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Sample records for cells reproduces gene

  1. Magnetofection: A Reproducible Method for Gene Delivery to Melanoma Cells

    PubMed Central

    Prosen, Lara; Prijic, Sara; Music, Branka; Lavrencak, Jaka; Cemazar, Maja; Sersa, Gregor

    2013-01-01

    Magnetofection is a nanoparticle-mediated approach for transfection of cells, tissues, and tumors. Specific interest is in using superparamagnetic iron oxide nanoparticles (SPIONs) as delivery system of therapeutic genes. Magnetofection has already been described in some proof-of-principle studies; however, fine tuning of the synthesis of SPIONs is necessary for its broader application. Physicochemical properties of SPIONs, synthesized by the co-precipitation in an alkaline aqueous medium, were tested after varying different parameters of the synthesis procedure. The storage time of iron(II) sulfate salt, the type of purified water, and the synthesis temperature did not affect physicochemical properties of SPIONs. Also, varying the parameters of the synthesis procedure did not influence magnetofection efficacy. However, for the pronounced gene expression encoded by plasmid DNA it was crucial to functionalize poly(acrylic) acid-stabilized SPIONs (SPIONs-PAA) with polyethyleneimine (PEI) without the adjustment of its elementary alkaline pH water solution to the physiological pH. In conclusion, the co-precipitation of iron(II) and iron(III) sulfate salts with subsequent PAA stabilization, PEI functionalization, and plasmid DNA binding is a robust method resulting in a reproducible and efficient magnetofection. To achieve high gene expression is important, however, the pH of PEI water solution for SPIONs-PAA functionalization, which should be in the alkaline range. PMID:23862136

  2. Reproducible, Scalable Fusion Gene Detection from RNA-Seq.

    PubMed

    Arsenijevic, Vladan; Davis-Dusenbery, Brandi N

    2016-01-01

    Chromosomal rearrangements resulting in the creation of novel gene products, termed fusion genes, have been identified as driving events in the development of multiple types of cancer. As these gene products typically do not exist in normal cells, they represent valuable prognostic and therapeutic targets. Advances in next-generation sequencing and computational approaches have greatly improved our ability to detect and identify fusion genes. Nevertheless, these approaches require significant computational resources. Here we describe an approach which leverages cloud computing technologies to perform fusion gene detection from RNA sequencing data at any scale. We additionally highlight methods to enhance reproducibility of bioinformatics analyses which may be applied to any next-generation sequencing experiment. PMID:26667464

  3. Single Cell Quantification of Reporter Gene Expression in Live Adult Caenorhabditis elegans Reveals Reproducible Cell-Specific Expression Patterns and Underlying Biological Variation.

    PubMed

    Mendenhall, Alexander R; Tedesco, Patricia M; Sands, Bryan; Johnson, Thomas E; Brent, Roger

    2015-01-01

    In multicellular organisms such as Caenorhabditis elegans, differences in complex phenotypes such as lifespan correlate with the level of expression of particular engineered reporter genes. In single celled organisms, quantitative understanding of responses to extracellular signals and of cell-to-cell variation in responses has depended on precise measurement of reporter gene expression. Here, we developed microscope-based methods to quantify reporter gene expression in cells of Caenorhabditis elegans with low measurement error. We then quantified expression in strains that carried different configurations of Phsp-16.2-fluorescent-protein reporters, in whole animals, and in all 20 cells of the intestine tissue, which is responsible for most of the fluorescent signal. Some animals bore more recently developed single copy Phsp-16.2 reporters integrated at defined chromosomal sites, others, "classical" multicopy reporter gene arrays integrated at random sites. At the level of whole animals, variation in gene expression was similar: strains with single copy reporters showed the same amount of animal-to-animal variation as strains with multicopy reporters. At the level of cells, in animals with single copy reporters, the pattern of expression in cells within the tissue was highly stereotyped. In animals with multicopy reporters, the cell-specific expression pattern was also stereotyped, but distinct, and somewhat more variable. Our methods are rapid and gentle enough to allow quantification of expression in the same cells of an animal at different times during adult life. They should allow investigators to use changes in reporter expression in single cells in tissues as quantitative phenotypes, and link those to molecular differences. Moreover, by diminishing measurement error, they should make possible dissection of the causes of the remaining, real, variation in expression. Understanding such variation should help reveal its contribution to differences in complex

  4. Evaluation of sensitivity, specificity, and reproducibility of an optimized method for detecting clonal rearrangements of immunoglobulin and T-cell receptor genes in formalin-fixed, paraffin-embedded sections.

    PubMed

    Slack, D N; McCarthy, K P; Wiedemann, L M; Sloane, J P

    1993-12-01

    Following our recent reports of detecting clonal immunoglobulin and T-cell receptor gene rearrangements by the polymerase chain reaction, we have improved and simplified the technique for use in diagnostic histopathology laboratories and determined, on coded samples, the sensitivity, specificity, and reproducibility of the modified methodology in distinguishing malignant lymphoma from reactive lymphoid hyperplasia and nonlymphoid tumors. Using only three primer pairs for the immunoglobulin heavy chain and T-cell receptor beta and gamma chain genes on well-characterized lesions of widely varying morphology and immunophenotype, clonal rearrangements were detected in 65% of B-cell lymphomas, and 77-82% of T-cell tumors. Specificity and observer consistency ranged from 93-97%. The method requires very careful control, particularly to avoid misinterpretation of results because of contamination and nonspecific amplification, but in its present form is relatively simple and inexpensive, and gives results on single paraffin-embedded sections within 24 h. PMID:8118599

  5. Analytic performance studies and clinical reproducibility of a real-time PCR assay for the detection of epidermal growth factor receptor gene mutations in formalin-fixed paraffin-embedded tissue specimens of non-small cell lung cancer

    PubMed Central

    2013-01-01

    Background Epidermal growth factor receptor (EGFR) gene mutations identify patients with non-small cell lung cancer (NSCLC) who have a high likelihood of benefiting from treatment with anti-EGFR tyrosine kinase inhibitors. Sanger sequencing is widely used for mutation detection but can be technically challenging, resulting in longer turn-around-time, with limited sensitivity for low levels of mutations. This manuscript details the technical performance verification studies and external clinical reproducibility studies of the cobas EGFR Mutation Test, a rapid multiplex real-time PCR assay designed to detect 41 mutations in exons 18, 19, 20 and 21. Methods The assay’s limit of detection was determined using 25 formalin-fixed paraffin-embedded tissue (FFPET)-derived and plasmid DNA blends. Assay performance for a panel of 201 specimens was compared against Sanger sequencing with resolution of discordant specimens by quantitative massively parallel pyrosequencing (MPP). Internal and external reproducibility was assessed using specimens tested in duplicate by different operators, using different reagent lots, instruments and at different sites. The effects on the performance of the cobas EGFR test of endogenous substances and nine therapeutic drugs were evaluated in ten FFPET specimens. Other tests included an evaluation of the effects of necrosis, micro-organisms and homologous DNA sequences on assay performance, and the inclusivity of the assay for less frequent mutations. Results A >95% hit rate was obtained in blends with >5% mutant alleles, as determined by MPP analysis, at a total DNA input of 150 ng. The overall percent agreement between Sanger sequencing and the cobas test was 96.7% (negative percent agreement 97.5%; positive percent agreement 95.8%). Assay repeatability was 98% when tested with two operators, instruments, and reagent lots. In the external reproducibility study, the agreement was > 99% across all sites, all operators and all reagent lots

  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

    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

    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

  9. 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

  10. Autoinduction of thyroid hormone receptor during metamorphosis is reproduced in Xenopus XTC-2 cells.

    PubMed

    Machuca, I; Tata, J R

    1992-09-01

    To determine if the autoinduction of thyroid hormone receptor (TR) alpha and beta mRNAs during metamorphosis in Xenopus tadpoles can be reproduced in cultured cells, we have screened four Xenopus cell lines (XTC-2, XL-177, XL2 and Kr) for receptor transcripts and their response to thyroid hormone. Exposure of XTC-2 cells to 10(-9) M triiodothyronine (T3) for 24 h upregulated TR alpha and beta mRNAs by 2-4- and 10-40-fold, respectively. In view of the marked similarity of the differential distribution of the two transcripts and their upregulation by T3 to the pattern of autoinduction seen in whole tadpoles, the process was studied in greater detail in XTC-2 cells. The time-course of autoinduction of TR alpha and beta mRNAs in these cells also resembled that in vivo, the two transcripts being significantly induced by 3-6 h after T3. Dose-response to T3, and the relative responses to its active and inactive analogs, confirmed that the process of autoinduction was initiated by thyroid hormone receptor with the same functional characteristics as that found in all amphibian and mammalian tissues. Experiments performed with cycloheximide suggested that intermediary protein(s) were involved in autoinduction, so that TR genes cannot be considered as 'immediate early' genes for this process. The possible advantages of studying thyroid hormone action in metamorphosis in XTC-2 cells are briefly discussed.

  11. Reproducibility, Fidelity, and Discriminant Validity of mRNA Amplification for Microarray Analysis from Primary Hematopoietic Cells

    PubMed Central

    Li, Liang; Roden, Joe; Shapiro, Bruce E.; Wold, Barbara J.; Bhatia, Smita; Forman, Stephen J.; Bhatia, Ravi

    2005-01-01

    Analysis of gene expression in clinical samples poses special challenges, including limited RNA availability and poor RNA quality. Quantitative information regarding reliability of RNA amplification methodologies applied to primary cells and representativeness of resulting gene expression profiles is limited. We evaluated four protocols for RNA amplification from peripheral blood mononuclear cells. Results obtained with 100 ng or 10 ng of RNA amplified using two rounds of cDNA synthesis and in vitro transcription were compared with control 2.5-μg RNA samples processed using a single round of in vitro transcription. Samples were hybridized to Affymetrix HG-U133A arrays. Considerable differences in results were obtained with different protocols. The optimal protocol resulted in highly reproducible gene expression profiles from amplified samples (r = 0.98) and good correlation between amplified and control samples (r = 0.94). Using the optimal protocol dissimilarities of gene expression between mononuclear cells from a normal individual and a patient with myelodysplastic syndrome were primarily maintained after amplification compared with controls. We conclude that small variations in methodology introduce considerable distortion of gene expression profiles obtained after RNA amplification from clinical samples and too strong a focus on a very small number of genes picked from an array analysis could be unduly influenced by seemingly acceptable methodologies. However, it is possible to obtain reproducible and representative results using optimized protocols. PMID:15681474

  12. Tumorigenic WAP-T Mouse Mammary Carcinoma Cells: A Model for a Self-Reproducing Homeostatic Cancer Cell System

    PubMed Central

    Otto, Benjamin; Gruner, Katharina; Heinlein, Christina; Kühl, Marion; Warnecke, Gabriele; Schumacher, Udo; Deppert, Wolfgang; Tolstonog, Genrich V.

    2010-01-01

    Background In analogy to normal stem cell differentiation, the current cancer stem cell (CSC) model presumes a hierarchical organization and an irreversible differentiation in tumor tissue. Accordingly, CSCs should comprise only a small subset of the tumor cells, which feeds tumor growth. However, some recent findings raised doubts on the general applicability of the CSC model and asked for its refinement. Methodology/Principal Findings In this study we analyzed the CSC properties of mammary carcinoma cells derived from transgenic (WAP-T) mice. We established a highly tumorigenic WAP-T cell line (G-2 cells) that displays stem-like traits. G-2 cells, as well as their clonal derivates, are closely related to primary tumors regarding histology and gene expression profiles, and reflect heterogeneity regarding their differentiation states. G-2 cultures comprise cell populations in distinct differentiation states identified by co-expression of cytoskeletal proteins (cytokeratins and vimentin), a combination of cell surface markers and a set of transcription factors. Cellular subsets sorted according to expression of CD24a, CD49f, CD61, Epcam, Sca1, and Thy1 cell surface proteins, or metabolic markers (e.g. ALDH activity) are competent to reconstitute the initial cellular composition. Repopulation efficiency greatly varies between individual subsets and is influenced by interactions with the respective complementary G-2 cellular subset. The balance between differentiation states is regulated in part by the transcription factor Sox10, as depletion of Sox10 led to up-regulation of Twist2 and increased the proportion of Thy1-expressing cells representing cells in a self-renewable, reversible, quasi-mesenchymal differentiation state. Conclusions/Significance G-2 cells constitute a self-reproducing cancer cell system, maintained by bi- and unidirectional conversion of complementary cellular subsets. Our work contributes to the current controversial discussion on the existence

  13. Safe, Efficient, and Reproducible Gene Therapy of the Brain in the Dog Models of Sanfilippo and Hurler Syndromes

    PubMed Central

    Ellinwood, N Matthew; Ausseil, Jérôme; Desmaris, Nathalie; Bigou, Stéphanie; Liu, Song; Jens, Jackie K; Snella, Elizabeth M; Mohammed, Eman EA; 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

    2011-01-01

    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. PMID:21139569

  14. Fly wing vein patterns have spatial reproducibility of a single cell

    PubMed Central

    Abouchar, Laurent; Petkova, Mariela D.; Steinhardt, Cynthia R.; Gregor, Thomas

    2014-01-01

    Developmental processes in multicellular organisms occur in fluctuating environments and are prone to noise, yet they produce complex patterns with astonishing reproducibility. We measure the left–right and inter-individual precision of bilaterally symmetric fly wings across the natural range of genetic and environmental conditions and find that wing vein patterns are specified with identical spatial precision and are reproducible to within a single-cell width. The early fly embryo operates at a similar degree of reproducibility, suggesting that the overall spatial precision of morphogenesis in Drosophila performs at the single-cell level. Could development be operating at the physical limit of what a biological system can achieve? PMID:24942847

  15. Activation-induced cytidine deaminase induces reproducible DNA breaks at many non-Ig loci in activated B cells

    PubMed Central

    Staszewski, Ori; Baker, Richard E.; Ucher, Anna J.; Martier, Raygene; Stavnezer, Janet; Guikema, Jeroen E.J.

    2011-01-01

    After immunization or infection, activation-induced cytidine deaminase (AID) initiates diversification of immunoglobulin (Ig) genes in B cells, introducing mutations within the antigen binding V regions (somatic hypermutation, SHM) and double-strand DNA breaks (DSBs) into switch (S) regions, leading to antibody class switch recombination (CSR). We asked if during B cell activation, AID also induces DNA breaks at genes other than IgH genes. Using a non-biased genome-wide approach, we have identified hundreds of reproducible AID-dependent DSBs in mouse splenic B cells shortly after induction of CSR in culture. Most interestingly, AID induces DSBs at sites syntenic with sites of translocations, deletions, and amplifications found in human B cell lymphomas, including within the oncogene B cell lymphoma11a (bcl11a)/evi9. Unlike AID-induced DSBs in Ig genes, genome-wide AID-dependent DSBs are not restricted to transcribed regions, and frequently occur within repeated sequence elements, including CA-repeats and non-CA tandem repeats, and SINEs. PMID:21255732

  16. Activation-induced cytidine deaminase induces reproducible DNA breaks at many non-Ig Loci in activated B cells.

    PubMed

    Staszewski, Ori; Baker, Richard E; Ucher, Anna J; Martier, Raygene; Stavnezer, Janet; Guikema, Jeroen E J

    2011-01-21

    After immunization or infection, activation-induced cytidine deaminase (AID) initiates diversification of immunoglobulin (Ig) genes in B cells, introducing mutations within the antigen-binding V regions (somatic hypermutation, SHM) and double-strand DNA breaks (DSBs) into switch (S) regions, leading to antibody class switch recombination (CSR). We asked if, during B cell activation, AID also induces DNA breaks at genes other than IgH genes. Using a nonbiased genome-wide approach, we have identified hundreds of reproducible, AID-dependent DSBs in mouse splenic B cells shortly after induction of CSR in culture. Most interestingly, AID induces DSBs at sites syntenic with sites of translocations, deletions, and amplifications found in human B cell lymphomas, including within the oncogene B cell lymphoma11a (bcl11a)/evi9. Unlike AID-induced DSBs in Ig genes, genome-wide AID-dependent DSBs are not restricted to transcribed regions and frequently occur within repeated sequence elements, including CA repeats, non-CA tandem repeats, and SINEs.

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

    PubMed

    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

    2013-12-24

    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.

  18. 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

  19. 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.

  20. Question 8: From a Set of Chemical Reactions to Reproducing Cells

    NASA Astrophysics Data System (ADS)

    Kaneko, Kunihiko

    2007-10-01

    As a prerequisite for a reproducing cell, we first discuss how non-equilibrium states are sustained endogenously in a catalytic reaction network. Negative correlation between abundances of resource chemicals and of catalysts for their consumption is shown to lead to hindrance of relaxation to equilibrium. Mutual reinforcement among such sustainment of non-equilibrium state, spatial structure formation, and reproduction of a compartment is discussed as a mechanism to further suppress the relaxation to equilibrium. As a next step to protocell, consistency between cell reproduction and replication of constituent molecules is theoretically studied, which leads to a power-law on abundance of molecules as well as log-normal distribution over cells, which are shown to be universal, and also confirmed experimentally in the present cells.

  1. 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.

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

    PubMed Central

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

    2014-01-01

    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. PMID:25084331

  3. Efficient and reproducible generation of tumour-infiltrating lymphocytes for renal cell carcinoma

    PubMed Central

    Baldan, V; Griffiths, R; Hawkins, R E; Gilham, D E

    2015-01-01

    Background: Tumour-infiltrating lymphocyte (TIL) therapy is showing great promise in the treatment of patients with advanced malignant melanoma. However, the translation of TIL therapy to non-melanoma tumours such as renal cell carcinoma has been less successful with a major constraint being the inability to reproducibly generate TILs from primary and metastatic tumour tissue. Methods: Primary and metastatic renal cell carcinoma biopsies were subjected to differential tumour disaggregation methods and procedures that stimulate the specific expansion of TILs tested to determine which reliably generated TIL maintained antitumour specificity. Results: Enzymatic or combined enzymatic/mechanical disaggregation resulted in equivalent numbers of TILs being liberated from renal cell carcinoma biopsies. Following mitogenic activation of the isolated TILs with anti-CD3/anti-CD28-coated paramagnetic beads, successful TIL expansion was achieved in 90% of initiated cultures. The frequency of T-cell recognition of autologous tumours was enhanced when tumours were disaggregated using the GentleMACS enzymatic/mechanical system. Conclusion: TILs can be consistently produced from renal cell carcinoma biopsies maintaining autologous tumour recognition after expansion in vitro. While the method of disaggregation has little impact on the success of TIL growth, methods that preserve the cell surface architecture facilitate TIL recognition of an autologous tumour, which is important in terms of characterising the functionality of the expanded TIL population. PMID:25867267

  4. 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.

  5. 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. PMID:26538323

  6. 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.

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

    PubMed Central

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

    2015-01-01

    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. Modular Analysis of Peripheral Blood Gene Expression in Rheumatoid Arthritis Captures Reproducible Gene Expression Changes in TNF Responders

    PubMed Central

    Oswald, Michaela; Curran, Mark; Lamberth, Sarah; Townsend, Robert; Hamilton, Jennifer D.; Chernoff, David N.; Carulli, John; Townsend, Michael; Weinblatt, Michael; Kern, Marlena; Pond, Cassandra; Lee, Annette; Gregersen, Peter K.

    2015-01-01

    Objective To establish whether the analysis of whole blood gene expression can be useful in predicting or monitoring response to anti-TNF therapy in RA. Methods Whole blood RNA (PAXgene) was obtained at baseline and 14 weeks on three independent cohorts with a combined total of 250 patients with rheumatoid arthritis beginning anti-TNF therapy. We employed an approach to gene expression analysis that is based on gene expression “modules”. Results Good and Moderate Responders by EULAR criteria exhibited highly significant and consistent changes in multiple gene expression modules using a hyper geometric analysis after 14 weeks of therapy. Strikingly, non responders exhibited very little change in any modules, despite exposure to TNF blockade. These patterns of change were highly consistent across all three cohorts, indicating that immunological changes after TNF treatment are specific to the combination of both drug exposure and responder status. In contrast, modular patterns of gene expression did not exhibit consistent differences between responders and non-responders at baseline in the three cohorts. Conclusions These data provide evidence that using gene expression modules related to inflammatory disease may provide a valuable method for objective monitoring of the response of RA patients who are treated with TNF inhibitors. PMID:25371395

  9. Developing an efficient and reproducible conjugation-based gene transfer system for bifidobacteria.

    PubMed

    Dominguez, Wilfredo; O'Sullivan, Daniel J

    2013-02-01

    Bifidobacteria are widely used as probiotics and have attracted increasing research interest worldwide. However, molecular techniques are still very scarce mainly due to the low efficiencies and strain-specific electroporation protocols that have been developed. Bacterial conjugation enables the transfer of genetic material among a relatively wide range of organisms and with virtually no size limitation. A conjugation protocol was developed based on the RP4 conjugative machinery in the Escherichia coli strain WM3064(pBB109). Using this machinery, the newly constructed transmissible E. coli-Bifidobacterium shuttle vector, pDOJHR-WD2, was successfully and consistently transferred into several strains representing four Bifidobacterium species at efficiencies which correlated with the E. coli to bifidobacteria ratios. Higher ratios were found to significantly improve transfer frequency per recipient, with almost 100 % transfer frequency occurring when the ratio was 10(5) : 1. The incompatible resident plasmid, pDOJH10S, in Bifidobacterium longum DJO10A was able to coexist, albeit at lower copy numbers, with the incoming vector pDOJHR-WD2 even though they possess the same ori. In some cases the copy number of this resident plasmid was too low to observe via gel electrophoresis, but it could be detected by Southern hybridization. Plasmid curing resulted in a strain, DJO10A-W3, that had lost both plasmids and this showed a one-log increase in conjugation efficiency due to the lack of plasmid incompatibility. In conclusion, this novel conjugative gene transfer protocol can be used for the introduction of genetic material (without size restriction) into Bifdobacterium species and is particularly useful for strains that are recalcitrant to electroporation.

  10. Reproducible Science▿

    PubMed Central

    Casadevall, Arturo; Fang, Ferric C.

    2010-01-01

    The reproducibility of an experimental result is a fundamental assumption in science. Yet, results that are merely confirmatory of previous findings are given low priority and can be difficult to publish. Furthermore, the complex and chaotic nature of biological systems imposes limitations on the replicability of scientific experiments. This essay explores the importance and limits of reproducibility in scientific manuscripts. PMID:20876290

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

    PubMed

    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

  12. 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

  13. 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

  14. Reproducible establishment of hemopoietic supportive stromal cell lines from murine bone marrow

    SciTech Connect

    Itoh, K.; Tezuka, H.; Sakoda, H.; Konno, M.; Nagata, K.; Uchiyama, T.; Uchino, H.; Mori, K.J.

    1989-02-01

    Stromal cell lines, designated MS-1, -2, -3, -4, -5, -6, and -7 were established by irradiating the adherent cells in long-term bone marrow cultures with 900-rad x-rays. Two of the cell lines, MS-1 and MS-5, have the capacity to support the growth of hemopoietic stem cells (spleen colony-forming cells and granulocyte-macrophage colony-forming cells) for greater than 2 months in vitro. These two cell lines were alkaline phosphatase-, peroxidase-, and factor VIII-negative and positive for periodic acid-Schiff and nonspecific esterase. Extracellular matrix proteins such as fibronectin, laminin, and collagen type I were produced by these two cell lines. Neither MS-1 cell- nor MS-5 cell-conditioned medium supported the growth of hemopoietic stem cells, and hemopoietic stem cells were found preferentially to be under and on MS-1 and MS-5 layers rather than in suspension. Close contact with the MS-1 cell layer or the MS-5 cell layer appears to be essential in maintaining hemopoiesis in vitro. Conditioned media from MS-1 cells and MS-5 cells stimulated granulocyte colony formation from murine bone marrow cells in semisolid culture.

  15. HeLa cell heterogeneity and coxsackievirus B3 cytopathic effect: implications for inter-laboratory reproducibility of results.

    PubMed

    Carson, Steven D; Pirruccello, Samuel J

    2013-04-01

    Concerns over cell line identities and contamination have led investigators to acquire fresh stocks of HeLa CCL-2 cells, but results with the HeLa CCL-2 cells do not always reproduce results with HeLa cells that have long history in the laboratory. When used for TCID(50) assays of Coxsackievirus B3/28 (CVB3/28), HeLa CCL-2 cells returned titers for CVB3/28 that were more than ten-fold lower than titers obtained using laboratory HeLa cells. The viral cytopathic effect was less distinct in the HeLa CCL-2 cultures, suggestive of a mixed population of cells with varied susceptibility to viral cytopathic effect. Analysis of short tandem repeat markers confirmed the identities of the cell lines as HeLa. Subpopulations in the HeLa CCL-2 culture, separated easily based on the speed with which they were released by trypsin-EDTA, differed in their susceptibilities to CVB3/28 cytopathic effect, and in their expression of the Coxsackievirus and adenovirus receptor (CAR). The distinctions between Lab HeLa and HeLa CCL-2 cells were less obvious when infected with CVB3/RD, a strain selected for growth in RD cells. Results that differ among laboratories may be due to the use of HeLa cell strains with different histories, and experiments using HeLa CCL-2 available from the American Type Culture Collection are probably incapable of reproducing many of the published studies of Coxsackievirus that have used HeLa cells with laboratory-dependent histories.

  16. Reproducibility of histological cell type in high-grade endometrial carcinoma.

    PubMed

    Han, Guangming; Sidhu, Davinder; Duggan, Máire A; Arseneau, Jocelyne; Cesari, Matthew; Clement, Philip B; Ewanowich, Carol A; Kalloger, Steve E; Köbel, Martin

    2013-12-01

    Subclassification of endometrial carcinoma according to histological type shows variable interobserver agreement. The aim of this study was to assess specifically the interobserver agreement of histological type in high-grade endometrial carcinomas, recorded by gynecological pathologists from five academic centers across Canada. In a secondary aim, the agreement of consensus diagnosis with immunohistochemical marker combinations was assessed including six routine (TP53, CDKN2A (p16), ER, PGR, Ki67, and VIM) and six experimental immunohistochemical markers (PTEN, ARID1A, CTNNB1, IGF2BP3, HNF1B, and TFF3). The paired interobserver agreement ranged from κ 0.50 to 0.63 (median 0.58) and the intraobserver agreement from κ 0.49 to 0.67 (median 0.61). Consensus about histological type based on morphological assessment was reached in 72% of high-grade endometrial carcinomas. A seven-marker immunohistochemical panel differentiated FIGO grade 3 endometrioid from serous carcinoma with a 100% concordance rate compared with the consensus diagnosis. More practically, a three-marker panel including TP53, ER, and CDKN2A (p16) can aid in the differential diagnosis of FIGO grade 3 endometrioid from endometrial serous carcinoma. Our study demonstrates that the inter- and intraobserver reproducibility of histological type based on morphology alone are mostly moderate. Ancillary techniques such as immunohistochemical marker panels are likely needed to improve diagnostic reproducibility of histological types within high-grade endometrial carcinomas.

  17. Elusive reproducibility.

    PubMed

    Gori, Gio Batta

    2014-08-01

    Reproducibility remains a mirage for many biomedical studies because inherent experimental uncertainties generate idiosyncratic outcomes. The authentication and error rates of primary empirical data are often elusive, while multifactorial confounders beset experimental setups. Substantive methodological remedies are difficult to conceive, signifying that many biomedical studies yield more or less plausible results, depending on the attending uncertainties. Real life applications of those results remain problematic, with important exceptions for counterfactual field validations of strong experimental signals, notably for some vaccines and drugs, and for certain safety and occupational measures. It is argued that industrial, commercial and public policies and regulations could not ethically rely on unreliable biomedical results; rather, they should be rationally grounded on transparent cost-benefit tradeoffs. PMID:24882687

  18. Elusive reproducibility.

    PubMed

    Gori, Gio Batta

    2014-08-01

    Reproducibility remains a mirage for many biomedical studies because inherent experimental uncertainties generate idiosyncratic outcomes. The authentication and error rates of primary empirical data are often elusive, while multifactorial confounders beset experimental setups. Substantive methodological remedies are difficult to conceive, signifying that many biomedical studies yield more or less plausible results, depending on the attending uncertainties. Real life applications of those results remain problematic, with important exceptions for counterfactual field validations of strong experimental signals, notably for some vaccines and drugs, and for certain safety and occupational measures. It is argued that industrial, commercial and public policies and regulations could not ethically rely on unreliable biomedical results; rather, they should be rationally grounded on transparent cost-benefit tradeoffs.

  19. 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

  20. 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

  1. 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.

  2. 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 %.

  3. 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 %. PMID:26388210

  4. Genotoxic effects of exposure to radiofrequency electromagnetic fields (RF-EMF) in HL-60 cells are not reproducible.

    PubMed

    Speit, Günter; Gminski, Richard; Tauber, Rudolf

    2013-08-15

    Conflicting results have been published regarding the induction of genotoxic effects by exposure to radiofrequency electromagnetic fields (RF-EMF). Various results indicating a genotoxic potential of RF-EMF were reported by the collaborative EU-funded REFLEX (Risk Evaluation of Potential Environmental Hazards From Low Energy Electromagnetic Field Exposure Using Sensitive in vitro Methods) project. There has been a long-lasting scientific debate about the reliability of the reported results and an attempt to reproduce parts of the results obtained with human fibroblasts failed. Another part of the REFLEX study was performed in Berlin with the human lymphoblastoid cell line HL-60; genotoxic effects of RF-EMF were measured by means of the comet assay and the micronucleus test. The plausibility and reliability of these results were also questioned. In order to contribute to a clarification of the biological significance of the reported findings, a repeat study was performed, involving scientists of the original study. Comet-assay experiments and micronucleus tests were performed under the same experimental conditions that had led to genotoxic effects in the REFLEX study. Here we report that the attempts to reproduce the induction of genotoxic effects by RF-EMF in HL-60 cells failed. No genotoxic effects of RF-EMF were measured in the repeat experiments. We could not find an explanation for the conflicting results. However, the negative repeat experiments suggest that the biological significance of genotoxic effects of RF-EMF reported by the REFLEX study should be re-assessed.

  5. 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.

  6. 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. PMID:27224960

  7. 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.

  8. Reduction in vitro of red cell glutathione reproduces defects of cellular sodium transport seen in oedematous malnutrition.

    PubMed

    Forrester, T; Golden, M; Brand, S; Swales, J

    1990-05-01

    Red cells in oedematous malnutrition (kwashiorkor) have an increased sodium content, 'leakiness' to sodium and enhanced sodium pumping. In non-oedematous malnutrition (marasmus) there is a reduction in the sodium pump activity. The explanation has hitherto been unknown but the glutathione content of red cells is low in kwashiorkor and normal in marasmus. We artificially lowered the glutathione content of normal red cells to values characteristic of mild oedematous malnutrition, using the enzyme inhibitors bischloronitrosourea (BCNU) and buthionine sulfoximine (BSOX). After preincubation, the cells were washed to remove the inhibitors and oxidized glutathione. Cellular content of sodium and potassium, and 86Rb influx were then measured. The reduction in glutathione reproduced the abnormalities of sodium content and flux observed in kwashiorkor. We suggest that oxidant stress in kwashiorkor, by reducing cellular glutathione, may affect cell membrane electrolyte transport. This may act through alterations in membrane sulfhydryl groups. Glutathione depletion may therefore play an important role in the clinical picture and natural history of oedematous malnutrition and may have relevance to other conditions where oxidant stress occurs.

  9. An on-chip microfluidic pressure regulator that facilitates reproducible loading of cells and hydrogels into microphysiological system platforms.

    PubMed

    Wang, Xiaolin; Phan, Duc T T; Zhao, Da; George, Steven C; Hughes, Christopher C W; Lee, Abraham P

    2016-03-01

    Coculturing multiple cell types together in 3-dimensional (3D) cultures better mimics the in vivo microphysiological environment, and has become widely adopted in recent years with the development of organ-on-chip systems. However, a bottleneck in set-up of these devices arises as a result of the delivery of the gel into the microfluidic chip being sensitive to pressure fluctuations, making gel confinement at a specific region challenging, especially when manual operation is performed. In this paper, we present a novel design of an on-chip regulator module with pressure-releasing safety microvalves that can facilitate stable gel delivery into designated microchannel regions while maintaining well-controlled, non-bursting gel interfaces. This pressure regulator design can be integrated into different microfluidic chip designs and is compatible with a wide variety of gel injection apparatuses operated automatically or manually at different flow rates. The sensitivity and working range of this pressure regulator can be adjusted by changing the width of its pressure releasing safety microvalve design. The effectiveness of the design is validated by its incorporation into a microfluidic platform we have developed for generating 3D vascularized micro-organs (VMOs). Reproducible gel loading is demonstrated for both an automatic syringe pump and a manually-operated micropipettor. This design allows for rapid and reproducible loading of hydrogels into microfluidic devices without the risk of bursting gel-air interfaces. PMID:26879519

  10. 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

  11. 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

  12. 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

  13. 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.

  14. 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. PMID:27635311

  15. 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, ...

  16. 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.

  17. 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. PMID:24299736

  18. Mouse thymic epithelial cell lines expressing "Aire" and peripheral tissue-specific antigens reproduce in vitro negative selection of T cells.

    PubMed

    Yamaguchi, Yoshitaka; Takayanagi, Atsushi; Chen, Jiabing; Sakai, Kosuke; Kudoh, Jun; Shimizu, Nobuyoshi

    2011-08-15

    In the human thymus, AIRE (autoimmune regulator) gene is expressed in a very limited type of medullary thymic epithelial cells (mTECs) and no cognate cell lines are available, hence the molecular analysis of AIRE gene function has been difficult. To improve this situation, we attempted to isolate Aire-expressing cells and established three cell lines (Aire⁺TEC1, Aire⁺TEC2, Aire⁺DC) from the abnormally enlarged thymus, which was developed in the transgenic mice expressing SV40 T-antigen driven by the mouse Aire gene promoter. When these Aire⁺ cell lines were co-cultured with fresh thymocytes, they adhered to the majority of thymocytes and induced apoptosis as if negative selection of T-cells in the thymus is occurring in vitro. Further analysis revealed that these Aire⁺ cell lines are derived from mTECs and exhibit characteristic natures of "antigen presenting cells" including several distinct abilities: to express a variety of peripheral tissue-specific antigens, to produce immunoproteasome and immunological synapse, and to express some of TNFSFs (tumor necrosis factor super families). Thus, the newly established Aire⁺ cell lines will be invaluable for the further detailed analysis of AIRE gene function in the central tolerance of immunity and autoimmune disease.

  19. Accurate comparison of antibody expression levels by reproducible transgene targeting in engineered recombination-competent CHO cells.

    PubMed

    Mayrhofer, Patrick; Kratzer, Bernhard; Sommeregger, Wolfgang; Steinfellner, Willibald; Reinhart, David; Mader, Alexander; Turan, Soeren; Qiao, Junhua; Bode, Juergen; Kunert, Renate

    2014-12-01

    Over the years, Chinese hamster ovary (CHO) cells have emerged as the major host for expressing biotherapeutic proteins. Traditional methods to generate high-producer cell lines rely on random integration(s) of the gene of interest but have thereby left the identification of bottlenecks as a challenging task. For comparison of different producer cell lines derived from various transfections, a system that provides control over transgene expression behavior is highly needed. This motivated us to develop a novel "DUKX-B11 F3/F" cell line to target different single-chain antibody fragments into the same chromosomal target site by recombinase-mediated cassette exchange (RMCE) using the flippase (FLP)/FLP recognition target (FRT) system. The RMCE-competent cell line contains a gfp reporter fused to a positive/negative selection system flanked by heterospecific FRT (F) variants under control of an external CMV promoter, constructed as "promoter trap". The expression stability and FLP accessibility of the tagged locus was demonstrated by successive rounds of RMCE. As a proof of concept, we performed RMCE using cassettes encoding two different anti-HIV single-chain Fc fragments, 3D6scFv-Fc and 2F5scFv-Fc. Both targeted integrations yielded homogenous cell populations with comparable intracellular product contents and messenger RNA (mRNA) levels but product related differences in specific productivities. These studies confirm the potential of the newly available "DUKX-B11 F3/F" cell line to guide different transgenes into identical transcriptional control regions by RMCE and thereby generate clones with comparable amounts of transgene mRNA. This new host is a prerequisite for cell biology studies of independent transfections and transgenes.

  20. An index of parameter reproducibility accounting for estimation uncertainty: theory and case study on β-cell responsivity and insulin sensitivity.

    PubMed

    Dalla Man, Chiara; Pillonetto, Gianluigi; Riz, Michela; Cobelli, Claudio

    2015-06-01

    Parameter reproducibility is necessary to perform longitudinal studies where parameters are assessed to monitor disease progression or effect of therapy but are also useful in powering the study, i.e., to define how many subjects should be studied to observe a given effect. The assessment of parameter reproducibility is usually accomplished by methods that do not take into account the fact that these parameters are estimated with uncertainty. This is particularly relevant in physiological and clinical studies where usually reproducibility cannot be assessed by multiple testing and is usually assessed from a single replication of the test. Working in a suitable stochastic framework, here we propose a new index (S) to measure reproducibility that takes into account parameter uncertainty and is particularly suited to handle the normal testing conditions of physiological and clinical investigations. Simulation results prove that S, by properly taking into account parameter uncertainty, is more accurate and robust than the methods available in the literature. The new metric is applied to assess reproducibility of insulin sensitivity and β-cell responsivity of a mixed-meal tolerance test from data obtained in the same subjects retested 1 wk apart. Results show that the indices of insulin sensitivity and β-cell responsivity to glucose are well reproducible. We conclude that the oral minimal models provide useful indices that can be used safely in prospective studies or to assess the efficacy of a given therapy.

  1. Convergence of gene and cell therapy.

    PubMed

    Bersenev, Alexey; Levine, Bruce L

    2012-11-01

    Gene therapy and cell therapy have followed similar roller coaster paths of rising public expectations and disappointment over the past two decades. There is now reason to believe that momentum in the field has reached the point where the successes will be more frequent. The use of gene-modified cells has opened new avenues for engineering desired cell properties, for the use of cells as vehicles for gene delivery, and for tracking cells and controlling cell persistence after transplantation. Some notable recent clinical developments in cellular engineering by gene transfer offer lessons on how the field has emerged, and hint at additional future clinical applications. PMID:23210811

  2. Purification of recombinant adeno-associated virus by iodixanol gradient ultracentrifugation allows rapid and reproducible preparation of vector stocks for gene transfer in the nervous system.

    PubMed

    Hermens, W T; ter Brake, O; Dijkhuizen, P A; Sonnemans, M A; Grimm, D; Kleinschmidt, J A; Verhaagen, J

    1999-07-20

    Recombinant adeno-associated virus (rAAV) vectors have become attractive tools for in vivo gene transfer. The production and purification of high-titer rAAV vector stocks for experimental and therapeutic gene transfer continue to undergo improvement. Standard rAAV vector purification protocols include the purification of the vector by cesium chloride (CsCl)-density gradient centrifugation followed by extensive desalination via dialysis against a physiological buffer for in vivo use. These procedures are extremely time consuming and frequently result in a substantial loss of the infectious vector titer. As an alternative to CsCl we have investigated the use of Iodixanol, an X-ray contrast solution, as the density-gradient medium. Purification of rAAV vectors by Iodixanol shortened the centrifugation period to 3 hr and resulted in reproducible concentration and purification of rAAV-vector stocks. We show that injection of rAAV derived from an Iodixanol gradient can be used for in vivo gene transfer applications in the brain and spinal cord without detectable cytopathic effects and directing stable transgene expression for at least 2 months.

  3. 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.

  4. Reproducibility of 18F-FDG PET uptake measurements in head and neck squamous cell carcinoma on both PET/CT and PET/MR

    PubMed Central

    Fischer, B M; Aznar, M C; Hansen, A E; Vogelius, I R; Löfgren, J; Andersen, F L; Loft, A; Kjaer, A; Højgaard, L; Specht, L

    2015-01-01

    Objective: To investigate reproducibility of fluorine-18 fludeoxyglucose (18F-FDG) uptake on 18F-FDG positron emission tomography (PET)/CT and 18F-FDG PET/MR scans in patients with head and neck squamous cell carcinoma (HNSCC). Methods: 30 patients with HNSCC were included in this prospective study. The patients were scanned twice before radiotherapy treatment with both PET/CT and PET/MR. Patients were scanned on the same scanners, 3 days apart and according to the same protocol. Metabolic tumour activity was measured by the maximum and peak standardized uptake value (SUVmax and SUVpeak, respectively), and total lesion glycolysis from the metabolic tumour volume defined from ≥50% SUVmax. Bland–Altman analysis with limits of agreement, coefficient of variation (CV) from the two modalities were performed in order to test the reproducibility. Furthermore, CVs from SUVmax and SUVpeak were compared. The area under the curve from cumulative SUV–volume histograms were measured and tested for reproducibility of the distribution of 18F-FDG uptake. Results: 24 patients had two pre-treatment PET/CT scans and 21 patients had two pre-treatment PET/MR scans available for further analyses. Mean difference for SUVmax, peak and mean was approximately 4% for PET/CT and 3% for PET/MR, with 95% limits of agreement less than ±20%. CV was small (5–7%) for both modalities. There was no significant difference in CVs between PET/CT and PET/MR (p = 0.31). SUVmax was not more reproducible than SUVpeak (p = 0.09). Conclusion: 18F-FDG uptake in PET/CT and PET/MR is highly reproducible and we found no difference in reproducibility between PET/CT and PET/MR. Advances in knowledge: This is the first report to test reproducibility of PET/CT and PET/MR. PMID:25634069

  5. Contributions of Gene Marking to Cell and Gene Therapies

    PubMed Central

    Barese, Cecilia N.

