positioning did not distinguish the active and inactive alleles, specific histone modifications were differentially enriched between the two alleles. Interestingly, expression levels of 8% of the monoallelically expressed genes remained similar between monoallelic and biallelic clones. These results support a model in which random monoallelic expression occurs stochastically during differentiation, and for some genes is compensated for by the cell to maintain the required transcriptional output of these genes. PMID:24576421

  17. Clock Genes in Glia Cells: A Rhythmic History.

    PubMed

    Chi-Castañeda, Donají; Ortega, Arturo

    2016-10-01

    Circadian rhythms are periodic patterns in biological processes that allow the organisms to anticipate changes in the environment. These rhythms are driven by the suprachiasmatic nucleus (SCN), the master circadian clock in vertebrates. At a molecular level, circadian rhythms are regulated by the so-called clock genes, which oscillate in a periodic manner. The protein products of clock genes are transcription factors that control their own and other genes' transcription, collectively known as "clock-controlled genes." Several brain regions other than the SCN express circadian rhythms of clock genes, including the amygdala, the olfactory bulb, the retina, and the cerebellum. Glia cells in these structures are expected to participate in rhythmicity. However, only certain types of glia cells may be called "glial clocks," since they express PER-based circadian oscillators, which depend of the SCN for their synchronization. This contribution summarizes the current information about clock genes in glia cells, their plausible role as oscillators and their medical implications.

  18. Constitutive expression of cell wall invertase genes increases grain yield and starch content in maize.

    PubMed

    Li, Bei; Liu, Hua; Zhang, Yue; Kang, Tao; Zhang, Li; Tong, Jianhua; Xiao, Langtao; Zhang, Hongxia

    2013-12-01

    Grain size, number and starch content are important determinants of grain yield and quality. One of the most important biological processes that determine these components is the carbon partitioning during the early grain filling, which requires the function of cell wall invertase. Here, we showed the constitutive expression of cell wall invertase-encoding gene from Arabidopsis, rice (Oryza sativa) or maize (Zea mays), driven by the cauliflower mosaic virus (CaMV) 35S promoter, all increased cell wall invertase activities in different tissues and organs, including leaves and developing seeds, and substantially improved grain yield up to 145.3% in transgenic maize plants as compared to the wild-type plants, an effect that was reproduced in our 2-year field trials at different locations. The dramatically increased grain yield is due to the enlarged ears with both enhanced grain size and grain number. Constitutive expression of the invertase-encoding gene also increased total starch content up to 20% in the transgenic kernels. Our results suggest that cell wall invertase gene can be genetically engineered to improve both grain yield and grain quality in crop plants.

  19. Runx Family Genes in Tissue Stem Cell Dynamics.

    PubMed

    Wang, Chelsia Qiuxia; Mok, Michelle Meng Huang; Yokomizo, Tomomasa; Tergaonkar, Vinay; Osato, Motomi

    2017-01-01

    The Runx family genes play important roles in development and cancer, largely via their regulation of tissue stem cell behavior. Their involvement in two organs, blood and skin, is well documented. This review summarizes currently known Runx functions in the stem cells of these tissues. The fundamental core mechanism(s) mediated by Runx proteins has been sought; however, it appears that there does not exist one single common machinery that governs both tissue stem cells. Instead, Runx family genes employ multiple spatiotemporal mechanisms in regulating individual tissue stem cell populations. Such specific Runx requirements have been unveiled by a series of cell type-, developmental stage- or age-specific gene targeting studies in mice. Observations from these experiments revealed that the regulation of stem cells by Runx family genes turned out to be far more complex than previously thought. For instance, although it has been reported that Runx1 is required for the endothelial-to-hematopoietic cell transition (EHT) but not thereafter, recent studies clearly demonstrated that Runx1 is also needed during the period subsequent to EHT, namely at perinatal stage. In addition, Runx1 ablation in the embryonic skin mesenchyme eventually leads to complete loss of hair follicle stem cells (HFSCs) in the adult epithelium, suggesting that Runx1 facilitates the specification of skin epithelial stem cells in a cell extrinsic manner. Further in-depth investigation into how Runx family genes are involved in stem cell regulation is warranted.

  20. Coordinate regulation of HOX genes in human hematopoietic cells

    SciTech Connect

    Magli, M.C.; Barba, P.; Celetti, A.; De Vita, G.; Cillo, C.; Boncinelli, E. )

    1991-07-15

    Hematopoiesis is a continuous process in which precursor cells proliferate and differentiate throughout life. However, the molecular mechanisms that govern this process are not clearly defined. Homeobox-containing genes, encoding DNA-binding homeodomains. are a network of genes highly conserved throughout evolution. They are organized in clusters expressed in the developing embryo with a positional hierarchy. The authors have analyzed expression of the four human HOX loci in erythroleukemic, promyelocytic, and monocytic cell lines to investigate whether the physical organization of human HOX genes reflects a regulatory hierarchy involved in the differentiation process of hematopoietic cells. The results demonstrate that cells representing various stages of hematopoietic differentiation display differential patterns of HOX gene expression and that HOX genes are coordinately switched on or off in blocks that may include entire loci. The entire HOX4 locus is silent in all lines analyzed and almost all the HOX2 genes are active in erythroleukemic cells and turned off in myeloid-restricted cells. The observations provide information about the regulation of HOX genes and suggest that the coordinate regulation of these genes may play an important role in lineage determination during early steps of hematopoiesis.

  1. Microbe-independent entry of oomycete RxLR effectors and fungal RxLR-like effectors into plant and animal cells is specific and reproducible.

    PubMed

    Tyler, Brett M; Kale, Shiv D; Wang, Qunqing; Tao, Kai; Clark, Helen R; Drews, Kelly; Antignani, Vincenzo; Rumore, Amanda; Hayes, Tristan; Plett, Jonathan M; Fudal, Isabelle; Gu, Biao; Chen, Qinghe; Affeldt, Katharyn J; Berthier, Erwin; Fischer, Gregory J; Dou, Daolong; Shan, Weixing; Keller, Nancy P; Martin, Francis; Rouxel, Thierry; Lawrence, Christopher B

    2013-06-01

    A wide diversity of pathogens and mutualists of plant and animal hosts, including oomycetes and fungi, produce effector proteins that enter the cytoplasm of host cells. A major question has been whether or not entry by these effectors can occur independently of the microbe or requires machinery provided by the microbe. Numerous publications have documented that oomycete RxLR effectors and fungal RxLR-like effectors can enter plant and animal cells independent of the microbe. A recent reexamination of whether the RxLR domain of oomycete RxLR effectors is sufficient for microbe-independent entry into host cells concluded that the RxLR domains of Phytophthora infestans Avr3a and of P. sojae Avr1b alone are NOT sufficient to enable microbe-independent entry of proteins into host and nonhost plant and animal cells. Here, we present new, more detailed data that unambiguously demonstrate that the RxLR domain of Avr1b does show efficient and specific entry into soybean root cells and also into wheat leaf cells, at levels well above background nonspecific entry. We also summarize host cell entry experiments with a wide diversity of oomycete and fungal effectors with RxLR or RxLR-like motifs that have been independently carried out by the seven different labs that coauthored this letter. Finally we discuss possible technical reasons why specific cell entry may have been not detected by Wawra et al. (2013).

  2. Predicting cell cycle regulated genes by causal interactions.

    PubMed

    Emmert-Streib, Frank; Dehmer, Matthias

    2009-08-18

    The fundamental difference between classic and modern biology is that technological innovations allow to generate high-throughput data to get insights into molecular interactions on a genomic scale. These high-throughput data can be used to infer gene networks, e.g., the transcriptional regulatory or signaling network, representing a blue print of the current dynamical state of the cellular system. However, gene networks do not provide direct answers to biological questions, instead, they need to be analyzed to reveal functional information of molecular working mechanisms. In this paper we propose a new approach to analyze the transcriptional regulatory network of yeast to predict cell cycle regulated genes. The novelty of our approach is that, in contrast to all other approaches aiming to predict cell cycle regulated genes, we do not use time series data but base our analysis on the prior information of causal interactions among genes. The major purpose of the present paper is to predict cell cycle regulated genes in S. cerevisiae. Our analysis is based on the transcriptional regulatory network, representing causal interactions between genes, and a list of known periodic genes. No further data are used. Our approach utilizes the causal membership of genes and the hierarchical organization of the transcriptional regulatory network leading to two groups of periodic genes with a well defined direction of information flow. We predict genes as periodic if they appear on unique shortest paths connecting two periodic genes from different hierarchy levels. Our results demonstrate that a classical problem as the prediction of cell cycle regulated genes can be seen in a new light if the concept of a causal membership of a gene is applied consequently. This also shows that there is a wealth of information buried in the transcriptional regulatory network whose unraveling may require more elaborate concepts than it might seem at first.

  3. Cell Pluripotency Levels Associated with Imprinted Genes in Human

    PubMed Central

    Yuan, Liyun; Tang, Xiaoyan; Zhang, Binyan; Ding, Guohui

    2015-01-01

    Pluripotent stem cells are exhibited similarly in the morphology, gene expression, growth properties, and epigenetic modification with embryonic stem cells (ESCs). However, it is still controversial that the pluripotency of induced pluripotent stem cell (iPSC) is much inferior to ESC, and the differentiation capacity of iPSC and ESC can also be separated by transcriptome and epigenetics. miRNAs, which act in posttranscriptional regulation of gene expression and are involved in many basic cellular processes, may reveal the answer. In this paper, we focused on identifying the hidden relationship between miRNAs and imprinted genes in cell pluripotency. Total miRNA expression patterns in iPSC and ES cells were comprehensively analysed and linked with human imprinted genes, which show a global picture of their potential function in pluripotent level. A new CPA4-KLF14 region which locates in chromosomal homologous segments (CHSs) within mammals and include both imprinted genes and significantly expressed miRNAs was first identified. Molecular network analysis showed genes interacted with imprinted genes closely and enriched in modules such as cancer, cell death and survival, and tumor morphology. This imprinted region may provide a new look for those who are interested in cell pluripotency of hiPSCs and hESCs. PMID:26504487

  4. Cell Pluripotency Levels Associated with Imprinted Genes in Human.

    PubMed

    Yuan, Liyun; Tang, Xiaoyan; Zhang, Binyan; Ding, Guohui

    2015-01-01

    Pluripotent stem cells are exhibited similarly in the morphology, gene expression, growth properties, and epigenetic modification with embryonic stem cells (ESCs). However, it is still controversial that the pluripotency of induced pluripotent stem cell (iPSC) is much inferior to ESC, and the differentiation capacity of iPSC and ESC can also be separated by transcriptome and epigenetics. miRNAs, which act in posttranscriptional regulation of gene expression and are involved in many basic cellular processes, may reveal the answer. In this paper, we focused on identifying the hidden relationship between miRNAs and imprinted genes in cell pluripotency. Total miRNA expression patterns in iPSC and ES cells were comprehensively analysed and linked with human imprinted genes, which show a global picture of their potential function in pluripotent level. A new CPA4-KLF14 region which locates in chromosomal homologous segments (CHSs) within mammals and include both imprinted genes and significantly expressed miRNAs was first identified. Molecular network analysis showed genes interacted with imprinted genes closely and enriched in modules such as cancer, cell death and survival, and tumor morphology. This imprinted region may provide a new look for those who are interested in cell pluripotency of hiPSCs and hESCs.

  5. Reproducibility of measurements of potential doubling time of tumour cells in the multicentre National Cancer Institute protocol T92-0045

    PubMed Central

    Wilson, G D; Paschoud, N; Pavy, J-J; Weber, K; Weber, P; Dubray, B; Coucke, P A

    1999-01-01

    We compared the flow cytometric measurement and analysis of the potential doubling time (Tpot) between three centres involved in the National Cancer Institute (NCI) protocol T92-0045. The primary purpose was to understand and minimize the variation within the measurement. A total of 102 specimens were selected at random from patients entered into the trial. Samples were prepared, stained, run and analysed in each centre and a single set of data analysed by all three centres. Analysis of the disc data set revealed that the measurement of labelling index (LI) was robust and reproducible. The estimation of duration of S-phase (Ts) was subject to errors of profile interpretation, particularly DNA ploidy status, and analysis. The LI dominated the variation in Tpot such that the level of final agreement, after removal of outliers and ploidy agreement, reached correlation coefficients of 0.9. The sample data showed poor agreement within each of the components of the measurement. There was some improvement when ploidy was in agreement, but correlation coefficients failed to exceed values of 0.5 for Tpot. The data suggest that observer-associated analysis of Ts and tissue processing and tumour heterogeneity were the major causes of variability in the Tpot measurement. The first two aspects can be standardized and minimized, but heterogeneity will remain a problem with biopsy techniques. © 1999 Cancer Research Campaign PMID:9888476

  6. Contextual sensitivity in scientific reproducibility

    PubMed Central

    Van Bavel, Jay J.; Mende-Siedlecki, Peter; Brady, William J.; Reinero, Diego A.

    2016-01-01

    In recent years, scientists have paid increasing attention to reproducibility. For example, the Reproducibility Project, a large-scale replication attempt of 100 studies published in top psychology journals found that only 39% could be unambiguously reproduced. There is a growing consensus among scientists that the lack of reproducibility in psychology and other fields stems from various methodological factors, including low statistical power, researcher’s degrees of freedom, and an emphasis on publishing surprising positive results. However, there is a contentious debate about the extent to which failures to reproduce certain results might also reflect contextual differences (often termed “hidden moderators”) between the original research and the replication attempt. Although psychologists have found extensive evidence that contextual factors alter behavior, some have argued that context is unlikely to influence the results of direct replications precisely because these studies use the same methods as those used in the original research. To help resolve this debate, we recoded the 100 original studies from the Reproducibility Project on the extent to which the research topic of each study was contextually sensitive. Results suggested that the contextual sensitivity of the research topic was associated with replication success, even after statistically adjusting for several methodological characteristics (e.g., statistical power, effect size). The association between contextual sensitivity and replication success did not differ across psychological subdisciplines. These results suggest that researchers, replicators, and consumers should be mindful of contextual factors that might influence a psychological process. We offer several guidelines for dealing with contextual sensitivity in reproducibility. PMID:27217556

  7. Towards Reproducibility in Computational Hydrology

    NASA Astrophysics Data System (ADS)

    Hutton, Christopher; Wagener, Thorsten; Freer, Jim; Han, Dawei

    2016-04-01

    The ability to reproduce published scientific findings is a foundational principle of scientific research. Independent observation helps to verify the legitimacy of individual findings; build upon sound observations so that we can evolve hypotheses (and models) of how catchments function; and move them from specific circumstances to more general theory. The rise of computational research has brought increased focus on the issue of reproducibility across the broader scientific literature. This is because publications based on computational research typically do not contain sufficient information to enable the results to be reproduced, and therefore verified. Given the rise of computational analysis in hydrology over the past 30 years, to what extent is reproducibility, or a lack thereof, a problem in hydrology? Whilst much hydrological code is accessible, the actual code and workflow that produced and therefore documents the provenance of published scientific findings, is rarely available. We argue that in order to advance and make more robust the process of hypothesis testing and knowledge creation within the computational hydrological community, we need to build on from existing open data initiatives and adopt common standards and infrastructures to: first make code re-useable and easy to find through consistent use of metadata; second, publish well documented workflows that combine re-useable code together with data to enable published scientific findings to be reproduced; finally, use unique persistent identifiers (e.g. DOIs) to reference re-useable and reproducible code, thereby clearly showing the provenance of published scientific findings. Whilst extra effort is require to make work reproducible, there are benefits to both the individual and the broader community in doing so, which will improve the credibility of the science in the face of the need for societies to adapt to changing hydrological environments.

  8. The ancestral gene repertoire of animal stem cells.

    PubMed

    Alié, Alexandre; Hayashi, Tetsutaro; Sugimura, Itsuro; Manuel, Michaël; Sugano, Wakana; Mano, Akira; Satoh, Nori; Agata, Kiyokazu; Funayama, Noriko

    2015-12-22

    Stem cells are pivotal for development and tissue homeostasis of multicellular animals, and the quest for a gene toolkit associated with the emergence of stem cells in a common ancestor of all metazoans remains a major challenge for evolutionary biology. We reconstructed the conserved gene repertoire of animal stem cells by transcriptomic profiling of totipotent archeocytes in the demosponge Ephydatia fluviatilis and by tracing shared molecular signatures with flatworm and Hydra stem cells. Phylostratigraphy analyses indicated that most of these stem-cell genes predate animal origin, with only few metazoan innovations, notably including several partners of the Piwi machinery known to promote genome stability. The ancestral stem-cell transcriptome is strikingly poor in transcription factors. Instead, it is rich in RNA regulatory actors, including components of the "germ-line multipotency program" and many RNA-binding proteins known as critical regulators of mammalian embryonic stem cells.

  9. The ancestral gene repertoire of animal stem cells

    PubMed Central

    Alié, Alexandre; Hayashi, Tetsutaro; Sugimura, Itsuro; Manuel, Michaël; Sugano, Wakana; Mano, Akira; Satoh, Nori; Agata, Kiyokazu; Funayama, Noriko

    2015-01-01

    Stem cells are pivotal for development and tissue homeostasis of multicellular animals, and the quest for a gene toolkit associated with the emergence of stem cells in a common ancestor of all metazoans remains a major challenge for evolutionary biology. We reconstructed the conserved gene repertoire of animal stem cells by transcriptomic profiling of totipotent archeocytes in the demosponge Ephydatia fluviatilis and by tracing shared molecular signatures with flatworm and Hydra stem cells. Phylostratigraphy analyses indicated that most of these stem-cell genes predate animal origin, with only few metazoan innovations, notably including several partners of the Piwi machinery known to promote genome stability. The ancestral stem-cell transcriptome is strikingly poor in transcription factors. Instead, it is rich in RNA regulatory actors, including components of the “germ-line multipotency program” and many RNA-binding proteins known as critical regulators of mammalian embryonic stem cells. PMID:26644562

  10. Definition of the ovalbumin gene promoter by transfer of an ovalglobin fusion gene into cultured cells.

    PubMed Central

    Knoll, B J; Zarucki-Schulz, T; Dean, D C; O'Malley, B W

    1983-01-01

    In order to study the initiation of transcription from the ovalbumin gene promoter, we constructed a hybrid gene (ovalglobin) in which 753 bps of ovalbumin gene 5'-flanking sequence were joined to the chicken adult beta-globin gene. When transfected into HeLa S3 cells, ovalglobin gene transcription initiated at the ovalbumin gene cap site, as measured by S1 nuclease and primer extension analysis. Deletion of 5'-flanking sequences to position -95 had little effect on transcription; deletion to -77 reduced transcription to about 20% of the wild type level and deletion to -48 reduced the level to about 2%. A deletion to -24, removing the sequence TATATAT, abolished transcription entirely. Hormonal regulation of the ovalglobin gene was observed when primary oviduct cells were used as recipients for DNA transfection. Under these conditions, addition of progesterone increased the level of ovalglobin transcripts to more than 10 times the uninduced level. Images PMID:6314256

  11. An Arabidopsis gene regulatory network for secondary cell wall synthesis

    SciTech Connect

    Taylor-Teeples, M.; Lin, L.; de Lucas, M.; Turco, G.; Toal, T. W.; Gaudinier, A.; Young, N. F.; Trabucco, G. M.; Veling, M. T.; Lamothe, R.; Handakumbura, P. P.; Xiong, G.; Wang, C.; Corwin, J.; Tsoukalas, A.; Zhang, L.; Ware, D.; Pauly, M.; Kliebenstein, D. J.; Dehesh, K.; Tagkopoulos, I.; Breton, G.; Pruneda-Paz, J. L.; Ahnert, S. E.; Kay, S. A.; Hazen, S. P.; Brady, S. M.

    2014-12-24

    The plant cell wall is an important factor for determining cell shape, function and response to the environment. Secondary cell walls, such as those found in xylem, are composed of cellulose, hemicelluloses and lignin and account for the bulk of plant biomass. The coordination between transcriptional regulation of synthesis for each polymer is complex and vital to cell function. A regulatory hierarchy of developmental switches has been proposed, although the full complement of regulators remains unknown. In this paper, we present a protein–DNA network between Arabidopsis thaliana transcription factors and secondary cell wall metabolic genes with gene expression regulated by a series of feed-forward loops. This model allowed us to develop and validate new hypotheses about secondary wall gene regulation under abiotic stress. Distinct stresses are able to perturb targeted genes to potentially promote functional adaptation. Finally, these interactions will serve as a foundation for understanding the regulation of a complex, integral plant component.

  12. Enrichment of cells exhibiting tetracycline regulated gene expression.

    PubMed

    Nahreini, Piruz; Hanson, Amy J; Prasad, Kedar N

    2003-05-01

    Tetracycline controlled gene expression varies significantly among cells within a cell line. Chromosomal integration sites of the tetracycline transactivator (tTA) gene and/or the test gene presumably account for the variable efficacy of this system. We hypothesized that the efficacy of tetracycline regulated gene expression is more dependent on the level of tTA inside cells and less dependent on the integration sites of the tetracycline transcription units. To test this hypothesis, we established a TetOff regulatied expression of a short-lived enhanced GFP (d2EGFP) via retroviral vectors in a neuroblastoma cell line (NBP2). We then enriched for two populations of NBP2 cells; one expressing high levels of d2EGFP (HG) and the other expressing low levels of d2EGFP (LG) in the absence of doxycycline. We show that the tTA is more abundant in HG cells than in LG cells; the cAMP-mediated transactivation of tTA's promoter further increases the efficacy of the tetracycline system; and the efficient doxycycline regulated expression of a test gene (i.e., VP16CREB) is achieved in HG cells. Therefore, we have developed a simple method to enrich for a population of tetracycline-responsive cells with no need for screening for tetracycline-responsive clonal cell lines.

  13. A unique mechanism regulating gene expression in 1-cell embryos

    PubMed Central

    YAMAMOTO, Ryoma; AOKI, Fugaku

    2016-01-01

    After fertilization, the genome of zygotes is transcriptionally silent. The timing of the initiation of transcription is species-specific and occurs at the mid-1-cell stage in mice. Recent analyses using high-throughput sequencing (HTS) have identified thousands of genes transcribed at the 1-cell stage, and the pattern of expression among these genes appears to be unique. In this article, we show the result of an additional analysis using HTS data from a previous study, and present the hypothesis that an extremely loose chromatin structure causes promiscuous gene expression in 1-cell embryos. PMID:27867162

  14. Determining Physical Mechanisms of Gene Expression Regulation from Single Cell Gene Expression Data

    PubMed Central

    Moignard, Victoria; Göttgens, Berthold; Adryan, Boris

    2016-01-01

    Many genes are expressed in bursts, which can contribute to cell-to-cell heterogeneity. It is now possible to measure this heterogeneity with high throughput single cell gene expression assays (single cell qPCR and RNA-seq). These experimental approaches generate gene expression distributions which can be used to estimate the kinetic parameters of gene expression bursting, namely the rate that genes turn on, the rate that genes turn off, and the rate of transcription. We construct a complete pipeline for the analysis of single cell qPCR data that uses the mathematics behind bursty expression to develop more accurate and robust algorithms for analyzing the origin of heterogeneity in experimental samples, specifically an algorithm for clustering cells by their bursting behavior (Simulated Annealing for Bursty Expression Clustering, SABEC) and a statistical tool for comparing the kinetic parameters of bursty expression across populations of cells (Estimation of Parameter changes in Kinetics, EPiK). We applied these methods to hematopoiesis, including a new single cell dataset in which transcription factors (TFs) involved in the earliest branchpoint of blood differentiation were individually up- and down-regulated. We could identify two unique sub-populations within a seemingly homogenous group of hematopoietic stem cells. In addition, we could predict regulatory mechanisms controlling the expression levels of eighteen key hematopoietic transcription factors throughout differentiation. Detailed information about gene regulatory mechanisms can therefore be obtained simply from high throughput single cell gene expression data, which should be widely applicable given the rapid expansion of single cell genomics. PMID:27551778

  15. Reference gene for primary culture of prostate cancer cells.

    PubMed

    Souza, Aline Francielle Damo; Brum, Ilma Simoni; Neto, Brasil Silva; Berger, Milton; Branchini, Gisele

    2013-04-01

    Selection of reference genes to normalize mRNA levels between samples is critical for gene expression studies because their expression can vary depending on the tissues or cells used and the experimental conditions. We performed ten cell cultures from samples of prostate cancer. Cells were divided into three groups: control (with no transfection protocol), cells transfected with siRNA specific to knockdown the androgen receptor and cells transfected with inespecific siRNAs. After 24 h, mRNA was extracted and gene expression was analyzed by Real-time qPCR. Nine candidates to reference genes for gene expression studies in this model were analyzed (aminolevulinate, delta-, synthase 1 (ALAS1); beta-actin (ACTB); beta-2-microglobulin (B2M); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); hypoxanthine phosphoribosyltransferase 1 (HPRT1); succinate dehydrogenase complex, subunit A, flavoprotein (Fp) (SDHA); TATA box binding protein (TBP); ubiquitin C (UBC); tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ)). Expression stability was calculated NormFinder algorithm to find the most stable genes. NormFinder calculated SDHA as the most stable gene and the gene with the lowest intergroup and intragroup variation, and indicated GAPDH and SDHA as the best combination of two genes for the purpose of normalization. Androgen receptor mRNA expression was evaluated after normalization by each candidate gene and showed statistical difference in the transfected group compared to control group only when normalized by combination of GAPDH and SDHA. Based on the algorithm analysis, the combination of SDHA and GAPDH should be used to normalize target genes mRNA levels in primary culture of prostate cancer cells submitted to transfection with siRNAs.

  16. Reproducibility of 3D chromatin configuration reconstructions

    PubMed Central

    Segal, Mark R.; Xiong, Hao; Capurso, Daniel; Vazquez, Mariel; Arsuaga, Javier

    2014-01-01

    It is widely recognized that the three-dimensional (3D) architecture of eukaryotic chromatin plays an important role in processes such as gene regulation and cancer-driving gene fusions. Observing or inferring this 3D structure at even modest resolutions had been problematic, since genomes are highly condensed and traditional assays are coarse. However, recently devised high-throughput molecular techniques have changed this situation. Notably, the development of a suite of chromatin conformation capture (CCC) assays has enabled elicitation of contacts—spatially close chromosomal loci—which have provided insights into chromatin architecture. Most analysis of CCC data has focused on the contact level, with less effort directed toward obtaining 3D reconstructions and evaluating the accuracy and reproducibility thereof. While questions of accuracy must be addressed experimentally, questions of reproducibility can be addressed statistically—the purpose of this paper. We use a constrained optimization technique to reconstruct chromatin configurations for a number of closely related yeast datasets and assess reproducibility using four metrics that measure the distance between 3D configurations. The first of these, Procrustes fitting, measures configuration closeness after applying reflection, rotation, translation, and scaling-based alignment of the structures. The others base comparisons on the within-configuration inter-point distance matrix. Inferential results for these metrics rely on suitable permutation approaches. Results indicate that distance matrix-based approaches are preferable to Procrustes analysis, not because of the metrics per se but rather on account of the ability to customize permutation schemes to handle within-chromosome contiguity. It has recently been emphasized that the use of constrained optimization approaches to 3D architecture reconstruction are prone to being trapped in local minima. Our methods of reproducibility assessment provide a

  17. Emerging role of regulatory T cells in gene transfer.

    PubMed

    Cao, Ou; Furlan-Freguia, Christian; Arruda, Valder R; Herzog, Roland W

    2007-10-01

    Induction and maintenance of immune tolerance to therapeutic transgene products are key requirements for successful gene replacement therapies. Gene transfer may also be used to specifically induce immune tolerance and thereby augment other types of therapies. Similarly, gene therapies for treatment of autoimmune diseases are being developed in order to restore tolerance to self-antigens. Regulatory T cells have emerged as key players in many aspects of immune tolerance, and a rapidly increasing body of work documents induction and/or activation of regulatory T cells by gene transfer. Regulatory T cells may suppress antibody formation and cytotoxic T cell responses and may be critical for immune tolerance to therapeutic proteins. In this regard, CD4(+)CD25(+) regulatory T cells have been identified as important components of tolerance in several gene transfer protocols, including hepatic in vivo gene transfer. Augmentation of regulatory T cell responses should be a promising new tool to achieve tolerance and avoid immune-mediated rejection of gene therapy. During the past decade, it has become obvious that immune regulation is an important and integral component of tolerance to self-antigens and of many forms of induced tolerance. Gene therapy can only be successful if the immune system does not reject the therapeutic transgene product. Recent studies provide a rapidly growing body of evidence that regulatory T cells (T(reg)) are involved and often play a crucial role in tolerance to proteins expressed by means of gene transfer. This review seeks to provide an overview of these data and their implications for gene therapy.

  18. Identification of Hematopoietic Stem Cell Engraftment Genes in Gene Therapy Studies.

    PubMed

    Powers, John M; Trobridge, Grant D

    2013-09-01

    Hematopoietic stem cell (HSC) therapy using replication-incompetent retroviral vectors is a promising approach to provide life-long correction for genetic defects. HSC gene therapy clinical studies have resulted in functional cures for several diseases, but in some studies clonal expansion or leukemia has occurred. This is due to the dyregulation of endogenous host gene expression from vector provirus insertional mutagenesis. Insertional mutagenesis screens using replicating retroviruses have been used extensively to identify genes that influence oncogenesis. However, retroviral mutagenesis screens can also be used to determine the role of genes in biological processes such as stem cell engraftment. The aim of this review is to describe the potential for vector insertion site data from gene therapy studies to provide novel insights into mechanisms of HSC engraftment. In HSC gene therapy studies dysregulation of host genes by replication-incompetent vector proviruses may lead to enrichment of repopulating clones with vector integrants near genes that influence engraftment. Thus, data from HSC gene therapy studies can be used to identify novel candidate engraftment genes. As HSC gene therapy use continues to expand, the vector insertion site data collected will be of great interest to help identify novel engraftment genes and may ultimately lead to new therapies to improve engraftment.

  19. Regulation of cell-to-cell variability in divergent gene expression

    NASA Astrophysics Data System (ADS)

    Yan, Chao; Wu, Shuyang; Pocetti, Christopher; Bai, Lu

    2016-03-01

    Cell-to-cell variability (noise) is an important feature of gene expression that impacts cell fitness and development. The regulatory mechanism of this variability is not fully understood. Here we investigate the effect on gene expression noise in divergent gene pairs (DGPs). We generated reporters driven by divergent promoters, rearranged their gene order, and probed their expressions using time-lapse fluorescence microscopy and single-molecule fluorescence in situ hybridization (smFISH). We show that two genes in a co-regulated DGP have higher expression covariance compared with the separate, tandem and convergent configurations, and this higher covariance is caused by more synchronized firing of the divergent transcriptions. For differentially regulated DGPs, the regulatory signal of one gene can stochastically `leak' to the other, causing increased gene expression noise. We propose that the DGPs' function in limiting or promoting gene expression noise may enhance or compromise cell fitness, providing an explanation for the conservation pattern of DGPs.

  20. Role of Hox genes in stem cell differentiation.

    PubMed

    Seifert, Anne; Werheid, David F; Knapp, Silvana M; Tobiasch, Edda

    2015-04-26

    Hox genes are an evolutionary highly conserved gene family. They determine the anterior-posterior body axis in bilateral organisms and influence the developmental fate of cells. Embryonic stem cells are usually devoid of any Hox gene expression, but these transcription factors are activated in varying spatial and temporal patterns defining the development of various body regions. In the adult body, Hox genes are among others responsible for driving the differentiation of tissue stem cells towards their respective lineages in order to repair and maintain the correct function of tissues and organs. Due to their involvement in the embryonic and adult body, they have been suggested to be useable for improving stem cell differentiations in vitro and in vivo. In many studies Hox genes have been found as driving factors in stem cell differentiation towards adipogenesis, in lineages involved in bone and joint formation, mainly chondrogenesis and osteogenesis, in cardiovascular lineages including endothelial and smooth muscle cell differentiations, and in neurogenesis. As life expectancy is rising, the demand for tissue reconstruction continues to increase. Stem cells have become an increasingly popular choice for creating therapies in regenerative medicine due to their self-renewal and differentiation potential. Especially mesenchymal stem cells are used more and more frequently due to their easy handling and accessibility, combined with a low tumorgenicity and little ethical concerns. This review therefore intends to summarize to date known correlations between natural Hox gene expression patterns in body tissues and during the differentiation of various stem cells towards their respective lineages with a major focus on mesenchymal stem cell differentiations. This overview shall help to understand the complex interactions of Hox genes and differentiation processes all over the body as well as in vitro for further improvement of stem cell treatments in future regenerative

  1. Role of Hox genes in stem cell differentiation

    PubMed Central

    Seifert, Anne; Werheid, David F; Knapp, Silvana M; Tobiasch, Edda

    2015-01-01

    Hox genes are an evolutionary highly conserved gene family. They determine the anterior-posterior body axis in bilateral organisms and influence the developmental fate of cells. Embryonic stem cells are usually devoid of any Hox gene expression, but these transcription factors are activated in varying spatial and temporal patterns defining the development of various body regions. In the adult body, Hox genes are among others responsible for driving the differentiation of tissue stem cells towards their respective lineages in order to repair and maintain the correct function of tissues and organs. Due to their involvement in the embryonic and adult body, they have been suggested to be useable for improving stem cell differentiations in vitro and in vivo. In many studies Hox genes have been found as driving factors in stem cell differentiation towards adipogenesis, in lineages involved in bone and joint formation, mainly chondrogenesis and osteogenesis, in cardiovascular lineages including endothelial and smooth muscle cell differentiations, and in neurogenesis. As life expectancy is rising, the demand for tissue reconstruction continues to increase. Stem cells have become an increasingly popular choice for creating therapies in regenerative medicine due to their self-renewal and differentiation potential. Especially mesenchymal stem cells are used more and more frequently due to their easy handling and accessibility, combined with a low tumorgenicity and little ethical concerns. This review therefore intends to summarize to date known correlations between natural Hox gene expression patterns in body tissues and during the differentiation of various stem cells towards their respective lineages with a major focus on mesenchymal stem cell differentiations. This overview shall help to understand the complex interactions of Hox genes and differentiation processes all over the body as well as in vitro for further improvement of stem cell treatments in future regenerative

  2. Overexpression of a metacaspase gene stimulates cell growth and stress response in Schizosaccharomyces pombe.

    PubMed

    Lim, Hye-Won; Kim, Su-Jung; Park, Eun-Hee; Lim, Chang-Jin

    2007-08-01

    A unique gene named pca1(+), encoding a metacaspase, was cloned from the fission yeast Schizosaccharomyces pombe and was used to create a recombinant plasmid, pPMC. The metacaspase mRNA level was markedly elevated in the fission yeast cells harboring the plasmid pPMC. Overexpressed Pca1(+) appeared to stimulate the growth of the fission yeast cells instead of arresting their growth. Its expression was enhanced by stress-inducing agents such as H(2)O(2), sodium nitroprusside, and CdCl(2), and it conferred cytoprotection, especially against CdCl(2). However, such protection was not reproducible in the budding yeast Saccharomyces cerevisiae harboring pPMC. Taken together, these results propose that Pca1(+) may be involved in the growth and stress response of the fission yeast.

  3. Dynamics of gene regulatory networks with cell division cycle

    NASA Astrophysics Data System (ADS)

    Chen, Luonan; Wang, Ruiqi; Kobayashi, Tetsuya J.; Aihara, Kazuyuki

    2004-07-01

    This paper focuses on modeling and analyzing the nonlinear dynamics of gene regulatory networks with the consideration of a cell division cycle with duplication process of DNA , in particular for switches and oscillators of synthetic networks. We derive two models that may correspond to the eukaryotic and prokaryotic cells, respectively. A biologically plausible three-gene model ( lac,tetR , and cI ) and a repressilator as switch and oscillator examples are used to illustrate our theoretical results. We show that the cell cycle may play a significant role in gene regulation due to the nonlinear dynamics of a gene regulatory network although gene expressions are usually tightly controlled by transcriptional factors.

  4. REST regulation of gene networks in adult neural stem cells

    PubMed Central

    Mukherjee, Shradha; Brulet, Rebecca; Zhang, Ling; Hsieh, Jenny

    2016-01-01

    Adult hippocampal neural stem cells generate newborn neurons throughout life due to their ability to self-renew and exist as quiescent neural progenitors (QNPs) before differentiating into transit-amplifying progenitors (TAPs) and newborn neurons. The mechanisms that control adult neural stem cell self-renewal are still largely unknown. Conditional knockout of REST (repressor element 1-silencing transcription factor) results in precocious activation of QNPs and reduced neurogenesis over time. To gain insight into the molecular mechanisms by which REST regulates adult neural stem cells, we perform chromatin immunoprecipitation sequencing and RNA-sequencing to identify direct REST target genes. We find REST regulates both QNPs and TAPs, and importantly, ribosome biogenesis, cell cycle and neuronal genes in the process. Furthermore, overexpression of individual REST target ribosome biogenesis or cell cycle genes is sufficient to induce activation of QNPs. Our data define novel REST targets to maintain the quiescent neural stem cell state. PMID:27819263

  5. Using injectoporation to deliver genes to mechanosensory hair cells.

    PubMed

    Xiong, Wei; Wagner, Thomas; Yan, Linxuan; Grillet, Nicolas; Müller, Ulrich

    2014-10-01

    Mechanosensation, the transduction of mechanical force into electrochemical signals, allows organisms to detect touch and sound, to register movement and gravity, and to sense changes in cell volume and shape. The hair cells of the mammalian inner ear are the mechanosensors for the detection of sound and head movement. The analysis of gene function in hair cells has been hampered by the lack of an efficient gene transfer method. Here we describe a method termed injectoporation that combines tissue microinjection with electroporation to express cDNAs and shRNAs in mouse cochlear hair cells. Injectoporation allows for gene transfer into dozens of hair cells, and it is compatible with the analysis of hair cell function using imaging approaches and electrophysiology. Tissue dissection and injectoporation can be carried out within a few hours, and the tissue can be cultured for days for subsequent functional analyses.

  6. REST regulation of gene networks in adult neural stem cells.

    PubMed

    Mukherjee, Shradha; Brulet, Rebecca; Zhang, Ling; Hsieh, Jenny

    2016-11-07

    Adult hippocampal neural stem cells generate newborn neurons throughout life due to their ability to self-renew and exist as quiescent neural progenitors (QNPs) before differentiating into transit-amplifying progenitors (TAPs) and newborn neurons. The mechanisms that control adult neural stem cell self-renewal are still largely unknown. Conditional knockout of REST (repressor element 1-silencing transcription factor) results in precocious activation of QNPs and reduced neurogenesis over time. To gain insight into the molecular mechanisms by which REST regulates adult neural stem cells, we perform chromatin immunoprecipitation sequencing and RNA-sequencing to identify direct REST target genes. We find REST regulates both QNPs and TAPs, and importantly, ribosome biogenesis, cell cycle and neuronal genes in the process. Furthermore, overexpression of individual REST target ribosome biogenesis or cell cycle genes is sufficient to induce activation of QNPs. Our data define novel REST targets to maintain the quiescent neural stem cell state.

  7. Evolution vs the number of gene copies per primitive cell.

    PubMed

    Koch, A L

    1984-01-01

    Computer simulations are presented of the rate at which an advantageous mutant would displace the prototype in a replicating system without an accurate segregation mechanism. If the number of gene copies in the system is indefinitely large, Darwinian evolution is essentially stopped because there is no coupling of phenotype with genotype, i.e., there is no growth advantage to the advantageous gene relative to the prototype and therefore no "survival of the fittest." The inhibition of evolution due to a number of gene copies less than 100 would have been not insurmountable. Although the presence of multiple copies would have allowed replacement by an advantageous mutant, it provided a way for the primitive cell to conserve less immediately useful genes that could evolve into different or more effective genes. This possibility was lost as accurate segregation mechanisms evolved and cells with few copies of each gene, such as modern procaryotes, arose.

  8. Prospects and limitations of T cell receptor gene therapy.

    PubMed

    Jorritsma, Annelies; Schotte, Remko; Coccoris, Miriam; de Witte, Moniek A; Schumacher, Ton N M

    2011-08-01

    Adoptive transfer of antigen-specific T cells is an attractive means to provide cancer patients with immune cells of a desired specificity and the efficacy of such adoptive transfers has been demonstrated in several clinical trials. Because the T cell receptor is the single specificity-determining molecule in T cell function, adoptive transfer of TCR genes into patient T cells may be used as an alternative approach for the transfer of tumor-specific T cell immunity. On theoretical grounds, TCR gene therapy has two substantial advantages over conventional cellular transfer. First, it circumvents the demanding process of in vitro generation of large numbers of specific immune cells. Second, it allows the use of a set of particularly effective TCR genes in large patient groups. Conversely, TCR gene therapy may be associated with a number of specific problems that are not confronted during classical cellular therapy. Here we review our current understanding of the potential and possible problems of TCR gene therapy, as based on in vitro experiments, mouse model systems and phase I clinical trials. Furthermore, we discuss the prospects of widespread clinical application of this gene therapy approach for the treatment of human cancer.

  9. Consistent quantitative gene product expression: #2. Antigen intensities on bone marrow cells are invariant between individuals

    PubMed Central

    Voigt, Andrew P.; Eidenschink Brodersen, Lisa; Fritschle, Wayne; Menssen, Andrew J.; Meshinchi, Soheil; Wells, Denise A.

    2016-01-01

    Abstract Five reference populations in bone marrow specimens were identified by flow cytometry using specific combinations of reagents in order define the variation of gene product expression intensities both within and between individuals. Mature lymphocytes, uncommitted progenitor cells, promyelocytes, mature monocytes and mature neutrophils can be reproducibly identified as distinct clusters of events in heterogeneous, maturing bone marrow specimens. Support Vector Machines were used to identify the reference populations in order to reduce subjective bias in manually defining boundaries of these populations since they were not discretely separated from the remainder of the cells. Reference populations were identified in 50 randomly selected bone marrow aspirates obtained over a period spanning 3 years and 6 months from pediatric patients following chemotherapy for acute myeloid leukemia (AML). The quantitative expression of gene products (cell surface antigens) and light scattering characteristics on these stressed specimens were demonstrated to be tightly regulated both within individuals and between individuals. Within an individual most gene products (CD45, CD34, CD14, CD16, CD64, CD33) demonstrated limited variability with a standard deviation of <0.20 log units while CD13 and CD36 exhibited broader variation >0.25 log units. Surprisingly, with the exception of CD33, the variation of the mean intensities of each antigen between individuals was even less than the variation within an individual. These data confirm that the amounts of gene products expressed on normal developing cells are highly regulated but differ in intensities between different lineages and during the maturational pathway of those lineages. The amounts of gene products expressed at specific stages of development of each lineage are a biologic constant with minimal variation within or between individuals. © 2016 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of

  10. Rotary head type reproducing apparatus

    DOEpatents

    Takayama, Nobutoshi; Edakubo, Hiroo; Kozuki, Susumu; Takei, Masahiro; Nagasawa, Kenichi

    1986-01-01

    In an apparatus of the kind arranged to reproduce, with a plurality of rotary heads, an information signal from a record bearing medium having many recording tracks which are parallel to each other with the information signal recorded therein and with a plurality of different pilot signals of different frequencies also recorded one by one, one in each of the recording tracks, a plurality of different reference signals of different frequencies are simultaneously generated. A tracking error is detected by using the different reference signals together with the pilot signals which are included in signals reproduced from the plurality of rotary heads.

  11. Optimizing ribozymes for somatic cell gene therapy.

    PubMed

    Branch, A D; Klotman, P E

    1998-01-01

    Therapeutic ribozymes are created through a multistep process that requires trial and error. There are few established rules governing ribozyme design, but guidelines are emerging. It is not yet known whether hammerheads and hairpins, the two ribozymes most widely studied as potential gene therapy agents, have the inherent capability to ablate single genes. Their capacity for specificity and selectivity remains to be explored through rigorous experimentation. These experiments require a battery of control molecules, the characteristics of which are outlined here. Methods for completing the steps in the ribozyme development process, from the selection of a target gene to the quantitation of RNA levels, are also presented and discussed.

  12. Reproducible Volume Restoration and Efficient Long-term Volume Retention after Point-of-care Standardized Cell-enhanced Fat Grafting in Breast Surgery

    PubMed Central

    Dos Anjos, Severiano; Matas-Palau, Aina; Mercader, Josep; Katz, Adam J.

