WorldWideScience

Sample records for characterization defines human

  1. Comprehensive genomic characterization defines human glioblastoma genes and core pathways

    NARCIS (Netherlands)

    Chin, L.; Meyerson, M.; Aldape, K.; Bigner, D.; Mikkelsen, T.; VandenBerg, S.; Kahn, A.; Penny, R.; Gerhard, D. S.; Getz, G.; Brennan, C.; Taylor, B. S.; Winckler, W.; Park, P.; Ladanyi, M.; Hoadley, K. A.; Verhaak, R. G. W.; Hayes, D. N.; Spellman, Paul T.; Absher, D.; Weir, B. A.; Ding, L.; Wheeler, D.; Lawrence, M. S.; Cibulskis, K.; Mardis, E.; Zhang, Jinghui; Wilson, R. K.; Donehower, L.; Wheeler, D. A.; Purdom, E.; Wallis, J.; Laird, P. W.; Herman, J. G.; Schuebel, K. E.; Weisenberger, D. J.; Baylin, S. B.; Schultz, N.; Yao, Jun; Wiedemeyer, R.; Weinstein, J.; Sander, C.; Gibbs, R. A.; Gray, J.; Kucherlapati, R.; Lander, E. S.; Myers, R. M.; Perou, C. M.; McLendon, Roger; Friedman, Allan; Van Meir, Erwin G; Brat, Daniel J; Mastrogianakis, Gena Marie; Olson, Jeffrey J; Lehman, Norman; Yung, W. K. Alfred; Bogler, Oliver; Berger, Mitchel; Prados, Michael; Muzny, Donna; Morgan, Margaret; Scherer, Steve; Sabo, Aniko; Nazareth, Lynn; Lewis, Lora; Hall, Otis; Zhu, Yiming; Ren, Yanru; Alvi, Omar; Yao, Jiqiang; Hawes, Alicia; Jhangiani, Shalini; Fowler, Gerald; San Lucas, Anthony; Kovar, Christie; Cree, Andrew; Dinh, Huyen; Santibanez, Jireh; Joshi, Vandita; Gonzalez-Garay, Manuel L.; Miller, Christopher A.; Milosavljevic, Aleksandar; Sougnez, Carrie; Fennell, Tim; Mahan, Scott; Wilkinson, Jane; Ziaugra, Liuda; Onofrio, Robert; Bloom, Toby; Nicol, Rob; Ardlie, Kristin; Baldwin, Jennifer; Gabriel, Stacey; Fulton, Robert S.; McLellan, Michael D.; Larson, David E.; Shi, Xiaoqi; Abbott, Rachel; Fulton, Lucinda; Chen, Ken; Koboldt, Daniel C.; Wendl, Michael C.; Meyer, Rick; Tang, Yuzhu; Lin, Ling; Osborne, John R.; Dunford-Shore, Brian H.; Miner, Tracie L.; Delehaunty, Kim; Markovic, Chris; Swift, Gary; Courtney, William; Pohl, Craig; Abbott, Scott; Hawkins, Amy; Leong, Shin; Haipek, Carrie; Schmidt, Heather; Wiechert, Maddy; Vickery, Tammi; Scott, Sacha; Dooling, David J.; Chinwalla, Asif; Weinstock, George M.; O'Kelly, Michael; Robinson, Jim; Alexe, Gabriele; Beroukhim, Rameen; Carter, Scott; Chiang, Derek; Gould, Josh; Gupta, Supriya; Korn, Josh; Mermel, Craig; Mesirov, Jill; Monti, Stefano; Nguyen, Huy; Parkin, Melissa; Reich, Michael; Stransky, Nicolas; Garraway, Levi; Golub, Todd; Protopopov, Alexei; Perna, Ilana; Aronson, Sandy; Sathiamoorthy, Narayan; Ren, Georgia; Kim, Hyunsoo; Kong, Sek Won; Xiao, Yonghong; Kohane, Isaac S.; Seidman, Jon; Cope, Leslie; Pan, Fei; Van Den Berg, David; Van Neste, Leander; Yi, Joo Mi; Li, Jun Z.; Southwick, Audrey; Brady, Shannon; Aggarwal, Amita; Chung, Tisha; Sherlock, Gavin; Brooks, James D.; Jakkula, Lakshmi R.; Lapuk, Anna V.; Marr, Henry; Dorton, Shannon; Choi, Yoon Gi; Han, Ju; Ray, Amrita; Wang, Victoria; Durinck, Steffen; Robinson, Mark; Wang, Nicholas J.; Vranizan, Karen; Peng, Vivian; Van Name, Eric; Fontenay, Gerald V.; Ngai, John; Conboy, John G.; Parvin, Bahram; Feiler, Heidi S.; Speed, Terence P.; Socci, Nicholas D.; Olshen, Adam; Lash, Alex; Reva, Boris; Antipin, Yevgeniy; Stukalov, Alexey; Gross, Benjamin; Cerami, Ethan; Wang, Wei Qing; Qin, Li-Xuan; Seshan, Venkatraman E.; Villafania, Liliana; Cavatore, Magali; Borsu, Laetitia; Viale, Agnes; Gerald, William; Topal, Michael D.; Qi, Yuan; Balu, Sai; Shi, Yan; Wu, George; Bittner, Michael; Shelton, Troy; Lenkiewicz, Elizabeth; Morris, Scott; Beasley, Debbie; Sanders, Sheri; Sfeir, Robert; Chen, Jessica; Nassau, David; Feng, Larry; Hickey, Erin; Schaefer, Carl; Madhavan, Subha; Buetow, Ken; Barker, Anna; Vockley, Joseph; Compton, Carolyn; Vaught, Jim; Fielding, Peter; Collins, Francis; Good, Peter; Guyer, Mark; Ozenberger, Brad; Peterson, Jane; Thomson, Elizabeth

    2008-01-01

    Human cancer cells typically harbour multiple chromosomal aberrations, nucleotide substitutions and epigenetic modifications that drive malignant transformation. The Cancer Genome Atlas ( TCGA) pilot project aims to assess the value of large- scale multi- dimensional analysis of these molecular char

  2. Radioiodination of human growth hormone with characterization and minimization of the commonly defined 'damaged products'

    International Nuclear Information System (INIS)

    The radioiodination and chromatographic purification of human growth hormone (hGH) has been studied in order to better define and control the so-called 'preparation damage', which is often a cause of interferences, loss in specific activity and sensitivity, misclassification errors in radioligand assays, and a source of misinterpretation when the tracer is used in receptors or in vivo studies. A series of labelings and false labelings, with and without protein carrier in the buffer used for Sephadex purification, indicate that the 'preparation damage' peak is made up of two components: aggregated 125I-hGH and BSA-carried radioactivity. The former can be minimized by the use of recently extracted non-lyophilized hGH, and the latter by enzymatic labeling. Both components can be better resolved, and thus eliminated, when Sephadex G-100 is employed rather than G-75. (Auth.)

  3. Defining Human Enhancement

    DEFF Research Database (Denmark)

    Nordberg, Ana

    2016-01-01

    on the legitimacy to legislate and on whether the existing legal framework is appropriate to address the various concerns. In order to do so, it is crucial to establish clear legal definitions. Precise distinctions between interventions on the human body are intrinsically difficult to formulate. However, subject-matter...... definitions are vital legal tools to determine what is currently regulated in established fields of law and whether there is room for a new legal field – Enhancement Law. This paper provides a reflection on the relevance of establishing a legal definition of human enhancement and to what extent different...... legal fields and jurisdictions may warrant different understandings of such concept. It reviews a number of different and often divergent concepts and taxonomies of human enhancement and concludes with the proposal and analysis of a definition: Use of technological means with the intention to improve...

  4. Defining poverty as distinctively human

    Directory of Open Access Journals (Sweden)

    H.P.P. Lötter

    2007-05-01

    Full Text Available While it is relatively easy for most people to identify human beings suffering from poverty, it is rather more difficult to come to a proper understanding of poverty. In this article the author wants to deepen our understanding of poverty by interpreting the conventional definitions of poverty in a new light. The article starts with a defence of a claim that poverty is a concept uniquely applicable to humans. It then present a critical discussion of the distinction between absolute and relative poverty and it is then argued that a revision of this distinction can provide general standards applicable to humans everywhere.

  5. A new human natural killer leukemia cell line, IMC-1. A complex chromosomal rearrangement defined by spectral karyotyping: functional and cytogenetic characterization.

    Science.gov (United States)

    Chen, I-Ming; Whalen, Margaret; Bankhurst, Arthur; Sever, Cordelia E; Doshi, Rashmi; Hardekopf, David; Montgomery, Karen; Willman, Cheryl L

    2004-03-01

    A new human IL-2 dependent leukemic cell line with a natural killer (NK) cell phenotype, IMC-1, was established from an adult patient with aggressive NK cell leukemia. The IMC-1 cell line expresses the CD56, CD2, CD11a, CD38 and HLA-DR cell surface antigens, whereas the CD16 and CD8 antigens expressed on the primary leukemic blasts from which the cell line was derived were lost after 7 and 28 weeks of culture, respectively. The IMC-1 cell line displays functional NK cytotoxicity and lyses target cells in a non-MHC restricted, antibody-independent manner with equal or superior efficiency to freshly isolated NK cells. Cytogenetic analysis at presentation and after 55 weeks in culture revealed complex structural and numerical abnormalities, defined by classic G-banding and by spectral karyotyping (SKY). Three apparently intact copies of chromosome 8 occurred in the diagnostic bone marrow specimen; the cell line also contains three copies of chromosome 8 but each was structurally altered. The development and detailed characterization of this new NK leukemic cell line will facilitate biologic and functional studies of NK cells and chromosomal aberrations potentially important in leukemic transformation.

  6. Defining International Human Resource Development: A Proposal

    Science.gov (United States)

    McLean, Gary N.; Wang, Xiaohui

    2007-01-01

    From the beginning of the use of the term, there have been struggles over the meaning of human resource development (HRD). In recent years, there has been increased attention to the field's definition. This paper moves this exploration one more step to an exploration of the dilemma of defining international and cross-national HRD. A beginning…

  7. Characterization and definability in modal first-order fragments

    CERN Document Server

    Carreiro, Facundo

    2010-01-01

    Model theoretic results such as Characterization and Definability give important information about different logics. It is well known that the proofs of those results for several modal logics have, somehow, the same 'taste'. A general proof for most modal logics below first order is still too ambitious. In this thesis we plan to isolate sufficient conditions for the characterization and definability theorems to hold in a wide range of logics. Along with these conditions we will prove that, whichever logic that meets them, satisfies both theorems. Therefore, one could give an unifying proof for logics with already known results. Moreover, one will be able to prove characterization and definability results for logics that have not yet been investigated. In both cases, it is only needed to check that a logic meets the requirements to automatically derive the desired results.

  8. Derivation of human embryonic stem cells in defined conditions.

    Science.gov (United States)

    Ludwig, Tenneille E; Levenstein, Mark E; Jones, Jeffrey M; Berggren, W Travis; Mitchen, Erika R; Frane, Jennifer L; Crandall, Leann J; Daigh, Christine A; Conard, Kevin R; Piekarczyk, Marian S; Llanas, Rachel A; Thomson, James A

    2006-02-01

    We have previously reported that high concentrations of basic fibroblast growth factor (bFGF) support feeder-independent growth of human embryonic stem (ES) cells, but those conditions included poorly defined serum and matrix components. Here we report feeder-independent human ES cell culture that includes protein components solely derived from recombinant sources or purified from human material. We describe the derivation of two new human ES cell lines in these defined culture conditions.

  9. Defining Human Failure Events for Petroleum Risk Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Ronald L. Boring; Knut Øien

    2014-06-01

    In this paper, an identification and description of barriers and human failure events (HFEs) for human reliability analysis (HRA) is performed. The barriers, called target systems, are identified from risk significant accident scenarios represented as defined situations of hazard and accident (DSHAs). This report serves as the foundation for further work to develop petroleum HFEs compatible with the SPAR-H method and intended for reuse in future HRAs.

  10. Regulatory networks define phenotypic classes of human stem cell lines

    OpenAIRE

    Müller, Franz-Josef; Louise C. Laurent; Kostka, Dennis; Ulitsky, Igor; Williams, Roy; Lu, Christina; Park, In-Hyun; Rao, Mahendra S.; Shamir, Ron; Philip H. Schwartz; Schmidt, Nils O.; Loring, Jeanne F.

    2008-01-01

    Stem cells are defined as self-renewing cell populations that can differentiate into multiple distinct cell types. However, hundreds of different human cell lines from embryonic, fetal, and adult sources have been called stem cells, even though they range from pluripotent cells, typified by embryonic stem cells, which are capable of virtually unlimited proliferation and differentiation, to adult stem cell lines, which can generate a far more limited repertory of differentiated cell types. The...

  11. Defining human mesenchymal stem cell efficacy in vivo.

    Science.gov (United States)

    Bonfield, Tracey L; Nolan Koloze, Mary T; Lennon, Donald P; Caplan, Arnold I

    2010-01-01

    Allogeneic human mesenchymal stem cells (hMSCs) can suppress graft versus host disease (GvHD) and have profound anti-inflammatory and regenerative capacity in stroke, infarct, spinal cord injury, meniscus regeneration, tendinitis, acute renal failure, and heart disease in human and animal models of disease. There is significant clinical hMSC variability in efficacy and the ultimate response in vivo. The challenge in hMSC based therapy is defining the efficacy of hMSC in vivo. Models which may provide insight into hMSC bioactivity in vivo would provide a means to distinguish hMSCs for clinical utility. hMSC function has been described as both regenerative and trophic through the production of bioactive factors. The regenerative component involves the multi-potentiality of hMSC progenitor differentiation. The secreted factors generated by the hMSCs are milieu and injury specific providing unique niches for responses in vivo. These bioactive factors are anti-scarring, angiogenic, anti-apoptotic as well as regenerative. Further, from an immunological standpoint, hMSC's can avoid host immune response, providing xenographic applications. To study the in vivo immuno-regulatory effectiveness of hMSCs, we used the ovalbumin challenge model of acute asthma. This is a quick 3 week in vivo pulmonary inflammation model with readily accessible ways of measuring effectiveness of hMSCs. Our data show that there is a direct correlation between the traditional ceramic cube score to hMSCs attenuation of cellular recruitment due to ovalbumin challenge. The results from these studies verify the in vivo immuno-modulator effectiveness of hMSCs and support the potential use of the ovalbumin model as an in vivo model of hMSC potency and efficacy. Our data also support future directions toward exploring hMSCs as an alternative therapeutic for the treatment of airway inflammation associated with asthma. PMID:20974000

  12. Defining human mesenchymal stem cell efficacy in vivo

    Directory of Open Access Journals (Sweden)

    Lennon Donald P

    2010-10-01

    Full Text Available Abstract Allogeneic human mesenchymal stem cells (hMSCs can suppress graft versus host disease (GvHD and have profound anti-inflammatory and regenerative capacity in stroke, infarct, spinal cord injury, meniscus regeneration, tendinitis, acute renal failure, and heart disease in human and animal models of disease. There is significant clinical hMSC variability in efficacy and the ultimate response in vivo. The challenge in hMSC based therapy is defining the efficacy of hMSC in vivo. Models which may provide insight into hMSC bioactivity in vivo would provide a means to distinguish hMSCs for clinical utility. hMSC function has been described as both regenerative and trophic through the production of bioactive factors. The regenerative component involves the multi-potentiality of hMSC progenitor differentiation. The secreted factors generated by the hMSCs are milieu and injury specific providing unique niches for responses in vivo. These bioactive factors are anti-scarring, angiogenic, anti-apoptotic as well as regenerative. Further, from an immunological standpoint, hMSC's can avoid host immune response, providing xenographic applications. To study the in vivo immuno-regulatory effectiveness of hMSCs, we used the ovalbumin challenge model of acute asthma. This is a quick 3 week in vivo pulmonary inflammation model with readily accessible ways of measuring effectiveness of hMSCs. Our data show that there is a direct correlation between the traditional ceramic cube score to hMSCs attenuation of cellular recruitment due to ovalbumin challenge. The results from these studies verify the in vivo immuno-modulator effectiveness of hMSCs and support the potential use of the ovalbumin model as an in vivo model of hMSC potency and efficacy. Our data also support future directions toward exploring hMSCs as an alternative therapeutic for the treatment of airway inflammation associated with asthma.

  13. Criteria to define HLA haplotype loss in human solid tumors

    NARCIS (Netherlands)

    Ramal, LM; van der Zwan, AW; Collado, A; Lopez-Nevot, MA; Tilanus, M; Garrido, F

    2000-01-01

    Short tandem repeat (STR) markers are currently used to define loss of heterozygosity (LOH) of genes and chromosomes in tumors. Chromosome 6 and chromosome 15 STR markers are applied to define loss of HLA and related genes (e.g. TAP and beta(2)m) The number of STR identified in the HLA region is sti

  14. Defining And Characterizing Sample Representativeness For DWPF Melter Feed Samples

    Energy Technology Data Exchange (ETDEWEB)

    Shine, E. P.; Poirier, M. R.

    2013-10-29

    statisticians used carefully thought out designs that systematically and economically provided plans for data collection from the DWPF process. Key shared features of the sampling designs used at DWPF and the Gy sampling methodology were the specification of a standard for sample representativeness, an investigation that produced data from the process to study the sampling function, and a decision framework used to assess whether the specification was met based on the data. Without going into detail with regard to the seven errors identified by Pierre Gy, as excellent summaries are readily available such as Pitard [1989] and Smith [2001], SRS engineers understood, for example, that samplers can be biased (Gy's extraction error), and developed plans to mitigate those biases. Experiments that compared installed samplers with more representative samples obtained directly from the tank may not have resulted in systematically partitioning sampling errors into the now well-known error categories of Gy, but did provide overall information on the suitability of sampling systems. Most of the designs in this report are related to the DWPF vessels, not the large SRS Tank Farm tanks. Samples from the DWPF Slurry Mix Evaporator (SME), which contains the feed to the DWPF melter, are characterized using standardized analytical methods with known uncertainty. The analytical error is combined with the established error from sampling and processing in DWPF to determine the melter feed composition. This composition is used with the known uncertainty of the models in the Product Composition Control System (PCCS) to ensure that the wasteform that is produced is comfortably within the acceptable processing and product performance region. Having the advantage of many years of processing that meets the waste glass product acceptance criteria, the DWPF process has provided a considerable amount of data about itself in addition to the data from many special studies. Demonstrating representative

  15. Defining And Characterizing Sample Representativeness For DWPF Melter Feed Samples

    Energy Technology Data Exchange (ETDEWEB)

    Shine, E. P.; Poirier, M. R.

    2013-10-29

    statisticians used carefully thought out designs that systematically and economically provided plans for data collection from the DWPF process. Key shared features of the sampling designs used at DWPF and the Gy sampling methodology were the specification of a standard for sample representativeness, an investigation that produced data from the process to study the sampling function, and a decision framework used to assess whether the specification was met based on the data. Without going into detail with regard to the seven errors identified by Pierre Gy, as excellent summaries are readily available such as Pitard [1989] and Smith [2001], SRS engineers understood, for example, that samplers can be biased (Gy's extraction error), and developed plans to mitigate those biases. Experiments that compared installed samplers with more representative samples obtained directly from the tank may not have resulted in systematically partitioning sampling errors into the now well-known error categories of Gy, but did provide overall information on the suitability of sampling systems. Most of the designs in this report are related to the DWPF vessels, not the large SRS Tank Farm tanks. Samples from the DWPF Slurry Mix Evaporator (SME), which contains the feed to the DWPF melter, are characterized using standardized analytical methods with known uncertainty. The analytical error is combined with the established error from sampling and processing in DWPF to determine the melter feed composition. This composition is used with the known uncertainty of the models in the Product Composition Control System (PCCS) to ensure that the wasteform that is produced is comfortably within the acceptable processing and product performance region. Having the advantage of many years of processing that meets the waste glass product acceptance criteria, the DWPF process has provided a considerable amount of data about itself in addition to the data from many special studies. Demonstrating representative

  16. Defining And Characterizing Sample Representativeness For DWPF Melter Feed Samples

    International Nuclear Information System (INIS)

    statisticians used carefully thought out designs that systematically and economically provided plans for data collection from the DWPF process. Key shared features of the sampling designs used at DWPF and the Gy sampling methodology were the specification of a standard for sample representativeness, an investigation that produced data from the process to study the sampling function, and a decision framework used to assess whether the specification was met based on the data. Without going into detail with regard to the seven errors identified by Pierre Gy, as excellent summaries are readily available such as Pitard [1989] and Smith [2001], SRS engineers understood, for example, that samplers can be biased (Gy's extraction error), and developed plans to mitigate those biases. Experiments that compared installed samplers with more representative samples obtained directly from the tank may not have resulted in systematically partitioning sampling errors into the now well-known error categories of Gy, but did provide overall information on the suitability of sampling systems. Most of the designs in this report are related to the DWPF vessels, not the large SRS Tank Farm tanks. Samples from the DWPF Slurry Mix Evaporator (SME), which contains the feed to the DWPF melter, are characterized using standardized analytical methods with known uncertainty. The analytical error is combined with the established error from sampling and processing in DWPF to determine the melter feed composition. This composition is used with the known uncertainty of the models in the Product Composition Control System (PCCS) to ensure that the wasteform that is produced is comfortably within the acceptable processing and product performance region. Having the advantage of many years of processing that meets the waste glass product acceptance criteria, the DWPF process has provided a considerable amount of data about itself in addition to the data from many special studies. Demonstrating representative sampling

  17. Defining the nature of human pluripotent stem cell progeny

    Institute of Scientific and Technical Information of China (English)

    Michaela Patterson; David N Chan; Iris Ha; Dana Case; Yongyan Cui; Ben Van Handel; Hanna KA Mikkola; William E Lowry

    2012-01-01

    While it is clear that human pluripotent stem cells (hPSCs) can differentiate to generate a panoply of various cell types,it is unknown how closely in vitro development mirrors that which occurs in vivo.To determine whether human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) make equivalent progeny,and whether either makes cells that are analogous to tissue-derived cells,we performed comprehensive transcriptome profiling of purified PSC derivatives and their tissue-derived counterparts.Expression profiling demonstrated that hESCs and hiPSCs make nearly identical progeny for the neural,hepatic,and mesenchymal lineages,and an absence of re-expression from exogenous reprogramming factors in hiPSC progeny.However,when compared to a tissuederived counterpart,the progeny of both hESCs and hiPSCs maintained expression of a subset of genes normally associated with early mammalian development,regardless of the type of cell generated.While pluripotent genes (OCT4,SOX2,REX1,and NANOG) appeared to be silenced immediately upon differentiation from hPSCs,genes normally unique to early embryos (LIN28A,LIN28B,DPPA4,and others) were not fully silenced in hPSC derivatives.These data and evidence from expression patterns in early human fetal tissue (3-16 weeks of development) suggest that the differentiated progeny of hPSCs are reflective of very early human development (< 6 weeks).These findings provide support for the idea that hPSCs can serve as useful in vitro models of early human development,but also raise important issues for disease modeling and the clinical application of hPSC derivatives.