    2011-01-01

    Abstract The first human genetic modification studies used replication-incompetent integrating vector vectors to introduce marker genes into T lymphocytes and subsequently into hematopoietic stem cells. Such studies have provided numerous insights into the biology of hematopoiesis and immune reconstitution and contributed to clinical development of gene and cell therapies. Tracking of hematopoietic reconstitution and analysis of the origin of residual malignant disease after hematopoietic transplantation has been possible via gene marking. Introduction of selectable marker genes has enabled preselection of specific T-cell populations for tumor and viral immunotherapy and reduced the threat of graft-versus-host disease, improving the survival of patients after allogeneic marrow transplantation. Marking studies in humans, murine xenografts, and large animals have helped optimize conditions for gene transfer into CD34+ hematopoietic progenitors, contributing to the achievement of gene transfer efficiencies sufficient for clinical benefit in several serious genetic diseases such as X-linked severe combined immunodeficiency and adrenoleukodystropy. When adverse events linked to insertional mutagenesis arose in clinical gene therapy trials for inherited immunodeficiencies, additional animal studies using gene-marking vectors have greatly increased our understanding of genotoxicity. The knowledge gained from these studies is being translated into new vector designs and clinical protocols, which we hope will continue to improve the efficiency, effectiveness and safety of these promising therapeutic approaches. PMID:21261461

  6. Contributions of gene marking to cell and gene therapies.

    PubMed

    Barese, Cecilia N; Dunbar, Cynthia E

    2011-06-01

    The first human genetic modification studies used replication-incompetent integrating vector vectors to introduce marker genes into T lymphocytes and subsequently into hematopoietic stem cells. Such studies have provided numerous insights into the biology of hematopoiesis and immune reconstitution and contributed to clinical development of gene and cell therapies. Tracking of hematopoietic reconstitution and analysis of the origin of residual malignant disease after hematopoietic transplantation has been possible via gene marking. Introduction of selectable marker genes has enabled preselection of specific T-cell populations for tumor and viral immunotherapy and reduced the threat of graft-versus-host disease, improving the survival of patients after allogeneic marrow transplantation. Marking studies in humans, murine xenografts, and large animals have helped optimize conditions for gene transfer into CD34(+) hematopoietic progenitors, contributing to the achievement of gene transfer efficiencies sufficient for clinical benefit in several serious genetic diseases such as X-linked severe combined immunodeficiency and adrenoleukodystrophy. When adverse events linked to insertional mutagenesis arose in clinical gene therapy trials for inherited immunodeficiencies, additional animal studies using gene-marking vectors have greatly increased our understanding of genotoxicity. The knowledge gained from these studies is being translated into new vector designs and clinical protocols, which we hope will continue to improve the efficiency, effectiveness and safety of these promising therapeutic approaches.

  7. An improved method of electroporation for introducing biologically active foreign genes into cultured mammalian cells

    SciTech Connect

    Tatsuka, Masaaki; Orita, Satoshi; Yagi, Takashi; Kakunaga, Takeo )

    1988-09-01

    The authors have developed a modified, reproducible, and efficient method for introducing cloned genes into mammalian cells by using an electric field followed by treatment with sodium butyrate. Transfection frequencies with plasmid pSV2-neo, consisting of an antibiotic (G418) resistance gene and simian virus 40 (SV40) early promoter, by electroporation were higher than those by calcium phosphate DNA precipitation. Treatment with sodium butyrate following electroporation significantly increased the frequency of transfection in various types of cell lines and primary cultured cells including human skin fibroblasts. Treatment with sodium butyrate also increased the transient expression of the gene for chloramphenicol acetyltransferase when the gene was introduced into BALB/c 3T3 cells by eletroporation. Electroporation combined with sodium butyrate treatment is an improved method for stable and transient biochemical transformation of foreign genes in cultured mammalian cells.

  8. Gene copy number and cell cycle arrest

    NASA Astrophysics Data System (ADS)

    Ghosh, Bhaswar; Bose, Indrani

    2006-03-01

    The cell cycle is an orderly sequence of events which ultimately lead to the division of a single cell into two daughter cells. In the case of DNA damage by radiation or chemicals, the damage checkpoints in the G1 and G2 phases of the cell cycle are activated. This results in an arrest of the cell cycle so that the DNA damage can be repaired. Once this is done, the cell continues with its usual cycle of activity. We study a mathematical model of the DNA damage checkpoint in the G2 phase which arrests the transition from the G2 to the M (mitotic) phase of the cell cycle. The tumor suppressor protein p53 plays a key role in activating the pathways leading to cell cycle arrest in mammalian systems. If the DNA damage is severe, the p53 proteins activate other pathways which bring about apoptosis, i.e., programmed cell death. Loss of the p53 gene results in the proliferation of cells containing damaged DNA, i.e., in the growth of tumors which may ultimately become cancerous. There is some recent experimental evidence which suggests that the mutation of a single copy of the p53 gene (in the normal cell each gene has two identical copies) is sufficient to trigger the formation of tumors. We study the effect of reducing the gene copy number of the p53 and two other genes on cell cycle arrest and obtain results consistent with experimental observations.

  9. 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.

  10. 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

  11. 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

  12. Gene and cell therapy for heart failure.

    PubMed

    de Muinck, Ebo D

    2009-08-01

    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 Ca(2+) pump in the sarcoplasmic reticulum (SR), SRCa(2+)-ATPase has been shown to correct deficient Ca(2+) 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.

  13. Imaging gene expression in single living cells

    PubMed Central

    Shav-Tal, Yaron; Singer, Robert H.; Darzacq, Xavier

    2016-01-01

    Technical advances in the field of live-cell imaging have introduced the cell biologist to a new, dynamic, subcellular world. The static world of molecules in fixed cells has now been extended to the time dimension. This allows the visualization and quantification of gene expression and intracellular trafficking events of the studied molecules and the associated enzymatic processes in individual cells, in real time. PMID:15459666

  14. Reproducibility of protein identification of selected cell types in Barrett's esophagus analyzed by combining laser-capture microdissection and mass spectrometry.

    PubMed

    Stingl, Christoph; van Vilsteren, Frederike G I; Guzel, Coskun; Ten Kate, Fiebo J W; Visser, Mike; Krishnadath, Kausilia K; Bergman, Jacques J; Luider, Theo M

    2011-01-01

    Barrett's esophagus (BE) is associated with increased risk of esophageal adenocarcinoma (EAC) and characterized by replacement of normal esophageal squamous epithelium by columnar epithelium. These alterations are also reflected in changes in the protein-expression profiles of the cell types involved. To separately investigate the proteomes of selected cell-types we combined laser-capture microdissection (LCM) and liquid chromatography-mass spectrometry (LC-MS). Aims were to determine the sensitivity, specificity, and technical reproducibility of the sampling method, and the biological variability within and between biopsies and patients. Frozen biopsies were cryo-sectioned, samples of around 2000 epithelial or stroma cells microdissected, digested and measured by Orbitrap LC-MS. Proteins were then identified by MS/MS database search and quantified by label-free analysis. An average of 366 protein-groups were identified per sample, and more protein-groups were found in epithelial samples than in stromal samples (442 vs 301, p < 0.0001). Altogether, 1254 distinct protein-groups were found, 289 and 88 of them significantly more often in epithelial and stroma samples, respectively. We assessed five different types of reproducibilities (run-to-run, intrabiopsy, biopsy-to-biopsy, experiment-to-experiment, and patient-to-patient) for protein identification and protein quantification. Reproducibility of protein identification ranged from 78 to 57%, and standard deviation of protein quantification was on patient-to-patient level four times higher than for run-to-run. We conclude that sampling around 2000 cells requires groups of 32 samples to detect significant, over 10-fold differences in protein abundances and thus creates a successful compromise between throughput and quality of results. We therefore believe that this method is suitable for investigating protein-expression profiles during carcinogenesis.

  15. 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.

  16. Reproducible High Yields of Recombinant Adeno-Associated Virus Produced Using Invertebrate Cells in 0.02- to 200-Liter Cultures

    PubMed Central

    Cecchini, Sylvain; Virag, Tamas

    2011-01-01

    Abstract The large amounts of recombinant adeno-associated virus (rAAV) vector needed for clinical trials and eventual commercialization require robust, economical, reproducible, and scalable production processes compatible with current good manufacturing practice. rAAV produced using baculovirus and insect cells satisfies these conditions; however, recovering rAAV particles from 200-liter bioreactors is more complicated than bench-scale vector preparations. Using a variety of processing media, we developed a reliable and routine downstream procedure for rAAV production that is scalable from 0.02- to 200-liter cultures. To facilitate the upstream process, we adapted the titerless infected-cell preservation and scale-up process for rAAV production. Single-use aliquots of cryopreserved baculovirus-infected insect cells (BIIC) are thawed and added to the suspension culture to achieve the desired ratio of BIIC to rAAV-producer cells. By using conditions established with small-scale cultures, rAAV was produced in larger volume cultures. Strikingly consistent rAAV yields were attained in cultures ranging from 10 liters to 200 liters. Based on the final yield, each cell produced 18,000 ± 6,800 particles of purified rAAV in 10-, 20-, 100-, and 200-liter cultures. Thus, with an average cell density of 4.32 × 106 cells/ml, ≥1016 purified rAAV particles are produced from 100 to 200 liters. The downstream process resulted in about 20% recovery estimated from comparing the quantities of capsid protein antigen in the crude bioreactor material and in the final, purified product. The ease and reproducibility of rAAV production in 200-liter bioreactors suggest that the limit has not been reached, and 500-liter productions are planned. PMID:21381980

  17. Synthetic therapeutic gene circuits in mammalian cells.

    PubMed

    Ye, Haifeng; Fussenegger, Martin

    2014-08-01

    In the emerging field of synthetic biology, scientists are focusing on designing and creating functional devices, systems, and organisms with novel functions by engineering and assembling standardised biological building blocks. The progress of synthetic biology has significantly advanced the design of functional gene networks that can reprogram metabolic activities in mammalian cells and provide new therapeutic opportunities for future gene- and cell-based therapies. In this review, we describe the most recent advances in synthetic mammalian gene networks designed for biomedical applications, including how these synthetic therapeutic gene circuits can be assembled to control signalling networks and applied to treat metabolic disorders, cancer, and immune diseases. We conclude by discussing the various challenges and future prospects of using synthetic mammalian gene networks for disease therapy.

  18. 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

  19. 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

  20. 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. PMID:27513567

  1. Gene and stem cell therapy for diabetes.

    PubMed

    Calne, Roy Y; Ghoneim, Mohamed A; Lee, K O; Uin, Gan Shu

    2013-01-01

    Gene and stem cell therapy has been on the scientific agenda in many laboratories for more than 20 years. The literature is enormous, but practical applications have been few. Recently advances in stem cell biology and gene therapy are clarifying some of the issues. I have made a few observations concerning our own studies on bone marrow mesenchymal stem cells cultured to produce a small percentage of insulin-producing cells and human insulin gene engineered into Lenti and AA viruses. The aim of clinical application would still seem to be several years away, if all goes well. The first step will be to produce enough insulin-secreting cells to be of potential value to patients. The next crucial question will be how to persuade the cells to respond to blood glucose levels swiftly and appropriately. With both stem cell and gene therapy, another important factor will be to ensure that any positive results will continue long enough to be preferable to insulin injections. PMID:25095498

  2. Galvanic-cell-induced growth of Ag nanosheet-assembled structures as sensitive and reproducible SERS substrates.

    PubMed

    Li, Zhongbo; Meng, Guowen; Huang, Qing; Zhu, Chuhong; Zhang, Zhuo; Li, Xiangdong

    2012-11-19

    SERS up: Ag nanosheet-assembled structures with controlled morphologies were achieved on indium tin oxide substrates by galvanic-cell-induced growth (see figure). These structures exhibit a highly active and homogeneous surface-enhanced Raman scattering (SERS) effect, and show promising potential as reliable SERS substrates for detection of trace polychlorinated biphenyls.

  3. 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. PMID:25777046

  4. T Cell Receptor Gene Therapy for Cancer

    PubMed Central

    Schmitt, Thomas M.; Ragnarsson, Gunnar B.

    2009-01-01

    Abstract T cell-based adoptive immunotherapy has been shown to be a promising treatment for various types of cancer. However, adoptive T cell therapy currently requires the custom isolation and characterization of tumor-specific T cells from each patient—a process that can be not only difficult and time-consuming but also often fails to yield high-avidity T cells, which together have limited the broad application of this approach as a clinical treatment. Employing T cell receptor (TCR) gene therapy as a component of adoptive T cell therapy strategies can overcome many of these obstacles, allowing autologous T cells with a defined specificity to be generated in a much shorter time period. Initial studies using this approach have been hampered by a number of technical difficulties resulting in low TCR expression and acquisition of potentially problematic specificities due to mispairing of introduced TCR chains with endogenous TCR chains. The last several years have seen substantial progress in our understanding of the multiple facets of TCR gene therapy that will have to be properly orchestrated for this strategy to succeed. Here we outline the challenges of TCR gene therapy and the advances that have been made toward realizing the promise of this approach. PMID:19702439

  5. Novel micro-bioreactor high throughput technology for cell culture process development: Reproducibility and scalability assessment of fed-batch CHO cultures.

    PubMed

    Amanullah, Ashraf; Otero, Jose Manuel; Mikola, Mark; Hsu, Amy; Zhang, Jinyou; Aunins, John; Schreyer, H Brett; Hope, James A; Russo, A Peter

    2010-05-01

    With increasing timeline pressures to get therapeutic and vaccine candidates into the clinic, resource intensive approaches such as the use of shake flasks and bench-top bioreactors may limit the design space for experimentation to yield highly productive processes. The need to conduct large numbers of experiments has resulted in the use of miniaturized high-throughput (HT) technology for process development. One such high-throughput system is the SimCell platform, a robotically driven, cell culture bioreactor system developed by BioProcessors Corp. This study describes the use of the SimCell micro-bioreactor technology for fed-batch cultivation of a GS-CHO transfectant expressing a model IgG4 monoclonal antibody. Cultivations were conducted in gas-permeable chambers based on a micro-fluidic design, with six micro-bioreactors (MBs) per micro-bioreactor array (MBA). Online, non-invasive measurement of total cell density, pH and dissolved oxygen (DO) was performed. One hundred fourteen parallel MBs (19 MBAs) were employed to examine process reproducibility and scalability at shake flask, 3- and 100-L bioreactor scales. The results of the study demonstrate that the SimCell platform operated under fed-batch conditions could support viable cell concentrations up to least 12 x 10(6) cells/mL. In addition, both intra-MB (MB to MB) as well as intra-MBA (MBA to MBA) culture performance was found to be highly reproducible. The intra-MB and -MBA variability was calculated for each measurement as the coefficient of variation defined as CV (%) = (standard deviation/mean) x 100. The % CV values for most intra-MB and intra-MBA measurements were generally under 10% and the intra-MBA values were slightly lower than those for intra-MB. Cell growth, process parameters, metabolic and protein titer profiles were also compared to those from shake flask, bench-top, and pilot scale bioreactor cultivations and found to be within +/-20% of the historical averages.

  6. Vasa genes: Emerging roles in the germ line and in multipotent cells

    PubMed Central

    Gustafson, Eric A.; Wessel, Gary M.

    2011-01-01

    Sexually reproducing metazoans establish a cell lineage during development that is ultimately dedicated to gamete production. Work in a variety of animals suggests that a group of conserved molecular determinants function in this germ line maintenance and function. The most universal of these genes are vasa and vasa-like DEAD box RNA helicase genes. However, recent evidence indicates that vasa genes also function in other cell types, distinct from the germ line. Here we evaluate our current understanding of vasa function and its regulation during development, addressing vasa’s emerging role in multipotent cells. We also explore the evolutionary diversification of the amino-terminal domain of this gene and how this impacts the association of vasa with nuage-like perinuclear structures. PMID:20586054

  7. 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. PMID:23108858

  8. Analysis of the interlaboratory and intralaboratory reproducibility of the enhancement of simian adenovirus SA7 transformation of Syrian hamster embryo cells by model carcinogenic and noncarcinogenic compounds.

    PubMed

    Schechtman, L M; Hatch, G G; Anderson, T M; Putman, D L; Kouri, R E; Cameron, J W; Nims, R W; Spalding, J W; Tennant, R W; Lubet, R A

    1986-01-01

    The intralaboratory and interlaboratory reproducibility of a DNA virus (SA7) transformation enhancement assay was investigated using nine carcinogenic and noncarcinogenic compounds representing a variety of chemical classes. By the use of standardized procedures designed to limit assay variables, replicate assay data were collected in two independent laboratories and analyzed for concurrence. The carcinogens, 7,12-dimethylbenz(a)anthracene, benzo(a)pyrene, and N-methyl-N'-nitro-N-nitrosoguanidine yielded reproducible dose-dependent cytotoxicity and positive transformation effects (defined as statistically significant [p less than or equal to 0.05] enhancement of virus transformation at two or more consecutive dose levels) in all experiments in both laboratories. The carcinogens lead chromate, diethylnitrosamine, 4-nitroquinoline-N-oxide, and 2-acetylaminofluorene demonstrated enhancement of SA7 transformation at two or more dose levels in 40-50% of the assays. The noncarcinogenic structural analogs anthracene and pyrene consistently did not produce positive assay responses when tested at dose levels up to the limits of solubility. Good interlaboratory concurrence was demonstrated for these model compounds in the Syrian hamster embryo cell-SA7 assay. PMID:3089771

  9. Cloning to reproduce desired genotypes.

    PubMed

    Westhusin, M E; Long, C R; Shin, T; Hill, J R; Looney, C R; Pryor, J H; Piedrahita, J A

    2001-01-01

    Cloned sheep, cattle, goats, pigs and mice have now been produced using somatic cells for nuclear transplantation. Animal cloning is still very inefficient with on average less than 10% of the cloned embryos transferred resulting in a live offspring. However successful cloning of a variety of different species and by a number of different laboratory groups has generated tremendous interest in reproducing desired genotypes. Some of these specific genotypes represent animal cell lines that have been genetically modified. In other cases there is a significant demand for cloning animals characterized by their inherent genetic value, for example prize livestock, household pets and rare or endangered species. A number of different variables may influence the ability to reproduce a specific genotype by cloning. These include species, source of recipient ova, cell type of nuclei donor, treatment of donor cells prior to nuclear transfer, and the techniques employed for nuclear transfer. At present, there is no solid evidence that suggests cloning will be limited to only a few specific animals, and in fact, most data collected to date suggests cloning will be applicable to a wide variety of different animals. The ability to reproduce any desired genotype by cloning will ultimately depend on the amount of time and resources invested in research.

  10. 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.

  11. 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.

  12. Reproducibility of Dynamic Contrast-Enhanced MRI in Renal Cell Carcinoma: A Prospective Analysis on Intra- and Interobserver and Scan-Rescan Performance of Pharmacokinetic Parameters.

    PubMed

    Wang, Haiyi; Su, Zihua; Ye, Huiyi; Xu, Xiao; Sun, Zhipeng; Li, Lu; Duan, Feixue; Song, Yuanyuan; Lambrou, Tryphon; Ma, Lin

    2015-09-01

    The objective of this study was to investigate the intra- and interobserver as well as scan-rescan reproducibility of quantitative parameters of renal cell carcinomas (RCCs) with dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). A total of 21 patients with clear cell RCCs (17 men, 4 woman; age 37-69 years, mean age 54.6 years, mean size, 5.0 ± 2.2 cm) were prospectively recruited from September 2012 to November 2012. Patients underwent paired DCE-MRI studies on a 3.0 T MR system with an interval of 48 to 72 hours. The extended-Tofts model and population-based arterial input function were used to calculate kinetic parameters. Three observers defined the 2-dimensional whole-tumor region of interest at the slice with the maximum diameter of the RCC. Intraobserver and scan-rescan differences were assessed using paired t tests, whereas interobserver differences using two-way analysis of variance. Intra- and interobserver reproducibility and scan-rescan reproducibility were evaluated using within-subject coefficient of variation (wCoV) and intraclass correlation coefficient (ICC). There were no significant intra-, interobserver, or scan-rescan differences in parameters (all P > 0.05). All ICCs for intra- and interobserver agreements were >0.75 (P < 0.05), whereas the scan-rescan agreement was moderate to good; V(e) (0.764, 95% confidence interval [CI]: 0.378-0.925) and K(ep) (0.906, 95% CI: 0.710-0.972) had higher ICC than K(trans) (0.686; 95% CI: 0.212-0.898) and V(p) (0.657; 95% CI: 0.164-0.888). In intra- and interobserver variability analyses, all parameters except V(p) had low wCoV values. K(trans) and V(e) had slightly lower intraobserver wCoV (1.2% and 0.9%) compared with K(ep) (3.7%), whereas all 3 of these parameters had similar interobserver wCoV values (2.5%, 3.1%, and 2.9%, respectively). Regarding scan-rescan variability, K(trans) and K(ep) showed slightly higher variation (15.6% and 15.4%) than V(e) (10.1%). V(p) had the largest

  13. 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.

  14. HSP-72 Accelerated Expression in Mononuclear Cells Induced In Vivo by Acetyl Salicylic Acid Can Be Reproduced In Vitro when Combined with H2O2

    PubMed Central

    Sandoval-Montiel, Alvaro A.; Zentella-de-Piña, Martha; Ventura-Gallegos, José L.; Frías-González, Susana; López-Macay, Ambar; Zentella-Dehesa, Alejandro

    2013-01-01

    Background Among NSAIDs acetyl salicylic acid remains as a valuable tool because of the variety of benefic prophylactic and therapeutic effects. Nevertheless, the molecular bases for these responses have not been complete understood. We explored the effect of acetyl salicylic acid on the heat shock response. Results Peripheral blood mononuclear cells from rats challenged with acetyl salicylic acid presented a faster kinetics of expression of HSP-72 messenger RNA and protein in response to in vitro heat shock. This effect reaches its maximum 2 h after treatment and disappeared after 5 h. On isolated peripheral blood mononuclear cells from untreated rats, incubation with acetyl salicylic acid was ineffective to produce priming, but this effect was mimicked when the cells were incubated with the combination of H2O2+ ASA. Conclusions Administration of acetyl salicylic acid to rats alters HSP-72 expression mechanism in a way that it becomes more efficient in response to in vitro heat shock. The fact that in vitro acetyl salicylic acid alone did not induce this priming effect implies that in vivo other signals are required. Priming could be reproduces in vitro with the combination of acetyl salicylic acid+H2O2. PMID:23762376

  15. Optogenetics for gene expression in mammalian cells.

    PubMed

    Müller, Konrad; Naumann, Sebastian; Weber, Wilfried; Zurbriggen, Matias D

    2015-02-01

    Molecular switches that are controlled by chemicals have evolved as central research instruments in mammalian cell biology. However, these tools are limited in terms of their spatiotemporal resolution due to freely diffusing inducers. These limitations have recently been addressed by the development of optogenetic, genetically encoded, and light-responsive tools that can be controlled with the unprecedented spatiotemporal precision of light. In this article, we first provide a brief overview of currently available optogenetic tools that have been designed to control diverse cellular processes. Then, we focus on recent developments in light-controlled gene expression technologies and provide the reader with a guideline for choosing the most suitable gene expression system.

  16. Established epigenetic modifications determine the expression of developmentally regulated globin genes in somatic cell hybrids.

    PubMed Central

    Stanworth, S J; Roberts, N A; Sharpe, J A; Sloane-Stanley, J A; Wood, W G

    1995-01-01

    Somatic cell hybrids generated from transgenic mouse cells have been used to examine the developmental regulation of human gamma-to-beta-globin gene switching. In hybrids between mouse erythroleukemia (MEL) cells and transgenic erythroblasts taken at various stages of development, there was regulated expression of the human fetal gamma and adult beta genes, reproducing the in vivo pattern prior to fusion. Hybrids formed from embryonic blood cells produced predominantly gamma mRNA, whereas beta gene expression was observed in adult hybrids and a complete range of intermediate patterns was found in fetal liver hybrids. The adult environment of the MEL cells, therefore, did not appear to influence selective transcription from this gene complex. Irradiation of the embryonic erythroid cells prior to fusion resulted in hybrids containing only small fragments of donor chromosomes, but the pattern of gene expression did not differ from that of unirradiated hybrids. This finding suggests that continued expression of trans-acting factors from the donor erythroblasts is not necessary for continued expression of the human gamma gene in MEL cells. These results contrast with the lack of developmental regulation of the cluster after transfection of naked DNA into MEL cells and suggest that epigenetic processes established during normal development result in the gene cluster adopting a developmental stage-specific, stable conformation which is maintained through multiple rounds of replication and transcription in the MEL cell hybrids. On prolonged culture, hybrids that initially expressed only the gamma transgene switched to beta gene expression. The time period of switching, from approximately 10 to > 40 weeks, was similar to that seen previously in human fetal erythroblast x MEL cell hybrids but in this case bore no relationship to the time of in vivo switching. It seems unlikely, therefore, that switching in these hybrids is regulated by a developmental clock. PMID:7623793

  17. 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.

  18. Reproducibility of 'Intelligent' Contouring of Gross Tumor Volume in Non-Small-Cell Lung Cancer on PET/CT Images Using a Standardized Visual Method

    SciTech Connect

    Bayne, Michael; Hicks, Rodney J.; Everitt, Sarah; Fimmell, Natalie

    2010-07-15

    Purpose: Positron emission tomography/computed tomography (PET/CT) is increasingly used for delineating gross tumor volume (GTV) in non-small-cell lung cancer (NSCLC). The methodology for contouring tumor margins remains controversial. We developed a rigorous visual protocol for contouring GTV that uses all available clinical information and studied its reproducibility in patients from a prospective PET/CT planning trial. Methods and Materials: Planning PET/CT scans from 6 consecutive patients were selected. Six 'observers' (two radiation oncologists, two nuclear medicine physicians, and two radiologists) contoured GTVs for each patient using a predefined protocol and subsequently recontoured 2 patients. For the estimated GTVs and axial distances, least-squares means for each observer and for each case were calculated and compared, using the F test and pairwise t-tests. In five cases, tumor margins were also autocontoured using standardized uptake value (SUV) cutoffs of 2.5 and 3.5 and 40% SUV{sub max}. Results: The magnitude of variation between observers was small relative to the mean (coefficient of variation [CV] = 3%), and the total variation (intraclass correlation coefficient [ICC] = 3%). For estimation of superior/inferior (SI), left/right (LR), and anterior/posterior (AP) borders of the GTV, differences between observers were also small (AP, CV = 2%, ICC = 0.4%; LR, CV = 6%, ICC = 2%; SI, CV 4%, ICC = 2%). GTVs autocontoured generated using SUV 2.5, 3.5, and 40% SUV{sub max} differed widely in each case. An SUV contour of 2.5 was most closely correlated with the mean GTV defined by the human observers. Conclusions: Observer variation contributed little to total variation in the GTV and axial distances. A visual contouring protocol gave reproducible results for contouring GTV in NSCLC.

  19. Optimizing autologous cell grafts to improve stem cell gene therapy.

    PubMed

    Psatha, Nikoletta; Karponi, Garyfalia; Yannaki, Evangelia

    2016-07-01

    Over the past decade, stem cell gene therapy has achieved unprecedented curative outcomes for several genetic disorders. Despite the unequivocal success, clinical gene therapy still faces challenges. Genetically engineered hematopoietic stem cells are particularly vulnerable to attenuation of their repopulating capacity once exposed to culture conditions, ultimately leading to low engraftment levels posttransplant. This becomes of particular importance when transduction rates are low or/and competitive transplant conditions are generated by reduced-intensity conditioning in the absence of a selective advantage of the transduced over the unmodified cells. These limitations could partially be overcome by introducing megadoses of genetically modified CD34(+) cells into conditioned patients or by transplanting hematopoietic stem cells hematopoietic stem cells with high engrafting and repopulating potential. On the basis of the lessons gained from cord blood transplantation, we summarize the most promising approaches to date of increasing either the numbers of hematopoietic stem cells for transplantation or/and their engraftability, as a platform toward the optimization of engineered stem cell grafts. PMID:27106799

  20. Identifying gene expression modules that define human cell fates.

    PubMed

    Germanguz, I; Listgarten, J; Cinkornpumin, J; Solomon, A; Gaeta, X; Lowry, W E

    2016-05-01

    Using a compendium of cell-state-specific gene expression data, we identified genes that uniquely define cell states, including those thought to represent various developmental stages. Our analysis sheds light on human cell fate through the identification of core genes that are altered over several developmental milestones, and across regional specification. Here we present cell-type specific gene expression data for 17 distinct cell states and demonstrate that these modules of genes can in fact define cell fate. Lastly, we introduce a web-based database to disseminate the results.

  1. An efficient and reproducible protocol for the production of salt tolerant transgenic wheat plants expressing the Arabidopsis AtNHX1 gene

    PubMed Central

    Moghaieb, Reda EA; Sharaf, Ahmed N; Soliman, Mohamed H; El-Arabi, Nagwa I; Momtaz, Osama A

    2014-01-01

    We present an efficient method for the production of transgenic salt tolerant hexaploid wheat plants expressing the Arabidopsis AtNHX1 gene. Wheat mature zygotic embryos were isolated from two hexaploid bread wheat (Triticum aestivum) cultivars (namely: Gemmeiza 9 and Gemmeiza 10) and were transformed with the A. tumefaciens LBA4404 harboring the pBI-121 vector containing the AtNHX1 gene. Transgenic wheat lines that express the gus intron was obtained and used as control. The results confirmed that npt-II gene could be transmitted and expressed in the T2 following 3:1 Mendelian segregation while the control plant couldn't. The data indicate that, the AtNHX1 gene was integrated in a stable manner into the wheat genome and the corresponding transcripts were expressed. The transformation efficiency was 5.7 and 7.5% for cultivars Gemmeiza 10 and Gemmeiza 9, respectively. A greenhouse experiment was conducted to investigate the effect of AtNHX1 gene in wheat salt tolerance. The transgenic wheat lines could maintain high growth rate under salt stress condition (350 mM NaCl) while the control plant couldn’t. The results confirmed that Na+/H+ antiporter gene AtNHX1 increased salt tolerance by increasing Na+ accumulation and keeping K+/Na+ balance. Thus, transgenic plants showed high tolerance to salt stress and can be considered as a new genetic resource in breeding programs. PMID:25007249

  2. 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

  3. Glyphosate selected amplification of the 5-enolpyruvylshikimate-3-phosphate synthase gene in cultured carrot cells.

    PubMed

    Shyr, Y Y; Hepburn, A G; Widholm, J M

    1992-04-01

    CAR and C1, two carrot (Daucus carota L.) suspension cultures of different genotypes, were subjected to stepwise selection for tolerance to the herbicide glyphosate [(N-phosphonomethyl)glycine]. The specific activity of the target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), as well as the mRNA level and copy number of the structural gene increased with each glyphosate selection step. Therefore, the tolerance to glyphosate is due to stepwise amplification of the EPSPS genes. During the amplification process, DNA rearrangement did not occur within the EPSPS gene of the CAR cell line but did occur during the selection step from 28 to 35 mM glyphosate for the C1 cell line, as determined by Southern hybridization of selected cell DNA following EcoRI restriction endonuclease digestion. Two cell lines derived from a previously selected glyphosate-tolerant cell line (PR), which also had undergone EPSPS gene amplification but have been maintained in glyphosate-free medium for 2 and 5 years, have lost 36 and 100% of the increased EPSPS activity, respectively. Southern blot analysis of these lines confirms that the amplified DNA is relatively stable in the absence of selection. These studies demonstrate that stepwise selection for glyphosate resistance reproducibly produces stepwise amplification of the EPSPS genes. The relative stability of this amplification indicates that the amplified genes are not extrachromosomal.

  4. Establishment of medakafish as a model for stem cell-based gene therapy: efficient gene delivery and potential chromosomal integration by baculoviral vectors.

    PubMed

    Yan, Yan; Du, Juan; Chen, Tiansheng; Yi, Meisheng; Li, Mingyou; Wang, Shu; Li, Chang Ming; Hong, Yunhan

    2009-08-01

    Viral vectors hold promise and challenges in gene therapy. Specifically, we have previously shown that baculoviral (BV) vectors have a high efficiency of gene delivery in human embryonic stem (ES) cells. Here we report the development of a complementary system to further our evaluation by utilizing the laboratory fish medaka that has ES cell lines and tools for experimental analyses in vitro and in vivo. We show that BV vectors can give rise to almost 100% of transient gene delivery in the medaka ES cell line MES1. BV-transduced MES1 cells reproducibly (at approximately 10(-5)) produce GFP-expressing colonies that, upon manual isolation, develop into stable clones during 300 days of culture. Surprisingly, BV transduction can also mediate efficient gene integration in the medaka genome, as fluorescent in situ hybridization revealed the presence of the BV-delivered gfp transgene in multiple locations in nuclei and on various chromosomes of metaphase spreads. We show that BV transduction does not compromise the genome stability and pluripotency of MES1 cells. We conclude that BV can efficiently mediate gene delivery and chromosomal integration in medaka ES cells. Therefore, medaka provides a powerful system for analyzing the potential of BV-mediated gene delivery in stem cells and gene therapy.

  5. Concentration of bone marrow mononuclear cells for in vitro treatment and AB0-incompatible transplantation: a rapid and reproducible procedure using the haemonetics V50 cell separator.

    PubMed

    Wiesneth, M; Hertenstein, B; Koerner, K; Heimpel, H; Heit, W

    1988-01-01

    Forty-nine allogeneic and 14 autologous bone marrow grafts were processed with the Haemonetics V50 cell separator (Haemonetics Corp., Braintree, USA) for in vitro treatment with antibodies and cryopreservation respectively. The concentration of hemopoietic progenitor cells was performed without any sedimentation or density gradient agents. The recovery is given in percent (mean +/- sd) of the original marrow values: mononuclear cells (MNC) 74 +/- 10%, polymorphonuclear cells (PMC) 48 +/- 17%, red blood cells (RBC) 12 +/- 5%, granulocyte/monocyte progenitors (CFU-GM) 83 +/- 36%, and erythroid progenitors (BFU-E) 78 +/- 38%. The recovery of nucleated cells (NC) was 90 +/- 13% and the viability 82 +/- 11% after cryopreservation. The technique described provides a simple, rapid and efficient preparation of large bone marrow volumes for in vitro treatment and AB0-incompatible transplantation.

  6. Highly Efficient, Reproducible, Uniform (CH3 NH3 )PbI3 Layer by Processing Additive Dripping for Solution-Processed Planar Heterojunction Perovskite Solar Cells.

    PubMed

    Kim, Hansol; Jeong, Hanbin; Lee, Jae Kwan

    2016-09-01

    A processing additive dripping (PAD) approach to forming highly efficient (CH3 NH3 )PbI3 (MAPbI3 ) perovskite layers was investigated. A MAPbI3 (CB/DIO) perovskite film fabricated by this approach, which included briefly dripping chlorobenzene incorporating a small amount of diiodooctane (DIO) during casting of a MAPbI3 perovskite precursor dissolved in dimethylformamide, exhibited superior smooth, uniform morphologies with high crystallinity and large grains and revealed completely homogeneous surface coverage. The surface coverage and morphology of the substrate significantly affected the photovoltaic performance of planar heterojunction (PHJ) perovskite solar cells (PrSCs), resulting in a power conversion efficiency of 11.45 % with high open-circuit voltage of 0.91 V and the highest fill factor of 80.87 %. Moreover, the PAD approach could effectively provide efficient MAPbI3 (CB/DIO) perovskite layers for highly efficient, reproducible, uniform PHJ PrSC devices without performance loss or variation even over larger active areas. PMID:27414840

  7. The regulation of gene expression in hair cells.

    PubMed

    Ryan, Allen F; Ikeda, Ryoukichi; Masuda, Masatsugu

    2015-11-01

    No genes have been discovered for which expression is limited only to inner ear hair cells. This is hardly surprising, since the number of mammalian genes is estimated to be 20-25,000, and each gene typically performs many tasks in various locations. Many genes are expressed in inner ear sensory cells and not in other cells of the labyrinth. However, these genes are also expressed in other locations, often in other sensory or neuronal cell types. How gene transcription is directed specifically to hair cells is unclear. Key transcription factors that act during development can specify cell phenotypes, and the hair cell is no exception. The transcription factor ATOH1 is well known for its ability to transform nonsensory cells of the developing inner ear into hair cells. And yet, ATOH1 also specifies different sensory cells at other locations, neuronal phenotypes in the brain, and epithelial cells in the gut. How it specifies hair cells in the inner ear, but alternate cell types in other locations, is not known. Studies of regulatory DNA and transcription factors are revealing mechanisms that direct gene expression to hair cells, and that determine the hair cell identity. The purpose of this review is to summarize what is known about such gene regulation in this key auditory and vestibular cell type.

  8. 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

  9. Magnetogastrography (MGG) Reproducibility Assessments

    NASA Astrophysics Data System (ADS)

    de la Roca-Chiapas, J. M.; Córdova, T.; Hernández, E.; Solorio, S.; Solís Ortiz, S.; Sosa, M.

    2006-09-01

    Seven healthy subjects underwent a magnetic pulse of 32 mT for 17 ms, seven times in 90 minutes. The procedure was repeated one and two weeks later. Assessments of the gastric emptying were carried out for each one of the measurements and a statistical analysis of ANOVA was performed for every group of data. The gastric emptying time was 19.22 ± 5 min. Reproducibility estimation was above 85%. Therefore, magnetogastrography seems to be an excellent technique to be implemented in routine clinical trials.

  10. 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

  11. Rheumatoid factors, B cells and immunoglobulin genes.

    PubMed

    Jefferis, R

    1995-04-01

    The paradigm of self, non-self discrimination in the immune system is under review as autoreactive B or T cells are increasingly delineated within normal individuals. The products of autoreactive B cells are, mostly, polyspecific IgM antibodies of low affinity. These 'natural' antibodies include rheumatoid factors (RF) encoded by unmutated germline immunoglobulin genes. In rheumatoid arthritis (RA) the RF may be of the IgM, IgG or IgA isotype, show evidence of somatic mutation and have increased affinity; consistent with maturation of an antigen driven immune response. This response could be initiated or driven by an auto-immunogenic form of IgG or an exogenous cross-reactive antigen. Changes in galactosylation of IgG have been reported to be a valuable diagnostic and prognostic indicator in RA. Speculation that these changes may precipitate some of the disease processes is critically reviewed.

  12. 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

  13. Highly reproducible, efficient hysteresis-less CH3NH3PbI(3-x)Cl(x) planar hybrid solar cells without requiring heat-treatment.

    PubMed

    Heo, Jin Hyuck; Im, Sang Hyuk

    2016-02-01

    CH3NH3PbI(3-x)Cl(x)(MAPbI(3-x)Cl(x)) mixed halide perovskite powder with uniform composition was synthesized via simple solution chemistry, which demonstrates highly reproducible, efficient planar type MAPbI(3-x)Cl(x) mixed halide perovskite solar cells. Pure MAPbI(3-x)Cl(x) 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 MAPbI(3-x)Cl(x) mixed halide and a selective remover of unreacted MAI and MACl byproducts. Accordingly, we could deposit a pinhole-free dense MAPbI(3-x)Cl(x) mixed halide perovskite film on a TiO2/FTO substrate through a simple one step spin-coating of pure MAPbI(3-x)Cl(x) mixed halide perovskite powder in DMF solution with HI additive, without further long heat-treatment processes. The deposited MAPbI(3-x)Cl(x) 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 MAPbI(3-x)Cl(x) 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 MAPbI(3-x)Cl(x) mixed halide perovskite solar cells was 18.65% ± 0.30% and the champion cell had 1.11 V V(oc), 22.1 mA cm(-2) J(sc), 77% F.F., and 18.9% η for forward scan conditions and 1.11 V V(oc), 22.1 mA cm(-2) J(sc), 78% F.F., and 19.1% η for reverse scan conditions. Although the thickness of the MAPbI(3-x)Cl(x) mixed halide perovskite layer varied from ∼500 nm to ∼900 nm, the efficiency was within the range of 18.3%-19.0%.

  14. Reproducing in cities.

    PubMed

    Mace, Ruth

    2008-02-01

    Reproducing in cities has always been costly, leading to lower fertility (that is, lower birth rates) in urban than in rural areas. Historically, although cities provided job opportunities, initially residents incurred the penalty of higher infant mortality, but as mortality rates fell at the end of the 19th century, European birth rates began to plummet. Fertility decline in Africa only started recently and has been dramatic in some cities. Here it is argued that both historical and evolutionary demographers are interpreting fertility declines across the globe in terms of the relative costs of child rearing, which increase to allow children to outcompete their peers. Now largely free from the fear of early death, postindustrial societies may create an environment that generates runaway parental investment, which will continue to drive fertility ever lower.

  15. Identification of C/EBPβ Target Genes in ALK+ Anaplastic Large Cell Lymphoma (ALCL) by Gene Expression Profiling and Chromatin Immunoprecipitation

    PubMed Central

    Bonzheim, Irina; Irmler, Martin; Klier-Richter, Margit; Steinhilber, Julia; Anastasov, Nataša; Schäfer, Sabine; Adam, Patrick; Beckers, Johannes; Raffeld, Mark; Fend, Falko; Quintanilla-Martinez, Leticia

    2013-01-01

    C/EBPβ (CCAAT enhancer binding protein) is a transcription factor that plays a crucial role in survival and transformation of ALK+ anaplastic large cell lymphoma (ALCL). The aim of this study was to identify the downstream targets of C/EBPβ responsible for ALK-mediated oncogenesis. C/EBPβ was knocked down in ALK+ ALCL cell lines with a C/EBPβ-shRNA, followed by gene expression profiling (GEP). GEP analysis revealed a reproducible signature of genes that were significantly regulated by C/EBPβ. Classification into biological categories revealed overrepresentation of genes involved in the immune response, apoptosis and cell proliferation. Transcriptional regulation by C/EBPβ was found in 6 of 11 (BCL2A1, G0S2, TRIB1, S100A9, DDX21 and DDIT4) genes investigated by chromatin immunoprecipitation. We demonstrated that BCL2A1, G0S2 and DDX21 play a crucial role in survival and proliferation of ALK+ ALCL cells. DDX21, a gene involved in rRNA biogenesis, was found differentially overexpressed in primary ALK+ ALCL cases. All three candidate genes were validated in primary ALCL cases by either immunohistochemistry or RT-qPCR. In conclusion, we identified and validated several key C/EBPβ-regulated genes with major impact on survival and cell growth in ALK+ ALCL, supporting the central role of C/EBPβ in ALK-mediated oncogenesis. PMID:23741337

  16. 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. PMID:27505711

  17. 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.