    2015-01-01

    Background: Lipoaspirated fat grafts are used to reconstruct volume defects in breast surgery. Although intraoperative treatment decisions are influenced by volume changes observed immediately after grafting, clinical effect and patient satisfaction are dependent on volume retention over time. The study objectives were to determine how immediate breast volume changes correlate to implanted graft volumes, to understand long-term adipose graft volume changes, and to study the “dose” effect of adding autologous stromal vascular fraction (SVF) cells to fat grafts on long-term volume retention. Methods: A total of 74 patients underwent 77 cell-enhanced fat grafting procedures to restore breast volume deficits associated with cosmetic and reconstructive indications. Although all procedures used standardized fat grafts, 21 of the fat grafts were enriched with a low dose of SVF cells and 56 were enriched with a high SVF cell dose. Three-dimensional imaging was used to quantify volume retention over time Results: For each milliliter of injected fat graft, immediate changes in breast volume were shown to be lower than the actual volume implanted for all methods and clinical indications treated. Long-term breast volume changes stabilize by 90–120 days after grafting. Final volume retention in the long-term was higher with high cell-enhanced fat grafts. Conclusions: Intraoperative immediate breast volume changes do not correspond with implanted fat graft volumes. In the early postoperative period (7–21 days), breast volume increases more than the implanted volume and then rapidly decreases in the subsequent 30–60 days. High-dose cell-enhanced fat grafts decrease early postsurgical breast edema and significantly improve long-term volume retention. PMID:26579353

  13. RNA-based gene circuits for cell regulation

    PubMed Central

    KARAGIANNIS, Peter; FUJITA, Yoshihiko; SAITO, Hirohide

    2016-01-01

    A major goal of synthetic biology is to control cell behavior. RNA-mediated genetic switches (RNA switches) are devices that serve this purpose, as they can control gene expressions in response to input signals. In general, RNA switches consist of two domains: an aptamer domain, which binds to an input molecule, and an actuator domain, which controls the gene expression. An input binding to the aptamer can cause the actuator to alter the RNA structure, thus changing access to translation machinery. The assembly of multiple RNA switches has led to complex gene circuits for cell therapies, including the selective killing of pathological cells and purification of cell populations. The inclusion of RNA binding proteins, such as L7Ae, increases the repertoire and precision of the circuit. In this short review, we discuss synthetic RNA switches for gene regulation and their potential therapeutic applications. PMID:27840389

  14. Probing cell-free gene expression noise in femtoliter volumes.

    PubMed

    Karig, David K; Jung, Seung-Yong; Srijanto, Bernadeta; Collier, C Patrick; Simpson, Michael L

    2013-09-20

    Cell-free systems offer a simplified and flexible context that enables important biological reactions while removing complicating factors such as fitness, division, and mutation that are associated with living cells. However, cell-free expression in unconfined spaces is missing important elements of expression in living cells. In particular, the small volume of living cells can give rise to significant stochastic effects, which are negligible in bulk cell-free reactions. Here, we confine cell-free gene expression reactions to cell-relevant 20 fL volumes (between the volumes of Escherichia coli and Saccharomyces cerevisiae ), in polydimethylsiloxane (PDMS) containers. We demonstrate that expression efficiency varies widely among different containers, likely due to non-Poisson distribution of expression machinery at the observed scale. Previously, this phenomenon has been observed only in liposomes. In addition, we analyze gene expression noise. This analysis is facilitated by our use of cell-free systems, which allow the mapping of the measured noise properties to intrinsic noise models. In contrast, previous live cell noise analysis efforts have been complicated by multiple noise sources. Noise analysis reveals signatures of translational bursting, while noise dynamics suggest that overall cell-free expression is limited by a diminishing translation rate. In addition to offering a unique approach to understanding noise in gene circuits, our work contributes to a deeper understanding of the biophysical properties of cell-free expression systems, thus aiding efforts to harness cell-free systems for synthetic biology applications.

  15. Neural Stem Cell Gene Therapy Ameliorates Pathology and Function in a Mouse Model of Globoid Cell Leukodystrophy

    PubMed Central

    Neri, Margherita; Ricca, Alessandra; di Girolamo, Ilaria; Alcala'-Franco, Beatriz; Cavazzin, Chiara; Orlacchio, Aldo; Martino, Sabata; Naldini, Luigi; Gritti, Angela

    2011-01-01

    Murine neural stem cells (mNSCs), either naive or genetically modified to express supranormal levels of β-galactocerebrosidase (GALC), were transplanted into the brain of Twitcher mice, a murine model of globoid cell leukodystrophy, a severe sphingolipidosis. Cells engrafted long-term into the host cytoarchitecture, producing functional GALC. Levels of enzyme activity in brain and spinal cord tissues were enhanced when GALC-overexpressing NSC were used. Enzymatic correction correlated with reduced tissue storage, decreased activation of astroglia and microglia, delayed onset of symptoms, and longer lifespan. Mechanisms underlying the therapeutic effect of mNSC included widespread enzyme distribution, cross-correction of host cells, anti-inflammatory activity, and neuroprotection. Similar cell engraftment and metabolic correction were reproduced using human NSC. Thus, NSC gene therapy rapidly reconstitutes sustained and long-lasting enzyme activity in central nervous system tissues. Combining this approach with treatments targeting the systemic disease associated with leukodystrophies may provide significant therapeutic benefit. Stem Cells 2011;29:1559–1571 PMID:21809420

  16. From adult stem cells to cancer stem cells: Oct-4 Gene, cell-cell communication, and hormones during tumor promotion.

    PubMed

    Trosko, James E

    2006-11-01

    Carcinogenesis is characterized by "initiation," "promotion," and "progression" phases. The "stem cell theory" and "de-differentiation" theories are used to explain the origin of cancer. Growth control for stem cells, which lack functional gap junctional intercellular communication (GJIC), involves negative soluble or niche factors, while for progenitor cells, it involves GJIC. Tumor promoters, hormones, and growth factors inhibit GJIC reversibly. Oncogenes stably inhibit GJIC. Cancer cells, which lack growth control and the ability to terminally differentiate and to apoptose, lack GJIC. The Oct3/4 gene, a POU (Pit-Oct-Unc) family of transcription factors was thought to be expressed only in embryonic stem cells and in tumor cells. With the availability of normal adult human stem cells, tests for the expression of Oct3/4 gene and the stem cell theory in human carcinogenesis became possible. Human breast, liver, pancreas, kidney, mesenchyme, and gastric stem cells, HeLa and MCF-7 cells, and canine tumors were tested with antibodies and polymerase chain reaction (PCR) primers for Oct3/4. Adult human breast stem cells, immortalized nontumorigenic and tumor cell lines, but not the normal differentiated cells, expressed Oct3/4. Adult human differentiated cells lose their Oct-4 expression. Oct3/4 is expressed in a few cells found in the basal layer of human skin epidermis. The data demonstrate that normal adult stem cells and cancer stem cells maintain expression of Oct3/4, consistent with the stem cell hypothesis of carcinogenesis. These Oct-4 positive cells might represent the "cancer stem cells." A strategy to target "cancer stem cells" is to suppress the Oct-4 gene in order to cause the cells to differentiate.

  17. Allele-specific gene expression patterns in primary leukemic cells reveal regulation of gene expression by CpG site methylation

    PubMed Central

    Milani, Lili; Lundmark, Anders; Nordlund, Jessica; Kiialainen, Anna; Flaegstad, Trond; Jonmundsson, Gudmundur; Kanerva, Jukka; Schmiegelow, Kjeld; Gunderson, Kevin L.; Lönnerholm, Gudmar; Syvänen, Ann-Christine

    2009-01-01

    To identify genes that are regulated by cis-acting functional elements in acute lymphoblastic leukemia (ALL) we determined the allele-specific expression (ASE) levels of 2529 genes by genotyping a genome-wide panel of single nucleotide polymorphisms in RNA and DNA from bone marrow and blood samples of 197 children with ALL. Using a reproducible, quantitative genotyping method and stringent criteria for scoring ASE, we found that 16% of the analyzed genes display ASE in multiple ALL cell samples. For most of the genes, the level of ASE varied largely between the samples, from 1.4-fold overexpression of one allele to apparent monoallelic expression. For genes exhibiting ASE, 55% displayed bidirectional ASE in which overexpression of either of the two SNP alleles occurred. For bidirectional ASE we also observed overall higher levels of ASE and correlation with the methylation level of these sites. Our results demonstrate that CpG site methylation is one of the factors that regulates gene expression in ALL cells. PMID:18997001

  18. An Exercise to Estimate Differential Gene Expression in Human Cells

    ERIC Educational Resources Information Center

    Chaudhry, M. Ahmad

    2006-01-01

    The expression of genes in cells of various tissue types varies considerably and is correlated with the function of a particular organ. The pattern of gene expression changes in diseased tissues, in response to therapy or infection and exposure to environmental mutagens, chemicals, ultraviolet light, and ionizing radiation. To better understand…

  19. Tripartite meeting in gene and cell therapy, 2008: Irish Society for Gene and Cell Therapy, British Society for Gene Therapy, and International Society for Cell and Gene Therapy of Cancer.

    PubMed

    Guinn, Barbara; Casey, Garrett; Collins, Sara; O'Brien, Tim; Alexander, M Yvonne; Tangney, Mark

    2008-10-01

    The second annual meeting of the Irish Society for Gene and Cell Therapy was held in Cork, Ireland on May 15 and 16, 2008 (http://crr.ucc.ie/isgct/). The meeting was jointly organized with the British Society for Gene Therapy and the International Society for Cell and Gene Therapy of Cancer. Because of the location of the conference and the co-organization of this meeting with the British and International Gene Therapy societies, the meeting enjoyed a range of talks from some of the major leaders in the field. Particularly notable were the talented molecular and cell biologists from Ireland who have contributed cutting edge science to the field of gene therapy. Topics including cardiovascular disease, repair of single-gene disorders, and cancer gene therapy were discussed with presentations ranging from basic research to translation into the clinic. Here we describe some of the most exciting presentations and their potential impact on imminent clinical gene therapy trials.

  20. Invitations to Cells: Life's Building Blocks. Teacher-Friendly Science Activities with Reproducible Handouts in English and Spanish. Grades 3-5. Living Things Science Series.

    ERIC Educational Resources Information Center

    Camp, Carole Ann, Ed.

    This booklet, one of six in the Living Things Science series, presents activities about cells which address basic "Benchmarks" suggested by the American Association for the Advancement of Science for the Living Environment for grades 3-5. Contents include background information, vocabulary (in English and Spanish), materials, procedures,…

  1. Gene and stem cell therapy of the hair follicle.

    PubMed

    Hoffman, Robert M

    2005-01-01

    The hair follicle is a highly complex appendage of the skin containing a multiplicity of cell types. The follicle undergoes constant cycling through the life of the organism including growth and resorption with growth dependent on specific stem cells. The targeting of the follicle by genes and stem cells to change its properties, in particular, the nature of the hair shaft is discussed. Hair follicle delivery systems are described such as liposomes and viral vectors for gene therapy. The nature of the hair follicle stem cells is discussed, in particular, its pluripotency.

  2. Effects of cell cycle noise on excitable gene circuits

    NASA Astrophysics Data System (ADS)

    Veliz-Cuba, Alan; Gupta, Chinmaya; Bennett, Matthew R.; Josić, Krešimir; Ott, William

    2016-12-01

    We assess the impact of cell cycle noise on gene circuit dynamics. For bistable genetic switches and excitable circuits, we find that transitions between metastable states most likely occur just after cell division and that this concentration effect intensifies in the presence of transcriptional delay. We explain this concentration effect with a three-states stochastic model. For genetic oscillators, we quantify the temporal correlations between daughter cells induced by cell division. Temporal correlations must be captured properly in order to accurately quantify noise sources within gene networks.

  3. Aptamer-guided gene targeting in yeast and human cells

    PubMed Central

    Ruff, Patrick; Koh, Kyung Duk; Keskin, Havva; Pai, Rekha B.; Storici, Francesca

    2014-01-01

    Gene targeting is a genetic technique to modify an endogenous DNA sequence in its genomic location via homologous recombination (HR) and is useful both for functional analysis and gene therapy applications. HR is inefficient in most organisms and cell types, including mammalian cells, often limiting the effectiveness of gene targeting. Therefore, increasing HR efficiency remains a major challenge to DNA editing. Here, we present a new concept for gene correction based on the development of DNA aptamers capable of binding to a site-specific DNA binding protein to facilitate the exchange of homologous genetic information between a donor molecule and the desired target locus (aptamer-guided gene targeting). We selected DNA aptamers to the I-SceI endonuclease. Bifunctional oligonucleotides containing an I-SceI aptamer sequence were designed as part of a longer single-stranded DNA molecule that contained a region with homology to repair an I-SceI generated double-strand break and correct a disrupted gene. The I-SceI aptamer-containing oligonucleotides stimulated gene targeting up to 32-fold in yeast Saccharomyces cerevisiae and up to 16-fold in human cells. This work provides a novel concept and research direction to increase gene targeting efficiency and lays the groundwork for future studies using aptamers for gene targeting. PMID:24500205

  4. Gene, stem cell, and future therapies for orphan diseases.

    PubMed

    Phillips, M Ian

    2012-08-01

    There are an estimated 7,000 "orphan diseases," but treatments are currently available for only about 5% of them. Recent progress in the advanced platforms of gene therapy, stem cell therapy, gene modification, and gene correction offers possibilities for new therapies and cures for rare diseases. Many rare diseases are genetic in origin, and gene therapy is being successfully applied to treat them. Human stem cell therapy, apart from bone marrow transplants, is still experimental. Genetic modification of stem cells can make stem cell-based products more effective. Autologous induced pluripotent stem (iPS) cells, when combined with new classes of artificial nucleases, have great potential in the ex vivo repair of specific mutated DNA sequences (zinc-finger proteins and transactivator-like effector nucleases). Patient-specific iPS cells can be corrected and transplanted back into the patient. Stem cells secrete paracrine factors that could become new therapeutic tools in the treatment of orphan diseases. Gene therapy and stem cell therapy with DNA repair are promising approaches to the treatment of rare, intractable diseases.

  5. Gene network reconstruction reveals cell cycle and antiviral genes as major drivers of cervical cancer.

    PubMed

    Mine, Karina L; Shulzhenko, Natalia; Yambartsev, Anatoly; Rochman, Mark; Sanson, Gerdine F O; Lando, Malin; Varma, Sudhir; Skinner, Jeff; Volfovsky, Natalia; Deng, Tao; Brenna, Sylvia M F; Carvalho, Carmen R N; Ribalta, Julisa C L; Bustin, Michael; Matzinger, Polly; Silva, Ismael D C G; Lyng, Heidi; Gerbase-DeLima, Maria; Morgun, Andrey

    2013-01-01

    Although human papillomavirus was identified as an aetiological factor in cervical cancer, the key human gene drivers of this disease remain unknown. Here we apply an unbiased approach integrating gene expression and chromosomal aberration data. In an independent group of patients, we reconstruct and validate a gene regulatory meta-network, and identify cell cycle and antiviral genes that constitute two major subnetworks upregulated in tumour samples. These genes are located within the same regions as chromosomal amplifications, most frequently on 3q. We propose a model in which selected chromosomal gains drive activation of antiviral genes contributing to episomal virus elimination, which synergizes with cell cycle dysregulation. These findings may help to explain the paradox of episomal human papillomavirus decline in women with invasive cancer who were previously unable to clear the virus.

  6. All-optical regulation of gene expression in targeted cells

    NASA Astrophysics Data System (ADS)

    Wang, Yisen; He, Hao; Li, Shiyang; Liu, Dayong; Lan, Bei; Hu, Minglie; Cao, Youjia; Wang, Chingyue

    2014-06-01

    Controllable gene expression is always a challenge and of great significance to biomedical research and clinical applications. Recently, various approaches based on extra-engineered light-sensitive proteins have been developed to provide optogenetic actuators for gene expression. Complicated biomedical techniques including exogenous genes engineering, transfection, and material delivery are needed. Here we present an all-optical method to regulate gene expression in targeted cells. Intrinsic or exogenous genes can be activated by a Ca2+-sensitive transcription factor nuclear factor of activated T cells (NFAT) driven by a short flash of femtosecond-laser irradiation. When applied to mesenchymal stem cells, expression of a differentiation regulator Osterix can be activated by this method to potentially induce differentiation of them. A laser-induced ``Ca2+-comb'' (LiCCo) by multi-time laser exposure is further developed to enhance gene expression efficiency. This noninvasive method hence provides an encouraging advance of gene expression regulation, with promising potential of applying in cell biology and stem-cell science.

  7. Cloned Hemoglobin Genes Enhance Growth Of Cells

    NASA Technical Reports Server (NTRS)

    Khosla, Chaitan; Bailey, James E.

    1991-01-01

    Experiments show that portable deoxyribonucleic acid (DNA) sequences incorporated into host cells make them produce hemoglobins - oxygen-binding proteins essential to function of red blood cells. Method useful in several biotechnological applications. One, enhancement of growth of cells at higher densities. Another, production of hemoglobin to enhance supplies of oxygen in cells, for use in chemical reactions requiring oxygen, as additive to serum to increase transport of oxygen, and for binding and separating oxygen from mixtures of gases.

  8. Gene transfer in inner ear cells: a challenging race.

    PubMed

    Sacheli, R; Delacroix, L; Vandenackerveken, P; Nguyen, L; Malgrange, B

    2013-03-01

    Recent advances in human genomics led to the identification of numerous defective genes causing deafness, which represent novel putative therapeutic targets. Future gene-based treatment of deafness resulting from genetic or acquired sensorineural hearing loss may include strategies ranging from gene therapy to antisense delivery. For successful development of gene therapies, a minimal requirement involves the engineering of appropriate gene carrier systems. Transfer of exogenous genetic material into the mammalian inner ear using viral or non-viral vectors has been characterized over the last decade. The nature of inner ear cells targeted, as well as the transgene expression level and duration, are highly dependent on the vector type, the route of administration and the strength of the promoter driving expression. This review summarizes and discusses recent advances in inner ear gene-transfer technologies aimed at examining gene function or identifying new treatment for inner ear disorders.

  9. Reproducible Bioinformatics Research for Biologists

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This book chapter describes the current Big Data problem in Bioinformatics and the resulting issues with performing reproducible computational research. The core of the chapter provides guidelines and summaries of current tools/techniques that a noncomputational researcher would need to learn to pe...

  10. Reproducible research in computational science.

    PubMed

    Peng, Roger D

    2011-12-02

    Computational science has led to exciting new developments, but the nature of the work has exposed limitations in our ability to evaluate published findings. Reproducibility has the potential to serve as a minimum standard for judging scientific claims when full independent replication of a study is not possible.

  11. Autophagy protects human brain microvascular endothelial cells against methylglyoxal-induced injuries, reproducible in a cerebral ischemic model in diabetic rats.

    PubMed

    Fang, Lili; Li, Xue; Zhong, Yinbo; Yu, Jing; Yu, Lina; Dai, Haibin; Yan, Min

    2015-10-01

    Cerebral microvascular endothelial cells (ECs) are crucial for brain vascular repair and maintenance, but their physiological function may be impaired during ischemic stroke and diabetes. Methylglyoxal (MGO), a reactive dicarbonyl produced during glucose metabolism, could exacerbate ischemia-induced EC injury and dysfunction. We investigated the protective effect of autophagy on cultured human brain microvascular endothelial cells (HBMEC) that underwent MGO treatment. A further study was conducted to explore the underlying mechanisms of the protective effect. Autophagic activity was assessed by evaluating protein levels, using western blot. 3-methyladenine (3-MA), bafilomycin A1, ammonium chloride (AC), Beclin 1 siRNA, and chloroquine (CQ) were used to cause autophagy inhibition. Alarmar blue assay and lactate dehydrogenase release assay were used to evaluate cell viability. Streptozotocin was administered to induce type I diabetes in rats and post-permanent middle cerebral artery occlusion was performed to elicit cerebral ischemia. Blood-brain barrier permeability was also assessed. Our study found that MGO reduced HBMEC cell viability in a concentration- and time-dependent manner, and triggered the responsive autophagy activation. Autophagy inhibitors bafilomycin A1, AC, 3-MA, and BECN1 siRNA exacerbated MGO-induced HBMEC injury. FAK phosphorylation inhibitor PF573228 inhibited MGO-triggered autophagy and enhanced lactate dehydrogenase release. Meanwhile, similar autophagy activation in brain vascular ECs was observed during permanent middle cerebral artery occlusion-induced cerebral ischemia in diabetic rats, while chloroquine-induced autophagy inhibition enhanced blood-brain barrier permeability. Taken together, our study indicates that autophagy triggered by MGO defends HBMEC against injuries.

  12. Regulation of Cell and Gene Therapy Medicinal Products in Taiwan.

    PubMed

    Lin, Yi-Chu; Wang, Po-Yu; Tsai, Shih-Chih; Lin, Chien-Liang; Tai, Hsuen-Yung; Lo, Chi-Fang; Wu, Shiow-Ing; Chiang, Yu-Mei; Liu, Li-Ling

    2015-01-01

    Owing to the rapid and mature development of emerging biotechnology in the fields of cell culture, cell preservation, and recombinant DNA technology, more and more cell or gene medicinal therapy products have been approved for marketing, to treat serious diseases which have been challenging to treat with current medical practice or medicine. This chapter will briefly introduce the Taiwan Food and Drug Administration (TFDA) and elaborate regulation of cell and gene therapy medicinal products in Taiwan, including regulatory history evolution, current regulatory framework, application and review procedures, and relevant jurisdictional issues. Under the promise of quality, safety, and efficacy of medicinal products, it is expected the regulation and environment will be more flexible, streamlining the process of the marketing approval of new emerging cell or gene therapy medicinal products and providing diverse treatment options for physicians and patients.

  13. Gene expression analysis of terminal differentiation of human melanoma cells highlights global reductions in cell cycle-associated genes.

    PubMed

    Huynh, Kim Mai; Kim, Gyoungmi; Kim, Dong-Joon; Yang, Suk-Jin; Park, Seong-min; Yeom, Young-Il; Fisher, Paul B; Kang, Dongchul

    2009-03-15

    Defects in differentiation are frequently observed in cancer cells. By appropriate treatment specific tumor cell types can be induced to terminally differentiate. Metastatic HO-1 human melanoma cells treated with IFN-beta plus mezerein (MEZ) undergo irreversible growth arrest and terminal differentiation followed by apoptosis. In order to define the molecular changes associated with this process, changes in gene expression were analyzed by cDNA microarray hybridization and by semi-quantitative and quantitative RT-PCRs of representative 44 genes. The expression of 210 genes was changed more than two-fold at either 8 or 24 h post-treatment (166 up and 44 down). Major biological processes associated with the up-regulated genes were response to endogenous/exogenous stimuli (38%), cell proliferation (13%), cell death (16%) and development (30%). Approximately 34% of the down-regulated genes were associated with cell cycle, 9% in DNA replication and 11% in chromosome organization, respectively. Suppression of cell cycle associated genes appeared to directly correlate with growth arrest observed in the terminal differentiation process. Expression of Calpain 3 (CAPN3) variant 6 was suppressed by the combined treatment and maintained high in various melanoma cell lines. However, over-expression of the CAPN3 did not significantly affect growth kinetics and cell viability, suggesting that up-regulation of CAPN3 alone may not be a causative, but an associated change with melanoma development. This analysis provides further insights into the spectrum of up-regulated and the first detailed investigation of down-regulated gene changes associated with and potentially causative of induction of loss of proliferative capacity and terminal differentiation in human melanoma cells.

  14. Regulation of global gene expression and cell proliferation by APP

    PubMed Central

    Wu, Yili; Zhang, Si; Xu, Qin; Zou, Haiyan; Zhou, Weihui; Cai, Fang; Li, Tingyu; Song, Weihong

    2016-01-01

    Down syndrome (DS), caused by trisomy of chromosome 21, is one of the most common genetic disorders. Patients with DS display growth retardation and inevitably develop characteristic Alzheimer’s disease (AD) neuropathology, including neurofibrillary tangles and neuritic plaques. The expression of amyloid precursor protein (APP) is increased in both DS and AD patients. To reveal the function of APP and elucidate the pathogenic role of increased APP expression in DS and AD, we performed gene expression profiling using microarray method in human cells overexpressing APP. A set of genes are significantly altered, which are involved in cell cycle, cell proliferation and p53 signaling. We found that overexpression of APP inhibits cell proliferation. Furthermore, we confirmed that the downregulation of two validated genes, PSMA5 and PSMB7, inhibits cell proliferation, suggesting that the downregulation of PSMA5 and PSMB7 is involved in APP-induced cell proliferation impairment. Taken together, this study suggests that APP regulates global gene expression and increased APP expression inhibits cell proliferation. Our study provides a novel insight that APP overexpression may contribute to the growth impairment in DS patients and promote AD pathogenesis by inhibiting cell proliferation including neural stem cell proliferation and neurogenesis. PMID:26936520

  15. Regulation of global gene expression and cell proliferation by APP.

    PubMed

    Wu, Yili; Zhang, Si; Xu, Qin; Zou, Haiyan; Zhou, Weihui; Cai, Fang; Li, Tingyu; Song, Weihong

    2016-03-03

    Down syndrome (DS), caused by trisomy of chromosome 21, is one of the most common genetic disorders. Patients with DS display growth retardation and inevitably develop characteristic Alzheimer's disease (AD) neuropathology, including neurofibrillary tangles and neuritic plaques. The expression of amyloid precursor protein (APP) is increased in both DS and AD patients. To reveal the function of APP and elucidate the pathogenic role of increased APP expression in DS and AD, we performed gene expression profiling using microarray method in human cells overexpressing APP. A set of genes are significantly altered, which are involved in cell cycle, cell proliferation and p53 signaling. We found that overexpression of APP inhibits cell proliferation. Furthermore, we confirmed that the downregulation of two validated genes, PSMA5 and PSMB7, inhibits cell proliferation, suggesting that the downregulation of PSMA5 and PSMB7 is involved in APP-induced cell proliferation impairment. Taken together, this study suggests that APP regulates global gene expression and increased APP expression inhibits cell proliferation. Our study provides a novel insight that APP overexpression may contribute to the growth impairment in DS patients and promote AD pathogenesis by inhibiting cell proliferation including neural stem cell proliferation and neurogenesis.

  16. A gene expression fingerprint of mouse stomach ECL cells.

    PubMed

    Andersson, Niklas; Skrtic, Sofia Movérare; Håkanson, Rolf; Ohlsson, Claes

    2005-07-01

    Many of the endocrine cells in the stomach are poorly characterized with respect to physiological significance. In some cases, the anticipated hormone has not yet been identified. Global gene expression analysis of mouse stomach was performed in an attempt to identify the ECL-cell peptide/protein. Specific functional activation (omeprazole-induced hypergastrinaemia) was used as a tool to generate a gene expression fingerprint of the ECL cells. The proposed fingerprint includes 14 genes, among them six are known to be expressed by ECL cells (=positive controls), and some novel ones, which are likely to be ECL-cell-related. The known ECL-cell-related genes are those encoding histidine decarboxylase, chromogranin A and B, vesicular monoamine transporter 2, synaptophysin, and the cholecystokinin-B receptor. In addition, the fingerprint included five genes, which might be involved in the process of secretion and three ESTs with unknown function. Interestingly, parathyroid hormone-like hormone (Pthlh) was identified as a candidate ECL-cell peptide hormone.

  17. Imaging techniques: new avenues in cancer gene and cell therapy.

    PubMed

    Saadatpour, Z; Rezaei, A; Ebrahimnejad, H; Baghaei, B; Bjorklund, G; Chartrand, M; Sahebkar, A; Morovati, H; Mirzaei, H R; Mirzaei, H

    2017-01-01

    Cancer is one of the world's most concerning health problems and poses many challenges in the range of approaches associated with the treatment of cancer. Current understanding of this disease brings to the fore a number of novel therapies that can be useful in the treatment of cancer. Among them, gene and cell therapies have emerged as novel and effective approaches. One of the most important challenges for cancer gene and cell therapies is correct monitoring of the modified genes and cells. In fact, visual tracking of therapeutic cells, immune cells, stem cells and genetic vectors that contain therapeutic genes and the various drugs is important in cancer therapy. Similarly, molecular imaging, such as nanosystems, fluorescence, bioluminescence, positron emission tomography, single photon-emission computed tomography and magnetic resonance imaging, have also been found to be powerful tools in monitoring cancer patients who have received therapeutic cell and gene therapies or drug therapies. In this review, we focus on these therapies and their molecular imaging techniques in treating and monitoring the progress of the therapies on various types of cancer.

  18. Oxygen-regulated gene expression in murine cumulus cells.

    PubMed

    Kind, Karen L; Tam, Kimberley K Y; Banwell, Kelly M; Gauld, Ashley D; Russell, Darryl L; Macpherson, Anne M; Brown, Hannah M; Frank, Laura A; Peet, Daniel J; Thompson, Jeremy G

    2015-01-01

    Oxygen is an important component of the environment of the cumulus-oocyte complex (COC), both in vivo within the ovarian follicle and during in vitro oocyte maturation (IVM). Cumulus cells have a key role in supporting oocyte development, and cumulus cell function and gene expression are known to be altered when the environment of the COC is perturbed. Oxygen-regulated gene expression is mediated through the actions of the transcription factors, the hypoxia-inducible factors (HIFs). In the present study, the effect of oxygen on cumulus cell gene expression was examined following in vitro maturation of the murine COC at 2%, 5% or 20% oxygen. Increased expression of HIF-responsive genes, including glucose transporter-1, lactate dehydrogenase A and BCL2/adenovirus E1B interacting protein 3, was observed in cumulus cells matured at 2% or 5%, compared with 20% oxygen. Stabilisation of HIF1α protein in cumulus cells exposed to low oxygen was confirmed by western blot and HIF-mediated transcriptional activity was demonstrated using a transgenic mouse expressing green fluorescent protein under the control of a promoter containing hypoxia response elements. These results indicate that oxygen concentration influences cumulus cell gene expression and support a role for HIF1α in mediating the cumulus cell response to varying oxygen.

  19. Engineering Synthetic Gene Circuits in Living Cells with CRISPR Technology.

    PubMed

    Jusiak, Barbara; Cleto, Sara; Perez-Piñera, Pablo; Lu, Timothy K

    2016-07-01

    One of the goals of synthetic biology is to build regulatory circuits that control cell behavior, for both basic research purposes and biomedical applications. The ability to build transcriptional regulatory devices depends on the availability of programmable, sequence-specific, and effective synthetic transcription factors (TFs). The prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR) system, recently harnessed for transcriptional regulation in various heterologous host cells, offers unprecedented ease in designing synthetic TFs. We review how CRISPR can be used to build synthetic gene circuits and discuss recent advances in CRISPR-mediated gene regulation that offer the potential to build increasingly complex, programmable, and efficient gene circuits in the future.

  20. Myostatin gene targeting in cultured China Han ovine myoblast cells.

    PubMed

    Zhang, L; Yang, X; An, X; Chen, Y

    2007-11-01

    Myostatin (MSTN), a member of the transforming growth factor-β superfamily, has been shown to be a negative regulator of myogenesis. Natural mutation in beef cattle causes double-muscling phenotypes. We report an investigation designed to knockout the MSTN gene by gene targeting in ovine myoblast cells. Two promoter-trap targeting vectors MSTN-green fluorescent protein (GFP) and MSTN-neo were constructed and used to transfect foetal and neonatal ovine primary myoblast cells. Both GFP-expressing cells and drug-resistant cells were obtained. Targeted cells expressing GFP were confirmed by polymerase chain reaction (PCR) assay and drug-resistant cells were characterised by PCR and Southern blot after growing into cell clones.

  1. Mouse T-cell receptor variable gene segment families

    SciTech Connect

    Arden, B.; Kabelitz, D.; Clark, S.P.; Mak, T.W.

    1995-10-01

    All mouse T-cell receptor {alpha}/{delta}, {beta}, and {gamma} variable (Tcra/d-, b-, and g-V) gene segments were aligned to compare the sequences with one another, to group them into subfamilies, and to derive a name which complies with the standard nomenclature. it was necessary to change the names of some V gene segments because they conflicted with those of other segments. The traditional classification into subfamilies was re-evaluated using a much larger pool of sequences. In the mouse, most V gene segments can be grouped into subfamilies of closely related genes with significantly less similarity between different subfamilies. 118 refs., 11 figs., 4 tabs.

  2. Screening for the Most Suitable Reference Genes for Gene Expression Studies in Equine Milk Somatic Cells.

    PubMed

    Cieslak, Jakub; Mackowski, Mariusz; Czyzak-Runowska, Grazyna; Wojtowski, Jacek; Puppel, Kamila; Kuczynska, Beata; Pawlak, Piotr

    2015-01-01

    Apart from the well-known role of somatic cell count as a parameter reflecting the inflammatory status of the mammary gland, the composition of cells isolated from milk is considered as a valuable material for gene expression studies in mammals. Due to its unique composition, in recent years an increasing interest in mare's milk consumption has been observed. Thus, investigating the genetic background of horse's milk variability presents and interesting study model. Relying on 39 milk samples collected from mares representing three breeds (Polish Primitive Horse, Polish Cold-blooded Horse, Polish Warmblood Horse) we aimed to investigate the utility of equine milk somatic cells as a source of mRNA and to screen the best reference genes for RT-qPCR using geNorm and NormFinder algorithms. The results showed that despite relatively low somatic cell counts in mare's milk, the amount and the quality of the extracted RNA are sufficient for gene expression studies. The analysis of the utility of 7 potential reference genes for RT-qPCR experiments for the normalization of equine milk somatic cells revealed some differences between the outcomes of the applied algorithms, although in both cases the KRT8 and TOP2B genes were pointed as the most stable. Analysis by geNorm showed that the combination of 4 reference genes (ACTB, GAPDH, TOP2B and KRT8) is required for apropriate RT-qPCR experiments normalization, whereas NormFinder algorithm pointed the combination of KRT8 and RPS9 genes as the most suitable. The trial study of the relative transcript abundance of the beta-casein gene with the use of various types and numbers of internal control genes confirmed once again that the selection of proper reference gene combinations is crucial for the final results of each real-time PCR experiment.

  3. Probing cell-free gene expression noise in femtoliter volumes

    SciTech Connect

    Karig, David K; Jung, Seung-Yong; Srijanto, Bernadeta R; Collier, Pat; Simpson, Michael L

    2013-01-01

    Cell-free systems offer a simplified and flexible context that enables important biological reactions while removing complicating factors such as fitness, division, and mutation that are associated with living cells. However, cell-free expression in unconfined spaces is missing important elements of expression in living cells. In particular, the small volume of living cells can give rise to significant stochastic effects, which are negligible in bulk cell-free reactions. Here, we confine cell-free gene expression reactions to cell relevant 20 fL volumes (between the volumes of E. coli and S. cerevisiae), in polydimethylsiloxane (PDMS) containers. We demonstrate that expression efficiency varies widely at this volume, and we analyze gene expression noise. Noise analysis reveals signatures of translational bursting while noise dynamics suggest that overall cell-free expression is limited by a diminishing translation rate. In addition to offering a unique approach to understanding noise in gene circuits, our work contributes to a deeper understanding of the biophysical properties of cell-free expression systems, thus aiding efforts to harness cell-free systems for synthetic biology applications.

  4. Can dead bacterial cells be defined and are genes expressed after cell death?

    PubMed

    Trevors, J T

    2012-07-01

    There is a paucity of knowledge on gene expression in dead bacterial cells. Why would this knowledge be useful? The cells are dead. However, the time duration of gene expression following cell death is often unknown, and possibly in the order of minutes. In addition, it is a challenge to determine if bacterial cells are dead, or viable but non-culturable (VBNC), and what is an agreed upon correct definition of dead bacteria. Cells in the bacterial population or community may die at different rates or times and this complicates both the viability and gene expression analysis. In this article, the definition of dead bacterial cells is discussed and its significance in continued gene expression in cells following death. The definition of living and dead has implications for possible, completely, synthetic bacterial cells that may be capable of growth and division.

  5. Expression of cloned immunoglobulin genes introduced into mouse L cells.

    PubMed Central

    Gilles, S D; Tonegawa, S

    1983-01-01

    Functionally rearranged immunoglobulin heavy-chain (gamma 2b) and light-chain (lambda 1 and kappa) genes were introduced into mouse L tk- cells by co-transformation with the Herpes virus tk gene. Cloned cell lines were selected in HAT medium and tested for the presence of transfected immunoglobulin gene sequences by Southern blotting analysis. It was found that the gamma 2b gene was accurately transcribed at a low level in transfected mouse L cells and cytoplasmic gamma 2b, heavy-chain protein was detected by immunoprecipitation of cell extracts. Light-chain genes, on the other hand, were not accurately transcribed. Instead, lambda 1 or kappa RNA species were detected which were approximately 200 to 300 bases longer than the authentic mRNAs. These results suggest that the expression of rearranged heavy-chain and light-chain genes are controlled differently and that these differences can be seen in transfected, non-lymphoid cells. Images PMID:6316279

  6. Cell Cycle Programs of Gene Expression Control Morphogenetic Protein Localization

    PubMed Central

    Lord, Matthew; Yang, Melody C.; Mischke, Michelle; Chant, John

    2000-01-01

    Genomic studies in yeast have revealed that one eighth of genes are cell cycle regulated in their expression. Almost without exception, the significance of cell cycle periodic gene expression has not been tested. Given that many such genes are critical to cellular morphogenesis, we wanted to examine the importance of periodic gene expression to this process. The expression profiles of two genes required for the axial pattern of cell division, BUD3 and BUD10/AXL2/SRO4, are strongly cell cycle regulated. BUD3 is expressed close to the onset of mitosis. BUD10 is expressed in late G1. Through promotor-swap experiments, the expression profile of each gene was altered and the consequences examined. We found that an S/G2 pulse of BUD3 expression controls the timing of Bud3p localization, but that this timing is not critical to Bud3p function. In contrast, a G1 pulse of BUD10 expression plays a direct role in Bud10p localization and function. Bud10p, a membrane protein, relies on the polarized secretory machinery specific to G1 to be delivered to its proper location. Such a secretion-based targeting mechanism for membrane proteins provides cells with flexibility in remodeling their architecture or evolving new forms. PMID:11134078

  7. Modelling epigenetic regulation of gene expression in 12 human cell types reveals combinatorial patterns of cell-type-specific genes.

    PubMed

    Lu, Yiming; Qu, Wubin; Min, Bo; Liu, Zheyan; Chen, Changsheng; Zhang, Chenggang

    2014-06-01

    The maintenance of the diverse cell types in a multicellular organism is one of the fundamental mysteries of biology. Modelling the dynamic regulatory relationships between the histone modifications and the gene expression across the diverse cell types is essential for the authors to understand the mechanisms of the epigenetic regulation. Here, the authors thoroughly assessed the histone modification enrichment profiles at the promoters and constructed quantitative models between the histone modification abundances and the gene expression in 12 human cell types. The author's results showed that the histone modifications at the promoters exhibited remarkably cell-type-dependent variability in the cell-type-specific (CTS) genes. They demonstrated that the variable profiles of the modifications are highly predictive for the dynamic changes of the gene expression across all the cell types. Their findings revealed the close relationship between the combinatorial patterns of the histone modifications and the CTS gene expression. They anticipate that the findings and the methods they used in this study could provide useful information for the future studies of the regulatory roles of the histone modifications in the CTS genes.

  8. Stem cell and neurogenic gene-expression profiles link prostate basal cells to aggressive prostate cancer.

    PubMed

    Zhang, Dingxiao; Park, Daechan; Zhong, Yi; Lu, Yue; Rycaj, Kiera; Gong, Shuai; Chen, Xin; Liu, Xin; Chao, Hsueh-Ping; Whitney, Pamela; Calhoun-Davis, Tammy; Takata, Yoko; Shen, Jianjun; Iyer, Vishwanath R; Tang, Dean G

    2016-02-29

    The prostate gland mainly contains basal and luminal cells constructed as a pseudostratified epithelium. Annotation of prostate epithelial transcriptomes provides a foundation for discoveries that can impact disease understanding and treatment. Here we describe a genome-wide transcriptome analysis of human benign prostatic basal and luminal epithelial populations using deep RNA sequencing. Through molecular and biological characterizations, we show that the differential gene-expression profiles account for their distinct functional properties. Strikingly, basal cells preferentially express gene categories associated with stem cells, neurogenesis and ribosomal RNA (rRNA) biogenesis. Consistent with this profile, basal cells functionally exhibit intrinsic stem-like and neurogenic properties with enhanced rRNA transcription activity. Of clinical relevance, the basal cell gene-expression profile is enriched in advanced, anaplastic, castration-resistant and metastatic prostate cancers. Therefore, we link the cell-type-specific gene signatures to aggressive subtypes of prostate cancer and identify gene signatures associated with adverse clinical features.

  9. T-cell receptor variable region gene usage in T-cell populations.

    PubMed Central

    Garman, R D; Ko, J L; Vulpe, C D; Raulet, D H

    1986-01-01

    We have examined T-cell receptor alpha- and beta-chain variable (V) region gene usage in T-cell populations predicted to have different major histocompatibility complex-restriction specificities. Using a sensitive ribonuclease protection assay to measure T-cell receptor mRNA levels, we found no striking differences in the usage of three V alpha genes and three V beta genes in T-cell populations from three congeneic H-2-disparate strains of mice and between the mutually exclusive Ly2+ L3T4- and Ly2- L3T4+ T-cell subpopulations. These results suggest that major histocompatibility complex restriction cannot be explained by the differential usage of nonoverlapping V alpha or V beta gene pools. In contrast, striking but unpredictable differences were seen in V gene usage in populations of T cells selected by activation with particular alloantigens. Images PMID:3487085

  10. Stem cell and neurogenic gene-expression profiles link prostate basal cells to aggressive prostate cancer

    PubMed Central

    Zhang, Dingxiao; Park, Daechan; Zhong, Yi; Lu, Yue; Rycaj, Kiera; Gong, Shuai; Chen, Xin; Liu, Xin; Chao, Hsueh-Ping; Whitney, Pamela; Calhoun-Davis, Tammy; Takata, Yoko; Shen, Jianjun; Iyer, Vishwanath R.; Tang, Dean G.

    2016-01-01

    The prostate gland mainly contains basal and luminal cells constructed as a pseudostratified epithelium. Annotation of prostate epithelial transcriptomes provides a foundation for discoveries that can impact disease understanding and treatment. Here we describe a genome-wide transcriptome analysis of human benign prostatic basal and luminal epithelial populations using deep RNA sequencing. Through molecular and biological characterizations, we show that the differential gene-expression profiles account for their distinct functional properties. Strikingly, basal cells preferentially express gene categories associated with stem cells, neurogenesis and ribosomal RNA (rRNA) biogenesis. Consistent with this profile, basal cells functionally exhibit intrinsic stem-like and neurogenic properties with enhanced rRNA transcription activity. Of clinical relevance, the basal cell gene-expression profile is enriched in advanced, anaplastic, castration-resistant and metastatic prostate cancers. Therefore, we link the cell-type-specific gene signatures to aggressive subtypes of prostate cancer and identify gene signatures associated with adverse clinical features. PMID:26924072

  11. Human amniotic fluid stem cells as a model for functional studies of genes involved in human genetic diseases or oncogenesis.

    PubMed

    Rosner, Margit; Dolznig, Helmut; Schipany, Katharina; Mikula, Mario; Brandau, Oliver; Hengstschläger, Markus

    2011-09-01

    Besides their putative usage for therapies, stem cells are a promising tool for functional studies of genes involved in human genetic diseases or oncogenesis. For this purpose induced pluripotent stem (iPS) cells can be derived from patients harbouring specific mutations. In contrast to adult stem cells, iPS cells are pluripotent and can efficiently be grown in culture. However, iPS cells are modulated due to the ectopic induction of pluripotency, harbour other somatic mutations accumulated during the life span of the source cells, exhibit only imperfectly cleared epigenetic memory of the source cell, and are often genomically instable. In addition, iPS cells from patients only allow the investigation of mutations, which are not prenatally lethal. Embryonic stem (ES) cells have a high proliferation and differentiation potential, but raise ethical issues. Human embryos, which are not transferred in the course of in vitro fertilization, because of preimplantation genetic diagnosis of a genetic defect, are still rarely donated for the establishment of ES cell lines. In addition, their usage for studies on gene functions for oncogenesis is hampered by the fact the ES cells are already tumorigenic per se. In 2003 amniotic fluid stem (AFS) cells have been discovered, which meanwhile have been demonstrated to harbour the potential to differentiate into cells of all three germ layers. Monoclonal human AFS cell lines derived from amniocenteses have a high proliferative potential, are genomically stable and are not associated with ethical controversies. Worldwide amniocenteses are performed for routine human genetic diagnosis. We here discuss how generation and banking of monoclonal human AFS cell lines with specific chromosomal aberrations or monogenic disease mutations would allow to study the functional consequences of disease causing mutations. In addition, recently a protocol for efficient and highly reproducible siRNA-mediated long-term knockdown of endogenous gene

  12. Dissection of tumour and host cells from target organs of metastasis for testing gene expression directly ex vivo.

    PubMed Central

    Rocha, M.; Hexel, K.; Bucur, M.; Schirrmacher, V.; Umansky, V.