  18. Defining human insulin-like growth factor I gene regulation.

    Science.gov (United States)

    Mukherjee, Aditi; Alzhanov, Damir; Rotwein, Peter

    2016-08-01

    Growth hormone (GH) plays an essential role in controlling somatic growth and in regulating multiple physiological processes in humans and other species. Insulin-like growth factor I (IGF-I), a conserved, secreted 70-amino acid peptide, is a critical mediator of many of the biological effects of GH. Previous studies have demonstrated that GH rapidly and potently promotes IGF-I gene expression in rodents and in some other mammals through the transcription factor STAT5b, leading to accumulation of IGF-I mRNAs and production of IGF-I. Despite this progress, very little is known about how GH or other trophic factors control human IGF1 gene expression, in large part because of the absence of any cellular model systems that robustly express IGF-I. Here, we have addressed mechanisms of regulation of human IGF-I by GH after generating cells in which the IGF1 chromosomal locus has been incorporated into a mouse cell line. Using this model, we found that physiological levels of GH rapidly stimulate human IGF1 gene transcription and identify several potential transcriptional enhancers in chromatin that bind STAT5b in a GH-regulated way. Each of the putative enhancers also activates a human IGF1 gene promoter in reconstitution experiments in the presence of the GH receptor, STAT5b, and GH. Thus we have developed a novel experimental platform that now may be used to determine how human IGF1 gene expression is controlled under different physiological and pathological conditions. PMID:27406741

  19. Defining and determining the properties of the human sleep homeostat

    NARCIS (Netherlands)

    Zavada, Andrei

    2007-01-01

    To summarize, the principal findings upon which this thesis is built, are these: 1. The most accurate measure of the human chronotype on the 24-h scale relative to external time is the timepoint at a point in the 30–40% range of habitual sleep length on free days. 2. The free-day mid-sleep time corr

  20. Variations in Humanized and Defined Culture Conditions Supporting Derivation of New Human Embryonic Stem Cell Lines

    DEFF Research Database (Denmark)

    Fletcher, Judy M; Ferrier, Patricia M; Gardner, John O;

    2006-01-01

    The evolution of "humanized" (i.e., free of animal sourced reagents) and ultimately chemically defined culture systems for human embryo stem cell (hESC) isolation and culture is of importance to improving their efficacy and safety in research and therapeutic applications. This can be achieved...... serum-free medium (SFM) containing only human sourced and recombinant protein. Further, outgrowth of embryonic cells from whole blastocysts in both media could be achieved for up to 1 week without reliance on feeder cells. All variant conditions sustained undifferentiated cell status, a stable karyotype......, with a transitional requirement for human feeder cells. This represents another sequential step in the generation of therapeutic grade stem cells with reduced risk of zoonotic pathogen transmission....

  1. Defining and determining the properties of the human sleep homeostat

    OpenAIRE

    Zavada, Andrei

    2007-01-01

    To summarize, the principal findings upon which this thesis is built, are these: 1. The most accurate measure of the human chronotype on the 24-h scale relative to external time is the timepoint at a point in the 30–40% range of habitual sleep length on free days. 2. The free-day mid-sleep time correlates negatively with the gain constant from Achermann’s elaboration of the two-process model. Age is a confounding factor in this relationship, which itself is positively associated with the gain...

  2. Monoclonal antibody-defined human pancreatic cancer-associated antigens.

    Science.gov (United States)

    Schmiegel, W H; Kalthoff, H; Arndt, R; Gieseking, J; Greten, H; Klöppel, G; Kreiker, C; Ladak, A; Lampe, V; Ulrich, S

    1985-03-01

    Three pancreatic cancer-associated antigens were characterized by use of monoclonal antibodies in immunobinding studies with various cellular and soluble target antigens, in immunoprecipitation, and in immunoperoxidase staining. C54-0 represents a tumor-associated Mr 122,000 antigen, which appears to be widely distributed on various epithelial tumors and to a lower extent on normal tissue. C1-N3 antigen exhibited a more restricted distribution, reacting with pancreatic and various gastrointestinal tract tumors as well as with chronically inflamed pancreatic tissue. The most specific antigen expression was observed for C1-P83 antigen, found on all exocrine tumors of the pancreas, but not on normal or chronically inflamed pancreatic tissue.

  3. Characterizing the human hematopoietic CDome

    DEFF Research Database (Denmark)

    Barnkob, Mike Stein; Simon, Christian; Olsen, Lars Rønn

    2014-01-01

    In this study, we performed extensive semi-automated data collection from the primary and secondary literature in an effort to characterize the expression of all membrane proteins within the CD scheme on hematopoietic cells. Utilizing over 6000 data points across 305 CD molecules on 206 cell types......, we seek to give a preliminary characterization of the "human hematopoietic CDome." We encountered severe gaps in the knowledge of CD protein expression, mostly resulting from incomplete and unstructured data generation, which we argue inhibit both basic research as well as therapies seeking to target...

  4. Development of novel monoclonal antibodies that define differentiation stages of human stromal (mesenchymal) stem cells

    DEFF Research Database (Denmark)

    Andersen, Ditte Caroline; Kortesidis, Angela; Zannettino, Andrew C W;

    2011-01-01

    Human mesenchymal stem cells (hMSC) are currently being introduced for cell therapy, yet, antibodies specific for native and differentiated MSCs are required for their identification prior to clinical use. Herein, high quality antibodies against MSC surface proteins were developed by immunizing...... differentiation. Interestingly, undifferentiated cells revealed a sole cytoplasmic distribution pattern of Collagen VI, which however changed to an extracellular matrix appearance upon osteogenic- and adipogenic differentiation. In relation to this, we found that STRO-1(+/-)/Collagen VI(-) sorted hMSC contained...... fewer differentiated alkaline phosphatase(+) cells compared to STRO-1(+/-)/Collagen VI(+) hMSC, suggesting that Collagen VI on the cell membrane exclusively defines differentiated MSCs. In conclusion, we have generated a panel of high quality antibodies to be used for characterization of MSCs, and in...

  5. Characterization of Human Thymic Exosomes

    OpenAIRE

    Gabriel Skogberg; Judith Gudmundsdottir; Sjoerd van der Post; Kerstin Sandström; Sören Bruhn; Mikael Benson; Lucia Mincheva-Nilsson; Vladimir Baranov; Esbjörn Telemo; Olov Ekwall

    2013-01-01

    Exosomes are nanosized membrane-bound vesicles that are released by various cell types and are capable of carrying proteins, lipids and RNAs which can be delivered to recipient cells. Exosomes play a role in intercellular communication and have been described to mediate immunologic information. In this article we report the first isolation and characterization of exosomes from human thymic tissue. Using electron microscopy, particle size determination, density gradient measurement, flow cytom...

  6. On the significance of contaminant plume-scale and dose-response models in defining hydrogeological characterization needs

    Science.gov (United States)

    de Barros, F.; Rubin, Y.; Maxwell, R.; Bai, H.

    2007-12-01

    Defining rational and effective hydrogeological data acquisition strategies is of crucial importance since financial resources available for such efforts are always limited. Usually such strategies are developed with the goal of reducing uncertainty, but less often they are developed in the context of the impacts of uncertainty. This paper presents an approach for determining site characterization needs based on human health risk factors. The main challenge is in striking a balance between improved definition of hydrogeological, behavioral and physiological parameters. Striking this balance can provide clear guidance on setting priorities for data acquisition and for better estimating adverse health effects in humans. This paper addresses this challenge through theoretical developments and numerical testing. We will report on a wide range of factors that affect the site characterization needs including contaminant plume's dimensions, travel distances and other length scales that characterize the transport problem, as well as health risk models. We introduce a new graphical tool that allows one to investigate the relative impact of hydrogeological and physiological parameters in risk. Results show that the impact of uncertainty reduction in the risk-related parameters decreases with increasing distances from the contaminant source. Also, results indicate that human health risk becomes less sensitive to hydrogeological measurements when dealing with ergodic plumes. This indicates that under ergodic conditions, uncertainty reduction in human health risk may benefit from better understanding of the physiological component as opposed to a detailed hydrogeological characterization

  7. Involuntary Euthanasia and Current Attempts to Define Persons with Mental Retardation as Less Than Human.

    Science.gov (United States)

    Lusthaus, Evelyn W.

    1985-01-01

    The author examines current attempts to define mentally retarded persons as less than human and suggests that these ideologies are being used to justify euthanasia practices and to formulate euthanasia policies. (CL)

  8. Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions

    OpenAIRE

    Saha, Krishanu; Mei, Ying; Reisterer, Colin M.; Pyzocha, Neena Kenton; Yang, Jing; Muffat, Julien; Davies, Martyn C.; Alexander, Morgan R.; Langer, Robert; Anderson, Daniel G.; Jaenisch, Rudolf

    2011-01-01

    The current gold standard for the culture of human pluripotent stem cells requires the use of a feeder layer of cells. Here, we develop a spatially defined culture system based on UV/ozone radiation modification of typical cell culture plastics to define a favorable surface environment for human pluripotent stem cell culture. Chemical and geometrical optimization of the surfaces enables control of early cell aggregation from fully dissociated cells, as predicted from a numerical model of cell...

  9. Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells

    OpenAIRE

    Akopian, Veronika; Andrews, Peter W.; Beil, Stephen; Benvenisty, Nissim; Brehm, Jennifer; Christie, Megan; Ford, Angela; Fox, Victoria; Gokhale, Paul J; Healy, Lyn; Holm, Frida; Hovatta, Outi; Knowles, Barbara B; Ludwig, Tenneille E; Ronald D G McKay

    2010-01-01

    There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with pr...

  10. Human tumour antigens defined by cytotoxicity and proliferative responses of cultured lymphoid cells

    Science.gov (United States)

    Vose, Brent M.; Bonnard, Guy D.

    1982-03-01

    The long-term goal of many laboratories has been to develop cellular reagents having specific reactivity against human tumour cells. Such immune cells should prove useful for defining the antigenicity of human malignancies and may have important therapeutic potential, as has been clearly shown in some animal models1. Here we describe methods of initiating continued lymphocyte cultures (CLC) having specific anti-tumour reactivity using conditioned media containing interleukin-2 (IL-2).

  11. Inter-species chimeras of leukaemia inhibitory factor define a major human receptor-binding determinant.

    OpenAIRE

    Owczarek, C M; Layton, M. J.; Metcalf, D; Lock, P; Willson, T A; Gough, N M; Nicola, N A

    1993-01-01

    Human leukaemia inhibitory factor (hLIF) binds to both human and mouse LIF receptors (LIF-R), while mouse LIF (mLIF) binds only to mouse LIF-R. Moreover, hLIF binds with higher affinity to the mLIF-R than does mLIF. In order to define the regions of the hLIF molecule responsible for species-specific interaction with the hLIF-R and for the unusual high-affinity binding to the mLIF-R, a series of 15 mouse/human LIF hybrids has been generated. Perhaps surprisingly, both of these properties mappe...

  12. Dispersion measurement as a method of quantifying geologic characterization and defining reservoir heterogeneity. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Menzie, D.E.

    1995-05-01

    The main objective of this research project is to investigate dispersion as a method of quantifying geological characterization and defining reservoir heterogeneity in order to enhance crude oil recovery. The dispersion of flow of a reservoir rock (dispersion coefficient and dispersivity) was identified as one of the physical properties of a reservoir rock by measuring the mixing of two miscible fluids, one displacing the other in a porous medium. A rock was 100% saturated with a resident fluid and displaced by a miscible fluid of equal viscosity and equal density. Some specific experiments were performed with unequal densities. Produced fluid was analyzed by refractometer, nuclear reaction, electrical conductivity and X-ray scan. Several physical and flow characteristics were measured on the sand rock sample in order to establish correlations with the measured dispersion property. Absolute permeability, effective porosity, relative permeability, capillary pressure, the heterogeneity factor and electrical conductivity were used to better understand the flow system. Linear, transverse, 2-D and 3-D dispersions were measured and used to characterize the rock heterogeneity of the flow system. A new system of measuring dispersion was developed using a gas displacing gas system in a porous medium. An attempt was also made to determine the dispersion property of an actual reservoir from present day well log data on a producing well. 275 refs., 102 figs., 17 tabs.

  13. An experimental characterization of human torso motion

    Science.gov (United States)

    Cafolla, Daniele; Chen, I.-Ming; Ceccarelli, Marco

    2015-12-01

    The torso plays an important role in the human-like operation of humanoids. In this paper, a method is proposed to analyze the behavior of the human torso by using inertial and magnetic sensing tools. Experiments are conducted to characterize the motion performance of the human torso during daily routine operations. Furthermore, the forces acting on the human body during these operations are evaluated to design and validate the performance of a humanoid robot.

  14. A chemically-defined protocol for generating chondrocytes from human embryonic stem cells

    OpenAIRE

    sprotocols

    2015-01-01

    Progress in generating robust differentiation protocols for efficient and scalable production of defined cell lineages from human embryonic stem cells (hESc) has been slow. Amongst the obstacles to be addressed are those inherent to standard hESc culture and differentiation practices including the use of feeder cells, serum and animal-derived matrices. These components are biologically complex, undefined and highly variable between batches, inhibiting the development of consistently reproduci...

  15. Endothelial Interleukin-6 defines the tumorigenic potential of primary human cancer stem cells

    OpenAIRE

    Krishnamurthy, Sudha; Warner, Kristy A.; Dong, Zhihong; Imai, Atsushi; Nör, Carolina; Ward, Brent B.; Helman, Joseph I.; Taichman, Russell S.; Bellile, Emily L.; McCauley, Laurie K.; Polverini, Peter J.; Prince, Mark E.; Wicha, Max S.; Jacques E Nör

    2014-01-01

    Head and neck squamous cell carcinomas (HNSCC) contain a small sub-population of stem cells endowed with unique capacity to generate tumors. These cancer stem cells (CSC) are localized in perivascular niches and rely on crosstalk with endothelial cells for survival and self-renewal, but the mechanisms involved are unknown. Here, we report that stromal interleukin (IL)-6 defines the tumorigenic capacity of CSC sorted from primary human HNSCC and transplanted into mice. In search for the cellul...

  16. Endothelial interleukin-6 defines the tumorigenic potential of primary human cancer stem cells.

    Science.gov (United States)

    Krishnamurthy, Sudha; Warner, Kristy A; Dong, Zhihong; Imai, Atsushi; Nör, Carolina; Ward, Brent B; Helman, Joseph I; Taichman, Russell S; Bellile, Emily L; McCauley, Laurie K; Polverini, Peter J; Prince, Mark E; Wicha, Max S; Nör, Jacques E

    2014-11-01

    Head and neck squamous cell carcinomas (HNSCC) contain a small subpopulation of stem cells endowed with unique capacity to generate tumors. These cancer stem cells (CSC) are localized in perivascular niches and rely on crosstalk with endothelial cells for survival and self-renewal, but the mechanisms involved are unknown. Here, we report that stromal interleukin (IL)-6 defines the tumorigenic capacity of CSC sorted from primary human HNSCC and transplanted into mice. In search for the cellular source of Interleukin-6 (IL-6), we observed a direct correlation between IL-6 levels in tumor-associated endothelial cells and the tumorigenicity of CSC. In vitro, endothelial cell-IL-6 enhanced orosphere formation, p-STAT3 activation, survival, and self-renewal of human CSC. Notably, a humanized anti-IL-6R antibody (tocilizumab) inhibited primary human CSC-mediated tumor initiation. Collectively, these data demonstrate that endothelial cell-secreted IL-6 defines the tumorigenic potential of CSC, and suggest that HNSCC patients might benefit from therapeutic inhibition of IL-6/IL-6R signaling. PMID:25078284

  17. Defining the HLA class I-associated viral antigen repertoire from HIV-1-infected human cells

    DEFF Research Database (Denmark)

    Ternette, Nicola; Yang, Hongbing; Partridge, Thomas;

    2016-01-01

    Recognition and eradication of infected cells by cytotoxic T lymphocytes is a key defense mechanism against intracellular pathogens. High-throughput definition of HLA class I-associated immunopeptidomes by mass spectrometry is an increasingly important analytical tool to advance our understanding...... of the induction of T-cell responses against pathogens such as HIV-1. We utilized a liquid chromatography tandem mass spectrometry workflow including de novo-assisted database searching to define the HLA class I-associated immunopeptidome of HIV-1-infected human cells. We here report for the first time...... the identification of 75 HIV-1-derived peptides bound to HLA class I complexes that were purified directly from HIV-1-infected human primary CD4+ T cells and the C8166 human T-cell line. Importantly, one-third of eluted HIV-1 peptides had not been previously known to be presented by HLA class I. Over 82...

  18. A new region of conservation is defined between human and mouse X chromosomes

    Energy Technology Data Exchange (ETDEWEB)

    Dinulos, M.B.; Disteche, C.M. [Univ. of Washington, Seattle, WA (United States); Bassi, M.T. [Univ. of Siena (Italy)] [and others

    1996-07-01

    Comparative mapping of the X chromosome in eutherian mammals have revealed distinct regions of conservation as well as evolutionary rearrangements between human and mouse. Recently, we and others mapped the murine homologue of CLCN4 (Chloride channel 4) to band F4 of the X chromosome in Mus spretus but to chromosome 7 in laboratory strains. We now report the mapping of the murine homologues of APXL (Apical protein Xenopus laevis-like) and OA1 (Ocular albinism type I), two genes that are located on the human X chromosome at band p22.3 and in close proximity to CLCN4. Interestingly, Oa1 and Apxl map to bands F2-F3 in both M. spretus and the laboratory strain C57BL/6J, defining a new rearrangement between human and mouse X chromosomes. 17 refs., 2 figs., 1 tab.

  19. Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions.

    Science.gov (United States)

    Saha, Krishanu; Mei, Ying; Reisterer, Colin M; Pyzocha, Neena Kenton; Yang, Jing; Muffat, Julien; Davies, Martyn C; Alexander, Morgan R; Langer, Robert; Anderson, Daniel G; Jaenisch, Rudolf

    2011-11-15

    The current gold standard for the culture of human pluripotent stem cells requires the use of a feeder layer of cells. Here, we develop a spatially defined culture system based on UV/ozone radiation modification of typical cell culture plastics to define a favorable surface environment for human pluripotent stem cell culture. Chemical and geometrical optimization of the surfaces enables control of early cell aggregation from fully dissociated cells, as predicted from a numerical model of cell migration, and results in significant increases in cell growth of undifferentiated cells. These chemically defined xeno-free substrates generate more than three times the number of cells than feeder-containing substrates per surface area. Further, reprogramming and typical gene-targeting protocols can be readily performed on these engineered surfaces. These substrates provide an attractive cell culture platform for the production of clinically relevant factor-free reprogrammed cells from patient tissue samples and facilitate the definition of standardized scale-up friendly methods for disease modeling and cell therapeutic applications.

  20. Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions.

    Science.gov (United States)

    Saha, Krishanu; Mei, Ying; Reisterer, Colin M; Pyzocha, Neena Kenton; Yang, Jing; Muffat, Julien; Davies, Martyn C; Alexander, Morgan R; Langer, Robert; Anderson, Daniel G; Jaenisch, Rudolf

    2011-11-15

    The current gold standard for the culture of human pluripotent stem cells requires the use of a feeder layer of cells. Here, we develop a spatially defined culture system based on UV/ozone radiation modification of typical cell culture plastics to define a favorable surface environment for human pluripotent stem cell culture. Chemical and geometrical optimization of the surfaces enables control of early cell aggregation from fully dissociated cells, as predicted from a numerical model of cell migration, and results in significant increases in cell growth of undifferentiated cells. These chemically defined xeno-free substrates generate more than three times the number of cells than feeder-containing substrates per surface area. Further, reprogramming and typical gene-targeting protocols can be readily performed on these engineered surfaces. These substrates provide an attractive cell culture platform for the production of clinically relevant factor-free reprogrammed cells from patient tissue samples and facilitate the definition of standardized scale-up friendly methods for disease modeling and cell therapeutic applications. PMID:22065768

  1. Synthesis, characterization and self-assembly of well-defined linear heptablock quaterpolymers

    KAUST Repository

    Ntaras, Christos

    2016-05-17

    Two well-defined heptablock quaterpolymers of the ABCDCBA type [Α: polystyrene (PS), B: poly(butadiene) with ∼90% 1,4-microstructure (PB1,4), C: poly(isoprene) with ∼55% 3,4-microstructure (PI3,4) and D: poly(dimethylsiloxane) (PDMS)] were synthesized by combining anionic polymerization high vacuum techniques and hydrosilylation/chlorosilane chemistry. All intermediates and final products were characterized by size exclusion chromatography, membrane osmometry, and proton nuclear magnetic resonance spectroscopy. Fourier transform infrared spectroscopy was used to further verify the chemical modification reaction of the difunctional PDMS. The self-assembly in bulk of these novel heptablock quarterpolymers, studied by transmission electron microscopy and small angle X-ray scattering, revealed 3-phase 4-layer alternating lamellae morphology of PS, PB1,4, and mixed PI3,4/PDMS domains. Differential scanning calorimetry was used to further confirm the miscibility of PI3,4 and PDMS blocks. It is the first time that PDMS is the central segment in such multiblock polymers (≥3 chemically different blocks). © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2016, 54, 1443–1449. © 2016 Wiley Periodicals, Inc.

  2. Human papillomavirus-associated cancers as acquired immunodeficiency syndrome defining illnesses

    Directory of Open Access Journals (Sweden)

    Shohreh Shahabi

    2013-04-01

    Full Text Available The Centers for Disease Control currently report cervical, vulvar, vaginal, anal and some head and neck cancers as human papillomavirus (HPV-associated cancers. Only cervical cancer is listed amongst acquired immunodeficiency syndrome (AIDS defining illnesses. All of these cancers may represent progression of the immunocompromised state with the inability to eradicate viral infection. This study reports the case of a 27-year old HIV positive female presenting with a persistent right vulvar exophytic lesion. High-risk HPV analysis and immunostaining for P16 were both positive. A biopsy of the lesion revealed invasive squamous cell carcinoma. The patient underwent neoadjuvant radiation and chemotherapy followed by a radical vulvectomy. During treatment, her CD4 T-lymphocyte count decreased to 120 advancing her condition from HIV to AIDS. This case suggests that all HPV-associated cancers should be included as AIDS defining illnesses.

  3. Utility of cheiloscopy, rugoscopy, and dactyloscopy for human identification in a defined cohort

    Directory of Open Access Journals (Sweden)

    Vimi S Mutalik

    2013-01-01

    Full Text Available Background: Identification is of paramount importance in any forensic investigation. Positive identification of living or deceased using distinctive traits is a cornerstone of forensic science. The uniqueness of these patterns and subtle distinction between traits has offered worthy supplemental tools in establishing the true nature of facts. Aim: The first aim of our study was to determine the most common pattern of lip prints, palatal rugae, and finger prints in the study subjects. Secondly, to determine if any specific pattern of lip print, palatal rugae, or the finger print concurs in individuals, and thereby establish a database of these prototypes for human identification from a defined cohort. Materials and Methods: The sample size comprised 100 female students of a dental college staying together in the hostel. Lip prints were recorded on a white bond sheet using lipstick, palatal rugae on dental casts, and finger prints using printer′s blue ink. Results: Our observation suggested that the reticular pattern of lip print, the wavy pattern of palatal rugae, and the loop pattern of finger prints were the predominant patterns. Correlation of the three parameters did not reveal significant differences. Conclusions: This approach of human identification utilizing conventional techniques and relevant parameters is pertinent in defined groups. However, larger representative sample with robust analytical tools may provide a necessary blueprint of human identification.