  18. 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

  19. 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.

  20. Targeting the exogenous htPAm gene on goat somatic cell beta-casein locus for transgenic goat production.

    PubMed

    Shen, Wei; Lan, Guocheng; Yang, Xueyi; Li, Lan; Min, Lingjiang; Yang, Zhengtian; Tian, Liyuan; Wu, Xiaojie; Sun, Yujiang; Chen, Hong; Tan, Jinghe; Deng, Jixian; Pan, Qingjie

    2007-04-01

    Combining gene targeting of animal somatic cells with nuclear transfer technique has provided a powerful method to produce transgenic animal mammary gland bioreactor. The objective of this study is to make an efficient and reproducible gene targeting in goat fetal fibroblasts by inserting the exogenous htPAm cDNA into the beta-casein locus with liposomes or electroporation so that htPAm protein might be produced in gene-targeted goat mammary gland. By gene-targeting technique, the exogenous htPAm gene was inserted to milk goat beta-casein gene sequences. Fetal fibroblasts were isolated from Day 35 fetuses of Guanzhong milk goats, and transfected with linear gene-targeting vector pGBC4htPAm using Lipefectamin-2000 and electoporation, respectively. Forty-eight gene-targeted cell colonies with homologous recombination were obtained, and three cell colonies were verified by DNA sequence analysis within the homologous recombination region. Using gene-targeted cell lines as donor cells for nuclear transfer, a total of 600 reconstructed embryos had been obtained, and 146 developed cloned embryos were transferred to 16 recipient goats, and finally three goats showed pregnancy at Day 90.

  1. Activation of Developmentally Mutated Human Globin Genes by Cell Fusion

    NASA Astrophysics Data System (ADS)

    Papayannopoulou, Thalia; Enver, Tariq; Takegawa, Susumu; Anagnou, Nicholas P.; Stamatoyannopoulos, George

    1988-11-01

    Human fetal globin genes are not expressed in hybrid cells produced by the fusion of normal human lymphocytes with mouse erythroleukemia cells. In contrast, when lymphocytes from persons with globin gene developmental mutations (hereditary persistence of fetal hemoglobin) are used for these fusions, fetal globin is expressed in the hybrid cells. Thus, mutations of developmental origin can be reconstituted in vitro by fusing mutant lymphoid cells with differentiated cell lines of the proper lineage. This system can readily be used for analyses, such as globin gene methylation, that normally require large numbers of pure nucleated erythroid cells, which are difficult to obtain.

  2. Transient Gene Expression in Epidermal Cells of Plant Leaves by Biolistic DNA Delivery

    PubMed Central

    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. PMID:23104330

  3. Gene-marking and haemopoietic stem-cell transplantation.

    PubMed

    Heslop, H E; Rooney, C M; Brenner, M K

    1995-12-01

    Gene transfer has allowed a number of biological issues in haematopoietic stem-cell transplantation to be addressed. Gene-marking studies have shown that residual malignant cells in infused marrow may contribute to relapse in acute myeloid leukaemia, neuroblastoma and chronic myeloid leukaemia. Double gene-marking techniques with distinguishable retroviral vectors are being used to compare purging techniques and the reconstitution of different sources of stem cells. In allogeneic bone-marrow transplantation, gene-marking has demonstrated that adoptively transferred cytotoxic T cells can persist and reconstitute antiviral immunity.

  4. Reproducible Experiment Platform

    NASA Astrophysics Data System (ADS)

    Likhomanenko, Tatiana; Rogozhnikov, Alex; Baranov, Alexander; Khairullin, Egor; Ustyuzhanin, Andrey

    2015-12-01

    Data analysis in fundamental sciences nowadays is an essential process that pushes frontiers of our knowledge and leads to new discoveries. At the same time we can see that complexity of those analyses increases fast due to a) enormous volumes of datasets being analyzed, b) variety of techniques and algorithms one have to check inside a single analysis, c) distributed nature of research teams that requires special communication media for knowledge and information exchange between individual researchers. There is a lot of resemblance between techniques and problems arising in the areas of industrial information retrieval and particle physics. To address those problems we propose Reproducible Experiment Platform (REP), a software infrastructure to support collaborative ecosystem for computational science. It is a Python based solution for research teams that allows running computational experiments on shared datasets, obtaining repeatable results, and consistent comparisons of the obtained results. We present some key features of REP based on case studies which include trigger optimization and physics analysis studies at the LHCb experiment.

  5. 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.

  6. Assignment of the group A rotavirus NSP4 gene into genotypes using a hemi-nested multiplex PCR assay: a rapid and reproducible assay for strain surveillance studies.

    PubMed

    Bányai, Krisztián; Bogdán, Agnes; Szücs, György; Arista, Serenella; De Grazia, Simona; Kang, Gagandeep; Banerjee, Indrani; Iturriza-Gómara, Miren; Buonavoglia, Canio; Martella, Vito

    2009-03-01

    The rotavirus non-structural protein NSP4 has been implicated in a number of biological functions during the rotavirus cellular cycle and pathogenesis, and has been addressed as a target for vaccine development. The NSP4 gene has been classified into six genotypes (A-F). A semi-nested triplex PCR was developed for genotyping the major human NSP4 genotypes (A-C), which are common in human rotavirus strains but are also shared among most mammalian rotavirus strains. A total of 192 previously characterized human strains representing numerous G and P type specificities (such as G1P[8], G1P[4], G2P[4], G3P[3], G3P[8], G3P[9], G4P[6], G4P[8], G6P[4], G6P[9], G6P[14], G8P[10], G8P[14], G9P[8], G9P[11], G10P[11], G12P[6] and G12P[8]) were tested for NSP4 specificity by the collaborating laboratories. An additional 35 animal strains, including the reference laboratory strains SA11 (simian, G3P[2]), NCDV (bovine, G6P[1]), K9 and CU-1 (canine, G3P[3]), together with 31 field isolates (canine, G3P[3]; feline, G3P[9]; porcine, G2P[23], G3P[6], G4P[6], G5P[6], G5P[7], G5P[26], G5P[27], G9P[6] and G9P[7]) were also successfully NSP4-typed. Four human G3P[9] strains and one feline G3P[9] strain were found to possess an NSP4 A genotype, instead of NSP4 C, suggesting a reassortment event between heterologous strains. Routine NSP4 genotyping may help to determine the genomic constellation of rotaviruses of man and livestock, and identify interspecies transmission of heterologous strains. PMID:19208878

  7. Gene expression dynamics during cell differentiation: Cell fates as attractors and cell fate decisions as bifurcations

    NASA Astrophysics Data System (ADS)

    Huang, Sui

    2006-03-01

    During development of multicellular organisms, multipotent stem and progenitor cells undergo a series of hierarchically organized ``somatic speciation'' processes consisting of binary branching events to achieve the diversity of discretely distinct differentiated cell types in the body. Current paradigms of genetic regulation of development do not explain this discreteness, nor the time-irreversibility of differentiation. Each cell contains the same genome with the same N (˜ 25,000) genes and each cell type k is characterized by a distinct stable gene activation pattern, expressed as the cell state vector Sk(t) = xk1(t) ,.. xki(t),.. xkN(t), where xki is the activation state of gene i in cell type k. Because genes are engaged in a network of mutual regulatory interactions, the movement of Sk(t) in the N-dimensional state space is highly constrained and the organism can only realize a tiny fraction of all possible configurations Sk. Then, the trajectories of Sk reflect the diversifying developmental paths and the mature cell types are high-dimensional attractor states. Experimental results based on gene expression profile measurements during blood cell differentiation using DNA microarrays are presented that support the old idea that cell types are attractors. This basic notion is extended to treat binary fate decisions as bifurcations in the dynamics of networks circuits. Specifically, during cell fate decision, the metastable progenitor attractor is destabilized, poising the cell on a `watershed state' so that it can stochastically or in response to deterministic perturbations enter either one of two alternative fates. Overall, the model and supporting experimental data provide an overarching conceptual framework that helps explain how the specifics of gene network architecture produces discreteness and robustness of cell types, allows for both stochastic and deterministic cell fate decision and ensures directionality of organismal development.

  8. Screening of genes involved in cell migration in Dictyostelium.

    PubMed

    Nagasaki, Akira; Uyeda, Taro Q P

    2008-03-10

    A single cell of wild-type Dictyostelium discoideum forms a visible colony on a plastic dish in several days, but due to enhanced cell migration, amiB-null mutant cells scatter over a large area and do not form noticeable colonies. Here, with an aim to identify genes involved in cell migration, we isolated suppresser mutants of amiB-null mutants that restore the ability to form colonies. From REMI (restriction enzyme-mediated integration)-mutagenized pool of double-mutants, we identified 18 responsible genes from them. These genes can be categorized into several biological processes. One cell line, Sab16 (Suppressor of amiB) was chosen for further analysis, which had a disrupted phospholipase D pldB gene. To confirm the role of pldB gene in cell migration, we knocked out the pldB gene and over-expressed gfp-pldB in wild-type cells. GFP-PLDB localized to plasma membrane and on vesicles, and in migrating cells, at the protruding regions of pseudopodia. Migration speed of vegetative pldB-null cells was reduced to 73% of that of the wild-type. These results suggest that PLDB plays an important role in migration in Dictyostelium cells, and that our screening system is useful for the identification of genes involved in cell migration. PMID:18164290

  9. Osteoblast-specific gene expression after transplantation of marrow cells: Implications for skeletal gene therapy

    PubMed Central

    Hou, Zhen; Nguyen, Que; Frenkel, Baruch; Nilsson, Susan K.; Milne, Moira; van Wijnen, André J.; Stein, Janet L.; Quesenberry, Peter; Lian, Jane B.; Stein, Gary S.

    1999-01-01

    Somatic gene therapies require targeted transfer of the therapeutic gene(s) into stem cells that proliferate and then differentiate and express the gene in a tissue-restricted manner. We have developed an approach for gene therapy using marrow cells that takes advantage of the osteoblast specificity of the osteocalcin promoter to confine expression of chimeric genes to bone. Adherent marrow cells, carrying a reporter gene [chloramphenicol acetyltransferase (CAT)] under the control of a 1.7-kilobase rat osteocalcin gene promoter, were expanded ex vivo. After transplantation by intravenous infusion, engrafted donor cells in recipient mice were detected by the presence of the transgene in a broad spectrum of tissues. However, expression of the transgene was restricted to osteoblasts and osteocytes, as established by biochemical analysis of CAT activity and immunohistochemical analysis of CAT expression at the single cell level. Our data indicate that donor cells achieved long-term engraftment in various tissues of the recipients and that the CAT gene under control of the osteocalcin promoter is expressed specifically in bone. Thus, transplantation of multipotential marrow cells containing the osteocalcin promoter-controlled transgene provides an efficacious approach to deliver therapeutic gene expression to osteoblasts for treatment of bone disorders or tumor metastasis to the skeleton. PMID:10377408

  10. Freedom of expression: cell-type-specific gene profiling.

    PubMed

    Otsuki, Leo; Cheetham, Seth W; Brand, Andrea H

    2014-01-01

    Cell fate and behavior are results of differential gene regulation, making techniques to profile gene expression in specific cell types highly desirable. Many methods now enable investigation at the DNA, RNA and protein level. This review introduces the most recent and popular techniques, and discusses key issues influencing the choice between these such as ease, cost and applicability of information gained. Interdisciplinary collaborations will no doubt contribute further advances, including not just in single cell type but single-cell expression profiling.

  11. 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. PMID:27026484

  12. 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.

  13. Genes that Confer the Identity of the Renin Cell

    PubMed Central

    Brunskill, Eric W.; Sequeira-Lopez, Maria Luisa S.; Pentz, Ellen S.; Lin, Eugene; Yu, Jing; Aronow, Bruce J.; Potter, S. Steven

    2011-01-01

    Renin-expressing cells modulate BP, fluid-electrolyte homeostasis, and kidney development, but remarkably little is known regarding the genetic regulatory network that governs the identity of these cells. Here we compared the gene expression profiles of renin cells with most cells in the kidney at various stages of development as well as after a physiologic challenge known to induce the transformation of arteriolar smooth muscle cells into renin-expressing cells. At all stages, renin cells expressed a distinct set of genes characteristic of the renin phenotype, which was vastly different from other cell types in the kidney. For example, cells programmed to exhibit the renin phenotype expressed Akr1b7, and maturing cells expressed angiogenic factors necessary for the development of the kidney vasculature and RGS (regulator of G-protein signaling) genes, suggesting a potential relationship between renin cells and pericytes. Contrary to the plasticity of arteriolar smooth muscle cells upstream from the glomerulus, which can transiently acquire the embryonic phenotype in the adult under physiologic stress, the adult juxtaglomerular cell always possessed characteristics of both smooth muscle and renin cells. Taken together, these results identify the gene expression profile of renin-expressing cells at various stages of maturity, and suggest that juxtaglomerular cells maintain properties of both smooth muscle and renin-expressing cells, likely to allow the rapid control of body fluids and BP through both contractile and endocrine functions. PMID:22034642

  14. Genes that confer the identity of the renin cell.

    PubMed

    Brunskill, Eric W; Sequeira-Lopez, Maria Luisa S; Pentz, Ellen S; Lin, Eugene; Yu, Jing; Aronow, Bruce J; Potter, S Steven; Gomez, R Ariel

    2011-12-01

    Renin-expressing cells modulate BP, fluid-electrolyte homeostasis, and kidney development, but remarkably little is known regarding the genetic regulatory network that governs the identity of these cells. Here we compared the gene expression profiles of renin cells with most cells in the kidney at various stages of development as well as after a physiologic challenge known to induce the transformation of arteriolar smooth muscle cells into renin-expressing cells. At all stages, renin cells expressed a distinct set of genes characteristic of the renin phenotype, which was vastly different from other cell types in the kidney. For example, cells programmed to exhibit the renin phenotype expressed Akr1b7, and maturing cells expressed angiogenic factors necessary for the development of the kidney vasculature and RGS (regulator of G-protein signaling) genes, suggesting a potential relationship between renin cells and pericytes. Contrary to the plasticity of arteriolar smooth muscle cells upstream from the glomerulus, which can transiently acquire the embryonic phenotype in the adult under physiologic stress, the adult juxtaglomerular cell always possessed characteristics of both smooth muscle and renin cells. Taken together, these results identify the gene expression profile of renin-expressing cells at various stages of maturity, and suggest that juxtaglomerular cells maintain properties of both smooth muscle and renin-expressing cells, likely to allow the rapid control of body fluids and BP through both contractile and endocrine functions. PMID:22034642

  15. 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

  16. 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.

  17. 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.

  18. 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. PMID:26371251

  19. Analysis of simple sequence repeats in mammalian cell cycle genes.

    PubMed

    Trivedi, Seema; Wills, Christopher; Metzgar, David

    2014-01-01

    Simple sequence repeats (SSRs), or microsatellites are hyper-mutable and can lead to disorders. Here we explore SSR distribution in cell cycle-associated genes [grouped into: checkpoint; regulation; replication, repair, and recombination (RRR); and transition] in humans and orthologues of eight mammals. Among the gene groups studied, transition genes have the highest SSR density. Trinucleotide repeats are not abundant and introns have higher repeat density than exons. Many repeats in human genes are conserved; however, CG motifs are conserved only in regulation genes. SSR variability in cell cycle genes represents a genetic Achilles' heel, yet SSRs are common in all groups of genes. This tolerance many be due to i) positions in introns where they do not disrupt gene function, ii) essential roles in regulation, iii) specific value of adaptability, and/or iv) lack of negative selection pressure. Present study may be useful for further exploration of their medical relevance and potential functionality.

  20. T cells stimulate catabolic gene expression by the stromal cells from giant cell tumor of bone

    SciTech Connect

    Cowan, Robert W.; Ghert, Michelle; Singh, Gurmit

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer Two T cell lines stimulate PTHrP, RANKL, MMP13 gene expression in GCT cell cultures. Black-Right-Pointing-Pointer CD40 expressed by stromal cells; CD40L detected in whole tumor but not cultures. Black-Right-Pointing-Pointer Effect of CD40L treatment on GCT cells increased PTHrP and MMP13 gene expression. Black-Right-Pointing-Pointer PTHrP treatment increased MMP13 expression, while inhibition decreased expression. Black-Right-Pointing-Pointer T cells may stimulate GCT stromal cells and promote the osteolysis of the tumor. -- Abstract: The factors that promote the localized bone resorption by giant cell tumor of bone (GCT) are not fully understood. We investigated whether T cells could contribute to bone resorption by stimulating expression of genes for parathyroid hormone-related protein (PTHrP), matrix metalloproteinase (MMP)-13, and the receptor activator of nuclear-factor {kappa}B ligand (RANKL). Two cell lines, Jurkat clone E6-1 and D1.1, were co-cultured with isolated GCT stromal cells. Real-time PCR analyses demonstrated a significant increase of all three genes following 48 h incubation, and PTHrP and MMP-13 gene expression was also increased at 24 h. Further, we examined the expression of CD40 ligand (CD40L), a protein expressed by activated T cells, and its receptor, CD40, in GCT. Immunohistochemistry results revealed expression of the CD40 receptor in both the stromal cells and giant cells of the tumor. RNA collected from whole GCT tissues showed expression of CD40LG, which was absent in cultured stromal cells, and suggests that CD40L is expressed within GCT. Stimulation of GCT stromal cells with CD40L significantly increased expression of the PTHrP and MMP-13 genes. Moreover, we show that inhibition of PTHrP with neutralizing antibodies significantly decreased MMP13 expression by the stromal cells compared to IgG-matched controls, whereas stimulation with PTHrP (1-34) increased MMP-13 gene expression. These

  1. 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

  2. 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. PMID:26369430

  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. CFTR gene transfer to lung epithelium--on the trail of a target cell.

    PubMed

    O'Dea, S; Harrison, D J

    2002-05-01

    Cystic fibrosis (CF) is a lethal inherited disease that afflicts up to 1 in 2,500 people in the western world. Since 1989, when mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene were identified as responsible for the disease, intense effort has been applied to the development of replacement gene therapy strategies to cure CF. Problems with basic gene delivery techniques along with limited knowledge of the pathogenesis of CF have hindered progress so far. However, recent insights into the expression patterns and functions of CFTR in developing and adult lungs are now advancing our understanding of this disease. It is becoming apparent that progress in gene delivery to cure CF may be best served by identification of a target cell(s) around which gene transfer strategies can be specifically tailored to most closely reproduce the effects of normal CFTR expression. In fact, accurate restoration of endogenous expression patterns may be crucial, not only for disease reversal, but also to avoid potentially deleterious effects of inappropriate expression. This approach is in turn confounded however, by ill-defined stem and progenitor cell pathways within the lung epithelium. Nonetheless, studies to date suggest that these pathways are relatively plastic and may respond differently during homeostasis compared with repair following injury. It may therefore be feasible to target the lung epithelium in a non-cell specific manner and allow endogenous differentiation pathways to subsequently establish correct CFTR distribution patterns. In this review, emerging information on CFTR expression and function in developing and adult lungs is discussed in the context of putative stem cell populations and their potential for current gene delivery approaches. PMID:12109214

  5. ccmGDB: a database for cancer cell metabolism genes

    PubMed Central

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

    2016-01-01

    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. PMID:26519468

  6. 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.

  7. Genes and Gene Networks Involved in Sodium Fluoride-Elicited Cell Death Accompanying Endoplasmic Reticulum Stress in Oral Epithelial Cells

    PubMed Central

    Tabuchi, Yoshiaki; Yunoki, Tatsuya; Hoshi, Nobuhiko; Suzuki, Nobuo; Kondo, Takashi

    2014-01-01

    Here, to understand the molecular mechanisms underlying cell death induced by sodium fluoride (NaF), we analyzed gene expression patterns in rat oral epithelial ROE2 cells exposed to NaF using global-scale microarrays and bioinformatics tools. A relatively high concentration of NaF (2 mM) induced cell death concomitant with decreases in mitochondrial membrane potential, chromatin condensation and caspase-3 activation. Using 980 probe sets, we identified 432 up-regulated and 548 down-regulated genes, that were differentially expressed by >2.5-fold in the cells treated with 2 mM of NaF and categorized them into 4 groups by K-means clustering. Ingenuity® pathway analysis revealed several gene networks from gene clusters. The gene networks Up-I and Up-II included many up-regulated genes that were mainly associated with the biological function of induction or prevention of cell death, respectively, such as Atf3, Ddit3 and Fos (for Up-I) and Atf4 and Hspa5 (for Up-II). Interestingly, knockdown of Ddit3 and Hspa5 significantly increased and decreased the number of viable cells, respectively. Moreover, several endoplasmic reticulum (ER) stress-related genes including, Ddit3, Atf4 and Hapa5, were observed in these gene networks. These findings will provide further insight into the molecular mechanisms of NaF-induced cell death accompanying ER stress in oral epithelial cells. PMID:24853129

  8. Expression of homeobox genes in human erythroleukemia cells.

    PubMed

    Shen, W F; Largman, C; Lowney, P; Hack, F M; Lawrence, H J

    1989-01-01

    Because homeobox-containing genes play a major role in embryogenesis and tissue identity in Drosophila and because similar genes encode tissue-specific transcription factors in mammalian cells, we hypothesized that homeobox genes might plan a role in hematopoietic differentiation and lineage commitment. We therefore surveyed a number of human leukemic cell lines for expression of homeobox-containing genes by Northern gel analysis with probes from the Hox 2 cluster of homeobox genes on chromosome 17. We observed transcripts for Hox 2.1, 2.2, 2.3 and 2.6 in the erythroid line HEL and for Hox 2.3 and 2.6 in the erythroid line K562. Using homeobox-specific probes we confirmed that the transcripts visualized contained the homeodomains for each gene as well as the flanking sequences. The myeloid lines HL60, KG1 and U937 did not express specific transcripts for any of the 4 genes studied. However, all these cell lines demonstrated bands when probed at low stringency with certain Hox 2 probes, indicating the expression of other homologous but as yet unidentified homeobox genes. Expression of Hox 2.3 and 2.6 was seen in some T and B lymphoid cell lines. Induction of differentiation in HEL cells resulted in complex modulation of expression of the Hox 2 genes. We have therefore observed erythroid-restricted expression of certain Hox 2 homeobox containing genes in human erythroid cell lines and modulation of that expression with differentiation, suggesting a role for these genes in the regulation of hematopoiesis. Different homeobox genes appear to be expressed in non-erythroid leukemic cell lines.

  9. Geometry of the Gene Expression Space of Individual Cells.

    PubMed

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

    2015-07-01

    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

  10. 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

  11. Reproducibility in a multiprocessor system

    DOEpatents

    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.

  12. 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

  13. Magnetic field-controlled gene expression in encapsulated cells

    PubMed Central

    Ortner, Viktoria; Kaspar, Cornelius; Halter, Christian; Töllner, Lars; Mykhaylyk, Olga; Walzer, Johann; Günzburg, Walter H.; Dangerfield, John A.; Hohenadl, Christine; Czerny, Thomas

    2012-01-01

    Cell and gene therapies have an enormous range of potential applications, but as for most other therapies, dosing is a critical issue, which makes regulated gene expression a prerequisite for advanced strategies. Several inducible expression systems have been established, which mainly rely on small molecules as inducers, such as hormones or antibiotics. The application of these inducers is difficult to control and the effects on gene regulation are slow. Here we describe a novel system for induction of gene expression in encapsulated cells. This involves the modification of cells to express potential therapeutic genes under the control of a heat inducible promoter and the co-encapsulation of these cells with magnetic nanoparticles. These nanoparticles produce heat when subjected to an alternating magnetic field; the elevated temperatures in the capsules then induce gene expression. In the present study we define the parameters of such systems and provide proof-of-principle using reporter gene constructs. The fine-tuned heating of nanoparticles in the magnetic field allows regulation of gene expression from the outside over a broad range and within short time. Such a system has great potential for advancement of cell and gene therapy approaches. PMID:22197778

  14. Gene Expression in Mammalian Cells After Exposure to 95 MeV Argon Ions

    NASA Astrophysics Data System (ADS)

    Arenz, A.; Hellweg, C. E.; Baumstark-Khan, C.

    Cell response to genotoxic agents is complex and involves the participation of different classes of genes (DNA repair, cell cycle control, signal transduction, apoptosis and oncogenesis). The unique feature of the space radiation environment is the dominance of high-energy charged particles (HZE or high LET radiation) which present a significant hazard to space flight crews, and accelerator-based experiments are underway to quantify the health risks due to unavoidable radiation exposure. High linear energy transfer (LET) radiation has an increased relative biological effectiveness (RBE) as compared to X-rays for cell death induction, gene mutation, genomic instability, and carcinogenesis. The tumour suppressor gene p53 plays a crucial role in maintaining the integrity of the genome. The p53 protein acts as a transcription factor that mediates cell cycle arrest and apoptosis by binding to DNA and activating transcription of specific genes. It is also though to be involved in damage repair by transcriptional activation of the newly identified p53 dependent ribonuclease subunit R2 (p53R2) that is directly involved in the p53 cell cycle checkpoint for repair of damaged DNA. In that case it is responsible for nucleotide delivery for DNA repair synthesis. DNA damages of cultured human cells (e.g. MCF-7, AGS, A549) exposed to accelerated argon ions at the French heavy ion facility GANIL were analysed for expression levels of certain damage- and apoptosis-relevant genes. RNA was extracted from cells exposed to different particle fluences after various recovery times. A real-time QRT-PCR assay was applied, which employs both relative and absolute quantification of a candidate mRNA biomarker. The expressions of different DNA damage inducible genes (e.g. p53R2, GADD45, p21) were analysed. A reproducible up-regulation representing a twofold to fourfold change in p53R2 gene expression level was confirmed for X-irradiated and Ar-ion exposed cells dependent on dose. Kinetics of p

  15. 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

  16. 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. PMID:26504487

  17. Microfluidic devices for measuring gene network dynamics in single cells

    PubMed Central

    Bennett, Matthew R.; Hasty, Jeff

    2010-01-01

    The dynamics governing gene regulation have an important role in determining the phenotype of a cell or organism. From processing extracellular signals to generating internal rhythms, gene networks are central to many time-dependent cellular processes. Recent technological advances now make it possible to track the dynamics of gene networks in single cells under various environmental conditions using microfluidic ‘lab-on-a-chip’ devices, and researchers are using these new techniques to analyse cellular dynamics and discover regulatory mechanisms. These technologies are expected to yield novel insights and allow the construction of mathematical models that more accurately describe the complex dynamics of gene regulation. PMID:19668248

  18. 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

  19. 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.

  20. 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. PMID:26644562

  1. 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

  2. An Arabidopsis Gene Regulatory Network for Secondary Cell Wall Synthesis

    PubMed Central

    Taylor-Teeples, M; Lin, L; de Lucas, M; Turco, G; Toal, TW; Gaudinier, A; Young, NF; Trabucco, GM; Veling, MT; Lamothe, R; Handakumbura, PP; Xiong, G; Wang, C; Corwin, J; Tsoukalas, A; Zhang, L; Ware, D; Pauly, M; Kliebenstein, DJ; Dehesh, K; Tagkopoulos, I; Breton, G; Pruneda-Paz, JL; Ahnert, SE; Kay, SA; Hazen, SP; Brady, SM

    2014-01-01

    Summary 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. Here, we present a protein-DNA network between Arabidopsis 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. These interactions will serve as a foundation for understanding the regulation of a complex, integral plant component. PMID:25533953

  3. Expression of HOX C homeobox genes in lymphoid cells.

    PubMed

    Lawrence, H J; Stage, K M; Mathews, C H; Detmer, K; Scibienski, R; MacKenzie, M; Migliaccio, E; Boncinelli, E; Largman, C

    1993-08-01

    The class I homeobox genes located in four clusters in mammalian genomes (HOX A, HOX B, HOX C, and HOX D) appear to play a major role in fetal development. Previous surveys of homeobox gene expression in human leukemic cell lines have shown that certain HOX A genes are expressed only in myeloid cell lines, whereas HOX B gene expression is largely restricted to cells with erythroid potential. We now report a survey of the expression patterns of 9 homeobox genes from the HOX C locus in a panel of 24 human and 7 murine leukemic cell lines. The most striking observation is the lymphoid-specific pattern of expression of HOX C4, located at the 3' end of the locus. A major transcript of 1.9 kilobases is observed in both T-cell and B-cell lines. HOX C4 expression is also detected in normal human marrow and peripheral blood lymphocytes, but not in mature granulocytes or monocytes. HOX C8 is also expressed in human lymphoid cells but is expressed in other blood cell types as well. However, the HOX C8 transcript pattern is lineage specific. These data, in conjunction with earlier findings, suggest that homeobox gene expression influences lineage determination during hematopoiesis.

  4. ALDH isozymes downregulation affects cell growth, cell motility and gene expression in lung cancer cells

    PubMed Central

    Moreb, Jan S; Baker, Henry V; Chang, Lung-Ji; Amaya, Maria; Lopez, M Cecilia; Ostmark, Blanca; Chou, Wayne

    2008-01-01

    Background Aldehyde dehydrogenase isozymes ALDH1A1 and ALDH3A1 are highly expressed in non small cell lung cancer. Neither the mechanisms nor the biologic significance for such over expression have been studied. Methods We have employed oligonucleotide microarrays to analyze changes in gene profiles in A549 lung cancer cell line in which ALDH activity was reduced by up to 95% using lentiviral mediated expression of siRNA against both isozymes (Lenti 1+3). Stringent analysis methods were used to identify gene expression patterns that are specific to the knock down of ALDH activity and significantly different in comparison to wild type A549 cells (WT) or cells similarly transduced with green fluorescent protein (GFP) siRNA. Results We confirmed significant and specific down regulation of ALDH1A1 and ALDH3A1 in Lenti 1+3 cells and in comparison to 12 other ALDH genes detected. The results of the microarray analysis were validated by real time RT-PCR on RNA obtained from Lenti 1+3 or WT cells treated with ALDH activity inhibitors. Detailed functional analysis was performed on 101 genes that were significantly different (P < 0.001) and their expression changed by ≥ 2 folds in the Lenti 1+3 group versus the control groups. There were 75 down regulated and 26 up regulated genes. Protein binding, organ development, signal transduction, transcription, lipid metabolism, and cell migration and adhesion were among the most affected pathways. Conclusion These molecular effects of the ALDH knock-down are associated with in vitro functional changes in the proliferation and motility of these cells and demonstrate the significance of ALDH enzymes in cell homeostasis with a potentially significant impact on the treatment of lung cancer. PMID:19025616

  5. 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

  6. [From gene to disease: basal cell naevus syndrome].

    PubMed

    de Meij, T G J; Baars, M J H; Gille, J J P; Hack, W W M; Haasnoot, K; van Hagen, J M

    2005-01-01

    Nevoid basal cell carcinoma syndrome (NBCCS, basal cell naevus syndrome, Gorlin syndrome) is an autosomal dominant disorder, caused by mutations in the PTCH gene mapped to chromosome 9q22.3. It is characterised by multiple basal cell carcinomas, keratocysts of the jaws, palmar and plantar pits, cerebral ectopic calcification and several skeletal anomalies. Occasionally, patients with NBCCS develop other neoplasms, particularly medulloblastomas and ovarian fibromas, indicating that the PTCH gene is a tumor-suppressor gene. Early recognition and careful follow-up are needed. Guidelines for managing these patients are presented.

  7. Gene-modified hematopoietic stem cells for cancer immunotherapy.

    PubMed

    Larson, Sarah; De Oliveira, Satiro N

    2014-01-01

    The rapid expansion of available cancer immunotherapies has resulted in favorable early outcomes. Specifically the use of gene therapy to introduce chimeric antigen receptors (CARs) and T cell receptors (TCRs) in T cells creates new immunotherapy options for patients. While showing early success with these approaches, limitations remain that can be overcome by the use of modification of hematopoietic stem cells (HSCs) to express CARs and TCRs. With modern gene therapy technologies, increased safety and control of the modification of the HSCs can be achieved through the use of a suicide gene.

  8. 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.

  9. 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.

  10. Engineering Gene Circuits for Mammalian Cell-Based Applications.

    PubMed

    Ausländer, Simon; Fussenegger, Martin

    2016-01-01

    Synthetic gene switches are basic building blocks for the construction of complex gene circuits that transform mammalian cells into useful cell-based machines for next-generation biotechnological and biomedical applications. Ligand-responsive gene switches are cellular sensors that are able to process specific signals to generate gene product responses. Their involvement in complex gene circuits results in sophisticated circuit topologies that are reminiscent of electronics and that are capable of providing engineered cells with the ability to memorize events, oscillate protein production, and perform complex information-processing tasks. Microencapsulated mammalian cells that are engineered with closed-loop gene networks can be implanted into mice to sense disease-related input signals and to process this information to produce a custom, fine-tuned therapeutic response that rebalances animal metabolism. Progress in gene circuit design, in combination with recent breakthroughs in genome engineering, may result in tailored engineered mammalian cells with great potential for future cell-based therapies. PMID:27194045

  11. 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

  12. Positive selection of gene-modified cells increases the efficacy of pancreatic cancer suicide gene therapy.

    PubMed

    Martinez-Quintanilla, Jordi; Cascallo, Manel; Gros, Alena; Fillat, Cristina; Alemany, Ramon

    2009-11-01

    Thymidine kinase (TK)-mediated suicide gene therapy has been considered for the treatment of pancreatic cancer. However, despite a bystander effect, the proportion of transduced tumor cells has proven too low to result in efficacy. We propose the use of a drug-selectable marker (MDR1) to enrich TK-expressing cells using chemotherapy. This enrichment or positive selection phase may increase the efficacy of suicide gene therapy. To test this strategy, we generated stable NP18MDR/TK-GFP transfectants and showed docetaxel resistance in vivo. Mixed tumors of MDR/TK-expressing cells and parental NP18 cells were established and docetaxel was used to increase the proportion of TK-expressing cells. After this positive selection phase, suicide gene therapy with ganciclovir was applied. Upon positive selection, the proportion of TK-expressing cells increased from 4% to 22%. Subsequent suicide gene therapy was more effective compared with a control group without positive selection. Starting with 10% of TK-expressing cells the positive-negative selection strategy completely inhibited tumor growth. Taken together, these results suggest that a positive-negative selection strategy based on MDR and TK genes represents an efficient way to increase the proportion of TK-expressing cells in the tumor and the efficacy of TK-mediated suicide gene therapy.

  13. Crucial Genes and Pathways in Chicken Germ Stem Cell Differentiation

    PubMed Central

    Zhang, Zhentao; Elsayed, Ahmed Kamel; Shi, Qingqing; Zhang, Yani; Zuo, Qisheng; Li, Dong; Lian, Chao; Tang, Beibei; Xiao, Tianrong; Xu, Qi; Chang, Guobin; Chen, Guohong; Zhang, Lei; Wang, Kehua; Wang, Yingjie; Jin, Kai; Wang, Yilin; Song, Jiuzhou; Cui, Hengmi; Li, Bichun

    2015-01-01

    Male germ cell differentiation is a subtle and complex regulatory process. Currently, its regulatory mechanism is still not fully understood. In our experiment, we performed the first comprehensive genome and transcriptome-wide analyses of the crucial genes and signaling pathways in three kinds of crucial cells (embryonic stem cells, primordial germ cell, and spermatogonial stem cells) that are associated with the male germ cell differentiation. We identified thousands of differentially expressed genes in this process, and from these we chose 173 candidate genes, of which 98 genes were involved in cell differentiation, 19 were involved in the metabolic process, and 56 were involved in the differentiation and metabolic processes, like GAL9, AMH, PLK1, and PSMD7 and so on. In addition, we found that 18 key signaling pathways were involved mainly in cell proliferation, differentiation, and signal transduction processes like TGF-β, Notch, and Jak-STAT. Further exploration found that the candidate gene expression patterns were the same between in vitro induction experiments and transcriptome results. Our results yield clues to the mechanistic basis of male germ cell differentiation and provide an important reference for further studies. PMID:25847247

  14. Using injectoporation to deliver genes to mechanosensory hair cells

    PubMed Central

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

    2014-01-01

    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. PMID:25232939

  15. Pancreatic beta cells express a diverse set of homeobox genes.

    PubMed Central

    Rudnick, A; Ling, T Y; Odagiri, H; Rutter, W J; German, M S

    1994-01-01

    Homeobox genes, which are found in all eukaryotic organisms, encode transcriptional regulators involved in cell-type differentiation and development. Several homeobox genes encoding homeodomain proteins that bind and activate the insulin gene promoter have been described. In an attempt to identify additional beta-cell homeodomain proteins, we designed primers based on the sequences of beta-cell homeobox genes cdx3 and lmx1 and the Drosophila homeodomain protein Antennapedia and used these primers to amplify inserts by PCR from an insulinoma cDNA library. The resulting amplification products include sequences encoding 10 distinct homeodomain proteins; 3 of these proteins have not been described previously. In addition, an insert was obtained encoding a splice variant of engrailed-2, a homeodomain protein previously identified in the central nervous system. Northern analysis revealed a distinct pattern of expression for each homeobox gene. Interestingly, the PCR-derived clones do not represent a complete sampling of the beta-cell library because no inserts encoding cdx3 or lmx1 protein were obtained. Beta cells probably express additional homeobox genes. The abundance and diversity of homeodomain proteins found in beta cells illustrate the remarkable complexity and redundancy of the machinery controlling beta-cell development and differentiation. Images PMID:7991607

  16. Cancer Genes Hypermethylated in Human Embryonic Stem Cells

    PubMed Central

    Calvanese, Vincenzo; Horrillo, Angelica; Hmadcha, Abdelkrim; Suarez-Álvarez, Beatriz; Fernandez, Agustín F.; Lara, Ester; Casado, Sara; Menendez, Pablo; Bueno, Clara; Garcia-Castro, Javier; Rubio, Ruth; Lapunzina, Pablo; Alaminos, Miguel; Borghese, Lodovica; Terstegge, Stefanie; Harrison, Neil J.; Moore, Harry D.; Brüstle, Oliver; Lopez-Larrea, Carlos; Andrews, Peter W.; Soria, Bernat; Esteller, Manel; Fraga, Mario F.

    2008-01-01

    Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs) in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation. Our results indicate a role for DNA methylation in the control of gene expression in human stem cells and suggest that, for genes repressed by promoter hypermethylation in stem cells in vivo, the aberrant process in cancer could be understood as a defect in establishing an unmethylated promoter during differentiation, rather than as an anomalous process of de novo hypermethylation. PMID:18820729

  17. 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.

  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. Expression profiling of cell cycle genes in human pancreatic islets with and without type 2 diabetes.

    PubMed

    Taneera, Jalal; Fadista, Joao; Ahlqvist, Emma; Zhang, Mengze; Wierup, Nils; Renström, Erik; Groop, Leif

    2013-08-15

    Microarray gene expression data were used to analyze the expression pattern of cyclin, cyclin-dependent kinase (CDKs) and cyclin-dependent kinase inhibitor (CDKIs) genes from human pancreatic islets with and without type 2 diabetes (T2D). Of the cyclin genes, CCNI was the most expressed. Data obtained from microarray and qRT-PCR showed higher expression of CCND1 in diabetic islets. Among the CDKs, CDK4, CDK8 and CDK9 were highly expressed, while CDK1 was expressed at low level. High expression of CDK18 was observed in diabetic islets. Of the CDKIs, CDKN1A expression was higher in diabetic islets in both microarray and qRT-PCR. Expression of CDKN1A, CDKN2A, CCNI2, CDK3 and CDK16 was correlated with age. Finally, eight SNPs in these genes were associated with T2D in the DIAGRAM database. Our data provide a comprehensive expression pattern of cell cycle genes in human islets. More human studies are required to confirm and reproduce animal studies. PMID:23707792

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. T-cell activation and early gene response in dogs.

    PubMed

    Mortlock, Sally-Anne; Wei, Jerry; Williamson, Peter

    2015-01-01

    T-cells play a crucial role in canine immunoregulation and defence against invading pathogens. Proliferation is fundamental to T-cell differentiation, homeostasis and immune response. Initiation of proliferation following receptor mediated stimuli requires a temporally programmed gene response that can be identified as immediate-early, mid- and late phases. The immediate-early response genes in T-cell activation engage the cell cycle machinery and promote subsequent gene activation events. Genes involved in this immediate-early response in dogs are yet to be identified. The present study was undertaken to characterise the early T-cell gene response in dogs to improve understanding of the genetic mechanisms regulating immune function. Gene expression profiles were characterised using canine gene expression microarrays and quantitative reverse transcription PCR (qRT-PCR), and paired samples from eleven dogs. Significant functional annotation clusters were identified following stimulation with phytohemagluttinin (PHA) (5μg/ml), including the Toll-like receptor signaling pathway and phosphorylation pathways. Using strict statistical criteria, 13 individual genes were found to be differentially expressed, nine of which have ontologies that relate to proliferation and cell cycle control. These included, prostaglandin-endoperoxide synthase 2 (PTGS2/COX2), early growth response 1 (EGR1), growth arrest and DNA damage-inducible gene (GADD45B), phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), V-FOS FBJ murine osteosarcoma viral oncogene homolog (FOS), early growth response 2 (EGR2), hemogen (HEMGN), polo-like kinase 2 (PLK2) and polo-like kinase 3 (PLK3). Differential gene expression was re-examined using qRT-PCR, which confirmed that EGR1, EGR2, PMAIP1, PTGS2, FOS and GADD45B were significantly upregulated in stimulated cells and ALAS2 downregulated. PTGS2 and EGR1 showed the highest levels of response in these dogs. Both of these genes are involved in cell cycle

  5. 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

  6. 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.