    1996-01-01

    We report on a new methodology which allows the direct analysis ex vivo of tumour cells and host cells (lymphocytes, macrophages, endothelial cells) from a metastasised organ (liver or spleen) at any time point during the metastatic process and without any further in vitro culture. First, we used a tumour cell line transduced with the bacterial gene lacZ, which permits the detection of the procaryotic enzyme beta-galactosidase in eukaryotic cells at the single cell level thus allowing flow adhesion cell sorting (FACS) analysis of tumour cells from metastasised target organs. Second, we established a method for the separation and enrichment of tumour and host cells from target organs of metastasis with a high viability and reproducibility. As exemplified with the murine lymphoma ESb, this new methodology permits the study of molecules of importance for metastasis or anti-tumour immunity (adhesion, costimulatory and cytotoxic molecules, cytokines, etc.) at the RNA or protein level in tumour and host cells during the whole process of metastasis. This novel approach may open new possibilities of developing strategies for intervention in tumour progression, since it allows the determination of the optimal window in time for successful treatments. The possibility of direct analysis of tumour and host cell properties also provides a new method for the evaluation of the effects of immunisation with tumour vaccines or of gene therapy. Images Figure 3 PMID:8883407

  13. Pluripotent Stem Cells for Gene Therapy of Degenerative Muscle Diseases.

    PubMed

    Loperfido, Mariana; Steele-Stallard, Heather B; Tedesco, Francesco Saverio; VandenDriessche, Thierry

    2015-01-01

    Human pluripotent stem cells represent a unique source for cell-based therapies and regenerative medicine. The intrinsic features of these cells such as their easy accessibility and their capacity to be expanded indefinitely overcome some limitations of conventional adult stem cells. Furthermore, the possibility to derive patient-specific induced pluripotent stem (iPS) cells in combination with the current development of gene modification methods could be used for autologous cell therapies of some genetic diseases. In particular, muscular dystrophies are considered to be a good candidate due to the lack of efficacious therapeutic treatments for patients to date, and in view of the encouraging results arising from recent preclinical studies. Some hurdles, including possible genetic instability and their efficient differentiation into muscle progenitors through vector/transgene-free methods have still to be overcome or need further optimization. Additionally, engraftment and functional contribution to muscle regeneration in pre-clinical models need to be carefully assessed before clinical translation. This review offers a summary of the advanced methods recently developed to derive muscle progenitors from pluripotent stem cells, as well as gene therapy by gene addition and gene editing methods using ZFNs, TALENs or CRISPR/Cas9. We have also discussed the main issues that need to be addressed for successful clinical translation of genetically corrected patient-specific pluripotent stem cells in autologous transplantation trials for skeletal muscle disorders.

  14. Modification of Schwann cell gene expression by electroporation in vivo.

    PubMed

    Aspalter, Manuela; Vyas, Alka; Feiner, Jeffrey; Griffin, John; Brushart, Thomas; Redett, Richard

    2009-01-30

    Clinical outcomes of nerve grafting are often inferior to those of end-to-end nerve repair. This may be due, in part, to the routine use of cutaneous nerve to support motor axon regeneration. In previous work, we have demonstrated that Schwann cells express distinct sensory and motor phenotypes, and that these promote regeneration in a modality-specific fashion. Intra-operative modification of graft Schwann cell phenotype might therefore improve clinical outcomes. This paper demonstrates the feasibility of electroporating genes into intact nerve to modify Schwann cell gene expression. Initial trials established 70 V, 5 ms as optimum electroporation parameters. Intact, denervated, and reinnervated rat tibial nerves were electroporated with the YFP gene and evaluated serially by counting S-100 positive cells that expressed YFP. In intact nerve, a mean of 28% of Schwann cells expressed the gene at 3 days, falling to 20% at 7 days with little expression at later times. There were no significant differences among the three groups at each time period. Electronmicroscopic evaluation of treated, intact nerve revealed only occasional demyelination and axon degeneration. Intra-operative electroporation of nerve graft is thus a practical means of altering Schwann cell gene expression without the risks inherent in viral transfection.

  15. Gene expression profiles of bronchoalveolar cells in Pulmonary TB

    PubMed Central

    Raju, Bindu; Hoshino, Yoshihiko; Belitskaya-Lévy, Ilana; Dawson, Rod; Ress, Stanley; Gold, Jeffrey A.; Condos, Rany; Pine, Richard; Brown, Stuart; Nolan, Anna; Rom, William N.; Weiden, Michael D.

    2008-01-01

    The host response to Mycobacterium tuberculosis includes macrophage activation, inflammation with increased immune effector cells, tissue necrosis and cavity formation, and fibrosis, distortion, and bronchiectasis. To evaluate the molecular basis of the immune response in the lungs of patients with active pulmonary tuberculosis (TB), we used bronchoalveolar lavage to obtain cells at the site of infection. Affymetrix Genechip micro-arrays and cDNA nylon filter microarrays interrogated gene expression in BAL cells from 11 healthy controls and 17 patients with active pulmonary TB. We found altered gene expression for 69 genes in TB versus normal controls that included cell surface markers, cytokines, chemokines, receptors, transcription factors, and complement components. In addition, TB BAL cell gene expression patternssegregated into 2 groups: one suggestive of a T helper type 1 (Th1) cellular immune response with increased STAT-4, IFN-γ receptor, and MIG expression with increased IFN-γ protein levels in BAL fluid; the other group displayed characteristics of Th2 immunity with increased STAT-6, CD81, and IL-10 receptor expression. We were able to demonstrate that a Th2 presentation could change to a Th1 pattern after anti-tuberculous treatment in one TB patient studied serially. These gene expression data support the conclusion that pulmonary TB produces a global change in the BAL cell transcriptome with manifestations of either Th1 or Th2 immunity. PMID:17921069

  16. Isolating human DNA repair genes using rodent-cell mutants

    SciTech Connect

    Thompson, L.H.; Weber, C.A.; Brookman, K.W.; Salazar, E.P.; Stewart, S.A.; Mitchell, D.L.

    1987-03-23

    The DNA repair systems of rodent and human cells appear to be at least as complex genetically as those in lower eukaryotes and bacteria. The use of mutant lines of rodent cells as a means of identifying human repair genes by functional complementation offers a new approach toward studying the role of repair in mutagenesis and carcinogenesis. In each of six cases examined using hybrid cells, specific human chromosomes have been identified that correct CHO cell mutations affecting repair of damage from uv or ionizing radiations. This finding suggests that both the repair genes and proteins may be virtually interchangeable between rodent and human cells. Using cosmid vectors, human repair genes that map to chromosome 19 have cloned as functional sequences: ERCC2 and XRCC1. ERCC1 was found to have homology with the yeast excision repair gene RAD10. Transformants of repair-deficient cell lines carrying the corresponding human gene show efficient correction of repair capacity by all criteria examined. 39 refs., 1 fig., 1 tab.

  17. Single-Cell Isolation and Gene Analysis: Pitfalls and Possibilities

    PubMed Central

    Hodne, Kjetil; Weltzien, Finn-Arne

    2015-01-01

    During the last two decades single-cell analysis (SCA) has revealed extensive phenotypic differences within homogenous cell populations. These phenotypic differences are reflected in the stochastic nature of gene regulation, which is often masked by qualitatively and quantitatively averaging in whole tissue analyses. The ability to isolate transcripts and investigate how genes are regulated at the single cell level requires highly sensitive and refined methods. This paper reviews different strategies currently used for SCA, including harvesting, reverse transcription, and amplification of the RNA, followed by methods for transcript quantification. The review provides the historical background to SCA, discusses limitations, and current and future possibilities in this exciting field of research. PMID:26569222

  18. Stem-Cell-Based Gene Therapy for HIV Infection

    PubMed Central

    Zhen, Anjie; Kitchen, Scott

    2013-01-01

    Despite the enormous success of combined anti-retroviral therapy, HIV infection is still a lifelong disease and continues to spread rapidly worldwide. There is a pressing need to develop a treatment that will cure HIV infection. Recent progress in stem cell manipulation and advancements in humanized mouse models have allowed rapid developments of gene therapy for HIV treatment. In this review, we will discuss two aspects of HIV gene therapy using human hematopoietic stem cells. The first is to generate immune systems resistant to HIV infection while the second strategy involves enhancing anti-HIV immunity to eliminate HIV infected cells. PMID:24368413

  19. Stem-cell-based gene therapy for HIV infection.

    PubMed

    Zhen, Anjie; Kitchen, Scott

    2013-12-24

    Despite the enormous success of combined anti-retroviral therapy, HIV infection is still a lifelong disease and continues to spread rapidly worldwide. There is a pressing need to develop a treatment that will cure HIV infection. Recent progress in stem cell manipulation and advancements in humanized mouse models have allowed rapid developments of gene therapy for HIV treatment. In this review, we will discuss two aspects of HIV gene therapy using human hematopoietic stem cells. The first is to generate immune systems resistant to HIV infection while the second strategy involves enhancing anti-HIV immunity to eliminate HIV infected cells.

  20. Salivary epithelial cells: an unassuming target site for gene therapeutics

    PubMed Central

    Perez, Paola; Rowzee, Anne M.; Zheng, Changyu; Adriaansen, Janik; Baum, Bruce J.

    2010-01-01

    Salivary glands are classical exocrine glands whose external secretions result in the production of saliva. However, in addition to the secretion of exocrine proteins, salivary epithelial cells are also capable of secreting proteins internally, into the bloodstream. This brief review examines the potential for using salivary epithelial cells as a target site for in situ gene transfer, with an ultimate goal of producing therapeutic proteins for treating both systemic and upper gastrointestinal tract disorders. The review discusses the protein secretory pathways reported to be present in salivary epithelial cells, the viral gene transfer vectors shown useful for transducing these cells, model transgenic secretory proteins examined, and some clinical conditions that might benefit from such salivary gland gene transfer. PMID:20219693

  1. Modification of the apolipoprotein B gene in HepG2 cells by gene targeting.

    PubMed Central

    Farese, R V; Flynn, L M; Young, S G

    1992-01-01

    The HepG2 cell line has been used extensively to study the synthesis and secretion of apolipoprotein (apo) B. In this study, we tested whether gene-targeting techniques can be used to inactivate one of the apo B alleles in HepG2 cells by homologous recombination using a transfected gene-targeting vector. Our vector contained exons 1-7 of the apo B gene, in which exon 2 was interrupted by a promoterless neomycin resistance (neo(r)) gene. The recombination of this vector with the cognate gene would inactivate an apo B allele and enable the apo B promoter to activate the transcription of the neo(r) gene. To detect the rare homologous recombinant clone, we developed a novel solid phase RIA that uses the apo B-specific monoclonal antibody MB19 to analyze the apo B secreted by G418-resistant (G418r) clones. Antibody MB19 detects a two-allele genetic polymorphism in apo B by binding to the apo B allotypes MB19(1) and MB19(2) with high and low affinity, respectively. HepG2 cells normally secrete both the apo B MB19 allotypes. Using the MB19 immunoassay, we identified a G418r HepG2 clone that had lost the ability to secrete the MB19(1) allotype. The inactivation of an apo B allele of this clone was confirmed by the polymerase chain reaction amplification of an 865-bp fragment unique to the targeted apo B allele and by Southern blotting of genomic DNA. This study demonstrates that gene-targeting techniques can be used to modify the apo B gene in HepG2 cells and demonstrates the usefulness of a novel solid phase RIA system for detecting apo B gene targeting events in this cell line. Images PMID:1321843

  2. Stem cell based anti-HIV Gene therapy

    PubMed Central

    Kitchen, Scott G.; Shimizu, Saki; An, Dong Sung

    2011-01-01

    Human stem cell-based therapeutic intervention strategies for treating HIV infection have recently undergone a renaissance as a major focus of investigation. Unlike most conventional antiviral therapies, genetically engineered hematopoietic stem cells possess the capacity for prolonged self-renewal that would continuously produce protected immune cells to fight against HIV. A successful strategy therefore has the potential to stably control and ultimately eradicate HIV from patients by a single or minimal treatment. Recent progress in the development of new technologies and clinical trials sets the stage for the current generation of gene therapy approaches to combat HIV infection. In this review, we will discuss two major approaches that are currently underway in the development of stem cell-based gene therapy to target HIV: One that focuses on the protection of cells from productive infection with HIV, and the other that focuses on targeting immune cells to directly combat HIV infection. PMID:21247612

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

    PubMed

    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.

  4. HIV Cell-to-Cell Spread Results in Earlier Onset of Viral Gene Expression by Multiple Infections per Cell

    PubMed Central

    Boullé, Mikaël; Müller, Thorsten G.; Dähling, Sabrina; Jackson, Laurelle; Mahamed, Deeqa; Oom, Lance; Lustig, Gila

    2016-01-01

    Cell-to-cell spread of HIV, a directed mode of viral transmission, has been observed to be more rapid than cell-free infection. However, a mechanism for earlier onset of viral gene expression in cell-to-cell spread was previously uncharacterized. Here we used time-lapse microscopy combined with automated image analysis to quantify the timing of the onset of HIV gene expression in a fluorescent reporter cell line, as well as single cell staining for infection over time in primary cells. We compared cell-to-cell spread of HIV to cell-free infection, and limited both types of transmission to a two-hour window to minimize differences due to virus transit time to the cell. The mean time to detectable onset of viral gene expression in cell-to-cell spread was accelerated by 19% in the reporter cell line and by 35% in peripheral blood mononuclear cells relative to cell-free HIV infection. Neither factors secreted by infected cells, nor contact with infected cells in the absence of transmission, detectably changed onset. We recapitulated the earlier onset by infecting with multiple cell-free viruses per cell. Surprisingly, the acceleration in onset of viral gene expression was not explained by cooperativity between infecting virions. Instead, more rapid onset was consistent with a model where the fastest expressing virus out of the infecting virus pool sets the time for infection independently of the other co-infecting viruses. PMID:27812216

  5. Temporal order of gene replication in Chinese hamster ovary cells.

    PubMed Central

    Taljanidisz, J; Popowski, J; Sarkar, N

    1989-01-01

    To investigate the molecular basis of the regulatory mechanisms responsible for the orderly replication of the mammalian genome, we have developed an experimental system by which the replication order of various genes can be defined with relative ease and precision. Exponentially growing CHO-K1 cells were separated into populations representing various stages of the cell cycle by centrifugal elutriation and analyzed for cell cycle status flow cytometry. The replication of specific genes in each elutriated fraction was measured by labeling with 5-mercuri-dCTP and [3H]dTPP under conditions of optimal DNA synthesis after cell permeabilization with lysolecithin. Newly synthesized mercurated DNA from each elutriated fraction was purified by affinity chromatography on thiol-agarose and replicated with the large fragment of Escherichia coli DNA polymerase I by using [alpha-32P]dATP and random primers. The 32P-labeled DNA representative of various stages of the cell cycle was then hybridized with dot blots of plasmid DNA containing specific cloned genes. From these results, it was possible to deduce the nuclear DNA content at the time each specific gene replicated during S phase (C value). The C values of 29 genes, which included single-copy genes, multifamily genes, oncogenes, and repetitive sequences, were determined and found to be distributed over the entire S phase. Of the 28 genes studied, 19 had been examined by others using in vivo labeling techniques, with results which agreed with the replication pattern observed in this study. The replication times of nine other genes are described here for the first time. Our method of analysis is sensitive enough to determine the replication time of single-copy genes. The replication times of various genes and their levels of expression in exponentially growing CHO cells were compared. Although there was a general correlation between transcriptional activity and replication in the first half of S phase, examination of specific

  6. Gene array identification of Ipf1/Pdx1-/- regulated genes in pancreatic progenitor cells

    PubMed Central

    Svensson, Per; Williams, Cecilia; Lundeberg, Joakim; Rydén, Patrik; Bergqvist, Ingela; Edlund, Helena

    2007-01-01

    Background The homeodomain transcription factor IPF1/PDX1 exerts a dual role in the pancreas; Ipf1/Pdx1 global null mutants fail to develop a pancreas whereas conditional inactivation of Ipf1/Pdx1 in β-cells leads to impaired β-cell function and diabetes. Although several putative target genes have been linked to the β-cell function of Ipf1/Pdx1, relatively little is known with respect to genes regulated by IPF1/PDX1 in early pancreatic progenitor cells. Results Microarray analyses identified a total of 111 genes that were differentially expressed in e10.5 pancreatic buds of Ipf1/Pdx1-/- embryos. The expression of one of these, Spondin 1, which encodes an extracellular matrix protein, has not previously been described in the pancreas. Quantitative real-time RT-PCR analyses and immunohistochemical analyses also revealed that the expression of FgfR2IIIb, that encodes the receptor for FGF10, was down-regulated in Ipf1/Pdx1-/- pancreatic progenitor cells. Conclusion This microarray analysis has identified a number of candidate genes that are differentially expressed in Ipf1/Pdx1-/- pancreatic buds. Several of the differentially expressed genes were known to be important for pancreatic progenitor cell proliferation and differentiation whereas others have not previously been associated with pancreatic development. PMID:18036209

  7. Classification of dendritic cell phenotypes from gene expression data

    PubMed Central

    2011-01-01

    Background The selection of relevant genes for sample classification is a common task in many gene expression studies. Although a number of tools have been developed to identify optimal gene expression signatures, they often generate gene lists that are too long to be exploited clinically. Consequently, researchers in the field try to identify the smallest set of genes that provide good sample classification. We investigated the genome-wide expression of the inflammatory phenotype in dendritic cells. Dendritic cells are a complex group of cells that play a critical role in vertebrate immunity. Therefore, the prediction of the inflammatory phenotype in these cells may help with the selection of immune-modulating compounds. Results A data mining protocol was applied to microarray data for murine cell lines treated with various inflammatory stimuli. The learning and validation data sets consisted of 155 and 49 samples, respectively. The data mining protocol reduced the number of probe sets from 5,802 to 10, then from 10 to 6 and finally from 6 to 3. The performances of a set of supervised classification models were compared. The best accuracy, when using the six following genes --Il12b, Cd40, Socs3, Irgm1, Plin2 and Lgals3bp-- was obtained by Tree Augmented Naïve Bayes and Nearest Neighbour (91.8%). Using the smallest set of three genes --Il12b, Cd40 and Socs3-- the performance remained satisfactory and the best accuracy was with Support Vector Machine (95.9%). These data mining models, using data for the genes Il12b, Cd40 and Socs3, were validated with a human data set consisting of 27 samples. Support Vector Machines (71.4%) and Nearest Neighbour (92.6%) gave the worst performances, but the remaining models correctly classified all the 27 samples. Conclusions The genes selected by the data mining protocol proposed were shown to be informative for discriminating between inflammatory and steady-state phenotypes in dendritic cells. The robustness of the data mining

  8. Nucleus- and cell-specific gene expression in monkey thalamus.

    PubMed

    Murray, Karl D; Choudary, Prabhakara V; Jones, Edward G

    2007-02-06

    Nuclei of the mammalian thalamus are aggregations of neurons with unique architectures and input-output connections, yet the molecular determinants of their organizational specificity remain unknown. By comparing expression profiles of thalamus and cerebral cortex in adult rhesus monkeys, we identified transcripts that are unique to dorsal thalamus or to individual nuclei within it. Real-time quantitative PCR and in situ hybridization analyses confirmed the findings. Expression profiling of individual nuclei microdissected from the dorsal thalamus revealed additional subsets of nucleus-specific genes. Functional annotation using Gene Ontology (GO) vocabulary and Ingenuity Pathways Analysis revealed overrepresentation of GO categories related to development, morphogenesis, cell-cell interactions, and extracellular matrix within the thalamus- and nucleus-specific genes, many involved in the Wnt signaling pathway. Examples included the transcription factor TCF7L2, localized exclusively to excitatory neurons; a calmodulin-binding protein PCP4; the bone extracellular matrix molecules SPP1 and SPARC; and other genes involved in axon outgrowth and cell matrix interactions. Other nucleus-specific genes such as CBLN1 are involved in synaptogenesis. The genes identified likely underlie nuclear specification, cell phenotype, and connectivity during development and their maintenance in the adult thalamus.

  9. Rod photoreceptor-specific gene expression in human retinoblastoma cells.

    PubMed Central

    Di Polo, A; Farber, D B

    1995-01-01

    Retinoblastoma cells in culture have previously been shown to express cone-specific genes but not their rod counterparts. We have detected the messages for the rod alpha, beta, and gamma subunits of cGMP phosphodiesterase (PDE), the rod alpha subunit of transducin, rod opsin, and the cone alpha' subunit of PDE in RNA of human Y-79 retinoblastoma cells by reverse transcription-PCR. Quantitative analysis of the mRNAs for the rod alpha and cone alpha' PDE subunits revealed that they were expressed at comparable levels; however, the transcript encoding the rod beta PDE subunit was 10 times more abundant in these cells. Northern hybridization analysis of Y-79 cell RNA confirmed the presence of the transcripts for rod and cone PDE catalytic subunits. To test whether the transcriptional machinery required for the expression of rod-specific genes was endogenous in Y-79 retinoblastoma cells, cultures were transfected with a construct containing the promoter region of the rod beta PDE subunit gene attached to the firefly luciferase reporter vector. Significant levels of reporter enzyme activity were observed in the cell lysates. Our results demonstrate that the Y-79 retinoblastoma cell line is a good model system for the study of transcriptional regulation of rod-specific genes. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7732024

  10. Positron Emission Tomography Reporter Genes and Reporter Probes: Gene and Cell Therapy Applications

    PubMed Central

    Yaghoubi, Shahriar S.; Campbell, Dean O.; Radu, Caius G.; Czernin, Johannes

    2012-01-01

    Positron emission tomography (PET) imaging reporter genes (IRGs) and PET reporter probes (PRPs) are amongst the most valuable tools for gene and cell therapy. PET IRGs/PRPs can be used to non-invasively monitor all aspects of the kinetics of therapeutic transgenes and cells in all types of living mammals. This technology is generalizable and can allow long-term kinetics monitoring. In gene therapy, PET IRGs/PRPs can be used for whole-body imaging of therapeutic transgene expression, monitoring variations in the magnitude of transgene expression over time. In cell or cellular gene therapy, PET IRGs/PRPs can be used for whole-body monitoring of therapeutic cell locations, quantity at all locations, survival and proliferation over time and also possibly changes in characteristics or function over time. In this review, we have classified PET IRGs/PRPs into two groups based on the source from which they were derived: human or non-human. This classification addresses the important concern of potential immunogenicity in humans, which is important for expansion of PET IRG imaging in clinical trials. We have then discussed the application of this technology in gene/cell therapy and described its use in these fields, including a summary of using PET IRGs/PRPs in gene and cell therapy clinical trials. This review concludes with a discussion of the future direction of PET IRGs/PRPs and recommends cell and gene therapists collaborate with molecular imaging experts early in their investigations to choose a PET IRG/PRP system suitable for progression into clinical trials. PMID:22509201

  11. Cell-based gene therapy against HIV.

    PubMed

    Dey, R; Pillai, B

    2015-11-01

    The ability to integrate inside the host genome lays a strong foundation for HIV to play hide and seek with the host's immune surveillance mechanisms. Present anti-viral therapies, although successful in suppressing the virus to a certain level, fail to wipe it out completely. However, recent approaches in modifying stem cells and enabling them to give rise to potent/resistant T-cells against HIV holds immense hope for eradication of the virus from the host. In this review, we will briefly discuss previous landmark studies on engineering stem cells or T-cells that have been explored for therapeutic efficacy against HIV. We will also analyze potential benefits and pitfalls of some studies done recently and will share our opinion on emerging trends.

  12. Single-cell RNA-sequence analysis of mouse glomerular mesangial cells uncovers mesangial cell essential genes.

    PubMed

    Lu, Yuqiu; Ye, Yuting; Yang, Qianqian; Shi, Shaolin

    2017-03-17

    Mesangial cells are essential for the structure and function of glomeruli, but the mechanisms underlying these roles are not well understood. Here, we performed a single-cell RNA-sequence (RNA-seq) analysis of mouse mesangial cells using the Fluidigm C1 platform. We found that gene expression in individual mesangial cells was tremendously heterogeneous, with mean correlation coefficients of 0.20, and most mesangial genes were actually expressed in only a portion of mesangial cells and are therefore presumably dispensable. In contrast, 1,045 genes were expressed in every single mesangial cell and were considered mesangial cell essential genes. A gene ontology analysis revealed a significant enrichment of genes associated with the endothelium, supporting the inference that mesangial cells function as pericytes. Among 58 endothelium-associated genes, 18 encode proteins that are secreted and may be directly involved in endothelial homeostasis. Importantly, 11 (Angpt2, Anxa5, Axl, Ecm1, Eng, Fn1, Mfge8, Msn, Nrp1, Serpine2, and Sparc) were upregulated, while 2 (Apoe and Fgf1) were downregulated in various glomerulopathies. The enrichment of genes associated with other reported functions of mesangial cells was also found. Furthermore, we identified 173 genes specifically expressed in every mesangial cell in glomeruli from the mesangial cell essential gene list. Finally, based on single mesangial cell RNA-seq results, we found that commonly used glomerular cell type markers, including Fhl2, Igfbp5, Wt1, Tek/Tie2, Kdr/Flk1, Flt1/Vegfr1, and Cd34, are actually not specific. Thus, single mesangial cell RNA-seq analysis has provided insights into the functions and underlying mechanisms of mesangial cells.

  13. Expression and function of FERMT genes in colon carcinoma cells.

    PubMed

    Kiriyama, Kenji; Hirohashi, Yoshihiko; Torigoe, Toshihiko; Kubo, Terufumi; Tamura, Yasuaki; Kanaseki, Takayuki; Takahashi, Akari; Nakazawa, Emiri; Saka, Eri; Ragnarsson, Charlotte; Nakatsugawa, Munehide; Inoda, Satoko; Asanuma, Hiroko; Takasu, Hideo; Hasegawa, Tadashi; Yasoshima, Takahiro; Hirata, Koichi; Sato, Noriyuki

    2013-01-01

    Invasion into the matrix is one of hallmarks of malignant diseases and is the first step for tumor metastasis. Thus, analysis of the molecular mechanisms of invasion is essential to overcome tumor cell invasion. In the present study, we screened for colon carcinoma-specific genes using a cDNA microarray database of colon carcinoma tissues and normal colon tissues, and we found that fermitin family member-1 (FERMT1) is overexpressed in colon carcinoma cells. FRRMT1, FERMT2 and FERMT3 expression was investigated in colon carcinoma cells. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that only FERMT1 had cancer cell-specific expression. Protein expression of FERMT1 was confirmed by western blotting and immunohistochemical staining. To address the molecular functions of FERMT genes in colon carcinoma cells, we established FERMT1-, FERMT2- and FERMT3-overexpressing colon carcinoma cells. FERMT1-overexpressing cells exhibited greater invasive ability than did FERMT2- and FERMT3-overexpressing cells. On the other hand, FERMT1-, FERMT2- and FERMT3-overexpressing cells exhibited enhancement of cell growth. Taken together, the results of this study indicate that FERMT1 is expressed specifically in colon carcinoma cells, and has roles in matrix invasion and cell growth. These findings indicate that FERMT1 is a potential molecular target for cancer therapy.

  14. Deregulation of lipid metabolism pathway genes in nasopharyngeal carcinoma cells

    PubMed Central

    DAKER, MAELINDA; BHUVANENDRAN, SAATHEEYAVAANE; AHMAD, MUNIRAH; TAKADA, KENZO; KHOO, ALAN SOO-BENG

    2012-01-01

    Nasopharyngeal carcinoma (NPC) is a unique tumour of epithelial origin with a distinct geographical distribution, closely associated with the Epstein-Barr virus (EBV). EBV-encoded RNAs (EBERs) are small non-polyadenylated RNAs that are abundantly expressed in latent EBV-infected NPC cells. To study the role of EBERs in NPC, we established stable expression of EBERs in HK1, an EBV-negative NPC cell line. Cells expressing EBERs consistently exhibited an increased growth rate. However, EBERs did not confer resistance towards cisplatin-induced apoptosis or promote migration or invasion ability in the cells tested. Using microarray gene expression profiling, we identified potential candidate genes that were deregulated in NPC cells expressing EBERs. Gene Ontology analysis of the data set revealed that EBERs upregulate the cellular lipid metabolic process. Upregulation of low-density lipoprotein receptor (LDLR) and fatty acid synthase (FASN) was observed in EBER-expressing cells. NPC cells exhibited LDL-dependent cell proliferation. In addition, a polyphenolic flavonoid compound, quercetin, known to inhibit FASN, was found to inhibit proliferation of NPC cells. PMID:23292678

  15. Cell-specific targeting strategies for electroporation-mediated gene delivery in cells and animals.

    PubMed

    Dean, David A

    2013-10-01

    The use of electroporation to facilitate gene transfer is an extremely powerful and useful method for both in vitro and in vivo applications. One of its great strengths is that it induces functional destabilization and permeabilization of cell membranes throughout a tissue leading to widespread gene transfer to multiple cells and cell types within the electric field. While this is a strength, it can also be a limitation in terms of cell-specific gene delivery. The ability to restrict gene delivery and expression to particular cell types is of paramount importance for many types of gene therapy, since ectopic expression of a transgene could lead to deleterious host inflammatory responses or dysregulation of normal cellular functions. At present, there are relatively few ways to obtain cell-specific targeting of nonviral vectors, molecular probes, small molecules, and imaging agents. We have developed a novel means of restricting gene delivery to desired cell types based on the ability to control the transport of plasmids into the nuclei of desired cell types. In this article, we discuss the mechanisms of this approach and several applications in living animals to demonstrate the benefits of the combination of electroporation and selective nuclear import of plasmids for cell-specific gene delivery.

  16. Suicide genes: monitoring cells in patients with a safety switch.

    PubMed

    Eissenberg, Linda G; Rettig, Michael; Dehdashti, Farrokh; Piwnica-Worms, David; DiPersio, John F

    2014-01-01

    Clinical trials increasingly incorporate suicide genes either as direct lytic agents for tumors or as safety switches in therapies based on genetically modified cells. Suicide genes can also be used as non-invasive reporters to monitor the biological consequences of administering genetically modified cells to patients and gather information relevant to patient safety. These genes can monitor therapeutic outcomes addressable by early clinical intervention. As an example, our recent clinical trial used (18)F-9-(4-fluoro-3-hydroxymethylbutyl)guanine ((18)FHBG) and positron emission tomography (PET)/CT scans to follow T cells transduced with herpes simplex virus thymidine kinase after administration to patients. Guided by preclinical data we ultimately hope to discern whether a particular pattern of transduced T cell migration within patients reflects early development of graft vs. host disease. Current difficulties in terms of choice of suicide gene, biodistribution of radiolabeled tracers in humans vs. animal models, and threshold levels of genetically modified cells needed for detection by PET/CT are discussed. As alternative suicide genes are developed, additional radiolabel probes suitable for imaging in patients should be considered.

  17. Stem and progenitor cell-mediated tumor selective gene therapy.

    PubMed

    Aboody, K S; Najbauer, J; Danks, M K

    2008-05-01

    The poor prognosis for patients with aggressive or metastatic tumors and the toxic side effects of currently available treatments necessitate the development of more effective tumor-selective therapies. Stem/progenitor cells display inherent tumor-tropic properties that can be exploited for targeted delivery of anticancer genes to invasive and metastatic tumors. Therapeutic genes that have been inserted into stem cells and delivered to tumors with high selectivity include prodrug-activating enzymes (cytosine deaminase, carboxylesterase, thymidine kinase), interleukins (IL-2, IL-4, IL-12, IL-23), interferon-beta, apoptosis-promoting genes (tumor necrosis factor-related apoptosis-inducing ligand) and metalloproteinases (PEX). We and others have demonstrated that neural and mesenchymal stem cells can deliver therapeutic genes to elicit a significant antitumor response in animal models of intracranial glioma, medulloblastoma, melanoma brain metastasis, disseminated neuroblastoma and breast cancer lung metastasis. Most studies reported reduction in tumor volume (up to 90%) and increased survival of tumor-bearing animals. Complete cures have also been achieved (90% disease-free survival for >1 year of mice bearing disseminated neuroblastoma tumors). As we learn more about the biology of stem cells and the molecular mechanisms that mediate their tumor-tropism and we identify efficacious gene products for specific tumor types, the clinical utility of cell-based delivery strategies becomes increasingly evident.

  18. Identification of hepatic microvascular adhesion-related genes of human colon cancer cells using random homozygous gene perturbation.

    PubMed

    Márquez, Joana; Kohli, Manu; Arteta, Beatriz; Chang, Shaojing; Li, Wu-Bo; Goldblatt, Michael; Vidal-Vanaclocha, Fernando

    2013-11-01

    Random homozygous gene perturbation (RHGP), in combination with liver sinusoidal endothelial cell (LSEC) adhesion screening of clonal colon cancer cells with perturbed genes, was used to identify genes contributing to the hepatic microvascular adhesion of colon cancer cells. Plasmid vector encoding transactivator and gene search vector were transfected into HT-29 human colorectal cancer cells to create a HT-29 RHGP cell library; the adhesion of these library cells to primary cultured mouse LSEC significantly decreased in the presence of RSL1 ligand (inducer), indicating that most of the genes contributing to HT-29 adhesion to LSEC were altered. Next, HT-29 RHGP cell library fractions with upregulated or silenced LSEC adhesion-related genes were isolated. Around 160 clones having altered expression in LSEC adhesion-related genes were obtained, and nine relevant protein-coding genes were identified. Some were proadhesive genes detected because of their overexpression in adherent HT-29 cells (DGCR8 and EFEMP1 genes) and their silenced status in nonadherent HT-29 cells (DGKE, DPY19L1, KIAA0753, PVR and USP11 genes). Others were antiadhesive genes detected because of their overexpression in nonadherent HT-29 cells (ITPKC gene) and their silenced status in adherent HT-29 cells (PPP6R2 gene). Silencing of PVR, DGCR8 and EFEMP1 genes decreased adhesion to LSEC and hepatic microvascular retention of HT-29 cells. The results conclude that RHGP was a valuable strategy for the discovery of mechanisms regulating microvascular adhesion of circulating colon cancer cells before hepatic metastasis formation. Identified genes may contribute to understand the metastatic process of colon cancer and to discovering molecular targets for hepatic metastasis therapeutics.

  19. Restoration of Mitochondrial Gene Expression Using a Cloned Human Gene in Chinese Hamster Lung Cell Mutant

    PubMed Central

    Sherif, Zaki A; Broome, Carolyn W

    2015-01-01

    Background Gal−32 is a Chinese hamster lung cell nuclear mutant that is unable to grow in galactose due to a defect in mitochondrial protein synthesis. Since the product of the Gal−32 gene was unknown, it was imperative to use phenotypic complementation to clone a human gene that corrected the Gal−32 mutation. Results Recessive Gal−32 cells were co-transformed with pSV2-neo plasmid DNA and recombinant DNA from a human genomic library containing the dominant human Gal+ gene and a chloramphenicol-resistance (camr) gene present in the pSV13 vector. Primary transformants were selected by growth in galactose and the neomycin analog G418. In order to rescue the human Gal+ gene, a genomic library was constructed with primary transformant DNA and the pCV108 cosmid vector. The camr gene was used to identify clones with the nearby human sequences. DNA from two camr, Alu-hybridizing clones was able to transform the recessive Gal−32 cells to the Gal+ phenotype and to restore mitochondrial protein synthesis. Conclusion These data demonstrate the isolation of two pCV108-transformant recombinant clones containing a human gene that complements the Chinese hamster Gal−32 mutation and restores galactose metabolism. PMID:26052559

  20. Heterozygous inactivation of the Nf1 gene in myeloid cells enhances neointima formation via a rosuvastatin-sensitive cellular pathway.

    PubMed

    Stansfield, Brian K; Bessler, Waylan K; Mali, Raghuveer; Mund, Julie A; Downing, Brandon; Li, Fang; Sarchet, Kara N; DiStasi, Matthew R; Conway, Simon J; Kapur, Reuben; Ingram, David A

    2013-03-01

    Mutations in the NF1 tumor suppressor gene cause Neurofibromatosis type 1 (NF1). Neurofibromin, the protein product of NF1, functions as a negative regulator of Ras activity. Some NF1 patients develop cardiovascular disease, which represents an underrecognized disease complication and contributes to excess morbidity and mortality. Specifically, NF1 patients develop arterial occlusion resulting in tissue ischemia and sudden death. Murine studies demonstrate that heterozygous inactivation of Nf1 (Nf1(+/-)) in bone marrow cells enhances neointima formation following arterial injury. Macrophages infiltrate Nf1(+/-) neointimas, and NF1 patients have increased circulating inflammatory monocytes in their peripheral blood. Therefore, we tested the hypothesis that heterozygous inactivation of Nf1 in myeloid cells is sufficient for neointima formation. Specific ablation of a single copy of the Nf1 gene in myeloid cells alone mobilizes a discrete pro-inflammatory murine monocyte population via a cell autonomous and gene-dosage dependent mechanism. Furthermore, lineage-restricted heterozygous inactivation of Nf1 in myeloid cells is sufficient to reproduce the enhanced neointima formation observed in Nf1(+/-) mice when compared with wild-type controls, and homozygous inactivation of Nf1 in myeloid cells amplified the degree of arterial stenosis after arterial injury. Treatment of Nf1(+/-) mice with rosuvastatin, a stain with anti-inflammatory properties, significantly reduced neointima formation when compared with control. These studies identify neurofibromin-deficient myeloid cells as critical cellular effectors of Nf1(+/-) neointima formation and propose a potential therapeutic for NF1 cardiovascular disease.

  1. UV-induced changes in cell cycle and gene expression within rabbit lens epithelial cells

    SciTech Connect

    Sidjanin, D.; Grdina, D.; Woloschak, G.E.

    1994-11-01

    Damage to lens epithelial cells is a probable initiation process in cataract formation induced by ultraviolet radiation. These experiments investigated the ability of 254 nm radiation on cell cycle progression and gene expression in rabbit lens epithelial cell line N/N1003A. No changes in expression of c-fos, c-jun, alpha- tubulin, or vimentin was observed following UV exposure. Using flow cytometry, an accumulation of cells in G1/S phase of the cell cycle 1 hr following exposure. The observed changes in gene expression, especially the decreased histone transcripts reported here may play a role in UV induced inhibition of cell cycle progression.

  2. Gene Silencing in Insect Cells Using RNAi.

    PubMed

    Wu, Hsuan-Chen; March, John C; Bentley, William E

    2016-01-01

    A technique is described for synthesizing and transfecting double stranded RNA (dsRNA) for RNA interference (RNAi) in Sf-21 cell culture. Transfection with dsRNA only requires an hour and the cells usually recover within 12 h. Suggestions for designing dsRNA are included in the methods. Furthermore, websites are provided for rapid and effective dsRNA design. Three kits are essential for using the described methods: RNAqueous®-4PCR, Megascript™ T7 kit, and the Superscript™ III kit from Life Technologies, Inc.

  3. Harnessing single cell sorting to identify cell division genes and regulators in bacteria.

    PubMed

    Burke, Catherine; Liu, Michael; Britton, Warwick; Triccas, James A; Thomas, Torsten; Smith, Adrian L; Allen, Steven; Salomon, Robert; Harry, Elizabeth

    2013-01-01

    Cell division is an essential cellular process that requires an array of known and unknown proteins for its spatial and temporal regulation. Here we develop a novel, high-throughput screening method for the identification of bacterial cell division genes and regulators. The method combines the over-expression of a shotgun genomic expression library to perturb the cell division process with high-throughput flow cytometry sorting to screen many thousands of clones. Using this approach, we recovered clones with a filamentous morphology for the model bacterium, Escherichia coli. Genetic analysis revealed that our screen identified both known cell division genes, and genes that have not previously been identified to be involved in cell division. This novel screening strategy is applicable to a wide range of organisms, including pathogenic bacteria, where cell division genes and regulators are attractive drug targets for antibiotic development.

  4. Harnessing Single Cell Sorting to Identify Cell Division Genes and Regulators in Bacteria

    PubMed Central

    Burke, Catherine; Liu, Michael; Britton, Warwick; Triccas, James A.; Thomas, Torsten; Smith, Adrian L.; Allen, Steven; Salomon, Robert; Harry, Elizabeth

    2013-01-01

    Cell division is an essential cellular process that requires an array of known and unknown proteins for its spatial and temporal regulation. Here we develop a novel, high-throughput screening method for the identification of bacterial cell division genes and regulators. The method combines the over-expression of a shotgun genomic expression library to perturb the cell division process with high-throughput flow cytometry sorting to screen many thousands of clones. Using this approach, we recovered clones with a filamentous morphology for the model bacterium, Escherichia coli. Genetic analysis revealed that our screen identified both known cell division genes, and genes that have not previously been identified to be involved in cell division. This novel screening strategy is applicable to a wide range of organisms, including pathogenic bacteria, where cell division genes and regulators are attractive drug targets for antibiotic development. PMID:23565292

  5. Impact of the cell division cycle on gene circuits

    NASA Astrophysics Data System (ADS)

    Bierbaum, Veronika; Klumpp, Stefan

    2015-12-01

    In growing cells, protein synthesis and cell growth are typically not synchronous, and, thus, protein concentrations vary over the cell division cycle. We have developed a theoretical description of genetic regulatory systems in bacteria that explicitly considers the cell division cycle to investigate its impact on gene expression. We calculate the cell-to-cell variations arising from cells being at different stages in the division cycle for unregulated genes and for basic regulatory mechanisms. These variations contribute to the extrinsic noise observed in single-cell experiments, and are most significant for proteins with short lifetimes. Negative autoregulation buffers against variation of protein concentration over the division cycle, but the effect is found to be relatively weak. Stronger buffering is achieved by an increased protein lifetime. Positive autoregulation can strongly amplify such variation if the parameters are set to values that lead to resonance-like behaviour. For cooperative positive autoregulation, the concentration variation over the division cycle diminishes the parameter region of bistability and modulates the switching times between the two stable states. The same effects are seen for a two-gene mutual-repression toggle switch. By contrast, an oscillatory circuit, the repressilator, is only weakly affected by the division cycle.

  6. Porphyromonas gingivalis genes isolated by screening for epithelial cell attachment.

    PubMed Central

    Duncan, M J; Emory, S A; Almira, E C

    1996-01-01

    Porphyromonas gingivalis is associated with chronic and severe periodontitis in adults. P. gingivalis and the other periodontal pathogens colonize and interact with gingival epithelial cells, but the genes and molecular mechanisms involved are unknown. To dissect the first steps in these interactions, a P. gingivalis expression library was screened for clones which bound human oral epithelial cells. Insert DNA from the recombinant clones did not contain homology to the P. gingivalis fimA gene, encoding fimbrillin, the subunit protein of fimbriae, but showed various degrees of homology to certain cysteine protease-hemagglutinin genes. The DNA sequence of one insert revealed three putative open reading frames which appeared to be in an operon. The relationship between P. gingivalis attachment to epithelial cells and the activities identified by the screen is discussed. PMID:8751909

  7. On a fundamental structure of gene networks in living cells.

    PubMed

    Kravchenko-Balasha, Nataly; Levitzki, Alexander; Goldstein, Andrew; Rotter, Varda; Gross, A; Remacle, F; Levine, R D

    2012-03-20

    Computers are organized into hardware and software. Using a theoretical approach to identify patterns in gene expression in a variety of species, organs, and cell types, we found that biological systems similarly are comprised of a relatively unchanging hardware-like gene pattern. Orthogonal patterns of software-like transcripts vary greatly, even among tumors of the same type from different individuals. Two distinguishable classes could be identified within the hardware-like component: those transcripts that are highly expressed and stable and an adaptable subset with lower expression that respond to external stimuli. Importantly, we demonstrate that this structure is conserved across organisms. Deletions of transcripts from the highly stable core are predicted to result in cell mortality. The approach provides a conceptual thermodynamic-like framework for the analysis of gene-expression levels and networks and their variations in diseased cells.

  8. Large animal models of hematopoietic stem cell gene therapy.

    PubMed

    Trobridge, G D; Kiem, H-P

    2010-08-01

    Large animal models have been instrumental in advancing hematopoietic stem cell (HSC) gene therapy. Here we review the advantages of large animal models, their contributions to the field of HSC gene therapy and recent progress in this field. Several properties of human HSCs including their purification, their cell-cycle characteristics, their response to cytokines and the proliferative demands placed on them after transplantation are more similar in large animal models than in mice. Progress in the development and use of retroviral vectors and ex vivo transduction protocols over the last decade has led to efficient gene transfer in both dogs and nonhuman primates. Importantly, the approaches developed in these models have translated well to the clinic. Large animals continue to be useful to evaluate the efficacy and safety of gene therapy, and dogs with hematopoietic diseases have now been cured by HSC gene therapy. Nonhuman primates allow evaluation of aspects of transplantation as well as disease-specific approaches such as AIDS (acquired immunodeficiency syndrome) gene therapy that can not be modeled well in the dog. Finally, large animal models have been used to evaluate the genotoxicity of viral vectors by comparing integration sites in hematopoietic repopulating cells and monitoring clonality after transplantation.

  9. Genome-editing Technologies for Gene and Cell Therapy.

    PubMed

    Maeder, Morgan L; Gersbach, Charles A

    2016-03-01

    Gene therapy has historically been defined as the addition of new genes to human cells. However, the recent advent of genome-editing technologies has enabled a new paradigm in which the sequence of the human genome can be precisely manipulated to achieve a therapeutic effect. This includes the correction of mutations that cause disease, the addition of therapeutic genes to specific sites in the genome, and the removal of deleterious genes or genome sequences. This review presents the mechanisms of different genome-editing strategies and describes each of the common nuclease-based platforms, including zinc finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, and the CRISPR/Cas9 system. We then summarize the progress made in applying genome editing to various areas of gene and cell therapy, including antiviral strategies, immunotherapies, and the treatment of monogenic hereditary disorders. The current challenges and future prospects for genome editing as a transformative technology for gene and cell therapy are also discussed.