  4. Utility of cheiloscopy, rugoscopy, and dactyloscopy for human identification in a defined cohort

    Science.gov (United States)

    Mutalik, Vimi S.; Menon, Aparna; Jayalakshmi, N.; Kamath, Asha; Raghu, A. R.

    2013-01-01

    Background: Identification is of paramount importance in any forensic investigation. Positive identification of living or deceased using distinctive traits is a cornerstone of forensic science. The uniqueness of these patterns and subtle distinction between traits has offered worthy supplemental tools in establishing the true nature of facts. Aim: The first aim of our study was to determine the most common pattern of lip prints, palatal rugae, and finger prints in the study subjects. Secondly, to determine if any specific pattern of lip print, palatal rugae, or the finger print concurs in individuals, and thereby establish a database of these prototypes for human identification from a defined cohort. Materials and Methods: The sample size comprised 100 female students of a dental college staying together in the hostel. Lip prints were recorded on a white bond sheet using lipstick, palatal rugae on dental casts, and finger prints using printer's blue ink. Results: Our observation suggested that the reticular pattern of lip print, the wavy pattern of palatal rugae, and the loop pattern of finger prints were the predominant patterns. Correlation of the three parameters did not reveal significant differences. Conclusions: This approach of human identification utilizing conventional techniques and relevant parameters is pertinent in defined groups. However, larger representative sample with robust analytical tools may provide a necessary blueprint of human identification. PMID:23960407

  5. What Is Trophoblast? A Combination of Criteria Define Human First-Trimester Trophoblast

    Directory of Open Access Journals (Sweden)

    Cheryl Q.E. Lee

    2016-02-01

    Full Text Available Controversy surrounds reports describing the derivation of human trophoblast cells from placentas and embryonic stem cells (ESC, partly due to the difficulty in identifying markers that define cells as belonging to the trophoblast lineage. We have selected criteria that are characteristic of primary first-trimester trophoblast: a set of protein markers, HLA class I profile, methylation of ELF5, and expression of microRNAs (miRNAs from the chromosome 19 miRNA cluster (C19MC. We tested these criteria on cells previously reported to show some phenotypic characteristics of trophoblast: bone morphogenetic protein (BMP-treated human ESC and 2102Ep, an embryonal carcinoma cell line. Both cell types only show some, but not all, of the four trophoblast criteria. Thus, BMP-treated human ESC have not fully differentiated to trophoblast. Our study identifies a robust panel, including both protein and non-protein-coding markers that, in combination, can be used to reliably define cells as characteristic of early trophoblast.

  6. Defining the role of common variation in the genomic and biological architecture of adult human height

    DEFF Research Database (Denmark)

    Wood, Andrew R.; Esko, Tonu; Yang, Jian;

    2014-01-01

    Using genome-wide data from 253,288 individuals, we identified 697 variants at genome-wide significance that together explained one-fifth of the heritability for adult height. By testing different numbers of variants in independent studies, we show that the most strongly associated similar to 2,0......TOR, osteoglycin and binding of hyaluronic acid. Our results indicate a genetic architecture for human height that is characterized by a very large but finite number (thousands) of causal variants....

  7. Defining the Relationship Between Human Error Classes and Technology Intervention Strategies

    Science.gov (United States)

    Wiegmann, Douglas A.; Rantanen, Eas M.

    2003-01-01

    The modus operandi in addressing human error in aviation systems is predominantly that of technological interventions or fixes. Such interventions exhibit considerable variability both in terms of sophistication and application. Some technological interventions address human error directly while others do so only indirectly. Some attempt to eliminate the occurrence of errors altogether whereas others look to reduce the negative consequences of these errors. In any case, technological interventions add to the complexity of the systems and may interact with other system components in unforeseeable ways and often create opportunities for novel human errors. Consequently, there is a need to develop standards for evaluating the potential safety benefit of each of these intervention products so that resources can be effectively invested to produce the biggest benefit to flight safety as well as to mitigate any adverse ramifications. The purpose of this project was to help define the relationship between human error and technological interventions, with the ultimate goal of developing a set of standards for evaluating or measuring the potential benefits of new human error fixes.

  8. Characterizing humans on Riemannian manifolds.

    Science.gov (United States)

    Tosato, Diego; Spera, Mauro; Cristani, Marco; Murino, Vittorio

    2013-08-01

    In surveillance applications, head and body orientation of people is of primary importance for assessing many behavioral traits. Unfortunately, in this context people are often encoded by a few, noisy pixels so that their characterization is difficult. We face this issue, proposing a computational framework which is based on an expressive descriptor, the covariance of features. Covariances have been employed for pedestrian detection purposes, actually a binary classification problem on Riemannian manifolds. In this paper, we show how to extend to the multiclassification case, presenting a novel descriptor, named weighted array of covariances, especially suited for dealing with tiny image representations. The extension requires a novel differential geometry approach in which covariances are projected on a unique tangent space where standard machine learning techniques can be applied. In particular, we adopt the Campbell-Baker-Hausdorff expansion as a means to approximate on the tangent space the genuine (geodesic) distances on the manifold in a very efficient way. We test our methodology on multiple benchmark datasets, and also propose new testing sets, getting convincing results in all the cases.

  9. Sleeping Beauty mutagenesis in a mouse medulloblastoma model defines networks that discriminate between human molecular subgroups

    Science.gov (United States)

    Genovesi, Laura A.; Ng, Ching Ging; Davis, Melissa J.; Remke, Marc; Taylor, Michael D.; Adams, David J.; Rust, Alistair G.; Ward, Jerrold M.; Ban, Kenneth H.; Jenkins, Nancy A.; Copeland, Neal G.; Wainwright, Brandon J.

    2013-01-01

    The Sleeping Beauty (SB) transposon mutagenesis screen is a powerful tool to facilitate the discovery of cancer genes that drive tumorigenesis in mouse models. In this study, we sought to identify genes that functionally cooperate with sonic hedgehog signaling to initiate medulloblastoma (MB), a tumor of the cerebellum. By combining SB mutagenesis with Patched1 heterozygous mice (Ptch1lacZ/+), we observed an increased frequency of MB and decreased tumor-free survival compared with Ptch1lacZ/+ controls. From an analysis of 85 tumors, we identified 77 common insertion sites that map to 56 genes potentially driving increased tumorigenesis. The common insertion site genes identified in the mutagenesis screen were mapped to human orthologs, which were used to select probes and corresponding expression data from an independent set of previously described human MB samples, and surprisingly were capable of accurately clustering known molecular subgroups of MB, thereby defining common regulatory networks underlying all forms of MB irrespective of subgroup. We performed a network analysis to discover the likely mechanisms of action of subnetworks and used an in vivo model to confirm a role for a highly ranked candidate gene, Nfia, in promoting MB formation. Our analysis implicates candidate cancer genes in the deregulation of apoptosis and translational elongation, and reveals a strong signature of transcriptional regulation that will have broad impact on expression programs in MB. These networks provide functional insights into the complex biology of human MB and identify potential avenues for intervention common to all clinical subgroups. PMID:24167280

  10. Differentiation of human embryonic stem cells to regional specific neural precursors in chemically defined medium conditions.

    Directory of Open Access Journals (Sweden)

    Slaven Erceg

    Full Text Available BACKGROUND: Human embryonic stem cells (hESC provide a unique model to study early events in human development. The hESC-derived cells can potentially be used to replace or restore different tissues including neuronal that have been damaged by disease or injury. METHODOLOGY AND PRINCIPAL FINDINGS: The cells of two different hESC lines were converted to neural rosettes using adherent and chemically defined conditions. The progenitor cells were exposed to retinoic acid (RA or to human recombinant basic fibroblast growth factor (bFGF in the late phase of the rosette formation. Exposing the progenitor cells to RA suppressed differentiation to rostral forebrain dopamine neural lineage and promoted that of spinal neural tissue including motor neurons. The functional characteristics of these differentiated neuronal precursors under both, rostral (bFGF and caudalizing (RA signals were confirmed by patch clamp analysis. CONCLUSIONS/SIGNIFICANCE: These findings suggest that our differentiation protocol has the capacity to generate region-specific and electrophysiologically active neurons under in vitro conditions without embryoid body formation, co-culture with stromal cells and without presence of cells of mesodermal or endodermal lineages.

  11. Growth factor-defined culture medium for human mesenchymal stem cells.

    Science.gov (United States)

    Mimura, Sumiyo; Kimura, Naohiro; Hirata, Mitsuhi; Tateyama, Daiki; Hayashida, Midori; Umezawa, Akihiro; Kohara, Arihiro; Nikawa, Hiroki; Okamoto, Tetsuji; Furue, Miho K

    2011-01-01

    Human bone marrow-derived mesenchymal stem cells (hMSCs) are potential cellular sources of therapeutic stem cells as they have the ability to proliferate and differentiate into a wide array of mesenchymal cell types such as osteoblasts, chondroblasts and adipocytes. hMSCs have been used clinically to treat patients with graft vs. host disease, osteogenesis imperfect, or alveolar cleft, suggesting that transplantation of hMSCs is comparatively safe as a stem cell-based therapy. However, conventional culture medium for hMSCs contains fetal bovine serum (FBS). In the present study, we developed a growth factor-defined, serum-free medium for culturing hMSCs. Under these conditions, TGF-beta1 promoted proliferation of hMSCs. The expanded hMSC population expressed the human pluripotency markers SSEA-3, -4, NANOG, OCT3/4 and SOX2. Furthermore, double positive cells for SSEA-3 and a mesenchymal cell marker, CD105, were detected in the population. The potential to differentiate into osteoblasts and adipocytes was confirmed. This work provides a useful tool to understand the basic biological properties of hMSCs in culture. PMID:21305471

  12. The human pancreas proteome defined by transcriptomics and antibody-based profiling.

    Science.gov (United States)

    Danielsson, Angelika; Pontén, Fredrik; Fagerberg, Linn; Hallström, Björn M; Schwenk, Jochen M; Uhlén, Mathias; Korsgren, Olle; Lindskog, Cecilia

    2014-01-01

    The pancreas is composed of both exocrine glands and intermingled endocrine cells to execute its diverse functions, including enzyme production for digestion of nutrients and hormone secretion for regulation of blood glucose levels. To define the molecular constituents with elevated expression in the human pancreas, we employed a genome-wide RNA sequencing analysis of the human transcriptome to identify genes with elevated expression in the human pancreas. This quantitative transcriptomics data was combined with immunohistochemistry-based protein profiling to allow mapping of the corresponding proteins to different compartments and specific cell types within the pancreas down to the single cell level. Analysis of whole pancreas identified 146 genes with elevated expression levels, of which 47 revealed a particular higher expression as compared to the other analyzed tissue types, thus termed pancreas enriched. Extended analysis of in vitro isolated endocrine islets identified an additional set of 42 genes with elevated expression in these specialized cells. Although only 0.7% of all genes showed an elevated expression level in the pancreas, this fraction of transcripts, in most cases encoding secreted proteins, constituted 68% of the total mRNA in pancreas. This demonstrates the extreme specialization of the pancreas for production of secreted proteins. Among the elevated expression profiles, several previously not described proteins were identified, both in endocrine cells (CFC1, FAM159B, RBPJL and RGS9) and exocrine glandular cells (AQP12A, DPEP1, GATM and ERP27). In summary, we provide a global analysis of the pancreas transcriptome and proteome with a comprehensive list of genes and proteins with elevated expression in pancreas. This list represents an important starting point for further studies of the molecular repertoire of pancreatic cells and their relation to disease states or treatment effects.

  13. Scalable cultivation of human pluripotent stem cells on chemically-defined surfaces

    Science.gov (United States)

    Hsiung, Michael Chi-Wei

    Human stem cells (SCs) are classified as self-renewing cells possessing great ability in therapeutic applications due of their ability to differentiate along any major cell lineage in the human body. Despite their restorative potential, widespread use of SCs is hampered by strenuous control issues. Along with the need for strict xeno-free environments to sustain growth in culture, current methods for growing human pluripotent stem cells (hPSCs) rely on platforms which impede large-scale cultivation and therapeutic delivery. Hence, any progress towards development of large-scale culture systems is severely hindered. In a concentrated effort to develop a scheme that can serve as a model precursor for large scale SC propagation in clinical use, we have explored methods for cultivating hPSCs on completely defined surfaces. We discuss novel approaches with the potential to go beyond the limitations presented by current methods. In particular, we studied the cultivation of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) on surface which underwent synthetic or chemical modification. Current methods for hPSCs rely on animal-based extracellular matrices (ECMs) such as mouse embryonic fibroblasts (MEFs) or feeders and murine sacoma cell-derived substrates to facilitate their growth. While these layers or coatings can be used to maximize the output of hPSC production, they cannot be considered for clinical use because they risk introducing foreign pathogens into culture. We have identified and developed conditions for a completely defined xeno-free substrate used for culturing hPSCs. By utilizing coupling chemistry, we can functionalize ester groups on a given surface and conjugate synthetic peptides containing the arginine-glycine-aspartic acid (RGD) motif, known for their role in cell adhesion. This method offers advantages over traditional hPSC culture by keeping the modified substrata free of xenogenic response and can be scaled up in

  14. Application of value of information of tank waste characterization: A new paradigm for defining tank waste characterization requirements

    International Nuclear Information System (INIS)

    This report presents the rationale for adopting a recommended characterization strategy that uses a risk-based decision-making framework for managing the Tank Waste Characterization program at Hanford. The risk-management/value-of-information (VOI) strategy that is illustrated explicitly links each information-gathering activity to its cost and provides a mechanism to ensure that characterization funds are spent where they can produce the largest reduction in risk. The approach was developed by tailoring well-known decision analysis techniques to specific tank waste characterization applications. This report illustrates how VOI calculations are performed and demonstrates that the VOI approach can definitely be used for real Tank Waste Remediation System (TWRS) characterization problems

  15. Characterization of human pluripotent stem cells.

    Science.gov (United States)

    Gokhale, Paul J; Andrews, Peter W

    2013-12-18

    Human pluripotent stem cells (PSCs), whether embryonic stem cells or induced PSCs, offer enormous opportunities for regenerative medicine and other biomedical applications once we have developed the ability to harness their capacity for extensive differentiation. Central to this is our ability to identify and characterize such PSCs, but this is fraught with potential difficulties that arise from a tension between functional definitions of pluripotency and the more convenient use of 'markers', a problem exacerbated by ethical issues, our lack of knowledge of early human embryonic development, and differences from the mouse paradigm.

  16. Defining the role of common variation in the genomic and biological architecture of adult human height.

    Science.gov (United States)

    Wood, Andrew R; Esko, Tonu; Yang, Jian; Vedantam, Sailaja; Pers, Tune H; Gustafsson, Stefan; Chu, Audrey Y; Estrada, Karol; Luan, Jian'an; Kutalik, Zoltán; Amin, Najaf; Buchkovich, Martin L; Croteau-Chonka, Damien C; Day, Felix R; Duan, Yanan; Fall, Tove; Fehrmann, Rudolf; Ferreira, Teresa; Jackson, Anne U; Karjalainen, Juha; Lo, Ken Sin; Locke, Adam E; Mägi, Reedik; Mihailov, Evelin; Porcu, Eleonora; Randall, Joshua C; Scherag, André; Vinkhuyzen, Anna A E; Westra, Harm-Jan; Winkler, Thomas W; Workalemahu, Tsegaselassie; Zhao, Jing Hua; Absher, Devin; Albrecht, Eva; Anderson, Denise; Baron, Jeffrey; Beekman, Marian; Demirkan, Ayse; Ehret, Georg B; Feenstra, Bjarke; Feitosa, Mary F; Fischer, Krista; Fraser, Ross M; Goel, Anuj; Gong, Jian; Justice, Anne E; Kanoni, Stavroula; Kleber, Marcus E; Kristiansson, Kati; Lim, Unhee; Lotay, Vaneet; Lui, Julian C; Mangino, Massimo; Mateo Leach, Irene; Medina-Gomez, Carolina; Nalls, Michael A; Nyholt, Dale R; Palmer, Cameron D; Pasko, Dorota; Pechlivanis, Sonali; Prokopenko, Inga; Ried, Janina S; Ripke, Stephan; Shungin, Dmitry; Stancáková, Alena; Strawbridge, Rona J; Sung, Yun Ju; Tanaka, Toshiko; Teumer, Alexander; Trompet, Stella; van der Laan, Sander W; van Setten, Jessica; Van Vliet-Ostaptchouk, Jana V; Wang, Zhaoming; Yengo, Loïc; Zhang, Weihua; Afzal, Uzma; Arnlöv, Johan; Arscott, Gillian M; Bandinelli, Stefania; Barrett, Amy; Bellis, Claire; Bennett, Amanda J; Berne, Christian; Blüher, Matthias; Bolton, Jennifer L; Böttcher, Yvonne; Boyd, Heather A; Bruinenberg, Marcel; Buckley, Brendan M; Buyske, Steven; Caspersen, Ida H; Chines, Peter S; Clarke, Robert; Claudi-Boehm, Simone; Cooper, Matthew; Daw, E Warwick; De Jong, Pim A; Deelen, Joris; Delgado, Graciela; Denny, Josh C; Dhonukshe-Rutten, Rosalie; Dimitriou, Maria; Doney, Alex S F; Dörr, Marcus; Eklund, Niina; Eury, Elodie; Folkersen, Lasse; Garcia, Melissa E; Geller, Frank; Giedraitis, Vilmantas; Go, Alan S; Grallert, Harald; Grammer, Tanja B; Gräßler, Jürgen; Grönberg, Henrik; de Groot, Lisette C P G M; Groves, Christopher J; Haessler, Jeffrey; Hall, Per; Haller, Toomas; Hallmans, Goran; Hannemann, Anke; Hartman, Catharina A; Hassinen, Maija; Hayward, Caroline; Heard-Costa, Nancy L; Helmer, Quinta; Hemani, Gibran; Henders, Anjali K; Hillege, Hans L; Hlatky, Mark A; Hoffmann, Wolfgang; Hoffmann, Per; Holmen, Oddgeir; Houwing-Duistermaat, Jeanine J; Illig, Thomas; Isaacs, Aaron; James, Alan L; Jeff, Janina; Johansen, Berit; Johansson, Åsa; Jolley, Jennifer; Juliusdottir, Thorhildur; Junttila, Juhani; Kho, Abel N; Kinnunen, Leena; Klopp, Norman; Kocher, Thomas; Kratzer, Wolfgang; Lichtner, Peter; Lind, Lars; Lindström, Jaana; Lobbens, Stéphane; Lorentzon, Mattias; Lu, Yingchang; Lyssenko, Valeriya; Magnusson, Patrik K E; Mahajan, Anubha; Maillard, Marc; McArdle, Wendy L; McKenzie, Colin A; McLachlan, Stela; McLaren, Paul J; Menni, Cristina; Merger, Sigrun; Milani, Lili; Moayyeri, Alireza; Monda, Keri L; Morken, Mario A; Müller, Gabriele; Müller-Nurasyid, Martina; Musk, Arthur W; Narisu, Narisu; Nauck, Matthias; Nolte, Ilja M; Nöthen, Markus M; Oozageer, Laticia; Pilz, Stefan; Rayner, Nigel W; Renstrom, Frida; Robertson, Neil R; Rose, Lynda M; Roussel, Ronan; Sanna, Serena; Scharnagl, Hubert; Scholtens, Salome; Schumacher, Fredrick R; Schunkert, Heribert; Scott, Robert A; Sehmi, Joban; Seufferlein, Thomas; Shi, Jianxin; Silventoinen, Karri; Smit, Johannes H; Smith, Albert Vernon; Smolonska, Joanna; Stanton, Alice V; Stirrups, Kathleen; Stott, David J; Stringham, Heather M; Sundström, Johan; Swertz, Morris A; Syvänen, Ann-Christine; Tayo, Bamidele O; Thorleifsson, Gudmar; Tyrer, Jonathan P; van Dijk, Suzanne; van Schoor, Natasja M; van der Velde, Nathalie; van Heemst, Diana; van Oort, Floor V A; Vermeulen, Sita H; Verweij, Niek; Vonk, Judith M; Waite, Lindsay L; Waldenberger, Melanie; Wennauer, Roman; Wilkens, Lynne R; Willenborg, Christina; Wilsgaard, Tom; Wojczynski, Mary K; Wong, Andrew; Wright, Alan F; Zhang, Qunyuan; Arveiler, Dominique; Bakker, Stephan J L; Beilby, John; Bergman, Richard N; Bergmann, Sven; Biffar, Reiner; Blangero, John; Boomsma, Dorret I; Bornstein, Stefan R; Bovet, Pascal; Brambilla, Paolo; Brown, Morris J; Campbell, Harry; Caulfield, Mark J; Chakravarti, Aravinda; Collins, Rory; Collins, Francis S; Crawford, Dana C; Cupples, L Adrienne; Danesh, John; de Faire, Ulf; den Ruijter, Hester M; Erbel, Raimund; Erdmann, Jeanette; Eriksson, Johan G; Farrall, Martin; Ferrannini, Ele; Ferrières, Jean; Ford, Ian; Forouhi, Nita G; Forrester, Terrence; Gansevoort, Ron T; Gejman, Pablo V; Gieger, Christian; Golay, Alain; Gottesman, Omri; Gudnason, Vilmundur; Gyllensten, Ulf; Haas, David W; Hall, Alistair S; Harris, Tamara B; Hattersley, Andrew T; Heath, Andrew C; Hengstenberg, Christian; Hicks, Andrew A; Hindorff, Lucia A; Hingorani, Aroon D; Hofman, Albert; Hovingh, G Kees; Humphries, Steve E; Hunt, Steven C; Hypponen, Elina; Jacobs, Kevin B; Jarvelin, Marjo-Riitta; Jousilahti, Pekka; Jula, Antti M; Kaprio, Jaakko; Kastelein, John J P; Kayser, Manfred; Kee, Frank; Keinanen-Kiukaanniemi, Sirkka M; Kiemeney, Lambertus A; Kooner, Jaspal S; Kooperberg, Charles; Koskinen, Seppo; Kovacs, Peter; Kraja, Aldi T; Kumari, Meena; Kuusisto, Johanna; Lakka, Timo A; Langenberg, Claudia; Le Marchand, Loic; Lehtimäki, Terho; Lupoli, Sara; Madden, Pamela A F; Männistö, Satu; Manunta, Paolo; Marette, André; Matise, Tara C; McKnight, Barbara; Meitinger, Thomas; Moll, Frans L; Montgomery, Grant W; Morris, Andrew D; Morris, Andrew P; Murray, Jeffrey C; Nelis, Mari; Ohlsson, Claes; Oldehinkel, Albertine J; Ong, Ken K; Ouwehand, Willem H; Pasterkamp, Gerard; Peters, Annette; Pramstaller, Peter P; Price, Jackie F; Qi, Lu; Raitakari, Olli T; Rankinen, Tuomo; Rao, D C; Rice, Treva K; Ritchie, Marylyn; Rudan, Igor; Salomaa, Veikko; Samani, Nilesh J; Saramies, Jouko; Sarzynski, Mark A; Schwarz, Peter E H; Sebert, Sylvain; Sever, Peter; Shuldiner, Alan R; Sinisalo, Juha; Steinthorsdottir, Valgerdur; Stolk, Ronald P; Tardif, Jean-Claude; Tönjes, Anke; Tremblay, Angelo; Tremoli, Elena; Virtamo, Jarmo; Vohl, Marie-Claude; Amouyel, Philippe; Asselbergs, Folkert W; Assimes, Themistocles L; Bochud, Murielle; Boehm, Bernhard O; Boerwinkle, Eric; Bottinger, Erwin P; Bouchard, Claude; Cauchi, Stéphane; Chambers, John C; Chanock, Stephen J; Cooper, Richard S; de Bakker, Paul I W; Dedoussis, George; Ferrucci, Luigi; Franks, Paul W; Froguel, Philippe; Groop, Leif C; Haiman, Christopher A; Hamsten, Anders; Hayes, M Geoffrey; Hui, Jennie; Hunter, David J; Hveem, Kristian; Jukema, J Wouter; Kaplan, Robert C; Kivimaki, Mika; Kuh, Diana; Laakso, Markku; Liu, Yongmei; Martin, Nicholas G; März, Winfried; Melbye, Mads; Moebus, Susanne; Munroe, Patricia B; Njølstad, Inger; Oostra, Ben A; Palmer, Colin N A; Pedersen, Nancy L; Perola, Markus; Pérusse, Louis; Peters, Ulrike; Powell, Joseph E; Power, Chris; Quertermous, Thomas; Rauramaa, Rainer; Reinmaa, Eva; Ridker, Paul M; Rivadeneira, Fernando; Rotter, Jerome I; Saaristo, Timo E; Saleheen, Danish; Schlessinger, David; Slagboom, P Eline; Snieder, Harold; Spector, Tim D; Strauch, Konstantin; Stumvoll, Michael; Tuomilehto, Jaakko; Uusitupa, Matti; van der Harst, Pim; Völzke, Henry; Walker, Mark; Wareham, Nicholas J; Watkins, Hugh; Wichmann, H-Erich; Wilson, James F; Zanen, Pieter; Deloukas, Panos; Heid, Iris M; Lindgren, Cecilia M; Mohlke, Karen L; Speliotes, Elizabeth K; Thorsteinsdottir, Unnur; Barroso, Inês; Fox, Caroline S; North, Kari E; Strachan, David P; Beckmann, Jacques S; Berndt, Sonja I; Boehnke, Michael; Borecki, Ingrid B; McCarthy, Mark I; Metspalu, Andres; Stefansson, Kari; Uitterlinden, André G; van Duijn, Cornelia M; Franke, Lude; Willer, Cristen J; Price, Alkes L; Lettre, Guillaume; Loos, Ruth J F; Weedon, Michael N; Ingelsson, Erik; O'Connell, Jeffrey R; Abecasis, Goncalo R; Chasman, Daniel I; Goddard, Michael E; Visscher, Peter M; Hirschhorn, Joel N; Frayling, Timothy M