  7. Quantitative Real-Time Polymerase Chain Reaction Measurement of HLA-DRA Gene Expression in Whole Blood Is Highly Reproducible and Shows Changes That Reflect Dynamic Shifts in Monocyte Surface HLA-DR Expression during the Course of Sepsis

    PubMed Central

    Tina, Elisabet; Bäckman, Anders; Magnuson, Anders; Strålin, Kristoffer; Söderquist, Bo; Källman, Jan

    2016-01-01

    Introduction A decrease in the expression of monocyte surface protein HLA-DR (mHLA-DR), measured by flow cytometry (FCM), has been suggested as a marker of immunosuppression and negative outcome in severe sepsis. However, FCM is not always available due to sample preparation that limits its use to laboratory operational hours. In this prospective study we evaluated dynamic changes in mHLA-DR expression during sepsis in relation to changes in HLA-DRA gene expression and Class II transactivator (CIITA), measured by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). Aims The aims of this study were: 1. to validate the robustness of qRT-PCR measurement of HLA-DRA- and CIITA–mRNA expression, in terms of reproducibility; and 2. to see if changes in expression of these genes reflect changes in mHLA-DR expression during the course of severe and non-severe bacteraemic sepsis. Methods and Findings Blood samples were collected from 60 patients with bacteraemic sepsis on up to five occasions during Days 1–28 after hospital admission. We found the reproducibility of the qRT-PCR method to be high by demonstrating low threshold variations (<0.11 standard deviation (SD)) of the qRT-PCR system, low intra-assay variation of Ct-values within triplicates (≤0.15 SD) and low inter-assay variations (12%) of the calculated target gene ratios. Our results also revealed dynamic HLA-DRA expression patterns during the course of sepsis that reflected those of mHLA-DR measured by FCM. Furthermore, HLA-DRA and mHLA-DR recovery slopes in patients with non-severe sepsis differed from those in patients with severe sepsis, shown by mixed model for repeated measurements (p<0.05). However, during the first seven days of sepsis, PCR-measurements showed a higher magnitude of difference between the two sepsis groups. Mean differences (95% CI) between severe sepsis (n = 20) and non-severe sepsis (n = 40) were; on day 1–2, HLA-DRA 0.40 (0.28–0.59) p<0.001, CIITA 0.48 (0.32–0.72) p = 0

  8. 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

  9. Stem Cell Based Gene Therapy in Prostate Cancer

    PubMed Central

    Lee, Hong Jun; Song, Yun Seob

    2014-01-01

    Current prostate cancer treatment, especially hormone refractory cancer, may create profound iatrogenic outcomes because of the adverse effects of cytotoxic agents. Suicide gene therapy has been investigated for the substitute modality for current chemotherapy because it enables the treatment targeting the cancer cells. However the classic suicide gene therapy has several profound side effects, including immune-compromised due to viral vector. Recently, stem cells have been regarded as a new upgraded cellular vehicle or vector because of its homing effects. Suicide gene therapy using genetically engineered mesenchymal stem cells or neural stem cells has the advantage of being safe, because prodrug administration not only eliminates tumor cells but consequently kills the more resistant therapeutic stem cells as well. The attractiveness of prodrug cancer gene therapy by stem cells targeted to tumors lies in activating the prodrug directly within the tumor mass, thus avoiding systemic toxicity. Therapeutic achievements using stem cells in prostate cancer include the cytosine deaminase/5-fluorocytosine prodrug system, herpes simplex virus thymidine kinase/ganciclovir, carboxyl esterase/CPT11, and interferon-beta. The aim of this study is to review the stem cell therapy in prostate cancer including its proven mechanisms and also limitations. PMID:25121103

  10. Experimental gene therapy using p21Waf1 gene for esophageal squamous cell carcinoma by gene gun technology.

    PubMed

    Tanaka, Yuichi; Fujii, Teruhiko; Yamana, Hideaki; Kato, Seiya; Morimatsu, Minoru; Shirouzu, Kazuo

    2004-10-01

    In our previous study, the proliferation rate of esophageal squamous cell carcinoma cell lines, which poorly expressed p21Waf1, was found to be regulated by p21Waf1 gene transfection using adenovirus vector. In the present study, in order to examine the effect of p21Waf1 gene therapy in esophageal cancer, we used gene gun technology, which proved to be a powerful method to introduce the p21Waf1 gene into esophageal cancer cells. p21Waf1 transfection to KE3 and YES2 cells (weakly expressed p21Waf1 protein cells) showed a high expression of p21Waf1 protein after applying this gene gun technique. In KE3 and YES2 cells, statistical significant growth inhibition was observed after p21Waf1 transfection compared with LacZ transfection (KE3, p=0.0009; YES2, p<0.0001). In in vivo transfection experiments, on day 14, the estimated volume of KE3 tumors subjected to p21Waf1 gene transfection was 95% in comparison with the pretreatment volume on day 0, while the volume of KE3 tumors subjected to LacZ gene therapy increased to 268%. On day 14, the estimated volume of YES2 tumors subjected to either p21Waf1 or LacZ gene therapy increased to 474 and 686%, respectively. In KE3 and YES2 cells, significant growth inhibition was observed after combination therapy using p21Waf1 transfection and anticancer drug 5-fluorouracil (5Fu) compared with 5Fu alone (KE3, p<0.0001; YES2, p<0.0001). In conclusion, p21Waf1 gene therapy using the gene gun technique significantly inhibited the low basal p21Waf1 expressed esophageal cancer cell growth in vitro and in vivo. Furthermore, p21Waf1 transfection strongly enhanced the effect of 5Fu suggesting that p21Waf1 may prove beneficial in chemotherapy combined with gene therapy using gene gun technology in patients with esophageal cancer who have a low level of p21Waf1 expressed tumor.

  11. 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.

  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. Gene trapping identifies transiently induced survival genes during programmed cell death

    PubMed Central

    Wempe, Frank; Yang, Ji-Yeon; Hammann, Joanna; Melchner, Harald von

    2001-01-01

    Background The existence of a constitutively expressed machinery for death in individual cells has led to the notion that survival factors repress this machinery and, if such factors are unavailable, cells die by default. In many cells, however, mRNA and protein synthesis inhibitors induce apoptosis, suggesting that in some cases transcriptional activity might actually impede cell death. To identify transcriptional mechanisms that interfere with cell death and survival, we combined gene trap mutagenesis with site-specific recombination (Cre/loxP system) to isolate genes from cells undergoing apoptosis by growth factor deprivation. Results From an integration library consisting of approximately 2 × 106 unique proviral integrations obtained by infecting the interleukin-3 (IL-3)-dependent hematopoietic cell line - FLOXIL3 - with U3Cre gene trap virus, we have isolated 125 individual clones that converted to factor independence upon IL-3 withdrawal. Of 102 cellular sequences adjacent to U3Cre integration sites, 17% belonged to known genes, 11% matched single expressed sequence tags (ESTs) or full cDNAs with unknown function and 72% had no match within the public databases. Most of the known genes recovered in this analysis encoded proteins with survival functions. Conclusions We have shown that hematopoietic cells undergoing apoptosis after withdrawal of IL-3 activate survival genes that impede cell death. This results in reduced apoptosis and improved survival of cells treated with a transient apoptotic stimulus. Thus, apoptosis in hematopoietic cells is the end result of a conflict between death and survival signals, rather than a simple death by default. PMID:11516336

  14. 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.

  15. 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. PMID:26437076

  16. 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.

  17. 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.

  18. [Immunoglobulin genes in lymphoid cells and regulation of their transcription].

    PubMed

    Stepchenko, A G; Urakov, D N; Luchina, N N; Deev, S M; Polianovskiĭ, O L

    1990-01-01

    The hybridoma genomes contain polyploid sets of immunoglobulin genes. We have shown, that the hybridoma PTF-02 genome contains three genes of heavy chains and two genes of light chains. The genes responsible for antibody synthesis were cloned and their structure were determined. Investigation of the kappa gene transcription and its fragments which contain regulatory sequences revealed a nuclear factor. The latter interacts with the octanucleotide localized at the promoter region of the kappa gene. The purified factor activates the transcription of the kappa gene in a heterologous cell-free system. Together with the tissue-specific factor there is also an universal factor interacting with the octanucleotide sequence. We have shown an additional factor in lymphoid cells interact with the protein which binds to the octanucleotide sequence. We have shown an additional factor in lymphoid cells interacting with the protein which binds to the octanucleotide sequence. As a result, there is a family of factors which interact with ATTTGCAT sequence. One major factor (m.w. 60 +/- 2 kDa) is an obligatory component for the initiation of immunoglobulin genes transcription.

  19. Transcriptional control of MHC class II gene expression during differentiation from B cells to plasma cells.

    PubMed

    Dellabona, P; Latron, F; Maffei, A; Scarpellino, L; Accolla, R S

    1989-04-15

    In this study we investigated the molecular mechanisms responsible for the extinction of the constitutive MHC class II gene expression of human B cells on somatic cell hybridization with murine plasmocytoma cells. We found that this event is due to trans-acting suppressor functions of mouse origin pre-existing in the plasmocytoma cells and acting at transcriptional level. Transcription of the entire family of human class II genes is suppressed, including genes as DO beta for which a distinct regulation of expression in B cells had been previously demonstrated. Suppression appears specific for class II genes because in the hybrids expression of MHC class I genes of mouse is unaffected and of human only partially reduced. Interestingly, also murine invariant chain gene is expressed in both parental plasmocytoma and hybrid cells although at reduced amounts as compared to a murine class II positive B cell line. The class II negative phenotype of hybrid cells and parental plasmocytoma cells is highly stable and unaffected by treatment with protein synthesis inhibitors, suggesting that the transcriptional suppressor function is not mediated by rapid, labile turning-over proteins. Possible mechanisms responsible for transcriptional regulation of MHC class II gene expression during terminal differentiation of B cells to plasma cells are discussed. PMID:2495328

  20. 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

  1. Gene networks related to the cell death elicited by hyperthermia in human oral squamous cell carcinoma HSC-3 cells.

    PubMed

    Tabuchi, Yoshiaki; Wada, Shigehito; Furusawa, Yukihiro; Ohtsuka, Kenzo; Kondo, Takashi

    2012-03-01

    Local hyperthermia (HT) for various types of malignant tumors has shown promising antitumor effects. To confirm the detailed molecular mechanism underlying cell death induced by HT, gene expression patterns and gene networks in human oral squamous cell carcinoma (OSCC) cells were examined using a combination of DNA microarray and bioinformatics tools. OSCC HSC-3 cells were treated with HT at 44˚C for 90 min or mild hyperthermia (MHT) at 42˚C for 90 min, followed by culturing at 37˚C for 0-24 h. Treatment of cells with HT prevented cell proliferation (62%) and induced cell death (17%), whereas these alterations were not observed in cells treated with MHT. Microarray analysis revealed substantial differences with respect to gene expression patterns and biological function for the two different hyperthermic treatments. Moreover, we identified the temperature-specific gene networks D and H that were obtained from significantly up-regulated genes in the HT and MHT conditions, respectively, using Ingenuity pathway analysis tools. Gene network D, which contains 14 genes such as ATF3, DUSP1 and JUN, was associated with relevant biological functions including cell death and cellular movement. Gene network H, which contains 13 genes such as BAG3, DNAJB1 and HSPA1B, was associated with cellular function and maintenance and cellular assembly and organization. These findings provide a basis for understanding the detailed molecular mechanisms of cell death elicited by HT in human OSCC cells. PMID:22179328

  2. Transcriptional wiring of cell wall-related genes in Arabidopsis.

    PubMed

    Mutwil, Marek; Ruprecht, Colin; Giorgi, Federico M; Bringmann, Martin; Usadel, Björn; Persson, Staffan

    2009-09-01

    Transcriptional coordination, or co-expression, of genes may signify functional relatedness of the corresponding proteins. For example, several genes involved in secondary cell wall cellulose biosynthesis are co-expressed with genes engaged in the synthesis of xylan, which is a major component of the secondary cell wall. To extend these types of analyses, we investigated the co-expression relationships of all Carbohydrate-Active enZYmes (CAZy)-related genes for Arabidopsis thaliana. Thus, the intention was to transcriptionally link different cell wall-related processes to each other, and also to other biological functions. To facilitate easy manual inspection, we have displayed these interactions as networks and matrices, and created a web-based interface (http://aranet.mpimp-golm.mpg.de/corecarb) containing downloadable files for all the transcriptional associations.

  3. 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

  4. Adenoviral-vector-mediated gene transfer to dendritic cells.

    PubMed

    Song, W; Crystal, R G

    2001-01-01

    Dendritic cells (DC) are the most potent antigen presenting cells capable of initiating T-cell-dependent immune responses (1-5). This biologic potential can be harnessed to elicit effective antigen-specific immune responses by transferring the relevant antigens to the DC. Once the DC have been mobilized and purified, the relevant antigens can be transferred to the DC as intact proteins, or as peptides representing specific epitopes, or with gene transfer using sequences of DNA or RNA coding for the pertinent antigen(s) (6-15). Theoretically, genetically modifying DC with genes coding for specific antigens has potential advantages over pulsing the DC with peptides repeating the antigen or antigen fragment. First, the genetically modified DC may present previously unknown epitopes in association with different MHC molecules. Second, gene transfer to DC ensures that the gene product is endogenously processed, leading to the generation of MHC class I-restricted cytotoxic T lymphocytes (CTL), the effector arm of cell-mediated immune responses. Finally, in addition to genes coding for the antigen(s), genetic modification of the DC can induce genes coding for mediators relevant to generation of the immune response to the antigen(s), further boosting host responses to the antigens presented by the modified DC. Different gene transfer approaches have been explored to genetically modify DC, including retroviral vectors (16-18), recombinant vaccinia virus vectors (19), and recombinant adenovirus (Ad) vectors (19-23). The focus of this chapter is on using recombinant Ad vectors to transfer genes to murine DC. We have used a similar strategy to transfer genes to human DC (24). As an example of the power of this technology, we will describe the use of Ad-vector-modified DC to suppress the growth of tumor cells modified to express a specific antigen.

  5. 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.

  6. WHIM syndrome myelokathexis reproduced in the NOD/SCID mouse xenotransplant model engrafted with healthy human stem cells transduced with C-terminus–truncated CXCR4

    PubMed Central

    Kawai, Toshinao; Choi, Uimook; Cardwell, Lanise; DeRavin, Suk See; Naumann, Nora; Whiting-Theobald, Narda L.; Linton, Gilda F.; Moon, Jaehyun; Murphy, Philip M.; Malech, Harry L.

    2007-01-01

    WHIM(warts, hypogammaglobulinemia, recurrent bacterial infection, and myelokathexis) syndrome is a rare immunodeficiency caused in many cases by autosomal dominant C-terminal truncation mutations in the chemokine receptor CXCR4. A prominent and unexplained feature of WHIM is myelokathexis (hypercellularity with apoptosis of mature myeloid cells in bone marrow and neutropenia). We transduced healthy human CD34+ peripheral blood–mobilized stem cells (PBSCs) with retrovirus vector encoding wild-type (wt) CXCR4 or WHIM-type mutated CXCR4 and studied these cells ex vivo in culture and after engraftment in a nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse xenograft model. Neither wt CXCR4 nor mutated CXCR4 transgene expression itself enhanced apoptosis of neutrophils arising in transduced PBSC cultures even with stimulation by a CXCR4 agonist, stromal cell–derived factor-1 (SDF-1 [CXCL12]). Excess wt CXCR4 expression by transduced human PBSCs enhanced marrow engraftment, but did not affect bone marrow (BM) apoptosis or the release of transduced leukocytes into PB. However, mutated CXCR4 transgene expression further enhanced BM engraftment, but was associated with a significant increase in apoptosis of transduced cells in BM and reduced release of transduced leukocytes into PB. We conclude that increased apoptosis of mature myeloid cells in WHIM is secondary to a failure of marrow release and progression to normal myeloid cell senescence, and not a direct effect of activation of mutated CXCR4. PMID:16946301

  7. Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol.

    PubMed

    Fonoudi, Hananeh; Ansari, Hassan; Abbasalizadeh, Saeed; Blue, Gillian M; Aghdami, Nasser; Winlaw, David S; Harvey, Richard P; Bosman, Alexis; Baharvand, Hossein

    2016-01-01

    Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-β, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing > 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes. PMID:27500408

  8. Homing genes, cell therapy and stroke.

    PubMed

    Shyu, Woei-Cherng; Lee, Yih-Jing; Liu, Demeral David; Lin, Shinn-Zong; Li, Hung

    2006-01-01

    Stem cell therapies, such as bone marrow transplantation, are a promising strategy for the treatment of stroke. Bone marrow-derived stem cells (BMSCs) including both hematopoietic and mesenchymal stem cells (HSCs and MSCs) can exhibit tremendous cellular differentiation in numerous organs. BMSCs may also promote structural and functional repair in several organs such as the heart, liver, brain, and skeletal muscle via stem cell plasticity. Interestingly, ischemia is known to induce mobilization of BMSCs in both animal models and humans. The tissue injury is "sensed" by the stem cells and they migrate to the site of damage and undergo differentiation. The plasticity, differentiation, and migratory functions of BMSCs in a given tissue are dependent on the specific signals present in the local micro-environment of the damaged tissue. Therefore, the ischemic micro-environment has critical patho-biological functions that are essential for the seeding, expansion, survival, renewal, growth and differentiation of BMSCs in damaged brain remodeling. Recent studies have identified the specific molecular signals, such as SDF-1/CXCR4, required for the interaction of BMSCs and damaged host tissues. Understanding the exact molecular basis of stem cell plasticity in relation to local ischemic signals could offer new insights to permit better management of stroke and other ischemic disorders. The aim of this review is to summarize recent studies into how BMSCs reach, recognize, and function in cerebral ischemic tissues, with particular regard to phenotypical reprogramming of stem cells, or "stem cell plasticity". PMID:16146779

  9. 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.

  10. 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

  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. 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

  13. 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.

  14. Gene expression changes in BVDV2-infected MDBK cells.

    PubMed

    Neill, John D; Ridpath, Julia F

    2003-06-01

    Bovine viral diarrhoea virus (BVDV) is a ubiquitous viral pathogen of cattle. The virus exists as one of two biotypes, cytopathic and non-cytopathic, based on the ability to induce cytopathic effect in cell culture. The non-cytopathic biotypes are able to establish non-apparent, persistent infections in both cell culture and in bovine foetuses of fewer than 150 days gestation. The mechanism by which viral tolerance is established is unknown. To examine the changes in gene expression that occur following infection of host cells with BVDV, serial analysis of gene expression (SAGE), a global gene expression technology was used. SAGE, a sequence-based technology, allows quantification of virtually every transcript in a cell type without prior sequence information. Transcript expression levels and identities are determined by DNA sequencing of libraries composed of 14 base DNA fragments (tags) derived from the 3' end of each cellular mRNA transcript. Comparison of data obtained from non-infected and BVDV2-infected cell libraries revealed a number of changes in gene expression. Many of these transcriptional changes could be placed into distinct biochemical pathways or functions. Both alpha and beta tubulins were downregulated, indicating possible dysfunction in cell division and other functions where microtubules play a major role. Expression of several genes encoding proteins involved in energy metabolism were downregulated, indicating possible decreased ATP synthesis. Genes encoding proteins involved in protein translation and post-translational modifications were generally upregulated. These data indicate that following infection with BVDV, changes in gene expression occur that are beneficial for virus replication while placing the cell at a metabolic disadvantage.

  15. 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

  16. AAV-mediated gene targeting methods for human cells

    PubMed Central

    Khan, Iram F; Hirata, Roli K; Russell, David W

    2013-01-01

    Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types, with targeting frequencies ranging from 10−5 to 10−2 per infected cell. these targeting frequencies are 1–4 logs higher than those obtained by conventional transfection or electroporation approaches. a wide variety of different types of mutations can be introduced into chromosomal loci with high fidelity and without genotoxicity. Here we provide a detailed protocol for gene targeting in human cells with AAV vectors. We describe methods for vector design, stock preparation and titration. optimized transduction protocols are provided for human pluripotent stem cells, mesenchymal stem cells, fibroblasts and transformed cell lines, as well as a method for identifying targeted clones by southern blots. this protocol (from vector design through a single round of targeting and screening) can be completed in ~10 weeks; each subsequent round of targeting and screening should take an additional 7 weeks. PMID:21455185

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. [Expression of rice dwarf virus outer coat protein gene(S8) in insect cells].

    PubMed

    Li, S; Liu, H; Chen, Z; Li, Y

    2001-04-01

    Outer coat protein gene(S8) of RDV was cloned into the transfer vector pVL 1393 to construct a recombinant vector pVL1393-S8. The recombinant vector pVL1393-S8 and the linear baculovirus RP23. LacZ were cotransfected into sf9 cells to produce the recombinant virus RP23-S8. RP23-S8 infected sf9 cells were collected and analysed by SDS-PAGE and Western-blot. The results showed that the S8 gene of RDV was expressed in sf9 cells and the expression level of sf9 cells was higher between 72-96 h after infected.

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

    PubMed

    Daker, Maelinda; Bhuvanendran, Saatheeyavaane; Ahmad, Munirah; Takada, Kenzo; Khoo, Alan Soo-Beng

    2013-03-01

    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.

  4. Comparative Genomics of Natural Killer Cell Receptor Gene Clusters

    PubMed Central

    Kelley, James; Walter, Lutz; Trowsdale, John

    2005-01-01

    Many receptors on natural killer (NK) cells recognize major histocompatibility complex class I molecules in order to monitor unhealthy tissues, such as cells infected with viruses, and some tumors. Genes encoding families of NK receptors and related sequences are organized into two main clusters in humans: the natural killer complex on Chromosome 12p13.1, which encodes C-type lectin molecules, and the leukocyte receptor complex on Chromosome 19q13.4, which encodes immunoglobulin superfamily molecules. The composition of these gene clusters differs markedly between closely related species, providing evidence for rapid, lineage-specific expansions or contractions of sets of loci. The choice of NK receptor genes is polarized in the two species most studied, mouse and human. In mouse, the C-type lectin-related Ly49 gene family predominates. Conversely, the single Ly49 sequence is a pseudogene in humans, and the immunoglobulin superfamily KIR gene family is extensive. These different gene sets encode proteins that are comparable in function and genetic diversity, even though they have undergone species-specific expansions. Understanding the biological significance of this curious situation may be aided by studying which NK receptor genes are used in other vertebrates, especially in relation to species-specific differences in genes for major histocompatibility complex class I molecules. PMID:16132082

  5. A Lentiviral Vector Expressing Desired Gene Only in Transduced Cells: An Approach for Suicide Gene Therapy.

    PubMed

    Mohammadi, Zahra; Shariati, Laleh; Khanahmad, Hossein; Kolahdouz, Mahsa; Kianpoor, Fariborz; Ghanbari, Jahan Afrooz; Hejazi, Zahra; Salehi, Mansoor; Nikpour, Parvaneh; Tabatabaiefar, Mohammad Amin

    2015-09-01

    Suicide gene therapy is a therapeutic strategy, in which cell suicide inducing transgenes are introduced into target cells. Inserting a toxin-encoding gene into a lentiviral vector leads to decreased efficiency of virus production due to lethal effect of toxin on packaging cells. In this study, we designed and constructed a transfer vector to express the toxin in transduced cells but not in packaging cells. Plasmid pLenti-F/GFP was constructed by cutting out R 5'LTR-R 3'LTR fragment with the AflII restriction endonuclease from a plasmid pLenti4-GW/H1/TO-laminshRNA, followed by ligating R 5'LTR-R 3'LTR fragment, constructed by three PCR stages. The promoter and GFP CDS were inserted in opposite strand. For lentiviral production, the HEK293T cell line was co-transfected with the PMD2G, psPAX2, and pLenti-F/GFP plasmids (envelope, packaging, and transfer plasmids).Viral vector titers were assayed. The HEK293T cell line was transduced with this virus. PCR was performed to confirm the presence of the promoter fragment between the R and U5 in 3'LTR. The lentivirus titers were approximately 2 × 10(5). The GFP expression was seen in 51 % of the HEK293T cells transduced with lentivirus. The PCR product size was 1440 bp confirming the promoter fragment position between the R and U5 in 3'LTR. The strategy enables us to use a broad spectrum of toxin genes in gene therapy and helps avoid the death of the packaging cells with lentiviral vectors carrying a toxin-encoding gene, thereby increasing the efficiency of viral production in packaging cells.

  6. 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.

  7. Gene and stem cell therapy in peripheral arterial occlusive disease.

    PubMed

    Kalka, C; Baumgartner, Iris

    2008-01-01

    Peripheral arterial occlusive disease (PAOD) is a manifestation of systemic atherosclerosis strongly associated with a high risk of cardiovascular morbidity and mortality. In a considerable proportion of patients with PAOD, revascularization either by endovascular means or by open surgery combined with best possible risk factor modification does not achieve limb salvage or relief of ischaemic rest pain. As a consequence, novel therapeutic strategies have been developed over the last two decades aiming to promote neovascularization and remodelling of collaterals. Gene and stem cell therapy are the main directions for clinical investigation concepts. For both, preclinical studies have shown promising results using a wide variety of genes encoding for growth factors and populations of adult stem cells, respectively. As a consequence, clinical trials have been performed applying gene and stem cell-based concepts. However, it has become apparent that a straightforward translation into humans is not possible. While several trials reported relief of symptoms and functional improvement, other trials did not confirm this early promise of efficacy. Ongoing clinical trials with an improved study design are needed to confirm the potential that gene and cell therapy may have and to prevent the gaps in our scientific knowledge that will jeopardize the establishment of angiogenic therapy as an additional medical treatment of PAOD. This review summarizes the experimental background and presents the current status of clinical applications and future perspectives of the therapeutic use of gene and cell therapy strategies for PAOD.

  8. 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, extension…

  9. Comparison of assays for sensitive and reproducible detection of cell culture-infectious Cryptosporidium parvum and Cryptosporidium hominis in drinking water.

    PubMed

    Johnson, Anne M; Giovanni, George D Di; Rochelle, Paul A

    2012-01-01

    This study compared the three most commonly used assays for detecting Cryptosporidium sp. infections in cell culture: immunofluorescent antibody and microscopy assay (IFA), PCR targeting Cryptosporidium sp.-specific DNA, and reverse transcriptase PCR (RT-PCR) targeting Cryptosporidium sp.-specific mRNA. Monolayers of HCT-8 cells, grown in 8-well chamber slides or 96-well plates, were inoculated with a variety of viable and inactivated oocysts to assess assay performance. All assays detected infection with low doses of flow cytometry-enumerated Cryptosporidium parvum oocysts, including infection with one oocyst and three oocysts. All methods also detected infection with Cryptosporidium hominis. The RT-PCR assay, IFA, and PCR assay detected infection in 23%, 25%, and 51% of monolayers inoculated with three C. parvum oocysts and 10%, 9%, and 16% of monolayers inoculated with one oocyst, respectively. The PCR assay was the most sensitive, but it had the highest frequency of false positives with mock-infected cells and inactivated oocysts. IFA was the only infection detection assay that did not produce false positives with mock-infected monolayers. IFA was also the only assay that detected infections in all experiments with spiked oocysts recovered from Envirochek capsules following filtration of 1,000 liters of treated water. Consequently, cell culture with IFA detection is the most appropriate method for routine and sensitive detection of infectious Cryptosporidium parvum and Cryptosporidium hominis in drinking water.

  10. 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.

  11. Differential sensitivities of transcription factor target genes underlie cell type-specific gene expression profiles

    PubMed Central

    Johnson, Kirby D.; Kim, Shin-Il; Bresnick, Emery H.

    2006-01-01

    Changes in transcription factor levels and activities dictate developmental fate. Such a change might affect the full ensemble of target genes for a factor or only uniquely sensitive targets. We investigated the relationship among activity of the hematopoietic transcription factor GATA-1, chromatin occupancy, and target gene sensitivity. Graded activation of GATA-1 in GATA-1-null cells revealed high-, intermediate-, and low-sensitivity targets. GATA-1 activity requirements for occupancy and transcription often correlated. A GATA-1 amino-terminal deletion mutant severely deregulated the low-sensitivity gene Tac-2. Thus, cells expressing different levels of a cell type-specific activator can have qualitatively distinct target gene expression patterns, and factor mutations preferentially deregulate low-sensitivity genes. Unlike other target genes, GATA-1-mediated Tac-2 regulation was bimodal, with activation followed by repression, and the coregulator Friend of GATA-1 (FOG-1) selectively mediated repression. A GATA-1 mutant defective in FOG-1 binding occupied a Tac-2 regulatory region at levels higher than wild-type GATA-1, whereas FOG-1 facilitated chromatin occupancy at a distinct target site. These results indicate that FOG-1 is a determinant of GATA factor target gene sensitivity by either facilitating or opposing chromatin occupancy. PMID:17043224

  12. Latent fingermark pore area reproducibility.

    PubMed

    Gupta, A; Buckley, K; Sutton, R

    2008-08-01

    The study of the reproducibility of friction ridge pore detail in fingermarks is a measure of their usefulness in personal identification. Pore area in latent prints developed using cyanoacrylate and ninhydrin were examined and measured by photomicrography using appropriate software tools. The data were analysed statistically and the results showed that pore area is not reproducible in developed latent prints, using either of the development techniques. The results add further support to the lack of reliability of pore area in personal identification. PMID:18617339

  13. 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.

  14. 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.

  15. Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells.

    PubMed

    Holkers, Maarten; Maggio, Ignazio; Liu, Jin; Janssen, Josephine M; Miselli, Francesca; Mussolino, Claudio; Recchia, Alessandra; Cathomen, Toni; Gonçalves, Manuel A F V

    2013-03-01

    The array of genome editing strategies based on targeted double-stranded DNA break formation have recently been enriched through the introduction of transcription activator-like type III effector (TALE) nucleases (TALENs). To advance the testing of TALE-based approaches, it will be crucial to deliver these custom-designed proteins not only into transformed cell types but also into more relevant, chromosomally stable, primary cells. Viral vectors are among the most effective gene transfer vehicles. Here, we investigated the capacity of human immunodeficiency virus type 1- and adenovirus-based vectors to package and deliver functional TALEN genes into various human cell types. To this end, we attempted to assemble particles of these two vector classes, each encoding a monomer of a TALEN pair targeted to a bipartite sequence within the AAVS1 'safe harbor' locus. Vector DNA analyses revealed that adenoviral vectors transferred intact TALEN genes, whereas lentiviral vectors failed to do so, as shown by their heterogeneously sized proviruses in target cells. Importantly, adenoviral vector-mediated TALEN gene delivery resulted in site-specific double-stranded DNA break formation at the intended AAVS1 target site at similarly high levels in both transformed and non-transformed cells. In conclusion, we demonstrate that adenoviral, but not lentiviral, vectors constitute a valuable TALEN gene delivery platform.

  16. Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells

    PubMed Central

    Holkers, Maarten; Maggio, Ignazio; Liu, Jin; Janssen, Josephine M.; Miselli, Francesca; Mussolino, Claudio; Recchia, Alessandra; Cathomen, Toni; Gonçalves, Manuel A. F. V.

    2013-01-01

    The array of genome editing strategies based on targeted double-stranded DNA break formation have recently been enriched through the introduction of transcription activator-like type III effector (TALE) nucleases (TALENs). To advance the testing of TALE-based approaches, it will be crucial to deliver these custom-designed proteins not only into transformed cell types but also into more relevant, chromosomally stable, primary cells. Viral vectors are among the most effective gene transfer vehicles. Here, we investigated the capacity of human immunodeficiency virus type 1- and adenovirus-based vectors to package and deliver functional TALEN genes into various human cell types. To this end, we attempted to assemble particles of these two vector classes, each encoding a monomer of a TALEN pair targeted to a bipartite sequence within the AAVS1 ‘safe harbor’ locus. Vector DNA analyses revealed that adenoviral vectors transferred intact TALEN genes, whereas lentiviral vectors failed to do so, as shown by their heterogeneously sized proviruses in target cells. Importantly, adenoviral vector-mediated TALEN gene delivery resulted in site-specific double-stranded DNA break formation at the intended AAVS1 target site at similarly high levels in both transformed and non-transformed cells. In conclusion, we demonstrate that adenoviral, but not lentiviral, vectors constitute a valuable TALEN gene delivery platform. PMID:23275534

  17. Modeling of gene therapy for regenerative cells using intelligent agents.

    PubMed

    Adly, Aya Sedky; Aboutabl, Amal Elsayed; Ibrahim, M Shaarawy

    2011-01-01

    Gene therapy is an exciting field that has attracted much interest since the first submission of clinical trials. Preliminary results were very encouraging and prompted many investigators and researchers. However, the ability of stem cells to differentiate into specific cell types holds immense potential for therapeutic use in gene therapy. Realization of this potential depends on efficient and optimized protocols for genetic manipulation of stem cells. It is widely recognized that gain/loss of function approaches using gene therapy are essential for understanding specific genes functions, and such approaches would be particularly valuable in studies involving stem cells. A significant complexity is that the development stage of vectors and their variety are still not sufficient to be efficiently applied in stem cell therapy. The development of scalable computer systems constitutes one step toward understanding dynamics of its potential. Therefore, the primary goal of this work is to develop a computer model that will support investigations of virus' behavior and organization on regenerative tissues including genetically modified stem cells. Different simulation scenarios were implemented, and their results were encouraging compared to ex vivo experiments, where the error rate lies in the range of acceptable values in this domain of application.

  18. Gene transfer into neural cells in vitro using adenoviral vectors.

    PubMed

    Southgate, T D; Kingston, P A; Castro, M G

    2001-05-01

    Adenoviruses (Ads) have become a very attractive and versatile vector system for delivering genes into brain cells in vitro and in vivo. One of the main attractions of Ads is that they can mediate gene transfer into post-mitotic cells, i.e. neurons. Ads are easy to grow and manipulate, stable, and their biology is very well understood. This unit is designed to help newcomers into the field, to design, prepare and grow replication-defective recombinant adenovirus vectors with the aim of transferring genes into neurons and glial cells in primary culture. It provides step-by-step methods describing the preparation of brain cell cultures, their infection using recombinant adenovirus vectors and also the assessment of transgene expression using a variety of techniques including fluorescence immunocytochemistry and fluorescence activated cell-sorting (FACS) analysis. The methods described will be useful to scientists wishing to enter the adenovirus field to construct adenovirus vectors to be used for gene transfer into neural cells.

  19. 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.

  20. Impact of the cell division cycle on gene circuits.

    PubMed

    Bierbaum, Veronika; Klumpp, Stefan

    2015-09-25

    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.

  1. Use of bacterial and firefly luciferases as reporter genes in DEAE-dextran-mediated transfection of mammalian cells.

    PubMed

    Pazzagli, M; Devine, J H; Peterson, D O; Baldwin, T O

    1992-08-01

    The aim of this study was to compare three different luciferase genes by placing them in a single reporter vector and expressing them in the same mammalian cell type. The luciferase genes investigated were the luc genes from the fireflies Photinus pyralis (PP) and Luciola mingrelica (LM) and the lux AB5 gene, a translational fusion of the two subunits of the bacterial luciferase from Vibrio harveyi (VH). The chloramphenicol acetyltransferase (CAT) gene was also included in this study for comparison. The performances of the assay methods of the corresponding enzymes were evaluated using reference materials and the results of the expressed enzymes following transfection were calculated using calibration curves. All of the bioluminescent assays possess high reproducibility both within and between the batches (less than 15%). The comparison of the assay methods shows that firefly luciferases have the highest detection sensitivity (0.05 and 0.08 amol for PP and LM, respectively) whereas the VH bacterial luciferase has 5 amol and CAT 100 amol. On the other hand, the transfection of the various plasmids shows that the content of the expressed enzyme within the cells is much higher for CAT than for the other luciferase genes. VH luciferase is expressed at very low levels in mammalian cells due to the relatively high temperature of growing of the mammalian cells that seems to impair the correct folding of the active enzyme. PP and LM luciferases are both expressed at picomolar level but usually 10 to 70 times less in content with respect to CAT within the transfected cells. On the basis of these results the overall improvement in sensitivity related to the use of firefly luciferases as reporter genes in mammalian cells is about 30 to 50 times with respect to that of CAT. PMID:1443530

  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. PMID:26820874

  3. A reproducible technique combining tritiated thymidine autoradiography with immunodetection of bromodeoxyuridine for double labelling studies of cell proliferation in paraffin sections of tissues.

    PubMed

    Hume, W J

    1990-05-01

    A method is described to combine tritiated thymidine autoradiography with immunoperoxidase detection of bromodeoxyuridine on the same paraffin sections. It overcomes the varied technical artefacts we encountered when first attempting to combine these techniques and results in preparations with extremely low peroxidase and autoradiographic backgrounds. In particular, we find it is important to avoid the use of detergents during immunostaining, otherwise grain counts are reduced and autoradiograph exposures need to be greatly increased, and to avoid excessive peroxidase staining which makes it difficult to visualize silver grains in the overlying emulsion. The advantages of a method to remove emulsion films using acid-alcohol, allowing the same sections to be dipped twice with a long and a short autoradiographic exposure, are presented. The routine combination of high quality tritiated thymidine autoradiography with clean immunoperoxidase staining of bromodeoxyuridine-positive nuclei provides a new and powerful cell kinetic, double-labelling method to augment existing techniques e.g. by labelling the same cells undergoing DNA synthesis in successive cell cycles.

  4. 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

  5. CDNA microarray analysis of nerve growth factor-regulated gene expression profile in rat PC12 cells.

    PubMed

    Lee, Kyung-Hee; Ryu, Chun Jeih; Hong, Hyo Jeong; Kim, Jiyoung; Lee, Eunjoo H

    2005-04-01

    Nerve growth factor (NGF)-driven differentiation of PC12 cells into neuronal-like cells provides a representative model system for studying neuronal differentiation processes. Despite of extensive research, gene regulation associated with the differentiation program in PC12 cells still needs to be elucidated. We used cDNA microarray analysis to characterize the response of PC12 cells to NGF at mRNA expression. Forty-six genes were reproducibly influenced by 2-fold or more after NGF treatment for 5 days. Twenty-five of the regulated transcripts were matched to genes which have known functions. Among the microarray results confirmed with real-time reverse transcriptase assay, several genes have not previously known to be modulated by NGF. The results mostly reflected changes in molecules regulating neural plasticity, cytoskeletal organization, and lipid metabolism, which include neuritin, PDZ protein Mrt1, lipoprotein lipase, tropomodulin 1 and rhoB. These observed genetic changes may provide new information about molecular mechanisms underlying NGF-promoted differentiation of PC12 cells. PMID:16076023

  6. Microarray analysis of gene expression in adult retinal ganglion cells.

    PubMed

    Ivanov, Dmitry; Dvoriantchikova, Galina; Nathanson, Lubov; McKinnon, Stuart J; Shestopalov, Valery I

    2006-01-01

    Retinal ganglion cells (RGCs) transfer visual information to the brain and are known to be susceptible to selective degeneration in various neuropathies such as glaucoma. This selective vulnerability suggests that these highly specialized neurons possess a distinct gene expression profile that becomes altered by neuropathy-associated stresses, which lead to the RGC death. In this study, to identify genes expressed predominantly in adult RGCs, a global transcriptional profile of purified primary RGCs has been compared to that of the whole retina. To avoid alterations of the original gene expression profile by cell culture conditions, we isolated RNA directly from adult RGCs purified by immunopanning without prior sub-cultivation. Genes expressed predominantly in RGCs included: Nrg1, Rgn, 14-3-3 family (Ywhah, Ywhaz, Ywhab), Nrn1, Gap43, Vsnl1, Rgs4. Some of these genes may serve as novel markers for these neurons. Our analysis revealed enrichment in genes controlling the pro-survival pathways in RGCs as compared to other retinal cells. PMID:16376886

  7. 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

  8. RNA-seq analysis of impact of PNN on gene expression and alternative splicing in corneal epithelial cells

    PubMed Central

    Akin, Debra; Newman, Jeremy R.B.; McIntyre, Lauren M.