  10. Genome-editing Technologies for Gene and Cell Therapy

    PubMed Central

    Maeder, Morgan L; Gersbach, Charles A

    2016-01-01

    Gene therapy has historically been defined as the addition of new genes to human cells. However, the recent advent of genome-editing technologies has enabled a new paradigm in which the sequence of the human genome can be precisely manipulated to achieve a therapeutic effect. This includes the correction of mutations that cause disease, the addition of therapeutic genes to specific sites in the genome, and the removal of deleterious genes or genome sequences. This review presents the mechanisms of different genome-editing strategies and describes each of the common nuclease-based platforms, including zinc finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, and the CRISPR/Cas9 system. We then summarize the progress made in applying genome editing to various areas of gene and cell therapy, including antiviral strategies, immunotherapies, and the treatment of monogenic hereditary disorders. The current challenges and future prospects for genome editing as a transformative technology for gene and cell therapy are also discussed. PMID:26755333

  11. Identification and Characterization of Renal Cell Carcinoma Gene Markers

    PubMed Central

    Dalgin, Gul S.; Holloway, Dustin T.; Liou, Louis S.; DeLisi, Charles

    2007-01-01

    Microarray gene expression profiling has been used to distinguish histological subtypes of renal cell carcinoma (RCC), and consequently to identify specific tumor markers. The analytical procedures currently in use find sets of genes whose average differential expression across the two categories differ significantly. In general each of the markers thus identified does not distinguish tumor from normal with 100% accuracy, although the group as a whole might be able to do so. For the purpose of developing a widely used economically viable diagnostic signature, however, large groups of genes are not likely to be useful. Here we use two different methods, one a support vector machine variant, and the other an exhaustive search, to reanalyze data previously generated in our Lab (Lenburg et al. 2003). We identify 158 genes, each having an expression level that is higher (lower) in every tumor sample than in any normal sample, and each having a minimum differential expression across the two categories at a significance of 0.01. The set is highly enriched in cancer related genes (p = 1.6 × 10−12), containing 43 genes previously associated with either RCC or other types of cancer. Many of the biomarkers appear to be associated with the central alterations known to be required for cancer transformation. These include the oncogenes JAZF1, AXL, ABL2; tumor suppressors RASD1, PTPRO, TFAP2A, CDKN1C; and genes involved in proteolysis or cell-adhesion such as WASF2, and PAPPA. PMID:19455236

  12. Relevant principal factors affecting the reproducibility of insect primary culture.

    PubMed

    Ogata, Norichika; Iwabuchi, Kikuo

    2017-02-22

    The primary culture of insect cells often suffers from problems with poor reproducibility in the quality of the final cell preparations. The cellular composition of the explants (cell number and cell types), surgical methods (surgical duration and surgical isolation), and physiological and genetic differences between donors may be critical factors affecting the reproducibility of culture. However, little is known about where biological variation (interindividual differences between donors) ends and technical variation (variance in replication of culture conditions) begins. In this study, we cultured larval fat bodies from the Japanese rhinoceros beetle, Allomyrina dichotoma, and evaluated, using linear mixed models, the effect of interindividual variation between donors on the reproducibility of the culture. We also performed transcriptome analysis of the hemocyte-like cells mainly seen in the cultures using RNA sequencing and ultrastructural analyses of hemocytes using a transmission electron microscope, revealing that the cultured cells have many characteristics of insect hemocytes.

  13. Triploid planarian reproduces truly bisexually with euploid gametes produced through a different meiotic system between sex.

    PubMed

    Chinone, Ayako; Nodono, Hanae; Matsumoto, Midori

    2014-06-01

    Although polyploids are common among plants and some animals, polyploidization often causes reproductive failure. Triploids, in particular, are characterized by the problems of chromosomal pairing and segregation during meiosis, which may cause aneuploid gametes and results in sterility. Thus, they are generally considered to reproduce only asexually. In the case of the Platyhelminthes Dugesia ryukyuensis, populations with triploid karyotypes are normally found in nature as both fissiparous and oviparous triploids. Fissiparous triploids can also be experimentally sexualized if they are fed sexual planarians, developing both gonads and other reproductive organs. Fully sexualized worms begin reproducing by copulation rather than fission. In this study, we examined the genotypes of the offspring obtained by breeding sexualized triploids and found that the offspring inherited genes from both parents, i.e., they reproduced truly bisexually. Furthermore, meiotic chromosome behavior in triploid sexualized planarians differed significantly between male and female germ lines, in that female germ line cells remained triploid until prophase I, whereas male germ line cells appeared to become diploid before entry into meiosis. Oocytes at the late diplotene stage contained not only paired bivalents but also unpaired univalents that were suggested to produce diploid eggs if they remained in subsequent processes. Triploid planarians may therefore form euploid gametes by different meiotic systems in female and male germ lines and thus are be able to reproduce sexually in contrast to many other triploid organisms.

  14. Molecular Evolution of Drosophila Germline Stem Cell and Neural Stem Cell Regulating Genes.

    PubMed

    Choi, Jae Young; Aquadro, Charles F

    2015-10-27

    Here, we study the molecular evolution of a near complete set of genes that had functional evidence in the regulation of the Drosophila germline and neural stem cell. Some of these genes have previously been shown to be rapidly evolving by positive selection raising the possibility that stem cell genes as a group have elevated signatures of positive selection. Using recent Drosophila comparative genome sequences and population genomic sequences of Drosophila melanogaster, we have investigated both long- and short-term evolution occurring across these two different stem cell systems, and compared them with a carefully chosen random set of genes to represent the background rate of evolution. Our results showed an excess of genes with evidence of a recent selective sweep in both germline and neural stem cells in D. melanogaster. However compared with their control genes, both stem cell systems had no significant excess of genes with long-term recurrent positive selection in D. melanogaster, or across orthologous sequences from the melanogaster group. The evidence of long-term positive selection was limited to a subset of genes with specific functions in both the germline and neural stem cell system.

  15. Development of gene and stem cell therapy for ocular neurodegeneration

    PubMed Central

    Zhang, Jing-Xue; Wang, Ning-Li; Lu, Qing-Jun

    2015-01-01

    Retinal degenerative diseases pose a serious threat to eye health, but there is currently no effective treatment available. Recent years have witnessed rapid development of several cutting-edge technologies, such as gene therapy, stem cell therapy, and tissue engineering. Due to the special features of ocular structure, some of these technologies have been translated into ophthalmological clinic practice with fruitful achievements, setting a good example for other fields. This paper reviews the development of the gene and stem cell therapies in ophthalmology. PMID:26086019

  16. Potential gene therapy strategies for cancer stem cells.

    PubMed

    Sell, Stewart

    2006-10-01

    To be maximally effective, therapy of cancer must be directed against both the resting stem cells and the proliferating cells of the cancer. The cell populations of both normal and cancer tissues consist of resting stem cells, proliferating transit-amplifying cells, terminally differentiating cells and dying (apoptotic) cells. The difference between normal tissue renewal and growth of cancers is that some of the transit-amplifying cells in the cancer population do not mature into terminally differentiating cells, but instead continue to proliferate and do not die (maturation arrest). Because of this the number of cancer cells increase, whereas the cell population of normal tissues remains a relatively constant. Conventional radiation treatment and chemotherapy kill the actively proliferating transit- amplifying cells of the cancer. Differentiation therapy, using specific targeted inhibitors of activation, effectively induces differentiation of the proliferating transit-amplifying cancer cells. However, even if the proliferating cancer cells are completely inhibited or eliminated, the cancer stem cells may restore the transit-amplifying population, so that clinical remission is usually temporary. The hypothesis presented in this paper is that successful cancer therapy must be directed against both the resting stem cells and the proliferating cells of the cancer. This may be possible if specific stem cell signals are inhibited using gene therapy, while at the same time attacking proliferating cells by conventional radiation treatment or chemotherapy. With advances in approaches using specific inhibitory RNA, such combination therapy may now be possible, but critical problems in delivering the inhibitory effect specifically to the cancer stem cells have yet to be worked out.

  17. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells.

    PubMed

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes.

  18. Au nanoinjectors for electrotriggered gene delivery into the cell nucleus.

    PubMed

    Kang, Mijeong; Kim, Bongsoo

    2015-01-01

    Intracellular delivery of exogenous materials is an essential technique required for many fundamental biological researches and medical treatments. As our understanding of cell structure and function has been improved and diverse therapeutic agents with a subcellular site of action have been continuously developed, there is a demand to enhance the performance of delivering devices. Ideal intracellular delivery devices should convey various kinds of exogenous materials without deteriorating cell viability regardless of cell type and, furthermore, precisely control the location and the timing of delivery as well as the amount of delivered materials for advanced researches.In this chapter the development of a new intracellular delivery device, a nanoinjector made of a Au (gold) nanowire (a Au nanoinjector) is described in which delivery is triggered by external application of an electric pulse. As a model study, a gene was delivered directly into the nucleus of a neuroblastoma cell, and successful delivery without cell damage was confirmed by the expression of the delivered gene. The insertion of a Au nanoinjector directly into a cell can be generally applied to any kind of cell, and a high degree of surface modification of Au allows attachment of diverse materials such as proteins, small molecules, or nanoparticles as well as genes on Au nanoinjectors. This expands their applicability, and it is expected that they will provide important information on the effects of delivered exogenous materials and consequently contribute to the development of related therapeutic or clinical technologies.

  19. Cell-type specific gene expression profiles of leukocytes in human peripheral blood

    PubMed Central

    Palmer, Chana; Diehn, Maximilian; Alizadeh, Ash A; Brown, Patrick O

    2006-01-01

    Background Blood is a complex tissue comprising numerous cell types with distinct functions and corresponding gene expression profiles. We attempted to define the cell type specific gene expression patterns for the major constituent cells of blood, including B-cells, CD4+ T-cells, CD8+ T-cells, lymphocytes and granulocytes. We did this by comparing the global gene expression profiles of purified B-cells, CD4+ T-cells, CD8+ T-cells, granulocytes, and lymphocytes using cDNA microarrays. Results Unsupervised clustering analysis showed that similar cell populations from different donors share common gene expression profiles. Supervised analyses identified gene expression signatures for B-cells (427 genes), T-cells (222 genes), CD8+ T-cells (23 genes), granulocytes (411 genes), and lymphocytes (67 genes). No statistically significant gene expression signature was identified for CD4+ cells. Genes encoding cell surface proteins were disproportionately represented among the genes that distinguished among the lymphocyte subpopulations. Lymphocytes were distinguishable from granulocytes based on their higher levels of expression of genes encoding ribosomal proteins, while granulocytes exhibited characteristic expression of various cell surface and inflammatory proteins. Conclusion The genes comprising the cell-type specific signatures encompassed many of the genes already known to be involved in cell-type specific processes, and provided clues that may prove useful in discovering the functions of many still unannotated genes. The most prominent feature of the cell type signature genes was the enrichment of genes encoding cell surface proteins, perhaps reflecting the importance of specialized systems for sensing the environment to the physiology of resting leukocytes. PMID:16704732

  20. Viscumins functionally modulate cell motility-associated gene expression.

    PubMed

    Schötterl, Sonja; Hübner, Miriam; Armento, Angela; Veninga, Vivien; Wirsik, Naita Maren; Bernatz, Simon; Lentzen, Hans; Mittelbronn, Michel; Naumann, Ulrike

    2017-02-01

    In Europe extracts from Viscum album L., the European white-berry mistletoe, are widely used as a complementary cancer therapy. Viscumins (mistletoe lectins, ML) have been scrutinized as important active components of mistletoe and exhibit a variety of anticancer effects such as stimulation of the immune system, induction of cytotoxicity, reduction of tumor cell motility as well as changes in the expression of genes associated with cancer development and progression. By microarray expression analysis, quantitative RT-PCR and RT-PCR based validation of microarray data we demonstrate for the Viscum album extract Iscador Qu and for the lectins Aviscumine and ML-1 that in glioma cells these drugs differentially modulate the expression of genes involved in the regulation of cell migration and invasion, including processes modulating cell architecture and cell adhesion. A variety of differentially expressed genes in ML treated cells are associated with the transforming growth factor (TGF)-β signaling pathway or are targets of TGF-β. ML treatment downregulated the expression of TGF-β itself, of the TGF-β receptor II (TGFBR2), of the TGF-β intracellular signal transducer protein SMAD2, and of matrix-metalloproteinases (MMP) MMP-2 and MMP-14. Even if the changes in gene expression differ between Aviscumine, Iscador Qu and ML-1, the overall regulation of motility associated gene expression by all drugs showed functional effects since tumor cell motility was reduced in a ML-dependent manner. Therefore, ML containing compounds might provide clinical benefit as adjuvant therapeutics in the treatment of patients with invasively growing tumors such as glioblastomas.

  1. An Arabidopsis gene regulatory network for secondary cell wall synthesis

    DOE PAGES

    Taylor-Teeples, M.; Lin, L.; de Lucas, M.; ...

    2014-12-24

    The plant cell wall is an important factor for determining cell shape, function and response to the environment. Secondary cell walls, such as those found in xylem, are composed of cellulose, hemicelluloses and lignin and account for the bulk of plant biomass. The coordination between transcriptional regulation of synthesis for each polymer is complex and vital to cell function. A regulatory hierarchy of developmental switches has been proposed, although the full complement of regulators remains unknown. In this paper, we present a protein–DNA network between Arabidopsis thaliana transcription factors and secondary cell wall metabolic genes with gene expression regulated bymore » a series of feed-forward loops. This model allowed us to develop and validate new hypotheses about secondary wall gene regulation under abiotic stress. Distinct stresses are able to perturb targeted genes to potentially promote functional adaptation. Finally, these interactions will serve as a foundation for understanding the regulation of a complex, integral plant component.« less

  2. Gene expression in Fusarium graminearum grown on plant cell wall.

    PubMed

    Carapito, Raphaël; Hatsch, Didier; Vorwerk, Sonja; Petkovski, Elizabet; Jeltsch, Jean-Marc; Phalip, Vincent

    2008-05-01

    Fusarium graminearum is a phytopathogenic filamentous fungus attacking a wide range of plants including Humulus lupulus (hop). Transcriptional analysis of F. graminearum grown on minimal media containing hop cell wall or glucose as the sole carbon source was performed by applying a highly stringent method combining microarrays and a subtracted cDNA library. In addition to genes coding for various cell wall degrading enzymes (CWDE), several metabolic pathways were induced in response to the plant cell wall substrate. Many genes participating in these pathways are probably involved in cellular transport. But the most interesting was that all the genes composing the 4-aminobutyrate-shunt (GABA-shunt) were also up-regulated in the presence of plant cell wall material and were present in the cDNA library. This study provides a description of a part of the fungal gene expression profile when it is in contact with raw biological materials, and helps in understanding the plant cell wall degradation and the infection process.

  3. Identification and validation of reference genes for expression studies in human keratinocyte cell lines treated with and without interferon-γ - a method for qRT-PCR reference gene determination.

    PubMed

    Riemer, Angelika B; Keskin, Derin B; Reinherz, Ellis L

    2012-08-01

    Based on the exquisite sensitivity, reproducibility and wide dynamic range of quantitative reverse-transcription real-time polymerase chain reaction (qRT-PCR), it is currently the gold standard for gene expression studies. Target gene expression is calculated relative to a stably expressed reference gene. An ideal reference should be uniformly expressed during all experimental conditions within the given experimental system. However, no commonly applicable 'best' reference gene has been identified. Thus, endogenous controls must be determined for every experimental system. As no appropriate reference genes have been reported for immunological studies in keratinocytes, we aimed at identifying and validating a set of endogenous controls for these settings. An extensive validation of sixteen possible endogenous controls in a panel of 8 normal and transformed keratinocyte cell lines in experimental conditions with and without interferon-γ was performed. RNA and cDNA quality was stringently controlled. Candidate reference genes were assessed by TaqMan(®) qRT-PCR. Two different statistical algorithms were used to determine the most stably and reproducibly expressed housekeeping genes. mRNA abundance was compared and reference genes with widely different ranges of expression than possible target genes were excluded. Subsequent geNorm and NormFinder analyses identified GAPDH, PGK1, IPO8 and PPIA as the most stably expressed genes in the keratinocyte panel under the given experimental conditions. We conclude that the geometric means of expression values of these four genes represents a robust normalization factor for qRT-PCR analyses in interferon-γ-dependent gene expression studies in keratinocytes. The methodology and results herein may help other researchers by facilitating their choice of reference genes.

  4. Prediction of epigenetically regulated genes in breast cancer cell lines

    SciTech Connect

    Loss, Leandro A; Sadanandam, Anguraj; Durinck, Steffen; Nautiyal, Shivani; Flaucher, Diane; Carlton, Victoria EH; Moorhead, Martin; Lu, Yontao; Gray, Joe W; Faham, Malek; Spellman, Paul; Parvin, Bahram

    2010-05-04

    Methylation of CpG islands within the DNA promoter regions is one mechanism that leads to aberrant gene expression in cancer. In particular, the abnormal methylation of CpG islands may silence associated genes. Therefore, using high-throughput microarrays to measure CpG island methylation will lead to better understanding of tumor pathobiology and progression, while revealing potentially new biomarkers. We have examined a recently developed high-throughput technology for measuring genome-wide methylation patterns called mTACL. Here, we propose a computational pipeline for integrating gene expression and CpG island methylation profles to identify epigenetically regulated genes for a panel of 45 breast cancer cell lines, which is widely used in the Integrative Cancer Biology Program (ICBP). The pipeline (i) reduces the dimensionality of the methylation data, (ii) associates the reduced methylation data with gene expression data, and (iii) ranks methylation-expression associations according to their epigenetic regulation. Dimensionality reduction is performed in two steps: (i) methylation sites are grouped across the genome to identify regions of interest, and (ii) methylation profles are clustered within each region. Associations between the clustered methylation and the gene expression data sets generate candidate matches within a fxed neighborhood around each gene. Finally, the methylation-expression associations are ranked through a logistic regression, and their significance is quantified through permutation analysis. Our two-step dimensionality reduction compressed 90% of the original data, reducing 137,688 methylation sites to 14,505 clusters. Methylation-expression associations produced 18,312 correspondences, which were used to further analyze epigenetic regulation. Logistic regression was used to identify 58 genes from these correspondences that showed a statistically signifcant negative correlation between methylation profles and gene expression in the

  5. Neural stem cell-based gene therapy for brain tumors.

    PubMed

    Kim, Seung U

    2011-03-01

    Advances in gene-based medicine since 1990s have ushered in new therapeutic strategy of gene therapy for inborn error genetic diseases and cancer. Malignant brain tumors such as glioblastoma multiforme and medulloblastoma remain virtually untreatable and lethal. Currently available treatment for brain tumors including radical surgical resection followed by radiation and chemotherapy, have substantially improved the survival rate in patients suffering from these brain tumors; however, it remains incurable in large proportion of patients. Therefore, there is substantial need for effective, low-toxicity therapies for patients with malignant brain tumors, and gene therapy targeting brain tumors should fulfill this requirement. Gene therapy for brain tumors includes many therapeutic strategies and these strategies can be grouped in two major categories: molecular and immunologic. The widely used molecular gene therapy approach is suicide gene therapy based on the conversion of non-toxic prodrugs into active anticancer agents via introduction of enzymes and genetic immunotherapy involves the gene transfer of immune-stimulating cytokines including IL-4, IL-12 and TRAIL. For both molecular and immune gene therapy, neural stem cells (NSCs) can be used as delivery vehicle of therapeutic genes. NSCs possess an inherent tumor tropism that supports their use as a reliable delivery vehicle to target therapeutic gene products to primary brain tumors and metastatic cancers throughout the brain. Significance of the NSC-based gene therapy for brain tumor is that it is possible to exploit the tumor-tropic property of NSCs to mediate effective, tumor-selective therapy for primary and metastatic cancers in the brain and outside, for which no tolerated curative treatments are currently available.

  6. Differential expression and alternative splicing of cell cycle genes in imatinib-treated K562 cells.

    PubMed

    Liu, Jing; Lin, Jin; Huang, Lin-Feng; Huang, Bo; Xu, Yan-Mei; Li, Jing; Wang, Yan; Zhang, Jing; Yang, Wei-Ming; Min, Qing-Hua; Wang, Xiao-Zhong

    2015-09-01

    Cancer progression often involves the disorder of the cell cycle, and a number of effective chemotherapeutic drugs have been shown to induce cell cycle arrest. The purpose of this study was to comprehensively investigate the effects of imatinib on the expression profile of cell cycle genes in the chronic myeloid leukemia (CML) K562 cell line. In addition, we also investigated alternative splicing of the cell cycle genes affected by imatinib, since an important relationship has been shown to exist between RNA splicing and cell cycle progression. Exon array analysis was performed using total RNA purified from normal and imatinib-treated K562 cells. We identified 185 differentially expressed genes and 277 alternative splicing events between the two cell groups. A detailed analysis by reverse transcription-PCR (RT-PCR) of key genes confirmed the experimental results of the exon array. These results suggested that treatment of K562 cells with imatinib shifts the expression and alternative splicing profiles of several cell cycle-related genes. Importantly, these findings may help improve imatinib treatment strategies in patients with CML and may be useful for imatinib resistance research and CML drug development.

  7. Gene, Stem Cell, and Alternative Therapies for SCA 1

    PubMed Central

    Wagner, Jacob L.; O'Connor, Deirdre M.; Donsante, Anthony; Boulis, Nicholas M.

    2016-01-01

    Spinocerebellar ataxia 1 is an autosomal dominant disease characterized by neurodegeneration and motor dysfunction. In disease pathogenesis, polyglutamine expansion within Ataxin-1, a gene involved in transcriptional repression, causes protein nuclear inclusions to form. Most notably, neuronal dysfunction presents in Purkinje cells. However, the effect of mutant Ataxin-1 is not entirely understood. Two mouse models are employed to represent spinocerebellar ataxia 1, a B05 transgenic model that specifically expresses mutant Ataxin-1 in Purkinje cells, and a Sca1 154Q/2Q model that inserts the polyglutamine expansion into the mouse Ataxin-1 locus so that the mutant Ataxin-1 is expressed in all cells that express Ataxin-1. This review aims to summarize and evaluate the wide variety of therapies proposed for spinocerebellar ataxia 1, specifically gene and stem cell therapies. PMID:27570504

  8. Expression of a human placental alkaline phosphatase gene in transfected cells: Use as a reporter for studies of gene expression

    SciTech Connect

    Henthorn, P.; Zervos, P.; Raducha, M.; Harris, H.; Kadesch, T.

    1988-09-01

    The human placental alkaline phosphatase gene has been cloned and reintroduced into mammalian cells. When a plasmid carrying the gene under control of the simian virus 40 early promoter (pSV2Apap) is transfected into a variety of different cell types, placental alkaline phosphatase activity can readily be detected by using whole cell suspensions or cell lysates. Alkaline phosphatase activity can also be visualized directly in individual transfected cells by histochemical staining. The gene is appropriate for use as a reporter in studies of gene regulation since its expression is dependent on the presence of exogenous transcription control elements. The overall assay to detect the expression of the gene is quantitative, very rapid, and inexpensive. Cotransfections of cells with pSV2Apap and a related plasmid carrying the bacterial chloramphenicol acetyltransferase gene (pSV2Acat) indicate that transcription of these two genes is detected with roughly the same sensitivity.

  9. T-cell intracellular antigens function as tumor suppressor genes.

    PubMed

    Sánchez-Jiménez, C; Ludeña, M D; Izquierdo, J M

    2015-03-05

    Knockdown of T-cell intracellular antigens TIA1 and TIAR in transformed cells triggers cell proliferation and tumor growth. Using a tetracycline-inducible system, we report here that an increased expression of TIA1 or TIAR in 293 cells results in reduced rates of cell proliferation. Ectopic expression of these proteins abolish endogenous TIA1 and TIAR levels via the regulation of splicing of their pre-mRNAs, and partially represses global translation in a phospho-eukaryotic initiation factor 2 alpha-dependent manner. This is accompanied by cell cycle arrest at G1/S and cell death through caspase-dependent apoptosis and autophagy. Genome-wide profiling illustrates a selective upregulation of p53 signaling pathway-related genes. Nude mice injected with doxycycline-inducible cells expressing TIA1 or TIAR retard, or even inhibit, growth of xenotumors. Remarkably, low expressions of TIA1 and TIAR correlate with poor prognosis in patients with lung squamous cell carcinoma. These findings strongly support the concept that TIA proteins act as tumor suppressor genes.

  10. Genetic alterations of HER genes in chromophobe renal cell carcinoma

    PubMed Central

    WENG, WEN HUI; CHEN, YING TZU; YU, KAI JIE; CHANG, YING HSU; CHUANG, CHENG KENG; PANG, SEE TONG

    2016-01-01

    Chromophobe (ch) renal cell carcinoma (RCC) is the 3rd most common subtype of RCC and occurs in 5% of all RCCs. Although chRCC generally demonstrates more favorable outcomes compared with other subtypes of RCC, there is a 6–7% probability of tumor progression and metastasis in this disease. The subclassification of a more aggressive subtype of chRCC may be useful for the management of this cancer. The Erb-B2 receptor tyrosine kinase 2 [also known as human epidermal growth factor receptor (HER) 2] gene has been reported to be important in chRCC. The present study aimed to further investigate the abnormalities of the HER family genes and their potential association with chRCC. Fluorescence in situ hybridization was performed on 11 chRCC tissue specimens, and the Spearman's rank correlation coefficient analysis was used to assess the results. The loss of one copy of the HER2 and HER4 genes was observed to be the major alteration of the tumor cells in all chRCC cases. Statistical data indicated that loss of the HER2 gene was strongly correlated with loss of the HER4 gene (P=0.019). The findings of previous studies were also combined for analysis, and were consistent with those of the present study. In addition, the amplification of HER1 was also strongly correlated with the amplification of HER4 (P=0.004). Furthermore, a high percentage of genetic structural rearrangements was observed in HER3 genes, which was significantly associated with amplification of HER2 (P=0.005). Certain alterations in the HER gene family were also noted as a phenomenom in chRCC. Therefore, the characterization of the underlying aberrant functions of HER genes may be of interest for additional studies in the context of using HER genes to distinguish between RCC subtypes in order to establish improved treatment guidelines. PMID:26998131

  11. Identification and Validation of Housekeeping Genes for Gene Expression Analysis of Cancer Stem Cells.

    PubMed

    Lemma, Silvia; Avnet, Sofia; Salerno, Manuela; Chano, Tokuhiro; Baldini, Nicola

    2016-01-01

    The characterization of cancer stem cell (CSC) subpopulation, through the comparison of the gene expression signature in respect to the native cancer cells, is particularly important for the identification of novel and more effective anticancer strategies. However, CSC have peculiar characteristics in terms of adhesion, growth, and metabolism that possibly implies a different modulation of the expression of the most commonly used housekeeping genes (HKG), like b-actin (ACTB). Although it is crucial to identify which are the most stable HKG genes to normalize the data derived from quantitative Real-Time PCR analysis to obtain robust and consistent results, an exhaustive validation of reference genes in CSC is still missing. Here, we isolated CSC spheres from different musculoskeletal sarcomas and carcinomas as a model to investigate on the stability of the mRNA expression of 15 commonly used HKG, in respect to the native cells. The selected genes were analysed for the variation coefficient and compared using the popular algorithms NormFinder and geNorm to evaluate stability ranking. As a result, we found that: 1) Tata Binding Protein (TBP), Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ), Peptidylprolyl isomerase A (PPIA), and Hydroxymethylbilane synthase (HMBS) are the most stable HKG for the comparison between CSC and native cells; 2) at least four reference genes should be considered for robust results; 3) the use of ACTB should not be recommended, 4) specific HKG should be considered for studies that are focused only on a specific tumor type, like sarcoma or carcinoma. Our results should be taken in consideration for all the studies of gene expression analysis of CSC, and will substantially contribute for future investigations aimed to identify novel anticancer therapy based on CSC targeting.

  12. Ultrasound Gene Transfer into Fibroblast Cells using Microbubbles

    NASA Astrophysics Data System (ADS)

    Nakamura, Yoji; Hirayama, Kota; Yoshinaka, Kiyoshi; Tei, Yuichi; Takagi, Shu; Matsumoto, Yoichiro

    2009-04-01

    Ultrasound is widely applied in the medical field and offers the strong advantages of non-invasiveness and high-selectivity. Gene transfer using ultrasound, which is called sonoporation, is one application. Ultrasound has the potential to deliver therapeutic materials such as genes, drugs or proteins into cells. Microbubbles are known to be able to improve delivery efficiency. This is attributed to therapeutic materials passing through the cell membrane after permeability is increased by destruction or oscillation of microbubbles. The present study tried to deliver the GFP plasmids into fibroblast cells. Cells were cultured in 6-well culture plates and exposed to ultrasound (frequency, 2.1 MHz; wave pattern, duty cycle 10%; intensity, 0-26 W/cm2; time, 0-200 s) transmitted through medium containing microbubbles (Levovist® (void fraction, 8×10-5) or Sonazoid® (void fraction, 0-24×10-4)) and GFP plasmids at a concentration of 15 μg/mL. Density of microbubbles after ultrasound irradiation was measured. When ultrasound intensity was increased with Levovist® 8×10-4, transfection efficiency increased, cell viability decreased and microbubbles disappeared. With Sonazoid®, transfection efficiency and cell viability were basically unchanged and microbubbles decreased, but did not disappear. Transfection efficiency also improved with increased ultrasound irradiation time or microbubble density. Microbubble destruction appeared to have the main effect on gene transfection under Levovist® and microbubble oscillation had the main effect under Sonazoid®.

  13. Perspectives of gene therapy in stem cell tissue engineering.

    PubMed

    Goessler, Ulrich Reinhart; Riedel, Katrin; Hormann, Karl; Riedel, Frank

    2006-01-01

    Tissue engineering is an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain or improve tissue function. It is hoped that forming tissue de novo will overcome many problems in plastic surgery associated with such areas as wound healing and the immunogenicity of transplanted tissue that lead to dysfunctional repair. Gene therapy is the science of the transfer of genetic material into individuals for therapeutic purposes by altering cellular function or structure at the molecular level. Recently, tissue engineering has been used in conjunction with gene therapy as a hybrid approach. This combination of stem-cell-based tissue engineering with gene therapy has the potential to provide regenerative tissue cells within an environment of optimal regulatory protein expression and would have many benefits in various areas such as the transplantation of skin, cartilage or bone. The aim of this review is to outline tissue engineering and possible applications of gene therapy in the field of biomedical engineering as well as basic principles of gene therapy, vectors and gene delivery.

  14. Altered epigenetic regulation of homeobox genes in human oral squamous cell carcinoma cells

    PubMed Central

    Marcinkiewicz, Katarzyna M.; Gudas, Lorraine J.

    2013-01-01

    To gain insight into oral squamous cell carcinogenesis, we performed deep sequencing (RNAseq) of non-tumorigenic human OKF6-TERT1R and tumorigenic SCC-9 cells. Numerous homeobox genes are differentially expressed between OKF6-TERT1R and SCC-9 cells. Data from Oncomine, a cancer microarray database, also show that homeobox (HOX) genes are dysregulated in oral SCC patients. The activity of Polycomb repressive complexes (PRC), which causes epigenetic modifications, and retinoic acid (RA) signaling can control HOX gene transcription. HOXB7, HOXC10, HOXC13, and HOXD8 transcripts are higher in SCC-9 than in OKF6-TERT1R cells; using ChIP (chromatin immunoprecipitation) we detected PRC2 protein SUZ12 and the epigenetic H3K27me3 mark on histone H3 at these genes in OKF6-TERT1R, but not in SCC-9 cells. In contrast, IRX1, IRX4, SIX2 and TSHZ3 transcripts are lower in SCC-9 than in OKF6-TERT1R cells. We detected SUZ12 and the H3K27me3 mark at these genes in SCC-9, but not in OKF6-TERT1R cells. SUZ12 depletion increased HOXB7, HOXC10, HOXC13, and HOXD8 transcript levels and decreased the proliferation of OKF6-TERT1R cells. Transcriptional responses to RA are attenuated in SCC-9 versus OKF6-TERT1R cells. SUZ12 and H3K27me3 levels were not altered by RA at these HOX genes in SCC-9 and OKF6-TERT1R cells. We conclude that altered activity of PRC2 is associated with dysregulation of homeobox gene expression in human SCC cells, and that this dysregulation potentially plays a role in the neoplastic transformation of oral keratinocytes. PMID:24076275

  15. Altered epigenetic regulation of homeobox genes in human oral squamous cell carcinoma cells.

    PubMed

    Marcinkiewicz, Katarzyna M; Gudas, Lorraine J

    2014-01-01

    To gain insight into oral squamous cell carcinogenesis, we performed deep sequencing (RNAseq) of non-tumorigenic human OKF6-TERT1R and tumorigenic SCC-9 cells. Numerous homeobox genes are differentially expressed between OKF6-TERT1R and SCC-9 cells. Data from Oncomine, a cancer microarray database, also show that homeobox (HOX) genes are dysregulated in oral SCC patients. The activity of Polycomb repressive complexes (PRC), which causes epigenetic modifications, and retinoic acid (RA) signaling can control HOX gene transcription. HOXB7, HOXC10, HOXC13, and HOXD8 transcripts are higher in SCC-9 than in OKF6-TERT1R cells; using ChIP (chromatin immunoprecipitation) we detected PRC2 protein SUZ12 and the epigenetic H3K27me3 mark on histone H3 at these genes in OKF6-TERT1R, but not in SCC-9 cells. In contrast, IRX1, IRX4, SIX2 and TSHZ3 transcripts are lower in SCC-9 than in OKF6-TERT1R cells. We detected SUZ12 and the H3K27me3 mark at these genes in SCC-9, but not in OKF6-TERT1R cells. SUZ12 depletion increased HOXB7, HOXC10, HOXC13, and HOXD8 transcript levels and decreased the proliferation of OKF6-TERT1R cells. Transcriptional responses to RA are attenuated in SCC-9 versus OKF6-TERT1R cells. SUZ12 and H3K27me3 levels were not altered by RA at these HOX genes in SCC-9 and OKF6-TERT1R cells. We conclude that altered activity of PRC2 is associated with dysregulation of homeobox gene expression in human SCC cells, and that this dysregulation potentially plays a role in the neoplastic transformation of oral keratinocytes.

  16. Gene Therapy in a Patient with Sickle Cell Disease.

    PubMed

    Ribeil, Jean-Antoine; Hacein-Bey-Abina, Salima; Payen, Emmanuel; Magnani, Alessandra; Semeraro, Michaela; Magrin, Elisa; Caccavelli, Laure; Neven, Benedicte; Bourget, Philippe; El Nemer, Wassim; Bartolucci, Pablo; Weber, Leslie; Puy, Hervé; Meritet, Jean-François; Grevent, David; Beuzard, Yves; Chrétien, Stany; Lefebvre, Thibaud; Ross, Robert W; Negre, Olivier; Veres, Gabor; Sandler, Laura; Soni, Sandeep; de Montalembert, Mariane; Blanche, Stéphane; Leboulch, Philippe; Cavazzana, Marina

    2017-03-02

    Sickle cell disease results from a homozygous missense mutation in the β-globin gene that causes polymerization of hemoglobin S. Gene therapy for patients with this disorder is complicated by the complex cellular abnormalities and challenges in achieving effective, persistent inhibition of polymerization of hemoglobin S. We describe our first patient treated with lentiviral vector-mediated addition of an antisickling β-globin gene into autologous hematopoietic stem cells. Adverse events were consistent with busulfan conditioning. Fifteen months after treatment, the level of therapeutic antisickling β-globin remained high (approximately 50% of β-like-globin chains) without recurrence of sickle crises and with correction of the biologic hallmarks of the disease. (Funded by Bluebird Bio and others; HGB-205 ClinicalTrials.gov number, NCT02151526 .).

  17. Adenovirus as a gene therapy vector for hematopoietic cells.

    PubMed

    Marini, F C; Yu, Q; Wickham, T; Kovesdi, I; Andreeff, M

    2000-06-01

    Adenovirus (Adv)-mediated gene transfer has recently gained new attention as a means to deliver genes for hematopoietic stem cell (HSC) or progenitor cell gene therapy. In the past, HSCs have been regarded as poor Adv targets, mainly because they lack the specific Adv receptors required for efficient and productive Adv infection. In addition, the nonintegrating nature of Adv has prevented its application to HSC and bone marrow transduction protocols where long-term expression is required. There is even controversy as to whether Adv can infect hematopoietic cells at all. In fact, the ability of Adv to infect epithelium-based targets and its inability to effectively transfect HSCs have been used in the development of eradication schemes that use Adv to preferentially infect and "purge" tumor cell-contaminating HSC grafts. However, there are data supporting the existence of productive Adv infections into HSCs. Such protocols involve the application of cytokine mixtures, high multiplicities of infection, long incubation periods, and more recently, immunological and genetic modifications to Adv itself to enable it to efficiently transfer genes into HSCs. This is a rapidly growing field, both in terms of techniques and applications. This review examines the two sides of the Adv/CD34 controversy as well as the current developments in this field.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  19. Lentiviral hematopoietic stem cell gene therapy in inherited metabolic disorders.

    PubMed

    Wagemaker, Gerard

    2014-10-01

    After more than 20 years of development, lentiviral hematopoietic stem cell gene therapy has entered the stage of initial clinical implementation for immune deficiencies and storage disorders. This brief review summarizes the development and applications, focusing on the lysosomal enzyme deficiencies, especially Pompe disease.

  20. Lentiviral Hematopoietic Stem Cell Gene Therapy in Inherited Metabolic Disorders

    PubMed Central

    2014-01-01

    Abstract After more than 20 years of development, lentiviral hematopoietic stem cell gene therapy has entered the stage of initial clinical implementation for immune deficiencies and storage disorders. This brief review summarizes the development and applications, focusing on the lysosomal enzyme deficiencies, especially Pompe disease. PMID:25184354

  1. Tbx16 regulates hox gene activation in mesodermal progenitor cells

    PubMed Central

    Payumo, Alexander Y.; McQuade, Lindsey E.; Walker, Whitney J.; Yamazoe, Sayumi; Chen, James K.

    2016-01-01

    The transcription factor T-box 16 (Tbx16/Spadetail) is an essential regulator of paraxial mesoderm development in zebrafish (Danio rerio). Mesodermal progenitor cells (MPCs) fail to differentiate into trunk somites in tbx16 mutants and instead accumulate within the tailbud in an immature state. The mechanisms by which Tbx16 controls mesoderm patterning have remained enigmatic, and we describe here the application of photoactivatable morpholino oligonucleotides to determine the Tbx16 transcriptome in MPCs. We identify 124 Tbx16-regulated genes that are expressed in zebrafish gastrulae, including several developmental signaling proteins and regulators of gastrulation, myogenesis, and somitogenesis. Unexpectedly, we observe that loss of Tbx16 function precociously activates posterior hox genes in MPCs, and overexpression of a single posterior hox gene is sufficient to disrupt MPC migration. Our studies support a model in which Tbx16 regulates the timing of collinear hox gene activation to coordinate the anterior-posterior fates and positions of paraxial MPCs. PMID:27376691

  2. A Gene Expression Signature for Chemoradiosensitivity of Colorectal Cancer Cells

    SciTech Connect

    Spitzner, Melanie; Emons, Georg; Kramer, Frank; Gaedcke, Jochen; Rave-Fraenk, Margret; Scharf, Jens-Gerd; Burfeind, Peter; Becker, Heinz; Beissbarth, Tim; Ghadimi, B. Michael; Ried, Thomas; Grade, Marian

    2010-11-15

    Purpose: The standard treatment of patients with locally advanced rectal cancers comprises preoperative 5-fluorouracil-based chemoradiotherapy followed by standardized surgery. However, tumor response to multimodal treatment has varied greatly, ranging from complete resistance to complete pathologic regression. The prediction of the response is, therefore, an important clinical need. Methods and Materials: To establish in vitro models for studying the molecular basis of this heterogeneous tumor response, we exposed 12 colorectal cancer cell lines to 3 {mu}M of 5-fluorouracil and 2 Gy of radiation. The differences in treatment sensitivity were then correlated with the pretherapeutic gene expression profiles of these cell lines. Results: We observed a heterogeneous response, with surviving fractions ranging from 0.28 to 0.81, closely recapitulating clinical reality. Using a linear model analysis, we identified 4,796 features whose expression levels correlated significantly with the sensitivity to chemoradiotherapy (Q <.05), including many genes involved in the mitogen-activated protein kinase signaling pathway or cell cycle genes. These data have suggested a potential relevance of the insulin and Wnt signaling pathways for treatment response, and we identified STAT3, RASSF1, DOK3, and ERBB2 as potential therapeutic targets. The microarray measurements were independently validated for a subset of these genes using real-time polymerase chain reactions. Conclusion: We are the first to report a gene expression signature for the in vitro chemoradiosensitivity of colorectal cancer cells. We anticipate that this analysis will unveil molecular biomarkers predictive of the response of rectal cancers to chemoradiotherapy and enable the identification of genes that could serve as targets to sensitize a priori resistant primary tumors.

  3. Differentially expressed genes in giant cell tumor of bone.

    PubMed

    Babeto, Erica; Conceição, André Luis Giacometti; Valsechi, Marina Curado; Peitl Junior, Paulo; de Campos Zuccari, Débora Aparecida Pires; de Lima, Luiz Guilherme Cernaglia Aureliano; Bonilha, Jane Lopes; de Freitas Calmon, Marília; Cordeiro, José Antônio; Rahal, Paula

    2011-04-01

    Giant cells tumors of bone (GCTB) are benign in nature but cause osteolytic destruction with a number of particular characteristics. These tumors can have uncertain biological behavior often contain a significant proportion of highly multinucleated cells, and may show aggressive behavior. We have studied differential gene expression in GCTB that may give a better understanding of their physiopathology, and might be helpful in prognosis and treatment. Rapid subtractive hybridization (RaSH) was used to identify and measure novel genes that appear to be differentially expressed, including KTN1, NEB, ROCK1, and ZAK using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry in the samples of GCTBs compared to normal bone tissue. Normal bone was used in the methodology RaSH for comparison with the GCTB in identification of differentially expressed genes. Functional annotation indicated that these genes are involved in cellular processes related to their tumor phenotype. The differential expression of KTN1, ROCK1, and ZAK was independently confirmed by qRT-PCR and immunohistochemistry. The expression of the KTN1 and ROCK1 genes were increased in samples by qRT-PCR and immunohistochemistry, and ZAK had reduced expression. Since ZAK have CpG islands in their promoter region and low expression in tumor tissue, their methylation pattern was analyzed by MSP-PCR. The genes identified KTN1, ROCK1, and ZAK may be responsible for loss of cellular homeostasis in GCTB since they are responsible for various functions related to tumorigenesis such as cell migration, cytoskeletal organization, apoptosis, and cell cycle control and thus may contribute at some stage in the process of formation and development of GCTB.

  4. A survey of disease connections for CD4+ T cell master genes and their directly linked genes.

    PubMed

    Li, Wentian; Espinal-Enríquez, Jesús; Simpfendorfer, Kim R; Hernández-Lemus, Enrique

    2015-12-01

    Genome-wide association studies and other genetic analyses have identified a large number of genes and variants implicating a variety of disease etiological mechanisms. It is imperative for the study of human diseases to put these genetic findings into a coherent functional context. Here we use system biology tools to examine disease connections of five master genes for CD4+ T cell subtypes (TBX21, GATA3, RORC, BCL6, and FOXP3). We compiled a list of genes functionally interacting (protein-protein interaction, or by acting in the same pathway) with the master genes, then we surveyed the disease connections, either by experimental evidence or by genetic association. Embryonic lethal genes (also known as essential genes) are over-represented in master genes and their interacting genes (55% versus 40% in other genes). Transcription factors are significantly enriched among genes interacting with the master genes (63% versus 10% in other genes). Predicted haploinsufficiency is a feature of most these genes. Disease-connected genes are enriched in this list of genes: 42% of these genes have a disease connection according to Online Mendelian Inheritance in Man (OMIM) (versus 23% in other genes), and 74% are associated with some diseases or phenotype in a Genome Wide Association Study (GWAS) (versus 43% in other genes). Seemingly, not all of the diseases connected to genes surveyed were immune related, which may indicate pleiotropic functions of the master regulator genes and associated genes.

  5. Gene Therapy Inhibiting Neointimal Vascular Lesion: In vivo Transfer of Endothelial Cell Nitric Oxide Synthase Gene

    NASA Astrophysics Data System (ADS)

    von der Leyen, Heiko E.; Gibbons, Gary H.; Morishita, Ryuichi; Lewis, Neil P.; Zhang, Lunan; Nakajima, Masatoshi; Kaneda, Yasufumi; Cooke, John P.; Dzau, Victor J.

    1995-02-01

    It is postulated that vascular disease involves a disturbance in the homeostatic balance of factors regulating vascular tone and structure. Recent developments in gene transfer techniques have emerged as an exciting therapeutic option to treat vascular disease. Several studies have established the feasibility of direct in vivo gene transfer into the vasculature by using reporter genes such as β-galactosidase or luciferase. To date no study has documented therapeutic effects with in vivo gene transfer of a cDNA encoding a functional enzyme. This study tests the hypothesis that endothelium-derived nitric oxide is an endogenous inhibitor of vascular lesion formation. After denudation by balloon injury of the endothelium of rat carotid arteries, we restored endothelial cell nitric oxide synthase (ec-NOS) expression in the vessel wall by using the highly efficient Sendai virus/liposome in vivo gene transfer technique. ec-NOS gene transfection not only restored NO production to levels seen in normal untreated vessels but also increased vascular reactivity of the injured vessel. Neointima formation at day 14 after balloon injury was inhibited by 70%. These findings provide direct evidence that NO is an endogenous inhibitor of vascular lesion formation in vivo (by inhibiting smooth muscle cell proliferation and migration) and suggest the possibility of ec-NOS transfection as a potential therapeutic approach to treat neointimal hyperplasia.