    2014-11-01

    Using genome-wide data from 253,288 individuals, we identified 697 variants at genome-wide significance that together explained one-fifth of the heritability for adult height. By testing different numbers of variants in independent studies, we show that the most strongly associated ∼2,000, ∼3,700 and ∼9,500 SNPs explained ∼21%, ∼24% and ∼29% of phenotypic variance. Furthermore, all common variants together captured 60% of heritability. The 697 variants clustered in 423 loci were enriched for genes, pathways and tissue types known to be involved in growth and together implicated genes and pathways not highlighted in earlier efforts, such as signaling by fibroblast growth factors, WNT/β-catenin and chondroitin sulfate-related genes. We identified several genes and pathways not previously connected with human skeletal growth, including mTOR, osteoglycin and binding of hyaluronic acid. Our results indicate a genetic architecture for human height that is characterized by a very large but finite number (thousands) of causal variants.

  17. Defining the role of common variation in the genomic and biological architecture of adult human height

    Science.gov (United States)

    Chu, Audrey Y; Estrada, Karol; Luan, Jian’an; Kutalik, Zoltán; Amin, Najaf; Buchkovich, Martin L; Croteau-Chonka, Damien C; Day, Felix R; Duan, Yanan; Fall, Tove; Fehrmann, Rudolf; Ferreira, Teresa; Jackson, Anne U; Karjalainen, Juha; Lo, Ken Sin; Locke, Adam E; Mägi, Reedik; Mihailov, Evelin; Porcu, Eleonora; Randall, Joshua C; Scherag, André; Vinkhuyzen, Anna AE; Westra, Harm-Jan; Winkler, Thomas W; Workalemahu, Tsegaselassie; Zhao, Jing Hua; Absher, Devin; Albrecht, Eva; Anderson, Denise; Baron, Jeffrey; Beekman, Marian; Demirkan, Ayse; Ehret, Georg B; Feenstra, Bjarke; Feitosa, Mary F; Fischer, Krista; Fraser, Ross M; Goel, Anuj; Gong, Jian; Justice, Anne E; Kanoni, Stavroula; Kleber, Marcus E; Kristiansson, Kati; Lim, Unhee; Lotay, Vaneet; Lui, Julian C; Mangino, Massimo; Leach, Irene Mateo; Medina-Gomez, Carolina; Nalls, Michael A; Nyholt, Dale R; Palmer, Cameron D; Pasko, Dorota; Pechlivanis, Sonali; Prokopenko, Inga; Ried, Janina S; Ripke, Stephan; Shungin, Dmitry; Stancáková, Alena; Strawbridge, Rona J; Sung, Yun Ju; Tanaka, Toshiko; Teumer, Alexander; Trompet, Stella; van der Laan, Sander W; van Setten, Jessica; Van Vliet-Ostaptchouk, Jana V; Wang, Zhaoming; Yengo, Loïc; Zhang, Weihua; Afzal, Uzma; Ärnlöv, Johan; Arscott, Gillian M; Bandinelli, Stefania; Barrett, Amy; Bellis, Claire; Bennett, Amanda J; Berne, Christian; Blüher, Matthias; Bolton, Jennifer L; Böttcher, Yvonne; Boyd, Heather A; Bruinenberg, Marcel; Buckley, Brendan M; Buyske, Steven; Caspersen, Ida H; Chines, Peter S; Clarke, Robert; Claudi-Boehm, Simone; Cooper, Matthew; Daw, E Warwick; De Jong, Pim A; Deelen, Joris; Delgado, Graciela; Denny, Josh C; Dhonukshe-Rutten, Rosalie; Dimitriou, Maria; Doney, Alex SF; Dörr, Marcus; Eklund, Niina; Eury, Elodie; Folkersen, Lasse; Garcia, Melissa E; Geller, Frank; Giedraitis, Vilmantas; Go, Alan S; Grallert, Harald; Grammer, Tanja B; Gräßler, Jürgen; Grönberg, Henrik; de Groot, Lisette C.P.G.M.; Groves, Christopher J; Haessler, Jeffrey; Hall, Per; Haller, Toomas; Hallmans, Goran; Hannemann, Anke; Hartman, Catharina A; Hassinen, Maija; Hayward, Caroline; Heard-Costa, Nancy L; Helmer, Quinta; Hemani, Gibran; Henders, Anjali K; Hillege, Hans L; Hlatky, Mark A; Hoffmann, Wolfgang; Hoffmann, Per; Holmen, Oddgeir; Houwing-Duistermaat, Jeanine J; Illig, Thomas; Isaacs, Aaron; James, Alan L; Jeff, Janina; Johansen, Berit; Johansson, Åsa; Jolley, Jennifer; Juliusdottir, Thorhildur; Junttila, Juhani; Kho, Abel N; Kinnunen, Leena; Klopp, Norman; Kocher, Thomas; Kratzer, Wolfgang; Lichtner, Peter; Lind, Lars; Lindström, Jaana; Lobbens, Stéphane; Lorentzon, Mattias; Lu, Yingchang; Lyssenko, Valeriya; Magnusson, Patrik KE; Mahajan, Anubha; Maillard, Marc; McArdle, Wendy L; McKenzie, Colin A; McLachlan, Stela; McLaren, Paul J; Menni, Cristina; Merger, Sigrun; Milani, Lili; Moayyeri, Alireza; Monda, Keri L; Morken, Mario A; Müller, Gabriele; Müller-Nurasyid, Martina; Musk, Arthur W; Narisu, Narisu; Nauck, Matthias; Nolte, Ilja M; Nöthen, Markus M; Oozageer, Laticia; Pilz, Stefan; Rayner, Nigel W; Renstrom, Frida; Robertson, Neil R; Rose, Lynda M; Roussel, Ronan; Sanna, Serena; Scharnagl, Hubert; Scholtens, Salome; Schumacher, Fredrick R; Schunkert, Heribert; Scott, Robert A; Sehmi, Joban; Seufferlein, Thomas; Shi, Jianxin; Silventoinen, Karri; Smit, Johannes H; Smith, Albert Vernon; Smolonska, Joanna; Stanton, Alice V; Stirrups, Kathleen; Stott, David J; Stringham, Heather M; Sundström, Johan; Swertz, Morris A; Syvänen, Ann-Christine; Tayo, Bamidele O; Thorleifsson, Gudmar; Tyrer, Jonathan P; van Dijk, Suzanne; van Schoor, Natasja M; van der Velde, Nathalie; van Heemst, Diana; van Oort, Floor VA; Vermeulen, Sita H; Verweij, Niek; Vonk, Judith M; Waite, Lindsay L; Waldenberger, Melanie; Wennauer, Roman; Wilkens, Lynne R; Willenborg, Christina; Wilsgaard, Tom; Wojczynski, Mary K; Wong, Andrew; Wright, Alan F; Zhang, Qunyuan; Arveiler, Dominique; Bakker, Stephan JL; Beilby, John; Bergman, Richard N; Bergmann, Sven; Biffar, Reiner; Blangero, John; Boomsma, Dorret I; Bornstein, Stefan R; Bovet, Pascal; Brambilla, Paolo; Brown, Morris J; Campbell, Harry; Caulfield, Mark J; Chakravarti, Aravinda; Collins, Rory; Collins, Francis S; Crawford, Dana C; Cupples, L Adrienne; Danesh, John; de Faire, Ulf; den Ruijter, Hester M; Erbel, Raimund; Erdmann, Jeanette; Eriksson, Johan G; Farrall, Martin; Ferrannini, Ele; Ferrières, Jean; Ford, Ian; Forouhi, Nita G; Forrester, Terrence; Gansevoort, Ron T; Gejman, Pablo V; Gieger, Christian; Golay, Alain; Gottesman, Omri; Gudnason, Vilmundur; Gyllensten, Ulf; Haas, David W; Hall, Alistair S; Harris, Tamara B; Hattersley, Andrew T; Heath, Andrew C; Hengstenberg, Christian; Hicks, Andrew A; Hindorff, Lucia A; Hingorani, Aroon D; Hofman, Albert; Hovingh, G Kees; Humphries, Steve E; Hunt, Steven C; Hypponen, Elina; Jacobs, Kevin B; Jarvelin, Marjo-Riitta; Jousilahti, Pekka; Jula, Antti M; Kaprio, Jaakko; Kastelein, John JP; Kayser, Manfred; Kee, Frank; Keinanen-Kiukaanniemi, Sirkka M; Kiemeney, Lambertus A; Kooner, Jaspal S; Kooperberg, Charles; Koskinen, Seppo; Kovacs, Peter; Kraja, Aldi T; Kumari, Meena; Kuusisto, Johanna; Lakka, Timo A; Langenberg, Claudia; Le Marchand, Loic; Lehtimäki, Terho; Lupoli, Sara; Madden, Pamela AF; Männistö, Satu; Manunta, Paolo; Marette, André; Matise, Tara C; McKnight, Barbara; Meitinger, Thomas; Moll, Frans L; Montgomery, Grant W; Morris, Andrew D; Morris, Andrew P; Murray, Jeffrey C; Nelis, Mari; Ohlsson, Claes; Oldehinkel, Albertine J; Ong, Ken K; Ouwehand, Willem H; Pasterkamp, Gerard; Peters, Annette; Pramstaller, Peter P; Price, Jackie F; Qi, Lu; Raitakari, Olli T; Rankinen, Tuomo; Rao, DC; Rice, Treva K; Ritchie, Marylyn; Rudan, Igor; Salomaa, Veikko; Samani, Nilesh J; Saramies, Jouko; Sarzynski, Mark A; Schwarz, Peter EH; Sebert, Sylvain; Sever, Peter; Shuldiner, Alan R; Sinisalo, Juha; Steinthorsdottir, Valgerdur; Stolk, Ronald P; Tardif, Jean-Claude; Tönjes, Anke; Tremblay, Angelo; Tremoli, Elena; Virtamo, Jarmo; Vohl, Marie-Claude; Amouyel, Philippe; Asselbergs, Folkert W; Assimes, Themistocles L; Bochud, Murielle; Boehm, Bernhard O; Boerwinkle, Eric; Bottinger, Erwin P; Bouchard, Claude; Cauchi, Stéphane; Chambers, John C; Chanock, Stephen J; Cooper, Richard S; de Bakker, Paul IW; Dedoussis, George; Ferrucci, Luigi; Franks, Paul W; Froguel, Philippe; Groop, Leif C; Haiman, Christopher A; Hamsten, Anders; Hayes, M Geoffrey; Hui, Jennie; Hunter, David J.; Hveem, Kristian; Jukema, J Wouter; Kaplan, Robert C; Kivimaki, Mika; Kuh, Diana; Laakso, Markku; Liu, Yongmei; Martin, Nicholas G; März, Winfried; Melbye, Mads; Moebus, Susanne; Munroe, Patricia B; Njølstad, Inger; Oostra, Ben A; Palmer, Colin NA; Pedersen, Nancy L; Perola, Markus; Pérusse, Louis; Peters, Ulrike; Powell, Joseph E; Power, Chris; Quertermous, Thomas; Rauramaa, Rainer; Reinmaa, Eva; Ridker, Paul M; Rivadeneira, Fernando; Rotter, Jerome I; Saaristo, Timo E; Saleheen, Danish; Schlessinger, David; Slagboom, P Eline; Snieder, Harold; Spector, Tim D; Strauch, Konstantin; Stumvoll, Michael; Tuomilehto, Jaakko; Uusitupa, Matti; van der Harst, Pim; Völzke, Henry; Walker, Mark; Wareham, Nicholas J; Watkins, Hugh; Wichmann, H-Erich; Wilson, James F; Zanen, Pieter; Deloukas, Panos; Heid, Iris M; Lindgren, Cecilia M; Mohlke, Karen L; Speliotes, Elizabeth K; Thorsteinsdottir, Unnur; Barroso, Inês; Fox, Caroline S; North, Kari E; Strachan, David P; Beckmann, Jacques S.; Berndt, Sonja I; Boehnke, Michael; Borecki, Ingrid B; McCarthy, Mark I; Metspalu, Andres; Stefansson, Kari; Uitterlinden, André G; van Duijn, Cornelia M; Franke, Lude; Willer, Cristen J; Price, Alkes L.; Lettre, Guillaume; Loos, Ruth JF; Weedon, Michael N; Ingelsson, Erik; O’Connell, Jeffrey R; Abecasis, Goncalo R; Chasman, Daniel I; Goddard, Michael E

    2014-01-01

    Using genome-wide data from 253,288 individuals, we identified 697 variants at genome-wide significance that together explain one-fifth of heritability for adult height. By testing different numbers of variants in independent studies, we show that the most strongly associated ~2,000, ~3,700 and ~9,500 SNPs explained ~21%, ~24% and ~29% of phenotypic variance. Furthermore, all common variants together captured the majority (60%) of heritability. The 697 variants clustered in 423 loci enriched for genes, pathways, and tissue-types known to be involved in growth and together implicated genes and pathways not highlighted in earlier efforts, such as signaling by fibroblast growth factors, WNT/beta-catenin, and chondroitin sulfate-related genes. We identified several genes and pathways not previously connected with human skeletal growth, including mTOR, osteoglycin and binding of hyaluronic acid. Our results indicate a genetic architecture for human height that is characterized by a very large but finite number (thousands) of causal variants. PMID:25282103

  18. Inherited human sex reversal due to impaired nucleocytoplasmic trafficking of SRY defines a male transcriptional threshold.

    Science.gov (United States)

    Chen, Yen-Shan; Racca, Joseph D; Phillips, Nelson B; Weiss, Michael A

    2013-09-17

    Human testis determination is initiated by SRY (sex determining region on Y chromosome). Mutations in SRY cause gonadal dysgenesis with female somatic phenotype. Two subtle variants (V60L and I90M in the high-mobility group box) define inherited alleles shared by an XY sterile daughter and fertile father. Whereas specific DNA binding and bending are unaffected in a rat embryonic pre-Sertoli cell line, the variants exhibited selective defects in nucleocytoplasmic shuttling due to impaired nuclear import (V60L; mediated by Exportin-4) or export (I90M; mediated by chromosome region maintenance 1). Decreased shuttling limits nuclear accumulation of phosphorylated (activated) SRY, in turn reducing occupancy of DNA sites regulating Sertoli-cell differentiation [the testis-specific SRY-box 9 (Sox9) enhancer]. Despite distinct patterns of biochemical and cell-biological perturbations, V60L and I90M each attenuated Sox9 expression in transient transfection assays by twofold. Such attenuation was also observed in studies of V60A, a clinical variant associated with ovotestes and hence ambiguity between divergent cell fates. This shared twofold threshold is reminiscent of autosomal syndromes of transcription-factor haploinsufficiency, including XY sex reversal associated with mutations in SOX9. Our results demonstrate that nucleocytoplasmic shuttling of SRY is necessary for robust initiation of testicular development. Although also characteristic of ungulate orthologs, such shuttling is not conserved among rodents wherein impaired nuclear export of the high-mobility group box and import-dependent phosphorylation are compensated by a microsatellite-associated transcriptional activation domain. Human sex reversal due to subtle defects in the nucleocytoplasmic shuttling of SRY suggests that its transcriptional activity lies near the edge of developmental ambiguity. PMID:24003159

  19. Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming

    Science.gov (United States)

    Moreau, Thomas; Evans, Amanda L.; Vasquez, Louella; Tijssen, Marloes R.; Yan, Ying; Trotter, Matthew W.; Howard, Daniel; Colzani, Maria; Arumugam, Meera; Wu, Wing Han; Dalby, Amanda; Lampela, Riina; Bouet, Guenaelle; Hobbs, Catherine M.; Pask, Dean C.; Payne, Holly; Ponomaryov, Tatyana; Brill, Alexander; Soranzo, Nicole; Ouwehand, Willem H.; Pedersen, Roger A.; Ghevaert, Cedric

    2016-01-01

    The production of megakaryocytes (MKs)—the precursors of blood platelets—from human pluripotent stem cells (hPSCs) offers exciting clinical opportunities for transfusion medicine. Here we describe an original approach for the large-scale generation of MKs in chemically defined conditions using a forward programming strategy relying on the concurrent exogenous expression of three transcription factors: GATA1, FLI1 and TAL1. The forward programmed MKs proliferate and differentiate in culture for several months with MK purity over 90% reaching up to 2 × 105 mature MKs per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as 1 million starting hPSCs. The high cell purity and yield achieved by MK forward programming, combined with efficient cryopreservation and good manufacturing practice (GMP)-compatible culture, make this approach eminently suitable to both in vitro production of platelets for transfusion and basic research in MK and platelet biology. PMID:27052461

  20. Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming.

    Science.gov (United States)

    Moreau, Thomas; Evans, Amanda L; Vasquez, Louella; Tijssen, Marloes R; Yan, Ying; Trotter, Matthew W; Howard, Daniel; Colzani, Maria; Arumugam, Meera; Wu, Wing Han; Dalby, Amanda; Lampela, Riina; Bouet, Guenaelle; Hobbs, Catherine M; Pask, Dean C; Payne, Holly; Ponomaryov, Tatyana; Brill, Alexander; Soranzo, Nicole; Ouwehand, Willem H; Pedersen, Roger A; Ghevaert, Cedric

    2016-01-01

    The production of megakaryocytes (MKs)--the precursors of blood platelets--from human pluripotent stem cells (hPSCs) offers exciting clinical opportunities for transfusion medicine. Here we describe an original approach for the large-scale generation of MKs in chemically defined conditions using a forward programming strategy relying on the concurrent exogenous expression of three transcription factors: GATA1, FLI1 and TAL1. The forward programmed MKs proliferate and differentiate in culture for several months with MK purity over 90% reaching up to 2 × 10(5) mature MKs per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as 1 million starting hPSCs. The high cell purity and yield achieved by MK forward programming, combined with efficient cryopreservation and good manufacturing practice (GMP)-compatible culture, make this approach eminently suitable to both in vitro production of platelets for transfusion and basic research in MK and platelet biology. PMID:27052461

  1. Human serum-derived protein removes the need for coating in defined human pluripotent stem cell culture

    Science.gov (United States)

    Pijuan-Galitó, Sara; Tamm, Christoffer; Schuster, Jens; Sobol, Maria; Forsberg, Lars; Merry, Catherine L. R.; Annerén, Cecilia

    2016-01-01

    Reliable, scalable and time-efficient culture methods are required to fully realize the clinical and industrial applications of human pluripotent stem (hPS) cells. Here we present a completely defined, xeno-free medium that supports long-term propagation of hPS cells on uncoated tissue culture plastic. The medium consists of the Essential 8 (E8) formulation supplemented with inter-α-inhibitor (IαI), a human serum-derived protein, recently demonstrated to activate key pluripotency pathways in mouse PS cells. IαI efficiently induces attachment and long-term growth of both embryonic and induced hPS cell lines when added as a soluble protein to the medium at seeding. IαI supplementation efficiently supports adaptation of feeder-dependent hPS cells to xeno-free conditions, clonal growth as well as single-cell survival in the absence of Rho-associated kinase inhibitor (ROCKi). This time-efficient and simplified culture method paves the way for large-scale, high-throughput hPS cell culture, and will be valuable for both basic research and commercial applications. PMID:27405751

  2. Human Lsg1 defines a family of essential GTPases that correlates with the evolution of compartmentalization

    Directory of Open Access Journals (Sweden)

    Scheffzek Klaus

    2005-10-01

    Full Text Available Abstract Background Compartmentalization is a key feature of eukaryotic cells, but its evolution remains poorly understood. GTPases are the oldest enzymes that use nucleotides as substrates and they participate in a wide range of cellular processes. Therefore, they are ideal tools for comparative genomic studies aimed at understanding how aspects of biological complexity such as cellular compartmentalization evolved. Results We describe the identification and characterization of a unique family of circularly permuted GTPases represented by the human orthologue of yeast Lsg1p. We placed the members of this family in the phylogenetic context of the YlqF Related GTPase (YRG family, which are present in Eukarya, Bacteria and Archea and include the stem cell regulator Nucleostemin. To extend the computational analysis, we showed that hLsg1 is an essential GTPase predominantly located in the endoplasmic reticulum and, in some cells, in Cajal bodies in the nucleus. Comparison of localization and siRNA datasets suggests that all members of the family are essential GTPases that have increased in number as the compartmentalization of the eukaryotic cell and the ribosome biogenesis pathway have evolved. Conclusion We propose a scenario, consistent with our data, for the evolution of this family: cytoplasmic components were first acquired, followed by nuclear components, and finally the mitochondrial and chloroplast elements were derived from different bacterial species, in parallel with the formation of the nucleolus and the specialization of nuclear components.