    2016-01-01

    Purpose The specialized corneal epithelium requires differentiated properties, specific for its role at the anterior surface of the eye. Thus, tight maintenance of the differentiated qualities of the corneal epithelial is essential. Pinin (PNN) is an exon junction component (EJC) that has dramatic implications for corneal epithelial cell differentiation and may act as a stabilizer of the corneal epithelial cell phenotype. Our studies revealed that PNN is involved in transcriptional repression complexes and spliceosomal complexes, placing PNN at the fulcrum between chromatin and mRNA splicing. Transcriptome analysis of PNN-knockdown cells revealed clear and reproducible alterations in transcript profiles and splicing patterns of a subset of genes that would significantly impact the epithelial cell phenotype. We further investigated PNN’s role in the regulation of gene expression and alternative splicing (AS) in a corneal epithelial context. Methods Human corneal epithelial (HCET) cells that carry the doxycycline-inducible PNN-knockdown shRNA vector were used to perform RNA-seq to determine differential gene expression and differential AS events. Results Multiple genes and AS events were identified as differentially expressed between PNN-knockdown and control cells. Genes upregulated by PNN knockdown included a large proportion of genes that are associated with enhanced cell migration and ECM remodeling processes, such as MMPs, ADAMs, HAS2, LAMA3, CXCRs, and UNC5C. Genes downregulated in response to PNN depletion included IGFBP5, FGD3, FGFR2, PAX6, RARG, and SOX10. AS events in PNN-knockdown cells compared to control cells were also more likely to be detected, and upregulated. In particular, 60% of exon-skipping events, detected in only one condition, were detected in PNN-knockdown cells and of the shared exon-skipping events, 92% of those differentially expressed were more frequent in the PNN knockdown. Conclusions These data suggest that lowering of PNN levels in

  9. Efficient retrovirus-mediated transfer of cell-cycle control genes to transformed cells.

    PubMed

    Strauss, B E; Costanzi-Strauss, E

    1999-07-01

    The use of gene therapy continues to be a promising, yet elusive, alternative for the treatment of cancer. The origins of cancer must be well understood so that the therapeutic gene can be chosen with the highest chance of successful tumor regression. The gene delivery system must be tailored for optimum transfer of the therapeutic gene to the target tissue. In order to accomplish this, we study models of G1 cell-cycle control in both normal and transformed cells in order to understand the reasons for uncontrolled cellular proliferation. We then use this information to choose the gene to be delivered to the cells. We have chosen to study p16, p21, p53 and pRb gene transfer using the pCL-retrovirus. Described here are some general concepts and specific results of our work that indicate continued hope for the development of genetically based cancer treatments.

  10. 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...

  11. Reproducible Research in Computational Science

    PubMed Central

    Peng, Roger D.

    2012-01-01

    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. PMID:22144613

  12. Reproducible research in computational science.

    PubMed

    Peng, Roger D

    2011-12-01

    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.

  13. Epithelial Cell Gene Expression Induced by Intracellular Staphylococcus aureus

    PubMed Central

    Li, Xianglu; Fusco, William G.; Seo, Keun S.; Bayles, Kenneth W.; Mosley, Erin E.; McGuire, Mark A.; Bohach, Gregory A.

    2009-01-01

    HEp-2 cell monolayers were cocultured with intracellular Staphylococcus aureus, and changes in gene expression were profiled using DNA microarrays. Intracellular S. aureus affected genes involved in cellular stress responses, signal transduction, inflammation, apoptosis, fibrosis, and cholesterol biosynthesis. Transcription of stress response and signal transduction-related genes including atf3, sgk, map2k1, map2k3, arhb, and arhe was increased. In addition, elevated transcription of proinflammatory genes was observed for tnfa, il1b, il6, il8, cxcl1, ccl20, cox2, and pai1. Genes involved in proapoptosis and fibrosis were also affected at transcriptional level by intracellular S. aureus. Notably, intracellular S. aureus induced strong transcriptional down-regulation of several cholesterol biosynthesis genes. These results suggest that epithelial cells respond to intracellular S. aureus by inducing genes affecting immunity and in repairing damage caused by the organism, and are consistent with the possibility that the organism exploits an intracellular environment to subvert host immunity and promote colonization. PMID:20016671

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

    PubMed Central

    Choi, Jae Young; Aquadro, Charles F.

    2015-01-01

    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. PMID:26507797

  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. Performance reproducibility index for classification

    PubMed Central

    Yousefi, Mohammadmahdi R.; Dougherty, Edward R.

    2012-01-01

    Motivation: A common practice in biomarker discovery is to decide whether a large laboratory experiment should be carried out based on the results of a preliminary study on a small set of specimens. Consideration of the efficacy of this approach motivates the introduction of a probabilistic measure, for whether a classifier showing promising results in a small-sample preliminary study will perform similarly on a large independent sample. Given the error estimate from the preliminary study, if the probability of reproducible error is low, then there is really no purpose in substantially allocating more resources to a large follow-on study. Indeed, if the probability of the preliminary study providing likely reproducible results is small, then why even perform the preliminary study? Results: This article introduces a reproducibility index for classification, measuring the probability that a sufficiently small error estimate on a small sample will motivate a large follow-on study. We provide a simulation study based on synthetic distribution models that possess known intrinsic classification difficulties and emulate real-world scenarios. We also set up similar simulations on four real datasets to show the consistency of results. The reproducibility indices for different distributional models, real datasets and classification schemes are empirically calculated. The effects of reporting and multiple-rule biases on the reproducibility index are also analyzed. Availability: We have implemented in C code the synthetic data distribution model, classification rules, feature selection routine and error estimation methods. The source code is available at http://gsp.tamu.edu/Publications/supplementary/yousefi12a/. Supplementary simulation results are also included. Contact: edward@ece.tamu.edu Supplementary Information: Supplementary data are available at Bioinformatics online. PMID:22954625

  17. Improved retroviral suicide gene transfer in colon cancer cell lines after cell synchronization with methotrexate

    PubMed Central

    2011-01-01

    Background Cancer gene therapy by retroviral vectors is mainly limited by the level of transduction. Retroviral gene transfer requires target cell division. Cell synchronization, obtained by drugs inducing a reversible inhibition of DNA synthesis, could therefore be proposed to precondition target cells to retroviral gene transfer. We tested whether drug-mediated cell synchronization could enhance the transfer efficiency of a retroviral-mediated gene encoding herpes simplex virus thymidine kinase (HSV-tk) in two colon cancer cell lines, DHDK12 and HT29. Methods Synchronization was induced by methotrexate (MTX), aracytin (ara-C) or aphidicolin. Gene transfer efficiency was assessed by the level of HSV-TK expression. Transduced cells were driven by ganciclovir (GCV) towards apoptosis that was assessed using annexin V labeling by quantitative flow cytometry. Results DHDK12 and HT29 cells were synchronized in S phase with MTX but not ara-C or aphidicolin. In synchronized DHDK12 and HT29 cells, the HSV-TK transduction rates were 2 and 1.5-fold higher than those obtained in control cells, respectively. Furthermore, the rate of apoptosis was increased two-fold in MTX-treated DHDK12 cells after treatment with GCV. Conclusions Our findings indicate that MTX-mediated synchronization of target cells allowed a significant improvement of retroviral HSV-tk gene transfer, resulting in an increased cell apoptosis in response to GCV. Pharmacological control of cell cycle may thus be a useful strategy to optimize the efficiency of retroviral-mediated cancer gene therapy. PMID:21970612

  18. 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.

  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. 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.

  1. Carbon nanoparticles for gene transfection in eukaryotic cell lines.

    PubMed

    Zanin, H; Hollanda, L M; Ceragioli, H J; Ferreira, M S; Machado, D; Lancellotti, M; Catharino, R R; Baranauskas, V; Lobo, A O

    2014-06-01

    For the first time, oxygen terminated cellulose carbon nanoparticles (CCN) was synthesised and applied in gene transfection of pIRES plasmid. The CCN was prepared from catalytic of polyaniline by chemical vapour deposition techniques. This plasmid contains one gene that encodes the green fluorescent protein (GFP) in eukaryotic cells, making them fluorescent. This new nanomaterial and pIRES plasmid formed π-stacking when dispersed in water by magnetic stirring. The frequencies shift in zeta potential confirmed the plasmid strongly connects to the nanomaterial. In vitro tests found that this conjugation was phagocytised by NG97, NIH-3T3 and A549 cell lines making them fluorescent, which was visualised by fluorescent microscopy. Before the transfection test, we studied CCN in cell viability. Both MTT and Neutral Red uptake tests were carried out using NG97, NIH-3T3 and A549 cell lines. Further, we use metabolomics to verify if small amounts of nanomaterial would be enough to cause some cellular damage in NG97 cells. We showed two mechanisms of action by CCN-DNA complex, producing an exogenous protein by the transfected cell and metabolomic changes that contributed by better understanding of glioblastoma, being the major finding of this work. Our results suggested that this nanomaterial has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity, good transfection efficiency, and low cell damage in small amounts of nanomaterials in metabolomic tests.

  2. Progesterone Upregulates Gene Expression in Normal Human Thyroid Follicular Cells.

    PubMed

    Bertoni, Ana Paula Santin; Brum, Ilma Simoni; Hillebrand, Ana Caroline; Furlanetto, Tania Weber

    2015-01-01

    Thyroid cancer and thyroid nodules are more prevalent in women than men, so female sex hormones may have an etiological role in these conditions. There are no data about direct effects of progesterone on thyroid cells, so the aim of the present study was to evaluate progesterone effects in the sodium-iodide symporter NIS, thyroglobulin TG, thyroperoxidase TPO, and KI-67 genes expression, in normal thyroid follicular cells, derived from human tissue. NIS, TG, TPO, and KI-67 mRNA expression increased significantly after TSH 20 μUI/mL, respectively: 2.08 times, P < 0.0001; 2.39 times, P = 0.01; 1.58 times, P = 0.0003; and 1.87 times, P < 0.0001. In thyroid cells treated with 20 μUI/mL TSH plus 10 nM progesterone, RNA expression of NIS, TG, and KI-67 genes increased, respectively: 1.78 times, P < 0.0001; 1.75 times, P = 0.037; and 1.95 times, P < 0.0001, and TPO mRNA expression also increased, though not significantly (1.77 times, P = 0.069). These effects were abolished by mifepristone, an antagonist of progesterone receptor, suggesting that genes involved in thyroid cell function and proliferation are upregulated by progesterone. This work provides evidence that progesterone has a direct effect on thyroid cells, upregulating genes involved in thyroid function and growth. PMID:26089899

  3. Progesterone Upregulates Gene Expression in Normal Human Thyroid Follicular Cells

    PubMed Central

    Bertoni, Ana Paula Santin; Brum, Ilma Simoni; Hillebrand, Ana Caroline; Furlanetto, Tania Weber

    2015-01-01

    Thyroid cancer and thyroid nodules are more prevalent in women than men, so female sex hormones may have an etiological role in these conditions. There are no data about direct effects of progesterone on thyroid cells, so the aim of the present study was to evaluate progesterone effects in the sodium-iodide symporter NIS, thyroglobulin TG, thyroperoxidase TPO, and KI-67 genes expression, in normal thyroid follicular cells, derived from human tissue. NIS, TG, TPO, and KI-67 mRNA expression increased significantly after TSH 20 μUI/mL, respectively: 2.08 times, P < 0.0001; 2.39 times, P = 0.01; 1.58 times, P = 0.0003; and 1.87 times, P < 0.0001. In thyroid cells treated with 20 μUI/mL TSH plus 10 nM progesterone, RNA expression of NIS, TG, and KI-67 genes increased, respectively: 1.78 times, P < 0.0001; 1.75 times, P = 0.037; and 1.95 times, P < 0.0001, and TPO mRNA expression also increased, though not significantly (1.77 times, P = 0.069). These effects were abolished by mifepristone, an antagonist of progesterone receptor, suggesting that genes involved in thyroid cell function and proliferation are upregulated by progesterone. This work provides evidence that progesterone has a direct effect on thyroid cells, upregulating genes involved in thyroid function and growth. PMID:26089899

  4. New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling

    PubMed Central

    Dik, Willem A.; Pike-Overzet, Karin; Weerkamp, Floor; de Ridder, Dick; de Haas, Edwin F.E.; Baert, Miranda R.M.; van der Spek, Peter; Koster, Esther E.L.; Reinders, Marcel J.T.; van Dongen, Jacques J.M.; Langerak, Anton W.; Staal, Frank J.T.

    2005-01-01

    To gain more insight into initiation and regulation of T cell receptor (TCR) gene rearrangement during human T cell development, we analyzed TCR gene rearrangements by quantitative PCR analysis in nine consecutive T cell developmental stages, including CD34+ lin− cord blood cells as a reference. The same stages were used for gene expression profiling using DNA microarrays. We show that TCR loci rearrange in a highly ordered way (TCRD-TCRG-TCRB-TCRA) and that the initiating Dδ2-Dδ3 rearrangement occurs at the most immature CD34+CD38−CD1a− stage. TCRB rearrangement starts at the CD34+CD38+CD1a− stage and complete in-frame TCRB rearrangements were first detected in the immature single positive stage. TCRB rearrangement data together with the PTCRA (pTα) expression pattern show that human TCRβ-selection occurs at the CD34+CD38+CD1a+ stage. By combining the TCR rearrangement data with gene expression data, we identified candidate factors for the initiation/regulation of TCR recombination. Our data demonstrate that a number of key events occur earlier than assumed previously; therefore, human T cell development is much more similar to murine T cell development than reported before. PMID:15928199

  5. 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

  6. Differential gene expression in skeletal muscle cells after membrane depolarization.

    PubMed

    Juretić, Nevenka; Urzúa, Ulises; Munroe, David J; Jaimovich, Enrique; Riveros, Nora

    2007-03-01

    Skeletal muscle is a highly plastic tissue with a remarkable capacity to adapt itself to challenges imposed by contractile activity. Adaptive response, that include hypertrophy and activation of oxidative mechanisms have been associated with transient changes in transcriptional activity of specific genes. To define the set of genes regulated by a depolarizing stimulus, we used 22 K mouse oligonucleotide microarrays. Total RNA from C2C12 myotubes was obtained at 2, 4, 18, and 24 h after high K+ stimulation. cDNA from control and depolarized samples was labeled with cyanine 3 or 5 dyes prior to microarray hybridization. Loess normalization followed by statistical analysis resulted in 423 differentially expressed genes using an unadjusted P-value < or = 0.01 as cut off. Depolarization affects transcriptional activity of a limited number of genes, mainly associated with metabolism, cell communication and response to stress. A number of genes related to Ca2+ signaling pathways are induced at 4 h, reinforcing the potential role of Ca2+ in early steps of signal transduction that leads to gene expression. Significant changes in the expression of molecules involved in muscle cell structure were observed; K+-depolarization increased Tnni1 and Acta1 mRNA levels in both differentiated C2C12 and rat skeletal muscle cells in primary culture. Of these two, depolarization induced slow Ca2+ transients appear to have a role only in the regulation of Tnni1 transcriptional activity. We suggest that depolarization induced expression of a small set of genes may underlie Ca2+ dependent plasticity of skeletal muscle cells. PMID:17146758

  7. 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.

  8. Gene essentiality and synthetic lethality in haploid human cells.

    PubMed

    Blomen, Vincent A; Májek, Peter; Jae, Lucas T; Bigenzahn, Johannes W; Nieuwenhuis, Joppe; Staring, Jacqueline; Sacco, Roberto; van Diemen, Ferdy R; Olk, Nadine; Stukalov, Alexey; Marceau, Caleb; Janssen, Hans; Carette, Jan E; Bennett, Keiryn L; Colinge, Jacques; Superti-Furga, Giulio; Brummelkamp, Thijn R

    2015-11-27

    Although the genes essential for life have been identified in less complex model organisms, their elucidation in human cells has been hindered by technical barriers. We used extensive mutagenesis in haploid human cells to identify approximately 2000 genes required for optimal fitness under culture conditions. To study the principles of genetic interactions in human cells, we created a synthetic lethality network focused on the secretory pathway based exclusively on mutations. This revealed a genetic cross-talk governing Golgi homeostasis, an additional subunit of the human oligosaccharyltransferase complex, and a phosphatidylinositol 4-kinase β adaptor hijacked by viruses. The synthetic lethality map parallels observations made in yeast and projects a route forward to reveal genetic networks in diverse aspects of human cell biology. PMID:26472760

  9. Gene essentiality and synthetic lethality in haploid human cells.

    PubMed

    Blomen, Vincent A; Májek, Peter; Jae, Lucas T; Bigenzahn, Johannes W; Nieuwenhuis, Joppe; Staring, Jacqueline; Sacco, Roberto; van Diemen, Ferdy R; Olk, Nadine; Stukalov, Alexey; Marceau, Caleb; Janssen, Hans; Carette, Jan E; Bennett, Keiryn L; Colinge, Jacques; Superti-Furga, Giulio; Brummelkamp, Thijn R

    2015-11-27

    Although the genes essential for life have been identified in less complex model organisms, their elucidation in human cells has been hindered by technical barriers. We used extensive mutagenesis in haploid human cells to identify approximately 2000 genes required for optimal fitness under culture conditions. To study the principles of genetic interactions in human cells, we created a synthetic lethality network focused on the secretory pathway based exclusively on mutations. This revealed a genetic cross-talk governing Golgi homeostasis, an additional subunit of the human oligosaccharyltransferase complex, and a phosphatidylinositol 4-kinase β adaptor hijacked by viruses. The synthetic lethality map parallels observations made in yeast and projects a route forward to reveal genetic networks in diverse aspects of human cell biology.

  10. 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

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

    PubMed

    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

  12. Identification of novel Notch target genes in T cell leukaemia

    PubMed Central

    Chadwick, Nicholas; Zeef, Leo; Portillo, Virginia; Fennessy, Carl; Warrander, Fiona; Hoyle, Sarah; Buckle, Anne-Marie

    2009-01-01

    Background Dysregulated Notch signalling is believed to play an important role in the development and maintenance of T cell leukaemia. At a cellular level, Notch signalling promotes proliferation and inhibits apoptosis of T cell acute lymphoblastic leukaemia (T-ALL) cells. In this study we aimed to identify novel transcriptional targets of Notch signalling in the T-ALL cell line, Jurkat. Results RNA was prepared from Jurkat cells retrovirally transduced with an empty vector (GFP-alone) or vectors containing constitutively active forms of Notch (N1ΔE or N3ΔE), and used for Affymetrix microarray analysis. A subset of genes found to be regulated by Notch was chosen for real-time PCR validation and in some cases, validation at the protein level, using several Notch-transduced T-ALL and non-T-ALL leukaemic cell lines. As expected, several known transcriptional target of Notch, such as HES1 and Deltex, were found to be overexpressed in Notch-transduced cells, however, many novel transcriptional targets of Notch signalling were identified using this approach. These included the T cell costimulatory molecule CD28, the anti-apoptotic protein GIMAP5, and inhibitor of DNA binding 1 (1D1). Conclusion The identification of such downstream Notch target genes provides insights into the mechanisms of Notch function in T cell leukaemia, and may help identify novel therapeutic targets in this disease. PMID:19508709

  13. 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.

  14. A Review of Gene Delivery and Stem Cell Based Therapies for Regenerating Inner Ear Hair Cells

    PubMed Central

    Devarajan, Keerthana; Staecker, Hinrich; Detamore, Michael S.

    2011-01-01

    Sensory neural hearing loss and vestibular dysfunction have become the most common forms of sensory defects, affecting millions of people worldwide. Developing effective therapies to restore hearing loss is challenging, owing to the limited regenerative capacity of the inner ear hair cells. With recent advances in understanding the developmental biology of mammalian and non-mammalian hair cells a variety of strategies have emerged to restore lost hair cells are being developed. Two predominant strategies have developed to restore hair cells: transfer of genes responsible for hair cell genesis and replacement of missing cells via transfer of stem cells. In this review article, we evaluate the use of several genes involved in hair cell regeneration, the advantages and disadvantages of the different viral vectors employed in inner ear gene delivery and the insights gained from the use of embryonic, adult and induced pluripotent stem cells in generating inner ear hair cells. Understanding the role of genes, vectors and stem cells in therapeutic strategies led us to explore potential solutions to overcome the limitations associated with their use in hair cell regeneration. PMID:24956306

  15. Gene expression profiling of circulating tumor cells and peripheral blood mononuclear cells from breast cancer patients

    PubMed Central

    Hensler, Michal; Vančurová, Irena; Becht, Etienne; Palata, Ondřej; Strnad, Pavel; Tesařová, Petra; Čabiňaková, Michaela; Švec, David; Kubista, Mikael; Bartůňková, Jiřina; Špíšek, Radek; Sojka, Luděk

    2016-01-01

    ABSTRACT Circulating tumor cells (CTCs) are cancer cells that are released from a tumor into the bloodstream. The presence of CTCs in peripheral blood has been associated with metastasis formation in patients with breast cancer. Therefore, the molecular characterization of CTCs may improve diagnostics and support treatment decisions. We performed gene expression profiling to evaluate the enriched CTCs and peripheral blood mononuclear cells (PBMCs) of breast cancer patients using an expression panel of 55 breast cancer-associated genes. The study revealed several significantly differentially expressed genes in the CTC-positive samples, including a few that were exclusively expressed in these cells. However, the expression of these genes was barely detectable in the PBMC samples. Some genes were differentially expressed in PBMCs, and the expression of these genes was correlated with tumor grade and the formation of metastasis. In this study, we have shown that the enriched CTCs of breast cancer patients overexpress genes involved in proteolytic degradation of the extracellular matrix (ECM) as well as genes that play important roles in the epithelial-mesenchymal transition (EMT) process that may occur in these cells. PMID:27141386

  16. 40 CFR 799.9530 - TSCA in vitro mammalian cell gene mutation test.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 33 2012-07-01 2012-07-01 false TSCA in vitro mammalian cell gene... MIXTURE TESTING REQUIREMENTS Health Effects Test Guidelines § 799.9530 TSCA in vitro mammalian cell gene.... The in vitro mammalian cell gene mutation test can be used to detect gene mutations induced...

  17. 40 CFR 799.9530 - TSCA in vitro mammalian cell gene mutation test.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 32 2011-07-01 2011-07-01 false TSCA in vitro mammalian cell gene... MIXTURE TESTING REQUIREMENTS Health Effects Test Guidelines § 799.9530 TSCA in vitro mammalian cell gene.... The in vitro mammalian cell gene mutation test can be used to detect gene mutations induced...

  18. 40 CFR 799.9530 - TSCA in vitro mammalian cell gene mutation test.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 32 2014-07-01 2014-07-01 false TSCA in vitro mammalian cell gene... MIXTURE TESTING REQUIREMENTS Health Effects Test Guidelines § 799.9530 TSCA in vitro mammalian cell gene.... The in vitro mammalian cell gene mutation test can be used to detect gene mutations induced...

  19. 40 CFR 799.9530 - TSCA in vitro mammalian cell gene mutation test.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 33 2013-07-01 2013-07-01 false TSCA in vitro mammalian cell gene... MIXTURE TESTING REQUIREMENTS Health Effects Test Guidelines § 799.9530 TSCA in vitro mammalian cell gene.... The in vitro mammalian cell gene mutation test can be used to detect gene mutations induced...

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

    PubMed Central

    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. PMID:26894994

  1. 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.

  2. 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.

  3. Promoter DNA Hypermethylation and Gene Repression in Undifferentiated Arabidopsis Cells

    PubMed Central

    Berdasco, María; Alcázar, Rubén; García-Ortiz, María Victoria; Ballestar, Esteban; Fernández, Agustín F.; Roldán-Arjona, Teresa; Tiburcio, Antonio F.; Altabella, Teresa; Buisine, Nicolas; Quesneville, Hadi; Baudry, Antoine; Lepiniec, Loïc; Alaminos, Miguel; Rodríguez, Roberto; Lloyd, Alan; Colot, Vincent; Bender, Judith; Canal, María Jesús; Esteller, Manel; Fraga, Mario F.

    2008-01-01

    Maintaining and acquiring the pluripotent cell state in plants is critical to tissue regeneration and vegetative multiplication. Histone-based epigenetic mechanisms are important for regulating this undifferentiated state. Here we report the use of genetic and pharmacological experimental approaches to show that Arabidopsis cell suspensions and calluses specifically repress some genes as a result of promoter DNA hypermethylation. We found that promoters of the MAPK12, GSTU10 and BXL1 genes become hypermethylated in callus cells and that hypermethylation also affects the TTG1, GSTF5, SUVH8, fimbrin and CCD7 genes in cell suspensions. Promoter hypermethylation in undifferentiated cells was associated with histone hypoacetylation and primarily occurred at CpG sites. Accordingly, we found that the process specifically depends on MET1 and DRM2 methyltransferases, as demonstrated with DNA methyltransferase mutants. Our results suggest that promoter DNA methylation may be another important epigenetic mechanism for the establishment and/or maintenance of the undifferentiated state in plant cells. PMID:18827894

  4. 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®.

  5. Reproducibility of NIF hohlraum measurements

    NASA Astrophysics Data System (ADS)

    Moody, J. D.; Ralph, J. E.; Turnbull, D. P.; Casey, D. T.; Albert, F.; Bachmann, B. L.; Doeppner, T.; Divol, L.; Grim, G. P.; Hoover, M.; Landen, O. L.; MacGowan, B. J.; Michel, P. A.; Moore, A. S.; Pino, J. E.; Schneider, M. B.; Tipton, R. E.; Smalyuk, V. A.; Strozzi, D. J.; Widmann, K.; Hohenberger, M.

    2015-11-01

    The strategy of experimentally ``tuning'' the implosion in a NIF hohlraum ignition target towards increasing hot-spot pressure, areal density of compressed fuel, and neutron yield relies on a level of experimental reproducibility. We examine the reproducibility of experimental measurements for a collection of 15 identical NIF hohlraum experiments. The measurements include incident laser power, backscattered optical power, x-ray measurements, hot-electron fraction and energy, and target characteristics. We use exact statistics to set 1-sigma confidence levels on the variations in each of the measurements. Of particular interest is the backscatter and laser-induced hot-spot locations on the hohlraum wall. Hohlraum implosion designs typically include variability specifications [S. W. Haan et al., Phys. Plasmas 18, 051001 (2011)]. We describe our findings and compare with the specifications. This work was performed under the auspices of the U.S. Department of Energy by University of California, Lawrence Livermore National Laboratory under Contract W-7405-Eng-48.

  6. 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...

  7. 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

  8. Gene transfer into mammalian cells by particle bombardment.

    PubMed

    Heiser, W C

    1994-03-01

    Using COS-7 and Chinese hamster ovary cells as model systems, I have examined the efficiency of gene transfer into mammalian cells by particle bombardment. The most important parameters affecting transformation efficiency are the size of the particles, the target distance, and the extent of chamber vacuum. The size of the cell culture plate also affects transformation efficiency. Factors which have little effect on transformation efficiency are the helium pressure, the gap distance, and the macrocarrier travel distance. Compared to several other gene transfer techniques, particle bombardment has the advantage of requiring a low amount of DNA and a low number of cells for successful expression, measured as either transient or stable. I also describe transformation of several murine cell lines which have not been successfully transformed, or have been transformed at only low levels using other methods. These cell lines include preadipocytes (BMS-2), macrophages (J774), and transformed pre-B cells (38B9 and 70Z/3). Compared to transformation by electroporation, lipofection, and diethylaminoethyl dextran, particle bombardment was found to give 50- to 240-fold higher levels of transient expression as measured by luciferase activity in cell extracts. PMID:8203746

  9. 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.

  10. 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.

  11. Somatic embryogenesis in Arabidopsis thaliana is facilitated by mutations in genes repressing meristematic cell divisions.

    PubMed Central

    Mordhorst, A P; Voerman, K J; Hartog, M V; Meijer, E A; van Went, J; Koornneef, M; de Vries, S C

    1998-01-01

    Embryogenesis in plants can commence from cells other than the fertilized egg cell. Embryogenesis initiated from somatic cells in vitro is an attractive system for studying early embryonic stages when they are accessible to experimental manipulation. Somatic embryogenesis in Arabidopsis offers the additional advantage that many zygotic embryo mutants can be studied under in vitro conditions. Two systems are available. The first employs immature zygotic embryos as starting material, yielding continuously growing embryogenic cultures in liquid medium. This is possible in at least 11 ecotypes. A second, more efficient and reproducible system, employing the primordia timing mutant (pt allelic to hpt, cop2, and amp1), was established. A significant advantage of the pt mutant is that intact seeds, germinated in 2,4-dichlorophenoxyacetic acid (2, 4-D) containing liquid medium, give rise to stable embryonic cell cultures, circumventing tedious hand dissection of immature zygotic embryos. pt zygotic embryos are first distinguishable from wild type at early heart stage by a broader embryonic shoot apical meristem (SAM). In culture, embryogenic clusters originate from the enlarged SAMs. pt somatic embryos had all characteristic embryo pattern elements seen in zygotic embryos, but with higher and more variable numbers of cells. Embryogenic cell cultures were also established from seedling, of other mutants with enlarged SAMs, such as clavata (clv). pt clv double mutants showed additive effects on SAM size and an even higher frequency of seedlings producing embryogenic cell lines. pt clv double mutant plants had very short fasciated inflorescence stems and additive effects on the number of rosette leaves. This suggests that the PT and CLV genes act in independent pathways that control SAM size. An increased population of noncommitted SAM cells may be responsible for facilitated establishment of somatic embryogenesis in Arabidopsis. PMID:9611173

  12. Gene therapy inhibiting neointimal vascular lesion: in vivo transfer of endothelial cell nitric oxide synthase gene.

    PubMed Central

    von der Leyen, H E; Gibbons, G H; Morishita, R; Lewis, N P; Zhang, L; Nakajima, M; Kaneda, Y; Cooke, J P; Dzau, V J

    1995-01-01

    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 beta-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 vessels. 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. Images Fig. 1 Fig. 2 Fig. 5 PMID:7532305

  13. 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.

  14. Genotoxicity of retroviral hematopoietic stem cell gene therapy

    PubMed Central

    Trobridge, Grant D

    2012-01-01

    Introduction Retroviral vectors have been developed for hematopoietic stem cell (HSC) gene therapy and have successfully cured X-linked severe combined immunodeficiency (SCID-X1), adenosine deaminase deficiency (ADA-SCID), adrenoleukodystrophy, and Wiskott-Aldrich syndrome. However, in HSC gene therapy clinical trials, genotoxicity mediated by integrated vector proviruses has led to clonal expansion, and in some cases frank leukemia. Numerous studies have been performed to understand the molecular basis of vector-mediated genotoxicity with the aim of developing safer vectors and safer gene therapy protocols. These genotoxicity studies are critical to advancing HSC gene therapy. Areas covered This review provides an introduction to the mechanisms of retroviral vector genotoxicity. It also covers advances over the last 20 years in designing safer gene therapy vectors, and in integration site analysis in clinical trials and large animal models. Mechanisms of retroviral-mediated genotoxicity, and the risk factors that contribute to clonal expansion and leukemia in HSC gene therapy are introduced. Expert opinion Continued research on virus–host interactions and next-generation vectors should further improve the safety of future HSC gene therapy vectors and protocols. PMID:21375467

  15. Beta-cell gene expression and functional characterisation of the human insulinoma cell line CM.

    PubMed

    Baroni, M G; Cavallo, M G; Mark, M; Monetini, L; Stoehrer, B; Pozzilli, P

    1999-04-01

    Animal insulinoma cell lines are widely used to study physiological and pathophysiological mechanisms involved in glucose metabolism and to establish in vitro models for studies on beta-cells. In contrast, human insulinoma cell lines are rarely used because of difficulties in obtaining and culturing them for long periods. The aim of our study was to investigate, under different experimental conditions, the capacity of the human insulinoma cell line CM to retain beta-cell function, particularly the expression of constitutive beta-cell genes (insulin, the glucose transporters GLUT1 and GLUT2, glucokinase), intracellular and secreted insulin, beta-cell granules, and cAMP content. Results showed that CM cells from an early-passage express specific beta-cell genes in response to glucose stimulation, in particular the insulin and GLUT genes. Such capacity is lost at later passages when cells are cultured at standard glucose concentrations. However, if cultured at lower glucose concentration (0.8 mM) for a longer time, CM cells re-acquire the capacity to respond to glucose stimulation, as shown by the increased expression of beta-cell genes (insulin, GLUT2, glucokinase). Nonetheless, insulin secretion could not be restored under such experimental conditions despite the presence of intracellular insulin, although cAMP response to a potent activator of adenylate cyclase, forskolin, was present indicating a viable system. In conclusion, these data show that the human insulinoma cell line CM, at both early-passage and late-passage, posseses a functional glucose-signalling pathway and insulin mRNA expression similar to normal beta-cells, representing, therefore, a good model for studies concerning the signalling and expression of beta-cells. Furthermore, we have previously shown that it is also a good model for immunological studies. In this respect it is important to note that the CM cell line is one of the very few existing human beta-cell lines in long-term culture.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27010670

  17. Gene transfer by adenovirus in smooth muscle cells.

    PubMed

    Yu, M F; Ewaskiewicz, J I; Adda, S; Bailey, K; Harris, V; Sosnoski, D; Tomasic, M; Wilson, J; Kotlikoff, M I

    1996-08-01

    We report adenovirus-mediated gene transfer into airway smooth muscle cells in cultured cells and organ-cultured tracheal segments. Incubation of cultured rat tracheal myocytes with virus (5 x 10(8) pfu/ml) for 6 h resulted in beta-galactosidase expression in 94.8 +/- 2.5% of cells (n = 4). Following incubation of thin (less than 200 microns diameter) equine trachealis muscle segments with virus in organ culture (5 x 10(8)-5 x 10(10) pfu/ml) the average expression of the Lac Z gene was approximately 19 +/- 10% (n = 9). Expression was markedly improved, however, in segments from neonatal rats (13-21 days). In two experiments in which the mucosa and serosa were removed, nearly all cells expressed beta-galactosidase, whereas in a third experiment in which the tissue was not dissected, about 40% of cells were stained. Viral infection had no effect on tension development of strips following organ culture. In vitro gene transfer may provide a useful method to alter protein expression and examine the effect of this alteration on excitation/contraction coupling in smooth muscle.

  18. Microbubble-Enhanced Ultrasound Gene Transfer into Fibroblast Cells

    NASA Astrophysics Data System (ADS)

    Hirayama, Kota; Kaneko, Yukio; Tei, Yuichi; Matsumoto, Yoichiro

    2007-05-01

    Ultrasound finds many applications in the medical field, including ultrasound imaging, non-invasive treatment of tumors and lithotripsy. Ultrasound also has a potential to deliver some therapeutic materials, such as genes, drugs or proteins into cells. It is known that microbubbles can improve the delivery efficiency. It is believed that therapeutic materials can pass through the cell membrane whose permeability is increased by microbubble destruction or the ultrasound pressure. In this study, we investigated the delivery of GFP plasmid gene into the fibroblast cells. Ultrasound (frequency = 2.1 MHz, duty cycle = 10%) was used to irradiate the cultured cells through a medium that contains microbubbles and GFP plasmid. GFP plasmid transfection could be easily observed by fluorescence microscopy. Ultrasound irradiation under a variety of conditions resulted in successful GFP plasmid delivery. Microbubbles enhanced GFP transfection, and conclusions were drawn as to the relationship between gene transfection and various ultrasound exposure parameters. We also investigated the effect of ultrasound intensity on cell viability.

  19. Single-cell gene expression profiling reveals functional heterogeneity of undifferentiated human epidermal cells

    PubMed Central

    Tan, David W. M.; Jensen, Kim B.; Trotter, Matthew W. B.; Connelly, John T.; Broad, Simon; Watt, Fiona M.

    2013-01-01

    Human epidermal stem cells express high levels of β1 integrins, delta-like 1 (DLL1) and the EGFR antagonist LRIG1. However, there is cell-to-cell variation in the relative abundance of DLL1 and LRIG1 mRNA transcripts. Single-cell global gene expression profiling showed that undifferentiated cells fell into two clusters delineated by expression of DLL1 and its binding partner syntenin. The DLL1+ cluster had elevated expression of genes associated with endocytosis, integrin-mediated adhesion and receptor tyrosine kinase signalling. Differentially expressed genes were not independently regulated, as overexpression of DLL1 alone or together with LRIG1 led to the upregulation of other genes in the DLL1+ cluster. Overexpression of DLL1 and LRIG1 resulted in enhanced extracellular matrix adhesion and increased caveolin-dependent EGFR endocytosis. Further characterisation of CD46, one of the genes upregulated in the DLL1+ cluster, revealed it to be a novel cell surface marker of human epidermal stem cells. Cells with high endogenous levels of CD46 expressed high levels of β1 integrin and DLL1 and were highly adhesive and clonogenic. Knockdown of CD46 decreased proliferative potential and β1 integrin-mediated adhesion. Thus, the previously unknown heterogeneity revealed by our studies results in differences in the interaction of undifferentiated basal keratinocytes with their environment. PMID:23482486

  20. 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.

  1. 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.

  2. 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. PMID:17855129

  3. 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

  4. DNA methylation and differential gene regulation in photoreceptor cell death

    PubMed Central

    Farinelli, P; Perera, A; Arango-Gonzalez, B; Trifunovic, D; Wagner, M; Carell, T; Biel, M; Zrenner, E; Michalakis, S; Paquet-Durand, F; Ekström, P A R

    2014-01-01

    Retinitis pigmentosa (RP) defines a group of inherited degenerative retinal diseases causing progressive loss of photoreceptors. To this day, RP is still untreatable and rational treatment development will require a thorough understanding of the underlying cell death mechanisms. Methylation of the DNA base cytosine by DNA methyltransferases (DNMTs) is an important epigenetic factor regulating gene expression, cell differentiation, cell death, and survival. Previous studies suggested an involvement of epigenetic mechanisms in RP, and in this study, increased cytosine methylation was detected in dying photoreceptors in the rd1, rd2, P23H, and S334ter rodent models for RP. Ultrastructural analysis of photoreceptor nuclear morphology in the rd1 mouse model for RP revealed a severely altered chromatin structure during retinal degeneration that coincided with an increased expression of the DNMT isozyme DNMT3a. To identify disease-specific differentially methylated DNA regions (DMRs) on a genomic level, we immunoprecipitated methylated DNA fragments and subsequently analyzed them with a targeted microarray. Genome-wide comparison of DMRs between rd1 and wild-type retina revealed hypermethylation of genes involved in cell death and survival as well as cell morphology and nervous system development. When correlating DMRs with gene expression data, we found that hypermethylation occurred alongside transcriptional repression. Consistently, motif analysis showed that binding sites of several important transcription factors for retinal physiology were hypermethylated in the mutant model, which also correlated with transcriptional silencing of their respective target genes. Finally, inhibition of DNMTs in rd1 organotypic retinal explants using decitabine resulted in a substantial reduction of photoreceptor cell death, suggesting inhibition of DNA methylation as a potential novel treatment in RP. PMID:25476906

  5. Identifying genes that mediate anthracyline toxicity in immune cells.

    PubMed

    Frick, Amber; Suzuki, Oscar T; Benton, Cristina; Parks, Bethany; Fedoriw, Yuri; Richards, Kristy L; Thomas, Russell S; Wiltshire, Tim

    2015-01-01

    The role of the immune system in response to chemotherapeutic agents remains elusive. The interpatient variability observed in immune and chemotherapeutic cytotoxic responses is likely, at least in part, due to complex genetic differences. Through the use of a panel of genetically diverse mouse inbred strains, we developed a drug screening platform aimed at identifying genes underlying these chemotherapeutic cytotoxic effects on immune cells. Using genome-wide association studies (GWAS), we identified four genome-wide significant quantitative trait loci (QTL) that contributed to the sensitivity of doxorubicin and idarubicin in immune cells. Of particular interest, a locus on chromosome 16 was significantly associated with cell viability following idarubicin administration (p = 5.01 × 10(-8)). Within this QTL lies App, which encodes amyloid beta precursor protein. Comparison of dose-response curves verified that T-cells in App knockout mice were more sensitive to idarubicin than those of C57BL/6J control mice (p < 0.05). In conclusion, the cellular screening approach coupled with GWAS led to the identification and subsequent validation of a gene involved in T-cell viability after idarubicin treatment. Previous studies have suggested a role for App in in vitro and in vivo cytotoxicity to anticancer agents; the overexpression of App enhances resistance, while the knockdown of this gene is deleterious to cell viability. Further investigations should include performing mechanistic studies, validating additional genes from the GWAS, including Ppfia1 and Ppfibp1, and ultimately translating the findings to in vivo and human studies.

  6. Live-cell, temporal gene expression analysis of osteogenic differentiation in adipose-derived stem cells.

    PubMed

    Desai, Hetal V; Voruganti, Indu S; Jayasuriya, Chathuraka; Chen, Qian; Darling, Eric M

    2014-03-01

    Adipose-derived stem cells (ASCs) are a widely investigated type of mesenchymal stem cells with great potential for musculoskeletal regeneration. However, the use of ASCs is complicated by their cellular heterogeneity, which exists at both the population and single-cell levels. This study demonstrates a live-cell assay to investigate gene expression in ASCs undergoing osteogenesis using fluorescently tagged DNA hybridization probes called molecular beacons. A molecular beacon was designed to target the mRNA sequence for alkaline phosphatase (ALPL), a gene characteristically expressed during early osteogenesis. The percentage of cells expressing this gene in a population was monitored daily to quantify the uniformity of the differentiation process. Differentiating ASC populations were repeatedly measured in a nondestructive fashion over a 10-day period to obtain temporal gene expression data. Results showed consistent expression patterns for the investigated osteogenic genes in response to induction medium. Peak signal level, indicating when the most cells expressed ALPL at once, was observed on days 3-5. The differentiation response of sample populations was generally uniform when assessed on a well-by-well basis over time. The expression of alkaline phosphatase is consistent with previous studies of osteogenic differentiation, suggesting that molecular beacons are a viable means of monitoring the spatiotemporal gene expression of live, differentiating ASCs.