  6. PAX6 maintains β cell identity by repressing genes of alternative islet cell types

    PubMed Central

    Swisa, Avital; Avrahami, Dana; Eden, Noa; Zhang, Jia; Feleke, Eseye; Dahan, Tehila; Cohen-Tayar, Yamit; Stolovich-Rain, Miri; Kaestner, Klaus H.; Glaser, Benjamin; Ashery-Padan, Ruth

    2016-01-01

    Type 2 diabetes is thought to involve a compromised β cell differentiation state, but the mechanisms underlying this dysfunction remain unclear. Here, we report a key role for the TF PAX6 in the maintenance of adult β cell identity and function. PAX6 was downregulated in β cells of diabetic db/db mice and in WT mice treated with an insulin receptor antagonist, revealing metabolic control of expression. Deletion of Pax6 in β cells of adult mice led to lethal hyperglycemia and ketosis that were attributed to loss of β cell function and expansion of α cells. Lineage-tracing, transcriptome, and chromatin analyses showed that PAX6 is a direct activator of β cell genes, thus maintaining mature β cell function and identity. In parallel, we found that PAX6 binds promoters and enhancers to repress alternative islet cell genes including ghrelin, glucagon, and somatostatin. Chromatin analysis and shRNA-mediated gene suppression experiments indicated a similar function of PAX6 in human β cells. We conclude that reduced expression of PAX6 in metabolically stressed β cells may contribute to β cell failure and α cell dysfunction in diabetes. PMID:27941241

  7. Aldosterone does not modify gene expression in human endothelial cells.

    PubMed

    Verhovez, A; Williams, T A; Morello, F; Monticone, S; Brizzi, M F; Dentelli, P; Fallo, F; Fabris, B; Amenta, F; Gomez-Sanchez, C; Veglio, F; Mulatero, P

    2012-03-01

    The toxic effects of aldosterone on the vasculature, and in particular on the endothelial layer, have been proposed as having an important role in the cardiovascular pathology observed in mineralocorticoid-excess states. In order to characterize the genomic molecular mechanisms driving the aldosterone-induced endothelial dysfunction, we performed an expression microarray on transcripts obtained from both human umbilical vein endothelial cells and human coronary artery endothelial cells stimulated with 10 - 7 M aldosterone for 18 h. The results were then subjected to qRT-PCR confirmation, also including a group of genes known to be involved in the control of the endothelial function or previously described as regulated by aldosterone. The state of activation of the mineralocorticoid receptor was investigated by means of a luciferase-reporter assay using a plasmid encoding a mineralocorticoid and glucocorticoid-sensitive promoter. Aldosterone did not determine any significant change in gene expression in either cell type both in the microarray and in the qRT-PCR analysis. The luciferase-reporter assay showed no activation of the mineralocorticoid receptor following aldosterone stimulation. The status of nonfunctionality of the mineralocorticoid receptor expressed in cultured human umbilical and coronary artery endothelial cells does not allow aldosterone to modify gene expression and provides evidence against either a beneficial or harmful genomic effect of aldosterone on healthy endothelial cells.

  8. Let There Be Light: Gene and Cell Therapy for Blindness.

    PubMed

    Dalkara, Deniz; Goureau, Olivier; Marazova, Katia; Sahel, José-Alain

    2016-02-01

    Retinal degenerative diseases are a leading cause of irreversible blindness. Retinal cell death is the main cause of vision loss in genetic disorders such as retinitis pigmentosa, Stargardt disease, and Leber congenital amaurosis, as well as in complex age-related diseases such as age-related macular degeneration. For these blinding conditions, gene and cell therapy approaches offer therapeutic intervention at various disease stages. The present review outlines advances in therapies for retinal degenerative disease, focusing on the progress and challenges in the development and clinical translation of gene and cell therapies. A significant body of preclinical evidence and initial clinical results pave the way for further development of these cutting edge treatments for patients with retinal degenerative disorders.

  9. Let There Be Light: Gene and Cell Therapy for Blindness

    PubMed Central

    Dalkara, Deniz; Goureau, Olivier; Marazova, Katia; Sahel, José-Alain

    2016-01-01

    Retinal degenerative diseases are a leading cause of irreversible blindness. Retinal cell death is the main cause of vision loss in genetic disorders such as retinitis pigmentosa, Stargardt disease, and Leber congenital amaurosis, as well as in complex age-related diseases such as age-related macular degeneration. For these blinding conditions, gene and cell therapy approaches offer therapeutic intervention at various disease stages. The present review outlines advances in therapies for retinal degenerative disease, focusing on the progress and challenges in the development and clinical translation of gene and cell therapies. A significant body of preclinical evidence and initial clinical results pave the way for further development of these cutting edge treatments for patients with retinal degenerative disorders. PMID:26751519

  10. WT1-specific T cell receptor gene therapy: improving TCR function in transduced T cells.

    PubMed

    Stauss, Hans J; Thomas, Sharyn; Cesco-Gaspere, Michela; Hart, Daniel P; Xue, Shao-An; Holler, Angelika; King, Judy; Wright, Graham; Perro, Mario; Pospori, Constantina; Morris, Emma

    2008-01-01

    Adoptive transfer of antigen-specific T lymphocytes is an attractive form of immunotherapy for haematological malignancies and cancer. The difficulty of isolating antigen-specific T lymphocytes for individual patients limits the more widespread use of adoptive T cell therapy. The demonstration that cloned T cell receptor (TCR) genes can be used to produce T lymphocyte populations of desired specificity offers new opportunities for antigen-specific T cell therapy. The first trial in humans demonstrated that TCR gene-modified T cells persisted for an extended time period and reduced tumor burden in some patients. The WT1 protein is an attractive target for immunotherapy of leukemia and solid cancer since elevated expression has been demonstrated in AML, CML, MDS and in breast, colon and ovarian cancer. In the past, we have isolated high avidity CTL specific for a WT1-derived peptide presented by HLA-A2 and cloned the TCR alpha and beta genes of a WT1-specific CTL line. The genes were inserted into retroviral vectors for transduction of human peripheral blood T lymphocytes of leukemia patients and normal donors. The treatment of leukemia-bearing NOD/SCID mice with T cells transduced with the WT1-specific TCR eliminated leukemia cells in the bone marrow of most mice, while treatment with T cells transduced with a TCR of irrelevant specificity did not diminish the leukemia burden. In order to improve the safety and efficacy of TCR gene therapy, we have developed lentiviral TCR gene transfer. In addition, we employed strategies to enhance TCR expression while avoiding TCR mis-pairing. It may be possible to generate dominant TCR constructs that can suppress the expression of the endogenous TCR on the surface of transduced T cells. The development of new TCR gene constructs holds great promise for the safe and effective delivery of TCR gene therapy for the treatment of malignancies.

  11. All-in-one processing of heterogeneous human cell grafts for gene and cell therapy

    PubMed Central

    Lukianova-Hleb, Ekaterina Y; Yvon, Eric S; Shpall, Elizabeth J; Lapotko, Dmitri O

    2016-01-01

    Current cell processing technologies for gene and cell therapies are often slow, expensive, labor intensive and are compromised by high cell losses and poor selectivity thus limiting the efficacy and availability of clinical cell therapies. We employ cell-specific on-demand mechanical intracellular impact from laser pulse-activated plasmonic nanobubbles (PNB) to process heterogeneous human cell grafts ex vivo with dual simultaneous functionality, the high cell type specificity, efficacy and processing rate for transfection of target CD3+ cells and elimination of subsets of unwanted CD25+ cells. The developed bulk flow PNB system selectively processed human cells at a rate of up to 100 million cell/minute, providing simultaneous transfection of CD3+ cells with the therapeutic gene (FKBP12(V36)-p30Caspase9) with the efficacy of 77% and viability 95% (versus 12 and 60%, respectively, for standard electroporation) and elimination of CD25+ cells with 99% efficacy. PNB flow technology can unite and replace several methodologies in an all-in-one universal ex vivo simultaneous procedure to precisely and rapidly prepare a cell graft for therapy. PNB’s can process various cell systems including cord blood, stem cells, and bone marrow. PMID:27006970

  12. Optimizing T-cell receptor gene therapy for hematologic malignancies

    PubMed Central

    Morris, Emma C.

    2016-01-01

    Recent advances in genetic engineering have enabled the delivery of clinical trials using patient T cells redirected to recognize tumor-associated antigens. The most dramatic results have been seen with T cells engineered to express a chimeric antigen receptor (CAR) specific for CD19, a differentiation antigen expressed in B cells and B lineage malignancies. We propose that antigen expression in nonmalignant cells may contribute to the efficacy of T-cell therapy by maintaining effector function and promoting memory. Although CAR recognition is limited to cell surface structures, T-cell receptors (TCRs) can recognize intracellular proteins. This not only expands the range of tumor-associated self-antigens that are amenable for T-cell therapy, but also allows TCR targeting of the cancer mutagenome. We will highlight biological bottlenecks that potentially limit mutation-specific T-cell therapy and may require high-avidity TCRs that are capable of activating effector function when the concentrations of mutant peptides are low. Unexpectedly, modified TCRs with artificially high affinities function poorly in response to low concentration of cognate peptide but pose an increased safety risk as they may respond optimally to cross-reactive peptides. Recent gene-editing tools, such as transcription activator–like effector nucleases and clustered regularly interspaced short palindromic repeats, provide a platform to delete endogenous TCR and HLA genes, which removes alloreactivity and decreases immunogenicity of third-party T cells. This represents an important step toward generic off-the-shelf T-cell products that may be used in the future for the treatment of large numbers of patients. PMID:27207802

  13. Gene expression analysis of PTEN positive glioblastoma stem cells identifies DUB3 and Wee1 modulation in a cell differentiation model.

    PubMed

    Forte, Stefano; Pagliuca, Alfredo; Maniscalchi, Eugenia T; Gulino, Rosario; Calabrese, Giovanna; Ricci-Vitiani, Lucia; Pallini, Roberto; Signore, Michele; Parenti, Rosalba; De Maria, Ruggero; Gulisano, Massimo

    2013-01-01

    The term astrocytoma defines a quite heterogeneous group of neoplastic diseases that collectively represent the most frequent brain tumors in humans. Among them, glioblastoma multiforme represents the most malignant form and its associated prognosis is one of the poorest among tumors of the central nervous system. It has been demonstrated that a small population of tumor cells, isolated from the brain neoplastic tissue, can reproduce the parental tumor when transplanted in immunodeficient mouse. These tumor initiating cells are supposed to be involved in cancer development and progression and possess stem cell-like features; like their normal counterpart, these cells remain quiescent until they are committed to differentiation. Many studies have shown that the role of the tumor suppressor protein PTEN in cell cycle progression is fundamental for tumor dynamics: in low grade gliomas, PTEN contributes to maintain cells in G1 while the loss of its activity is frequently observed in high grade gliomas. The mechanisms underlying the above described PTEN activity have been studied in many tumors, but those involved in the maintenance of tumor initiating cells quiescence remain to be investigated in more detail. The aim of the present study is to shed light on the role of PTEN pathway on cell cycle regulation in Glioblastoma stem cells, through a cell differentiation model. Our results suggest the existence of a molecular mechanism, that involves DUB3 and WEE1 gene products in the regulation of Cdc25a, as functional effector of the PTEN/Akt pathway.

  14. Gene Expression Analysis of PTEN Positive Glioblastoma Stem Cells Identifies DUB3 and Wee1 Modulation in a Cell Differentiation Model

    PubMed Central

    Forte, Stefano; Pagliuca, Alfredo; Maniscalchi, Eugenia T.; Gulino, Rosario; Calabrese, Giovanna; Ricci-Vitiani, Lucia; Pallini, Roberto; Signore, Michele; Parenti, Rosalba; De Maria, Ruggero; Gulisano, Massimo

    2013-01-01

    The term astrocytoma defines a quite heterogeneous group of neoplastic diseases that collectively represent the most frequent brain tumors in humans. Among them, glioblastoma multiforme represents the most malignant form and its associated prognosis is one of the poorest among tumors of the central nervous system. It has been demonstrated that a small population of tumor cells, isolated from the brain neoplastic tissue, can reproduce the parental tumor when transplanted in immunodeficient mouse. These tumor initiating cells are supposed to be involved in cancer development and progression and possess stem cell-like features; like their normal counterpart, these cells remain quiescent until they are committed to differentiation. Many studies have shown that the role of the tumor suppressor protein PTEN in cell cycle progression is fundamental for tumor dynamics: in low grade gliomas, PTEN contributes to maintain cells in G1 while the loss of its activity is frequently observed in high grade gliomas. The mechanisms underlying the above described PTEN activity have been studied in many tumors, but those involved in the maintenance of tumor initiating cells quiescence remain to be investigated in more detail. The aim of the present study is to shed light on the role of PTEN pathway on cell cycle regulation in Glioblastoma stem cells, through a cell differentiation model. Our results suggest the existence of a molecular mechanism, that involves DUB3 and WEE1 gene products in the regulation of Cdc25a, as functional effector of the PTEN/Akt pathway. PMID:24349068

  15. Transplantation of NGF-gene-modified bone marrow stromal cells into a rat model of Alzheimer' disease.

    PubMed

    Li, Li-Yan; Li, Jin-Tao; Wu, Qing-Ying; Li, Jin; Feng, Zhong-Tang; Liu, Su; Wang, Ting-Hua

    2008-02-01

    It is well known that bone marrow stromal cells (BMSC) grafted into the hippocampus of the rat model of Alzheimer's disease (AD) could survive and differentiate into cholinergic neurons as well as contribute towards functional restoration. The present study evaluated the effects of BMSC as a seed cell modified by nerve growth factor (NGF) gene into the hippocampus of AD rats. The beta-amyloid protein was injected bilaterally into the rat hippocampus to reproduce the AD model. After the human total RNA was extracted, the NGF gene was amplified by reverse transcription-polymerase chain reaction, then cloned into the pcDNA3. BMSC derived from a green fluorescence protein transgenic mouse were isolated, cultured, identified, and transfected by the NGF recombinant. The NGF-gene-modified BMSC were then transplanted into the hippocampus of AD rats. The results showed that implanted BMSC survived, migrated and expressed NGF as well as differentiated into ChAT-positive neurons. A significant improvement in learning and memory in AD rats was also seen in NGF-gene-modified BMSC group, when compared with the BMSC group. The present findings suggested that BMSC provided an effective carrier for delivery of NGF into AD rats, and the administration of NGF-gene-modified BMSC may be considered as a potential strategy for the development of effective therapies for the treatment of AD.

  16. Type 1 Diabetes Candidate Genes Linked to Pancreatic Islet Cell Inflammation and Beta-Cell Apoptosis

    PubMed Central

    Størling, Joachim; Pociot, Flemming

    2017-01-01

    Type 1 diabetes (T1D) is a chronic immune-mediated disease resulting from the selective destruction of the insulin-producing pancreatic islet β-cells. Susceptibility to the disease is the result of complex interactions between environmental and genetic risk factors. Genome-wide association studies (GWAS) have identified more than 50 genetic regions that affect the risk of developing T1D. Most of these susceptibility loci, however, harbor several genes, and the causal variant(s) and gene(s) for most of the loci remain to be established. A significant part of the genes located in the T1D susceptibility loci are expressed in human islets and β cells and mounting evidence suggests that some of these genes modulate the β-cell response to the immune system and viral infection and regulate apoptotic β-cell death. Here, we discuss the current status of T1D susceptibility loci and candidate genes with focus on pancreatic islet cell inflammation and β-cell apoptosis. PMID:28212332

  17. Type 1 Diabetes Candidate Genes Linked to Pancreatic Islet Cell Inflammation and Beta-Cell Apoptosis.

    PubMed

    Størling, Joachim; Pociot, Flemming

    2017-02-16

    Type 1 diabetes (T1D) is a chronic immune-mediated disease resulting from the selective destruction of the insulin-producing pancreatic islet β-cells. Susceptibility to the disease is the result of complex interactions between environmental and genetic risk factors. Genome-wide association studies (GWAS) have identified more than 50 genetic regions that affect the risk of developing T1D. Most of these susceptibility loci, however, harbor several genes, and the causal variant(s) and gene(s) for most of the loci remain to be established. A significant part of the genes located in the T1D susceptibility loci are expressed in human islets and β cells and mounting evidence suggests that some of these genes modulate the β-cell response to the immune system and viral infection and regulate apoptotic β-cell death. Here, we discuss the current status of T1D susceptibility loci and candidate genes with focus on pancreatic islet cell inflammation and β-cell apoptosis.

  18. Stage-dependent gene expression profiles during natural killer cell development.

    PubMed

    Kang, Hyung-Sik; Kim, Eun-Mi; Lee, Sanggyu; Yoon, Suk-Ran; Kawamura, Toshihiko; Lee, Young-Cheol; Kim, Sangsoo; Myung, Pyung-Keun; Wang, San Ming; Choi, Inpyo

    2005-11-01

    Natural killer (NK) cells develop from hematopoietic stem cells (HSCs) in the bone marrow. To understand the molecular regulation of NK cell development, serial analysis of gene expression (SAGE) was applied to HSCs, NK precursor (pNK) cells, and mature NK cells (mNK) cultured without or with OP9 stromal cells. From 170,464 total individual tags from four SAGE libraries, 35,385 unique genes were identified. A set of genes was expressed in a stage-specific manner: 15 genes in HSCs, 30 genes in pNK cells, and 27 genes in mNK cells. Among them, lipoprotein lipase induced NK cell maturation and cytotoxic activity. Identification of genome-wide profiles of gene expression in different stages of NK cell development affords us a fundamental basis for defining the molecular network during NK cell development.

  19. Effects of whole genome duplication on cell size and gene expression in mouse embryonic stem cells

    PubMed Central

    IMAI, Hiroyuki; FUJII, Wataru; KUSAKABE, Ken Takeshi; KISO, Yasuo; KANO, Kiyoshi

    2016-01-01

    Alterations in ploidy tend to influence cell physiology, which in the long-term, contribute to species adaptation and evolution. Polyploid cells are observed under physiological conditions in the nerve and liver tissues, and in tumorigenic processes. Although tetraploid cells have been studied in mammalian cells, the basic characteristics and alterations caused by whole genome duplication are still poorly understood. The purpose of this study was to acquire basic knowledge about the effect of whole genome duplication on the cell cycle, cell size, and gene expression. Using flow cytometry, we demonstrate that cell cycle subpopulations in mouse tetraploid embryonic stem cells (TESCs) were similar to those in embryonic stem cells (ESCs). We performed smear preparations and flow cytometric analysis to identify cell size alterations. These indicated that the relative cell volume of TESCs was approximately 2.2–2.5 fold that of ESCs. We also investigated the effect of whole genome duplication on the expression of housekeeping and pluripotency marker genes using quantitative real-time PCR with external RNA. We found that the target transcripts were 2.2 times more abundant in TESCs than those in ESCs. This indicated that gene expression and cell volume increased in parallel. Our findings suggest the existence of a homeostatic mechanism controlling the cytoplasmic transcript levels in accordance with genome volume changes caused by whole genome duplication. PMID:27569766

  20. T-cell suicide gene therapy prompts thymic renewal in adults after hematopoietic stem cell transplantation.

    PubMed

    Vago, Luca; Oliveira, Giacomo; Bondanza, Attilio; Noviello, Maddalena; Soldati, Corrado; Ghio, Domenico; Brigida, Immacolata; Greco, Raffaella; Lupo Stanghellini, Maria Teresa; Peccatori, Jacopo; Fracchia, Sergio; Del Fiacco, Matteo; Traversari, Catia; Aiuti, Alessandro; Del Maschio, Alessandro; Bordignon, Claudio; Ciceri, Fabio; Bonini, Chiara

    2012-08-30

    The genetic modification of T cells with a suicide gene grants a mechanism of control of adverse reactions, allowing safe infusion after partially incompatible hematopoietic stem cell transplantation (HSCT). In the TK007 clinical trial, 22 adults with hematologic malignancies experienced a rapid and sustained immune recovery after T cell-depleted HSCT and serial infusions of purified donor T cells expressing the HSV thymidine kinase suicide gene (TK+ cells). After a first wave of circulating TK+ cells, the majority of T cells supporting long-term immune reconstitution did not carry the suicide gene and displayed high numbers of naive lymphocytes, suggesting the thymus-dependent development of T cells, occurring only upon TK+ -cell engraftment. Accordingly, after the infusions, we documented an increase in circulating TCR excision circles and CD31+ recent thymic emigrants and a substantial expansion of the active thymic tissue as shown by chest tomography scans. Interestingly, a peak in the serum level of IL-7 was observed after each infusion of TK+ cells, anticipating the appearance of newly generated T cells. The results of the present study show that the infusion of genetically modified donor T cells after HSCT can drive the recovery of thymic activity in adults, leading to immune reconstitution.

  1. Effects of space flight exposure on cell growth, tumorigenicity and gene expression in cancer cells

    NASA Astrophysics Data System (ADS)

    Yang, Cheng; Li, Yuehui; Zhang, Zhijie; Luo, Chen; Tong, Yongqing; Zhou, Guohua; Xie, Pingli; Hu, Jinyue; Li, Guancheng

    2008-12-01

    It is well recognized that harsh outer space environment, consisting of microgravity and radiation, poses significant health risks for human cells. To investigate potential effects of the space environment exposure on cancer cells we examined the biological changes in Caski cells carried by the "Shen Zhou IV" spaceship. After exposure for 7 days in spaceflight, 1440 survival subclonal cell lines were established and 4 cell lines were screened. 44F10 and 17E3 were selected because of their increased cell proliferation and tumorigenesis, while 48A9 and 31F2 had slower cytological events. Experiments with cell proliferation assay, flow cytometry, soft agar assay, tumorigenesis assay and DNA microarray analysis have shown that selected cell lines presented multiple biological changes in cell morphology, cell growth, tumorigenicity and gene expression. These results suggest that space environment exposure can make significant biological impact on cancer cells and provide an entry point to find the immunological target of tumorigenesis.

  2. Recombinant cells that highly express chromosomally-integrated heterologous gene

    DOEpatents

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    2007-03-20

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  3. Recombinant cells that highly express chromosomally-integrated heterologous genes

    DOEpatents

    Ingram, L.O.; Ohta, Kazuyoshi; Wood, B.E.

    1998-10-13

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol. 13 figs.

  4. Recombinant cells that highly express chromosomally-integrated heterologous genes

    DOEpatents

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    2000-08-22

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  5. Recombinant cells that highly express chromosomally-integrated heterologous genes

    DOEpatents

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    1998-01-01

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  6. RNAi and overexpression of genes in ovarian somatic cells.

    PubMed

    Saito, Kuniaki

    2014-01-01

    Emerging evidence indicates that PIWI proteins, in collaboration with PIWI-interacting RNAs (piRNAs), play a critical role in retrotransposon silencing in Drosophila gonadal somatic and germ-line cells. The recent establishment of female germ-line stem cells/ovarian somatic sheet and its derivative cell line, ovarian somatic cells (OSCs), allows researchers to study the molecular functions of several protein factors involved in the primary piRNA pathway in Drosophila. Although transgene expression is difficult to achieve in gonad-derived cell lines, transfection of both expression vectors and knockdown reagents is highly effective in OSCs. Here, I focus on techniques that knockdown or overexpress genes of interest in OSCs.

  7. NKL homeobox gene activities in hematopoietic stem cells, T-cell development and T-cell leukemia

    PubMed Central

    Pommerenke, Claudia; Scherr, Michaela; Meyer, Corinna; Kaufmann, Maren; Battmer, Karin; MacLeod, Roderick A. F.; Drexler, Hans G.

    2017-01-01

    T-cell acute lymphoblastic leukemia (T-ALL) cells represent developmentally arrested T-cell progenitors, subsets of which aberrantly express homeobox genes of the NKL subclass, including TLX1, TLX3, NKX2-1, NKX2-5, NKX3-1 and MSX1. Here, we analyzed the transcriptional landscape of all 48 members of the NKL homeobox gene subclass in CD34+ hematopoietic stem and progenitor cells (HSPCs) and during lymphopoiesis, identifying activities of nine particular genes. Four of these were expressed in HSPCs (HHEX, HLX1, NKX2-3 and NKX3-1) and three in common lymphoid progenitors (HHEX, HLX1 and MSX1). Interestingly, our data indicated downregulation of NKL homeobox gene transcripts in late progenitors and mature T-cells, a phenomenon which might explain the oncogenic impact of this group of genes in T-ALL. Using MSX1-expressing T-ALL cell lines as models, we showed that HHEX activates while HLX1, NKX2-3 and NKX3-1 repress MSX1 transcription, demonstrating the mutual regulation and differential activities of these homeobox genes. Analysis of a public T-ALL expression profiling data set comprising 117 patient samples identified 20 aberrantly activated members of the NKL subclass, extending the number of known NKL homeobox oncogene candidates. While 7/20 genes were also active during hematopoiesis, the remaining 13 showed ectopic expression. Finally, comparative analyses of T-ALL patient and cell line profiling data of NKL-positive and NKL-negative samples indicated absence of shared target genes but instead highlighted deregulation of apoptosis as common oncogenic effect. Taken together, we present a comprehensive survey of NKL homeobox genes in early hematopoiesis, T-cell development and T-ALL, showing that these genes generate an NKL-code for the diverse stages of lymphoid development which might be fundamental for regular differentiation. PMID:28151996

  8. HMGA1 drives stem cell, inflammatory pathway, and cell cycle progression genes during lymphoid tumorigenesis

    PubMed Central

    2011-01-01

    Background Although the high mobility group A1 (HMGA1) gene is widely overexpressed in diverse cancers and portends a poor prognosis in some tumors, the molecular mechanisms that mediate its role in transformation have remained elusive. HMGA1 functions as a potent oncogene in cultured cells and induces aggressive lymphoid tumors in transgenic mice. Because HMGA1 chromatin remodeling proteins regulate transcription, HMGA1 is thought to drive malignant transformation by modulating expression of specific genes. Genome-wide studies to define HMGA1 transcriptional networks during tumorigenesis, however, are lacking. To define the HMGA1 transcriptome, we analyzed gene expression profiles in lymphoid cells from HMGA1a transgenic mice at different stages in tumorigenesis. Results RNA from lymphoid samples at 2 months (before tumors develop) and 12 months (after tumors are well-established) was screened for differential expression of > 20,000 unique genes by microarray analysis (Affymetrix) using a parametric and nonparametric approach. Differential expression was confirmed by quantitative RT-PCR in a subset of genes. Differentially expressed genes were analyzed for cellular pathways and functions using Ingenuity Pathway Analysis. Early in tumorigenesis, HMGA1 induced inflammatory pathways with NFkappaB identified as a major node. In established tumors, HMGA1 induced pathways involved in cell cycle progression, cell-mediated immune response, and cancer. At both stages in tumorigenesis, HMGA1 induced pathways involved in cellular development, hematopoiesis, and hematologic development. Gene set enrichment analysis showed that stem cell and immature T cell genes are enriched in the established tumors. To determine if these results are relevant to human tumors, we knocked-down HMGA1 in human T-cell leukemia cells and identified a subset of genes dysregulated in both the transgenic and human lymphoid tumors. Conclusions We found that HMGA1 induces inflammatory pathways early in

  9. Zscan4: a novel gene expressed exclusively in late 2-cell embryos and embryonic stem cells

    PubMed Central

    Falco, Geppino; Lee, Sung-Lim; Stanghellini, Ilaria; Bassey, Uwem C.; Hamatani, Toshio; Ko, Minoru S. H.

    2007-01-01

    The first wave of transcription, called zygotic genome activation (ZGA), begins during the 2-cell stage in mouse preimplantation development and marks a vital transition from the maternal genetic to the embryonic genetic program. Utilizing DNA microarray data, we looked for genes that are expressed only during ZGA and found Zscan4, whose expression is restricted to late 2-cell stage embryos. Sequence analysis of genomic DNA and cDNA clones revealed nine paralogous genes tightly clustered in 0.85 Mb on mouse Chromosome 7. Three genes are not transcribed and are thus considered pseudogenes. Among the six expressed genes named Zscan4a-Zscan4f, three -- Zscan4c, Zscan4d, and Zscan4f -- encode full-length ORFs with 506 amino acids. Zscan4d is a predominant transcript at the late 2-cell stage, whereas Zscan4c is a predominant transcript in embryonic stem (ES) cells. No transcripts of any Zscan4 genes are detected in any other cell types. Reduction of Zscan4 transcript levels by siRNAs delays the progression from the 2-cell to the 4-cell stage and produces blastocysts that fail to implant or proliferate in blastocyst outgrowth culture. Zscan4 thus seems to be essential for preimplantation development. PMID:17553482

  10. Gene-specific cell labeling using MiMIC transposons

    PubMed Central

    Gnerer, Joshua P.; Venken, Koen J. T.; Dierick, Herman A.

    2015-01-01

    Binary expression systems such as GAL4/UAS, LexA/LexAop and QF/QUAS have greatly enhanced the power of Drosophila as a model organism by allowing spatio-temporal manipulation of gene function as well as cell and neural circuit function. Tissue-specific expression of these heterologous transcription factors relies on random transposon integration near enhancers or promoters that drive the binary transcription factor embedded in the transposon. Alternatively, gene-specific promoter elements are directly fused to the binary factor within the transposon followed by random or site-specific integration. However, such insertions do not consistently recapitulate endogenous expression. We used Minos-Mediated Integration Cassette (MiMIC) transposons to convert host loci into reliable gene-specific binary effectors. MiMIC transposons allow recombinase-mediated cassette exchange to modify the transposon content. We developed novel exchange cassettes to convert coding intronic MiMIC insertions into gene-specific binary factor protein-traps. In addition, we expanded the set of binary factor exchange cassettes available for non-coding intronic MiMIC insertions. We show that binary factor conversions of different insertions in the same locus have indistinguishable expression patterns, suggesting that they reliably reflect endogenous gene expression. We show the efficacy and broad applicability of these new tools by dissecting the cellular expression patterns of the Drosophila serotonin receptor gene family. PMID:25712101

  11. Gene-specific cell labeling using MiMIC transposons.

    PubMed

    Gnerer, Joshua P; Venken, Koen J T; Dierick, Herman A

    2015-04-30

    Binary expression systems such as GAL4/UAS, LexA/LexAop and QF/QUAS have greatly enhanced the power of Drosophila as a model organism by allowing spatio-temporal manipulation of gene function as well as cell and neural circuit function. Tissue-specific expression of these heterologous transcription factors relies on random transposon integration near enhancers or promoters that drive the binary transcription factor embedded in the transposon. Alternatively, gene-specific promoter elements are directly fused to the binary factor within the transposon followed by random or site-specific integration. However, such insertions do not consistently recapitulate endogenous expression. We used Minos-Mediated Integration Cassette (MiMIC) transposons to convert host loci into reliable gene-specific binary effectors. MiMIC transposons allow recombinase-mediated cassette exchange to modify the transposon content. We developed novel exchange cassettes to convert coding intronic MiMIC insertions into gene-specific binary factor protein-traps. In addition, we expanded the set of binary factor exchange cassettes available for non-coding intronic MiMIC insertions. We show that binary factor conversions of different insertions in the same locus have indistinguishable expression patterns, suggesting that they reliably reflect endogenous gene expression. We show the efficacy and broad applicability of these new tools by dissecting the cellular expression patterns of the Drosophila serotonin receptor gene family.

  12. Targeted gene therapy and cell reprogramming in Fanconi anemia

    PubMed Central

    Rio, Paula; Baños, Rocio; Lombardo, Angelo; Quintana-Bustamante, Oscar; Alvarez, Lara; Garate, Zita; Genovese, Pietro; Almarza, Elena; Valeri, Antonio; Díez, Begoña; Navarro, Susana; Torres, Yaima; Trujillo, Juan P; Murillas, Rodolfo; Segovia, Jose C; Samper, Enrique; Surralles, Jordi; Gregory, Philip D; Holmes, Michael C; Naldini, Luigi; Bueren, Juan A

    2014-01-01

    Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies. PMID:24859981

  13. Cooperative functions of Hes/Hey genes in auditory hair cell and supporting cell development.

    PubMed

    Tateya, Tomoko; Imayoshi, Itaru; Tateya, Ichiro; Ito, Juichi; Kageyama, Ryoichiro

    2011-04-15

    Notch-mediated lateral inhibition has been reported to regulate auditory hair cell and supporting cell development from common precursors. While the Notch effector genes Hes1, Hes5 and Hey1 are expressed in the developing cochlea, inactivation of either of them causes only mild abnormality, suggesting their functional redundancy. To explore the roles of Hes/Hey genes in cochlear development, we examined compound heterozygous or homozygous mutant mice that lacked Hes1, Hes5 and Hey1 alleles. We found that a reduction in Hes/Hey gene dosage led to graded increase of hair cell formation. However, if at least one allele of Hes1, Hes5 or Hey1 was intact, excessive hair cells were accompanied by overproduction of supporting cells, suggesting that the hair cell increase does not occur at the expense of supporting cells, and that each Hes/Hey gene functions to induce supporting cells. By contrast, when all alleles of Hes1, Hes5 and Hey1 were inactivated, the number of hair cells increased more drastically, whereas that of supporting cells was unchanged compared with control, suggesting that supporting cell formation was balanced by their overproduction and fate conversion into hair cells. The increase of the cell numbers seemed to occur after the prosensory domain formation in the mutants because the proliferation state and the size of the prosensory domain were not affected. Thus, Hes1, Hes5 and Hey1 cooperatively inhibit hair cell formation, and one allele of Hes1, Hes5 or Hey1 is sufficient for supporting cell production probably by lateral inhibition in the sensory epithelium. Strikingly, Hes/Hey mutations lead to disorganized cell alignment and polarity and to hearing loss despite hair cell overproduction. These results suggest that Hes/Hey gene dosage is essential not only for generation of appropriate numbers of hair cells and supporting cells by controlling cell proliferation and lateral inhibition but also for the hearing ability by regulating the cell alignment

  14. Epigenetic Gene Regulation in Stem Cells and Correlation to Cancer

    PubMed Central

    Mathews, Lesley A.; Crea, Francesco; Farrar, W. L.

    2009-01-01

    Through the classic study of genetics, much has been learned about the regulation and progression of human disease. Specifically, cancer has been defined as a disease driven by genetic alterations, including mutations in tumor-suppressor genes and oncogenes, as well as chromosomal abnormalities. However, the study of normal human development has identified that in addition to classical genetics, regulation of gene expression is also modified by ‘epigenetic’ alterations including chromatin remodeling and histone variants, DNA methylation, the regulation of polycomb group proteins and the epigenetic function of non-coding RNA. These changes are modifications inherited both during meiosis and mitosis, yet they do not result in alterations of the actual DNA sequence. A number of biological questions are directly influenced by epigenetics, such as how does a cell know when to divide, differentiate or remain quiescent, and more importantly, what happens when these pathways become altered? Do these alterations lead to the development and/or progression of cancer? This review will focus on summarizing the limited current literature involving epigenetic alterations in the context of human cancer stems cells (CSCs). The extent to which epigenetic changes define cell fate, identity, and phenotype are still under intense investigation, and many questions remain largely unanswered. Before discussing epigenetic gene silencing in CSCs, the different classifications of stem cells and their properties will be introduced. This will be followed by an introduction to the different epigenetic mechanisms Finally, there will be a discussion of the current knowledge of epigenetic modifications in stem cells, specifically what is known from rodent systems and established cancer cell lines, and how they are leading us to understand human stem cells. PMID:19443100

  15. Identifying Francisella tularensis genes required for growth in host cells.

    PubMed

    Brunton, J; Steele, S; Miller, C; Lovullo, E; Taft-Benz, S; Kawula, T

    2015-08-01

    Francisella tularensis is a highly virulent Gram-negative intracellular pathogen capable of infecting a vast diversity of hosts, ranging from amoebae to humans. A hallmark of F. tularensis virulence is its ability to quickly grow to high densities within a diverse set of host cells, including, but not limited to, macrophages and epithelial cells. We developed a luminescence reporter system to facilitate a large-scale transposon mutagenesis screen to identify genes required for growth in macrophage and epithelial cell lines. We screened 7,454 individual mutants, 269 of which exhibited reduced intracellular growth. Transposon insertions in the 269 growth-defective strains mapped to 68 different genes. FTT_0924, a gene of unknown function but highly conserved among Francisella species, was identified in this screen to be defective for intracellular growth within both macrophage and epithelial cell lines. FTT_0924 was required for full Schu S4 virulence in a murine pulmonary infection model. The ΔFTT_0924 mutant bacterial membrane is permeable when replicating in hypotonic solution and within macrophages, resulting in strongly reduced viability. The permeability and reduced viability were rescued when the mutant was grown in a hypertonic solution, indicating that FTT_0924 is required for resisting osmotic stress. The ΔFTT_0924 mutant was also significantly more sensitive to β-lactam antibiotics than Schu S4. Taken together, the data strongly suggest that FTT_0924 is required for maintaining peptidoglycan integrity and virulence.

  16. Identifying Francisella tularensis Genes Required for Growth in Host Cells

    PubMed Central

    Brunton, J.; Steele, S.; Miller, C.; Lovullo, E.; Taft-Benz, S.

    2015-01-01

    Francisella tularensis is a highly virulent Gram-negative intracellular pathogen capable of infecting a vast diversity of hosts, ranging from amoebae to humans. A hallmark of F. tularensis virulence is its ability to quickly grow to high densities within a diverse set of host cells, including, but not limited to, macrophages and epithelial cells. We developed a luminescence reporter system to facilitate a large-scale transposon mutagenesis screen to identify genes required for growth in macrophage and epithelial cell lines. We screened 7,454 individual mutants, 269 of which exhibited reduced intracellular growth. Transposon insertions in the 269 growth-defective strains mapped to 68 different genes. FTT_0924, a gene of unknown function but highly conserved among Francisella species, was identified in this screen to be defective for intracellular growth within both macrophage and epithelial cell lines. FTT_0924 was required for full Schu S4 virulence in a murine pulmonary infection model. The ΔFTT_0924 mutant bacterial membrane is permeable when replicating in hypotonic solution and within macrophages, resulting in strongly reduced viability. The permeability and reduced viability were rescued when the mutant was grown in a hypertonic solution, indicating that FTT_0924 is required for resisting osmotic stress. The ΔFTT_0924 mutant was also significantly more sensitive to β-lactam antibiotics than Schu S4. Taken together, the data strongly suggest that FTT_0924 is required for maintaining peptidoglycan integrity and virulence. PMID:25987704

  17. Programmable cells: Interfacing natural and engineered gene networks

    NASA Astrophysics Data System (ADS)

    Kobayashi, Hideki; Kærn, Mads; Araki, Michihiro; Chung, Kristy; Gardner, Timothy S.; Cantor, Charles R.; Collins, James J.

    2004-06-01

    Novel cellular behaviors and characteristics can be obtained by coupling engineered gene networks to the cell's natural regulatory circuitry through appropriately designed input and output interfaces. Here, we demonstrate how an engineered genetic circuit can be used to construct cells that respond to biological signals in a predetermined and programmable fashion. We employ a modular design strategy to create Escherichia coli strains where a genetic toggle switch is interfaced with: (i) the SOS signaling pathway responding to DNA damage, and (ii) a transgenic quorum sensing signaling pathway from Vibrio fischeri. The genetic toggle switch endows these strains with binary response dynamics and an epigenetic inheritance that supports a persistent phenotypic alteration in response to transient signals. These features are exploited to engineer cells that form biofilms in response to DNA-damaging agents and cells that activate protein synthesis when the cell population reaches a critical density. Our work represents a step toward the development of "plug-and-play" genetic circuitry that can be used to create cells with programmable behaviors. heterologous gene expression | synthetic biology | Escherichia coli

  18. Gene mutations and actionable genetic lesions in mantle cell lymphoma

    PubMed Central

    Ahmed, Makhdum; Zhang, Leo; Nomie, Krystle; Lam, Laura; Wang, Michael

    2016-01-01

    Mutations and epigenetic alterations are key events in transforming normal cells to cancer cells. Mantle cell lymphoma (MCL), a non-Hodgkin's lymphoma of the B-cell, is an aggressive malignancy with poor prognosis especially for those patients who are resistant to the frontline drugs. There is a great need to describe the molecular basis and mechanism of drug resistance in MCL to develop new strategies for treatment. We reviewed frequent somatic mutations and mutations involving the B-cell pathways in MCL and discussed clinical trials that attempted to disrupt these gene pathways and/or epigenetic events. Recurrent gene mutations were discussed in the light of prognostic and therapeutic opportunity and also the challenges of targeting these lesions. Mutations in the ATM, CCND1, TP53, MLL2, TRAF2 and NOTCH1 were most frequently encountered in mantle cell lymphoma. Translational models should be built that would assess mutations longitudinally to identify important compensatory, pro-survival and anti-apoptic pathways and actionable genetic targets. PMID:27449094

  19. Global gene expression response to telomerase in bovine adrenocortical cells

    SciTech Connect

    Perrault, Steven D.; Hornsby, Peter J.; Betts, Dean H. . E-mail: bettsd@uoguelph.ca

    2005-09-30

    The infinite proliferative capability of most immortalized cells is dependent upon the presence of the enzyme telomerase and its ability to maintain telomere length and structure. However, telomerase may be involved in a greater system than telomere length regulation, as recent evidence has shown it capable of increasing wound healing in vivo, and improving cellular proliferation rate and survival from apoptosis in vitro. Here, we describe the global gene expression response to ectopic telomerase expression in an in vitro bovine adrenocortical cell model. Telomerase-immortalized cells showed an increased ability for proliferation and survival in minimal essential medium above cells transgenic for GFP. cDNA microarray analyses revealed an altered cell state indicative of increased adrenocortical cell proliferation regulated by the IGF2 pathway and alterations in members of the TGF-B family. As well, we identified alterations in genes associated with development and wound healing that support a model that high telomerase expression induces a highly adaptable, progenitor-like state.

  20. Differential transformation of mammary epithelial cells by Wnt genes.

    PubMed Central

    Wong, G T; Gavin, B J; McMahon, A P

    1994-01-01

    The mouse Wnt family includes at least 10 genes that encode structurally related secreted glycoproteins. Wnt-1 and Wnt-3 were originally identified as oncogenes activated by the insertion of mouse mammary tumor virus in virus-induced mammary adenocarcinomas, although they are not expressed in the normal mammary gland. However, five other Wnt genes are differentially expressed during development of adult mammary tissue, suggesting that they may play distinct roles in various phases of mammary gland growth and development. Induction of transformation by Wnt-1 and Wnt-3 may be due to interference with these normal regulatory events; however, there is no direct evidence for this hypothesis. We have tested Wnt family members for the ability to induce transformation of cultured mammary cells. The results demonstrate that the Wnt gene family can be divided into three groups depending on their ability to induce morphological transformation and altered growth characteristics of the C57MG mammary epithelial cell line. Wnt-1, Wnt-3A, and Wnt-7A were highly transforming and induced colonies which formed and shed balls of cells. Wnt-2, Wnt-5B, and Wnt-7B also induced transformation but with a lower frequency and an apparent decrease in saturation density. In contrast, Wnt-6 and two other family members which are normally expressed in C57MG cells, Wnt-4 and Wnt-5A, failed to induce transformation. These data demonstrate that the Wnt genes have distinct effects on cell growth and should not be regarded as functionally equivalent. Images PMID:8065359

  1. Pigment-cell-specific genes from fibroblasts are transactivated after chromosomal transfer into melanoma cells

    SciTech Connect

    Powers, T.P.; Davidson, R.L.; Shows, T.B.

    1994-02-01

    Human and mouse fibroblast chromosomes carrying tyrosinase or b-locus genes were introduced, by microcell hybridization, into pigmented Syrian hamster melanoma cells, and the microcell hybrids were tested for transactivation of the fibroblast tyrosinase and b-locus genes. By using species-specific PCR amplification to distinguish fibroblast and melanoma cDNAs, it was demonstrated that the previously silent fibroblast tyrosinase and b-locus genes were transactivated following chromosomal transfer into pigmented melanoma cells. However, transactivation of the mouse fibroblast tyrosinase gene was unstable in microcell hybrid subclones and possibly dependent on a second fibroblast locus that could have segregated in the subclones. This second locus was not necessary for transactivation of the fibroblast b-locus gene, thus demonstrating noncoordinate transactivation of fibroblast tyrosinase and b-locus genes. Transactivation of the fibroblast tyrosinase gene in microcell hybrids apparently is dependent on the absence of a putative fibroblast extinguisher locus for tyrosinase gene expression, which presumably is responsible for the extinction of pigmentation in hybrids between karyotypically complete fibroblasts and melanoma cells. 46 refs., 5 figs., 2 tabs.