  3. Polymorphic Expression of a Human Superficial Bladder Tumor Antigen Defined by Mouse Monoclonal Antibodies

    Science.gov (United States)

    Fradet, Yves; Islam, Nazrul; Boucher, Lucie; Parent-Vaugeois, Carmen; Tardif, Marc

    1987-10-01

    Three mouse monoclonal antibodies (mAbs), which define a highly restricted antigen, were obtained by simultaneous immunizations with superficial papillary bladder tumor cells and mouse polyclonal serum against normal urothelium. The antigen was detected by the avidin/biotin/peroxidase method in 30/44 superficial bladder tumors (68%) but in only 4/27 infiltrating urothelial cancers (with much less intensity). No normal adult or fetal tissues tested expressed the antigen, including normal urothelium from 40 individuals, 13 of whom had a bladder tumor positive for the antigen. Only 1 of 45 nonbladder tumors showed some reactivity with one of the three mAbs. Serological tests on a large panel of human cancer cell lines and normal cultured cells were negative. The antigen is highly stable and well preserved on paraffin-embedded tissues. Electrophoretic transfer blot experiments with fresh tumor extracts showed that all three mAbs react with a determinant on a component of 300,000 Mr (pI 9.5) and 62,000 Mr (pI 6.5). The antigen shows polymorphic expression at the cellular level on tissue sections and also at a molecular level on immunoblots where the two bands are differentially detected on extracts of a series of tumors but are not visualized on normal urothelium extracts. The characteristics of this antigenic system suggest that it may provide some insights about the biology of bladder cancer. Specific detection of the antigen on 70% of superficial bladder tumors with normal cytology may be useful for their diagnosis and follow-up.

  4. Derivation of transgene-free human induced pluripotent stem cells from human peripheral T cells in defined culture conditions.

    Directory of Open Access Journals (Sweden)

    Yoshikazu Kishino

    Full Text Available Recently, induced pluripotent stem cells (iPSCs were established as promising cell sources for revolutionary regenerative therapies. The initial culture system used for iPSC generation needed fetal calf serum in the culture medium and mouse embryonic fibroblast as a feeder layer, both of which could possibly transfer unknown exogenous antigens and pathogens into the iPSC population. Therefore, the development of culture systems designed to minimize such potential risks has become increasingly vital for future applications of iPSCs for clinical use. On another front, although donor cell types for generating iPSCs are wide-ranging, T cells have attracted attention as unique cell sources for iPSCs generation because T cell-derived iPSCs (TiPSCs have a unique monoclonal T cell receptor genomic rearrangement that enables their differentiation into antigen-specific T cells, which can be applied to novel immunotherapies. In the present study, we generated transgene-free human TiPSCs using a combination of activated human T cells and Sendai virus under defined culture conditions. These TiPSCs expressed pluripotent markers by quantitative PCR and immunostaining, had a normal karyotype, and were capable of differentiating into cells from all three germ layers. This method of TiPSCs generation is more suitable for the therapeutic application of iPSC technology because it lowers the risks associated with the presence of undefined, animal-derived feeder cells and serum. Therefore this work will lead to establishment of safer iPSCs and extended clinical application.

  5. Proteomics Analysis of HRAS-Mediated Oncogenic Transformation in a Genetically Defined Human Ovarian Cancer Model*

    OpenAIRE

    Young, Travis; Mei, Fang; Liu, Jinsong; Bast, Robert C.; Kurosky, Alexander; Cheng, Xiaodong

    2005-01-01

    Ras is a small GTP binding protein mutated in approximately 30% human cancer. Despite its important role in the initiation and progression of human cancer, the underlying mechamism of RAS-induced human epithelial transformation remains elusive. In this study, we probe the cellular and molecular mechanisms of RAS-mediated transformation, by profiling two human ovarian epithelial cell lines. One cell line was immortalized with SV40 T/t antigens and the human catalytic subunit of telomerase (T29...

  6. MicroRNAs define distinct human neuroblastoma cell phenotypes and regulate their differentiation and tumorigenicity

    International Nuclear Information System (INIS)

    Neuroblastoma (NB) is the most common extracranial solid tumor in children. NB tumors and derived cell lines are phenotypically heterogeneous. Cell lines are classified by phenotype, each having distinct differentiation and tumorigenic properties. The neuroblastic phenotype is tumorigenic, has neuronal features and includes stem cells (I-cells) and neuronal cells (N-cells). The non-neuronal phenotype (S-cell) comprises cells that are non-tumorigenic with features of glial/smooth muscle precursor cells. This study identified miRNAs associated with each distinct cell phenotypes and investigated their role in regulating associated differentiation and tumorigenic properties. A miRNA microarray was performed on the three cell phenotypes and expression verified by qRT-PCR. miRNAs specific for certain cell phenotypes were modulated using miRNA inhibitors or stable transfection. Neuronal differentiation was induced by RA; non-neuronal differentiation by BrdU. Changes in tumorigenicity were assayed by soft agar colony forming ability. N-myc binding to miR-375 promoter was assayed by chromatin-immunoprecipitation. Unsupervised hierarchical clustering of miRNA microarray data segregated neuroblastic and non-neuronal cell lines and showed that specific miRNAs define each phenotype. qRT-PCR validation confirmed that increased levels of miR-21, miR-221 and miR-335 are associated with the non-neuronal phenotype, whereas increased levels of miR-124 and miR-375 are exclusive to neuroblastic cells. Downregulation of miR-335 in non-neuronal cells modulates expression levels of HAND1 and JAG1, known modulators of neuronal differentiation. Overexpression of miR-124 in stem cells induces terminal neuronal differentiation with reduced malignancy. Expression of miR-375 is exclusive for N-myc-expressing neuroblastic cells and is regulated by N-myc. Moreover, miR-375 downregulates expression of the neuronal-specific RNA binding protein HuD. Thus, miRNAs define distinct NB cell phenotypes

  7. Clonal characterization of rat muscle satellite cells: proliferation, metabolism and differentiation define an intrinsic heterogeneity.

    Directory of Open Access Journals (Sweden)

    Carlo A Rossi

    Full Text Available Satellite cells (SCs represent a distinct lineage of myogenic progenitors responsible for the postnatal growth, repair and maintenance of skeletal muscle. Distinguished on the basis of their unique position in mature skeletal muscle, SCs were considered unipotent stem cells with the ability of generating a unique specialized phenotype. Subsequently, it was demonstrated in mice that opposite differentiation towards osteogenic and adipogenic pathways was also possible. Even though the pool of SCs is accepted as the major, and possibly the only, source of myonuclei in postnatal muscle, it is likely that SCs are not all multipotent stem cells and evidences for diversities within the myogenic compartment have been described both in vitro and in vivo. Here, by isolating single fibers from rat flexor digitorum brevis (FDB muscle we were able to identify and clonally characterize two main subpopulations of SCs: the low proliferative clones (LPC present in major proportion (approximately 75% and the high proliferative clones (HPC, present instead in minor amount (approximately 25%. LPC spontaneously generate myotubes whilst HPC differentiate into adipocytes even though they may skip the adipogenic program if co-cultured with LPC. LPC and HPC differ also for mitochondrial membrane potential (DeltaPsi(m, ATP balance and Reactive Oxygen Species (ROS generation underlying diversities in metabolism that precede differentiation. Notably, SCs heterogeneity is retained in vivo. SCs may therefore be comprised of two distinct, though not irreversibly committed, populations of cells distinguishable for prominent differences in basal biological features such as proliferation, metabolism and differentiation. By these means, novel insights on SCs heterogeneity are provided and evidences for biological readouts potentially relevant for diagnostic purposes described.

  8. Defining Advancement Career Paths and Succession Plans: Critical Human Capital Retention Strategies for High-Performing Advancement Divisions

    Science.gov (United States)

    Croteau, Jon Derek; Wolk, Holly Gordon

    2010-01-01

    There are many factors that can influence whether a highly talented staff member will build a career within an institution or use it as a stepping stone. This article defines and explores the notions of developing career paths and succession planning and why they are critical human capital investment strategies in retaining the highest performers…

  9. DEFINING ASPECTS OF HUMAN RESOURCE MANAGEMENT STRATEGY WITHIN THE GENERAL STRATEGY OF THE MODERN ORGANIZATION

    OpenAIRE

    Emanoil MUSCALU; Silvana Nicoleta MUNTEAN

    2013-01-01

    The field of human resources requires the presence and action of several categories of persons and managerial structures interested in the quality of human resources and the activities developed by them. Besides managers and employees there are also the shareholders, the unions, the customers, the different national or local agencies, the local community, etc., with major interests regarding decisions in the human resources area. In order to harmonize their activities and achieve an optimal p...

  10. Human colonic goblet cells. Demonstration of distinct subpopulations defined by mucin-specific monoclonal antibodies.

    OpenAIRE

    Podolsky, D K; Fournier, D A; Lynch, K E

    1986-01-01

    We studied glycoprotein content of human colonic goblet cells, using a library of monoclonal antibodies (MAbs) directed against purified human colonic mucin (HCM). Using indirect immunofluorescence (IIF), we found that 17 of 23 anti-HCM MAbs stained some or all goblet cells of normal human colonic mucosa. We observed a variety of cellular staining patterns, including (a) diffuse (homogeneous) staining of intracellular mucin, (b) speckled (inhomogeneous) staining of mucin droplets, (c) periphe...

  11. CD146/MCAM defines functionality of human bone marrow stromal stem cell populations

    DEFF Research Database (Denmark)

    Harkness, Linda; Zaher, Walid; Ditzel, Nicholas;

    2016-01-01

    significantly increased migration ability as demonstrated by bioluminescence imaging. Conclusion Our studies demonstrate that CD146 defines a subpopulation of hMSCs capable of bone formation and in vivo trans-endothelial migration and thus represents a population of hMSCs suitable for use in clinical protocols...

  12. NIH Human Microbiome Project defines normal bacterial makeup of the body

    Science.gov (United States)

    Microbes inhabit just about every part of the human body, living on the skin, in the gut, and up the nose. Sometimes they cause sickness, but most of the time, microorganisms live in harmony with their human hosts, providing vital functions essential for

  13. DEFINING ASPECTS OF HUMAN RESOURCE MANAGEMENT STRATEGY WITHIN THE GENERAL STRATEGY OF THE MODERN ORGANIZATION

    Directory of Open Access Journals (Sweden)

    Emanoil MUSCALU

    2013-07-01

    Full Text Available The field of human resources requires the presence and action of several categories of persons and managerial structures interested in the quality of human resources and the activities developed by them. Besides managers and employees there are also the shareholders, the unions, the customers, the different national or local agencies, the local community, etc., with major interests regarding decisions in the human resources area. In order to harmonize their activities and achieve an optimal perspective within the evolution of Human Resource Management, special attention is paid to the strategy of human resources management. According to many specialists, strategies in the field of Human Resource Management show, in the first place, that personnel function adopts a broader perspective and a more dynamic view of human resources, which enables its full integration within the other functions of the organization. In the second place, strategies in the field of Human Resource Management designate the assembly of long term objectives concerning human resources, the main modalities of achieving them and the necessary resources which guarantee that the organization’s structure, value and culture as well as the utilization of its personnel contribute to fulfilling the general objectives of the organization. Therefore, we approached in this paper the problems of grounding and elaborating the Human Resource Management strategy, and we outlined their specific traits, as these are necessary aspects in order to emphasise at the end of our paper the correlation between the strategy in the field of Human Resource Management and the general strategy of the organization. Taking into account specialists and practitioners’ increased interest in knowing, substantiating and implementing strategies in the area of Human Resource Management, we consider that the aspects presented in this paper are modern issues and a starting pointing in solving the great problems of

  14. The relationship between human T-lymphocyte subsets defined by monoclonal antibodies and by avidity differences to sheep erythrocytes

    DEFF Research Database (Denmark)

    Hokland, P; Hokland, M; Heron, I

    1982-01-01

    By E-rosette sedimentation procedures and fluorescence activated cell sorting of monoclonal antibody-coated T lymphocytes, we have investigated the relationship between the human T lymphocyte subsets defined by the monoclonal antibodies against the T4 and T8 antigens and those defined by avidity...... differences to sheep erythrocytes. Through a correlation was demonstrated between the T4+ (inducer) cells and the high avidity ("active") T cells and between the T8+ (suppressor) cells and low avidity T cells, these subsets were far from identical, and it is concluded that the application of monoclonal...

  15. Chemically Defined and Xeno-Free Cryopreservation of Human Adipose-Derived Stem Cells.

    Science.gov (United States)

    López, Melany; Bollag, Roni J; Yu, Jack C; Isales, Carlos M; Eroglu, Ali

    2016-01-01

    The stromal compartment of adipose tissue harbors multipotent cells known as adipose-derived stem cells (ASCs). These cells can differentiate into various lineages including osteogenic, chrondrogenic, adipogenic, and neurogenic; this cellular fraction may be easily obtained in large quantities through a clinically safe liposuction procedure. Therefore, ASCs offer exceptional opportunities for tissue engineering and regenerative medicine. However, current practices involving ASCs typically use fetal bovine serum (FBS)-based cryopreservation solutions that are associated with risks of immunological reactions and of transmitting infectious diseases and prions. To realize clinical applications of ASCs, serum- and xeno-free defined cryopreservation methods are needed. To this end, an animal product-free chemically defined cryopreservation medium was formulated by adding two antioxidants (reduced glutathione and ascorbic acid 2-phosphate), two polymers (PVA and ficoll), two permeating cryoprotectants (ethylene glycol and dimethylsulfoxide), a disaccharide (trehalose), and a calcium chelator (EGTA) to HEPES-buffered DMEM/F12. To limit the number of experimental groups, the concentration of trehalose, both polymers, and EGTA was fixed while the presence of the permeating CPAs and antioxidants was varied. ASCs suspended either in different versions of the defined medium or in the conventional undefined cryopreservation medium (10% dimethylsulfoxide+10% DMEM/F12+80% serum) were cooled to -70°C at 1°C/min before being plunged into liquid nitrogen. Samples were thawed either in air or in a water bath at 37°C. The presence of antioxidants along with 3.5% concentration of each penetrating cryoprotectant improved the freezing outcome to the level of the undefined cryopreservation medium, but the plating efficiency was still lower than that of unfrozen controls. Subsequently, increasing the concentration of both permeating cryoprotectants to 5% further improved the plating

  16. Chemically Defined and Xeno-Free Cryopreservation of Human Adipose-Derived Stem Cells

    Science.gov (United States)

    López, Melany; Bollag, Roni J.; Yu, Jack C.; Isales, Carlos M.; Eroglu, Ali

    2016-01-01

    The stromal compartment of adipose tissue harbors multipotent cells known as adipose-derived stem cells (ASCs). These cells can differentiate into various lineages including osteogenic, chrondrogenic, adipogenic, and neurogenic; this cellular fraction may be easily obtained in large quantities through a clinically safe liposuction procedure. Therefore, ASCs offer exceptional opportunities for tissue engineering and regenerative medicine. However, current practices involving ASCs typically use fetal bovine serum (FBS)-based cryopreservation solutions that are associated with risks of immunological reactions and of transmitting infectious diseases and prions. To realize clinical applications of ASCs, serum- and xeno-free defined cryopreservation methods are needed. To this end, an animal product-free chemically defined cryopreservation medium was formulated by adding two antioxidants (reduced glutathione and ascorbic acid 2-phosphate), two polymers (PVA and ficoll), two permeating cryoprotectants (ethylene glycol and dimethylsulfoxide), a disaccharide (trehalose), and a calcium chelator (EGTA) to HEPES-buffered DMEM/F12. To limit the number of experimental groups, the concentration of trehalose, both polymers, and EGTA was fixed while the presence of the permeating CPAs and antioxidants was varied. ASCs suspended either in different versions of the defined medium or in the conventional undefined cryopreservation medium (10% dimethylsulfoxide+10% DMEM/F12+80% serum) were cooled to -70°C at 1°C/min before being plunged into liquid nitrogen. Samples were thawed either in air or in a water bath at 37°C. The presence of antioxidants along with 3.5% concentration of each penetrating cryoprotectant improved the freezing outcome to the level of the undefined cryopreservation medium, but the plating efficiency was still lower than that of unfrozen controls. Subsequently, increasing the concentration of both permeating cryoprotectants to 5% further improved the plating

  17. Defining the genomic signature of totipotency and pluripotency during early human development.

    Directory of Open Access Journals (Sweden)

    Amparo Galan

    Full Text Available The genetic mechanisms governing human pre-implantation embryo development and the in vitro counterparts, human embryonic stem cells (hESCs, still remain incomplete. Previous global genome studies demonstrated that totipotent blastomeres from day-3 human embryos and pluripotent inner cell masses (ICMs from blastocysts, display unique and differing transcriptomes. Nevertheless, comparative gene expression analysis has revealed that no significant differences exist between hESCs derived from blastomeres versus those obtained from ICMs, suggesting that pluripotent hESCs involve a new developmental progression. To understand early human stages evolution, we developed an undifferentiation network signature (UNS and applied it to a differential gene expression profile between single blastomeres from day-3 embryos, ICMs and hESCs. This allowed us to establish a unique signature composed of highly interconnected genes characteristic of totipotency (61 genes, in vivo pluripotency (20 genes, and in vitro pluripotency (107 genes, and which are also proprietary according to functional analysis. This systems biology approach has led to an improved understanding of the molecular and signaling processes governing human pre-implantation embryo development, as well as enabling us to comprehend how hESCs might adapt to in vitro culture conditions.

  18. Direct conversion of mouse and human fibroblasts to functional melanocytes by defined factors

    Science.gov (United States)

    Yang, Ruifeng; Zheng, Ying; Li, Ling; Liu, Shujing; Burrows, Michelle; Wei, Zhi; Nace, Arben; Herlyn, Meenhard; Cui, Rutao; Guo, Wei; Cotsarelis, George; Xu, Xiaowei

    2015-01-01

    Direct reprogramming provides a fundamentally new approach for the generation of patient-specific cells. Here, by screening a pool of candidate transcription factors, we identify that a combination of three factors, MITF, SOX10 and PAX3, directly converts mouse and human fibroblasts to functional melanocytes. Induced melanocytes (iMels) activate melanocyte-specific networks, express components of pigment production and delivery system, and produce melanosomes. Human iMels properly integrate into the dermal-epidermal junction, and produce and deliver melanin pigment to surrounding keratinocytes in a 3D organotypic skin reconstruct. Human iMels generate pigmented epidermis and hair follicles in skin reconstitution assays in vivo. The generation of iMels has important implications for studies of melanocyte lineage commitment, pigmentation disorders and cell replacement therapies. PMID:25510211

  19. Direct conversion of mouse and human fibroblasts to functional melanocytes by defined factors.

    Science.gov (United States)

    Yang, Ruifeng; Zheng, Ying; Li, Ling; Liu, Shujing; Burrows, Michelle; Wei, Zhi; Nace, Arben; Herlyn, Meenhard; Cui, Rutao; Guo, Wei; Cotsarelis, George; Xu, Xiaowei

    2014-01-01

    Direct reprogramming provides a fundamentally new approach for the generation of patient-specific cells. Here, by screening a pool of candidate transcription factors, we identify that a combination of the three factors, MITF, SOX10 and PAX3, directly converts mouse and human fibroblasts to functional melanocytes. Induced melanocytes (iMels) activate melanocyte-specific networks, express components of pigment production and delivery system and produce melanosomes. Human iMels properly integrate into the dermal-epidermal junction and produce and deliver melanin pigment to surrounding keratinocytes in a 3D organotypic skin reconstruct. Human iMels generate pigmented epidermis and hair follicles in skin reconstitution assays in vivo. The generation of iMels has important implications for studies of melanocyte lineage commitment, pigmentation disorders and cell replacement therapies. PMID:25510211

  20. Chemically Defined and Xeno-Free Cryopreservation of Human Adipose-Derived Stem Cells.

    Directory of Open Access Journals (Sweden)

    Melany López

    Full Text Available The stromal compartment of adipose tissue harbors multipotent cells known as adipose-derived stem cells (ASCs. These cells can differentiate into various lineages including osteogenic, chrondrogenic, adipogenic, and neurogenic; this cellular fraction may be easily obtained in large quantities through a clinically safe liposuction procedure. Therefore, ASCs offer exceptional opportunities for tissue engineering and regenerative medicine. However, current practices involving ASCs typically use fetal bovine serum (FBS-based cryopreservation solutions that are associated with risks of immunological reactions and of transmitting infectious diseases and prions. To realize clinical applications of ASCs, serum- and xeno-free defined cryopreservation methods are needed. To this end, an animal product-free chemically defined cryopreservation medium was formulated by adding two antioxidants (reduced glutathione and ascorbic acid 2-phosphate, two polymers (PVA and ficoll, two permeating cryoprotectants (ethylene glycol and dimethylsulfoxide, a disaccharide (trehalose, and a calcium chelator (EGTA to HEPES-buffered DMEM/F12. To limit the number of experimental groups, the concentration of trehalose, both polymers, and EGTA was fixed while the presence of the permeating CPAs and antioxidants was varied. ASCs suspended either in different versions of the defined medium or in the conventional undefined cryopreservation medium (10% dimethylsulfoxide+10% DMEM/F12+80% serum were cooled to -70°C at 1°C/min before being plunged into liquid nitrogen. Samples were thawed either in air or in a water bath at 37°C. The presence of antioxidants along with 3.5% concentration of each penetrating cryoprotectant improved the freezing outcome to the level of the undefined cryopreservation medium, but the plating efficiency was still lower than that of unfrozen controls. Subsequently, increasing the concentration of both permeating cryoprotectants to 5% further improved the

  1. Synthesis and Characterization of Well-Defined Regular Star Polyisoprenes with 3, 4, 6 and 8 Arms

    KAUST Repository

    Ratkanthwar, Kedar R.

    2013-01-01

    Three series of regular well-defined star polyisoprenes (PIs) with 3, 4 and 6 arms (each series: same arm molecular weight) have been synthesized by anionic polymerization high vacuum techniques and chlorosilane chemistry. In addition, three linear PIs with practically the double arm molecular weight of the corresponding series (2-arm star PIs) have been synthesized, as well as one 8-arm star PI. All intermediate (arms) and final (stars) products have been characterized by size exclusion chromatography (SEC), SEC-multi-angle laser light scattering (SEC-MALLS) and nuclear magnetic resonance (NMR) spectroscopy. The Tg of the star PIs was determined by differential scanning calorimetry. These model polymeric materials are essential for polymer physics and polymer physical chemistry in order to establish the structure/property relationships.

  2. Generation of primitive neural stem cells from human fibroblasts using a defined set of factors

    Directory of Open Access Journals (Sweden)

    Takumi Miura

    2015-11-01

    Full Text Available In mice, leukemia inhibitory factor (LIF-dependent primitive neural stem cells (NSCs have a higher neurogenic potential than bFGF-dependent definitive NSCs. Therefore, expandable primitive NSCs are required for research and for the development of therapeutic strategies for neurological diseases. There is a dearth of suitable techniques for the generation of human long-term expandable primitive NSCs. Here, we have described a method for the conversion of human fibroblasts to LIF-dependent primitive NSCs using a strategy based on techniques for the generation of induced pluripotent stem cells (iPSCs. These LIF-dependent induced NSCs (LD-iNSCs can be expanded for >100 passages. Long-term cultured LD-iNSCs demonstrated multipotent neural differentiation potential and could generate motor neurons and dopaminergic neurons, as well as astrocytes and oligodendrocytes, indicating a high level of plasticity. Furthermore, LD-iNSCs easily reverted to human iPSCs, indicating that LD-iNSCs are in an intermediate iPSC state. This method may facilitate the generation of patient-specific human neurons for studies and treatment of neurodegenerative diseases.