  7. Live-Cell, Temporal Gene Expression Analysis of Osteogenic Differentiation in Adipose-Derived Stem Cells

    PubMed Central

    Desai, Hetal V.; Voruganti, Indu S.; Jayasuriya, Chathuraka; Chen, Qian

    2014-01-01

    Adipose-derived stem cells (ASCs) are a widely investigated type of mesenchymal stem cells with great potential for musculoskeletal regeneration. However, the use of ASCs is complicated by their cellular heterogeneity, which exists at both the population and single-cell levels. This study demonstrates a live-cell assay to investigate gene expression in ASCs undergoing osteogenesis using fluorescently tagged DNA hybridization probes called molecular beacons. A molecular beacon was designed to target the mRNA sequence for alkaline phosphatase (ALPL), a gene characteristically expressed during early osteogenesis. The percentage of cells expressing this gene in a population was monitored daily to quantify the uniformity of the differentiation process. Differentiating ASC populations were repeatedly measured in a nondestructive fashion over a 10-day period to obtain temporal gene expression data. Results showed consistent expression patterns for the investigated osteogenic genes in response to induction medium. Peak signal level, indicating when the most cells expressed ALPL at once, was observed on days 3–5. The differentiation response of sample populations was generally uniform when assessed on a well-by-well basis over time. The expression of alkaline phosphatase is consistent with previous studies of osteogenic differentiation, suggesting that molecular beacons are a viable means of monitoring the spatiotemporal gene expression of live, differentiating ASCs. PMID:24367991

  8. Arginine-Rich Polyplexes for Gene Delivery to Neuronal Cells

    PubMed Central

    Morris, Viola B.; Labhasetwar, Vinod

    2015-01-01

    Neuronal gene therapy potentially offers an effective therapeutic intervention to cure or slow the progression of neurological diseases. However, neuronal cells are difficult to transfect with nonviral vectors, and in vivo their transport across the blood-brain barrier (BBB) is inefficient. We synthesized a series of arginine-rich oligopeptides, grafted with polyethyleneimine (PEI) and modified with a short-chain polyethylene glycol (PEG). We hypothesized that the arginine would enhance cellular uptake and transport of these polyplexes across the BBB, with PEG imparting biocompatibility and “stealth” properties and PEI facilitating DNA condensation and gene transfection. The optimized composition of the polyplexes demonstrated hemocompatibility with red blood cells, causing no lysis or aggregation, and showed significantly better cytocompatibility than PEI in vitro. Polyplexes formulated with luciferase-expressing plasmid DNA could transfect rat primary astrocytes and neurons in vitro. Confocal imaging data showed efficient cellular uptake of DNA and its sustained intracellular retention and nuclear localization with polyplexes. Intravenous administration of the optimized polyplexes in mice led to gene expression in the brain, which upon further immunohistochemical analysis demonstrated gene expression in neurons. In conclusion, we have successfully designed a nonviral vector for in vitro and in vivo neuronal gene delivery. PMID:26000961

  9. Firefly luciferase gene: structure and expression in mammalian cells.

    PubMed Central

    de Wet, J R; Wood, K V; DeLuca, M; Helinski, D R; Subramani, S

    1987-01-01

    The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5' end of the luciferase mRNA was determined by both S1 nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5' sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell lines stably expressing firefly luciferase were isolated. Deleting a portion of the 5'-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5' deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30- to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression. Images PMID:3821727

  10. Human clusterin gene expression is confined to surviving cells during in vitro programmed cell death.

    PubMed Central

    French, L E; Wohlwend, A; Sappino, A P; Tschopp, J; Schifferli, J A

    1994-01-01

    Clusterin is a serum glycoprotein endowed with cell aggregating, complement inhibitory, and lipid binding properties, and is also considered as a specific marker of dying cells, its expression being increased in various tissues undergoing programmed cell death (PCD). However, no study has so far directly shown that cells expressing clusterin in these tissues are actually apoptotic as defined by morphological and biochemical criteria. We have studied cellular clusterin gene expression in vitro using three different models of PCD: (a) ultraviolet B (UV-B) irradiation of human U937, HeLa, and A431 cell lines, (b) in vitro aging of human peripheral blood neutrophils (PMNs), and (c) dexamethasone-induced cell death of the human lymphoblastoid cell line CEM-C7. In all three models, the classical morphological and biochemical features of PCD observed did not correlate with an increase, but with either a marked decrease or an absence of clusterin gene expression as assessed by Northern blot analysis. In situ hybridization of U937 and A431 cells after UV-B irradiation revealed, in addition, that only morphologically normal cells that are surviving continue to express the clusterin gene. Our results demonstrate that in the human myeloid, lymphoid, and epithelial cell types studied, clusterin gene expression is not a prerequisite to their death by apoptosis. In addition, and most interestingly, in situ hybridization of U937 and A431 cells revealed that only surviving cells express the clusterin gene after the induction of PCD, thus providing novel evidence suggesting that clusterin may be associated with cell survival within tissues regressing as a consequence of PCD. Images PMID:8113419

  11. 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.

  12. 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. PMID:24402417

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. The planar cell-polarity gene stbm regulates cell behaviour and cell fate in vertebrate embryos.

    PubMed

    Park, Maiyon; Moon, Randall T

    2002-01-01

    The gene strabismus (stbm)/Van Gogh (Vang) functions in the planar cell-polarity pathway in Drosophila. As the existence of such a pathway in vertebrates has not been firmly established, we investigated the functions and signalling activities encoded by stbm in vertebrate embryos. In regard to cell fate, inhibition of Stbm function in zebrafish embryos leads to reduction of anterior neural markers, whereas gain of function leads to a rise in the levels of these markers. In regard to cell behaviour, both gain-of-function and loss-of-function assays reveal a role for Stbm in mediating cell movements during gastrulation. Mechanistically, Stbm inhibits Wnt-mediated activation of beta-catenin-dependent transcription while promoting phosphorylation of c-Jun- and AP-1-dependent transcription. This complex effect on intracellular signalling pathways probably involves dishevelled (dsh), as Stbm was found to interact with the Dsh protein, and as Dsh is known to function in both planar cell-polarity and beta-catenin pathways in Drosophila.

  18. 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.

  19. Isolation and characterization of a novel B cell activation gene

    SciTech Connect

    Hong, J.X.; Wilson, G.L.; Fox, C.H.; Kehrl, J.H. )

    1993-05-01

    Using subtractive cDNA cloning, the authors have isolated a series of cDNA clones that are differentially expressed between B and T lymphocytes. Whereas some of the isolated cDNA are from known B cell-specific genes, many of them represent previously uncharacterized genes. One of these unknown genes was denoted as BL34. Northern blot analysis performed with the BL34 cDNA revealed a 1.6-kb mRNA transcript that was present at low levels in RNA extracted from resting B lymphocytes, but whose expression was markedly increased in RNA prepared from mitogen-activated B cells. Similarly, RNA prepared from several B cell lines treated with phorbol myristate acetate (PMA) contained high levels of BL34 mRNA. In contrast, RNA from purified T cells treated with phytohemagglutinin and PMA had undetectable amounts of BL34 mRNA. In addition, high levels of BL34 mRNA were detected in RNA purified from PBMC of a patient with B cell acute lymphocytic leukemia. Southern blot analysis of human DNA from various tissues and cells lines demonstrated that BL34 is a single-copy gene without evidence of rearrangement. Two full length BL34 cDNA were sequenced, and an open reading frame of 588 bp was identified that was predicted to encode for a 196 amino acid protein. Searches of several protein data bases failed to find any homologous proteins. To directly analyze the expression of BL34 mRNA in lymphoid tissues in situ, hybridization studies with human tonsil tissue sections were performed. BL34 mRNA was detected in a portion of the cells in the germinal center region and adjacent to the mantle region. Further characterization of the BL34 gene and its protein should lead to insights to its role in B cell function and the consequences of its over-expression in acute lymphocytic leukemia. 26 refs., 6 figs., 1 tab.

  20. T Cells and Gene Regulation: The Switching On and Turning Up of Genes after T Cell Receptor Stimulation in CD8 T Cells

    PubMed Central

    Conley, James M.; Gallagher, Michael P.; Berg, Leslie J.

    2016-01-01

    Signaling downstream of the T cell receptor (TCR) is directly regulated by the dose and affinity of peptide antigen. The strength of TCR signaling drives a multitude of T cell functions from development to differentiation. CD8 T cells differentiate into a diverse pool of effector and memory cells after activation, a process that is critical for pathogen clearance and is highly regulated by TCR signal strength. T cells rapidly alter their gene expression upon activation. Multiple signaling pathways downstream of the TCR activate transcription factors, which are critical for this process. The dynamics between proximal TCR signaling, transcription factor activation and CD8 T cell function are discussed here. We propose that inducible T cell kinase (ITK) acts as a rheostat for gene expression. This unique regulation of TCR signaling by ITK provides a possible signaling mechanism for the promotion of a diverse T cell repertoire in response to pathogen. PMID:26973653

  1. Identification of genes associated with renal cell carcinoma using gene expression profiling analysis

    PubMed Central

    YAO, TING; WANG, QINFU; ZHANG, WENYONG; BIAN, AIHONG; ZHANG, JINPING

    2016-01-01

    Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults and accounts for ~80% of all kidney cancer cases. However, the pathogenesis of RCC has not yet been fully elucidated. To interpret the pathogenesis of RCC at the molecular level, gene expression data and bio-informatics methods were used to identify RCC associated genes. Gene expression data was downloaded from Gene Expression Omnibus (GEO) database and identified differentially coexpressed genes (DCGs) and dysfunctional pathways in RCC patients compared with controls. In addition, a regulatory network was constructed using the known regulatory data between transcription factors (TFs) and target genes in the University of California Santa Cruz (UCSC) Genome Browser (http://genome.ucsc.edu) and the regulatory impact factor of each TF was calculated. A total of 258,0427 pairs of DCGs were identified. The regulatory network contained 1,525 pairs of regulatory associations between 126 TFs and 1,259 target genes and these genes were mainly enriched in cancer pathways, ErbB and MAPK. In the regulatory network, the 10 most strongly associated TFs were FOXC1, GATA3, ESR1, FOXL1, PATZ1, MYB, STAT5A, EGR2, EGR3 and PELP1. GATA3, ERG and MYB serve important roles in RCC while FOXC1, ESR1, FOXL1, PATZ1, STAT5A and PELP1 may be potential genes associated with RCC. In conclusion, the present study constructed a regulatory network and screened out several TFs that may be used as molecular biomarkers of RCC. However, future studies are needed to confirm the findings of the present study. PMID:27347102

  2. 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

  3. Chagas’ disease parasite-derived neurotrophic factor activates cholinergic gene expression in neuronal PC12 cells

    PubMed Central

    Akpan, Nsikan; Caradonna, Kacey; Chuenkova, Marina V.; PereiraPerrin, Mercio

    2008-01-01

    A parasite-derived neurotrophic factor (PDNF) produced by the Chagas’ disease parasite Trypanosoma cruzi binds nerve growth factor (NGF) receptor TrkA, increasing receptor autophosphorylation, activating phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK/Erk) pathways, and transcription factor CREB. The end-result is enhanced survival and neuritogenesis of various types of neurons. PDNF also enhances the expression and activity of tyrosine hydroxylase, a rate limiting enzyme in the synthesis of dopamine and other catecholamine neurotransmitters. It remains unknown, however, if PDNF alters expression and metabolism of acetylcholine (ACh), a neurotransmitter thought to play a role in Chagas’ disease progression. Here we demonstrate that PDNF stimulates mRNA and protein expression of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT), which are critical for synthesis and storage of ACh. Stimulation requires functional TrkA because it did not occur in cell mutants that lack the receptor and in TrkA-expressing wild-type cells treated with K252a, an inhibitor of TrkA kinase activity. It also requires TrkA-dependent PI3K and MAPK/Erk signaling pathways because PDNF stimulation of cholinergic transcripts is abolished by specific pharmacological inhibitors. Furthermore, the cholinergic actions of PDNF were reproduced by PDNF-expressing extracellular T. cruzi trypomastigotes at the start of host cell invasion. In contrast, host cells bearing intracellular T. cruzi showed decreased, rather than increased, cholinergic gene expression. These results suggest that T. cruzi invasion of the nervous system alters cholinergic gene expression and that could play a role in neuropathology, and/or lack thereof, in Chagas’ disease patients. PMID:18502403

  4. Chagas' disease parasite-derived neurotrophic factor activates cholinergic gene expression in neuronal PC12 cells.

    PubMed

    Akpan, Nsikan; Caradonna, Kacey; Chuenkova, Marina V; PereiraPerrin, Mercio

    2008-06-27

    A parasite-derived neurotrophic factor (PDNF) produced by the Chagas' disease parasite Trypanosoma cruzi binds nerve growth factor (NGF) receptor TrkA, increasing receptor autophosphorylation, and activating phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK/Erk) pathways, and transcription factor CREB. The end-result is enhanced survival and neuritogenesis of various types of neurons. PDNF also enhances the expression and activity of tyrosine hydroxylase, a rate limiting enzyme in the synthesis of dopamine and other catecholamine neurotransmitters. It remains unknown, however, if PDNF alters expression and metabolism of acetylcholine (ACh), a neurotransmitter thought to play a role in Chagas' disease progression. Here we demonstrate that PDNF stimulates mRNA and protein expression of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT), which are critical for synthesis and storage of ACh. Stimulation requires functional TrkA because it did not occur in cell mutants that lack the receptor and in TrkA-expressing wild-type cells treated with K252a, an inhibitor of TrkA kinase activity. It also requires TrkA-dependent PI3K and MAPK/Erk signaling pathways because PDNF stimulation of cholinergic transcripts is abolished by specific pharmacological inhibitors. Furthermore, the cholinergic actions of PDNF were reproduced by PDNF-expressing extracellular T. cruzi trypomastigotes at the start of host cell invasion. In contrast, host cells bearing intracellular T. cruzi showed decreased, rather than increased, cholinergic gene expression. These results suggest that T. cruzi invasion of the nervous system alters cholinergic gene expression and that could play a role in neuropathology, and/or lack thereof, in Chagas' disease patients. PMID:18502403

  5. 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

  6. Repression of the albumin gene in Novikoff hepatoma cells

    SciTech Connect

    Capetanaki, Y.G.; Flytzanis, C.N.; Alonso, A.

    1982-03-01

    Novikoff hepatoma cells have lost their capacity to synthesize albumin. As a first approach to study the mechanisms underlying this event, in vitro translation in a reticulocyte system was performed using total polyadenylated mRNA from rat liver and Novikoff hepatoma cells. Immunoprecipitation of the in vitro translation products with albumin-specific antibody revealed a total lack of albumin synthesis in Novikoff hepatoma, suggesting the absence of functional albumin mRNA in these cells. Titration experiments using as probe albumin cDNA cloned in pBR322 plasmid demonstrated the absence of albumin-specific sequences in both polysomal and nuclear polyadenylated and total RNA from Novikoff cells. This albumin recombinant plasmid was obtained by screening a rat liver cDNA library with albumin (/sup 32/P)cDNA reverse transcribed from immuno-precipitated mRNA. The presence of an albumin-specific gene insert was documented with translation assays as well as by restriction mapping. Repression of the albumin gene at the transcriptional level was further demonstrated by RNA blotting experiments using the cloned albumin cDNA probe. Genomic DNA blots using the cloned albumin cDNA as probe did not reveal any large-scale deletions, insertions, or rearrangements in the albumin gene, suggesting that the processes involved in the suppression of albumin mRNA synthesis do not involve extensive genomic rearrangements.

  7. How do B cells differ from T cells in terms of gene expression?

    PubMed

    Lefkovits, I

    1995-01-01

    As part of an ongoing program to identify the genes that distinguish B cells from T cells, we have analyzed 910 molecular species of polypeptides detectable in mouse lymphocytes by 2D gel electrophoresis. Of these 910 polypeptides, 488 were present in both B and T cells, 185 in B but not T cells, and 237 in T but not B cells. The detected set of polypeptides accounts for more than 95% of the protein mass of lymphocytes. There are about 2000 other polypeptides that are below the threshold of detection. We have started to identify and to retrieve cDNA clones belonging to the B and T cell subset.

  8. 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.

  9. Neuregulin-regulated gene expression in mammary carcinoma cells.

    PubMed

    Amin, Dhara N; Tuck, David; Stern, David F

    2005-09-10

    Recent studies have suggested that autocrine production of Neuregulin (NRG), a growth factor that activates members of the Epidermal Growth Factor Receptor/ErbB family of proto-oncogenes, is sufficient for breast tumor initiation and progression. To elucidate the molecular mechanisms regulating these events, we undertook a global analysis of genes regulated by NRG in luminal mammary epithelial cell lines. Gene expression profiling of estrogen receptor-positive T47D cells exposed to NRG-1 revealed both previously identified and novel targets of NRG activation. Profiling of other estrogen receptor-positive breast cancer cell lines, MCF7 and SUM44, yielded a group of twenty-one genes whose transcripts are upregulated by NRG in all three lines tested. The NRG targets are FBJ murine osteosarcoma viral oncogene homolog B, Early growth response 1, v-jun avian sarcoma virus 17 oncogene homolog, Activating transcription factor 3, Homo sapiens cDNA FLJ31636 fis, Jun B proto-oncogene, Forkhead box C1, Platelet/endothelial cell adhesion molecule 1, NADPH-dependent retinol dehydrogenase/reductase, Dual specificity phosphatase 5, NGF inducible protein TIS21, Connective tissue growth factor, Jun D proto-oncogene, Serum response factor, Cullin 1, v-myc avian myelocytomatosis viral oncogene, Transient receptor potential channel 1, Low density lipoprotein receptor, Transforming growth factor beta 1, Nucleoporin 88 kDa, and Pleckstrin homology-like domain A1. Since NRG activation of these cells induces resistance to anti-hormonal therapy, the identified genes may provide clues to molecular events regulating mammary tumor progression and hormone independence.

  10. Pigment-cell-specific genes from fibroblasts are transactivated after chromosomal transfer into melanoma cells.

    PubMed Central

    Powers, T P; Shows, T B; Davidson, R L

    1994-01-01

    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. Images PMID:8289799

  11. 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.

  12. 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.

  13. Direct gene transfer into human cultured cells facilitated by laser micropuncture of the cell membrane

    SciTech Connect

    Tao, W.; Wilkinson, J.; Stanbridge, E.J.; Berns, M.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 media containing an aminoglycoside antibiotic, G418. Integration of the neo gene into individual 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 8x10-4-3x10-3. This method appears to be 100-fold more efficient than the standard calcium phosphate-mediated method of DNA transfer.

  14. Embryo quality predictive models based on cumulus cells gene expression

    PubMed Central

    Burnik Papler, T; Verdenik, I; Fon Tacer, K; Vrtačnik Bokal, E

    2016-01-01

    Abstract Since the introduction of in vitro fertilization (IVF) in clinical practice of infertility treatment, the indicators for high quality embryos were investigated. Cumulus cells (CC) have a specific gene expression profile according to the developmental potential of the oocyte they are surrounding, and therefore, specific gene expression could be used as a biomarker. The aim of our study was to combine more than one biomarker to observe improvement in prediction value of embryo development. In this study, 58 CC samples from 17 IVF patients were analyzed. This study was approved by the Republic of Slovenia National Medical Ethics Committee. Gene expression analysis [quantitative real time polymerase chain reaction (qPCR)] for five genes, analyzed according to embryo quality level, was performed. Two prediction models were tested for embryo quality prediction: a binary logistic and a decision tree model. As the main outcome, gene expression levels for five genes were taken and the area under the curve (AUC) for two prediction models were calculated. Among tested genes, AMHR2 and LIF showed significant expression difference between high quality and low quality embryos. These two genes were used for the construction of two prediction models: the binary logistic model yielded an AUC of 0.72 ± 0.08 and the decision tree model yielded an AUC of 0.73 ± 0.03. Two different prediction models yielded similar predictive power to differentiate high and low quality embryos. In terms of eventual clinical decision making, the decision tree model resulted in easy-to-interpret rules that are highly applicable in clinical practice. PMID:27785402

  15. 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.

  16. Lactobacillus acidophilus L-92 Cells Activate Expression of Immunomodulatory Genes in THP-1 Cells

    PubMed Central

    YANAGIHARA, Sae; GOTO, Hiroaki; HIROTA, Tatsuhiko; FUKUDA, Shinji; OHNO, Hiroshi; YAMAMOTO, Naoyuki

    2014-01-01

    To understand the immunomodulatory effects of Lactobacillus acidophilus L-92 cells suggested from our previous study of in vivo anti-allergy and anti-virus effects, host immune responses in macrophage-like THP-1 cells after 4 h (the early phase) and 24 h (the late phase) of cocultivation with L-92 cells were investigated by transcriptome analysis. In the early phase of L-92 treatment, various transcription regulator genes, such as, NFkB1, NFkB2, JUN, HIVEP2 and RELB, and genes encoding chemokines and cytokines, such as CCL4, CXCL11, CCL3 and TNF, were upregulated. Two transmembrane receptor genes, TLR7 and ICAM1, were also upregulated in the early phase of treatment. In contrast, many transmembrane receptor genes, such as IL7R, CD80, CRLF2, CD86, CD5, HLA-DQA1, IL2RA, IL15RA and CSF2RA, and some cytokine genes, including IL6, IL23A and CCL22, were significantly upregulated in the late phase after L-92 exposure. Some genes encoding cytokines, such as IL1A, IL1B and IL8, and the enzyme IDO1 were upregulated at both the early and the late phases of treatment. These results suggest that probiotic L-92 might promote Th1 and regulatory T-cell responses by activation of the MAPK signaling pathway, followed by the NOD-like receptor signaling pathway in THP-1 cells. PMID:25379363

  17. A proteomic chronology of gene expression through the cell cycle in human myeloid leukemia cells

    PubMed Central

    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 PMID:24596151

  18. 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.

  19. 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

  20. Stimulated stromal cells induce gamma-globin gene expression in erythroid cells via nitric oxide production

    PubMed Central

    Čokić, Vladan P.; Beleslin-Čokić, Bojana B.; Smith, Reginald D.; Economou, Antaeus P.; Wahl, Larry M.; Noguchi, Constance T.; Schechter, Alan N.

    2009-01-01

    Objective We have previously shown that nitric oxide (NO) is involved in the hydroxyurea-induced increase of gamma-globin gene expression in cultured human erythroid progenitor cells and that hydroxyurea increases NO production in endothelial cells via endothelial NO synthase (NOS). We have now expanded those studies to demonstrate that the stimulation of gamma-globin gene expression is also mediated by NOS induction in stromal cells within the bone marrow microenvironment. Materials and Methods Using NO analyzer, we measured NO production in endothelial and macrophage cell cultures. In co-culture studies of erythroid and stromal cells we measured globin gene expression during stimulation by NO inducers. Results Hydroxyurea (30–100 μM) induced NOS-dependent production of NO in human macrophages (up to 1.2 μM). Co-culture studies of human macrophages with erythroid progenitor cells also resulted in induction of gamma-globin mRNA expression (up to 3 fold) in the presence of hydroxyurea. NOS-dependent stimulation of NO by lipopolysaccharide (up to 0.6 μM) has been observed in human macrophages. We found that lipopolysaccharide and interferon-gamma together increased gamma-globin gene expression (up to 2 fold) in human macrophage/erythroid cell co-cultures. Co-culture of human bone marrow endothelial cells with erythroid progenitor cells also induced gamma-globin mRNA expression (2.4 fold) in the presence of hydroxyurea (40 μM). Conclusion These results demonstrate an arrangement by which NO and fetal hemoglobin inducers may stimulate globin genes in erythroid cells via the common paracrine effect of bone marrow stromal cells. PMID:19576950

  1. Hypoxia-mediated regulation of gene expression in mammalian cells

    PubMed Central

    Shih, Shu-Ching; Claffey, Kevin P.

    1998-01-01

    The molecular mechanism underlying oxygen sensing in mammalian cells has been extensively investigated in the areas of glucose transport, glycolysis, erythropoiesis, angiogenesis and catecholamine metabolism. Expression of functionally operative representative proteins in these specific areas, such as the glucose transporter 1, glycolytic enzymes, erythropoietin, vascular endothelial growth factor and tyrosine hydroxylase are all induced by hypoxia. Recent studies demonstrated that both transcriptional activation and post-transcriptional mechanisms are important to the hypoxia-mediated regulation of gene expression. In this article, the cis-acting elements and trans-acting factors involved in the transcriptional activation of gene expression will be reviewed. In addition, the mechanisms of post-transcriptional mRNA stabilization will also be addressed. We will discuss whether these two processes of regulation of hypoxia-responsive genes are mechanistically linked and co-operative in nature. PMID:10319016

  2. 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.

  3. Gene expression profiling analysis of osteosarcoma cell lines

    PubMed Central

    SUN, LU; LI, JIE; YAN, BING

    2015-01-01

    Osteosarcoma (OS) is the most common type of primary bone malignancy and has a poor prognosis. To investigate the mechanisms of osteosarcoma, the present analyzed the GSE28424 microarray. GSE28424 was downloaded from the Gene Expression Omnibus, and included a collective of 19 OS cell lines and four normal bone cell lines, which were used as controls. Subsequently, the differentially expressed genes (DEGs) were screened using the Limma package in Bioconductor. Gene Ontology (GO) and pathway enrichment analysis of the DEGs was performed using the Database for Annotation, Visualization and Integrated Discovery, interactions between the proteins encoded by the DEGs were identified using STRING, and the protein-protein interaction (PPI) network was visualized using Cytoscape. In addition, modular analysis of the PPI network was performed using the Clique Percolation Method (CPM) in CFinder. A total of 1,170 DEGs were screened, including 530 upreguated and 640 downregulated genes. The enriched functions included organelle fission, immune response and response to wounding. In addition, RPL8 was observed to be involved with the ribosomal pathway in module A of the PPI network of the DEGs. PLCG1, SYK and PLCG2 were also involved in the B-cell receptor signaling pathway in module B and the Fc-epsilon RI signaling pathway in module C. In addition, AURKA (degree=39), MAD2L1 (degree=38), CDCA8 (degree=38), BUB1 (degree=37) and MELK (degree=37) exhibited higher degrees of connectivity in module F. The results of the present study suggested that the RPL8, PLCG1, PLCG2, SYK, MAD2L1, AURKA, CDCA8, BUB1 and MELK genes may be involved in OS. PMID:26096802

  4. Genes and plant cell walls: a difficult relationship.

    PubMed

    Wojtaszek, P

    2000-08-01

    Chemical information, carried by genes, is one of several types of information important for the functioning of cells and organisms. While genes govern the two-dimensional flow of information, the cell walls are at the basis of a structural, three-dimensional framework of plant form and growth. Recent data show the walls to be a cellular 'organelle' undergoing dynamic changes in response to a plethora of stimuli. In this review, an integrated approach, rooted in the organismal perspective, is taken to consider the role of cell walls in the biology of plants. First, the complexity of molecular and biochemical events leading to the biosynthesis of wall components is described within the framework of its spatial cellular organisation, and the major regulatory check-points are characterised. Second, cell walls form a structural and functional continuum within the whole plant and thus could be defined in relation to the protoplasts that produce them and in relation to the plant itself. Model systems of suspension-cultured cells are used to reveal the existence of a bidirectional exchange of information between the protoplast and its walls. The 'plasticity' of plant cell reactions, seen in defence responses or in changes in wall composition, to e.g. stress, plant growth regulators or chemical agents as well as the role of cell walls and/or wall components in somatic embryogenesis are also discussed. Third, being a continuum within the plant body, the walls fulfil vital functions in plant growth and development. The examples characterised include the determination of cellular polarity and the plane of cell division, cytokinesis, and the role of plasmodesmata in cell-to-cell communication and the formation of functional symplastic domains. Fourth, the exocellular control of morphogenetic processes is described and the potential of cell walls as determinants or reservoirs of positional information is indicated. Particular emphasis is put on the (bio)chemical signals coming

  5. Identification of novel fusion genes in testicular germ cell tumors

    PubMed Central

    Hoff, Andreas M.; Alagaratnam, Sharmini; Zhao, Sen; Bruun, Jarle; Andrews, Peter W.; Lothe, Ragnhild A.; Skotheim, Rolf I.

    2015-01-01

    Testicular germ cell tumors (TGCT) are the most frequently diagnosed solid tumors in young men ages 15 to 44 years. Embryonal carcinomas (EC) comprise a subset of TGCTs that exhibit pluripotent characteristics similar to embryonic stem (ES) cells, but the genetic drivers underlying malignant transformation of ECs are unknown. To elucidate the abnormal genetic events potentially contributing to TGCT malignancy, such as the existence of fusion genes or aberrant fusion transcript expression, we performed RNA sequencing of EC cell lines and their non-malignant ES cell line counterparts. We identified eight novel fusion transcripts and one gene with alternative promoter usage, ETV6. Four out of nine transcripts were found recurrently expressed in an extended panel of primary TGCTs and additional EC cell lines, but not in normal parenchyma of the testis, implying tumor-specific expression. Two of the recurrent transcripts involved an intrachromosomal fusion between RCC1 and HENMT1 located 80 Mbp apart and an interchromosomal fusion between RCC1 and ABHD12B. RCC1-ABHD12B and the ETV6 transcript variant were found to be preferentially expressed in the more undifferentiated TGCT subtypes. In vitro differentiation of the NTERA2 EC cell line resulted in significantly reduced expression of both fusion transcripts involving RCC1 and the ETV6 transcript variant, indicating that they are markers of pluripotency in a malignant setting. In conclusion, we identified eight novel fusion transcripts that, to our knowledge, are the first fusion genes described in TGCT and may therefore potentially serve as genomic biomarkers of malignant progression. PMID:26659575

  6. 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.

  7. Diversity of Gene Expression in Hepatocellular Carcinoma Cells

    PubMed Central

    Zhang, Fan; Cui, Li; Kuo, Michael D.

    2016-01-01

    Understanding tumor diversity has been a long-lasting and challenging question for researchers in the field of cancer heterogeneity or tumor evolution. Studies have reported that compared to normal cells, there is a higher genetic diversity in tumor cells, while higher genetic diversity is associated with higher progression risks of tumor. We thus hypothesized that tumor diversity also holds true at the gene expression level. To test this hypothesis, we used t-test to compare the means of Simpson’s diversity index for gene expression (SDIG) between tumor and non-tumor samples. We found that the mean SDIG in tumor tissues is significantly higher than that in the non-tumor or normal tissues (P < 0.05) for most datasets. We also combined microarrays and next-generation sequencing data for validation. This cross-platform and cross-experimental validation greatly increased the reliability of our results. PMID:26779818

  8. Spatial reconstruction of single-cell gene expression

    PubMed Central

    Satija, Rahul; Farrell, Jeffrey A.; Gennert, David; Schier, Alexander F.; Regev, Aviv

    2015-01-01

    Spatial localization is a key determinant of cellular fate and behavior, but spatial RNA assays traditionally rely on staining for a limited number of RNA species. In contrast, single-cell RNA-seq allows for deep profiling of cellular gene expression, but established methods separate cells from their native spatial context. Here we present Seurat, a computational strategy to infer cellular localization by integrating single-cell RNA-seq data with in situ RNA patterns. We applied Seurat to spatially map 851 single cells from dissociated zebrafish (Danio rerio) embryos, inferring a transcriptome-wide map of spatial patterning. We confirmed Seurat’s accuracy using several experimental approaches, and used it to identify a set of archetypal expression patterns and spatial markers. Additionally, Seurat correctly localizes rare subpopulations, accurately mapping both spatially restricted and scattered groups. Seurat will be applicable to mapping cellular localization within complex patterned tissues in diverse systems. PMID:25867923

  9. Essential gene profiles in breast, pancreas and ovarian cancer cells

    PubMed Central

    Sayad, Azin; Karamboulas, Konstantina; Krzyzanowski, Paul M.; Sircoulomb, Fabrice; Medrano, Mauricio; Fedyshyn, Yaroslav; Koh, Judice L.Y.; van Dyk, Dewald; Fedyshyn, Bodhana; Luhova, Marianna; Brito, Glauber C.; Vizeacoumar, Franco J.; Vizeacoumar, Frederick S.; Datti, Alessandro; Kasimer, Dahlia; Buzina, Alla; Mero, Patricia; Misquitta, Christine; Normand, Josee; Haider, Maliha; Ketela, Troy; Wrana, Jeffrey L.; Rottapel, Robert; Neel, Benjamin G.; Moffat, Jason

    2016-01-01

    Genomic analyses are yielding a host of new information on the multiple genetic abnormalities associated with specific types of cancer. A comprehensive description of cancer-associated genetic abnormalities can improve our ability to classify tumors into clinically relevant subgroups, and, on occasion, identify mutant genes that drive the cancer phenotype (“drivers”). More often, though, the functional significance of cancer-associated mutations is difficult to discern. Genome-wide pooled shRNA screens enable global identification of the genes essential for cancer cell survival and proliferation, providing a “functional genomic” map of human cancer to complement genomic studies. Using a lentiviral shRNA library targeting ~16,000 genes and a newly developed, dynamic scoring approach, we identified essential gene profiles in 72 breast, pancreatic, and ovarian cancer cell lines. Integrating our results with current and future genomic data should facilitate the systematic identification of drivers, unanticipated synthetic lethal relationships, and functional vulnerabilities of these tumor types. PMID:22585861

  10. 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

  11. [Gene-stem Cell therapy for ischemic stroke].

    PubMed

    Abe, Koji

    2009-09-01

    Besides blood flow restoration, neuroprotection is essential for treating strokes at an acute stage. Both neurotrophic factors (NTFs) and free radical scavengers can act as neuroprotective agents with abilities to inhibit cell death and facilitate cell survival under cerebral ischemia. For example, topical application of glial cell line-derived neurotrophic factor (GDNF) remarkably reduced infarct size and brain edema after middle cerebral artery (MCA) occlusion in rats. Reduction in the infarct size was not found to be related to a change in the cerebral blood flow (CBF), but was accompanied by marked reduction in BrdU-positive cells in the affected area after TdT-mediated dUTP-biotin nick end labeling (TUNEL) for caspses. Thus, GDNF elicited a direct protective effect against ischemic brain damage, but without improving CBF. Sendai virus vectors harboring the GDNF gene led to a remarkable reduction in infract volume without affecting regional CBF but reduced the translocation of apoptosis inducible factor (AIF) from the mitochondria to cytoplasm. Regenerative therapy involving neural stem cells which are intrinsically activated or exogenously transplanted, is an important treatment strategy. To facilitate stem cell migration, an artificial scaffold can be implanted into the injured brain for promoting ischemic brain repair. Addition of NTFs greatly enhanced an intrinsic migration or invasion of stem cells into the scaffold: this strategy could be used in the future for enhancing regenerative potential of brain cells after chronic ischemia-induced brain damage. PMID:19803403

  12. Interchromosomal gene conversion at an endogenous human cell locus.

    PubMed Central

    Quintana, P J; Neuwirth, E A; Grosovsky, A J

    2001-01-01

    To examine the relationship between gene conversion and reciprocal exchange at an endogenous chromosomal locus, we developed a reversion assay in a thymidine kinase deficient mutant, TX545, derived from the human lymphoblastoid cell line TK6. Selectable revertants of TX545 can be generated through interchromosomal gene conversion at the site of inactivating mutations on each tk allele or by reciprocal exchange that alters the linkage relationships of inactivating polymorphisms within the tk locus. Analysis of loss of heterozygosity (LOH) at intragenic polymorphisms and flanking microsatellite markers was used to initially evaluate allelotypes in TK(+) revertants for patterns associated with either gene conversion or crossing over. The linkage pattern in a subset of convertants was then unambiguously established, even in the event of prereplicative recombinational exchanges, by haplotype analysis of flanking microsatellite loci in tk(-/-) LOH mutants collected from the tk(+/-) parental convertant. Some (7/38; 18%) revertants were attributable to easily discriminated nonrecombinational mechanisms, including suppressor mutations within the tk coding sequence. However, all revertants classified as a recombinational event (28/38; 74%) were attributed to localized gene conversion, representing a highly significant preference (P < 0.0001) over gene conversion with associated reciprocal exchange, which was never observed. PMID:11404339

  13. 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.

  14. Transcriptional regulation of cathelicidin genes in chicken bone marrow cells.

    PubMed

    Lee, Sang In; Jang, Hyun June; Jeon, Mi-hyang; Lee, Mi Ock; Kim, Jeom Sun; Jeon, Ik-Soo; Byun, Sung June

    2016-04-01

    Cathelicidins form a family of vertebrate-specific immune molecules with an evolutionarily conserved gene structure. We analyzed the expression patterns of cathelicidin genes (CAMP, CATH3, and CATHB1) in chicken bone marrow cells (BMCs) and chicken embryonic fibroblasts (CEFs). We found that CAMP and CATHB1 were significantly up-regulated in BMCs, whereas the expression of CATH3 did not differ significantly between BMCs and CEFs. To study the mechanism underlying the up-regulation of cathelicidin genes in BMCs, we predicted the transcription factors (TFs) that bind to the 5'-flanking regions of cathelicidin genes. CEBPA, EBF1, HES1, MSX1, and ZIC3 were up-regulated in BMCs compared to CEFs. Subsequently, when a siRNA-mediated knockdown assay was performed for MSX1, the expression of CAMP and CATHB1 was decreased in BMCs. We also showed that the transcriptional activity of the CAMP promoter was decreased by mutation of the MSX1-binding sites present within the 5'-flanking region of CAMP. These results increase our understanding of the regulatory mechanisms controlling cathelicidin genes in BMCs.

  15. Reproducibility of sterilized rubber impressions.

    PubMed

    Abdelaziz, Khalid M; Hassan, Ahmed M; Hodges, J S

    2004-01-01

    Impressions, dentures and other dental appliances may be contaminated with oral micro-flora or other organisms of varying pathogenicity from patient's saliva and blood. Several approaches have been tried to control the transmission of infectious organisms via dental impressions and because disinfection is less effective and has several drawbacks for impression characterization, several sterilization methods have been suggested. This study evaluated the reproducibility of rubber impressions after sterilization by different methods. Dimensional accuracy and wettability of two rubber impression materials (vinyl polysiloxane and polyether) were evaluated after sterilization by each of three well-known methods (immersion in 2% glutaraldehyde for 10 h, autoclaving and microwave radiation). Non-sterilized impressions served as control. The effect of the tray material on impression accuracy and the effect of topical surfactant on the wettability were also evaluated. One-way ANOVA with Dunnett's method was used for statistical analysis. All sterilizing methods reduced the reproducibility of rubber impressions, although not always significantly. Microwave sterilization had a small effect on both accuracy and wettability. The greater effects of the other methods could usually be overcome by using ceramic trays and by spraying impression surfaces with surfactant before pouring the gypsum mix. There was one exception: glutaraldehyde still degraded dimensional accuracy even with ceramic trays and surfactant. We conclude that a) sterilization of rubber impressions made on acrylic trays was usually associated with a degree of dimensional change; b) microwave energy seems to be a suitable technique for sterilizing rubber impressions; c) topical surfactant application helped restore wettability of sterilized impressions. PMID:15798825

  16. Evaluation of guidewire path reproducibility.

    PubMed

    Schafer, Sebastian; Hoffmann, Kenneth R; Noël, Peter B; Ionita, Ciprian N; Dmochowski, Jacek

    2008-05-01

    The number of minimally invasive vascular interventions is increasing. In these interventions, a variety of devices are directed to and placed at the site of intervention. The device used in almost all of these interventions is the guidewire, acting as a monorail for all devices which are delivered to the intervention site. However, even with the guidewire in place, clinicians still experience difficulties during the interventions. As a first step toward understanding these difficulties and facilitating guidewire and device guidance, we have investigated the reproducibility of the final paths of the guidewire in vessel phantom models on different factors: user, materials and geometry. Three vessel phantoms (vessel diameters approximately 4 mm) were constructed having tortuousity similar to the internal carotid artery from silicon tubing and encased in Sylgard elastomer. Several trained users repeatedly passed two guidewires of different flexibility through the phantoms under pulsatile flow conditions. After the guidewire had been placed, rotational c-arm image sequences were acquired (9 in. II mode, 0.185 mm pixel size), and the phantom and guidewire were reconstructed (512(3), 0.288 mm voxel size). The reconstructed volumes were aligned. The centerlines of the guidewire and the phantom vessel were then determined using region-growing techniques. Guidewire paths appear similar across users but not across materials. The average root mean square difference of the repeated placement was 0.17 +/- 0.02 mm (plastic-coated guidewire), 0.73 +/- 0.55 mm (steel guidewire) and 1.15 +/- 0.65 mm (steel versus plastic-coated). For a given guidewire, these results indicate that the guidewire path is relatively reproducible in shape and position.

  17. 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

  18. [Single-cell detection of EGFR gene mutation in circulating tumor cells in lung cancer].

    PubMed

    Shuai, Sun; Yuliang, Deng

    2015-12-01

    Circulating tumor cells (CTCs) are cells that shed from a primary tumor and enter the peripheral blood circulation. The CTCs are closely associated with tumor development and metastasis because of its high heterogeneity. However, there are still no effective methods to detect single-cell heterogeneity of the CTCs. To this end, we developed a method to detect gene mutation in CTCs at the single-cell level and applied it to the detection of EGFR gene mutation in single lung cancer CTC. Specifically, the rare CTCs were captured from blood using an integrated microfluidic system, and then were released into a microchip with thousands of nanoliter wells to isolate single CTC. The single CTC was then transferred into a PCR tube under the microscope for single-cell genome amplification and detection of EGFR gene mutation. We firstly modified chip and capillary and optimized PCR conditions (annealing temperature, number of cycles) using non-small-cell lung cancer (NSCLC) cell lines A549, NCI-H1650 and NCI-H1975 as samples, which showed maximal amplification after 30 cycles with an annealing temperature at 59℃. We then successfully detected blood samples from NSCLC patients using this method. 5 CTCs were obtained from 2 mL patient's blood and the sequencing of EGFR exons 18, 19, 20 and 21 showed no mutations. Our results demonstrated that this method is sensitive enough to detect gene mutation in single CTC and has guiding significance in clinic research. PMID:26704950

  19. 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.