  2. Direct Gene Transfer into Human Cultured Cells Facilitated by Laser Micropuncture of the Cell Membrane

    NASA Astrophysics Data System (ADS)

    Tao, Wen; Wilkinson, Joyce; Stanbridge, Eric J.; Berns, Michael W.

    1987-06-01

    The selective alteration of the cellular genome by laser microbeam irradiation has been extensively applied in cell biology. We report here the use of the third harmonic (355 nm) of an yttrium-aluminum garnet laser to facilitate the direct transfer of the neo gene into cultured human HT1080-6TG cells. The resultant transformants were selected in medium containing an aminoglycoside antibiotic, G418. Integration of the neo gene into individual human chromosomes and expression of the gene were demonstrated by Southern blot analyses, microcell-mediated chromosome transfer, and chromosome analyses. The stability of the integrated neo gene in the transformants was shown by a comparative growth assay in selective and nonselective media. Transformation and incorporation of the neo gene into the host genome occurred at a frequency of 8 × 10-4-3 × 10-3. This method appears to be 100-fold more efficient than the standard calcium phosphate-mediated method of DNA transfer.

  3. Cell Sorting and Noise-Induced Cell Plasticity Coordinate to Sharpen Boundaries between Gene Expression Domains

    PubMed Central

    2017-01-01

    A fundamental question in biology is how sharp boundaries of gene expression form precisely in spite of biological variation/noise. Numerous mechanisms position gene expression domains across fields of cells (e.g. morphogens), but how these domains are refined remains unclear. In some cases, domain boundaries sharpen through differential adhesion-mediated cell sorting. However, boundaries can also sharpen through cellular plasticity, with cell fate changes driven by up- or down-regulation of gene expression. In this context, we have argued that noise in gene expression can help cells transition to the correct fate. Here we investigate the efficacy of cell sorting, gene expression plasticity, and their combination in boundary sharpening using multi-scale, stochastic models. We focus on the formation of hindbrain segments (rhombomeres) in the developing zebrafish as an example, but the mechanisms investigated apply broadly to many tissues. Our results indicate that neither sorting nor plasticity is sufficient on its own to sharpen transition regions between different rhombomeres. Rather the two have complementary strengths and weaknesses, which synergize when combined to sharpen gene expression boundaries. PMID:28135279

  4. A proteomic chronology of gene expression through the cell cycle in human myeloid leukemia cells.

    PubMed

    Ly, Tony; Ahmad, Yasmeen; Shlien, Adam; Soroka, Dominique; Mills, Allie; Emanuele, Michael J; Stratton, Michael R; Lamond, Angus I

    2014-01-01

    Technological advances have enabled the analysis of cellular protein and RNA levels with unprecedented depth and sensitivity, allowing for an unbiased re-evaluation of gene regulation during fundamental biological processes. Here, we have chronicled the dynamics of protein and mRNA expression levels across a minimally perturbed cell cycle in human myeloid leukemia cells using centrifugal elutriation combined with mass spectrometry-based proteomics and RNA-Seq, avoiding artificial synchronization procedures. We identify myeloid-specific gene expression and variations in protein abundance, isoform expression and phosphorylation at different cell cycle stages. We dissect the relationship between protein and mRNA levels for both bulk gene expression and for over ∼6000 genes individually across the cell cycle, revealing complex, gene-specific patterns. This data set, one of the deepest surveys to date of gene expression in human cells, is presented in an online, searchable database, the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd/). DOI: http://dx.doi.org/10.7554/eLife.01630.001.

  5. Generation of hypoxanthine phosphoribosyltransferase gene knockout rabbits by homologous recombination and gene trapping through somatic cell nuclear transfer.

    PubMed

    Yin, Mingru; Jiang, Weihua; Fang, Zhenfu; Kong, Pengcheng; Xing, Fengying; Li, Yao; Chen, Xuejin; Li, Shangang

    2015-11-02

    The rabbit is a common animal model that has been employed in studies on various human disorders, and the generation of genetically modified rabbit lines is highly desirable. Female rabbits have been successfully cloned from cumulus cells, and the somatic cell nuclear transfer (SCNT) technology is well established. The present study generated hypoxanthine phosphoribosyltransferase (HPRT) gene knockout rabbits using recombinant adeno-associated virus-mediated homologous recombination and SCNT. Gene trap strategies were employed to enhance the gene targeting rates. The male and female gene knockout fibroblast cell lines were derived by different strategies. When male HPRT knockout cells were used for SCNT, no live rabbits were obtained. However, when female HPRT(+/-) cells were used for SCNT, live, healthy rabbits were generated. The cloned HPRT(+/-) rabbits were fertile at maturity. We demonstrate a new technique to produce gene-targeted rabbits. This approach may also be used in the genetic manipulation of different genes or in other species.

  6. Altered epigenetic regulation of homeobox genes in human oral squamous cell carcinoma cells

    SciTech Connect

    Marcinkiewicz, Katarzyna M.; Gudas, Lorraine J.

    2014-01-01

    To gain insight into oral squamous cell carcinogenesis, we performed deep sequencing (RNAseq) of non-tumorigenic human OKF6-TERT1R and tumorigenic SCC-9 cells. Numerous homeobox genes are differentially expressed between OKF6-TERT1R and SCC-9 cells. Data from Oncomine, a cancer microarray database, also show that homeobox (HOX) genes are dysregulated in oral SCC patients. The activity of Polycomb repressive complexes (PRC), which causes epigenetic modifications, and retinoic acid (RA) signaling can control HOX gene transcription. HOXB7, HOXC10, HOXC13, and HOXD8 transcripts are higher in SCC-9 than in OKF6-TERT1R cells; using ChIP (chromatin immunoprecipitation) we detected PRC2 protein SUZ12 and the epigenetic H3K27me3 mark on histone H3 at these genes in OKF6-TERT1R, but not in SCC-9 cells. In contrast, IRX1, IRX4, SIX2 and TSHZ3 transcripts are lower in SCC-9 than in OKF6-TERT1R cells. We detected SUZ12 and the H3K27me3 mark at these genes in SCC-9, but not in OKF6-TERT1R cells. SUZ12 depletion increased HOXB7, HOXC10, HOXC13, and HOXD8 transcript levels and decreased the proliferation of OKF6-TERT1R cells. Transcriptional responses to RA are attenuated in SCC-9 versus OKF6-TERT1R cells. SUZ12 and H3K27me3 levels were not altered by RA at these HOX genes in SCC-9 and OKF6-TERT1R cells. We conclude that altered activity of PRC2 is associated with dysregulation of homeobox gene expression in human SCC cells, and that this dysregulation potentially plays a role in the neoplastic transformation of oral keratinocytes. - Highlights: • RNAseq elucidates differences between non-tumorigenic and tumorigenic oral keratinocytes. • Changes in HOX mRNA in SCC-9 vs. OKF6-TERT1R cells are a result of altered epigenetic regulation. • RNAseq shows that retinoic acid (RA) influences gene expression in both OKF6-TERT1R and SCC-9 cells.

  7. Comparative Gene Expression Profiling of Primary and Metastatic Renal Cell Carcinoma Stem Cell-Like Cancer Cells

    PubMed Central

    Czarnecka, Anna M.; Lewicki, Sławomir; Helbrecht, Igor; Brodaczewska, Klaudia; Koch, Irena; Zdanowski, Robert; Król, Magdalena; Szczylik, Cezary

    2016-01-01

    Background Recent advancement in cancer research has shown that tumors are highly heterogeneous, and multiple phenotypically different cell populations are found in a single tumor. Cancer development and tumor growth are driven by specific types of cells—stem cell-like cancer cells (SCLCCs)—which are also responsible for metastatic spread and drug resistance. This research was designed to verify the presence of SCLCCs in renal cell cancer cell lines. Subsequently, we aimed to characterize phenotype and cell biology of CD105+ cells, defined previously as renal cell carcinoma tumor-initiating cells. The main goal of the project was to describe the gene-expression profile of stem cell-like cancer cells of primary tumor and metastatic origin. Materials and Methods Real-time PCR analysis of stemness genes (Oct-4, Nanog and Ncam) and soft agar colony formation assay were conducted to check the stemness properties of renal cell carcinoma (RCC) cell lines. FACS analysis of CD105+ and CD133+ cells was performed on RCC cells. Isolated CD105+ cells were verified for expression of mesenchymal markers—CD24, CD146, CD90, CD73, CD44, CD11b, CD19, CD34, CD45, HLA-DR and alkaline phosphatase. Hanging drop assay was used to investigate CD105+ cell-cell cohesion. Analysis of free-floating 3D spheres formed by isolated CD105+ was verified, as spheres have been hypothesized to contain undifferentiated multipotent progenitor cells. Finally, CD105+ cells were sorted from primary (Caki-2) and metastatic (ACHN) renal cell cancer cell lines. Gene-expression profiling of sorted CD105+ cells was performed with Agilent’s human GE 4x44K v2 microarrays. Differentially expressed genes were further categorized into canonical pathways. Network analysis and downstream analysis were performed with Ingenuity Pathway Analysis. Results Metastatic RCC cell lines (ACHN and Caki-1) demonstrated higher colony-forming ability in comparison to primary RCC cell lines. Metastatic RCC cell lines harbor

  8. Curcumin alters gene expression-associated DNA damage, cell cycle, cell survival and cell migration and invasion in NCI-H460 human lung cancer cells in vitro.

    PubMed

    Chiang, I-Tsang; Wang, Wei-Shu; Liu, Hsin-Chung; Yang, Su-Tso; Tang, Nou-Ying; Chung, Jing-Gung

    2015-10-01

    Lung cancer is the most common cause of cancer mortality and new cases are on the increase worldwide. However, the treatment of lung cancer remains unsatisfactory. Curcumin has been shown to induce cell death in many human cancer cells, including human lung cancer cells. However, the effects of curcumin on genetic mechanisms associated with these actions remain unclear. Curcumin (2 µM) was added to NCI-H460 human lung cancer cells and the cells were incubated for 24 h. Total RNA was extracted from isolated cells for cDNA synthesis, labeling, microarray hybridization and flour‑labeled cDNA hybridized on chip. Localized concentrations of fluorescent molecules were detected and quantified using Expression Console software (Affymetrix) with default RMA parameters. GeneGo software was used for the key genes involved and their possible interaction pathways. The results showed that ~170 genes were significantly upregulated and 577 genes were significantly downregulated in curcumin‑treated cells. Specifically, the up‑ and downregulated genes included CCNE2, associated with DNA damage; ID3, associated with cell survival and 146 genes with a >2- to 3-fold change including the TP53INP1 gene, associated with DNA damage; CDC6, CDCA5, TAKMIP2, CDK14, CDK5, CDCA76, CDC25A, CDC5L and SKP2, associated with cell cycle; the CARD6, ID1 and ID2 genes, associated with cell survival and the BRMS1L, associated with cell migration and invasion. Additionally, 59 downregulated genes exhibited a >4-fold change, including the DDIT3 gene, associated with DNA damage; while 97 genes had a >3- to 4-fold change including the DDIT4 gene, associated with DNA damage; the CCPG1 gene, associated with cell cycle and 321 genes with a >2- to 3-fold including the GADD45A and CGREF1 genes, associated with DNA damage; the CCPG1 gene, associated with cell cycle, the TNFRSF10B, GAS5, TSSC1 and TNFRSF11B gene, associated with cell survival and the ARHAP29 and CADM2 genes, associated with cell migration

  9. Single-cell epigenomics: powerful new methods for understanding gene regulation and cell identity.

    PubMed

    Clark, Stephen J; Lee, Heather J; Smallwood, Sébastien A; Kelsey, Gavin; Reik, Wolf

    2016-04-18

    Emerging single-cell epigenomic methods are being developed with the exciting potential to transform our knowledge of gene regulation. Here we review available techniques and future possibilities, arguing that the full potential of single-cell epigenetic studies will be realized through parallel profiling of genomic, transcriptional, and epigenetic information.

  10. Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy.

    PubMed

    Biffi, Alessandra; Montini, Eugenio; Lorioli, Laura; Cesani, Martina; Fumagalli, Francesca; Plati, Tiziana; Baldoli, Cristina; Martino, Sabata; Calabria, Andrea; Canale, Sabrina; Benedicenti, Fabrizio; Vallanti, Giuliana; Biasco, Luca; Leo, Simone; Kabbara, Nabil; Zanetti, Gianluigi; Rizzo, William B; Mehta, Nalini A L; Cicalese, Maria Pia; Casiraghi, Miriam; Boelens, Jaap J; Del Carro, Ubaldo; Dow, David J; Schmidt, Manfred; Assanelli, Andrea; Neduva, Victor; Di Serio, Clelia; Stupka, Elia; Gardner, Jason; von Kalle, Christof; Bordignon, Claudio; Ciceri, Fabio; Rovelli, Attilio; Roncarolo, Maria Grazia; Aiuti, Alessandro; Sessa, Maria; Naldini, Luigi

    2013-08-23

    Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disease caused by arylsulfatase A (ARSA) deficiency. Patients with MLD exhibit progressive motor and cognitive impairment and die within a few years of symptom onset. We used a lentiviral vector to transfer a functional ARSA gene into hematopoietic stem cells (HSCs) from three presymptomatic patients who showed genetic, biochemical, and neurophysiological evidence of late infantile MLD. After reinfusion of the gene-corrected HSCs, the patients showed extensive and stable ARSA gene replacement, which led to high enzyme expression throughout hematopoietic lineages and in cerebrospinal fluid. Analyses of vector integrations revealed no evidence of aberrant clonal behavior. The disease did not manifest or progress in the three patients 7 to 21 months beyond the predicted age of symptom onset. These findings indicate that extensive genetic engineering of human hematopoiesis can be achieved with lentiviral vectors and that this approach may offer therapeutic benefit for MLD patients.

  11. [Alteration of isozyme gene expression during cell differentiation and oncogenesis].

    PubMed

    Yamada, K; Noguchi, T

    1995-05-01

    Rat pyruvate kinase (PK) has four isozymes, called the M1-, M2-, L-, and R-types. The M1- and M2-type isozymes of PK are produced from the PKM gene by alternative splicing, whereas the L- and R-type isozymes of PK are produced from the PKL gene by use of different tissue-specific promoters. In early development, only M2-type PK expresses in all tissues. After late morphogenesis, M1-, L-, and R-type PK express tissue-specifically. In contrast, cell proliferation such as regenerating liver and oncogenesis lead to decrease or cessation of the expression of tissue-specific PK isozymes and to stimulation of the expression of M2-type PK. These phenomena from the point of view transcriptional regulatory apparatus of the PKM and PKL gene are discussed.

  12. Enrichment of human hematopoietic stem/progenitor cells facilitates transduction for stem cell gene therapy.

    PubMed

    Baldwin, Kismet; Urbinati, Fabrizia; Romero, Zulema; Campo-Fernandez, Beatriz; Kaufman, Michael L; Cooper, Aaron R; Masiuk, Katelyn; Hollis, Roger P; Kohn, Donald B

    2015-05-01

    Autologous hematopoietic stem cell (HSC) gene therapy for sickle cell disease has the potential to treat this illness without the major immunological complications associated with allogeneic transplantation. However, transduction efficiency by β-globin lentiviral vectors using CD34-enriched cell populations is suboptimal and large vector production batches may be needed for clinical trials. Transducing a cell population more enriched for HSC could greatly reduce vector needs and, potentially, increase transduction efficiency. CD34(+) /CD38(-) cells, comprising ∼1%-3% of all CD34(+) cells, were isolated from healthy cord blood CD34(+) cells by fluorescence-activated cell sorting and transduced with a lentiviral vector expressing an antisickling form of beta-globin (CCL-β(AS3) -FB). Isolated CD34(+) /CD38(-) cells were able to generate progeny over an extended period of long-term culture (LTC) compared to the CD34(+) cells and required up to 40-fold less vector for transduction compared to bulk CD34(+) preparations containing an equivalent number of CD34(+) /CD38(-) cells. Transduction of isolated CD34(+) /CD38(-) cells was comparable to CD34(+) cells measured by quantitative PCR at day 14 with reduced vector needs, and average vector copy/cell remained higher over time for LTC initiated from CD34(+) /38(-) cells. Following in vitro erythroid differentiation, HBBAS3 mRNA expression was similar in cultures derived from CD34(+) /CD38(-) cells or unfractionated CD34(+) cells. In vivo studies showed equivalent engraftment of transduced CD34(+) /CD38(-) cells when transplanted in competition with 100-fold more CD34(+) /CD38(+) cells. This work provides initial evidence for the beneficial effects from isolating human CD34(+) /CD38(-) cells to use significantly less vector and potentially improve transduction for HSC gene therapy.

  13. EP300-ZNF384 fusion gene product up-regulates GATA3 gene expression and induces hematopoietic stem cell gene expression signature in B-cell precursor acute lymphoblastic leukemia cells.

    PubMed

    Yaguchi, Akinori; Ishibashi, Takeshi; Terada, Kazuki; Ueno-Yokohata, Hitomi; Saito, Yuya; Fujimura, Junya; Shimizu, Toshiaki; Ohki, Kentaro; Manabe, Atsushi; Kiyokawa, Nobutaka

    2017-04-04

    ZNF384-related fusion genes are associated with a distinct subgroup of B-cell precursor acute lymphoblastic leukemias in childhood, with a frequency of approximately 3-4%. We previously identified a novel EP300-ZNF384 fusion gene. Patients with the ZNF384-related fusion gene exhibit a hematopoietic stem cell (HSC) gene expression signature and characteristic immunophenotype with negative or low expression of CD10 and aberrant expression of myeloid antigens, such as CD33 and CD13. However, the molecular basis of this pathogenesis remains completely unknown. In the present study, we examined the biological effects of EP300-ZNF384 expression induced by retrovirus-mediated gene transduction in an REH B-cell precursor acute lymphoblastic leukemia cell line, and observed the acquisition of the HSC gene expression signature and an up-regulation of GATA3 gene expression, as assessed by microarray analysis. In contrast, the gene expression profile induced by wild-type ZNF384 in REH cells was significantly different from that by EP300-ZNF384 expression. Together with the results of reporter assays, which revealed the enhancement of GATA3-promoter activity by EP300-ZNF384 expression, these findings suggest that EP300-ZNF384 mediates GATA3 gene expression and may be involved in the acquisition of the HSC gene expression signature and characteristic immunophenotype in B-cell precursor acute lymphoblastic leukemia cells.

  14. Concomitant T-cell receptor alpha and delta gene rearrangements in individual T-cell precursors.

    PubMed Central

    Thompson, S D; Pelkonen, J; Hurwitz, J L

    1990-01-01

    A debate has recently surfaced concerning the degree of precommitment attained by alpha beta and gamma delta T-cell precursors prior to T-cell receptor (TCR) gene rearrangement. It has been suggested that precursors may be precommitted to rearrange either alpha or delta genes, but not both, thus giving rise to alpha beta- and gamma delta-producing T cells, respectively. Alternatively, the precursors may be flexible with regard to potential TCR gene rearrangements. To address this controversy, the gene rearrangements among a group of T-cell hybridomas from fetal, newborn, and early postnatal mouse thymi were examined. Six probes spanning the delta and alpha loci were used in Southern blot analyses to characterize the rearrangements which occurred on homologous chromosomes in each cell. Although homologous chromosomes often rearranged in synchrony within the alpha locus, a number of hybridomas were found which had retained a delta rearrangement on one chromosome and an alpha rearrangement on the second. Results show that a precommitment by T cells to rearrange delta or alpha genes in a mutually exclusive manner is not an absolute feature of mouse thymocyte development. Images PMID:2164690

  15. Gene editing for cell engineering: trends and applications.

    PubMed

    Gupta, Sanjeev K; Shukla, Pratyoosh

    2016-08-18

    Gene editing with all its own advantages in molecular biology applications has made easy manipulation of various production hosts with the discovery and implementation of modern gene editing tools such as Crispr (Clustered regularly interspaced short palindromic repeats), TALENs (Transcription activator-like effector nucleases) and ZFNs (Zinc finger nucleases). With the advent of these modern tools, it is now possible to manipulate the genome of industrial production hosts such as yeast and mammalian cells which allows developing a potential and cost effective recombinant therapeutic protein. These tools also allow single editing to multiple genes for knocking-in or knocking-out of a host genome quickly in an efficient manner. A recent study on "multiplexed" gene editing revolutionized the knock-out and knock-in events of yeast and CHO, mammalian cells genome for metabolic engineering as well as high, stable, and consistent expression of a transgene encoding complex therapeutic protein such as monoclonal antibody. The gene of interest can either be integrated or deleted at single or multiple loci depending on the strategy and production requirement. This review will give a gist of all the modern tools with a brief description and advances in genetic manipulation using three major tools being implemented for the modification of such hosts with the emphasis on the use of Crispr-Cas9 for the "multiplexing gene-editing approach" for genetic manipulation of yeast and CHO mammalian hosts that ultimately leads to a fast track product development with consistent, improved product yield, quality, and thus affordability for a population at large.

  16. Engineering blood vessels by gene and cell therapy.

    PubMed

    Zarbiv, Gabriel; Preis, Meir; Ben-Yosef, Yaara; Flugelman, Moshe Y

    2007-08-01

    Cardiovascular-related syndromes are the leading cause of morbidity and mortality worldwide. Arterial narrowing and blockage due to atherosclerosis cause reduced blood flow to the brain, heart and legs. Bypass surgery to improve blood flow to the heart and legs in these patients is performed in hundreds of thousands of patients every year. Autologous grafts, such as the internal thoracic artery and saphenous vein, are used in most patients, but in a significant number of patients such grafts are not available and synthetic grafts are used. Synthetic grafts have higher failure rates than autologous grafts due to thrombosis and scar formation within graft lumen. Cell and gene therapy combined with tissue engineering hold a great promise to provide grafts that will be biocompatible and durable. This review describes the field of vascular grafts in the context of tissue engineering using cell and gene therapies.

  17. Androgen regulates ADAMTS15 gene expression in prostate cancer cells.

    PubMed

    Molokwu, Chidi N; Adeniji, Olajumoke O; Chandrasekharan, Shankar; Hamdy, Freddie C; Buttle, David J

    2010-08-01

    Prostate cancer is a major cause of mortality, largely as a consequence of metastases and transformation to androgen-independent growth. Metalloproteinases are implicated in cancer progression. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) are expressed in prostate cancer cells, with ADAMTS-1 and ADAMTS-15 being the most abundant. ADAMTS-15 but not ADAMTS-1 expression was downregulated by androgen in LNCaP prostate cancer cells, possibly through androgen response elements associated with the gene. ADAMTS-15 expression is predictive for survival in breast cancer, and the situation may be similar in prostate cancer, as androgen independence is usually due to aberrant signaling through its receptor.

  18. [VEGF gene expression in transfected human multipotent stromal cells].

    PubMed

    Smirnikhina, S A; Lavrov, A V; Bochkov, N P

    2011-01-01

    Dynamics of VEGF gene expression in transfected multipotent stromal cells from adipose tissue was examined using electroporation and lipofection. Differences in the potency and dynamics of plasmid elimination (up to day 9) between cell cultures were observed. All cultures were divided into fast and slow plasmid-eliminating ones. Interculture differences in VEGF expression were detected. The possibility of a 5-6-fold increase of VEGF expression was shown. There were no differences in transfection potency, plasmid elimination dynamics, and VEGF expression after transfection by both nonviral methods.

  19. Effect of Hypergravity on Endothelial Cell Function and Gene Expression

    NASA Astrophysics Data System (ADS)

    Morbidelli, Lucia; Marziliano, Nicola; Basile, Venere; Pezzatini, Silvia; Romano, Giovanni; Conti, Antonio; Monici, Monica

    2009-01-01

    It is well known that endothelial cells (ECs), which play a major role in cardiovascular system functioning, are very sensitive to mechanical stimuli. It has been demonstrated that changes in inertial conditions (i.e. microgravity and hypergravity) can affect both phenotypic and genotypic expression in ECs. In this report we describe the effects of hypergravity on ECs isolated from bovine aorta (BAECs). ECs were repeatedly exposed to discontinuous hypergravity conditions (5 × 10 min at 10× g with 10 min at 1× g between sets), simulated in a hyperfuge. Then, cell morphology and metabolism were analyzed by autofluorescence techniques. The phenotypic expression of cytoskeleton constituents ( β-actin, vimentin, tubulin), adhesion and survival signals (integrins), mediators of inflammation and angiogenesis was evaluated by immunocytofluorescence. Quantitative PCR (Q-PCR) with Low Density Arrays (LDAs) was used to evaluate modifications in gene expression. After hypergravity exposure, no significant changes were observed in cell morphology and energy metabolism. Cells remained adherent to the substratum, but integrin distribution was modified. Accordingly, the cytoskeletal network reorganized, documenting cell activation. There was a reduction in expression of genes controlling vasoconstriction and inflammation. Proapoptotic signals were downregulated. On the whole, the results documented that hypergravity exposure maintained EC survival and function by activation of adaptive mechanisms.

  20. Fasting-induced changes in ECL cell gene expression.

    PubMed

    Lambrecht, Nils W G; Yakubov, Iskandar; Sachs, George

    2007-10-22

    Gastric enterochromaffin-like (ECL) cells release histamine in response to food because of elevation of gastrin and neural release of pituitary adenylate cyclase-activating peptide (PACAP). Acid secretion is at a basal level in the absence of food but is rapidly stimulated with feeding. Rats fasted for 24 h showed a significant decrease of mucosal histamine despite steady-state expression of the histamine-synthesizing enzyme histidine decarboxylase (HDC). Comparative transcriptomal analysis using gene expression oligonucleotide microarrays of 95% pure ECL cells from fed and 24-h fasted rats, thereby eliminating mRNA contamination from other gastric mucosal cell types, identified significantly increased gene expression of the enzymes histidase and urocanase catabolizing the HDC substrate L-histidine but significantly decreased expression of the cellular L-histidine uptake transporter SN2 and of the vesicular monoamine transporter 2 (VMAT-2) responsible for histamine uptake into secretory vesicles. This was confirmed by reverse transcriptase-quantitative polymerase chain reaction of gastric fundic mucosal samples from fed and 24-h fasted rats. The decrease of VMAT-2 gene expression was also shown by a decrease in VMAT-2 protein content in protein extracts from fed and 24-h fasted rats compared with equal amounts of HDC protein and Na-K-ATPase alpha(1)-subunit protein content. These results indicate that rat gastric ECL cells regulate their histamine content during 24-h fasting not by a change in HDC gene or protein expression but by regulation of substrate concentration for HDC and a decreased histamine secretory pool.

  1. Generation of Gene Knockout Mice by ES Cell Microinjection

    PubMed Central

    Longenecker, Glenn; Kulkarni, Ashok B

    2009-01-01

    This unit lists and describes protocols used in the production of chimeric mice leading to the generation of gene knockout mice. These protocols include the collection of blastocyst embryos, ES cell injection, and uterine transfer of injected blastocysts. Support protocols in the superovulation of blastocyst donor mice, generation of pseudopregnant recipients, fabrication of glass pipettes, and generation of germline mice are also included. Practical tips and solutions are mentioned to help troubleshoot problems that may occur. PMID:19731226

  2. Ca2+ signaling, genes and the cell cycle

    PubMed Central

    Machaca, Khaled

    2013-01-01

    Changes in the concentration and spatial distribution of Ca2+ ions in the cytoplasm constitute a ubiquitous intracellular signaling module in cellular physiology. With the advent of Ca2+ dyes that allow direct visualization of Ca2+ transients, combined with powerful experimental tools such as electrophysiological recordings, intracellular Ca2+ transients have been implicated in practically every aspect of cellular physiology, including cellular proliferation. Ca2+ signals are associated with different phases of the cell cycle and interfering with Ca2+ signaling or downstream pathways often disrupts progression of the cell cycle. Although there exists a dependence between Ca2+ signals and the cell cycle the mechanisms involved are not well defined and given the cross-talk between Ca2+ and other signaling modules, it is difficult to assess the exact role of Ca2+ signals in cell cycle progression. Two exceptions however, include fertilization and T-cell activation, where well-defined roles for Ca2+ signals in mediating progression through specific stages of the cell cycle have been clearly established. In the case of T-cell activation Ca2+ regulates entry into the cell cycle through the induction of gene transcription. PMID:21084120

  3. Human T-cell receptor variable gene segment families

    SciTech Connect

    Arden, B.; Kabelitz, D.; Clark, S.P.; Mak, T.W.

    1995-10-01

    Multiple DNA and protein sequence alignments have been constructed for the human T-cell receptor {alpha}/{delta}, {beta}, and {gamma} (TCRA/D, B, and G) variable (V) gene segments. The traditional classification into subfamilies was confirmed using a much larger pool of sequences. For each sequence, a name was derived which complies with the standard nomenclature. The traditional numbering of V gene segments in the order of their discovery was continued and changed when in conflict with names of other segments. By discriminating between alleles at the same locus versus genes from different loci, we were able to reduce the number of more than 150 different TCRBV sequences in the database to a repertoire of only 47 functional TCRBV gene segments. An extension of this analysis to the over 100 TCRAV sequences results in a predicted repertoire of 42 functional TCRAV gene segments. Our alignment revealed two residues that distinguish between the highly homologous V{delta} and V{alpha}, one at a site that in V{sub H} contacts the constant region, the other at the interface between immunoglobulin V{sub H} and V{sub L}. This site may be responsible for restricted pairing between certain V{delta} and V{gamma} chains. On the other hand, V{beta} and V{gamma} appear to be related by the fact that their CDR2 length is increased by four residues as compared with that of V{alpha}/{delta} peptides. 150 refs., 12 figs., 5 tabs.

  4. MGMT enrichment and second gene co-expression in hematopoietic progenitor cells using separate or dual-gene lentiviral vectors.

    PubMed

    Roth, Justin C; Alberti, Michael O; Ismail, Mourad; Lingas, Karen T; Reese, Jane S; Gerson, Stanton L

    2015-01-22

    The DNA repair gene O(6)-methylguanine-DNA methyltransferase (MGMT) allows efficient in vivo enrichment of transduced hematopoietic stem cells (HSC). Thus, linking this selection strategy to therapeutic gene expression offers the potential to reconstitute diseased hematopoietic tissue with gene-corrected cells. However, different dual-gene expression vector strategies are limited by poor expression of one or both transgenes. To evaluate different co-expression strategies in the context of MGMT-mediated HSC enrichment, we compared selection and expression efficacies in cells cotransduced with separate single-gene MGMT and GFP lentivectors to those obtained with dual-gene vectors employing either encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) or foot and mouth disease virus (FMDV) 2A elements for co-expression strategies. Each strategy was evaluated in vitro and in vivo using equivalent multiplicities of infection (MOI) to transduce 5-fluorouracil (5-FU) or Lin(-)Sca-1(+)c-kit(+) (LSK)-enriched murine bone marrow cells (BMCs). The highest dual-gene expression (MGMT(+)GFP(+)) percentages were obtained with the FMDV-2A dual-gene vector, but half of the resulting gene products existed as fusion proteins. Following selection, dual-gene expression percentages in single-gene vector cotransduced and dual-gene vector transduced populations were similar. Equivalent MGMT expression levels were obtained with each strategy, but GFP expression levels derived from the IRES dual-gene vector were significantly lower. In mice, vector-insertion averages were similar among cells enriched after dual-gene vectors and those cotransduced with single-gene vectors. These data demonstrate the limitations and advantages of each strategy in the context of MGMT-mediated selection, and may provide insights into vector design with respect to a particular therapeutic gene or hematologic defect.

  5. Nonhomologous end joining-mediated gene replacement in plant cells.

    PubMed

    Weinthal, Dan Michael; Taylor, Roslyn Ann; Tzfira, Tzvi

    2013-05-01

    Stimulation of the homologous recombination DNA-repair pathway via the induction of genomic double-strand breaks (DSBs) by zinc finger nucleases (ZFNs) has been deployed for gene replacement in plant cells. Nonhomologous end joining (NHEJ)-mediated repair of DSBs, on the other hand, has been utilized for the induction of site-specific mutagenesis in plants. Since NHEJ is the dominant DSB repair pathway and can also lead to the capture of foreign DNA molecules, we suggest that it can also be deployed for gene replacement. An acceptor DNA molecule in which a green fluorescent protein (GFP) coding sequence (gfp) was flanked by ZFN recognition sequences was used to produce transgenic target plants. A donor DNA molecule in which a promoterless hygromycin B phosphotransferase-encoding gene (hpt) was flanked by ZFN recognition sequences was constructed. The donor DNA molecule and ZFN expression cassette were delivered into target plants. ZFN-mediated site-specific mutagenesis and complete removal of the GFP coding sequence resulted in the recovery of hygromycin-resistant plants that no longer expressed GFP and in which the hpt gene was unlinked to the acceptor DNA. More importantly, ZFN-mediated digestion of both donor and acceptor DNA molecules resulted in NHEJ-mediated replacement of the gfp with hpt and recovery of hygromycin-resistant plants that no longer expressed GFP and in which the hpt gene was physically linked to the acceptor DNA. Sequence and phenotypical analyses, and transmission of the replacement events to the next generation, confirmed the stability of the NHEJ-induced gene exchange, suggesting its use as a novel method for transgene replacement and gene stacking in plants.

  6. Expression of insulin-like growth factor family genes in clear cell renal cell carcinoma

    PubMed Central

    Białożyt, Michał; Plato, Marta; Mazurek, Urszula; Braczkowska, Bogumiła

    2016-01-01

    Aim of the study Despite significant progress in the pathology of clear cell renal cell carcinoma (ccRCC), diagnostic and predictive factors of major importance have not been discovered. Some hopes are associated with insulin-like growth factors. The aim of the study was to compare the expression of genes for insulin-like growth factor family in tumours and in tissue of kidneys without cancer. Material and methods Fifty-two patients years with clear cell renal cell cancer were qualified to the study group; patients nephrectomised because of hydronephrosis were included in the control group. Expression of genes were evaluated by RT-PCR. Results Expression of IGFR-1 gene in tumour accounts for about 60% of cases. The incidence is higher than in corresponding adjacent non-cancerous kidney tissues and higher (but with no statistical significance) than in kidney without cancer. Expression of IGFR-2 gene in tumours has not been established. The incidence of the expression in corresponding adjacent non-cancerous kidney tissues is small. Expression of this gene has been present in all specimens from kidneys without cancer. Expression of IGFBP-3 gene ascertained in all (except four) cases of ccRCC and in the majority of clippings from adjacent tissue. It was not found in kidneys from the control group. IGF-1, IGF-2, and IGFR-1 mRNA copy numbers in ccRCC were higher than in the material from the control group PMID:27358591

  7. Genomic instability, driver genes and cell selection: Projections from cancer to stem cells.

    PubMed

    Ben-David, Uri

    2015-04-01

    Cancer cells and stem cells share many traits, including a tendency towards genomic instability. Human cancers exhibit tumor-specific genomic aberrations, which often affect their malignancy and drug response. During their culture propagation, human pluripotent stem cells (hPSCs) also acquire characteristic genomic aberrations, which may have significant impact on their molecular and cellular phenotypes. These aberrations vary in size from single nucleotide alterations to copy number alterations to whole chromosome gains. A prominent challenge in both cancer and stem cell research is to identify "driver aberrations" that confer a selection advantage, and "driver genes" that underlie the recurrence of these aberrations. Following principles that are already well-established in cancer research, candidate driver genes have also been suggested in hPSCs. Experimental validation of the functional role of such candidates can uncover whether these are bona fide driver genes. The identification of driver genes may bring us closer to a mechanistic understanding of the genomic instability of stem cells. Guided by terminologies and methodologies commonly applied in cancer research, such understanding may have important ramifications for both stem cell and cancer biology. This article is part of a Special Issue entitled: Stress as a fundamental theme in cell plasticity.

  8. Differential gene expression in stromal cells of human giant cell tumor of bone.

    PubMed

    Wuelling, M; Delling, G; Kaiser, E

    2004-12-01

    Giant cell tumor (GCT) offers a unique model for the hematopoietic-stromal cell interaction in human bone marrow. Evidence has been presented that GCT stromal cells (GCTSCs) promote accumulation, size and activity of the giant cells. Although GCTSCs are considered the neoplastic component of GCT, little is known about their genetic basis and, to date, a tumor-specific gene expression pattern has not been characterized. Mesenchymal stem cells (MSCs) have been identified as the origin of the GCT neoplastic stromal cell. Using state of the art array technology, expression profiling was applied to enriched stromal cell populations from five different GCTs and two primary MSCs as controls. Of the 29 differentially expressed genes found, 25 showed an increased expression. Differential mRNA expression was verified by real-time polymerase chain reaction analysis of 10 selected genes, supporting the validity of cDNA arrays as a tool to identify tumor-related genes in GCTSCs. Increased expression of two oncogenes, JUN and NME2, was substantiated at the protein level, utilizing immunohistochemical evaluation of GCT sections and Western-blot analysis. Increased phosphorylation of JUN Ser-63 was also found.

  9. Patterns of gene expression in peripheral blood mononuclear cells and outcomes from patients with sepsis secondary to community acquired pneumonia.

    PubMed

    Severino, Patricia; Silva, Eliézer; Baggio-Zappia, Giovana Lotici; Brunialti, Milena Karina Coló; Nucci, Laura Alejandra; Rigato, Otelo; da Silva, Ismael Dale Cotrim Guerreiro; Machado, Flávia Ribeiro; Salomao, Reinaldo

    2014-01-01

    Mechanisms governing the inflammatory response during sepsis have been shown to be complex, involving cross-talk between diverse signaling pathways. Current knowledge regarding the mechanisms underlying sepsis provides an incomplete picture of the syndrome, justifying additional efforts to understand this condition. Microarray-based expression profiling is a powerful approach for the investigation of complex clinical conditions such as sepsis. In this study, we investigate whole-genome expression profiles in mononuclear cells from survivors (n = 5) and non-survivors (n = 5) of sepsis. To circumvent the heterogeneity of septic patients, only patients admitted with sepsis caused by community-acquired pneumonia were included. Blood samples were collected at the time of sepsis diagnosis and seven days later to evaluate the role of biological processes or genes possibly involved in patient recovery. Principal Components Analysis (PCA) profiling discriminated between patients with early sepsis and healthy individuals. Genes with differential expression were grouped according to Gene Ontology, and most genes related to immune defense were up-regulated in septic patients. Additionally, PCA in the early stage was able to distinguish survivors from non-survivors. Differences in oxidative phosphorylation seem to be associated with clinical outcome because significant differences in the expression profile of genes related to mitochondrial electron transport chain (ETC) I-V were observed between survivors and non-survivors at the time of patient enrollment. Global gene expression profiles after seven days of sepsis progression seem to reproduce, to a certain extent, patterns collected at the time of diagnosis. Gene expression profiles comparing admission and follow-up samples differed between survivors and non-survivors, with decreased expression of genes related to immune functions in non-survivors. In conclusion, genes related to host defense and inflammatory response

  10. From the Cover: Design of artificial cell-cell communication using gene and metabolic networks

    NASA Astrophysics Data System (ADS)

    Bulter, Thomas; Lee, Sun-Gu; Waichun Wong, Wilson; Fung, Eileen; Connor, Michael R.; Liao, James C.

    2004-02-01

    Artificial transcriptional networks have been used to achieve novel, nonnative behavior in bacteria. Typically, these artificial circuits are isolated from cellular metabolism and are designed to function without intercellular communication. To attain concerted biological behavior in a population, synchronization through intercellular communication is highly desirable. Here we demonstrate the design and construction of a gene-metabolic circuit that uses a common metabolite to achieve tunable artificial cell-cell communication. This circuit uses a threshold concentration of acetate to induce gene expression by acetate kinase and part of the nitrogen-regulation two-component system. As one application of the cell-cell communication circuit we created an artificial quorum sensor. Engineering of carbon metabolism in Escherichia coli made acetate secretion proportional to cell density and independent of oxygen availability. In these cells the circuit induced gene expression in response to a threshold cell density. This threshold can be tuned effectively by controlling pH over the cell membrane, which determines the partition of acetate between medium and cells. Mutagenesis of the enhancer sequence of the glnAp2 promoter produced variants of the circuit with changed sensitivity demonstrating tunability of the circuit by engineering of its components. The behavior of the circuit shows remarkable predictability based on a mathematical design model.

  11. Regulation of proliferation and gene expression in cultured human aortic smooth muscle cells by resveratrol and standardized grape extracts

    SciTech Connect

    Wang Zhirong; Chen Yan; Labinskyy, Nazar; Hsieh Tzechen; Ungvari, Zoltan; Wu, Joseph M. . E-mail: Joseph_Wu@nymc.edu

    2006-07-21

    Epidemiologic studies suggest that low to moderate consumption of red wine is inversely associated with the risk of coronary heart disease; the protection is in part attributed to grape-derived polyphenols, notably trans-resveratrol, present in red wine. It is not clear whether the cardioprotective effects of resveratrol can be reproduced by standardized grape extracts (SGE). In the present studies, we determined, using cultured human aortic smooth muscle cells (HASMC), growth and specific gene responses to resveratrol and SGE provided by the California Table Grape Commission. Suppression of HASMC proliferation by resveratrol was accompanied by a dose-dependent increase in the expression of tumor suppressor gene p53 and heat shock protein HSP27. Using resveratrol affinity chromatography and biochemical fractionation procedures, we showed by immunoblot analysis that treatment of HASMC with resveratrol increased the expression of quinone reductase I and II, and also altered their subcellular distribution. Growth of HASMC was significantly inhibited by 70% ethanolic SGE; however, gene expression patterns in various cellular compartments elicited in response to SGE were substantially different from those observed in resveratrol-treated cells. Further, SGE also differed from resveratrol in not being able to induce relaxation of rat carotid arterial rings. These results indicate that distinct mechanisms are involved in the regulation of HASMC growth and gene expression by SGE and resveratrol.

  12. Identification of genes needed for regeneration, stem cell function, and tissue homeostasis by systematic gene perturbation in planaria.

    PubMed

    Reddien, Peter W; Bermange, Adam L; Murfitt, Kenneth J; Jennings, Joya R; Sánchez Alvarado, Alejandro

    2005-05-01

    Planarians have been a classic model system for the study of regeneration, tissue homeostasis, and stem cell biology for over a century, but they have not historically been accessible to extensive genetic manipulation. Here we utilize RNA-mediated genetic interference (RNAi) to introduce large-scale gene inhibition studies to the classic planarian system. 1065 genes were screened. Phenotypes associated with the RNAi of 240 genes identify many specific defects in the process of regeneration and define the major categories of defects planarians display following gene perturbations. We assessed the effects of inhibiting genes with RNAi on tissue homeostasis in intact animals and stem cell (neoblast) proliferation in amputated animals identifying candidate stem cell, regeneration, and homeostasis regulators. Our study demonstrates the great potential of RNAi for the systematic exploration of gene function in understudied organisms and establishes planarians as a powerful model for the molecular genetic study of stem cells, regeneration, and tissue homeostasis.