  3. Experimental Approaches for Defining Functional Roles of Microbes in the Human Gut

    DEFF Research Database (Denmark)

    Dantas, Gautam; Sommer, Morten; Degnan, Patrick H.;

    2013-01-01

    The complex and intimate relationship between humans and their gut microbial communities is becoming less obscure, due in part to large-scale gut microbial genome-sequencing projects and culture-independent surveys of the composition and gene content of these communities.These studies build upon,...

  4. Effects of selenium, calcium and calcium ionophore on human oocytes in vitro maturation in a chemically defined medium

    OpenAIRE

    Hessam Roustaei; Mohammad Hasan Fallahian; Fatemeh Peyghambari; Raheleh Aram; Mohammad Ali Sabbaghi; Elham Saboori; Mehri Makki; Abbas Ahmadi

    2012-01-01

    Background: In vitro maturation (IVM) of human oocytes is an emerging procedure quickly incorporated into the world of assisted reproductive technologies. As an effective method of in vitro maturation, several studies have reported the critical role of differentions on activating the complex process involved in both gamete maturation and fertilization. Objective: In this study, we supplemented a chemically defined medium with different combinations of selenium, calcium and calcium ionophore c...

  5. Defining the molecular pathologies in cloaca malformation: similarities between mouse and human

    Directory of Open Access Journals (Sweden)

    Laura A. Runck

    2014-04-01

    Full Text Available Anorectal malformations are congenital anomalies that form a spectrum of disorders, from the most benign type with excellent functional prognosis, to very complex, such as cloaca malformation in females in which the rectum, vagina and urethra fail to develop separately and instead drain via a single common channel into the perineum. The severity of this phenotype suggests that the defect occurs in the early stages of embryonic development of the organs derived from the cloaca. Owing to the inability to directly investigate human embryonic cloaca development, current research has relied on the use of mouse models of anorectal malformations. However, even studies of mouse embryos lack analysis of the earliest stages of cloaca patterning and morphogenesis. Here we compared human and mouse cloaca development and retrospectively identified that early mis-patterning of the embryonic cloaca might underlie the most severe forms of anorectal malformation in humans. In mouse, we identified that defective sonic hedgehog (Shh signaling results in early dorsal-ventral epithelial abnormalities prior to the reported defects in septation. This is manifested by the absence of Sox2 and aberrant expression of keratins in the embryonic cloaca of Shh knockout mice. Shh knockout embryos additionally develop a hypervascular stroma, which is defective in BMP signaling. These epithelial and stromal defects persist later, creating an indeterminate epithelium with molecular alterations in the common channel. We then used these animals to perform a broad comparison with patients with mild-to-severe forms of anorectal malformations including cloaca malformation. We found striking parallels with the Shh mouse model, including nearly identical defective molecular identity of the epithelium and surrounding stroma. Our work strongly suggests that early embryonic cloacal epithelial differentiation defects might be the underlying cause of severe forms of anorectal malformations

  6. Defining the protein interaction network of human malaria parasite Plasmodium falciparum

    KAUST Repository

    Ramaprasad, Abhinay

    2012-02-01

    Malaria, caused by the protozoan parasite Plasmodium falciparum, affects around 225. million people yearly and a huge international effort is directed towards combating this grave threat to world health and economic development. Considerable advances have been made in malaria research triggered by the sequencing of its genome in 2002, followed by several high-throughput studies defining the malaria transcriptome and proteome. A protein-protein interaction (PPI) network seeks to trace the dynamic interactions between proteins, thereby elucidating their local and global functional relationships. Experimentally derived PPI network from high-throughput methods such as yeast two hybrid (Y2H) screens are inherently noisy, but combining these independent datasets by computational methods tends to give a greater accuracy and coverage. This review aims to discuss the computational approaches used till date to construct a malaria protein interaction network and to catalog the functional predictions and biological inferences made from analysis of the PPI network. © 2011 Elsevier Inc.

  7. CD146/MCAM defines functionality of human bone marrow stromal stem cell populations

    DEFF Research Database (Denmark)

    Harkness, Linda; Zaher, Walid; Ditzel, Nicholas;

    2016-01-01

    BACKGROUND: Identification of surface markers for prospective isolation of functionally homogenous populations of human skeletal (stromal, mesenchymal) stem cells (hMSCs) is highly relevant for cell therapy protocols. Thus, we examined the possible use of CD146 to subtype a heterogeneous h......MSC population. METHODS: Using flow cytometry and cell sorting, we isolated two distinct hMSC-CD146(+) and hMSC-CD146(-) cell populations from the telomerized human bone marrow-derived stromal cell line (hMSC-TERT). Cells were examined for differences in their size, shape and texture by using high......-content analysis and additionally for their ability to differentiate toward osteogenesis in vitro and form bone in vivo, and their migrational ability in vivo and in vitro was investigated. RESULTS: In vitro, the two cell populations exhibited similar growth rate and differentiation capacity to osteoblasts...

  8. Defining cell-type specificity at the transcriptional level in human disease

    OpenAIRE

    Ju, Wenjun; Greene, Casey S; Eichinger, Felix; Nair, Viji; Hodgin, Jeffrey B.; Bitzer, Markus; Lee, Young-Suk; Zhu, Qian; Kehata, Masami; Li, Min; Jiang, Song; Rastaldi, Maria Pia; Cohen, Clemens D; Troyanskaya, Olga G.; Kretzler, Matthias

    2013-01-01

    Cell-lineage–specific transcripts are essential for differentiated tissue function, implicated in hereditary organ failure, and mediate acquired chronic diseases. However, experimental identification of cell-lineage–specific genes in a genome-scale manner is infeasible for most solid human tissues. We developed the first genome-scale method to identify genes with cell-lineage–specific expression, even in lineages not separable by experimental microdissection. Our machine-learning–based approa...

  9. Why do humans keep animals? Does the answer help to define standards for organic animal husbandry?

    OpenAIRE

    Rahmann, Gerold (Ed.)

    2003-01-01

    Domestic livestock, such as farm animals and pets, are part of the cultural heritage of mankind. Animals have been used for food, services, non-food or religious purposes or just as a hobby for thousands of years. Man-made breeding has created livestock that are adapted for different purposes and functions. Today, thousand of breeds exist: adapted to the various conditions in human households. The discussion of animal welfare and the definition of standards for livestock keeping have to...

  10. Re-defining the Human: Triumphs and Tribulations of Homo xeroxiens

    Directory of Open Access Journals (Sweden)

    Munawar A. Anees

    1999-12-01

    Full Text Available In the endless human quest for understanding the nature of life, cloning represents a new fundamental paradigm. Making a major departure from the normative mode of reproduction, it forces a new division of genetic endowment. Consequently, biological identity and individuality come to acquire new meanings. The inherent instrumentalism of cloning thus advances the dependence of moral choices in society. Ultimately, it manifests itself in the trinity of instrumentalism, namely: objectification, reductionism, and determinism. This, more than anything else, is the epic of contingency of the episteme. A contingent episteme in turn assumes the role of a moral arbitrator. This paper argues that cloning is the most potent catalyst for the emergence of neo-Genesis. The technological reincarnation then is merely a function of the inevitable evolution of the technique. Legislative controls on the technique itself simply betray the underlying moral contingency. It is asserted that irrespective of these controls, eventually, cloned human beings would be a reality. The dilemma faced by us, therefore, is not the challenge of technique but judgement on the moral future of human society. Is the "new birth" also the birth of a new norm? An unfolding of a new world of identity, rights, responsibilities, and a worldview?

  11. Identification of copy number variants defining genomic differences among major human groups.

    Directory of Open Access Journals (Sweden)

    Lluís Armengol

    Full Text Available BACKGROUND: Understanding the genetic contribution to phenotype variation of human groups is necessary to elucidate differences in disease predisposition and response to pharmaceutical treatments in different human populations. METHODOLOGY/PRINCIPAL FINDINGS: We have investigated the genome-wide profile of structural variation on pooled samples from the three populations studied in the HapMap project by comparative genome hybridization (CGH in different array platforms. We have identified and experimentally validated 33 genomic loci that show significant copy number differences from one population to the other. Interestingly, we found an enrichment of genes related to environment adaptation (immune response, lipid metabolism and extracellular space within these regions and the study of expression data revealed that more than half of the copy number variants (CNVs translate into gene-expression differences among populations, suggesting that they could have functional consequences. In addition, the identification of single nucleotide polymorphisms (SNPs that are in linkage disequilibrium with the copy number alleles allowed us to detect evidences of population differentiation and recent selection at the nucleotide variation level. CONCLUSIONS: Overall, our results provide a comprehensive view of relevant copy number changes that might play a role in phenotypic differences among major human populations, and generate a list of interesting candidates for future studies.

  12. Defined plant extracts can protect human cells against combined xenobiotic effects

    Directory of Open Access Journals (Sweden)

    Clair Emilie

    2011-01-01

    Full Text Available Abstract Background Pollutants representative of common environmental contaminants induce intracellular toxicity in human cells, which is generally amplified in combinations. We wanted to test the common pathways of intoxication and detoxification in human embryonic and liver cell lines. We used various pollutants such as Roundup residues, Bisphenol-A and Atrazine, and five precise medicinal plant extracts called Circ1, Dig1, Dig2, Sp1, and Uro1 in order to understand whether specific molecular actions took place or not. Methods Kidney and liver are major detoxification organs. We have studied embryonic kidney and hepatic human cell lines E293 and HepG2. The intoxication was induced on the one hand by a formulation of one of the most common herbicides worldwide, Roundup 450 GT+ (glyphosate and specific adjuvants, and on the other hand by a mixture of Bisphenol-A and Atrazine, all found in surface waters, feed and food. The prevention and curative effects of plant extracts were also measured on mitochondrial succinate dehydrogenase activity, on the entry of radiolabelled glyphosate (in Roundup in cells, and on cytochromes P450 1A2 and 3A4 as well as glutathione-S-transferase. Results Clear toxicities of pollutants were observed on both cell lines at very low sub-agricultural dilutions. The prevention of such phenomena took place within 48 h with the plant extracts tested, with success rates ranging between 25-34% for the E293 intoxicated by Roundup, and surprisingly up to 71% for the HepG2. By contrast, after intoxication, no plant extract was capable of restoring E293 viability within 48 h, however, two medicinal plant combinations did restore the Bisphenol-A/Atrazine intoxicated HepG2 up to 24-28%. The analysis of underlying mechanisms revealed that plant extracts were not capable of preventing radiolabelled glyphosate from entering cells; however Dig2 did restore the CYP1A2 activity disrupted by Roundup, and had only a mild preventive effect

  13. Improvement of Traceability of Widely-Defined Measurements in the Field of Humanities

    Science.gov (United States)

    Sapozhnikova, K.; Taymanov, R.

    2010-01-01

    In the last decades, a tendency to extend the domain of "fuzzy" measurements of multiparametric quantities to the field of humanities has been observed. In the measurement process, the "fuzzy" measurements should meet the requirements of metrological traceability. The paper deals with the approach proposed for developing a measurement model of "fuzzy" measurements. The approach suggested is illustrated by an example of a model for measuring the emotions contained in musical fragments. The model is based on the hypothesis that permits to explain the origination of emotions in the evolution process.

  14. The analysis of the human hemopexin promoter defines a new class of liver-specific genes.

    OpenAIRE

    Poli, V.; Silengo, L; Altruda, F; Cortese, R

    1989-01-01

    Hemopexin (Hpx) is a plasma glycoprotein which is expressed only in the liver. It is synthesized at a lower rate in the fetal liver than in the adult, and its level increases during acute infections. As shown here, a fragment of the human hemopexin promoter spanning from positions -130 to +22 relative to the cap site is sufficient to direct cell-specific transcription of a reporter gene. Within this segment a short sequence, located between positions -120 and -104, is responsible for this eff...

  15. NKp80 Defines a Critical Step during Human Natural Killer Cell Development

    Directory of Open Access Journals (Sweden)

    Aharon G. Freud

    2016-07-01

    Full Text Available Human natural killer (NK cells develop in secondary lymphoid tissues (SLTs through distinct stages. We identified two SLT lineage (Lin−CD34−CD117+/−CD94+CD16− “stage 4” subsets according to expression of the C-type lectin-like surface-activating receptor, NKp80: NKp80− (stage “4a” and NKp80+ (stage “4b”. Whereas stage 4b cells expressed more of the transcription factors T-BET and EOMES, produced interferon-gamma, and were cytotoxic, stage 4a cells expressed more of the transcription factors RORγt and AHR and produced interleukin-22, similar to SLT Lin−CD34−CD117+CD94−CD16− “stage 3” cells, whose phenotype overlaps with that of group 3 innate lymphoid cells (ILC3s. Co-culture with dendritic cells or transplantation into immunodeficient mice produced mature NK cells from stage 3 and stage 4a populations. These data identify NKp80 as a marker of NK cell maturity in SLTs and support a model of human NK cell development through a stage 4a intermediate with ILC3-associated features.

  16. NKp80 Defines a Critical Step during Human Natural Killer Cell Development.

    Science.gov (United States)

    Freud, Aharon G; Keller, Karen A; Scoville, Steven D; Mundy-Bosse, Bethany L; Cheng, Stephanie; Youssef, Youssef; Hughes, Tiffany; Zhang, Xiaoli; Mo, Xiaokui; Porcu, Pierluigi; Baiocchi, Robert A; Yu, Jianhua; Carson, William E; Caligiuri, Michael A

    2016-07-12

    Human natural killer (NK) cells develop in secondary lymphoid tissues (SLTs) through distinct stages. We identified two SLT lineage (Lin)(-)CD34(-)CD117(+/-)CD94(+)CD16(-) "stage 4" subsets according to expression of the C-type lectin-like surface-activating receptor, NKp80: NKp80(-) (stage "4a") and NKp80(+) (stage "4b"). Whereas stage 4b cells expressed more of the transcription factors T-BET and EOMES, produced interferon-gamma, and were cytotoxic, stage 4a cells expressed more of the transcription factors RORγt and AHR and produced interleukin-22, similar to SLT Lin(-)CD34(-)CD117(+)CD94(-)CD16(-) "stage 3" cells, whose phenotype overlaps with that of group 3 innate lymphoid cells (ILC3s). Co-culture with dendritic cells or transplantation into immunodeficient mice produced mature NK cells from stage 3 and stage 4a populations. These data identify NKp80 as a marker of NK cell maturity in SLTs and support a model of human NK cell development through a stage 4a intermediate with ILC3-associated features.

  17. CD161 Defines a Transcriptional and Functional Phenotype across Distinct Human T Cell Lineages

    Directory of Open Access Journals (Sweden)

    Joannah R. Fergusson

    2014-11-01

    Full Text Available The C-type lectin CD161 is expressed by a large proportion of human T lymphocytes of all lineages, including a population known as mucosal-associated invariant T (MAIT cells. To understand whether different T cell subsets expressing CD161 have similar properties, we examined these populations in parallel using mass cytometry and mRNA microarray approaches. The analysis identified a conserved CD161++/MAIT cell transcriptional signature enriched in CD161+CD8+ T cells, which can be extended to CD161+ CD4+ and CD161+TCRγδ+ T cells. Furthermore, this led to the identification of a shared innate-like, TCR-independent response to interleukin (IL-12 plus IL-18 by different CD161-expressing T cell populations. This response was independent of regulation by CD161, which acted as a costimulatory molecule in the context of T cell receptor stimulation. Expression of CD161 hence identifies a transcriptional and functional phenotype, shared across human T lymphocytes and independent of both T cell receptor (TCR expression and cell lineage.

  18. Wide-field retinotopy defines human cortical visual area v6.

    Science.gov (United States)

    Pitzalis, Sabrina; Galletti, Claudio; Huang, Ruey-Song; Patria, Fabiana; Committeri, Giorgia; Galati, Gaspare; Fattori, Patrizia; Sereno, Martin I

    2006-07-26

    The retinotopic organization of a newly identified visual area near the midline in the dorsalmost part of the human parieto-occipital sulcus was mapped using high-field functional magnetic resonance imaging, cortical surface-based analysis, and wide-field retinotopic stimulation. This area was found in all 34 subjects that were mapped. It represents the contralateral visual hemifield in both hemispheres of all subjects, with upper fields located anterior and medial to areas V2/V3, and lower fields medial and slightly anterior to areas V3/V3A. It contains a representation of the center of gaze distinct from V3A, a large representation of the visual periphery, and a mirror-image representation of the visual field. Based on similarity in position, visuotopic organization, and relationship with the neighboring extrastriate visual areas, we suggest it might be the human homolog of macaque area V6, and perhaps of area M (medial) or DM (dorsomedial) of New World primates.

  19. NKp80 Defines a Critical Step during Human Natural Killer Cell Development.

    Science.gov (United States)

    Freud, Aharon G; Keller, Karen A; Scoville, Steven D; Mundy-Bosse, Bethany L; Cheng, Stephanie; Youssef, Youssef; Hughes, Tiffany; Zhang, Xiaoli; Mo, Xiaokui; Porcu, Pierluigi; Baiocchi, Robert A; Yu, Jianhua; Carson, William E; Caligiuri, Michael A

    2016-07-12

    Human natural killer (NK) cells develop in secondary lymphoid tissues (SLTs) through distinct stages. We identified two SLT lineage (Lin)(-)CD34(-)CD117(+/-)CD94(+)CD16(-) "stage 4" subsets according to expression of the C-type lectin-like surface-activating receptor, NKp80: NKp80(-) (stage "4a") and NKp80(+) (stage "4b"). Whereas stage 4b cells expressed more of the transcription factors T-BET and EOMES, produced interferon-gamma, and were cytotoxic, stage 4a cells expressed more of the transcription factors RORγt and AHR and produced interleukin-22, similar to SLT Lin(-)CD34(-)CD117(+)CD94(-)CD16(-) "stage 3" cells, whose phenotype overlaps with that of group 3 innate lymphoid cells (ILC3s). Co-culture with dendritic cells or transplantation into immunodeficient mice produced mature NK cells from stage 3 and stage 4a populations. These data identify NKp80 as a marker of NK cell maturity in SLTs and support a model of human NK cell development through a stage 4a intermediate with ILC3-associated features. PMID:27373165

  20. Defined conditions for the isolation and expansion of basal prostate progenitor cells of mouse and human origin.

    Science.gov (United States)

    Höfner, Thomas; Eisen, Christian; Klein, Corinna; Rigo-Watermeier, Teresa; Goeppinger, Stephan M; Jauch, Anna; Schoell, Brigitte; Vogel, Vanessa; Noll, Elisa; Weichert, Wilko; Baccelli, Irène; Schillert, Anja; Wagner, Steve; Pahernik, Sascha; Sprick, Martin R; Trumpp, Andreas

    2015-03-10

    Methods to isolate and culture primary prostate epithelial stem/progenitor cells (PESCs) have proven difficult and ineffective. Here, we present a method to grow and expand both murine and human basal PESCs long term in serum- and feeder-free conditions. The method enriches for adherent mouse basal PESCs with a Lin(-)SCA-1(+)CD49f(+)TROP2(high) phenotype. Progesterone and sodium selenite are additionally required for the growth of human Lin(-)CD49f(+)TROP2(high) PESCs. The gene-expression profiles of expanded basal PESCs show similarities to ESCs, and NF-kB function is critical for epithelial differentiation of sphere-cultured PESCs. When transplanted in combination with urogenital sinus mesenchyme, expanded mouse and human PESCs generate ectopic prostatic tubules, demonstrating their stem cell activity in vivo. This novel method will facilitate the molecular, genomic, and functional characterization of normal and pathologic prostate glands of mouse and human origin. PMID:25702639

  1. The chemical interactome space between the human host and the genetically defined gut metabotypes

    DEFF Research Database (Denmark)

    Jacobsen, Ulrik Plesner; Nielsen, Henrik Bjørn; Hildebrand, Falk;

    2013-01-01

    The bacteria that colonize the gastrointestinal tracts of mammals represent a highly selected microbiome that has a profound influence on human physiology by shaping the host’s metabolic and immune system activity. Despite the recent advances on the biological principles that underlie microbial...... symbiosis in the gut of mammals, mechanistic understanding of the contributions of the gut microbiome and how variations in the metabotypes are linked to the host health are obscure. Here, we mapped the entire metabolic potential of the gut microiome based solely on metagenomics sequencing data derived from...... fecal samples of 124 Europeans (healthy, obese and with inflammatory bowel disease). Interestingly, three distinct clusters of individuals with high, medium and low metabolic potential were observed. By illustrating these results in the context of bacterial population, we concluded that the abundance...

  2. Derivation of human differential photoreceptor-like cells from the iris by defined combinations of CRX, RX and NEUROD.

    Directory of Open Access Journals (Sweden)

    Yuko Seko

    Full Text Available Examples of direct differentiation by defined transcription factors have been provided for beta-cells, cardiomyocytes and neurons. In the human visual system, there are four kinds of photoreceptors in the retina. Neural retina and iris-pigmented epithelium (IPE share a common developmental origin, leading us to test whether human iris cells could differentiate to retinal neurons. We here define the transcription factor combinations that can determine human photoreceptor cell fate. Expression of rhodopsin, blue opsin and green/red opsin in induced photoreceptor cells were dependent on combinations of transcription factors: A combination of CRX and NEUROD induced rhodopsin and blue opsin, but did not induce green opsin; a combination of CRX and RX induced blue opsin and green/red opsin, but did not induce rhodopsin. Phototransduction-related genes as well as opsin genes were up-regulated in those cells. Functional analysis; i.e. patch clamp recordings, clearly revealed that generated photoreceptor cells, induced by CRX, RX and NEUROD, responded to light. The response was an inward current instead of the typical outward current. These data suggest that photosensitive photoreceptor cells can be generated by combinations of transcription factors. The combination of CRX and RX generate immature photoreceptors: and additional NEUROD promotes maturation. These findings contribute substantially to a major advance toward eventual cell-based therapy for retinal degenerative diseases.

  3. Defining the proteome of human iris, ciliary body, retinal pigment epithelium, and choroid.

    Science.gov (United States)

    Zhang, Pingbo; Kirby, David; Dufresne, Craig; Chen, Yan; Turner, Randi; Ferri, Sara; Edward, Deepak P; Van Eyk, Jennifer E; Semba, Richard D

    2016-04-01

    The iris is a fine structure that controls the amount of light that enters the eye. The ciliary body controls the shape of the lens and produces aqueous humor. The retinal pigment epithelium and choroid (RPE/choroid) are essential in supporting the retina and absorbing light energy that enters the eye. Proteins were extracted from iris, ciliary body, and RPE/choroid tissues of eyes from five individuals and fractionated using SDS-PAGE. After in-gel digestion, peptides were analyzed using LC-MS/MS on an Orbitrap Elite mass spectrometer. In iris, ciliary body, and RPE/choroid, we identified 2959, 2867, and 2755 nonredundant proteins with peptide and protein false-positive rates of database of the human iris, ciliary body, and RPE/choroid may serve as a valuable resource for future investigations of the eye in health and disease. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD001424 and PXD002194. PMID:26834087

  4. Gene expression correlations in human cancer cell lines define molecular interaction networks for epithelial phenotype.