  20. Human respiratory epithelial cells from nasal turbinate expressed stem cell genes even after serial passaging.

    PubMed

    Ruszymah, B H I; Izham, B A Azrul; Heikal, M Y Mohd; Khor, S F; Fauzi, M B; Aminuddin, B S

    2011-12-01

    Current development in the field of tissue engineering led to the idea of repairing and regenerating the respiratory airway through in vitro reconstruction using autologous respiratory epithelial (RE). To ensure the capability of proliferation, the stem cell property of RE cells from the nasal turbinate should be evaluated. Respiratory epithelial cells from six human nasal turbinates were harvested and cultured in vitro. The gene expression of FZD-9 and BST-1 were expressed in passage 2 (P2) and passage 4 (P4). The levels of expression were not significant between both passages. The RE cells exhibit the stem cell properties, which remains even after serial passaging.

  1. Identification of genes needed for regeneration, stem cell function, and tissue homeostasis by systematic gene perturbation in planaria

    PubMed Central

    Reddien, Peter W.; Bermange, Adam L.; Murfitt, Kenneth J.; Jennings, Joya R.; Alvarado, Alejandro Sánchez

    2007-01-01

    Summary Planarians have been a classic model system for the study of regeneration, tissue homeostasis, and stem cell biology for over a century, but 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. PMID:15866156

  2. Multiplexed transposon-mediated stable gene transfer in human cells

    PubMed Central

    Kahlig, Kristopher M.; Saridey, Sai K.; Kaja, Aparna; Daniels, Melissa A.; George, Alfred L.; Wilson, Matthew H.

    2010-01-01

    Generation of cultured human cells stably expressing one or more recombinant gene sequences is a widely used approach in biomedical research, biotechnology, and drug development. Conventional methods are not efficient and have severe limitations especially when engineering cells to coexpress multiple transgenes or multiprotein complexes. In this report, we harnessed the highly efficient, nonviral, and plasmid-based piggyBac transposon system to enable concurrent genomic integration of multiple independent transposons harboring distinct protein-coding DNA sequences. Flow cytometry of cell clones derived from a single multiplexed transfection demonstrated approximately 60% (three transposons) or approximately 30% (four transposons) stable coexpression of all delivered transgenes with selection for a single marker transposon. We validated multiplexed piggyBac transposon delivery by coexpressing large transgenes encoding a multisubunit neuronal voltage-gated sodium channel (SCN1A) containing a pore-forming subunit and two accessory subunits while using two additional genes for selection. Previously unobtainable robust sodium current was demonstrated through 38 passages, suitable for use on an automated high-throughput electrophysiology platform. Cotransfection of three large (up to 10.8 kb) piggyBac transposons generated a heterozygous SCN1A stable cell line expressing two separate alleles of the pore-forming subunit and two accessory subunits (total of four sodium channel subunits) with robust functional expression. We conclude that the piggyBac transposon system can be used to perform multiplexed stable gene transfer in cultured human cells, and this technology may be valuable for applications requiring concurrent expression of multiprotein complexes. PMID:20080581

  3. 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

  4. [Experimental gene therapy using p21/WAF1 gene in esophageal squamous cell carcinoma--adenovirus infection and gene gun technology].

    PubMed

    Fujii, T; Tanaka, Y; Tanaka, T; Matono, S; Sueyoshi, S; Fujita, H; Shirouzu, K; Kato, S; Yamana, H

    2001-10-01

    p21/WAF1 (p21) inhibits the activity of the cyclin/cdk complex and controls the G1 to S cell phase transition. In the present study, we used a recombinant adenoviral approach and gene gun technology to introduce p21 into esophageal cancer cells in order to assess the effect of p21 on cell growth. Infection with the p21 adenovirus (AdV) using gene gun technology resulted in inhibition of TE9 and KE3 cell growth. The levels of involucrin, which is a marker of squamous epithelium differentiation, markedly increased at 48 h and 72 h after p21 AdV infection in TE9 cells. These results indicate that p21 plays an important role in esophageal cancer cell proliferation. Overexpression of the p21 gene can inhibit cell growth and induce differentiation in esophageal cancer cells. p21 gene therapy may prove beneficial in the treatment of esophageal cancer.

  5. 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.

  6. 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.

  7. Cell of origin associated classification of B-cell malignancies by gene signatures of the normal B-cell hierarchy.

    PubMed

    Johnsen, Hans Erik; Bergkvist, Kim Steve; Schmitz, Alexander; Kjeldsen, Malene Krag; Hansen, Steen Møller; Gaihede, Michael; Nørgaard, Martin Agge; Bæch, John; Grønholdt, Marie-Louise; Jensen, Frank Svendsen; Johansen, Preben; Bødker, Julie Støve; Bøgsted, Martin; Dybkær, Karen

    2014-06-01

    Recent findings have suggested biological classification of B-cell malignancies as exemplified by the "activated B-cell-like" (ABC), the "germinal-center B-cell-like" (GCB) and primary mediastinal B-cell lymphoma (PMBL) subtypes of diffuse large B-cell lymphoma and "recurrent translocation and cyclin D" (TC) classification of multiple myeloma. Biological classification of B-cell derived cancers may be refined by a direct and systematic strategy where identification and characterization of normal B-cell differentiation subsets are used to define the cancer cell of origin phenotype. Here we propose a strategy combining multiparametric flow cytometry, global gene expression profiling and biostatistical modeling to generate B-cell subset specific gene signatures from sorted normal human immature, naive, germinal centrocytes and centroblasts, post-germinal memory B-cells, plasmablasts and plasma cells from available lymphoid tissues including lymph nodes, tonsils, thymus, peripheral blood and bone marrow. This strategy will provide an accurate image of the stage of differentiation, which prospectively can be used to classify any B-cell malignancy and eventually purify tumor cells. This report briefly describes the current models of the normal B-cell subset differentiation in multiple tissues and the pathogenesis of malignancies originating from the normal germinal B-cell hierarchy. PMID:23998255

  8. Immortalized neural progenitor cells for CNS gene transfer and repair.

    PubMed

    Martínez-Serrano, A; Björklund, A

    1997-11-01

    Immortalized multipotent neural stem and progenitor cells have emerged as a highly convenient source of tissue for genetic manipulation and ex vivo gene transfer to the CNS. Recent studies show that these cells, which can be maintained and genetically transduced as cell lines in culture, can survive, integrate and differentiate into both neurons and glia after transplantation to the intact or damaged brain. Progenitors engineered to secrete trophic factors, or to produce neurotransmitter-related or metabolic enzymes can be made to repopulate diseased or injured brain areas, thus providing a new potential therapeutic tool for the blockade of neurodegenerative processes and reversal of behavioural deficits in animal models of neurodegenerative diseases. With further technical improvements, the use of immortalized neural progenitors may bring us closer to the challenging goal of targeted and effective CNS repair.

  9. Transposon mutagenesis identifies genes that transform neural stem cells into glioma-initiating cells.

    PubMed

    Koso, Hideto; Takeda, Haruna; Yew, Christopher Chin Kuan; Ward, Jerrold M; Nariai, Naoki; Ueno, Kazuko; Nagasaki, Masao; Watanabe, Sumiko; Rust, Alistair G; Adams, David J; Copeland, Neal G; Jenkins, Nancy A

    2012-10-30

    Neural stem cells (NSCs) are considered to be the cell of origin of glioblastoma multiforme (GBM). However, the genetic alterations that transform NSCs into glioma-initiating cells remain elusive. Using a unique transposon mutagenesis strategy that mutagenizes NSCs in culture, followed by additional rounds of mutagenesis to generate tumors in vivo, we have identified genes and signaling pathways that can transform NSCs into glioma-initiating cells. Mobilization of Sleeping Beauty transposons in NSCs induced the immortalization of astroglial-like cells, which were then able to generate tumors with characteristics of the mesenchymal subtype of GBM on transplantation, consistent with a potential astroglial origin for mesenchymal GBM. Sequence analysis of transposon insertion sites from tumors and immortalized cells identified more than 200 frequently mutated genes, including human GBM-associated genes, such as Met and Nf1, and made it possible to discriminate between genes that function during astroglial immortalization vs. later stages of tumor development. We also functionally validated five GBM candidate genes using a previously undescribed high-throughput method. Finally, we show that even clonally related tumors derived from the same immortalized line have acquired distinct combinations of genetic alterations during tumor development, suggesting that tumor formation in this model system involves competition among genetically variant cells, which is similar to the Darwinian evolutionary processes now thought to generate many human cancers. This mutagenesis strategy is faster and simpler than conventional transposon screens and can potentially be applied to any tissue stem/progenitor cells that can be grown and differentiated in vitro.

  10. Targeting the Gdnf Gene in peritubular myoid cells disrupts undifferentiated spermatogonial cell development

    PubMed Central

    Chen, Liang-Yu; Willis, William D.; Eddy, Edward M.

    2016-01-01

    Spermatogonial stem cells (SSCs) are a subpopulation of undifferentiated spermatogonia located in a niche at the base of the seminiferous epithelium delimited by Sertoli cells and peritubular myoid (PM) cells. SSCs self-renew or differentiate into spermatogonia that proliferate to give rise to spermatocytes and maintain spermatogenesis. Glial cell line-derived neurotrophic factor (GDNF) is essential for this process. Sertoli cells produce GDNF and other growth factors and are commonly thought to be responsible for regulating SSC development, but limited attention has been paid to the role of PM cells in this process. A conditional knockout (cKO) of the androgen receptor gene in PM cells resulted in male infertility. We found that testosterone (T) induces GDNF expression in mouse PM cells in vitro and neonatal spermatogonia (including SSCs) co-cultured with T-treated PM cells were able to colonize testes of germ cell-depleted mice after transplantation. This strongly suggested that T-regulated production of GDNF by PM cells is required for spermatogonial development, but PM cells might produce other factors in vitro that are responsible. In this study, we tested the hypothesis that production of GDNF by PM cells is essential for spermatogonial development by generating mice with a cKO of the Gdnf gene in PM cells. The cKO males sired up to two litters but became infertile due to collapse of spermatogenesis and loss of undifferentiated spermatogonia. These studies show for the first time, to our knowledge, that the production of GDNF by PM cells is essential for undifferentiated spermatogonial cell development in vivo. PMID:26831079

  11. Targeting the Gdnf Gene in peritubular myoid cells disrupts undifferentiated spermatogonial cell development.

    PubMed

    Chen, Liang-Yu; Willis, William D; Eddy, Edward M

    2016-02-16

    Spermatogonial stem cells (SSCs) are a subpopulation of undifferentiated spermatogonia located in a niche at the base of the seminiferous epithelium delimited by Sertoli cells and peritubular myoid (PM) cells. SSCs self-renew or differentiate into spermatogonia that proliferate to give rise to spermatocytes and maintain spermatogenesis. Glial cell line-derived neurotrophic factor (GDNF) is essential for this process. Sertoli cells produce GDNF and other growth factors and are commonly thought to be responsible for regulating SSC development, but limited attention has been paid to the role of PM cells in this process. A conditional knockout (cKO) of the androgen receptor gene in PM cells resulted in male infertility. We found that testosterone (T) induces GDNF expression in mouse PM cells in vitro and neonatal spermatogonia (including SSCs) co-cultured with T-treated PM cells were able to colonize testes of germ cell-depleted mice after transplantation. This strongly suggested that T-regulated production of GDNF by PM cells is required for spermatogonial development, but PM cells might produce other factors in vitro that are responsible. In this study, we tested the hypothesis that production of GDNF by PM cells is essential for spermatogonial development by generating mice with a cKO of the Gdnf gene in PM cells. The cKO males sired up to two litters but became infertile due to collapse of spermatogenesis and loss of undifferentiated spermatogonia. These studies show for the first time, to our knowledge, that the production of GDNF by PM cells is essential for undifferentiated spermatogonial cell development in vivo.

  12. Mutation and gene transfer of neutral amino acid transport System L genes in mammalian cells

    SciTech Connect

    El-Gewely, M.R.; Collarini, E.J.; Campbell, G.S.; Oxender, D.L.

    1987-05-01

    The authors are attempting to clone the genes coding for amino acid transport System L. Chinese hamster ovary (CHO) cell mutants that are temperature sensitive in their leucyl-tRNA synthetase show temperature-dependent regulation of System L. Temperature resistant mutants isolated from these cells have constitutively derepressed System L activity. Somatic cell fusion studies using these mutants have suggested that a trans-acting element controls regulation of System L. Mutants with reduced transport activity were isolated by a TH-suicide selection. The growth of these mutant cells is limited by the transport defect. CHO mutants were transformed with a human cosmid library, followed by selection at high temperatures and low leucine concentrations. Some transformants have increased levels of System L activity, suggesting that human genes coding for leucine transport have been incorporated into the CHO genome. Human sequences were rescued by a lambda in vitro packaging system. These sequences hybridize to vector and total human DNA. Experiments are being done to confirm that these sequences indeed code for transport System L. They are also attempting to label membrane components of amino acid transporters by group-specific modifying reagents.

  13. Gene transfer into mammalian cells by jet injection.

    PubMed

    Furth, P A; Shamay, A; Hennighausen, L

    1995-04-01

    Jet injection of DNA in solution is a technique that can be used to transfer DNA into tissues of living animals, where the introduced genes are expressed. The muscle, fat, skin, and mammary tissue of mice, and the fat, skin, and mammary tissue of sheep can be transfected with DNA. A jet injector, such as the Ped-o-jet (Stirn Industries, Dayton, NJ), is used to form a jet from 100 to 300 microliters of a DNA solution. This jet has sufficient force to travel into and through tissues of adult and juvenile animals. The introduced DNA is found in cells surrounding the path of the jet. When jet injection is performed through the surface of intact skin, underlying muscle, mammary, and fat, cells up to 2 cm distant from the point of injection are transfected with DNA. In this study, we demonstrate that the efficiency of DNA transfer is dependent upon the force of injection. Jet injection is an alternative to needle injection, lipofection, and particle bombardment for the introduction of "naked" DNA into the tissues of animals. This technique has potential for the introduction of genes into living organisms for genetic vaccination and gene therapy. PMID:7590772

  14. Differential gene expression in pulmonary artery endothelial cells exposed to sickle cell plasma.

    PubMed

    Klings, Elizabeth S; Safaya, Surinder; Adewoye, Adeboye H; Odhiambo, Adam; Frampton, Garrett; Lenburg, Marc; Gerry, Norman; Sebastiani, Paola; Steinberg, Martin H; Farber, Harrison W

    2005-05-11

    Clinical variability in sickle cell disease (SCD) suggests a role for extra-erythrocytic factors in the pathogenesis of vasoocclusion. We hypothesized that endothelial cell (EC) dysfunction, one possible modifier of disease variability, results from induction of phenotypic changes by circulating factors. Accordingly, we analyzed gene expression in cultured human pulmonary artery ECs (HPAEC) exposed to plasma from 1) sickle acute chest syndrome (ACS) patients, 2) SCD patients at steady state, 3) normal volunteers, and 4) serum-free media, using whole genome microarrays (U133A-B GeneChip, Affymetrix). Data were analyzed by Bayesian analysis of differential gene expression (BADGE). Differential expression was defined by the probability of >1.5 fold change in signal intensity greater than 0.999 and a predicted score of 70-100, measured by cross-validation. Compared with normal plasma, plasma from SCD patients (steady state) resulted in differential expression of 50 genes in HPAEC. Of these genes, molecules involved in cholesterol biosynthesis and lipid transport, the cellular stress response, and extracellular matrix proteins were most prominent. Another 58 genes were differentially expressed in HPAEC exposed to plasma from ACS patients. The pattern of altered gene expression suggests that plasma from SCD patients induces an EC phenotype which is anti-apoptotic and favors cholesterol biosynthesis. An altered EC phenotype elicited by SCD plasma may contribute to the pathogenesis of sickle vasoocclusion.

  15. 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.

  16. Heterogeneity of single cell cytokine gene expression in clonal T cell populations.

    PubMed

    Bucy, R P; Panoskaltsis-Mortari, A; Huang, G Q; Li, J; Karr, L; Ross, M; Russell, J H; Murphy, K M; Weaver, C T

    1994-10-01

    T helper type 0 (Th0), Th1, and Th2 CD4+ T cell clones derived from a T cell receptor alpha/beta (TCR-alpha/beta) transgenic mouse were activated by antigen presented on "artificial" antigen-presenting cells that expressed or lacked the costimulatory molecule B7-1, and were analyzed for single cell cytokine mRNA expression by in situ hybridization. There was significant heterogeneity in the frequency of T cells that expressed individual cytokine mRNAs within each clonal population, suggesting that transcriptional control of each of the cytokine genes was not coordinate within an individual cell. The majority of antigen-stimulated Th1 cells expressed mRNA for interferon gamma (IFN-gamma), but far fewer cells in the same population expressed interleukin 2 (IL-2). Similarly, the frequency of IL-4-expressing cells was greater than that of IL-5- or IL-10-expressing cells in the same Th2 population, but the difference in expression frequencies was more variable between clones. The expression frequencies of each of the cytokines was quite heterogeneous in the antigen-activated Th0 population. The principal effect of increased antigen on the activation of individual cytokine genes in each of the clonal populations was to increase recruitment of mRNA-positive cells, with little or no effect on the level of cytokine mRNA expression in individual positive cells. The effects of B7 costimulation were variable depending on the cytokine gene analyzed. B7 costimulation markedly increased the frequency and the level of IL-2 mRNA expression in individual positive cells in the Th1 and Th0 populations, with less effect on the recruitment and single cell expression level of IFN-gamma. IL-4 frequencies were modestly increased by B7 costimulation of the Th2 clones, but there was no detectable increase in single cell IL-4 expression level. The observed patterns of cytokine mRNA expression favor a model of T cell activation in which all-or-none, rather than graded, responses of cytokine

  17. Combined Single-Cell Functional and Gene Expression Analysis Resolves Heterogeneity within Stem Cell Populations

    PubMed Central

    Wilson, Nicola K.; Kent, David G.; Buettner, Florian; Shehata, Mona; Macaulay, Iain C.; Calero-Nieto, Fernando J.; Sánchez Castillo, Manuel; Oedekoven, Caroline A.; Diamanti, Evangelia; Schulte, Reiner; Ponting, Chris P.; Voet, Thierry; Caldas, Carlos; Stingl, John; Green, Anthony R.; Theis, Fabian J.; Göttgens, Berthold

    2015-01-01

    Summary Heterogeneity within the self-renewal durability of adult hematopoietic stem cells (HSCs) challenges our understanding of the molecular framework underlying HSC function. Gene expression studies have been hampered by the presence of multiple HSC subtypes and contaminating non-HSCs in bulk HSC populations. To gain deeper insight into the gene expression program of murine HSCs, we combined single-cell functional assays with flow cytometric index sorting and single-cell gene expression assays. Through bioinformatic integration of these datasets, we designed an unbiased sorting strategy that separates non-HSCs away from HSCs, and single-cell transplantation experiments using the enriched population were combined with RNA-seq data to identify key molecules that associate with long-term durable self-renewal, producing a single-cell molecular dataset that is linked to functional stem cell activity. Finally, we demonstrated the broader applicability of this approach for linking key molecules with defined cellular functions in another stem cell system. PMID:26004780

  18. 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. PMID:14970588

  19. 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.

  20. A systematic study on drug-response associated genes using baseline gene expressions of the Cancer Cell Line Encyclopedia

    PubMed Central

    Liu, Xiaoming; Yang, Jiasheng; Zhang, Yi; Fang, Yun; Wang, Fayou; Wang, Jun; Zheng, Xiaoqi; Yang, Jialiang

    2016-01-01

    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. PMID:26960563

  1. 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.

  2. Rearrangement of variable region T cell receptor gamma genes in acute lymphoblastic leukemia. V gamma gene usage differs in mature and immature T cells.

    PubMed Central

    Hara, J; Benedict, S H; Yumura, K; Ha-Kawa, K; Gelfand, E W

    1989-01-01

    Using probes recognizing variable regions (V gamma) and joining regions (J gamma) of the T cell receptor (TCR) gamma gene, we have analyzed the usage of V gamma genes in 24 patients with T cell acute lymphoblastic leukemia (ALL) and 36 patients with B-precursor ALL. In CD3- T-ALL derived from immature T cells, V gamma genes more proximal to J gamma were frequently rearranged; V gamma 8, V gamma 9, V gamma 10, and V gamma 11 were used in 19 of 24 rearrangements. In contrast, CD3+ T-ALL derived from a more mature stage of T cell ontogeny, showed a high frequency of rearrangements involving V gamma genes distal to J gamma; V gamma 2, V gamma 3, V gamma 4, and V gamma 5 were used in 17 of 25 rearrangements. In B-precursor ALL, no notable bias of V gamma gene usage was observed. This probably reflects the possibility that TCR genes may not rearrange according to a T cell hierarchy when under control of a B cell gene program. Furthermore, deletions of those V gamma genes located 3' to rearranged V gamma genes were observed in all patients analyzed. This supports the theory that loop deletion is a major mechanism for TCR-gamma gene rearrangement. Images PMID:2522937

  3. Early BrdU-responsive genes constitute a novel class of senescence-associated genes in human cells

    SciTech Connect

    Minagawa, Sachi; Nakabayashi, Kazuhiko; Fujii, Michihiko; Scherer, Stephen W.; Ayusawa, Dai . E-mail: dayusawa@yokohama-cu.ac.jp

    2005-04-01

    We identified genes that immediately respond to 5-bromodeoxyuridine (BrdU) in SUSM-1, an immortal fibroblastic line, with DNA microarray and Northern blot analysis. At least 29 genes were found to alter gene expression greater than twice more or less than controls within 36 h after addition of BrdU. They took several different expression patterns upon addition of BrdU, and the majority showed a significant alteration within 12 h. When compared among SUSM-1, HeLa, and TIG-7 normal human fibroblasts, 19 genes behaved similarly upon addition of BrdU. In addition, 14 genes, 9 of which are novel as regards senescence, behaved similarly in senescent TIG-7 cells. The genes do not seem to have a role in proliferation or cell cycle progression. These results suggest that the early BrdU-responsive genes represent early signs of cellular senescence and can be its new biomarkers.

  4. Utilizing cell-matrix interactions to modulate gene transfer to stem cells inside hyaluronic acid hydrogels.

    PubMed

    Gojgini, Shiva; Tokatlian, Talar; Segura, Tatiana

    2011-10-01

    The effective delivery of DNA locally would increase the applicability of gene therapy in tissue regeneration, where diseased tissue is to be repaired in situ. One promising approach is to use hydrogel scaffolds to encapsulate and deliver plasmid DNA in the form of nanoparticles to the diseased tissue, so that cells infiltrating the scaffold are transfected to induce regeneration. This study focuses on the design of a DNA nanoparticle-loaded hydrogel scaffold. In particular, this study focuses on understanding how cell-matrix interactions affect gene transfer to adult stem cells cultured inside matrix metalloproteinase (MMP) degradable hyaluronic acid (HA) hydrogel scaffolds. HA was cross-linked to form a hydrogel material using a MMP degradable peptide and Michael addition chemistry. Gene transfer inside these hydrogel materials was assessed as a function of polyplex nitrogen to phosphate ratio (N/P = 5 to 12), matrix stiffness (100-1700 Pa), RGD (Arg-Gly-Asp) concentration (10-400 μM), and RGD presentation (0.2-4.7 RGDs per HA molecule). All variables were found to affect gene transfer to mouse mensenchymal stem cells culture inside the DNA loaded hydrogels. As expected, higher N/P ratios lead to higher gene transfer efficiency but also higher toxicity; softer hydrogels resulted in higher transgene expression than stiffer hydrogels, and an intermediate RGD concentration and RGD clustering resulted in higher transgene expression. We believe that the knowledge gained through this in vitro model can be utilized to design better scaffold-mediated gene delivery for local gene therapy.

  5. 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.

  6. Killer cell immunoglobulin-like receptor gene association with cryptorchidism.

    PubMed

    Niepiekło-Miniewska, Wanda; Kuśnierczyk, Piotr; Havrylyuk, Anna; Kamieniczna, Marzena; Nakonechnyy, Andrij; Chopyak, Valentyna; Kurpisz, Maciej

    2015-12-01

    Cryptorchidism is a condition where a testis persists in the abdominal cavity. Thus, due to elevated temperature we may expect induction of aberrant immune reactions depending on genetic constitution of individual. This may be reflected by development of anti-sperm antibodies (ASA) in cryptorchid males. Also, natural killer (NK) cells which belong to innate immunity may control adaptive immunity. Therefore, the gene system encoding polymorphic NK cell immunoglobulin receptors (KIRs) has been studied. 109 prepubertal boys with cryptorchidism and 136 ethnically matched young male donors were selected to study NK cell KIRs. DNA was isolated using automatic Maxwell(®) system from the peripheral venous blood drawn onto anticoagulant. Olerup SSP KIR Genotyping kit including Taq polymerase was used for detection of KIR genes. Human leukocyte antigen-C (HLA-C) groups, C1 and C2 were established using a Olerup SSP KIR HLA Ligand kit. KIR2DL2 (killer immunoglobulin-like receptor two-domain long 2) and KIR2DS2 (killer immunoglobulin-like receptor two-domain short 2) genes were less frequent in patients than in control individuals (corrected p values: 0.0110 and 0.0383, respectively). However, no significant differences were observed between ASA-positive and ASA-negative patients, or between bilateral or unilateral cryptorchidism. No association between KIR ligands C1 and C2, alone or together with KIR2DL2, was found. However, the results suggest that KIR2DL2+/KIR2DS2+ genotype may be, to some extent, protective against cryptorchidism.

  7. 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.

  8. 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

  9. Baculovirus and insect cell gene expression: review of baculovirus biotechnology.

    PubMed

    Patterson, R M; Selkirk, J K; Merrick, B A

    1995-01-01

    The BEVS continues to evolve as a powerful, flexible tool for molecular biology, protein function, and biomedical research. Future developments offer the promise of replacement of hazardous chemical insecticides with environmentally safe biopesticides, construction of baculovirus vectors which encode genes for specific post-translational modifications, and establishment of efficient, stably transformed insect cell lines. FDA approval of BEVS-produced products offer the prospect of new biopharmaceuticals, in particular human therapeutics and vaccines, to improve human health and increase the quality of life for millions of people.

  10. vasa-related genes and their expression in stem cells of colonial parasitic rhizocephalan barnacle Polyascus polygenea (Arthropoda: Crustacea: Cirripedia: Rhizocephala).

    PubMed

    Shukalyuk, Andrey I; Golovnina, Kseniya A; Baiborodin, Sergei I; Gunbin, Konstantin V; Blinov, Alexander G; Isaeva, Valeria V

    2007-02-01

    vasa (vas)-related genes are members of the DEAD-box protein family and are expressed in the germ cells of many Metazoa. We cloned vasa-related genes (PpVLG, CpVLG) and other DEAD-box family related genes (PpDRH1, PpDRH2, CpDRH, AtDRHr) from the colonial parasitic rhizocephalan barnacle Polyascus polygenea, the non-colonial Clistosaccus paguri (Crustacea: Cirripedia: Rhizocephala), and the parasitic isopodan Athelgis takanoshimensis (Crustacea: Isopoda). The colonial Polyascus polygenea, a parasite of the coastal crabs Hemigrapsus sanguineus and Hemigrapsus longitarsis was used as a model object for further detailed investigations. Phylogenetic analysis suggested that PpVLG and CpVLG are closely related to vasa-like genes of other Arthropoda. The rest of the studied genes form their own separate branch on the phylogenetic tree and have a common ancestry with the p68 and PL10 subfamilies. We suppose this group may be a new subfamily of the DEAD-box RNA helicases that is specific for parasitic Crustacea. We found PpVLG and PpDRH1 expression products in stem cells from stolons and buds of internae, during asexual reproduction of colonial P. polygenea, and in germ cells from sexually reproducing externae, including male spermatogenic cells and female oogenic cells.

  11. 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.

  12. Gene expression analysis uncovers novel Hedgehog interacting protein (HHIP) effects in human bronchial epithelial cells

    PubMed Central

    Zhou, Xiaobo; Qiu, Weiliang; Sathirapongsasuti, J. Fah.; Cho, Michael H.; Mancini, John D.; Lao, Taotao; Thibault, Derek M.; Litonjua, Gus; Bakke, Per S.; Gulsvik, Amund; Lomas, David A.; Beaty, Terri H.; Hersh, Craig P.; Anderson, Christopher; Geigenmuller, Ute; Raby, Benjamin A.; Rennard, Stephen I.; Perrella, Mark A.; Choi, Augustine M.K.; Quackenbush, John; Silverman, Edwin K.

    2013-01-01

    Hedgehog Interacting Protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis. PMID:23459001

  13. A gold nanoparticle pentapeptide: gene fusion to induce therapeutic gene expression in mesenchymal stem cells.

    PubMed

    Muroski, Megan E; Morgan, Thomas J; Levenson, Cathy W; Strouse, Geoffrey F

    2014-10-22

    Mesenchymal stem cells (MSC) have been identified as having great potential as autologous cell therapeutics to treat traumatic brain injury and spinal injury as well as neuronal and cardiac ischemic events. All future clinical applications of MSC cell therapies must allow the MSC to be harvested, transfected, and induced to express a desired protein or selection of proteins to have medical benefit. For the full potential of MSC cell therapy to be realized, it is desirable to systematically alter the protein expression of therapeutically beneficial biomolecules in harvested MSC cells with high fidelity in a single transfection event. We have developed a delivery platform on the basis of the use of a solid gold nanoparticle that has been surface modified to produce a fusion containing a zwitterionic, pentapeptide designed from Bax inhibiting peptide (Ku70) to enhance cellular uptake and a linearized expression vector to induce enhanced expression of brain-derived neurotrophic factor (BDNF) in rat-derived MSCs. Ku70 is observed to effect >80% transfection following a single treatment of femur bone marrow isolated rat MSCs with efficiencies for the delivery of a 6.6 kbp gene on either a Au nanoparticle (NP) or CdSe/ZnS quantum dot (QD). Gene expression is observed within 4 d by optical measurements, and secretion is observed within 10 d by Western Blot analysis. The combination of being able to selectively engineer the NP, to colocalize biological agents, and to enhance the stability of those agents has provided the strong impetus to utilize this novel class of materials to engineer primary MSCs. PMID:25198921

  14. Abnormal gene expression profile reveals the common key signatures associated with clear cell renal cell carcinoma: a meta-analysis.

    PubMed

    Zhang, H J; Sun, Z Q; Qian, W Q; Sheng, L

    2015-01-01

    The aims of this study were to identify the common gene signatures of clear cell renal cell carcinoma (CCRCC), and to expand the respective protein-protein interaction networks associated with CCRCC regulation. For the latter, we utilized multiple gene expression data sets from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO), with which we could analyze the aberrant gene expression patterns at the transcriptome level that distinguish cancer from normal samples. We obtained the GSE781 and GSE6344 clear cell renal cell carcinoma gene expression datasets from GEO, which contained a total of 37 cancer and 37 normal samples. Subsequent R language analysis allowed identification of the differentially expressed genes. The genes that exhibited significant up or downregulation in cancers were entered into the Database for Annotation, Visualization, and Integrated Discovery to perform analysis of gene functional annotations, resulting in the generation of two protein-protein interaction networks that included the most significantly up or downregulated genes in CCRCC. These allowed us to identify the key factor genes, which could potentially be utilized to separate cancer versus normal samples. The differentially regulated genes are also highly likely to be functionally important regulatory factors in renal cell carcinoma: cell functions showing enrichment of these genes include amine biosynthetic and vitamin metabolic processes, ion binding, extracellular transport function, and regulation of biosynthesis. Together, the results from our study offer further reason to pursue diagnosis and therapy of CCRCC at the molecular level. PMID:25867368

  15. Simvastatin Modulates Mesenchymal Stromal Cell Proliferation and Gene Expression

    PubMed Central

    Zanette, Dalila Lucíola; Lorenzi, Julio Cesar Cetrulo; Panepucci, Rodrigo Alexandre; Palma, Patricia Vianna Bonini; dos Santos, Daiane Fernanda; Prata, Karen Lima; Silva, Wilson Araújo

    2015-01-01

    Statins are widely used hypocholesterolemic drugs that block the mevalonate pathway, responsible for the biosysnthesis of cholesterol. However, statins also have pleiotropic effects that interfere with several signaling pathways. Mesenchymal stromal cells (MSC) are a heterogeneous mixture of cells that can be isolated from a variety of tissues and are identified by the expression of a panel of surface markers and by their ability to differentiate in vitro into osteocytes, adipocytes and chondrocytes. MSC were isolated from amniotic membranes and bone marrows and characterized based on ISCT (International Society for Cell Therapy) minimal criteria. Simvastatin-treated cells and controls were directly assayed by CFSE (Carboxyfluorescein diacetate succinimidyl ester) staining to assess their cell proliferation and their RNA was used for microarray analyses and quantitative PCR (qPCR). These MSC were also evaluated for their ability to inhibit PBMC (peripheral blood mononuclear cells) proliferation. We show here that simvastatin negatively modulates MSC proliferation in a dose-dependent way and regulates the expression of proliferation-related genes. Importantly, we observed that simvastatin increased the percentage of a subset of smaller MSC, which also were actively proliferating. The association of MSC decreased size with increased pluripotency and the accumulating evidence that statins may prevent cellular senescence led us to hypothesize that simvastatin induces a smaller subpopulation that may have increased ability to maintain the entire pool of MSC and also to protect them from cellular senescence induced by long-term cultures/passages in vitro. These results may be important to better understand the pleiotropic effects of statins and its effects on the biology of cells with regenerative potential. PMID:25874574

  16. A simple and reproducible breast cancer prognostic test

    PubMed Central

    2013-01-01

    Background A small number of prognostic and predictive tests based on gene expression are currently offered as reference laboratory tests. In contrast to such success stories, a number of flaws and errors have recently been identified in other genomic-based predictors and the success rate for developing clinically useful genomic signatures is low. These errors have led to widespread concerns about the protocols for conducting and reporting of computational research. As a result, a need has emerged for a template for reproducible development of genomic signatures that incorporates full transparency, data sharing and statistical robustness. Results Here we present the first fully reproducible analysis of the data used to train and test MammaPrint, an FDA-cleared prognostic test for breast cancer based on a 70-gene expression signature. We provide all the software and documentation necessary for researchers to build and evaluate genomic classifiers based on these data. As an example of the utility of this reproducible research resource, we develop a simple prognostic classifier that uses only 16 genes from the MammaPrint signature and is equally accurate in predicting 5-year disease free survival. Conclusions Our study provides a prototypic example for reproducible development of computational algorithms for learning prognostic biomarkers in the era of personalized medicine. PMID:23682826

  17. Mesenchymal stem cell: a new horizon in cancer gene therapy.

    PubMed

    Mohammadi, M; Jaafari, M R; Mirzaei, H R; Mirzaei, H

    2016-09-01

    Cancer is one of the main problems in public health worldwide. Despite rapid advances in the diagnosis and treatment of cancer, the efficacy of current treatment strategies is still limited. There are promising new results in animal models whereby mesenchymal stem cells (MSCs) can be used as vehicles for targeted therapies. The use of MSCs as therapeutic biological vehicles in cell therapy has several advantages, including immune-silence, tumor tropism, easy and rapid isolation, ex vivo expansion, multilineage differentiation and the capacity to deliver a number of therapeutic agents. Some studies have shown that the microenvironment of the tumor provides a preferential niche for homing and survival of MSCs. Here, we have highlighted various applications of MSCs in cancer gene therapy. PMID:27650780

  18. Retinoid-mediated transcriptional regulaton of keratin genes in human epidermal and squamous cell carcinoma cells

    SciTech Connect

    Stellmach, V.; Leask, A.; Fuchs, E. )

    1991-06-01

    Vitamin A and other retinoids profoundly inhibit morphological and biochemical heatures of epidermal differentiation in vivo and in vitro. To elucidate the molecular mechanisms underlying the differential expression of epidermal keratins and their regulation by retinoids, the authors retinoid-mediated changes in total protein expression, protein synthesis, mRNA expression, and transcription in cultured human keratinocytes and in squamous cell carcinoma (SCC-13) cells of epidermal origin. The studies revealed that the epidermal keratins, K5, K6, K14, and K16, their mRNAs, and their transcripts were diminished relative to actin as a consequence of retinoic acid (RA) treatment. The effects were most pronounced in SCC-13 and were detected as early as 6 hr post-RA treatment, with enhancement over an additional 24-48 hr. Repression was also observed when 5{prime} upstream sequences of K14 or K5 genes were used to drive expression of a chloramphenicol acetyltransferase reporter gene in SCC-13 keratinocytes. Both cell types were found to express mRNAs for the RA receptors {alpha} and {gamma}, which may be involved in the RA-mediated transcriptional changes in these cells. The rapid transcriptional changes in epidermal keratin genes were in striking contrast to the previously reported slow transcriptional changes in simple epithelial keratin genes.

  19. 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

  20. Reprogramming resistant genes: in-depth comparison of gene expressions among iPS, ES, and somatic cells

    PubMed Central

    Polouliakh, Natalia

    2012-01-01

    Transcription factor-based reprogramming reverts adult cells to an embryonic state, yielding potential for generating different tissue types. However, recent reports indicated the substantial differences in pattern of gene expression between induced pluripotent stem (iPS) cells and embryonic stem cells (ESC). In this study, we compare gene expression signatures of different iPS and ES cell lines and relate expression profiles of differently expressed genes to their expression status in somatic cells. As a result, we discovered that genes resistant to reprogramming comprise two major clusters, which are reprogramming dependent “Induced Genes” and somatic origin “Inherited Genes,” both exhibiting preferences in methylation marks. Closer look into the Induced Genes by means of the transcription regulation analysis predicted several groups of genes with various roles in reprogramming and transcription factor DNA binding model. We believe that our results are a helpful source for biologists for further improvement of iPS cell technology. PMID:23386832

  1. Predicting stochastic gene expression dynamics in single cells.

    PubMed

    Mettetal, Jerome T; Muzzey, Dale; Pedraza, Juan M; Ozbudak, Ertugrul M; van Oudenaarden, Alexander

    2006-05-01

    Fluctuations in protein numbers (noise) due to inherent stochastic effects in single cells can have large effects on the dynamic behavior of gene regulatory networks. Although deterministic models can predict the average network behavior, they fail to incorporate the stochasticity characteristic of gene expression, thereby limiting their relevance when single cell behaviors deviate from the population average. Recently, stochastic models have been used to predict distributions of steady-state protein levels within a population but not to predict the dynamic, presteady-state distributions. In the present work, we experimentally examine a system whose dynamics are heavily influenced by stochastic effects. We measure population distributions of protein numbers as a function of time in the Escherichia coli lactose uptake network (lac operon). We then introduce a dynamic stochastic model and show that prediction of dynamic distributions requires only a few noise parameters in addition to the rates that characterize a deterministic model. Whereas the deterministic model cannot fully capture the observed behavior, our stochastic model correctly predicts the experimental dynamics without any fit parameters. Our results provide a proof of principle for the possibility of faithfully predicting dynamic population distributions from deterministic models supplemented by a stochastic component that captures the major noise sources. PMID:16648266

  2. Aging: a portrait from gene expression profile in blood cells.

    PubMed

    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

    2016-08-01

    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

  3. 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

  4. Gene and cell therapy for chronic ischaemic heart disease.

    PubMed

    Poh, Kian-Keong

    2007-01-01

    Viable treatment options are becoming available for the 'no-option' patient with chronic ischaemic heart disease. Instead of revascularising the highly diseased epicardial coronary arteries, scientists and clinicians have been looking at augmenting mother nature's way of providing biological bypass in an attempt to provide symptomatic relief in these patients. The novel use of gene and cell therapies for myocardial neovascularisation has exploded into a flurry of early clinical trials. This translational research has been motivated by an improved understanding of the biological mechanisms involved in tissue repair after ischaemic injury. While safety concerns will be top in priority in these trials, different types or combination of therapies, dose and route of delivery are being tested before further optimisation and establishment. With cautious optimism, a new era in the treatment of ischaemic heart disease is being entered. This article reviews the present state in gene and cell therapies for ischaemic heart disease, the modalities of their delivery, novel imaging techniques and future perspectives.

  5. Altered gene expression in conjunctival squamous cell carcinoma.

    PubMed

    Mahale, Alka; Alkatan, Hind; Alwadani, Saeed; Othman, Maha; Suarez, Maria J; Price, Antoinette; Al-Hussain, Hailah; Jastaneiah, Sabah; Yu, Wayne; Maktabi, Azza; Deepak, Edward P; Eberhart, Charles G; Asnaghi, Laura

    2016-05-01

    Conjunctival squamous cell carcinoma is a malignancy of the ocular surface. The molecular drivers responsible for the development and progression of this disease are not well understood. We therefore compared the transcriptional profiles of eight snap-frozen conjunctival squamous cell carcinomas and one in situ lesion with normal conjunctival specimens in order to identify diagnostic markers or therapeutic targets. RNA was analyzed using oligonucleotide microarrays, and a wide range of transcripts with altered expression identified, including many dysregulated in carcinomas arising at other sites. Among the upregulated genes, we observed more than 30-fold induction of the matrix metalloproteinases, MMP-9 and MMP-11, as well as a prominent increase in the mRNA level of a calcium-binding protein important for the intracellular calcium signaling, S100A2, which was induced over 20-fold in the tumor cohort. Clusterin was the most downregulated gene, with an approximately 180-fold reduction in the mRNA expression. These alterations were all confirmed by qPCR in the samples used for initial microarray analysis. In addition, immunohistochemical analysis confirmed the overexpression of MMP-11 and S100A2, as well as reductions in clusterin, in several independent in situ carcinomas of conjunctiva. These data identify a number of alterations, including upregulation of MMP-9, MMP-11, and S100A2, as well as downregulation of clusterin, associated with epithelial tumorigenesis in the ocular surface. PMID:26916071

  6. Reporter gene technologies for imaging cell fates in hematopoiesis.

    PubMed

    Kusy, Sophie; Contag, Christopher H

    2014-01-01

    protocols for gene delivery into hematopoietic cells for the purposes of applying these specific labels to cell trafficking. The goal of this chapter is to provide adequate background information to allow the design and implementation of an experimental system for in vivo imaging in mice. PMID:24473775

  7. Screening glioma stem cells in U251 cells based on the P1 promoter of the CD133 gene

    PubMed Central

    Wang, Xiaofeng; Chen, Lu; Xiao, Zhongdi; Wang, Yali; Liu, Tiemei; Zhang, Tianfu; Zhang, Yucheng

    2016-01-01

    Cluster of differentiation (CD)133 is an important cell surface marker of glioma stem cells (GSCs). The transcription of the CD133 gene is controlled by five alternative promoters (P1, P2, P3, P4 and P5), which are expressed in a tissue-specific manner. In the present study, gene recombination technology was used to construct two types of gene expression vectors that contained the P1 promoter of the CD133 gene, which regulated either the neomycin-resistance gene or the herpes simplex virus thymidine kinase (HSV-TK) gene. Following the stable transfection of U251 glioblastoma cells with these two gene vectors, the cells expressing the P1 promoter that regulated the neomycin-resistance gene were named CD133 (+) cells, while the cells expressing the P1 promoter regulating the HSV-TK gene were called CD133 (−) cells. The expression of CD133 was detected by flow cytometry and reverse transcription-quantitative polymerase chain reaction. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to assess cell proliferation ability, while the cell cycle was analyzed by flow cytometry, and a clone formation test was performed to evaluate the invasive capability of the cells. The results demonstrated that, due to CD133 expression, the cell proliferation ability and the invasive capability of CD133 (+) cells were significantly higher than those of CD133 (−) cells. In conclusion, the present study successfully established a novel method of screening GSCs in U251 cells based on the P1 promoter of the CD133 gene.