  13. Microarray Analysis of Cell Cycle Gene Expression in Adult Human Corneal Endothelial Cells

    PubMed Central

    Ha Thi, Binh Minh; Campolmi, Nelly; He, Zhiguo; Pipparelli, Aurélien; Manissolle, Chloé; Thuret, Jean-Yves; Piselli, Simone; Forest, Fabien; Peoc'h, Michel; Garraud, Olivier; Gain, Philippe; Thuret, Gilles

    2014-01-01

    Corneal endothelial cells (ECs) form a monolayer that controls the hydration of the cornea and thus its transparency. Their almost nil proliferative status in humans is responsible, in several frequent diseases, for cell pool attrition that leads to irreversible corneal clouding. To screen for candidate genes involved in cell cycle arrest, we studied human ECs subjected to various environments thought to induce different proliferative profiles compared to ECs in vivo. Donor corneas (a few hours after death), organ-cultured (OC) corneas, in vitro confluent and non-confluent primary cultures, and an immortalized EC line were compared to healthy ECs retrieved in the first minutes of corneal grafts. Transcriptional profiles were compared using a cDNA array of 112 key genes of the cell cycle and analysed using Gene Ontology classification; cluster analysis and gene map presentation of the cell cycle regulation pathway were performed by GenMAPP. Results were validated using qRT-PCR on 11 selected genes. We found several transcripts of proteins implicated in cell cycle arrest and not previously reported in human ECs. Early G1-phase arrest effectors and multiple DNA damage-induced cell cycle arrest-associated transcripts were found in vivo and over-represented in OC and in vitro ECs. Though highly proliferative, immortalized ECs also exhibited overexpression of transcripts implicated in cell cycle arrest. These new effectors likely explain the stress-induced premature senescence that characterizes human adult ECs. They are potential targets for triggering and controlling EC proliferation with a view to increasing the cell pool of stored corneas or facilitating mass EC culture for bioengineered endothelial grafts. PMID:24747418

  14. Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible Lhx2 expression

    PubMed Central

    Richter, Karin; Wirta, Valtteri; Dahl, Lina; Bruce, Sara; Lundeberg, Joakim; Carlsson, Leif; Williams, Cecilia

    2006-01-01

    Background Expression of the LIM-homeobox gene Lhx2 in murine hematopoietic cells allows for the generation of hematopoietic stem cell (HSC)-like cell lines. To address the molecular basis of Lhx2 function, we generated HSC-like cell lines where Lhx2 expression is regulated by a tet-on system and hence dependent on the presence of doxycyclin (dox). These cell lines efficiently down-regulate Lhx2 expression upon dox withdrawal leading to a rapid differentiation into various myeloid cell types. Results Global gene expression of these cell lines cultured in dox was compared to different time points after dox withdrawal using microarray technology. We identified 267 differentially expressed genes. The majority of the genes overlapping with HSC-specific databases were those down-regulated after turning off Lhx2 expression and a majority of the genes overlapping with those defined as late progenitor-specific genes were the up-regulated genes, suggesting that these cell lines represent a relevant model system for normal HSCs also at the level of global gene expression. Moreover, in situ hybridisations of several genes down-regulated after dox withdrawal showed overlapping expression patterns with Lhx2 in various tissues during embryonic development. Conclusion Global gene expression analysis of HSC-like cell lines with inducible Lhx2 expression has identified genes putatively linked to self-renewal / differentiation of HSCs, and function of Lhx2 in organ development and stem / progenitor cells of non-hematopoietic origin. PMID:16600034

  15. Somatic cell gene mutations in humans: biomarkers for genotoxicity.

    PubMed Central

    Albertini, R J; Nicklas, J A; O'Neill, J P

    1993-01-01

    Somatic cell gene mutations arising in vivo in humans provide biomarkers for genotoxicity. Four assays, each measuring changes in a different "recorder" gene, are available for detecting mutations of the hemoglobin (Hb) and glycophorin A (gpa) genes in red blood cells and the hypoxanthine-guanine phosphoribosyltransferase (hprt) and HLA genes in T-lymphocytes. Mean adult background mutant frequencies have been established; i.e., approximately 4 x 10(-8) (Hb), 5-10 x 10(-6) (hprt), 10-20 x 10(-6) (gpa) and 30 x 10(-6) (HLA). All the assays have now been used in studies of individuals exposed to physical and/or chemical genotoxic agents, and all have shown elevated values following exposures; examples are presented. In addition to quantitation, the lymphocyte assays allow molecular analyses of in vivo mutations, the definition of background and induced mutational spectra, and the search for unique changes for characterizing specific mutagens. The HPRT system currently has the largest database in this regard. Approximately 15% of adult background hprt mutations are due to gross structural alterations (primarily deletions) having random breakpoints; 85% result from "point" changes detected only by sequencing. In contrast, a specific intragenic deletion due to DNA cleavage at specific sites characterizes fetal hprt mutations, implicating a developmental mistake in their genesis. (This kind of developmental mistake in other genes is frequently observed in lymphoid malignancies.) Mutational spectra are just beginning to be defined for induced hprt mutations, e.g., ionizing radiation produces large deletions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8143616

  16. Treating hearing disorders with cell and gene therapy

    NASA Astrophysics Data System (ADS)

    Gillespie, Lisa N.; Richardson, Rachael T.; Nayagam, Bryony A.; Wise, Andrew K.

    2014-12-01

    Hearing loss is an increasing problem for a substantial number of people and, with an aging population, the incidence and severity of hearing loss will become more significant over time. There are very few therapies currently available to treat hearing loss, and so the development of new therapeutic strategies for hearing impaired individuals is of paramount importance to address this unmet clinical need. Most forms of hearing loss are progressive in nature and therefore an opportunity exists to develop novel therapeutic approaches to slow or halt hearing loss progression, or even repair or replace lost hearing function. Numerous emerging technologies have potential as therapeutic options. This paper details the potential of cell- and gene-based therapies to provide therapeutic agents to protect sensory and neural cells from various insults known to cause hearing loss; explores the potential of replacing lost sensory and nerve cells using gene and stem cell therapy; and describes the considerations for clinical translation and the challenges that need to be overcome.

  17. Hematopoietic stem cell mobilization strategies for gene therapy of beta thalassemia and sickle cell disease.

    PubMed

    Yannaki, Evangelia; Stamatoyannopoulos, George

    2010-08-01

    Effective gene therapy for hemoglobinopathies will require high numbers of autologous gene-engineered hematopoetic stem cells to be reintroduced into the patients. Stem cell mobilization using G-CSF is the most convenient and effective approach to achieve this goal, but it can have severe side effects in sickle cell anemia and be potentially harmful in the case of severe thalassemia. Hence, the optimal way of collection of hematopoetic stem cells from patients with thalassemia and sickle cell disease needs to be determined. In this paper, we review the possible risks of G-CSF mobilization in hemoglobinopathies and we outline the approaches used in an on-going clinical trial in which pretreatment with hydroxyurea is used to reduce potential risks of G-CSF administration to patients with severe beta thalassemia.

  18. Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation.

    PubMed

    Itaka, Keiji; Uchida, Satoshi; Matsui, Akitsugu; Yanagihara, Kayoko; Ikegami, Masaru; Endo, Taisuke; Ishii, Takehiko; Kataoka, Kazunori

    2015-07-31

    To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 µm diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

  19. Autophagy genes function sequentially to promote apoptotic cell corpse degradation in the engulfing cell

    PubMed Central

    Li, Wei; Zou, Wei; Yang, Yihong; Chai, Yongping; Chen, Baohui; Cheng, Shiya; Tian, Dong

    2012-01-01

    Apoptotic cell degradation is a fundamental process for organism development, and impaired clearance causes inflammatory or autoimmune disease. Although autophagy genes were reported to be essential for exposing the engulfment signal on apoptotic cells, their roles in phagocytes for apoptotic cell removal are not well understood. In this paper, we develop live-cell imaging techniques to study apoptotic cell clearance in the Caenorhabditis elegans Q neuroblast lineage. We show that the autophagy proteins LGG-1/LC3, ATG-18, and EPG-5 were sequentially recruited to internalized apoptotic Q cells in the phagocyte. In atg-18 or epg-5 mutants, apoptotic Q cells were internalized but not properly degraded; this phenotype was fully rescued by the expression of autophagy genes in the phagocyte. Time-lapse analysis of autophagy mutants revealed that recruitment of the small guanosine triphosphatases RAB-5 and RAB-7 to the phagosome and the formation of phagolysosome were all significantly delayed. Thus, autophagy genes act within the phagocyte to promote apoptotic cell degradation. PMID:22451698

  20. Pinhole-Free and Surface-Nanostructured NiOx Film by Room-Temperature Solution Process for High-Performance Flexible Perovskite Solar Cells with Good Stability and Reproducibility.

    PubMed

    Zhang, Hong; Cheng, Jiaqi; Lin, Francis; He, Hexiang; Mao, Jian; Wong, Kam Sing; Jen, Alex K-Y; Choy, Wallace C H

    2016-01-26

    Recently, researchers have focused on the design of highly efficient flexible perovskite solar cells (PVSCs), which enables the implementation of portable and roll-to-roll fabrication in large scale. While NiOx is a promising material for hole transport layer (HTL) candidate for fabricating efficient PVSCs on a rigid substrate, the reported NiOx HTLs are formed using different multistep treatments (such as 300-500 °C annealing, O2-plasma, UVO, etc.), which hinders the development of flexible PVSCs based on NiOx. Meanwhile, the features of nanostructured morphology and flawless film quality are very important for the film to function as highly effective HTL of PVSCs. However, it is difficult to have the two features coexist natively, particularly in a solution process that flawless film will usually come with smooth morphology. Here, we demonstrate the flawless and surface-nanostructured NiOx film from a simple and controllable room-temperature solution process for achieving high performance flexible PVSCs with good stability and reproducibility. The power conversion efficiency (PCE) can reaches a promising value of 14.53% with no obvious hysteresis (and a high PCE of 17.60% for PVSC on ITO glass). Furthermore, the NiOx-based PVSCs show markedly improved air stability. Regarding the performance improvement, the flawless and surface-nanostructured NiOx film can make the interfacial recombination and monomolecular Shockley-Read-Hall recombination of PVSC reduce. In addition, the formation of an intimate junction of large interfacial area at NiOx film/the perovskite layer improve the hole extraction and thus PVSC performances. This work contributes to the evolution of flexible PVSCs with simple fabrication process and high device performances.

  1. An X chromosome gene regulates hematopoietic stem cell kinetics

    PubMed Central

    Abkowitz, Janis L.; Taboada, Monica; Shelton, Grady H.; Catlin, Sandra N.; Guttorp, Peter; Kiklevich, J. Veronika

    1998-01-01

    Females are natural mosaics for X chromosome-linked genes. As X chromosome inactivation occurs randomly, the ratio of parental phenotypes among blood cells is approximately 1:1. Recently, however, ratios of greater than 3:1 have been observed in 38–56% of women over age 60. This could result from a depletion of hematopoietic stem cells (HSCs) with aging (and the maintenance of hematopoiesis by a few residual clones) or from myelodysplasia (the dominance of a neoplastic clone). Each possibility has major implications for chemotherapy and for transplantation in elderly patients. We report similar findings in longitudinal studies of female Safari cats and demonstrate that the excessive skewing that develops with aging results from a third mechanism that has no pathologic consequence, hemizygous selection. We show that there is a competitive advantage for all HSCs with a specific X chromosome phenotype and, thus, demonstrate that an X chromosome gene (or genes) regulates HSC replication, differentiation, and/or survival. PMID:9520458

  2. Benzyl isothiocyanate alters the gene expression with cell cycle regulation and cell death in human brain glioblastoma GBM 8401 cells.

    PubMed

    Tang, Nou-Ying; Chueh, Fu-Shin; Yu, Chien-Chih; Liao, Ching-Lung; Lin, Jen-Jyh; Hsia, Te-Chun; Wu, King-Chuen; Liu, Hsin-Chung; Lu, Kung-Wen; Chung, Jing-Gung

    2016-04-01

    Glioblastoma multiforme (GBM) is a highly malignant devastating brain tumor in adults. Benzyl isothiocyanate (BITC) is one of the isothiocyanates that have been shown to induce human cancer cell apoptosis and cell cycle arrest. Herein, the effect of BITC on cell viability and apoptotic cell death and the genetic levels of human brain glioblastoma GBM 8401 cells in vitro were investigated. We found that BITC induced cell morphological changes, decreased cell viability and the induction of cell apoptosis in GBM 8401 cells was time-dependent. cDNA microarray was used to examine the effects of BITC on GBM 8401 cells and we found that numerous genes associated with cell death and cell cycle regulation in GBM 8401 cells were altered after BITC treatment. The results show that expression of 317 genes was upregulated, and two genes were associated with DNA damage, the DNA-damage-inducible transcript 3 (DDIT3) was increased 3.66-fold and the growth arrest and DNA-damage-inducible α (GADD45A) was increased 2.34-fold. We also found that expression of 182 genes was downregulated and two genes were associated with receptor for cell responses to stimuli, the EGF containing fibulin-like extracellular matrix protein 1 (EFEMP1) was inhibited 2.01-fold and the TNF receptor-associated protein 1 (TRAP1) was inhibited 2.08-fold. BITC inhibited seven mitochondria ribosomal genes, the mitochondrial ribosomal protein; tumor protein D52 (MRPS28) was inhibited 2.06-fold, the mitochondria ribosomal protein S2 (MRPS2) decreased 2.07-fold, the mitochondria ribosomal protein L23 (MRPL23) decreased 2.08-fold, the mitochondria ribosomal protein S2 (MRPS2) decreased 2.07-fold, the mitochondria ribosomal protein S12 (MRPS12) decreased 2.08-fold, the mitochondria ribosomal protein L12 (MRPL12) decreased 2.25-fold and the mitochondria ribosomal protein S34 (MRPS34) was decreased 2.30-fold in GBM 8401 cells. These changes of gene expression can provide the effects of BITC on the genetic level and are

  3. Language-Agnostic Reproducible Data Analysis Using Literate Programming.

    PubMed

    Vassilev, Boris; Louhimo, Riku; Ikonen, Elina; Hautaniemi, Sampsa

    2016-01-01

    A modern biomedical research project can easily contain hundreds of analysis steps and lack of reproducibility of the analyses has been recognized as a severe issue. While thorough documentation enables reproducibility, the number of analysis programs used can be so large that in reality reproducibility cannot be easily achieved. Literate programming is an approach to present computer programs to human readers. The code is rearranged to follow the logic of the program, and to explain that logic in a natural language. The code executed by the computer is extracted from the literate source code. As such, literate programming is an ideal formalism for systematizing analysis steps in biomedical research. We have developed the reproducible computing tool Lir (literate, reproducible computing) that allows a tool-agnostic approach to biomedical data analysis. We demonstrate the utility of Lir by applying it to a case study. Our aim was to investigate the role of endosomal trafficking regulators to the progression of breast cancer. In this analysis, a variety of tools were combined to interpret the available data: a relational database, standard command-line tools, and a statistical computing environment. The analysis revealed that the lipid transport related genes LAPTM4B and NDRG1 are coamplified in breast cancer patients, and identified genes potentially cooperating with LAPTM4B in breast cancer progression. Our case study demonstrates that with Lir, an array of tools can be combined in the same data analysis to improve efficiency, reproducibility, and ease of understanding. Lir is an open-source software available at github.com/borisvassilev/lir.

  4. Language-Agnostic Reproducible Data Analysis Using Literate Programming

    PubMed Central

    Vassilev, Boris; Louhimo, Riku; Ikonen, Elina; Hautaniemi, Sampsa

    2016-01-01

    A modern biomedical research project can easily contain hundreds of analysis steps and lack of reproducibility of the analyses has been recognized as a severe issue. While thorough documentation enables reproducibility, the number of analysis programs used can be so large that in reality reproducibility cannot be easily achieved. Literate programming is an approach to present computer programs to human readers. The code is rearranged to follow the logic of the program, and to explain that logic in a natural language. The code executed by the computer is extracted from the literate source code. As such, literate programming is an ideal formalism for systematizing analysis steps in biomedical research. We have developed the reproducible computing tool Lir (literate, reproducible computing) that allows a tool-agnostic approach to biomedical data analysis. We demonstrate the utility of Lir by applying it to a case study. Our aim was to investigate the role of endosomal trafficking regulators to the progression of breast cancer. In this analysis, a variety of tools were combined to interpret the available data: a relational database, standard command-line tools, and a statistical computing environment. The analysis revealed that the lipid transport related genes LAPTM4B and NDRG1 are coamplified in breast cancer patients, and identified genes potentially cooperating with LAPTM4B in breast cancer progression. Our case study demonstrates that with Lir, an array of tools can be combined in the same data analysis to improve efficiency, reproducibility, and ease of understanding. Lir is an open-source software available at github.com/borisvassilev/lir. PMID:27711123

  5. Reliability and reproducibility of Kienbock's disease staging.

    PubMed

    Goeminne, S; Degreef, I; De Smet, L

    2010-09-01

    We evaluated the interobserver reliability and intraobserver reproducibility of the Lichtman et al. classification for Kienböck's disease by getting four observers with different experience to look at 70 sets of wrist radiographs at different points in time. These observers staged each set of radiographs. Paired comparisons of the observations identified an agreement in 63% of cases and a mean weighted kappa coefficient of 0.64 confirming interobserver reliability. The stage of the involved lunate was reproduced in 78% of the observations with a mean weighted kappa coefficient of 0.81 showing intraobserver reproducibility. This classification for Kienböck's disease has good reliability and reproducibility.

  6. Co-culture with periodontal ligament stem cells enhances osteogenic gene expression in de-differentiated fat cells.

    PubMed

    Tansriratanawong, Kallapat; Tamaki, Yuichi; Ishikawa, Hiroshi; Sato, Soh

    2014-10-01

    In recent decades, de-differentiated fat cells (DFAT cells) have emerged in regenerative medicine because of their trans-differentiation capability and the fact that their characteristics are similar to bone marrow mesenchymal stem cells. Even so, there is no evidence to support the osteogenic induction using DFAT cells in periodontal regeneration and also the co-culture system. Consequently, this study sought to evaluate the DFAT cells co-culture with periodontal ligament stem cells (PDLSCs) in vitro in terms of gene expression by comparing runt-related transcription factor 2 (RUNX2) and Peroxisome proliferator-activated receptor gamma 2 (PPARγ2) genes. We isolated DFAT cells from mature adipocytes and compared proliferation with PDLSCs. After co-culture with PDLSCs, we analyzed transcriptional activity implying by DNA methylation in all adipogenic gene promoters using combined bisulfite restriction analysis. We compared gene expression in RUNX2 gene with the PPARγ2 gene using quantitative RT-PCR. After being sub-cultured, DFAT cells demonstrated morphology similar to fibroblast-like cells. At the same time, PDLSCs established all stem cell characteristics. Interestingly, the co-culture system attenuated proliferation while enhancing osteogenic gene expression in RUNX2 gene. Using the co-culture system, DFAT cells could trans-differentiate into osteogenic lineage enhancing, but conversely, their adipogenic characteristic diminished. Therefore, DFAT cells and the co-culture system might be a novel cell-based therapy for promoting osteogenic differentiation in periodontal regeneration.

  7. Surface engineering of lentiviral vectors for gene transfer into gene therapy target cells.

    PubMed

    Lévy, Camille; Verhoeyen, Els; Cosset, François-Loïc

    2015-10-01

    Since they allow gene integration into their host genome, lentiviral vectors (LVs) have strong therapeutic potentials, as emphasized by recent clinical trials. The surface-display of the pantropic vesicular stomatitis virus G glycoprotein (VSV-G) on LVs resulted in powerful tools for fundamental and clinical research. However, improved LVs are required either to genetically modify cell types not permissive to classical VSV-G-LVs or to restrict entry to specific cell types. Incorporation of heterologous viral glycoproteins (gps) on LVs often require modification of their cytoplasmic tails and ligands can be inserted into their ectodomain to target LVs to specific receptors. Recently, measles virus (MV) gps have been identified as strong candidates for LV-retargeting to multiple cell types, with the potential to evolve toward clinical applications.

  8. Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells.

    PubMed

    Fan, Lianchun; Kadura, Ibrahim; Krebs, Lara E; Hatfield, Christopher C; Shaw, Margaret M; Frye, Christopher C

    2012-04-01

    Although Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins, one of the major challenges in CHO cell line generation (CLG) is how to efficiently identify those rare, high-producing clones among a large population of low- and non-productive clones. It is not unusual that several hundred individual clones need to be screened for the identification of a commercial clonal cell line with acceptable productivity and growth profile making the cell line appropriate for commercial application. This inefficiency makes the process of CLG both time consuming and laborious. Currently, there are two main CHO expression systems, dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection and glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection, that have been in wide industrial use. Since selection of recombinant cell lines in the GS-CHO system is based on the balance between the expression of the GS gene introduced by the expression plasmid and the addition of the GS inhibitor, L-MSX, the expression of GS from the endogenous GS gene in parental CHOK1SV cells will likely interfere with the selection process. To study endogenous GS expression's potential impact on selection efficiency, GS-knockout CHOK1SV cell lines were generated using the zinc finger nuclease (ZFN) technology designed to specifically target the endogenous CHO GS gene. The high efficiency (∼2%) of bi-allelic modification on the CHO GS gene supports the unique advantages of the ZFN technology, especially in CHO cells. GS enzyme function disruption was confirmed by the observation of glutamine-dependent growth of all GS-knockout cell lines. Full evaluation of the GS-knockout cell lines in a standard industrial cell culture process was performed. Bulk culture productivity improved two- to three-fold through the use of GS-knockout cells as parent cells. The selection stringency was

  9. Three T cell hybridomas do not contain detectable heavy chain variable gene transcripts

    PubMed Central

    1983-01-01

    We attempted to determine whether T cells express any VH gene segments. cDNA libraries were constructed from one suppressor and two helper T cell hybridomas. Both the library construction and screening were designed to maximize detection of a wide range of VH gene segments. One screening method should detect about half of the sequenced VH genes, while the second should detect most of these genes. The probability of detecting a VH gene homologous to the probes and present at 10 copies per cell was 77% for one helper cell cDNA library, 88% for the second helper cell library, and greater than 99% for the suppressor cell library. No cDNA clones with VH gene segments were detected. From this result, we conclude that VH gene segments are not likely to encode the antigen-specific receptor in the cells we tested. PMID:6190978

  10. Adenovirus-mediated gene transfer to tumor cells.

    PubMed

    Cascalló, Manel; Alemany, Ramon

    2004-01-01

    Cell transduction in vitro is only the first step toward proving that a genetherapy vector can be useful to treat tumors. However, tumor targeting in vivo is now the milestone for gene therapy to succeed against disseminated cancer. Therefore, most valuable information is obtained from studies of vector biodistribution. Owing to the hepatotropism of adenoviral vectors, a particularly important parameter is the tumor/liver ratio. This ratio can be given at the level of gene expression if the amount of transgene expression is measured. To optimize the targeting, however, the levels of viral particles that reach the tumor compared to other organs must be studied. Most of this chapter deals with methods to quantify the virus fate in tumor-bearing animals. We present a radioactive labeling method that can be used to study biodistribution. After a small section dealing with tumor models, we describe methods to quantify different parameters related to adenovirus-mediated tumor targeting.

  11. A systematic study on drug-response associated genes using baseline gene expressions of the Cancer Cell Line Encyclopedia.

    PubMed

    Liu, Xiaoming; Yang, Jiasheng; Zhang, Yi; Fang, Yun; Wang, Fayou; Wang, Jun; Zheng, Xiaoqi; Yang, Jialiang

    2016-03-10

    We have studied drug-response associated (DRA) gene expressions by applying a systems biology framework to the Cancer Cell Line Encyclopedia data. More than 4,000 genes are inferred to be DRA for at least one drug, while the number of DRA genes for each drug varies dramatically from almost 0 to 1,226. Functional enrichment analysis shows that the DRA genes are significantly enriched in genes associated with cell cycle and plasma membrane. Moreover, there might be two patterns of DRA genes between genders. There are significantly shared DRA genes between male and female for most drugs, while very little DRA genes tend to be shared between the two genders for a few drugs targeting sex-specific cancers (e.g., PD-0332991 for breast cancer and ovarian cancer). Our analyses also show substantial difference for DRA genes between young and old samples, suggesting the necessity of considering the age effects for personalized medicine in cancers. Lastly, differential module and key driver analyses confirm cell cycle related modules as top differential ones for drug sensitivity. The analyses also reveal the role of TSPO, TP53, and many other immune or cell cycle related genes as important key drivers for DRA network modules. These key drivers provide new drug targets to improve the sensitivity of cancer therapy.

  12. A systematic study on drug-response associated genes using baseline gene expressions of the Cancer Cell Line Encyclopedia

    NASA Astrophysics Data System (ADS)

    Liu, Xiaoming; Yang, Jiasheng; Zhang, Yi; Fang, Yun; Wang, Fayou; Wang, Jun; Zheng, Xiaoqi; Yang, Jialiang

    2016-03-01

    We have studied drug-response associated (DRA) gene expressions by applying a systems biology framework to the Cancer Cell Line Encyclopedia data. More than 4,000 genes are inferred to be DRA for at least one drug, while the number of DRA genes for each drug varies dramatically from almost 0 to 1,226. Functional enrichment analysis shows that the DRA genes are significantly enriched in genes associated with cell cycle and plasma membrane. Moreover, there might be two patterns of DRA genes between genders. There are significantly shared DRA genes between male and female for most drugs, while very little DRA genes tend to be shared between the two genders for a few drugs targeting sex-specific cancers (e.g., PD-0332991 for breast cancer and ovarian cancer). Our analyses also show substantial difference for DRA genes between young and old samples, suggesting the necessity of considering the age effects for personalized medicine in cancers. Lastly, differential module and key driver analyses confirm cell cycle related modules as top differential ones for drug sensitivity. The analyses also reveal the role of TSPO, TP53, and many other immune or cell cycle related genes as important key drivers for DRA network modules. These key drivers provide new drug targets to improve the sensitivity of cancer therapy.

  13. Evaluation of 16S rRNA Gene Primer Pairs for Monitoring Microbial Community Structures Showed High Reproducibility within and Low Comparability between Datasets Generated with Multiple Archaeal and Bacterial Primer Pairs

    PubMed Central

    Fischer, Martin A.; Güllert, Simon; Neulinger, Sven C.; Streit, Wolfgang R.; Schmitz, Ruth A.

    2016-01-01

    The application of next-generation sequencing technology in microbial community analysis increased our knowledge and understanding of the complexity and diversity of a variety of ecosystems. In contrast to Bacteria, the archaeal domain was often not particularly addressed in the analysis of microbial communities. Consequently, established primers specifically amplifying the archaeal 16S ribosomal gene region are scarce compared to the variety of primers targeting bacterial sequences. In this study, we aimed to validate archaeal primers suitable for high throughput next generation sequencing. Three archaeal 16S primer pairs as well as two bacterial and one general microbial 16S primer pairs were comprehensively tested by in-silico evaluation and performing an experimental analysis of a complex microbial community of a biogas reactor. The results obtained clearly demonstrate that comparability of community profiles established using different primer pairs is difficult. 16S rRNA gene data derived from a shotgun metagenome of the same reactor sample added an additional perspective on the community structure. Furthermore, in-silico evaluation of primers, especially those for amplification of archaeal 16S rRNA gene regions, does not necessarily reflect the results obtained in experimental approaches. In the latter, archaeal primer pair ArchV34 showed the highest similarity to the archaeal community structure compared to observed by the metagenomic approach and thus appears to be the appropriate for analyzing archaeal communities in biogas reactors. However, a disadvantage of this primer pair was its low specificity for the archaeal domain in the experimental application leading to high amounts of bacterial sequences within the dataset. Overall our results indicate a rather limited comparability between community structures investigated and determined using different primer pairs as well as between metagenome and 16S rRNA gene amplicon based community structure analysis

  14. Quantitative analysis of cell-type specific gene expression in the green alga Volvox carteri

    PubMed Central

    Nematollahi, Ghazaleh; Kianianmomeni, Arash; Hallmann, Armin

    2006-01-01

    Background The multicellular alga Volvox carteri possesses only two cell types: mortal, motile somatic cells and potentially immortal, immotile reproductive cells. It is therefore an attractive model system for studying how cell-autonomous cytodifferentiation is programmed within a genome. Moreover, there are ongoing genome projects both in Volvox carteri and in the closely related unicellular alga Chlamydomonas reinhardtii. However, gene sequencing is only the beginning. To identify cell-type specific expression and to determine relative expression rates, we evaluate the potential of real-time RT-PCR for quantifying gene transcript levels. Results Here we analyze a diversified pool of 39 target genes by real-time RT-PCR for each cell type. This gene pool contains previously known genes with unknown localization of cellular expression, 28 novel genes which are described in this study for the first time, and a few known, cell-type specific genes as a control. The respective gene products are, for instance, part of photosynthesis, cellular regulation, stress response, or transport processes. We provide expression data for all these genes. Conclusion The results show that quantitative real-time RT-PCR is a favorable approach to analyze cell-type specific gene expression in Volvox, which can be extended to a much larger number of genes or to developmental or metabolic mutants. Our expression data also provide a basis for a detailed analysis of individual, previously unknown, cell-type specifically expressed genes. PMID:17184518

  15. TMPRSS2-ERG gene fusion in small cell carcinoma of the prostate.

    PubMed

    Guo, Charles C; Dancer, Jane Y; Wang, Yan; Aparicio, Ana; Navone, Nora M; Troncoso, Patricia; Czerniak, Bogdan A

    2011-01-01

    Recent studies have shown that most prostate cancers carry the TMPRSS2-ERG gene fusion. Here we evaluated the TMPRSS2-ERG gene fusion in small cell carcinoma of the prostate (n = 12) in comparison with small cell carcinoma of the urinary bladder (n = 12) and lung (n = 11). Fluorescence in situ hybridization demonstrated rearrangement of the ERG gene in 8 cases of prostatic small cell carcinoma (67%), and the rearrangement was associated with deletion of the 5' ERG gene in 7 cases, but rearrangement of the ERG gene was not present in any small cell carcinoma of the urinary bladder or lung. Next we evaluated the TMPRSS2-ERG gene fusion in nude mouse xenografts that were derived from 2 prostatic small cell carcinomas carrying the TMPRSS2-ERG gene fusion. Two transcripts encoded by the TMPRSS2-ERG gene fusion were detected by reverse transcriptase polymerase chain reaction, and DNA sequencing demonstrated that the 2 transcripts were composed of fusions of exon 1 of the TMPRSS2 gene to exon 4 or 5 of the ERG gene. Our study demonstrates the specific presence of TMPRSS2-ERG gene fusion in prostatic small cell carcinoma, which may be helpful in distinguishing small cell carcinoma of prostatic origin from nonprostatic origins. The high prevalence of the TMPRSS2-ERG gene fusion in prostatic small cell carcinoma as well as adenocarcinoma implies that small cell carcinoma may share a common pathogenic pathway with adenocarcinoma in the prostate.

  16. Optimizing A Lipocomplex-Based Gene Transfer Method into HeLa Cell Line

    PubMed Central

    Asgharian, Alimohammad; Banan, Mehdi; Najmabadi, Hossein

    2014-01-01

    One of the most significant steps in gene expression studies is transferring genes into cell cultures. Despite there are different methods for gene delivery such as viral and non-viral producers, some cationic lipid reagents have recently developed to transfect into mam- malian cell lines. The main aim of this study was optimizing and improving lipocomplex based transient transfection procedures into HeLa cell line which is being used widely as a typical cell in biological studies. This study was an experimental research. In this work, pCMV β-Gal DNA plasmid was used as a reporter DNA for determining the rate of gene transfection into HeLa cells. To accomplish the highest gene delivery into HeLa cells, optimizing experiments were carried out in different volumes of FuGENE-HD, LipofectamineTM2000 and X-tremeGENE. Also, we investigated tranasfection efficiency in presence of various cell densities of HeLa cells. Then, transfection efficiency and cell toxicity were measured by beta gal staining and trypan blue methods, respectively. Using FuGENE-HD in volume of 4µl along with 105 HeLa cells, transfection efficiency was higher (43.66 ± 1.52%) in comparison with the cationic lipids LipofectamineTM2000 and X-tremeGENE. In addition, the rate of cell toxicity in presence of FuGENE-HD was less than 5%. In summary, the cationic lipid FuGENE-HD indicates a suitable potential to transfer DNA into HeLa cells and it can be an efficient reagent for gene delivery for HeLa cells in vitro. Moreover, it is worth designing and optimizing gene transfer experiments for other cell lines with FuGENE-HD due to its low toxicity and high efficiency. PMID:24381863

  17. Optimizing A Lipocomplex-Based Gene Transfer Method into HeLa Cell Line.

    PubMed

    Asgharian, Alimohammad; Banan, Mehdi; Najmabadi, Hossein

    2014-01-01

    One of the most significant steps in gene expression studies is transferring genes into cell cultures. Despite there are different methods for gene delivery such as viral and non-viral producers, some cationic lipid reagents have recently developed to transfect into mam- malian cell lines. The main aim of this study was optimizing and improving lipocomplex based transient transfection procedures into HeLa cell line which is being used widely as a typical cell in biological studies. This study was an experimental research. In this work, pCMV β-Gal DNA plasmid was used as a reporter DNA for determining the rate of gene transfection into HeLa cells. To accomplish the highest gene delivery into HeLa cells, optimizing experiments were car- ried out in different volumes of FuGENE-HD, Lipofectamine(TM)2000 and X-tremeGENE. Also, we investigated tranasfection efficiency in presence of various cell densities of HeLa cells. Then, transfection efficiency and cell toxicity were measured by beta gal staining and trypan blue methods, respectively. Using FuGENE-HD in volume of 4µl along with 10(5) HeLa cells, transfection efficiency was higher (43.66 ± 1.52%) in comparison with the cationic lipids Lipofectamine(TM)2000 and X-tremeGENE. In addition, the rate of cell toxicity in presence of FuGENE-HD was less than 5%. In summary, the cationic lipid FuGENE-HD indicates a suitable potential to transfer DNA into HeLa cells and it can be an efficient reagent for gene delivery for HeLa cells in vitro. Moreover, it is worth designing and optimizing gene transfer experiments for other cell lines with FuGENE-HD due to its low toxicity and high efficiency.

  18. Functional characterization of ecdysone receptor gene switches in mammalian cells.

    PubMed

    Panguluri, Siva K; Kumar, Prasanna; Palli, Subba R

    2006-12-01

    Regulated expression of transgene is essential in basic research as well as for many therapeutic applications. The main purpose of the present study is to understand the functioning of the ecdysone receptor (EcR)-based gene switch in mammalian cells and to develop improved versions of EcR gene switches. We utilized EcR mutants to develop new EcR gene switches that showed higher ligand sensitivity and higher magnitude of induction of reporter gene expression in the presence of ligand. We also developed monopartite versions of EcR gene switches with reduced size of the components that are accommodated into viral vectors. Ligand binding assays revealed that EcR alone could not bind to the nonsteroidal ligand, RH-2485. The EcR's heterodimeric partner, ultraspiracle, is required for efficient binding of EcR to the ligand. The essential role of retinoid X receptor (RXR) or its insect homolog, ultraspiracle, in EcR function is shown by RXR knockdown experiments using RNAi. Chromatin immunoprecipitation assays demonstrated that VP16 (activation domain, AD):GAL4(DNA binding domain, DBD):EcR(ligand binding domain, LBD) or GAL4(DBD):EcR(LBD) fusion proteins can bind to GAL4 response elements in the absence of ligand. The VP16(AD) fusion protein of a chimera between human and locust RXR could heterodimerize with GAL4(DBD):EcR(LBD) in the absence of ligand but the VP16(AD) fusion protein of Homo sapiens RXR requires ligand for its heterodimerization with GAL4(DBD):EcR(LBD).

  19. Transfer of nonselectable genes into mouse teratocarcinoma cells and transcription of the transferred human. beta. -globin gene

    SciTech Connect

    Wagner, E.F.; Mintz, B.

    1982-02-01

    Teratocarcinoma (TCC) stem cells can function as vehicles for the introduction of specific recombinant genes into mice. Because most genes do not code for a selectable marker, the authors investigated the transformation efficiency of vectors with a linked selectable gene. In one series, TCC cells first selected for thymidine kinase deficiency were treated with DNA from the plasmid vector PtkH..beta..1 containing the human genomic ..beta..-globin gene and the thymidine kinase gene of herpes simplex virus. A high transformation frequency was obtained after selection in hypoxanthine-aminopterin-thymidine medium. Hybridization tests revealed that the majority of transformants had intact copies of the human gene among three to six total copies per cell. These were associated with cellular DNA sequences as judged from the presence of additional new restriction fragments and from stability of the sequences in tumors produced by injecting the cells subcutaneously. Total polyadenylate-containing RNA from cell cultures of two out of four transformants examined showed hybridization to the human gene probe: one RNA species resembled mature human ..beta..-globin mRNA transcripts; the others were of larger size. In differentiating tumors, various tissues, including hematopoietic cells of TCC provenance could be found. In a second model set of experiments, wild-type TCC cells were used to test a dominant-selection scheme with pSV-gpt vectors. Numerous transformants were isolated, and their transfected DNA was apparently stably integrated. Thus, any gene of choice can be transferred into TCC stem cells even without mutagenesis of the cells, and selected cell clones can be characterized. Cells of interest may then be introduced into early embryos to produce new mouse strains with predetermined genetic changes.

  20. Gene expression profiling in multipotent DFAT cells derived from mature adipocytes

    SciTech Connect

    Ono, Hiromasa; Oki, Yoshinao; Bono, Hidemasa; Kano, Koichiro

    2011-04-15

    Highlights: {yields} Adipocyte dedifferentiation is evident in a significant decrease in typical genes. {yields} Cell proliferation is strongly related to adipocyte dedifferentiation. {yields} Dedifferentiated adipocytes express several lineage-specific genes. {yields} Comparative analyses using publicly available datasets boost the interpretation. -- Abstract: Cellular dedifferentiation signifies the withdrawal of cells from a specific differentiated state to a stem cell-like undifferentiated state. However, the mechanism of dedifferentiation remains obscure. Here we performed comparative transcriptome analyses during dedifferentiation in mature adipocytes (MAs) to identify the transcriptional signatures of multipotent dedifferentiated fat (DFAT) cells derived from MAs. Using microarray systems, we explored similarly expressed as well as significantly differentially expressed genes in MAs during dedifferentiation. This analysis revealed significant changes in gene expression during this process, including a significant reduction in expression of genes for lipid metabolism concomitantly with a significant increase in expression of genes for cell movement, cell migration, tissue developmental processes, cell growth, cell proliferation, cell morphogenesis, altered cell shape, and cell differentiation. Our observations indicate that the transcriptional signatures of DFAT cells derived from MAs are summarized in terms of a significant decrease in functional phenotype-related genes and a parallel increase in cell proliferation, altered cell morphology, and regulation of the differentiation of related genes. A better understanding of the mechanisms involved in dedifferentiation may enable scientists to control and possibly alter the plasticity of the differentiated state, which may lead to benefits not only in stem cell research but also in regenerative medicine.

  1. Transcriptional Regulation of Tlr11 Gene Expression in Epithelial Cells*

    PubMed Central

    Cai, Zhenyu; Shi, Zhongcheng; Sanchez, Amir; Zhang, Tingting; Liu, Mingyao; Yang, Jianghua; Wang, Fen; Zhang, Dekai

    2009-01-01

    As sensors of invading microorganisms, Toll-like receptors (TLRs) are expressed not only on macrophages and dendritic cells (DCs) but also on epithelial cells. In the TLR family, Tlr11 appears to have the unique feature in that it is expressed primarily on epithelial cells, although it is also expressed on DCs and macrophages. Here, we demonstrate that transcription of the Tlr11 gene is regulated through two cis-acting elements, one Ets-binding site and one interferon regulatory factor (IRF)-binding site. The Ets element interacts with the epithelium-specific transcription factors, ESE-1 and ESE-3, and the IRF motif interacts with IRF-8. Thus, Tlr11 expression on epithelial cells is regulated by the transcription factors that are presumably distinct from transcription factors that regulate the expression of TLRs in innate immune cells such as macrophages and DCs. Our results imply that the distinctive transcription regulatory machinery for TLRs on epithelium may represent a promising new avenue for the development of epithelia-specific therapeutic interventions. PMID:19801549

  2. Release testing of retroviral vectors and gene-modified cells.

    PubMed

    Nordling, Diana; Kaiser, Anne; Reeves, Lilith

    2009-01-01

    This chapter will review the design and execution of release testing requirements for retroviral vectors and gene-modified cells consistent with ensuring the success of the clinical trial on the basis of current US regulatory requirements. It is the ethical and legal responsibility of the clinical trial sponsor(s) to ensure safety of the patients through proper evaluation of the drug products prior to use. Any clinical trial drug product used in human subjects must be produced and evaluated for safety, quality, purity, and effectiveness according to Current Good Manufacturing Practices appropriate for the stage of clinical development.

  3. Reporter gene expression in dendritic cells after gene gun administration of plasmid DNA.

    PubMed

    Watkins, Craig; Hopkins, John; Harkiss, Gordon

    2005-07-21

    Dendritic cells (DC) play an integral role in plasmid DNA vaccination. However, the interaction between plasmid DNA and DC in vivo is incompletely understood. In this report, we utilise the sheep pseudoafferent cannulation model to examine the interaction between plasmid DNA encoding enhanced green fluorescent protein (pEGFP) and afferent lymph DC (ALDC) following gene gun administration. The results show that peaks of fluorescent ALDC tended to appear around days 1-4 and 9-13, then erratically thereafter for up to 2 months. Phenotypic analysis showed that EGFP+ ALDC expressed MHC class II, WC6, CD1b, and SIRPalpha markers. Plasmid, detected by PCR, was found in lymph cells and cell-free plasma on a daily basis, and was present variably for up to 2 months. Plasmid was also detected in purified CD1b+ ALDC, but the presence of plasmid did not correlate with EGFP expression by ALDC. Free EGFP in afferent lymph plasma was detectable by luminometry only after three administrations of the plasmid. The results show that gene gun administered pEGFP persisted for extended periods after a single administration, leeching out of skin on a daily basis. The plasmid was associated with both the cellular and fluid components of afferent lymph. EGFP protein appeared in afferent lymph in a pulsatile manner, but associated only with ALDC.

  4. Comprehensive Gene Expression Analysis of Human Embryonic Stem Cells during Differentiation into Neural Cells

    PubMed Central

    Fathi, Ali; Hatami, Maryam; Hajihosseini, Vahid; Fattahi, Faranak; Kiani, Sahar; Baharvand, Hossein; Salekdeh, Ghasem Hosseini

    2011-01-01

    Global gene expression analysis of human embryonic stem cells (hESCs) that differentiate into neural cells would help to further define the molecular mechanisms involved in neurogenesis in humans. We performed a comprehensive transcripteome analysis of hESC differentiation at three different stages: early neural differentiation, neural ectoderm, and differentiated neurons. We identified and validated time-dependent gene expression patterns and showed that the gene expression patterns reflect early ESC differentiation. Sets of genes are induced in primary ectodermal lineages and then in differentiated neurons, constituting consecutive waves of known and novel genes. Pathway analysis revealed dynamic expression patterns of members of several signaling pathways, including NOTCH, mTOR and Toll like receptors (TLR), during neural differentiation. An interaction network analysis revealed that the TGFβ family of genes, including LEFTY1, ID1 and ID2, are possible key players in the proliferation and maintenance of neural ectoderm. Collectively, these results enhance our understanding of the molecular dynamics underlying neural commitment and differentiation. PMID:21829537

  5. Use of Nascent RNA Microarrays to Study Inducible Gene Expression in Breast Cancer Cells

    DTIC Science & Technology

    2005-09-01

    detect inducible gene expression following activation of a transcription factor we used the p53 mutant lung cancer cell line H1299 /tsp53 expressing a...temperature-sensitive p53 gene and a control cell line H1299 /neo expressing a neo control vector. To activate the transcription factor p53 we lowered...expression in H1299 +tsp53 cells nascent RNA gene expression in H1299 +neo cells. Nascent RNA was collected 3 hours after switching to the permissive

  6. Explorations in statistics: statistical facets of reproducibility.

    PubMed

    Curran-Everett, Douglas

    2016-06-01

    Learning about statistics is a lot like learning about science: the learning is more meaningful if you can actively explore. This eleventh installment of Explorations in Statistics explores statistical facets of reproducibility. If we obtain an experimental result that is scientifically meaningful and statistically unusual, we would like to know that our result reflects a general biological phenomenon that another researcher could reproduce if (s)he repeated our experiment. But more often than not, we may learn this researcher cannot replicate our result. The National Institutes of Health and the Federation of American Societies for Experimental Biology have created training modules and outlined strategies to help improve the reproducibility of research. These particular approaches are necessary, but they are not sufficient. The principles of hypothesis testing and estimation are inherent to the notion of reproducibility in science. If we want to improve the reproducibility of our research, then we need to rethink how we apply fundamental concepts of statistics to our science.

  7. Selection of individual VH genes occurs at the pro-B to pre-B cell transition*

    PubMed Central

    Meng, Wenzhao; Yunk, Lenka; Wang, Li-San; Maganty, Avinash; Xue, Emily; Cohen, Philip L.; Eisenberg, Robert A.; Weigert, Martin G.; Mancini, Stephane J.C.; Luning Prak, Eline T.

    2011-01-01

    B cells are subjected to selection at multiple checkpoints during their development. The selection of antibody heavy chains is difficult to study because of the large diversity of the CDR3. In order to study the selection of individual antibody heavy chain variable region genes (VH), we performed CDR3 spectratyping of 75–300 rearrangements per individual VH in C57BL6/J mice. We measured the fraction of rearrangements that were in-frame (IF fraction) in B cell DNA. We demonstrate that individual VH genes have different IF fractions, ranging from 10% to 90%, and that these IF fractions are reproducible in different mice. For most VHs, the IF fraction in pro-B cells approximated 33% and then shifted to the nearly final (mature) B cell value by the cycling pre-B cell stage. The frequency of high IF VH usage increased in cycling pre-B cells compared to pro-B cells, whereas this did not occur for low IF VHs. The IF fraction did not shift as much in BCR-expressing B cells and was minimally affected by light chain usage for most VH. High IF clan II/III VHs share more positively charged CDR2 sequences, whereas high IF clan I J558 CDR2 sequences are diverse. These data indicate that individual VHs are subjected to differential selection, that VH IF fraction is mainly established through pre-BCR mediated selection, that it may operate differently in clan I vs. II/III VHs, and that it has a lasting influence on the antibody repertoire. PMID:21746964

  8. Myelomatous plasma cells display an aberrant gene expression pattern similar to that observed in normal memory B cells

    PubMed Central

    Báez, Alicia; Piruat, José I; Caballero-Velázquez, Teresa; Sánchez-Abarca, Luís I; Álvarez-Laderas, Isabel; Barbado, M Victoria; García-Guerrero, Estefanía; Millán-Uclés, África; Martín-Sánchez, Jesús; Medrano, Mayte; Pérez-Simón, José Antonio

    2015-01-01

    Memory B cells (MBCs) remain in a quiescent state for years, expressing pro-survival and anti-apoptotic factors while repressing cell proliferation and activation genes. During their differentiation into plasma cells (PCs), their expression pattern is reversed, with a higher expression of genes related to cell proliferation and activation, and a lower expression of pro-survival genes. To determine whether myelomatous PCs (mPCs) share characteristics with normal PCs and MBCs and to identify genes involved in the pathophysiology of multiple myeloma (MM), we compared gene expression patterns in these three cell sub-types. We observed that mPCs had features intermediate between those of MBCs and normal PCs, and identified 3455 genes differentially expressed in mPCs relative to normal PCs but with a similar expression pattern to that in MBCs. Most of these genes are involved in cell death and survival, cell growth and proliferation and protein synthesis. According to our findings, mPCs have a gene expression pattern closer to a MBC than a PC with a high expression of genes involved in cell survival. These genes should be physiologically inactivated in the transit from MBC to PC, but remain overexpressed in mPCs and thus may play a role in the pathophysiology of the disease. PMID:25628947

  9. Positions of pluripotency genes and hepatocyte-specific genes in the nucleus before and after mouse ES cell differentiation.