    Directory of Open Access Journals (Sweden)

    Kurt W Kohn

    Full Text Available Using gene expression data to enhance our knowledge of control networks relevant to cancer biology and therapy is a challenging but urgent task. Based on the premise that genes that are expressed together in a variety of cell types are likely to functions together, we derived mutually correlated genes that function together in various processes in epithelial-like tumor cells. Expression-correlated genes were derived from data for the NCI-60 human tumor cell lines, as well as data from the Broad Institute's CCLE cell lines. NCI-60 cell lines that selectively expressed a mutually correlated subset of tight junction genes served as a signature for epithelial-like cancer cells. Those signature cell lines served as a seed to derive other correlated genes, many of which had various other epithelial-related functions. Literature survey yielded molecular interaction and function information about those genes, from which molecular interaction maps were assembled. Many of the genes had epithelial functions unrelated to tight junctions, demonstrating that new function categories were elicited. The most highly correlated genes were implicated in the following epithelial functions: interactions at tight junctions (CLDN7, CLDN4, CLDN3, MARVELD3, MARVELD2, TJP3, CGN, CRB3, LLGL2, EPCAM, LNX1; interactions at adherens junctions (CDH1, ADAP1, CAMSAP3; interactions at desmosomes (PPL, PKP3, JUP; transcription regulation of cell-cell junction complexes (GRHL1 and 2; epithelial RNA splicing regulators (ESRP1 and 2; epithelial vesicle traffic (RAB25, EPN3, GRHL2, EHF, ADAP1, MYO5B; epithelial Ca(+2 signaling (ATP2C2, S100A14, BSPRY; terminal differentiation of epithelial cells (OVOL1 and 2, ST14, PRSS8, SPINT1 and 2; maintenance of apico-basal polarity (RAB25, LLGL2, EPN3. The findings provide a foundation for future studies to elucidate the functions of regulatory networks specific to epithelial-like cancer cells and to probe for anti-cancer drug targets.

  5. Defining global syndromes of fire and the relationship of these to biomes, climate and human activity

    Science.gov (United States)

    Lehmann, C.; Archibald, S.; Gomez-Dans, J.; Bradstock, R.

    2012-12-01

    . There were, however, striking correlations between particular pyromes and biomes. For example frequent, intense, large fires were associated with grasslands and tropical savanna in 75% of instances. Rare intense, large fires were dominantly associated with boreal forests. Crucially, we identified a fifth pyrome that we consider to represent human-engineered modifications to fire characteristics. This pyrome, characterised by infrequent, cool, small fires that can occur throughout the year, occurs within all biomes, and was dominant in regions of extensive land transformation. Our research presents a conceptual framework that may help develop capacity to predict future fire, as our analysis suggests that pathways of change in future fire regimes are unlikely to be unilaterally responsive to climate in a deterministic way.

  6. Characterizing metabolic changes in human colorectal cancer.

    Science.gov (United States)

    Williams, Michael D; Zhang, Xing; Park, Jeong-Jin; Siems, William F; Gang, David R; Resar, Linda M S; Reeves, Raymond; Hill, Herbert H

    2015-06-01

    Colorectal cancer (CRC) remains a leading cause of cancer death worldwide, despite the fact that it is a curable disease when diagnosed early. The development of new screening methods to aid in early diagnosis or identify precursor lesions at risk for progressing to CRC will be vital to improving the survival rate of individuals predisposed to CRC. Metabolomics is an advancing area that has recently seen numerous applications to the field of cancer research. Altered metabolism has been studied for many years as a means to understand and characterize cancer. However, further work is required to establish standard procedures and improve our ability to identify distinct metabolomic profiles that can be used to diagnose CRC or predict disease progression. The present study demonstrates the use of direct infusion traveling wave ion mobility mass spectrometry to distinguish metabolic profiles from CRC samples and matched non-neoplastic epithelium as well as metastatic and primary tumors at different stages of disease (T1-T4). By directly infusing our samples, the analysis time was reduced significantly, thus increasing the speed and efficiency of this method compared to traditional metabolomics platforms. Partial least squares discriminant analysis was used to visualize differences between the metabolic profiles of sample types and to identify the specific m/z features that led to this differentiation. Identification of the distinct m/z features was made using the human metabolome database. We discovered alterations in fatty acid biosynthesis and oxidative, glycolytic, and polyamine pathways that distinguish tumors from non-malignant colonic epithelium as well as various stages of CRC. Although further studies are needed, our results indicate that colonic epithelial cells undergo metabolic reprogramming during their evolution to CRC, and the distinct metabolites could serve as diagnostic tools or potential targets in therapy or primary prevention. Graphical Abstract

  7. Transforming human gait for signature signals characterization

    International Nuclear Information System (INIS)

    An integrated wireless inductive sensor is reported based on a system for monitoring human movement and body size. The proposed system senses the presence of human beings using electromagnetic field by making use of basic inductive coupling approach, hence analysing the performance of human monitoring. The amalgamation of the integrated system proposed will help in providing better services to the elderly people resided in healthcare centres. The developed sensing system is of low cost, flexible, robust, and easily implantable and capable of inductive sensing through marking signature waveforms as a result of human movements

  8. A Small Molecule that Promotes Cardiac Differentiation of Human Pluripotent Stem Cells under Defined, Cytokine- and Xeno-free Conditions

    Directory of Open Access Journals (Sweden)

    Itsunari Minami

    2012-11-01

    Full Text Available Human pluripotent stem cells (hPSCs, including embryonic stem cells and induced pluripotent stem cells, are potentially useful in regenerative therapies for heart disease. For medical applications, clinical-grade cardiac cells must be produced from hPSCs in a defined, cost-effective manner. Cell-based screening led to the discovery of KY02111, a small molecule that promotes differentiation of hPSCs to cardiomyocytes. Although the direct target of KY02111 remains unknown, results of the present study suggest that KY02111 promotes differentiation by inhibiting WNT signaling in hPSCs but in a manner that is distinct from that of previously studied WNT inhibitors. Combined use of KY02111 and WNT signaling modulators produced robust cardiac differentiation of hPSCs in a xeno-free, defined medium, devoid of serum and any kind of recombinant cytokines and hormones, such as BMP4, Activin A, or insulin. The methodology has potential as a means for the practical production of human cardiomyocytes for regeneration therapies.

  9. Generation and characterization of human insulin-releasing cell lines

    OpenAIRE

    Joffé Elisa; Machado Marcel CC; Buchanan Cecilia; Terra Letícia F; Stigliano Iván; Krogh Karin; Peters Maria G; Labriola Leticia; Puricelli Lydia; Sogayar Mari C

    2009-01-01

    Abstract Background The in vitro culture of insulinomas provides an attractive tool to study cell proliferation and insulin synthesis and secretion. However, only a few human beta cell lines have been described, with long-term passage resulting in loss of insulin secretion. Therefore, we set out to establish and characterize human insulin-releasing cell lines. Results We generated ex-vivo primary cultures from two independent human insulinomas and from a human nesidioblastosis, all of which w...

  10. A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation

    Science.gov (United States)

    Lei, Yuguo; Schaffer, David V.

    2013-12-01

    Human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, are promising for numerous biomedical applications, such as cell replacement therapies, tissue and whole-organ engineering, and high-throughput pharmacology and toxicology screening. Each of these applications requires large numbers of cells of high quality; however, the scalable expansion and differentiation of hPSCs, especially for clinical utilization, remains a challenge. We report a simple, defined, efficient, scalable, and good manufacturing practice-compatible 3D culture system for hPSC expansion and differentiation. It employs a thermoresponsive hydrogel that combines easy manipulation and completely defined conditions, free of any human- or animal-derived factors, and entailing only recombinant protein factors. Under an optimized protocol, the 3D system enables long-term, serial expansion of multiple hPSCs lines with a high expansion rate (∼20-fold per 5-d passage, for a 1072-fold expansion over 280 d), yield (∼2.0 × 107 cells per mL of hydrogel), and purity (∼95% Oct4+), even with single-cell inoculation, all of which offer considerable advantages relative to current approaches. Moreover, the system enabled 3D directed differentiation of hPSCs into multiple lineages, including dopaminergic neuron progenitors with a yield of ∼8 × 107 dopaminergic progenitors per mL of hydrogel and ∼80-fold expansion by the end of a 15-d derivation. This versatile system may be useful at numerous scales, from basic biological investigation to clinical development.

  11. Defining antigen-specific plasmablast and memory B cell subsets in human blood after viral infection or vaccination.

    Science.gov (United States)

    Ellebedy, Ali H; Jackson, Katherine J L; Kissick, Haydn T; Nakaya, Helder I; Davis, Carl W; Roskin, Krishna M; McElroy, Anita K; Oshansky, Christine M; Elbein, Rivka; Thomas, Shine; Lyon, George M; Spiropoulou, Christina F; Mehta, Aneesh K; Thomas, Paul G; Boyd, Scott D; Ahmed, Rafi

    2016-10-01

    Antigen-specific B cells bifurcate into antibody-secreting cells (ASCs) and memory B cells (MBCs) after infection or vaccination. ASCs (plasmablasts) have been extensively studied in humans, but less is known about B cells that become activated but do not differentiate into plasmablasts. Here we have defined the phenotype and transcriptional program of a subset of antigen-specific B cells, which we have called 'activated B cells' (ABCs), that were distinct from ASCs and were committed to the MBC lineage. We detected ABCs in humans after infection with Ebola virus or influenza virus and also after vaccination. By simultaneously analyzing antigen-specific ASCs and ABCs in human blood after vaccination against influenza virus, we investigated the clonal overlap and extent of somatic hypermutation (SHM) in the ASC (effector) and ABC (memory) lineages. Longitudinal tracking of vaccination-induced hemagglutinin (HA)-specific clones revealed no overall increase in SHM over time, which suggested that repeated annual immunization might have limitations in enhancing the quality of influenza-virus-specific antibody. PMID:27525369

  12. Structural characterization of the interaction of human lactoferrin with calmodulin.

    Directory of Open Access Journals (Sweden)

    Jessica L Gifford

    Full Text Available Lactoferrin (Lf is an 80 kDa, iron (Fe(3+-binding immunoregulatory glycoprotein secreted into most exocrine fluids, found in high concentrations in colostrum and milk, and released from neutrophil secondary granules at sites of infection and inflammation. In a number of cell types, Lf is internalized through receptor-mediated endocytosis and targeted to the nucleus where it has been demonstrated to act as a transcriptional trans-activator. Here we characterize human Lf's interaction with calmodulin (CaM, a ubiquitous, 17 kDa regulatory calcium (Ca(2+-binding protein localized in the cytoplasm and nucleus of activated cells. Due to the size of this intermolecular complex (∼100 kDa, TROSY-based NMR techniques were employed to structurally characterize Ca(2+-CaM when bound to intact apo-Lf. Both CaM's backbone amides and the ε-methyl group of key methionine residues were used as probes in chemical shift perturbation and cross-saturation experiments to define the binding interface of apo-Lf on Ca(2+-CaM. Unlike the collapsed conformation through which Ca(2+-CaM binds the CaM-binding domains of its classical targets, Ca(2+-CaM assumes an extended structure when bound to apo-Lf. Apo-Lf appears to interact predominantly with the C-terminal lobe of Ca(2+-CaM, enabling the N-terminal lobe to potentially bind another target. Our use of intact apo-Lf has made possible the identification of a secondary interaction interface, removed from CaM's primary binding domain. Secondary interfaces play a key role in the target's response to CaM binding, highlighting the importance of studying intact complexes. This solution-based approach can be applied to study other regulatory calcium-binding EF-hand proteins in intact intermolecular complexes.

  13. Defined Conditions for the Isolation and Expansion of Basal Prostate Progenitor Cells of Mouse and Human Origin

    Directory of Open Access Journals (Sweden)

    Thomas Höfner

    2015-03-01

    Full Text Available Methods to isolate and culture primary prostate epithelial stem/progenitor cells (PESCs have proven difficult and ineffective. Here, we present a method to grow and expand both murine and human basal PESCs long term in serum- and feeder-free conditions. The method enriches for adherent mouse basal PESCs with a Lin−SCA-1+CD49f+TROP2high phenotype. Progesterone and sodium selenite are additionally required for the growth of human Lin−CD49f+TROP2high PESCs. The gene-expression profiles of expanded basal PESCs show similarities to ESCs, and NF-kB function is critical for epithelial differentiation of sphere-cultured PESCs. When transplanted in combination with urogenital sinus mesenchyme, expanded mouse and human PESCs generate ectopic prostatic tubules, demonstrating their stem cell activity in vivo. This novel method will facilitate the molecular, genomic, and functional characterization of normal and pathologic prostate glands of mouse and human origin.

  14. Defined Conditions for the Isolation and Expansion of Basal Prostate Progenitor Cells of Mouse and Human Origin

    Science.gov (United States)

    Höfner, Thomas; Eisen, Christian; Klein, Corinna; Rigo-Watermeier, Teresa; Goeppinger, Stephan M.; Jauch, Anna; Schoell, Brigitte; Vogel, Vanessa; Noll, Elisa; Weichert, Wilko; Baccelli, Irène; Schillert, Anja; Wagner, Steve; Pahernik, Sascha; Sprick, Martin R.; Trumpp, Andreas

    2015-01-01

    Summary Methods to isolate and culture primary prostate epithelial stem/progenitor cells (PESCs) have proven difficult and ineffective. Here, we present a method to grow and expand both murine and human basal PESCs long term in serum- and feeder-free conditions. The method enriches for adherent mouse basal PESCs with a Lin−SCA-1+CD49f+TROP2high phenotype. Progesterone and sodium selenite are additionally required for the growth of human Lin−CD49f+TROP2high PESCs. The gene-expression profiles of expanded basal PESCs show similarities to ESCs, and NF-kB function is critical for epithelial differentiation of sphere-cultured PESCs. When transplanted in combination with urogenital sinus mesenchyme, expanded mouse and human PESCs generate ectopic prostatic tubules, demonstrating their stem cell activity in vivo. This novel method will facilitate the molecular, genomic, and functional characterization of normal and pathologic prostate glands of mouse and human origin. PMID:25702639

  15. Molecular characterization of human thyroid hormone receptor β isoform 4.

    Science.gov (United States)

    Moriyama, Kenji; Yamamoto, Hiroyuki; Futawaka, Kumi; Atake, Asami; Kasahara, Masato; Tagami, Tetsuya

    2016-01-01

    Thyroid hormone exerts a pleiotropic effect on development, differentiation, and metabolism through thyroid hormone receptor (TR). A novel thyroid hormone receptor β isoform (TRβ4) was cloned using PCR from a human pituitary cDNA library as a template. We report here the characterization of TRβ4 from a molecular basis. Temporal expression of TRβ4 during the fetal period is abundant in the brain and kidney, comparable with the adult pattern. Western blot analysis revealed that TRs are ubiquitination labile proteins, while TRβ1 is potentially stable. TRβ1, peroxisome proliferator-activated receptors (PPAR), and vitamin D receptor (VDR), which belong to class II transcription factors that function via the formation of heterodimeric complexes with retinoid X receptor (RXR), were suppressed by TRβ4 in a dose-dependent manner. Thus, TRβ4 exhibits ligand-independent transcriptional silencing, possibly as a substitute for dimerized RXR. In this study, TRβ1 and TRβ4 transcripts were detected in several cell lines. Quantitative RT-PCR assay showed that the expression of TRβ4 in human embryonic carcinoma cells of the testis was suppressed by sex hormone in a reciprocal manner to TRβ1. In contrast, TRβ4 was expressed under a high dose of triiodothyronine (T3) in a reciprocal manner to TRβ1. Finally, in transiently transfected NIH-3T3 cells, green fluorescence protein (GFP)-tagged TRβ4 was mostly nuclear in both the absence and the presence of T3. By mutating defined regions of both TRβs, we found that both TRβ1 and TRβ4 had altered nuclear/cytoplasmic distribution as compared with wild-type, and different to T3 and the nuclear receptor corepressor (NCoR). Thus, site-specific DNA binding is not essential for maintaining TRβs within the nucleus. PMID:26513165

  16. Defining as a Mathematical Activity: A Framework for Characterizing Progress from Informal to More Formal Ways of Reasoning

    Science.gov (United States)

    Zandieh, Michelle; Rasmussen, Chris

    2010-01-01

    The purpose of this paper is to further the notion of defining as a mathematical activity by elaborating a framework that structures the role of defining in student progress from informal to more formal ways of reasoning. The framework is the result of a retrospective account of a significant learning experience that occurred in an undergraduate…

  17. Defining microbiota for developing new probiotics

    OpenAIRE

    Collado, Maria Carmen; Bäuerl, Christine; Pérez-Martínez, Gaspar

    2012-01-01

    The human body harbors complex communities of microbes that play a prominent role in human health. Detailed characterization of the microbiota in the target population forms the basis of probiotic use. Probiotics are defined as live bacterial preparations with clinically documented health effects in humans and independently of their genus and species, probiotic strains are unique and their beneficial properties on human health have to be assessed in a case-by-case manner. Understanding the me...

  18. Characterization and partial purification of phospholipase D from human placenta

    DEFF Research Database (Denmark)

    Vinggaard, Anne Marie; Hansen, Harald S.

    1995-01-01

    We report the existence in the human placenta of a phosphatidylcholine- hydrolyzing phospholipase D (PLD) activity, which has been characterized and partially purified. Triton X-100 effectively solubilized PLD from the particulate fraction of human placenta in a dose-dependent manner. However...

  19. Characterization and crystallization of human uroporphyrinogen decarboxylase.

    OpenAIRE

    Phillips, J. D.; Whitby, F. G.; Kushner, J. P.; Hill, C. P.

    1997-01-01

    The cytosolic enzyme uroporphyrinogen decarboxylase (URO-D) catalyzes the fifth step in the heme biosynthetic pathway, converting uroporphyrinogen to coproporphyrinogen by decarboxylating the four acetate side chains of the substrate. Recombinant human URO-D has been expressed in Escherichia coli with a histidine tag and has been purified to homogeneity. Purified protein was determined to be a monodisperse dimer by dynamic light scattering. Equilibrium sedimentation analysis confirmed that th...

  20. Using the Human Eye to Characterize Displays

    Science.gov (United States)

    Gille, Jennifer; Larimer, James; Rutkowski, Michael (Technical Monitor)

    2001-01-01

    Monitor characterization has taken on new importance for non-professional users, who are not usually equipped to make photometric measurements. We studied adjusting brightness to set the black level, banding effects due to digitization, and gamma estimation in the light and in the dark, and a color-matching task in the light, on a desktop CRT (Cathode-Ray Tube) and a laptop LCD (Liquid Crystal Display). Observers demonstrated the sensitivity of the visual system for comparative judgments in black-level adjustment, banding visibility, and gamma estimation. The results of the color-matching task were ambiguous. In the brightness adjustment task, the action of the adjustment was not as presumed; however, perceptual judgments were as expected under the actual conditions. When the gamma estimates of observers were compared to photometric measurements, problems with the definition of gamma were identified. Information about absolute light levels that would be important for characterizing a displays, given the shortcomings of gamma in measuring apparent contrast, are not measurable by eye alone. The LCD was not studied as extensively as the CRT because of viewing-angle problems, and its transfer function did not follow a power law, rendering gamma estimation meaningless.

  1. Neurochemical organization of the human basal ganglia: anatomofunctional territories defined by the distributions of calcium-binding proteins and SMI-32.

    Science.gov (United States)

    Morel, Anne; Loup, Fabienne; Magnin, Michel; Jeanmonod, Daniel

    2002-01-28

    The distribution of the calcium-binding proteins calbindin-D28K (CB), parvalbumin (PV) and calretinin (CR), and of the nonphosphorylated neurofilament protein (with SMI-32) was investigated in the human basal ganglia to identify anatomofunctional territories. In the striatum, gradients of neuropil immunostaining define four major territories: The first (T1) includes all but the rostroventral half of the putamen and is characterized by enhanced matriceal PV and SMI-32 immunoreactivity (-ir). The second territory (T2) encompasses most part of the caudate nucleus (Cd) and rostral putamen (PuT), which show enhanced matriceal CB-ir. The third and fourth territories (T3 and T4) comprise rostroventral parts of Cd and PuT characterized by complementary patch/matrix distributions of CB- and CR-ir, and the accumbens nucleus (Acb), respectively. The latter is separated into lateral (prominently enhanced in CB-ir) and medial (prominently enhanced in CR-ir) subdivisions. In the pallidum, parallel gradients also delimit four territories, T1 in the caudal half of external (GPe) and internal (GPi) divisions, characterized by enhanced PV- and SMI-32-ir; T2 in their rostral half, characterized by enhanced CB-ir; and T3 and T4 in their rostroventral pole and in the subpallidal area, respectively, both expressing CB- and CR-ir but with different intensities. The subthalamic nucleus (STh) shows contrasting patterns of dense PV-ir (sparing only the most medial part) and low CB-ir. Expression of CR-ir is relatively low, except in the medial, low PV-ir, part of the nucleus, whereas SMI-32-ir is moderate across the whole nucleus. The substantia nigra is characterized by complementary patterns of high neuropil CB- and SMI-32-ir in pars reticulata (SNr) and high CR-ir in pars compacta (SNc) and in the ventral tegmental area (VTA). The compartmentalization of calcium-binding proteins and SMI-32 in the human basal ganglia, in particular in the striatum and pallidum, delimits anatomofunctional

  2. SS18 together with animal-specific factors defines human BAF-type SWI/SNF complexes.

    Directory of Open Access Journals (Sweden)

    Evelien Middeljans

    Full Text Available BACKGROUND: Nucleosome translocation along DNA is catalyzed by eukaryotic SNF2-type ATPases. One class of SNF2-ATPases is distinguished by the presence of a C-terminal bromodomain and is conserved from yeast to man and plants. This class of SNF2 enzymes forms rather large protein complexes that are collectively called SWI/SNF complexes. They are involved in transcription and DNA repair. Two broad types of SWI/SNF complexes have been reported in the literature; PBAF and BAF. These are distinguished by the inclusion or not of polybromo and several ARID subunits. Here we investigated human SS18, a protein that is conserved in plants and animals. SS18 is a putative SWI/SNF subunit which has been implicated in the etiology of synovial sarcomas by virtue of being a target for oncogenic chromosomal translocations that underlie synovial sarcomas. METHODOLOGY/PRINCIPAL FINDINGS: We pursued a proteomic approach whereby the SS18 open reading frame was fused to a tandem affinity purification tag and expressed in amenable human cells. The fusion permitted efficient and exclusive purification of so-called BAF-type SWI/SNF complexes which bear ARID1A/BAF250a or ARID1B/BAF250b subunits. This demonstrates that SS18 is a BAF subtype-specific SWI/SNF complex subunit. The same result was obtained when using the SS18-SSX1 oncogenic translocation product. Furthermore, SS18L1, DPF1, DPF2, DPF3, BRD9, BCL7A, BCL7B and BCL7C were identified. 'Complex walking' showed that they all co-purify with each other, defining human BAF-type complexes. By contrast,we demonstrate that human PHF10 is part of the PBAF complex, which harbors both ARID2/BAF200 and polybromo/BAF180 subunits, but not SS18 and nor the above BAF-specific subunits. CONCLUSIONS/SIGNIFICANCE: SWI/SNF complexes are found in most eukaryotes and in the course of evolution new SWI/SNF subunits appeared. SS18 is found in plants as well as animals. Our results suggest that in both protostome and deuterostome animals

  3. The characterization of twenty sequenced human genomes.

    Directory of Open Access Journals (Sweden)

    Kimberly Pelak

    2010-09-01

    Full Text Available We present the analysis of twenty human genomes to evaluate the prospects for identifying rare functional variants that contribute to a phenotype of interest. We sequenced at high coverage ten "case" genomes from individuals with severe hemophilia A and ten "control" genomes. We summarize the number of genetic variants emerging from a study of this magnitude, and provide a proof of concept for the identification of rare and highly-penetrant functional variants by confirming that the cause of hemophilia A is easily recognizable in this data set. We also show that the number of novel single nucleotide variants (SNVs discovered per genome seems to stabilize at about 144,000 new variants per genome, after the first 15 individuals have been sequenced. Finally, we find that, on average, each genome carries 165 homozygous protein-truncating or stop loss variants in genes representing a diverse set of pathways.