  8. 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.

  9. Inferring single-cell gene expression mechanisms using stochastic simulation

    PubMed Central

    Daigle, Bernie J.; Soltani, Mohammad; Petzold, Linda R.; Singh, Abhyudai

    2015-01-01

    Motivation: Stochastic promoter switching between transcriptionally active (ON) and inactive (OFF) states is a major source of noise in gene expression. It is often implicitly assumed that transitions between promoter states are memoryless, i.e. promoters spend an exponentially distributed time interval in each of the two states. However, increasing evidence suggests that promoter ON/OFF times can be non-exponential, hinting at more complex transcriptional regulatory architectures. Given the essential role of gene expression in all cellular functions, efficient computational techniques for characterizing promoter architectures are critically needed. Results: We have developed a novel model reduction for promoters with arbitrary numbers of ON and OFF states, allowing us to approximate complex promoter switching behavior with Weibull-distributed ON/OFF times. Using this model reduction, we created bursty Monte Carlo expectation-maximization with modified cross-entropy method (‘bursty MCEM2’), an efficient parameter estimation and model selection technique for inferring the number and configuration of promoter states from single-cell gene expression data. Application of bursty MCEM2 to data from the endogenous mouse glutaminase promoter reveals nearly deterministic promoter OFF times, consistent with a multi-step activation mechanism consisting of 10 or more inactive states. Our novel approach to modeling promoter fluctuations together with bursty MCEM2 provides powerful tools for characterizing transcriptional bursting across genes under different environmental conditions. Availability and implementation: R source code implementing bursty MCEM2 is available upon request. Contact: absingh@udel.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25573914

  10. 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.

  11. Cell types differ in global coordination of splicing and proportion of highly expressed genes.

    PubMed

    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

  12. 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

  13. 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.

  14. 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.

  15. Multimodality imaging of reporter gene expression using a novel fusion vector in living cells and animals

    DOEpatents

    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.

  16. Differences in the expression of chromosome 1 genes between lung telocytes and other cells: mesenchymal stem cells, fibroblasts, alveolar type II cells, airway epithelial cells and lymphocytes

    PubMed Central

    Sun, Xiaoru; Zheng, Minghuan; Zhang, Miaomiao; Qian, Mengjia; Zheng, Yonghua; Li, Meiyi; Cretoiu, Dragos; Chen, Chengshui; Chen, Luonan; Popescu, Laurentiu M; Wang, Xiangdong

    2014-01-01

    Telocytes (TCs) are a unique type of interstitial cells with specific, extremely long prolongations named telopodes (Tps). Our previous study showed that TCs are distinct from fibroblasts (Fbs) and mesenchymal stem cells (MSCs) as concerns gene expression and proteomics. The present study explores patterns of mouse TC-specific gene profiles on chromosome 1. We investigated the network of main genes and the potential functional correlations. We compared gene expression profiles of mouse pulmonary TCs, MSCs, Fbs, alveolar type II cells (ATII), airway basal cells (ABCs), proximal airway cells (PACs), CD8+ T cells from bronchial lymph nodes (T-BL) and CD8+ T cells from lungs (T-LL). The functional and feature networks were identified and compared by bioinformatics tools. Our data showed that on TC chromosome 1, there are about 25% up-regulated and 70% down-regulated genes (more than onefold) as compared with the other cells respectively. Capn2, Fhl2 and Qsox1 were over-expressed in TCs compared to the other cells, indicating that biological functions of TCs are mainly associated with morphogenesis and local tissue homoeostasis. TCs seem to have important roles in the prevention of tissue inflammation and fibrogenesis development in lung inflammatory diseases and as modulators of immune cell response. In conclusion, TCs are distinct from the other cell types. PMID:24826900

  17. Direct gene transfer into human cultured cells facilitated by laser micropuncture of the cell membrane

    SciTech Connect

    Tao, W.; Wilkinson, J.; Stanbridge, E.J.; Berns, M.W.

    1987-06-01

    The selective alteration of the cellular genome by laser microbeam irradiation has been extensively applied in cell biology. The authors 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 x 10 /sup -4/-3 x 10/sup -3/. This method appears to be 100-fold more efficient than the standard calcium phosphate-mediated method of DNA transfer.

  18. Large Scale Identification of Genes Involved in Cell Surface Biosynthesis and Architecture in Saccharomyces Cerevisiae

    PubMed Central

    Lussier, M.; White, A. M.; Sheraton, J.; di-Paolo, T.; Treadwell, J.; Southard, S. B.; Horenstein, C. I.; Chen-Weiner, J.; Ram, AFJ.; Kapteyn, J. C.; Roemer, T. W.; Vo, D. H.; Bondoc, D. C.; Hall, J.; Wei Zhong, W.; Sdicu, A. M.; Davies, J.; Klis, F. M.; Robbins, P. W.; Bussey, H.

    1997-01-01

    The sequenced yeast genome offers a unique resource for the analysis of eukaryotic cell function and enables genome-wide screens for genes involved in cellular processes. We have identified genes involved in cell surface assembly by screening transposon-mutagenized cells for altered sensitivity to calcofluor white, followed by supplementary screens to further characterize mutant phenotypes. The mutated genes were directly retrieved from genomic DNA and then matched uniquely to a gene in the yeast genome database. Eighty-two genes with apparent perturbation of the cell surface were identified, with mutations in 65 of them displaying at least one further cell surface phenotype in addition to their modified sensitivity to calcofluor. Fifty of these genes were previously known, 17 encoded proteins whose function could be anticipated through sequence homology or previously recognized phenotypes and 15 genes had no previously known phenotype. PMID:9335584

  19. Induction of cell cycle progression by hepatitis B virus HBx gene expression in quiescent mouse fibroblasts.

    PubMed Central

    Koike, K; Moriya, K; Yotsuyanagi, H; Iino, S; Kurokawa, K

    1994-01-01

    The HBx gene of hepatitis B virus has been shown to induce hepatic tumors in transgenic mice and is implicated in hepatocarcinogenesis in human hepatitis B virus infection. To further characterize the role of HBx gene in carcinogenesis, we established mouse fibroblast cell lines in which the expression of HBx gene could be controlled by glucocorticoid hormone and examined the effect of HBx gene expression on cell growth in vitro. Along with the expression of HBx gene, most cells in the G0/G1 phase moved into the S phase in 24 h, and the cell cycle progressed further toward 48 h. Induction of DNA synthesis was also demonstrated by bromo-deoxyuridine labeling analysis. These results indicate that HBx gene has a function to trigger the synthesis of cellular DNA and suggest that HBx gene may play a role in hepatocarcinogenesis in human infection by driving deregulated cell cycle progression. Images PMID:8040286

  20. 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...

  1. 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

  2. Gene set enrichment analysis and ingenuity pathway analysis of metastatic clear cell renal cell carcinoma cell line.

    PubMed

    Khan, Mohammed I; Dębski, Konrad J; Dabrowski, Michał; Czarnecka, Anna M; Szczylik, Cezary

    2016-08-01

    In recent years, genome-wide RNA expression analysis has become a routine tool that offers a great opportunity to study and understand the key role of genes that contribute to carcinogenesis. Various microarray platforms and statistical approaches can be used to identify genes that might serve as prognostic biomarkers and be developed as antitumor therapies in the future. Metastatic renal cell carcinoma (mRCC) is a serious, life-threatening disease, and there are few treatment options for patients. In this study, we performed one-color microarray gene expression (4×44K) analysis of the mRCC cell line Caki-1 and the healthy kidney cell line ASE-5063. A total of 1,921 genes were differentially expressed in the Caki-1 cell line (1,023 upregulated and 898 downregulated). Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) approaches were used to analyze the differential-expression data. The objective of this research was to identify complex biological changes that occur during metastatic development using Caki-1 as a model mRCC cell line. Our data suggest that there are multiple deregulated pathways associated with metastatic clear cell renal cell carcinoma (mccRCC), including integrin-linked kinase (ILK) signaling, leukocyte extravasation signaling, IGF-I signaling, CXCR4 signaling, and phosphoinositol 3-kinase/AKT/mammalian target of rapamycin signaling. The IPA upstream analysis predicted top transcriptional regulators that are either activated or inhibited, such as estrogen receptors, TP53, KDM5B, SPDEF, and CDKN1A. The GSEA approach was used to further confirm enriched pathway data following IPA.

  3. Whole Cell Formaldehyde Cross-Linking Simplifies Purification of Mitochondrial Nucleoids and Associated Proteins Involved in Mitochondrial Gene Expression

    PubMed Central

    Rajala, Nina; Hensen, Fenna; Wessels, Hans J. C. T.; Ives, Daniel; Gloerich, Jolein; Spelbrink, Johannes N.

    2015-01-01

    Mitochondrial DNA/protein complexes (nucleoids) appear as discrete entities inside the mitochondrial network when observed by live-cell imaging and immunofluorescence. This somewhat trivial observation in recent years has spurred research towards isolation of these complexes and the identification of nucleoid-associated proteins. Here we show that whole cell formaldehyde crosslinking combined with affinity purification and tandem mass-spectrometry provides a simple and reproducible method to identify potential nucleoid associated proteins. The method avoids spurious mitochondrial isolation and subsequent multifarious nucleoid enrichment protocols and can be implemented to allow for label-free quantification (LFQ) by mass-spectrometry. Using expression of a Flag-tagged Twinkle helicase and appropriate controls we show that this method identifies many previously identified nucleoid associated proteins. Using LFQ to compare HEK293 cells with and without mtDNA, but both expressing Twinkle-FLAG, identifies many proteins that are reduced or absent in the absence of mtDNA. This set not only includes established mtDNA maintenance proteins but also many proteins involved in mitochondrial RNA metabolism and translation and therefore represents what can be considered an mtDNA gene expression proteome. Our data provides a very valuable resource for both basic mitochondrial researchers as well as clinical geneticists working to identify novel disease genes on the basis of exome sequence data. PMID:25695250

  4. Activation of the c-fos gene in prodynorphin- and proenkephalin-expressing cells of nucleus tractus solitarius after seizures.

    PubMed

    Kanter, R K; Erickson, J T; Millhorn, D E

    1994-10-01

    We performed studies to determine the anatomical regions and chemical phenotypes of neurons within the rat medulla oblongata activated by pentylenetetrazole-induced seizures. Activated cells were identified by their expression of the c-fos gene, detected by in situ hybridization for c-fos mRNA and immunocytochemistry for Fos protein. Activated cells were located predominantly in nucleus tractus solitarius (NTS), with c-fos mRNA appearing within 20 min after seizures (peak at 1-2 h), followed by Fos immunoreactivity visible at 1 h (peak at 2-4 h). Neither nonspecific noxious stimulation by intraperitoneal injection of saline nor brief exposure to hypoxic or hypercapnic gas mixtures to stimulate chemoreceptors reproduced this pattern of labeling. Prodynorphin or proenkephalin mRNA, detected by in situ hybridization, was colocalized with Fos immunoreactivity in many NTS cells. Thus, seizures activate neuronal pathways in the medulla oblongata which express genes for endogenous opioids. Potential long-term effects of seizures are suggested by the in situ hybridization finding that NTS prodynorphin mRNA increased 24 h after seizures compared to control levels. PMID:7957742

  5. 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

  6. 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

    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

  7. 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

  8. 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

    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

  9. 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

  10. An in situ hybridization-based screen for heterogeneously expressed genes in mouse ES cells.

    PubMed

    Carter, Mark G; Stagg, Carole A; Falco, Geppino; Yoshikawa, Toshiyuki; Bassey, Uwem C; Aiba, Kazuhiro; Sharova, Lioudmila V; Shaik, Nabeebi; Ko, Minoru S H

    2008-02-01

    We previously reported that Zscan4 showed heterogeneous expression patterns in mouse embryonic stem (ES) cells. To identify genes that show similar expression patterns, we carried out high-throughput in situ hybridization assays on ES cell cultures for 244 genes. Most of the genes are involved in transcriptional regulation, and were selected using microarray-based comparisons of gene expression profiles in ES and embryonal carcinoma (EC) cells versus differentiated cell types. Pou5f1 (Oct4, Oct3/4) and Krt8 (EndoA) were used as controls. Hybridization signals were detected on ES cell colonies for 147 genes (60%). The majority (136 genes) of them showed relatively homogeneous expression in ES cell colonies. However, we found that two genes unequivocally showed Zscan4-like spotted expression pattern (spot-in-colony pattern; Whsc2 and Rhox9). We also found that nine genes showed relatively heterogeneous expression pattern (mosaic-in-colony pattern: Zfp42/Rex1, Rest, Atf4, Pa2g4, E2f2, Nanog, Dppa3/Pgc7/Stella, Esrrb, and Fscn1). Among these genes, Zfp42/Rex1 showed unequivocally heterogeneous expression in individual ES cells prepared by the CytoSpin. These results show the presence of different types or states of cells within ES cell cultures otherwise thought to be undifferentiated and homogeneous, suggesting a previously unappreciated complexity in ES cell cultures.

  11. Expression of Cell Cycle–Related Genes With Cytokine-Induced Cell Cycle Progression of Primitive Hematopoietic Stem Cells

    PubMed Central

    Dooner, Gerri J.; Del Tatto, Michael; Colvin, Gerald A.; Johnson, Kevin; Dooner, Mark S.

    2010-01-01

    Primitive marrow lineage-negative rhodamine low and Hoechst low (LRH) stem cells isolated on the basis of quiescence respond to the cytokines thrombopoietin, FLT3L, and steel factor by synchronously progressing through cell cycle. We have now profiled the mRNA expression, as determined by real-time RT-PCR, of 47 hematopoietic or cell cycle-related genes, focusing on the variations in the cell cycle regulators with cycle transit. LRH stem cells, at isolation, showed expression of all interrogated genes, but at relatively low levels. In our studies, there was a good deal of consistency with regard to cell cycle regulatory genes involved in the G1/S progression point of LRH murine stem cells. The observed pattern of expression of cyclin A2 is consistent with actions at these phases of cell cycle. Minimal elevations were seen at 16 h with higher elevations at 24, 32, 40, and 48 h times encompassing S, G2, and M phases. CDK2 expression pattern was also consistent with a role in G1/S transition with a modest elevation at 24 h and more substantial elevation at 32 h. The observed pattern of expression of cyclin F mRNA with marked elevations at 16–40 h was also consistent with actions in S and G2 phases. Cyclin D1 expression pattern was less consistent with its known role in G1 progression. The alterations in multiple other cell cycle regulators were consistent with previous information obtained in other cell systems. The cycle regulatory mechanics appears to be preserved across broad ranges of cell types. PMID:19788373

  12. Volatile anesthetics modulate gene expression in breast and brain tumor cells.

    PubMed

    Huitink, Johannes M; Heimerikxs, Mike; Nieuwland, Marja; Loer, Stephan A; Brugman, Wim; Velds, Arno; Sie, Daoud; Kerkhoven, Ron M

    2010-12-01

    Gene expression is increasingly used for diagnostic, prognostic, and therapeutic purposes in clinical practice. We tested the hypothesis that volatile anesthetics (VA) affect gene expression of tumor cells. Cells from the neuronal cell line SH-SY5Y and from the breast cell line MCF-7 were exposed ex vivo to enflurane, isoflurane, desflurane, halothane, sevoflurane, or nitrous oxide. Microarray gene expression profiles were studied. We observed significant differences in gene expression levels of cell cultures and response in time when exposed to different VA. Some genes used for predictive genetic fingerprints for breast cancer were affected by VA. Our findings suggest that VA modulate gene expression in breast and brain tumor cell cultures in a unique and time-dependent manner. PMID:20889943

  13. Direct Imaging of Gene-Carrier Complexes in Animal Cells

    NASA Astrophysics Data System (ADS)

    Lin, Alison J.; Slack, Nelle L.; Ahmad, Ayesha; Matsumoto, Brian; Safinya, Cyrus R.

    1998-03-01

    Cationic lipids are promising gene carriers for DNA transfection. Establishing the correlations between structures of cationic lipid/DNA complexes (CL-DNA) and pathways of transfection will greatly aid us in achieving the optimal CL-DNA transfections. Our first step is to determine the uptake mechanism of DNA by studying the interactions and structures of DNA and cationic lipids. X-ray diffraction shows that the CL-DNA undergoes structural phase transitions from lamellar( J. Raedler, I. Koltover, T. Salditt, C. R. Safinya, Science 275, 810 (1997).) to inverted hexagonal self-assemblies as we change the lipid composition. X-ray diffraction and optical microscopy techniques are used to directly image the progress of the CL-DNA in mouse L-cells and unravel the complex structure in-situ. Fluorescence and confocal optical microscopy techniques allow us to monitor the interactions between the complexes and different organelles in the cell cytoplasm. Current results indicate that once inside cells, complexes containing DOPE follow a different pathway from those containing DOPC. This research is funded by NSF-DMR-9624091, PRF-31352-AC7, and Los Alamos-STB/UC:96-108.

  14. Using Magnetic Nanoparticles for Gene Transfer to Neural Stem Cells: Stem Cell Propagation Method Influences Outcomes

    PubMed Central

    Pickard, Mark R.; Adams, Christopher F.; Barraud, Perrine; Chari, Divya M.

    2015-01-01

    Genetically engineered neural stem cell (NSC) transplants offer a key strategy to augment neural repair by releasing therapeutic biomolecules into injury sites. Genetic modification of NSCs is heavily reliant on viral vectors but cytotoxic effects have prompted development of non-viral alternatives, such as magnetic nanoparticle (MNPs). NSCs are propagated in laboratories as either 3-D suspension “neurospheres” or 2-D adherent “monolayers”. MNPs deployed with oscillating magnetic fields (“magnetofection technology”) mediate effective gene transfer to neurospheres but the efficacy of this approach for monolayers is unknown. It is important to address this issue as oscillating magnetic fields dramatically enhance MNP-based transfection in transplant cells (e.g., astrocytes and oligodendrocyte precursors) propagated as monolayers. We report for the first time that oscillating magnetic fields enhanced MNP-based transfection with reporter and functional (basic fibroblast growth factor; FGF2) genes in monolayer cultures yielding high transfection versus neurospheres. Transfected NSCs showed high viability and could re-form neurospheres, which is important as neurospheres yield higher post-transplantation viability versus monolayer cells. Our results demonstrate that the combination of oscillating magnetic fields and a monolayer format yields the highest efficacy for MNP-mediated gene transfer to NSCs, offering a viable non-viral alternative for genetic modification of this important neural cell transplant population. PMID:25918990

  15. Using magnetic nanoparticles for gene transfer to neural stem cells: stem cell propagation method influences outcomes.

    PubMed

    Pickard, Mark R; Adams, Christopher F; Barraud, Perrine; Chari, Divya M

    2015-04-24

    Genetically engineered neural stem cell (NSC) transplants offer a key strategy to augment neural repair by releasing therapeutic biomolecules into injury sites. Genetic modification of NSCs is heavily reliant on viral vectors but cytotoxic effects have prompted development of non-viral alternatives, such as magnetic nanoparticle (MNPs). NSCs are propagated in laboratories as either 3-D suspension "neurospheres" or 2-D adherent "monolayers". MNPs deployed with oscillating magnetic fields ("magnetofection technology") mediate effective gene transfer to neurospheres but the efficacy of this approach for monolayers is unknown. It is important to address this issue as oscillating magnetic fields dramatically enhance MNP-based transfection in transplant cells (e.g., astrocytes and oligodendrocyte precursors) propagated as monolayers. We report for the first time that oscillating magnetic fields enhanced MNP-based transfection with reporter and functional (basic fibroblast growth factor; FGF2) genes in monolayer cultures yielding high transfection versus neurospheres. Transfected NSCs showed high viability and could re-form neurospheres, which is important as neurospheres yield higher post-transplantation viability versus monolayer cells. Our results demonstrate that the combination of oscillating magnetic fields and a monolayer format yields the highest efficacy for MNP-mediated gene transfer to NSCs, offering a viable non-viral alternative for genetic modification of this important neural cell transplant population.

  16. Assessment of high resolution melt analysis feasibility for evaluation of beta-globin gene mutations as a reproducible, cost-efficient and fast alternative to the present conventional method

    PubMed Central

    Ramezanzadeh, Mahboubeh; Salehi, Mansour; Salehi, Rasoul

    2016-01-01

    Background: Beta-thalassemia is the most prevalent monogenic disease throughout the world. It was the first genetic disorder nominated for nation-wide prevention programs involving population screening for heterozygotes and prenatal diagnosis (PND) in Iran. Due to the high prevalence of beta-thalassemia, the shift from conventional mutation detection methods to more recently developed techniques based on novel innovative technologies are essential. We aimed to develop a real-time polymerase chain reaction (PCR) based protocol using high resolution melting (HRM) analysis for diagnosis of common beta-thalassemia mutations. Materials and Methods: Forty DNA samples extracted from peripheral blood of suspected beta-thalassemia carriers participated in this study were subjected to amplification refractory mutation system (ARMS). We then used 20 of these samples for HRM optimization. When 100% sensitivity and specificity was obtained with HRM procedure, we applied the technique for mutation detection on another remaining 20 samples as thalassemia cases with unknown mutations (detected mutations with ARMS-PCR kept confidential). Finally, the HRM procedure applied on 2 chorionic villous sample (CVS) biopsied from 12 weeks gestational age pregnant women for routine PND analysis. Results: In the first step of study, Fr 8/9 (+G), IVSI-1 (G > A), IVSI-5 (G > C), IVSI-110 (G > A), and CD44 (−C) mutations were diagnosed in samples under study using ARMS-PCR technique. Finally, the HRM procedure applied on 20 unknown samples and 2 CVS The results of HRM were in complete concordance with ARMS and confirmed by sequencing. Conclusions: The advantages of HRM analysis over conventional methods is high throughput, rapid, accurate, cost-effective, and reproducible. PMID:27169102

  17. Meteorite Atmospheric Entry Reproduced in Plasmatron

    NASA Astrophysics Data System (ADS)

    Pittarello, L.; McKibbin, S.; Goderis, S.; Soens, B.; Bariselli, F.; Barros Dias, B. R.; Zavalan, F. L.; Magin, T.; Claeys, Ph.

    2016-08-01

    Plasmatron facility allows experimental conditions that reproduce atmospheric entry of meteorites. Tests on basalt, as meteorite analogue, have been performed. Preliminary results have highlighted melting and evaporation effects.

  18. Circadian Clock Genes Modulate Human Bone Marrow Mesenchymal Stem Cell Differentiation, Migration and Cell Cycle

    PubMed Central

    Boucher, Helene; Vanneaux, Valerie; Domet, Thomas; Parouchev, Alexandre; Larghero, Jerome

    2016-01-01

    Many of the components that regulate the circadian clock have been identified in organisms and humans. The influence of circadian rhythm (CR) on the regulation of stem cells biology began to be evaluated. However, little is known on the role of CR on human mesenchymal stem cell (hMSCs) properties. The objective of this study was to investigate the influence of CR on the differentiation capacities of bone marrow hMSCs, as well as the regulation of cell cycle and migration capabilities. To that, we used both a chemical approach with a GSK-3β specific inhibitor (2’E,3’Z-6-bromoindirubin-3’-oxime, BIO) and a knockdown of CLOCK and PER2, two of the main genes involved in CR regulation. In these experimental conditions, a dramatic inhibition of adipocyte differentiation was observed, while osteoblastic differentiation capacities were not modified. In addition, cell migration was decreased in PER2-/- cells. Lastly, downregulation of circadian clock genes induced a modification of the hMSCs cell cycle phase distribution, which was shown to be related to a change of the cyclin expression profile. Taken together, these data showed that CR plays a role in the regulation of hMSCs differentiation and division, and likely represent key factor in maintaining hMSCs properties. PMID:26741371

  19. Graded Smad2/3 Activation Is Converted Directly into Levels of Target Gene Expression in Embryonic Stem Cells

    PubMed Central

    Mavrakis, Konstantinos J.; Goggolidou, Paraskevi; Norris, Dominic P.; Episkopou, Vasso

    2009-01-01

    The Transforming Growth Factor (TGF) β signalling family includes morphogens, such as Nodal and Activin, with important functions in vertebrate development. The concentration of the morphogen is critical for fate decisions in the responding cells. Smad2 and Smad3 are effectors of the Nodal/Activin branch of TGFβ signalling: they are activated by receptors, enter the nucleus and directly transcribe target genes. However, there have been no studies correlating levels of Smad2/3 activation with expression patterns of endogenous target genes in a developmental context over time. We used mouse Embryonic Stem (ES) cells to create a system whereby levels of activated Smad2/3 can be manipulated by an inducible constitutively active receptor (Alk4*) and an inhibitor (SB-431542) that blocks specifically Smad2/3 activation. The transcriptional responses were analysed by microarrays at different time points during activation and repression. We identified several genes that follow faithfully and reproducibly the Smad2/3 activation profile. Twenty-seven of these were novel and expressed in the early embryo downstream of Smad2/3 signalling. As they responded to Smad2/3 activation in the absence of protein synthesis, they were considered direct. These immediate responsive genes included negative intracellular feedback factors, like SnoN and I-Smad7, which inhibit the transcriptional activity of Smad2/3. However, their activation did not lead to subsequent repression of target genes over time, suggesting that this type of feedback is inefficient in ES cells or it is counteracted by mechanisms such as ubiquitin-mediated degradation by Arkadia. Here we present an ES cell system along with a database containing the expression profile of thousands of genes downstream of Smad2/3 activation patterns, in the presence or absence of protein synthesis. Furthermore, we identify primary target genes that follow proportionately and with high sensitivity changes in Smad2/3 levels over 15–30

  20. Complete TCR-α gene locus control region activity in T cells derived in vitro from embryonic stem cells.

    PubMed

    Lahiji, Armin; Kucerová-Levisohn, Martina; Lovett, Jordana; Holmes, Roxanne; Zúñiga-Pflücker, Juan Carlos; Ortiz, Benjamin D

    2013-07-01

    Locus control regions (LCRs) are cis-acting gene regulatory elements with the unique, integration site-independent ability to transfer the characteristics of their locus-of-origin's gene expression pattern to a linked transgene in mice. LCR activities have been discovered in numerous T cell lineage-expressed gene loci. These elements can be adapted to the design of stem cell gene therapy vectors that direct robust therapeutic gene expression to the T cell progeny of engineered stem cells. Currently, transgenic mice provide the only experimental approach that wholly supports all the critical aspects of LCR activity. In this study, we report the manifestation of all key features of mouse TCR-α gene LCR function in T cells derived in vitro from mouse embryonic stem cells. High-level, copy number-related TCR-α LCR-linked reporter gene expression levels are cell type restricted in this system, and upregulated during the expected stage transition of T cell development. We also report that de novo introduction of TCR-α LCR-linked transgenes into existing T cell lines yields incomplete LCR activity. These data indicate that establishing full TCR-α LCR activity requires critical molecular events occurring prior to final T lineage determination. This study also validates a novel, tractable, and more rapid approach for the study of LCR activity in T cells, and its translation to therapeutic genetic engineering.

  1. Cell-Specific Promoters Enable Lipid-Based Nanoparticles to Deliver Genes to Specific Cells of the Retina In Vivo

    PubMed Central

    Wang, Yuhong; Rajala, Ammaji; Cao, Binrui; Ranjo-Bishop, Michelle; Agbaga, Martin-Paul; Mao, Chuanbin; Rajala, Raju V.S.

    2016-01-01

    Non-viral vectors, such as lipid-based nanoparticles (liposome-protamine-DNA complex [LPD]), could be used to deliver a functional gene to the retina to correct visual function and treat blindness. However, one of the limitations of LPD is the lack of cell specificity, as the retina is composed of seven types of cells. If the same gene is expressed in multiple cell types or is absent from one desired cell type, LPD-mediated gene delivery to every cell may have off-target effects. To circumvent this problem, we have tested LPD-mediated gene delivery using various generalized, modified, and retinal cell-specific promoters. We achieved retinal pigment epithelium cell specificity with vitelliform macular dystrophy (VMD2), rod cell specificity with mouse rhodopsin, cone cell specificity with red/green opsin, and ganglion cell specificity with thymocyte antigen promoters. Here we show for the first time that cell-specific promoters enable lipid-based nanoparticles to deliver genes to specific cells of the retina in vivo. This work will inspire investigators in the field of lipid nanotechnology to couple cell-specific promoters to drive expression in a cell- and tissue-specific manner. PMID:27446487

  2. Cell-Specific Promoters Enable Lipid-Based Nanoparticles to Deliver Genes to Specific Cells of the Retina In Vivo.

    PubMed

    Wang, Yuhong; Rajala, Ammaji; Cao, Binrui; Ranjo-Bishop, Michelle; Agbaga, Martin-Paul; Mao, Chuanbin; Rajala, Raju V S

    2016-01-01

    Non-viral vectors, such as lipid-based nanoparticles (liposome-protamine-DNA complex [LPD]), could be used to deliver a functional gene to the retina to correct visual function and treat blindness. However, one of the limitations of LPD is the lack of cell specificity, as the retina is composed of seven types of cells. If the same gene is expressed in multiple cell types or is absent from one desired cell type, LPD-mediated gene delivery to every cell may have off-target effects. To circumvent this problem, we have tested LPD-mediated gene delivery using various generalized, modified, and retinal cell-specific promoters. We achieved retinal pigment epithelium cell specificity with vitelliform macular dystrophy (VMD2), rod cell specificity with mouse rhodopsin, cone cell specificity with red/green opsin, and ganglion cell specificity with thymocyte antigen promoters. Here we show for the first time that cell-specific promoters enable lipid-based nanoparticles to deliver genes to specific cells of the retina in vivo. This work will inspire investigators in the field of lipid nanotechnology to couple cell-specific promoters to drive expression in a cell- and tissue-specific manner. PMID:27446487

  3. Meta-analysis of differentiating mouse embryonic stem cell gene expression kinetics reveals early change of a small gene set.

    PubMed

    Glover, Clive H; Marin, Michael; Eaves, Connie J; Helgason, Cheryl D; Piret, James M; Bryan, Jennifer

    2006-11-24

    Stem cell differentiation involves critical changes in gene expression. Identification of these should provide endpoints useful for optimizing stem cell propagation as well as potential clues about mechanisms governing stem cell maintenance. Here we describe the results of a new meta-analysis methodology applied to multiple gene expression datasets from three mouse embryonic stem cell (ESC) lines obtained at specific time points during the course of their differentiation into various lineages. We developed methods to identify genes with expression changes that correlated with the altered frequency of functionally defined, undifferentiated ESC in culture. In each dataset, we computed a novel statistical confidence measure for every gene which captured the certainty that a particular gene exhibited an expression pattern of interest within that dataset. This permitted a joint analysis of the datasets, despite the different experimental designs. Using a ranking scheme that favored genes exhibiting patterns of interest, we focused on the top 88 genes whose expression was consistently changed when ESC were induced to differentiate. Seven of these (103728_at, 8430410A17Rik, Klf2, Nr0b1, Sox2, Tcl1, and Zfp42) showed a rapid decrease in expression concurrent with a decrease in frequency of undifferentiated cells and remained predictive when evaluated in additional maintenance and differentiating protocols. Through a novel meta-analysis, this study identifies a small set of genes whose expression is useful for identifying changes in stem cell frequencies in cultures of mouse ESC. The methods and findings have broader applicability to understanding the regulation of self-renewal of other stem cell types.

  4. Robust Prognostic Gene Expression Signatures in Bladder Cancer and Lung Adenocarcinoma Depend on Cell Cycle Related Genes

    PubMed Central

    Dancik, Garrett M.; Theodorescu, Dan

    2014-01-01

    Few prognostic biomarkers are approved for clinical use primarily because their initial performance cannot be repeated in independent datasets. We posited that robust biomarkers could be obtained by identifying deregulated biological processes shared among tumor types having a common etiology. We performed a gene set enrichment analysis in 20 publicly available gene expression datasets comprising 1968 patients having one of the three most common tobacco-related cancers (lung, bladder, head and neck) and identified cell cycle related genes as the most consistently prognostic class of biomarkers in bladder (BL) and lung adenocarcinoma (LUAD). We also found the prognostic value of 13 of 14 published BL and LUAD signatures were dependent on cell cycle related genes, supporting the importance of cell cycle related biomarkers for prognosis. Interestingly, no prognostic gene classes were identified in squamous cell lung carcinoma or head and neck squamous cell carcinoma. Next, a specific 31 gene cell cycle proliferation (CCP) signature, previously derived in prostate tumors was evaluated and found predictive of outcome in BL and LUAD cohorts in univariate and multivariate analyses. Specifically, CCP score significantly enhanced the predictive ability of multivariate models based on standard clinical variables for progression in BL patients and survival in LUAD patients in multiple cohorts. We then generated random CCP signatures of various sizes and found sets of 10–15 genes had robust performance in these BL and LUAD cohorts, a finding that was confirmed in an independent cohort. Our work characterizes the importance of cell cycle related genes in prognostic signatures for BL and LUAD patients and identifies a specific signature likely to survive additional validation. PMID:24465512

  5. Neural differentiation potential of human bone marrow-derived mesenchymal stromal cells: misleading marker gene expression

    PubMed Central

    Montzka, Katrin; Lassonczyk, Nina; Tschöke, Beate; Neuss, Sabine; Führmann, Tobias; Franzen, Rachelle; Smeets, Ralf; Brook, Gary A; Wöltje, Michael

    2009-01-01

    Background In contrast to pluripotent embryonic stem cells, adult stem cells have been considered to be multipotent, being somewhat more restricted in their differentiation capacity and only giving rise to cell types related to their tissue of origin. Several studies, however, have reported that bone marrow-derived mesenchymal stromal cells (MSCs) are capable of transdifferentiating to neural cell types, effectively crossing normal lineage restriction boundaries. Such reports have been based on the detection of neural-related proteins by the differentiated MSCs. In order to assess the potential of human adult MSCs to undergo true differentiation to a neural lineage and to determine the degree of homogeneity between donor samples, we have used RT-PCR and immunocytochemistry to investigate the basal expression of a range of neural related mRNAs and proteins in populations of non-differentiated MSCs obtained from 4 donors. Results The expression analysis revealed that several of the commonly used marker genes from other studies like nestin, Enolase2 and microtubule associated protein 1b (MAP1b) are already expressed by undifferentiated human MSCs. Furthermore, mRNA for some of the neural-related transcription factors, e.g. Engrailed-1 and Nurr1 were also strongly expressed. However, several other neural-related mRNAs (e.g. DRD2, enolase2, NFL and MBP) could be identified, but not in all donor samples. Similarly, synaptic vesicle-related mRNA, STX1A could only be detected in 2 of the 4 undifferentiated donor hMSC samples. More significantly, each donor sample revealed a unique expression pattern, demonstrating a significant variation of marker expression. Conclusion The present study highlights the existence of an inter-donor variability of expression of neural-related markers in human MSC samples that has not previously been described. This donor-related heterogeneity might influence the reproducibility of transdifferentiation protocols as well as contributing to the

  6. Surface cell density effects on Escherichia coli gene expression during cell attachment.

    PubMed

    Mauter, Meagan S; Mauter, Meagan; Fait, Aaron; Elimelech, Menachem; Herzberg, Moshe

    2013-06-18

    Escherichia coli attachment to a surface initiates a complex series of interconnected signaling and regulation pathways that promote biofilm formation and maturation. The present work investigates the effect of deposited cell density on E. coli cell physiology, metabolic activity, and gene expression in the initial stages of biofilm development. Deposited cell density is controlled by exploiting the relationship between ionic strength and bacterial attachment efficiency in a packed bed column. Distinct differences in cell transcriptome are analyzed by comparing sessile cultures at two different cell surface densities and differentiating ionic strength effects by analyzing planktonic cultures in parallel. Our results indicate that operons regulating trypotophan production and the galactitol phosphotransferase system (including dihydroxyacetone phosphate synthesis) are strongly affected by cell density on the surface. Additional transcriptome and metabolomic impacts of cell density on succinate, proline, and pyroglutamic acid systems are also reported. These results are consistent with the hypothesis that surface cell density plays a major role in sessile cell physiology, commencing with the first stage of biofilm formation. These findings improve our understanding of biofilm formation in natural and engineered environmental systems and will contribute to future work ranging from pathogen migration in the environment to control of biofouling on engineered surfaces.

  7. Using intron splicing trick for preferential gene expression in transduced cells: an approach for suicide gene therapy.

    PubMed

    Pourzadegan, F; Shariati, L; Taghizadeh, R; Khanahmad, H; Mohammadi, Z; Tabatabaiefar, M A

    2016-01-01

    Suicide gene therapy is one of the most innovative approaches in which a potential toxic gene is delivered to the targeted cancer cell by different target delivery methods. We constructed a transfer vector to express green fluorescent protein (GFP) in transduced cells but not in packaging cells. We placed gfp under the control of the cytomegalovirus (CMV) promoter, which is positioned between the two long-terminal repeats in reverse direction. The intron-2 sequence of the human beta globin gene with two poly-A signals and several stop codons on the antisense strand was placed on the leading strand between the CMV promoter and gfp. For lentiviral production, the HEK293T and line were co-transfected with the PMD2G, psPAX2 and pLentiGFP-Ins2 plasmids. The HEK293T and line were transduced with this virus. PCR was performed for evaluation of intron splicing in transduced cells. The GFP expression was seen in 65% of the cells transduced. The PCR amplification of the genomic DNA of transduced cells confirmed the splicing of intron 2. The strategy is significant to accomplish our goal for preserving the packaging cells from the toxic gene expression during viral assembly and the resultant reduction in viral titration. Also it serves to address several other issues in the gene therapy.

  8. 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

  9. Kisspeptin regulation of genes involved in cell invasion and angiogenesis in first trimester human trophoblast cells.

    PubMed

    Francis, Víctor A; Abera, Aron B; Matjila, Mushi; Millar, Robert P; Katz, Arieh A

    2014-01-01

    The precise regulation of extravillous trophoblast invasion of the uterine wall is a key process in successful pregnancies. Kisspeptin (KP) has been shown to inhibit cancer cell metastasis and placental trophoblast cell migration. In this study primary cultures of first trimester human trophoblast cells have been utilized in order to study the regulation of invasion and angiogenesis-related genes by KP. Trophoblast cells were isolated from first trimester placenta and their identity was confirmed by immunostaining for cytokeratin-7. Real-time quantitative RT-PCR demonstrated that primary trophoblast cells express higher levels of GPR54 (KP receptor) and KP mRNA than the trophoblast cell line HTR8Svneo. Furthermore, trophoblast cells also expressed higher GPR54 and KP protein levels. Treating primary trophoblast cells with KP induced ERK1/2 phosphorylation, while co-treating the cells with a KP antagonist almost completely blocked the activation of ERK1/2 and demonstrated that KP through its cognate GPR54 receptor can activate ERK1/2 in trophoblast cells. KP reduced the migratory capability of trophoblast cells in a scratch-migration assay. Real-time quantitative RT-PCR demonstrated that KP treatment reduced the expression of matrix metalloproteinase 1, 2, 3, 7, 9, 10, 14 and VEGF-A, and increased the expression of tissue inhibitors of metalloproteinases 1 and 3. These results suggest that KP can inhibit first trimester trophoblast cells invasion via inhibition of cell migration and down regulation of the metalloproteinase system and VEGF-A. PMID:24923321

  10. Surface-Engineered Viral Vectors for Selective and Cell Type-Specific Gene Delivery.

    PubMed

    Buchholz, Christian J; Friedel, Thorsten; Büning, Hildegard

    2015-12-01

    Recent progress in gene transfer technology enables the delivery of genes precisely to the application-relevant cell type ex vivo on cultivated primary cells or in vivo on local or systemic administration. Gene vectors based on lentiviruses or adeno-associated viruses can be engineered such that they use a cell surface marker of choice for cell entry instead of their natural receptors. Binding to the surface marker is mediated by a targeting ligand displayed on the vector particle surface, which can be a peptide, single-chain antibody, or designed ankyrin repeat protein. Examples include vectors that deliver genes to specialized endothelial cells or lymphocytes, tumor cells, or particular cells of the nervous system with potential applications in gene function studies and molecular medicine.

  11. Cytokeratin gene expression in hepatoma hybrid