    PubMed

    Udagawa, K; Ohyama, T

    2014-03-24

    Spatial positioning of genes in the cell nucleus plays an important role in the regulation of genomic functions. Evidence for changes in gene positioning associated with transcriptional activity has been reported. However, our understanding of this phenomenon is still quite limited. We examined how pluripotency genes and hepatocyte-specific genes behave during the differentiation of mouse embryonic stem (ES) cells into hepatocytes, by targeting the loci of the Klf4, Nanog, Oct4, Sox2, Cyp7α1, Pck1, Tat, and Tdo2 genes, and using three-dimensional fluorescence in situ hybridization analyses. We found that each gene has a distinctly inherent localization profile in the ES cell nucleus. During differentiation, the Klf4, Nanog, Oct4, Cyp7α1, Pck1, and Tat loci shifted toward the nuclear center, while the Sox2 and Tdo2 loci shifted toward the periphery. The Klf4, Nanog, Oct4, and Tdo2 seem to prefer the outer regions, rather than the inner regions, when they are active. We also found that the radial positioning of the focused genes in the hepatocyte cell nucleus was highly correlated with the local GC content and the gene density of the surrounding region, but not with gene activity.

  10. The core planar cell polarity gene, Vangl2, directs adult corneal epithelial cell alignment and migration

    PubMed Central

    Findlay, Amy S.; Panzica, D. Alessio; Walczysko, Petr; Holt, Amy B.; Henderson, Deborah J.; West, John D.; Rajnicek, Ann M.

    2016-01-01

    This study shows that the core planar cell polarity (PCP) genes direct the aligned cell migration in the adult corneal epithelium, a stratified squamous epithelium on the outer surface of the vertebrate eye. Expression of multiple core PCP genes was demonstrated in the adult corneal epithelium. PCP components were manipulated genetically and pharmacologically in human and mouse corneal epithelial cells in vivo and in vitro. Knockdown of VANGL2 reduced the directional component of migration of human corneal epithelial (HCE) cells without affecting speed. It was shown that signalling through PCP mediators, dishevelled, dishevelled-associated activator of morphogenesis and Rho-associated protein kinase directs the alignment of HCE cells by affecting cytoskeletal reorganization. Cells in which VANGL2 was disrupted tended to misalign on grooved surfaces and migrate across, rather than parallel to the grooves. Adult corneal epithelial cells in which Vangl2 had been conditionally deleted showed a reduced rate of wound-healing migration. Conditional deletion of Vangl2 in the mouse corneal epithelium ablated the normal highly stereotyped patterns of centripetal cell migration in vivo from the periphery (limbus) to the centre of the cornea. Corneal opacity owing to chronic wounding is a major cause of degenerative blindness across the world, and this study shows that Vangl2 activity is required for directional corneal epithelial migration. PMID:27853583

  11. Stem Cell Gene Therapy for Fanconi Anemia: Report from the 1st International Fanconi Anemia Gene Therapy Working Group Meeting

    PubMed Central

    Tolar, Jakub; Adair, Jennifer E; Antoniou, Michael; Bartholomae, Cynthia C; Becker, Pamela S; Blazar, Bruce R; Bueren, Juan; Carroll, Thomas; Cavazzana-Calvo, Marina; Clapp, D Wade; Dalgleish, Robert; Galy, Anne; Gaspar, H Bobby; Hanenberg, Helmut; Von Kalle, Christof; Kiem, Hans-Peter; Lindeman, Dirk; Naldini, Luigi; Navarro, Susana; Renella, Raffaele; Rio, Paula; Sevilla, Julián; Schmidt, Manfred; Verhoeyen, Els; Wagner, John E; Williams, David A; Thrasher, Adrian J

    2011-01-01

    Survival rates after allogeneic hematopoietic cell transplantation (HCT) for Fanconi anemia (FA) have increased dramatically since 2000. However, the use of autologous stem cell gene therapy, whereby the patient's own blood stem cells are modified to express the wild-type gene product, could potentially avoid the early and late complications of allogeneic HCT. Over the last decades, gene therapy has experienced a high degree of optimism interrupted by periods of diminished expectation. Optimism stems from recent examples of successful gene correction in several congenital immunodeficiencies, whereas diminished expectations come from the realization that gene therapy will not be free of side effects. The goal of the 1st International Fanconi Anemia Gene Therapy Working Group Meeting was to determine the optimal strategy for moving stem cell gene therapy into clinical trials for individuals with FA. To this end, key investigators examined vector design, transduction method, criteria for large-scale clinical-grade vector manufacture, hematopoietic cell preparation, and eligibility criteria for FA patients most likely to benefit. The report summarizes the roadmap for the development of gene therapy for FA. PMID:21540837

  12. Single-cell RNA-seq reveals dynamic, random monoallelic gene expression in mammalian cells.

    PubMed

    Deng, Qiaolin; Ramsköld, Daniel; Reinius, Björn; Sandberg, Rickard

    2014-01-10

    Expression from both alleles is generally observed in analyses of diploid cell populations, but studies addressing allelic expression patterns genome-wide in single cells are lacking. Here, we present global analyses of allelic expression across individual cells of mouse preimplantation embryos of mixed background (CAST/EiJ × C57BL/6J). We discovered abundant (12 to 24%) monoallelic expression of autosomal genes and that expression of the two alleles occurs independently. The monoallelic expression appeared random and dynamic because there was considerable variation among closely related embryonic cells. Similar patterns of monoallelic expression were observed in mature cells. Our allelic expression analysis also demonstrates the de novo inactivation of the paternal X chromosome. We conclude that independent and stochastic allelic transcription generates abundant random monoallelic expression in the mammalian cell.

  13. Aristaless Related Homeobox Gene, Arx, Is Implicated in Mouse Fetal Leydig Cell Differentiation Possibly through Expressing in the Progenitor Cells

    PubMed Central

    Miyabayashi, Kanako; Katoh-Fukui, Yuko; Ogawa, Hidesato; Baba, Takashi; Shima, Yuichi; Sugiyama, Noriyuki; Kitamura, Kunio; Morohashi, Ken-ichirou

    2013-01-01

    Development of the testis begins with the expression of the SRY gene in pre-Sertoli cells. Soon after, testis cords containing Sertoli and germ cells are formed and fetal Leydig cells subsequently develop in the interstitial space. Studies using knockout mice have indicated that multiple genes encoding growth factors and transcription factors are implicated in fetal Leydig cell differentiation. Previously, we demonstrated that the Arx gene is implicated in this process. However, how ARX regulates Leydig cell differentiation remained unknown. In this study, we examined Arx KO testes and revealed that fetal Leydig cell numbers largely decrease throughout the fetal life. Since our study shows that fetal Leydig cells rarely proliferate, this decrease in the KO testes is thought to be due to defects of fetal Leydig progenitor cells. In sexually indifferent fetal gonads of wild type, ARX was expressed in the coelomic epithelial cells and cells underneath the epithelium as well as cells at the gonad-mesonephros border, both of which have been described to contain progenitors of fetal Leydig cells. After testis differentiation, ARX was expressed in a large population of the interstitial cells but not in fetal Leydig cells, raising the possibility that ARX-positive cells contain fetal Leydig progenitor cells. When examining marker gene expression, we observed cells as if they were differentiating into fetal Leydig cells from the progenitor cells. Based on these results, we propose that ARX acts as a positive factor for differentiation of fetal Leydig cells through functioning at the progenitor stage. PMID:23840809

  14. Notochordal Cells Influence Gene Expression of Inflammatory Mediators of Annulus Fibrosus Cells in Proinflammatory Cytokines Stimulation

    PubMed Central

    Moon, Hong Joo; Joe, Hoon; Kwon, Taek Hyun; Choi, Hye-Kyoung; Park, Youn Kwan

    2010-01-01

    Objective Notochordal cells in the intervertebral disc interact with nucleus pulposus (NP) cells and support the maintenance of disc homeostasis by regulation of matrix production. However, the influence of notochordal cells has not been evaluated in the annulus fibrosus (AF), which is the primary pain generator in the disc. We hypothesized that the notochordal cell has the capacity to modulate inflammatory mediators secreted by AF cells secondary to stimulation. Methods Notochordal and AF cells were isolated from adult New Zealand white rabbits. AF pellets were cultured with notochordal cell clusters or in notochordal cell-conditioned media (NCCM) for 24 or 48 hours with proinflammatory cytokines at varying concentrations. Gene expression in AF pellets were assayed for nitric oxide synthase (iNOS), cyclo-oxygenase (COX)-2, and interleukin (IL)-6 by real time reverse transcriptase polymerase chain reaction (RT-PCR). Results AF pellet in NCCM significantly decreased the iNOS and COX-2 messenger ribonucleic acid (mRNA) levels compared to AF pellets alone and AF pellets with notochordal cells (p < 0.05). AF pellet resulted in dose-dependent iNOS and COX-2 expression in response to IL-1β, stimulation, demonstrating that 1 ng/ml for 24 hours yielded a maximal response. AF pellet in NCCM significantly decreased the expression of iNOS and COX-2 in response to 1ng/ml IL-1β, stimulation at 24 hours (p < 0.05). There was no difference in IL-6 expression compared to AF pellets alone or AF pellets with notochordal cell clusters. Conclusion We conclude that soluble factors from notochordal cells mitigate the gene expression of inflammatory mediators in stimulated AF, as expected after annular injury, suggesting that notochordal cells could serve as a novel therapeutic approach in symptomatic disc development. PMID:20717505

  15. Histone gene expression and chromatin structure in mammalian cell hybrids

    PubMed Central

    1980-01-01

    DNA isolated from mammalian cell nuclear reveals discrete size patterns when partially digested with micrococcal nuclease. The DNA repeat lengths from different tissues within a species or from different species may vary. These differences have been attributed to the presence of different species of histone H1. To examine the nature of regulation of DNA repeat lengths and their possible relationship to histone H1, we have selected several mouse and human cell lines that differ in their DNA repeat lengths and examined them and their cell hybrids. 24 mouse X human and five mouse X mouse hybrid cell lines were analyzed. All the interspecific hybrids exhibited the repeat pattern characteristic of the murine parent. The mouse intraspecific hybrids had a repeat pattern of only one of the parents. We conclude that the partial human chromosome complements retained in the hybrids assume the repeat lengths exhibited by the mouse cells. Because H1 histones have been implicated in the determination of DNA repeat lengths, we also investigated the regulation of H1 histone expression in these cell hybrids. Purified H1 histones were radioactively labeled in vitro, and individual subfractions were subjected to proteolysis followed by gel electrophoresis. The resulting partial peptide maps off H1 histone subfractions A and B were distinguishable from one another and from different cell lines. In the mouse X human hybrids analyzed, only the mouse H1 histones were detected. These observations were extended to H2b by analysis of the hybrid cell histone by Triton-acid-urea gels. Neither the DNA repeat length nor histone expression is affected by the presence of any specific human chromosome. The fact that human genes are expressed in these hybrids suggests that the H1 histones of one species is able to interact with the chromatin of another species in a biologically funtional conformation. Analysis of the intraspecific PG19 X B82 (mouse X mouse) hybrids reveals the presence of H1

  16. Expression of Endogenous Retroviral Genes in Leukemic Guinea Pig Cells

    PubMed Central

    Davis, A. R.; Nayak, D. P.

    1977-01-01

    The expression of guinea pig retrovirus (5-bromodeoxyuridine[BUdR]-induced GPV) was studied in guinea pig L2C leukemic lymphoblasts by use of molecular hybridization of viral complementary DNA (cDNA) to cellular RNA. It was found that L2C leukemic lymphoblasts, leukemic spleen, and BUdR-induced virus-producing cells contain virus-specific RNA: 0.05% (800 to 960 copies per cell), 0.02% (360 copies per cell), and 0.3% (5,120 copies per cell), respectively. Adult normal liver and spleen, on the other hand, contain less than 0.2 copy of viral RNA per cell. Both BUdR-induced cells and L2C leukemic lymphoblasts contained 14S, 22S, 35S, and 70S RNA species of total and cytoplasmic virus-specific RNA as determined by sucrose velocity gradient analysis and hybridization of sucrose gradient fractions to cDNA. Virus-specific mRNA was identified in both BUdR-induced cells and L2C leukemic lymphoblasts by the criterion that it cosedimented with purified polyribosomes in a sucrose gradient and that it changed to a lower sedimentation value if polyribosomes were disaggregated with EDTA prior to centrifugation. Virus-specific mRNA obtained from either the polyribosome region of purified polyribosomes or the released messenger region of EDTA-disaggregated purified polyribosomes consisted of 14S, 20S, and 35S species in both BUdR-induced cells and L2C leukemic lymphoblasts. Hybridization of cDNA to the RNA of L2C leukemic lymphoblasts and BUdR-induced cells was essentially complete. Additionally, leukemic lymphoblast RNA could displace 95% of the hybridization of BUdR-induced GPV 70S RNA to guinea pig DNA. The midpoints of thermal denaturation of hybrids formed between GPV cDNA and the RNA of either L2C leukemic lymphoblasts or the 70S RNA of BUdR-induced GPV were both 89°C in 2× concentrated 0.15 M NaCl plus 0.015 M sodium citrate. These results show that BUdR-induced GPV genes are essentially completely expressed in L2C leukemic lymphoblasts and that virus-specific mRNA is

  17. Aging: a portrait from gene expression profile in blood cells

    PubMed Central

    Calabria, Elisa; Mazza, Emilia Maria Cristina; Dyar, Kenneth Allen; Pogliaghi, Silvia; Bruseghini, Paolo; Morandi, Carlo; Salvagno, Gian Luca; Gelati, Matteo; Guidi, Gian Cesare; Bicciato, Silvio; Schiaffino, Stefano; Schena, Federico; Capelli, Carlo

    2016-01-01

    The availability of reliable biomarkers of aging is important not only to monitor the effect of interventions and predict the timing of pathologies associated with aging but also to understand the mechanisms and devise appropriate countermeasures. Blood cells provide an easily available tissue and gene expression profiles from whole blood samples appear to mirror disease states and some aspects of the aging process itself. We report here a microarray analysis of whole blood samples from two cohorts of healthy adult and elderly subjects, aged 43±3 and 68±4 years, respectively, to monitor gene expression changes in the initial phase of the senescence process. A number of significant changes were found in the elderly compared to the adult group, including decreased levels of transcripts coding for components of the mitochondrial respiratory chain, which correlate with a parallel decline in the maximum rate of oxygen consumption (VO2max), as monitored in the same subjects. In addition, blood cells show age-related changes in the expression of several markers of immunosenescence, inflammation and oxidative stress. These findings support the notion that the immune system has a major role in tissue homeostasis and repair, which appears to be impaired since early stages of the aging process. PMID:27545843

  18. A novel gene delivery system for mammalian cells.

    PubMed

    Gibson, Brian; Duffy, Angela M; Gould Fogerite, Susan; Krause-Elsmore, Sara; Lu, Ruying; Shang, Gaofeng; Chen, Zi-Wei; Mannino, Raphael J; Bouchier-Hayes, David J; Harmey, Judith H

    2004-01-01

    Although gene therapy holds great promise for the treatment of both acquired and genetic diseases, its development has been limited by practical considerations. Non-viral efficacy of delivery remains quite poor. We are investigating the feasibility of a novel lipid-based delivery system, cochleates, to deliver transgenes to mammalian cells. Rhodamine-labelled empty cochleates were incubated with two cell-lines (4T1 adenocarcinoma and H36.12 macrophage hybridoma) and primary macrophages in vitro and in vivo. Cochleates containing green fluorescent protein (GFP) expression plasmid were incubated with 4T1 adenocarcinoma cells. Cellular uptake of labelled cochleates or transgene GFP expression were visualised with fluorescence microscopy. 4T1 and H36.12 lines showed 39% and 23.1% uptake of rhodamine-cochleates, respectively. Human monocyte-derived macrophages and mouse peritoneal macrophages had 48+/-5.38% and 51.46+/-15.6% uptake of rhodamine-cochleates in vitro. In vivo 25.69+/-0.127% of peritoneal macrophages were rhodamine-positive after intra-peritoneal injection of rhodamine-cochleates. 19.49+/-10.12% of 4T1 cells expressed GFP. Cochleates may therefore be an effective, non-toxic and non-immunogenic method to introduce transgenes in vitro and in vivo.

  19. Gene regulatory networks governing haematopoietic stem cell development and identity.

    PubMed

    Pimanda, John E; Göttgens, Berthold

    2010-01-01

    Development can be viewed as a dynamic progression through regulatory states which characterise the various cell types within a given differentiation cascade. To understand the progression of regulatory states that define the origin and subsequent development of haematopoietic stem cells, the first imperative is to understand the ontogeny of haematopoiesis. We are fortunate that the ontogeny of blood development is one of the best characterized mammalian developmental systems. However, the field is still in its infancy with regard to the reconstruction of gene regulatory networks and their interactions with cell signalling cascades that drive a mesodermal progenitor to adopt the identity of a haematopoietic stem cell and beyond. Nevertheless, a framework to dissect these networks and comprehend the logic of its circuitry does exist and although they may not as yet be available, a sense for the tools that will be required to achieve this aim is also emerging. In this review we cover the fundamentals of network architecture, methods used to reconstruct networks, current knowledge of haematopoietic and related transcriptional networks, current challenges and future outlook.

  20. Bach2 represses plasma cell gene regulatory network in B cells to promote antibody class switch.

    PubMed

    Muto, Akihiko; Ochiai, Kyoko; Kimura, Yoshitaka; Itoh-Nakadai, Ari; Calame, Kathryn L; Ikebe, Dai; Tashiro, Satoshi; Igarashi, Kazuhiko

    2010-12-01

    Two transcription factors, Pax5 and Blimp-1, form a gene regulatory network (GRN) with a double-negative loop, which defines either B-cell (Pax5 high) or plasma cell (Blimp-1 high) status as a binary switch. However, it is unclear how this B-cell GRN registers class switch DNA recombination (CSR), an event that takes place before the terminal differentiation to plasma cells. In the absence of Bach2 encoding a transcription factor required for CSR, mouse splenic B cells more frequently and rapidly expressed Blimp-1 and differentiated to IgM plasma cells as compared with wild-type cells. Genetic loss of Blimp-1 in Bach2(-/-) B cells was sufficient to restore CSR. These data with mathematical modelling of the GRN indicate that Bach2 achieves a time delay in Blimp-1 induction, which inhibits plasma cell differentiation and promotes CSR (Delay-Driven Diversity model for CSR). Reduction in mature B-cell numbers in Bach2(-/-) mice was not rescued by Blimp-1 ablation, indicating that Bach2 regulates B-cell differentiation and function through Blimp-1-dependent and -independent GRNs.

  1. CD133-targeted gene transfer into long-term repopulating hematopoietic stem cells.

    PubMed

    Brendel, Christian; Goebel, Benjamin; Daniela, Abriss; Brugman, Martijn; Kneissl, Sabrina; Schwäble, Joachim; Kaufmann, Kerstin B; Müller-Kuller, Uta; Kunkel, Hana; Chen-Wichmann, Linping; Abel, Tobias; Serve, Hubert; Bystrykh, Leonid; Buchholz, Christian J; Grez, Manuel

    2015-01-01

    Gene therapy for hematological disorders relies on the genetic modification of CD34(+) cells, a heterogeneous cell population containing about 0.01% long-term repopulating cells. Here, we show that the lentiviral vector CD133-LV, which uses a surface marker on human primitive hematopoietic stem cells (HSCs) as entry receptor, transfers genes preferentially into cells with high engraftment capability. Transduction of unstimulated CD34(+) cells with CD133-LV resulted in gene marking of cells with competitive proliferative advantage in vitro and in immunodeficient mice. The CD133-LV-transduced population contained significantly more cells with repopulating capacity than cells transduced with vesicular stomatitis virus (VSV)-LV, a lentiviral vector pseudotyped with the vesicular stomatitis virus G protein. Upon transfer of a barcode library, CD133-LV-transduced cells sustained gene marking in vivo for a prolonged period of time with a 6.7-fold higher recovery of barcodes compared to transduced control cells. Moreover, CD133-LV-transduced cells were capable of repopulating secondary recipients. Lastly, we show that this targeting strategy can be used for transfer of a therapeutic gene into CD34(+) cells obtained from patients suffering of X-linked chronic granulomatous disease. In conclusion, direct gene transfer into CD133(+) cells allows for sustained long-term engraftment of gene corrected cells.

  2. Combination treatment with 6-mercaptopurine and allopurinol in HepG2 and HEK293 cells – Effects on gene expression levels and thiopurine metabolism

    PubMed Central

    Haglund, Sofie; Vikingsson, Svante; Almer, Sven; Söderman, Jan

    2017-01-01

    Combination treatment with low-dose thiopurine and allopurinol (AP) has successfully been used in patients with inflammatory bowel disease with a so called skewed thiopurine metabolite profile. In red blood cells in vivo, it reduces the concentration of methylated metabolites and increases the concentration of the phosphorylated ones, which is associated with improved therapeutic efficacy. This study aimed to investigate the largely unknown mechanism of AP on thiopurine metabolism in cells with an active thiopurine metabolic pathway using HepG2 and HEK293 cells. Cells were treated with 6-mercaptopurine (6MP) and AP or its metabolite oxypurinol. The expression of genes known to be associated with thiopurine metabolism, and the concentration of thiopurine metabolites were analyzed. Gene expression levels were only affected by AP in the presence of 6MP. The addition of AP to 6MP affected the expression of in total 19 genes in the two cell lines. In both cell lines the expression of the transporter SLC29A2 was reduced by the combined treatment. Six regulated genes in HepG2 cells and 8 regulated genes in HEK293 cells were connected to networks with 18 and 35 genes, respectively, present at known susceptibility loci for inflammatory bowel disease, when analyzed using a protein-protein interaction database. The genes identified as regulated as well as the disease associated interacting genes represent new candidates for further investigation in the context of combination therapy with thiopurines and AP. However, no differences in absolute metabolite concentrations were observed between 6MP+AP or 6MP+oxypurinol vs. 6MP alone in either of the two cell lines. In conclusion; the effect of AP on gene expression levels requires the presence of 6MP, at least in vitro. Previously described AP-effects on metabolite concentrations observed in red blood cells in vivo could not be reproduced in our cell lines in vitro. AP’s effects in relation to thiopurine metabolism are complex

  3. An Efficient Antipodal Cell Isolation Method for Screening of Cell Type-Specific Genes in Arabidopsis thaliana

    PubMed Central

    Sun, Meng-xiang

    2016-01-01

    In flowering plants, the mature embryo sac consists of seven cells, namely two synergid cells and an egg cell at the micropylar end, one central cell, and three antipodal cells at the chalazal end. Excluding the antipodal cell, as a model for the study of cell fate determination and cell type specification, the roles of these embryo sac component cells in fertilization and seed formation have been widely investigated. At this time, little is known regarding the function of antipodal cells and their cell type-specific gene expression patterns. One reason for this is difficulties related to the observation and isolation of cells for detailed functional analyses. Here, we report a method for antipodal cell isolation and transcriptome analysis. We identified antipodal cell-specific marker line K44-1, and based on this marker line, established a procedure allowing us to isolate antipodal cells with both high quality and quantity. PCR validation of antipodal-specific genes from antipodal cell cDNA showed that the isolated cells are qualified and can be used for transcriptome analysis and screening of cell type-specific marker genes. The isolated cells could keep viable for a week in culture condition. This method can be used to efficiently isolate antipodal cells of high quality and will promote the functional investigation of antipodal cells in Arabidopsis thaliana. This increases our understanding of the molecular regulatory mechanism of antipodal cell specification. PMID:27875553

  4. Expression profiles of genes in DJ-1-knockdown and L 166 P DJ-1 mutant cells.

    PubMed

    Nishinaga, Hiromi; Takahashi-Niki, Kazuko; Taira, Takahiro; Andreadis, Athena; Iguchi-Ariga, Sanae M M; Ariga, Hiroyoshi

    2005-12-16

    DJ-1 is a novel oncogene and a causative gene for the familial form of Parkinson's disease (PD). DJ-1 has been shown to play roles in anti-oxidative stress by eliminating reactive oxygen species and in transcriptional regulation of genes. Loss of these functions of DJ-1 is thought to trigger the onset of PD. In this study, to identify genes for which expressions are regulated by DJ-1, DNA microarray analyses were carried out using two mouse NIH3T3 cell lines, DJ-1-knockdown cells and cells harboring an exogenously added L 166 P DJ-1 mutant found in PD patients. In both cell lines, drastic changes in expressions of genes, including genes related to stress, apoptosis, oxidative stress and neurotoxicity, were observed and changes in expressions were confirmed by RT-PCR. Of the genes identified, expression level of the extracellular superoxide dismutase (SOD 3) gene was found to decrease in DJ-1-knockdown cells, while expressions of SOD 1 and SOD 2 genes did not change. Furthermore, expression of the tau gene, a gene whose product gives cells neurotoxicity by aggregation, was found to increase at its promoter level in L 166 P DJ-1 cells. These findings suggest that DJ-1 regulates expressions of genes for which functions are thought to be related to cell death or neurodegeneration.

  5. Cell types differ in global coordination of splicing and proportion of highly expressed genes

    PubMed Central

    Trakhtenberg, Ephraim F.; Pho, Nam; Holton, Kristina M.; Chittenden, Thomas W.; Goldberg, Jeffrey L.; Dong, Lingsheng

    2016-01-01

    Balance in the transcriptome is regulated by coordinated synthesis and degradation of RNA molecules. Here we investigated whether mammalian cell types intrinsically differ in global coordination of gene splicing and expression levels. We analyzed RNA-seq transcriptome profiles of 8 different purified mouse cell types. We found that different cell types vary in proportion of highly expressed genes and the number of alternatively spliced transcripts expressed per gene, and that the cell types that express more variants of alternatively spliced transcripts per gene are those that have higher proportion of highly expressed genes. Cell types segregated into two clusters based on high or low proportion of highly expressed genes. Biological functions involved in negative regulation of gene expression were enriched in the group of cell types with low proportion of highly expressed genes, and biological functions involved in regulation of transcription and RNA splicing were enriched in the group of cell types with high proportion of highly expressed genes. Our findings show that cell types differ in proportion of highly expressed genes and the number of alternatively spliced transcripts expressed per gene, which represent distinct properties of the transcriptome and may reflect intrinsic differences in global coordination of synthesis, splicing, and degradation of RNA molecules. PMID:27577089

  6. Multimodality imaging of reporter gene expression using a novel fusion vector in living cells and animals

    DOEpatents

    Gambhir; Sanjiv , Pritha; Ray

    2009-04-28

    Novel double and triple fusion reporter gene constructs harboring distinct imageable reporter genes are provided, as well as applications for the use of such double and triple fusion constructs in living cells and in living animals using distinct imaging technologies.

  7. Multimodality imaging of reporter gene expression using a novel fusion vector in living cells and animals

    DOEpatents

    Gambhir, Sanjiv; Pritha, Ray

    2011-06-07

    Novel double and triple fusion reporter gene constructs harboring distinct imagable reporter genes are provided, as well as applications for the use of such double and triple fusion constructs in living cells and in living animals using distinct imaging technologies.

  8. Multimodality imaging of reporter gene expression using a novel fusion vector in living cells and animals

    SciTech Connect

    Gambhir, Sanjiv; Pritha, Ray

    2015-07-14

    Novel double and triple fusion reporter gene constructs harboring distinct imagable reporter genes are provided, as well as applications for the use of such double and triple fusion constructs in living cells and in living animals using distinct imaging technologies.

  9. The Economics of Reproducibility in Preclinical Research.

    PubMed

    Freedman, Leonard P; Cockburn, Iain M; Simcoe, Timothy S

    2015-06-01

    Low reproducibility rates within life science research undermine cumulative knowledge production and contribute to both delays and costs of therapeutic drug development. An analysis of past studies indicates that the cumulative (total) prevalence of irreproducible preclinical research exceeds 50%, resulting in approximately US$28,000,000,000 (US$28B)/year spent on preclinical research that is not reproducible-in the United States alone. We outline a framework for solutions and a plan for long-term improvements in reproducibility rates that will help to accelerate the discovery of life-saving therapies and cures.

  10. Gene expression profiling identifies genes predictive of oral squamous cell carcinoma.

    PubMed

    Chen, Chu; Méndez, Eduardo; Houck, John; Fan, Wenhong; Lohavanichbutr, Pawadee; Doody, Dave; Yueh, Bevan; Futran, Neal D; Upton, Melissa; Farwell, D Gregory; Schwartz, Stephen M; Zhao, Lue Ping

    2008-08-01

    Oral squamous cell carcinoma (OSCC) is associated with substantial mortality and morbidity. To identify potential biomarkers for the early detection of invasive OSCC, we compared the gene expressions of incident primary OSCC, oral dysplasia, and clinically normal oral tissue from surgical patients without head and neck cancer or preneoplastic oral lesions (controls), using Affymetrix U133 2.0 Plus arrays. We identified 131 differentially expressed probe sets using a training set of 119 OSCC patients and 35 controls. Forward and stepwise logistic regression analyses identified 10 successive combinations of genes which expression differentiated OSCC from controls. The best model included LAMC2, encoding laminin-gamma2 chain, and COL4A1, encoding collagen, type IV alpha1 chain. Subsequent modeling without these two markers showed that COL1A1, encoding collagen, type I alpha1 chain, and PADI1, encoding peptidyl arginine deiminase, type 1, could also distinguish OSCC from controls. We validated these two models using an internal independent testing set of 48 invasive OSCC and 10 controls and an external testing set of 42 head and neck squamous cell carcinoma cases and 14 controls (GEO GSE6791), with sensitivity and specificity above 95%. These two models were also able to distinguish dysplasia (n = 17) from control (n = 35) tissue. Differential expression of these four genes was confirmed by quantitative reverse transcription-PCR. If confirmed in larger studies, the proposed models may hold promise for monitoring local recurrence at surgical margins and the development of second primary oral cancer in patients with OSCC.

  11. 75 FR 54351 - Cell and Gene Therapy Clinical Trials in Pediatric Populations; Public Workshop

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-07

    ... HUMAN SERVICES Food and Drug Administration Cell and Gene Therapy Clinical Trials in Pediatric... public workshop entitled ``Cell and Gene Therapy Clinical Trials in Pediatric Populations.'' The purpose... therapy clinical researchers, and other stakeholders regarding best practices related to cell and...

  12. Semi-automated closed system manufacturing of lentivirus gene-modified haematopoietic stem cells for gene therapy

    PubMed Central

    Adair, Jennifer E.; Waters, Timothy; Haworth, Kevin G.; Kubek, Sara P.; Trobridge, Grant D.; Hocum, Jonah D.; Heimfeld, Shelly; Kiem, Hans-Peter

    2016-01-01

    Haematopoietic stem cell (HSC) gene therapy has demonstrated potential to treat many diseases. However, current state of the art requires sophisticated ex vivo gene transfer in a dedicated Good Manufacturing Practices facility, limiting availability. An automated process would improve the availability and standardized manufacture of HSC gene therapy. Here, we develop a novel program for semi-automated cell isolation and culture equipment to permit complete benchtop generation of gene-modified CD34+ blood cell products for transplantation. These cell products meet current manufacturing quality standards for both mobilized leukapheresis and bone marrow, and reconstitute human haematopoiesis in immunocompromised mice. Importantly, nonhuman primate autologous gene-modified CD34+ cell products are capable of stable, polyclonal multilineage reconstitution with follow-up of more than 1 year. These data demonstrate proof of concept for point-of-care delivery of HSC gene therapy. Given the many target diseases for gene therapy, there is enormous potential for this approach to treat patients on a global scale. PMID:27762266

  13. The human desmin promoter drives robust gene expression for skeletal muscle stem cell-mediated gene therapy.

    PubMed

    Jonuschies, Jacqueline; Antoniou, Michael; Waddington, Simon; Boldrin, Luisa; Muntoni, Francesco; Thrasher, Adrian; Morgan, Jennifer

    2014-01-01

    Lentiviral vectors (LVs) represent suitable candidates to mediate gene therapy for muscular dystrophies as they infect dividing and non-dividing cells and integrate their genetic material into the host genome, thereby theoretically mediating longterm expression. We evaluated the ability of LVs where a GFP reporter gene was under the control of five different promoters, to transduce and mediate expression in myogenic and non-myogenic cells in vitro and in skeletal muscle fibres and stem (satellite) cells in vivo. We further analysed lentivirally-transduced satellite cell-derived myoblasts following their transplantation into dystrophic, immunodeficient mouse muscles. The spleen focus-forming virus promoter mediated the highest gene expression in all cell types; the CBX3-HNRPA2B1 ubiquitously-acting chromatin opening element (UCOE) promoter was also active in all cells, whereas the human desmin promoter in isolation or fused with UCOE had lower activity in non-muscle cells. Surprisingly, the human skeletal muscle actin promoter was also active in immune cells. The human desmin promoter mediated robust, persistent reporter gene expression in myogenic cells in vitro, and satellite cells and muscle fibres in vivo. The human desmin promoter combined with UCOE did not significantly increase transgene expression. Therefore, our data indicate that the desmin promoter is suitable for the development of therapeutic purposes.

  14. In Vitro Generation of Human NK cells Expressing Chimeric Antigen Receptor through Differentiation of Gene-Modified Hematopoietic Stem Cells

    PubMed Central

    Lowe, Emily; Truscott, Laurel C.; De Oliveira, Satiro N.

    2016-01-01

    Summary NK cells represent a very promising source for adoptive cellular approaches for cancer immunotherapy, and extensive research has been conducted, including clinical trials. Gene modification of NK cells can direct their specificity and enhance their function, but the efficiency of gene transfer techniques is very limited. Here we describe two protocols designed to generate mature human NK cells from gene-modified hematopoietic stem cells. These protocols use chimeric antigen receptor as the transgene, but could potentially be modified for the expression any particular transgene in human NK cells. PMID:27177671

  15. Pressure Stabilizer for Reproducible Picoinjection in Droplet Microfluidic Systems

    PubMed Central

    Rhee, Minsoung; Light, Yooli K.; Yilmaz, Suzan; Adams, Paul D.; Saxena, Deepak

    2014-01-01

    Picoinjection is a promising technique to add reagents into pre-formed emulsion droplets on chip; however, it is sensitive to pressure fluctuation, making stable operation of the picoinjector challenging. We present a chip architecture using a simple pressure stabilizer for consistent and highly reproducible picoinjection in multi-step biochemical assays with droplets. Incorporation of the stabilizer immediately upstream of a picoinjector or a combination of injectors greatly reduces pressure fluctuations enabling reproducible and effective picoinjection in systems where the pressure varies actively during operation. We demonstrate the effectiveness of the pressure stabilizer for an integrated platform for on-demand encapsulation of bacterial cells followed by picoinjection of reagents for lysing the encapsulated cells. The pressure stabilizer was also used for picoinjection of multiple displacement amplification (MDA) reagents to achieve genomic DNA amplification of lysed bacterial cells. PMID:25270338

  16. Pressure stabilizer for reproducible picoinjection in droplet microfluidic systems.

    PubMed

    Rhee, Minsoung; Light, Yooli K; Yilmaz, Suzan; Adams, Paul D; Saxena, Deepak; Meagher, Robert J; Singh, Anup K

    2014-12-07

    Picoinjection is a promising technique to add reagents into pre-formed emulsion droplets on chip however, it is sensitive to pressure fluctuation, making stable operation of the picoinjector challenging. We present a chip architecture using a simple pressure stabilizer for consistent and highly reproducible picoinjection in multi-step biochemical assays with droplets. Incorporation of the stabilizer immediately upstream of a picoinjector or a combination of injectors greatly reduces pressure fluctuations enabling reproducible and effective picoinjection in systems where the pressure varies actively during operation. We demonstrate the effectiveness of the pressure stabilizer for an integrated platform for on-demand encapsulation of bacterial cells followed by picoinjection of reagents for lysing the encapsulated cells. The pressure stabilizer was also used for picoinjection of multiple displacement amplification (MDA) reagents to achieve genomic DNA amplification of lysed bacterial cells.

  17. [Isolation and gene modification of amniotic fluid derived progenitor cells].

    PubMed

    Yang, Chenmin; Fan, Shuyue; Tang, Huixiang; Gong, Zhijuan; Gong, Xiuli; Ren, Zhaorui; Zeng, Fanyi

    2014-03-01

    We established methods to isolate human amniotic fluid-derived progenitor cells (hAFPCs), and analyze the ability of hAFPCs to secrete human coagulation factor IX (hFIX) after gene modification. The hAFPCs were manually isolated by selection for attachment to gelatin coated culture dish. hFIX cDNA was transfected into hAPFCs by using a lentiviral vector. The hFIX protein concentration and activity produced from hAFPCs were determined by enzyme-linked immunosorbent assay (ELISA) and clotting assay. The isolated spindle-shaped cells showed fibroblastoid morphology after three culture passages. The doubling time in culture was 39.05 hours. Immunocytochemistry staining of the fibroblast-like cells from amniotic fluid detected expression of stem cell markers such as SSEA4 and TRA1-60. Quantitative PCR analysis demonstrated the expression of NANOG, OCT4 and SOX2 mRNAs. Transfected hAFPCs could produce and secrete hFIX into the culture medium. The observed concentration of secreted hFIX was 20.37% +/- 2.77% two days after passage, with clotting activity of 16.42% +/- 1.78%. The amount of hFIX:Ag reached a plateau of 50.35% +/- 5.42%, with clotting activity 45.34% +/- 4.67%. In conclusion, this study established method to isolate and culture amniotic fluid progenitor cells. Transfected hAFPCs can produce hFIX at stable levels in vitro, and clotting activity increases with higher hFIX concentration. Genetically engineered hAFPC are a potential method for prenatal treatment of hemophilia B.

  18. Reproducible research in vadose zone sciences

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A significant portion of present-day soil and Earth science research is computational, involving complex data analysis pipelines, advanced mathematical and statistical models, and sophisticated computer codes. Opportunities for scientific progress are greatly diminished if reproducing and building o...

  19. Thou Shalt Be Reproducible! A Technology Perspective

    PubMed Central

    Mair, Patrick

    2016-01-01

    This article elaborates on reproducibility in psychology from a technological viewpoint. Modern open source computational environments are shown and explained that foster reproducibility throughout the whole research life cycle, and to which emerging psychology researchers should be sensitized, are shown and explained. First, data archiving platforms that make datasets publicly available are presented. Second, R is advocated as the data-analytic lingua franca in psychology for achieving reproducible statistical analysis. Third, dynamic report generation environments for writing reproducible manuscripts that integrate text, data analysis, and statistical outputs such as figures and tables in a single document are described. Supplementary materials are provided in order to get the reader started with these technologies. PMID:27471486

  20. Monitoring cell physiology by expression profiles and discovering cell type-specific genes by compiled expression profiles

    SciTech Connect

    Okubo, Kousaku; Itoh, Kouichi; Fukushima, Atsushi; Yoshii, Junji; Matsubara, Kenichi

    1995-11-20

    A gene expression profile is the list showing the expressed gene species and the abundance of their transcripts in a given cell or tissue. This list is made by constructing 3{prime}-directed cDNA libraries consisting of only the 3{prime}-termini of mRNA and sequencing randomly selected clones from such libraries: genes are identified by the sequences, and the composition of mRNA, which reflects gene activities, is measured from the frequency of appearance of the gene transcripts. For practical reasons, the number of sequenced clones has been limited to approximately 1000 per library at present, but the resulting profile covers almost all highly or moderately expressed genes, along with many less active genes. We constructed expression profiles from the HL60 human promyelocytic cell line and two of its derivatives, granulocytoids induced by DMSO and monocytoids induced by TPA. In HL60, a significant fraction of the abundantly expressed genes was for protein synthesis. Upon induction, these genes were partially or totally silenced; transcripts for proteins that characterize the granulocytes and monocyte-macrophages became abundant. By compiling and comparing different expression profiles, genes can be categorized into those expressed in diverse cell types and those active only in limited cell types. Although at present, the number of expression profiles that can be compiled is limited and this categorization is applicable only to abundantly expressed genes, 13 novel genes that may represent granulocyte- or monocyte-specific functions have been discovered. 37 refs., 1 fig., 2 tabs.

  1. Potency Biomarker Signature Genes from Multiparametric Osteogenesis Assays: Will cGMP Human Bone Marrow Mesenchymal Stromal Cells Make Bone?

    PubMed Central

    Murgia, Alba; Veronesi, Elena; Candini, Olivia; Caselli, Anna; D’souza, Naomi; Rasini, Valeria; Giorgini, Andrea; Catani, Fabio; Iughetti, Lorenzo

    2016-01-01

    In skeletal regeneration approaches using human bone marrow derived mesenchymal stromal cells (hBM-MSC), functional evaluation before implantation has traditionally used biomarkers identified using fetal bovine serum-based osteogenic induction media and time courses of at least two weeks. However, emerging pre-clinical evidence indicates donor-dependent discrepancies between these ex vivo measurements and the ability to form bone, calling for improved tests. Therefore, we adopted a multiparametric approach aiming to generate an osteogenic potency assay with improved correlation. hBM-MSC populations from six donors, each expanded under clinical-grade (cGMP) conditions, showed heterogeneity for ex vivo growth response, mineralization and bone-forming ability in a murine xenograft assay. A subset of literature-based biomarker genes was reproducibly upregulated to a significant extent across all populations as cells responded to two different osteogenic induction media. These 12 biomarkers were also measurable in a one-week assay, befitting clinical cell expansion time frames and cGMP growth conditions. They were selected for further challenge using a combinatorial approach aimed at determining ex vivo and in vivo consistency. We identified five globally relevant osteogenic signature genes, notably TGF-ß1 pathway interactors; ALPL, COL1A2, DCN, ELN and RUNX2. Used in agglomerative cluster analysis, they correctly grouped the bone-forming cell populations as distinct. Although donor #6 cells were correlation slope outliers, they contrastingly formed bone without showing ex vivo mineralization. Mathematical expression level normalization of the most discrepantly upregulated signature gene COL1A2, sufficed to cluster donor #6 with the bone-forming classification. Moreover, attenuating factors causing genuine COL1A2 gene down-regulation, restored ex vivo mineralization. This suggested that the signature gene had an osteogenically influential role; nonetheless no single

  2. The MOC31PE immunotoxin reduces cell migration and induces gene expression and cell death in ovarian cancer cells

    PubMed Central

    2014-01-01

    Background The standard treatment of ovarian cancer with chemotherapy often leads to drug resistance and relapse of the disease, and the need for development of novel therapy alternatives is obvious. The MOC31PE immunotoxin binds to the cell surface antigen EpCAM, which is expressed by the majority of epithelial cancers including ovarian carcinomas, and we studied the cytotoxic effects of MOC31PE in ovarian cancer cells. Methods Investigation of the effects of MOC31PE treatment on protein synthesis, cell viability, proliferation and gene expression of the ovarian cancer cell lines B76 and HOC7. Results MOC31PE treatment for 24 h caused a dose-dependent reduction of protein synthesis with ID50 values of less than 10 ng/ml, followed by reduced cell viability. In a gene expression array monitoring the expression of 84 key genes in cancer pathways, 13 of the genes were differentially expressed by MOC31PE treatment in comparison to untreated cells. By combining MOC31PE and the immune suppressor cyclosporin A (CsA) the MOC31PE effect on protein synthesis inhibition and cell viability increased tenfold. Cell migration was also reduced, both in the individual MOC31PE and CsA treatment, but even more when combining MOC31PE and CsA. In tumor metastasis PCR arrays, 23 of 84 genes were differentially expressed comparing CsA versus MOC31PE + CsA treatment. Increased expression of the tumor suppressor KISS1 and the nuclear receptor NR4A3 was observed, and the differential candidate gene expression was confirmed in complementary qPCR analyses. For NR4A3 this was not accompanied by increased protein expression. However, a subcellular fractionation assay revealed increased mitochondrial NR4A3 in MOC31PE treated cells, suggesting a role for this protein in MOC31PE-induced apoptotic cell death. Conclusion The present study demonstrates that MOC31PE may become a new targeted therapy for ovarian cancer and that the MOC31PE anti-cancer effect is potentiated by CsA. PMID:24528603

  3. Poly(Dimethylsiloxane) (PDMS) Affects Gene Expression in PC12 Cells Differentiating into Neuronal-Like Cells

    PubMed Central

    Łopacińska, Joanna M.; Emnéus, Jenny; Dufva, Martin

    2013-01-01

    Introduction Microfluidics systems usually consist of materials like PMMA - poly(methyl methacrylate) and PDMS - poly(dimethylsiloxane) and not polystyrene (PS), which is usually used for cell culture. Cellular and molecular responses