  4. Echoendoscopic characterization of the human colon

    Directory of Open Access Journals (Sweden)

    Fernando M. Castro-Poças

    2015-08-01

    Full Text Available Purpose: To characterize colon and rectum walls, pericolic and perirectal spaces, using endoscopic ultrasonography miniprobes. Methods: Sixty individuals (50% males, aged 18-80, were included. Using 12 and 20 MHz endoscopic ultrasonography miniprobes, all different colon segments (ascending, transverse, descending, sigmoid and rectum were evaluated according to the number and thickness of the different layers in intestinal wall, to the presence and (largest diameter of vessels in the submucosa and of peri-intestinal nodes. Results: The 20 MHz miniprobe identified a higher number of layers than the 12 MHz miniprobe, with medians of 7 and 5 respectively (p < 0.001. The rectal wall (p = 0.001, its muscularis propria (p < 0.001 and mucosa (p = 0.01 were significantly thicker than the different segments of the colon, which had no significant differences between them. Patients aged 41-60 presented thicker colonic wall and muscularis propria in descending (p = 0.001 and p = 0.004 and rectum (p=0.01 and p=0.01. Submucosal vessels were identified in 30% of individuals in descending and rectum, and in 12% in ascending. Adenopathies were observed in 9% of the colon segments and 5% in rectum. Conclusions: A higher frequency enabled the identification of a higher number of layers. Rectal wall is thicker than the one from all the segments of the colon and there are no differences between these, namely in the ascending colon. Moreover, peri-intestinal adenopathies were rarely identified but present in asymptomatic individuals. All together, these results describe for the first time features which are relevant during staging and therapeutic management of colonic lesions.

  5. Complete Genome Sequence of Germline Chromosomally Integrated Human Herpesvirus 6A and Analyses Integration Sites Define a New Human Endogenous Virus with Potential to Reactivate as an Emerging Infection.

    Science.gov (United States)

    Tweedy, Joshua; Spyrou, Maria Alexandra; Pearson, Max; Lassner, Dirk; Kuhl, Uwe; Gompels, Ursula A

    2016-01-15

    Human herpesvirus-6A and B (HHV-6A, HHV-6B) have recently defined endogenous genomes, resulting from integration into the germline: chromosomally-integrated "CiHHV-6A/B". These affect approximately 1.0% of human populations, giving potential for virus gene expression in every cell. We previously showed that CiHHV-6A was more divergent than CiHHV-6B by examining four genes in 44 European CiHHV-6A/B cardiac/haematology patients. There was evidence for gene expression/reactivation, implying functional non-defective genomes. To further define the relationship between HHV-6A and CiHHV-6A we used next-generation sequencing to characterize genomes from three CiHHV-6A cardiac patients. Comparisons to known exogenous HHV-6A showed CiHHV-6A genomes formed a separate clade; including all 85 non-interrupted genes and necessary cis-acting signals for reactivation as infectious virus. Greater single nucleotide polymorphism (SNP) density was defined in 16 genes and the direct repeats (DR) terminal regions. Using these SNPs, deep sequencing analyses demonstrated superinfection with exogenous HHV-6A in two of the CiHHV-6A patients with recurrent cardiac disease. Characterisation of the integration sites in twelve patients identified the human chromosome 17p subtelomere as a prevalent site, which had specific repeat structures and phylogenetically related CiHHV-6A coding sequences indicating common ancestral origins. Overall CiHHV-6A genomes were similar, but distinct from known exogenous HHV-6A virus, and have the capacity to reactivate as emerging virus infections.

  6. Can play be defined?

    DEFF Research Database (Denmark)

    Eichberg, Henning

    2015-01-01

    Can play be defined? There is reason to raise critical questions about the established academic demand that at phenomenon – also in humanist studies – should first of all be defined, i.e. de-lineated and by neat lines limited to a “little box” that can be handled. The following chapter develops....... Human beings can very well understand play – or whatever phenomenon in human life – without defining it....

  7. Dynamic Asia: Coupling of climate, tectonics, rivers, and people defines risk and opportunity for the world's largest human populations

    Science.gov (United States)

    Goodbred, S. L., Jr.; Steckler, M. S.; Gilligan, J. M.; Ackerly, B.; Ayers, J. C.; Wilson, C.; Small, C.; Seeber, L.

    2014-12-01

    Coupling between the Himalayan-Tibetan uplift and intense Asian monsoon yields tremendous regional runoff and sediment supply. This vigorous mass-transfer system sustains 7 of the world's 10 largest riverine sediment loads, which in turn have constructed vast, fertile fluvial-deltaic lowlands. These environments across south and east Asia host about 1/3 of all people on Earth. Such large and dense populations have flourished amidst the region's generally abundant water supplies, fisheries, and agricultural production. Yet the same environmental attributes that are so rich in resources also define a uniquely dynamic region, where rates of change are rapid and punctuated by frequent, intense events. Indeed, 8 of the world's 10 deadliest natural disasters have occurred in this region, involving a combination of earthquakes, tropical cyclones, river floods, and tsunamis. Other stresses that regularly impact the region include periods of monsoon collapse and drought, widespread arsenic contamination of groundwater, relative sea-level rise and coastal inundation, and groundwater salinization. Thus the communities of this region persistently face the challenge of balancing the carrying capacity of a resource-rich environment with its associated hazards and challenges. One important concept that has become increasingly more apparent is the connection within watersheds that transmits local effects both upstream and downstream within the system. Here we emphasize two additional points that we believe are essential in developing plausible strategies for sustaining health, resilience, and stability of the region. First, problems related to the natural environment are closely coupled with human activities and our concurrent responses to environmental change. Thus resulting issues are complex and multifaceted in ways that require natural scientists to better engage with researchers in the humanities and social sciences. Second, despite similar risks affecting many millions of

  8. Structural characterization of human Uch37

    Energy Technology Data Exchange (ETDEWEB)

    Burgie, E. Sethe; Bingman, Craig A.; Soni, Ameet B.; Phillips, Jr., George N. (UW)

    2012-06-28

    Uch37 is a deubiquitylating enzyme (DUB) that is functionally linked with multiple protein complexes and signal transduction pathways. Uch37 associates with the 26S proteasome through Rpn13 where it serves to remove distal ubiquitin moeities from polyubiquitylated proteins. Uch37's proteasome associated activity was shown to liberate proteins from destruction. However, Uch37 may also specifically facilitate the destruction of inducible nitric oxide synthase and I{kappa}B-{alpha} at the proteasome. Wicks et al. established Uch37's potential to modulate the transforming growth factor-{beta}(TGF-{beta}) signaling cascade, through tis interaction with SMAD7. Yao et al. demonstrated that Uch37 also associates with the Ino80 chromatin-remodeling complex (Ino80 complex), which is involved in DNA repair and transcriptional regulation. Uch37's importance in metazoan development was underscored recently as Uch37 knockouts in mice result in prenatal lethality, where mutant embryos had severe defects in brain development. Protein ubiquitylation is an ATP-dependent post-translational modification that serves to signal a wide variety of cellular processes in eukaryotes. A protein cascade, generally comprising three enzymes, functions to activate, transport and specifically transfer ubiquitin to the targeted protein, culminating in an isopeptide linkage between the {epsilon}-amino group of a target protein's lysysl residue and the ubiquitin's terminal carboxylate. Monoubiquitination plays an important role in histone regulation, endocytosis, and viral budding. Further processing of the target protein may be accomplished by ubiquitylation of the protein on a different lysine, or through the formation of polyubiquitin chains, where the best-characterized outcome is destruction of the polyubiquitin-labeled protein in the proteasome. DUBs catalyze the removal of ubiquitin from proteins. This activity serves to reverse the effects of ubiquitination, permit

  9. Characterization of adhesive interactions between human endothelial cells and megakaryocytes.

    OpenAIRE

    Avraham, H; Cowley, S; Chi, S. Y.; Jiang, S.; Groopman, J E

    1993-01-01

    Cell-cell adhesion is essential for many immunological functions and is believed to be important in the regulation of hematopoiesis. Adhesive interactions between human endothelial cells and megakaryocytes were characterized in vitro using the CMK megakaryocytic cell line as well as marrow megakaryocytes. Although there was no adhesion between unactivated human umbilical vein endothelial cells (HUVEC) and megakaryocytes, treatment of HUVEC with inflammatory cytokines such as IL-1 beta, tumor ...

  10. Molecular Cloning, Overexpression and Characterization of Human Interleukin 1α

    OpenAIRE

    Rajalingam, Dakshinamurthy; Kacer, Doreen; Prudovsky, Igor; Kumar, Thallapuranam Krishnaswamy Suresh

    2007-01-01

    Interleukin-1 alpha (IL-1α) regulates a wide range of important cellular processes. In this study for the first time we report the cloning, expression, biophysical and biological characterization of the human interleukin-1α. Human IL-1α has been expressed in Escherichia coli in high yields (~ 4 mg per liter of the bacterial culture). The protein was purified to homogeneity (~ 98% purity) using affinity chromatography and size exclusion chromatography. Results of the steady state fluorescence ...

  11. Co-regulated transcripts associated to cooperating eSNPs define Bi-fan motifs in human gene networks.

    Directory of Open Access Journals (Sweden)

    Anat Kreimer

    2014-09-01

    Full Text Available Associations between the level of single transcripts and single corresponding genetic variants, expression single nucleotide polymorphisms (eSNPs, have been extensively studied and reported. However, most expression traits are complex, involving the cooperative action of multiple SNPs at different loci affecting multiple genes. Finding these cooperating eSNPs by exhaustive search has proven to be statistically challenging. In this paper we utilized availability of sequencing data with transcriptional profiles in the same cohorts to identify two kinds of usual suspects: eSNPs that alter coding sequences or eSNPs within the span of transcription factors (TFs. We utilize a computational framework for considering triplets, each comprised of a SNP and two associated genes. We examine pairs of triplets with such cooperating source eSNPs that are both associated with the same pair of target genes. We characterize such quartets through their genomic, topological and functional properties. We establish that this regulatory structure of cooperating quartets is frequent in real data, but is rarely observed in permutations. eSNP sources are mostly located on different chromosomes and away from their targets. In the majority of quartets, SNPs affect the expression of the two gene targets independently of one another, suggesting a mutually independent rather than a directionally dependent effect. Furthermore, the directions in which the minor allele count of the SNP affects gene expression within quartets are consistent, so that the two source eSNPs either both have the same effect on the target genes or both affect one gene in the opposite direction to the other. Same-effect eSNPs are observed more often than expected by chance. Cooperating quartets reported here in a human system might correspond to bi-fans, a known network motif of four nodes previously described in model organisms. Overall, our analysis offers insights regarding the fine motif structure

  12. Structural characterization of human and bovine lung surfactant protein D

    DEFF Research Database (Denmark)

    Leth-Larsen, Rikke; Holmskov, U; Højrup, P

    1999-01-01

    was characterized in human SP-D. The carbohydrate was determined as a complex type bi-antennary structure, with a small content of mono-antennary and tri-antennary structures. No sialic acid residues were present on the glycan, but some had an attached fucose and/or an N-acetylglucosamine residue linked to the core...

  13. Molecular characterization of apocrine carcinoma of the breast: validation of an apocrine protein signature in a well-defined cohort

    DEFF Research Database (Denmark)

    Celis, J.E.; Cabezon, T.; Moreira, José;

    2009-01-01

    1), in addition to a set of categorizing markers that are consistently expressed (AR, CD24) or not expressed (ERalpha, PgR, Bcl-2, and GATA-3) by apocrine metaplasia in benign breast lesions and apocrine sweat glands. This panel was used to analyze a well-defined cohort consisting of 14 apocrine...... that IACs correspond to a distinct, even if heterogeneous, molecular subgroup of breast carcinomas that can be readily identified in an unbiased way using a combination of markers that recapitulate the phenotype of apocrine sweat glands (15-PGDH(+), ACSM1(+), AR(+), CD24(+), ERalpha(-), PgR(-), Bcl-2...

  14. Nanomechanical characterization of human hair using nanoindentation and SEM

    Energy Technology Data Exchange (ETDEWEB)

    Wei Guohua [Nanotribology Laboratory for Information Storage and MEMS/NEMS (NLIM), Ohio State University, Columbus, OH 43210 (United States); Bhushan, Bharat [Nanotribology Laboratory for Information Storage and MEMS/NEMS (NLIM), Ohio State University, Columbus, OH 43210 (United States)]. E-mail: bhushan.2@osu.edu; Torgerson, Peter M. [Procter and Gamble Company, Sharon Woods Technical Center, Cincinnati, OH 45241 (United States)

    2005-11-15

    Human hair is a nanocomposite biological fiber with well-characterized microstructures. Nanomechanical characterization of human hair can help to evaluate the effect of cosmetic products on hair surface, can provide a better understanding of the physicochemical properties of a wide variety of composite biological systems, and can provide the dermatologists with some useful markers for the diagnosis of hair disorders. The paper presents systematic studies of nanomechanical properties of human hair including hardness, elastic modulus and creep, using the nanoindentation technique. The samples include Caucasian, Asian and African hair at virgin, chemo-mechanically damaged and treated conditions. Hair morphology was studied using scanning electron microscopy (SEM). Indentation experiments were performed on both the surface and cross-section of the hair, and the indents were studied using SEM. The nanomechanical properties of hair as a function of hair composition, microstructure, ethnicity, damage and treatment are discussed.

  15. Comparison of the material modelling performing characterization of weld microstructure of S355N steel and defining of residual stresses

    OpenAIRE

    Kuitunen, Mikko

    2016-01-01

    The objective of this Master thesis was to answer the following questions. Do the characterized microstructures of VTT S355N samples with their mechanical properties and the experimental results of TIG welds of the Voss‘s doctoral thesis correspond to the modelled material results? Is the mixing of the investigated weld samples of LAHW and MAG welds even? Do X-ray diffraction measured residual stresses of S355N butt weld samples correspond with the measured residual stresses of Contour method...

  16. Re-Defining the Subsurface Biosphere: Characterization of Fungal Populations from Energy Limited Deep Marine Subsurface Sediments

    Science.gov (United States)

    Reese, B. K.; Ariza, M.; St. Peter, C.; Hoffman, C.; Edwards, K. J.; Mills, H. J.

    2012-12-01

    The detection and characterization of metabolically active fungal populations within the deep marine subsurface will alter current ecosystem models that are limited to bacterial and archaeal populations. Although marine fungi have been studied for over fifty years, a detailed description of fungal populations within the deep subsurface is lacking. Fungi possess metabolic pathways capable of utilizing previously considered non-bioavailable energy reserves. Therefore, metabolically active fungi would occupy a unique niche within subsurface ecosystems, with the potential to provide an organic carbon source for heterotrophic prokaryotic populations not currently being considered in subsurface energy budgets. Sediments from the South Pacific Gyre subsurface, one of the most energy-limited environments on Earth, were collected during the Integrated Ocean Drilling Program (IODP) Expedition 329. Anaerobic and aerobic sediment slurry cultures using fresh sediment began directly following the completion of the Expedition (December 2010). From these cultures, multiple fungal lineages have been isolated on several media types that vary in carbon concentrations. Physical growth parameters of these subsurface fungal isolates were determined and compared to previously characterized lineages. Additionally, the overall diversity of metabolically active and dormant fungal populations was determined using high throughput sequencing of nucleic acids extracted from in situ cryopreserved South Pacific Gyre sediments. This project provides a robust step in determining the importance and impact of fungal populations within the marine subsurface biosphere.

  17. Proteogenomics: Integrating Next-Generation Sequencing and Mass Spectrometry to Characterize Human Proteomic Variation

    Science.gov (United States)

    Sheynkman, Gloria M.; Shortreed, Michael R.; Cesnik, Anthony J.; Smith, Lloyd M.

    2016-06-01

    Mass spectrometry-based proteomics has emerged as the leading method for detection, quantification, and characterization of proteins. Nearly all proteomic workflows rely on proteomic databases to identify peptides and proteins, but these databases typically contain a generic set of proteins that lack variations unique to a given sample, precluding their detection. Fortunately, proteogenomics enables the detection of such proteomic variations and can be defined, broadly, as the use of nucleotide sequences to generate candidate protein sequences for mass spectrometry database searching. Proteogenomics is experiencing heightened significance due to two developments: (a) advances in DNA sequencing technologies that have made complete sequencing of human genomes and transcriptomes routine, and (b) the unveiling of the tremendous complexity of the human proteome as expressed at the levels of genes, cells, tissues, individuals, and populations. We review here the field of human proteogenomics, with an emphasis on its history, current implementations, the types of proteomic variations it reveals, and several important applications.

  18. Characterization of ATPase Activity of Recombinant Human Pif1

    Institute of Scientific and Technical Information of China (English)

    Yu HUANG; Deng-Hong ZHANG; Jin-Qiu ZHOU

    2006-01-01

    Saccharomyces cerevisiae Pif1p helicase is the founding member of the Pif1 subfamily that is conserved from yeast to human. The potential human homolog of the yeast PIF1 gene has been cloned from the cDNA library of the Hek293 cell line. Here, we described a purification procedure of glutathione Stransferase (GST)-fused N terminal truncated human Pif1 protein (hPif1△N) from yeast and characterized the enzymatic kinetics of its ATP hydrolysis activity. The ATPase activity of human Pif1 is dependent on divalent cation, such as Mg2+, Ca2+ and single-stranded DNA. Km for ATP for the ATPase activity is approximately 200 μM. As the ATPase activity is essential for hPif1's helicase activity, these results will facilitate the further investigation on hPif1.

  19. Isolation and Characterization of Well-Defined Silica-Supported Azametallacyclopentane: A Key Intermediate in Catalytic Hydroaminoalkylation Reactions

    KAUST Repository

    Hamzaoui, Bilel

    2015-09-25

    Intermolecular catalytic hydroaminoalkylation of unactivated alkene occurs with silica-supported azazirconacyclopropane [[TRIPLE BOND]Si[BOND]O[BOND]Zr(HNMe2)(η2-NMeCH2)(NMe2)]. Mechanistic studies were conducted using surface organometallic chemistry (SOMC) concepts to identify the key surface intermediates. The azametallacyclopentene intermediate {[TRIPLE BOND]Si[BOND]O[BOND]Zr(HNMe2)[η2-NMeCH2CH(Me)CH2](NMe2)} was isolated after treating with 1-propylene and characterized by FT-IR spectroscopy, elemental analysis, 1H 13C HETCOR, DARR SS-NMR and DQ TQ SS-NMR. The regeneration of the catalyst was conducted by dimethylamine protonolysis to yield the pure amine.

  20. Characterization and histologic localization of human growth hormone-variant gene expression in the placenta.

    OpenAIRE

    Liebhaber, S A; Urbanek, M; Ray, J.; Tuan, R.S.; Cooke, N E

    1989-01-01

    The human growth hormone-variant (hGH-V) gene is one of five highly similar growth hormone-related genes clustered on the short arm of chromosome 17. Although the pattern of expression of the adjacent normal growth hormone (hGH-N) and chorionic somatomammotropin (hCS) genes in this cluster are well characterized, the expression of the hGH-V gene remains to be defined. In previous studies, we have demonstrated that the hGH-V gene is transcribed in the term placenta and expressed as two alterna...

  1. In Vivo Characterization of Human APOA5 Haplotypes

    Energy Technology Data Exchange (ETDEWEB)

    Ahituv, Nadav; Akiyama, Jennifer; Chapman-Helleboid, Audrey; Fruchart, Jamila; Pennacchio, Len A.

    2006-10-01

    Increased plasma triglycerides concentrations are an independent risk factor for cardiovascular disease. Numerous studies support a reproducible genetic association between two minor haplotypes in the human apolipoprotein A5 gene (APOA5) and increased plasma triglyceride concentrations. We thus sought to investigate the effect of these minor haplotypes (APOA5*2 and APOA5*3) on ApoAV plasma levels through the precise insertion of single-copy intact APOA5 haplotypes at a targeted location in the mouse genome. While we found no difference in the amount of human plasma ApoAV in mice containing the common APOA5*1 and minor APOA5*2 haplotype, the introduction of the single APOA5*3 defining allele (19W) resulted in 3-fold lower ApoAV plasma levels consistent with existing genetic association studies. These results indicate that S19W polymorphism is likely to be functional and explain the strong association of this variant with plasma triglycerides supporting the value of sensitive in vivo assays to define the functional nature of human haplotypes.

  2. Define Project

    DEFF Research Database (Denmark)

    Munk-Madsen, Andreas

    2005-01-01

    "Project" is a key concept in IS management. The word is frequently used in textbooks and standards. Yet we seldom find a precise definition of the concept. This paper discusses how to define the concept of a project. The proposed definition covers both heavily formalized projects and informally ...

  3. Characterization of Side Population Cells from Human Airway Epithelium

    OpenAIRE

    Hackett, Tillie-Louise; Shaheen, Furquan; Johnson, Andrew; Wadsworth, Samuel; Pechkovsky, Dmitri V; Jacoby, David B.; Kicic, Anthony; Stick, Stephen M.; Knight, Darryl A.

    2008-01-01

    The airway epithelium is the first line of contact with the inhaled external environment and is continuously exposed to and injured by pollutants, allergens, and viruses. However, little is known about epithelial repair and in particular the identity and role of tissue resident stem/progenitor cells that may contribute to epithelial regeneration. The aims of the present study were to identify, isolate, and characterize side population (SP) cells in human tracheobronchial epithelium. Epithelia...

  4. Dynamic Propagation Channel Characterization and Modeling for Human Body Communication

    OpenAIRE

    Lei Wang; Jingjing Ma; Zhicheng Li; Hong Chen; Zedong Nie

    2012-01-01

    This paper presents the first characterization and modeling of dynamic propagation channels for human body communication (HBC). In-situ experiments were performed using customized transceivers in an anechoic chamber. Three HBC propagation channels, i.e., from right leg to left leg, from right hand to left hand and from right hand to left leg, were investigated under thirty-three motion scenarios. Snapshots of data (2,800,000) were acquired from five volunteers. Various path gains caused by di...

  5. Generation and characterization of human insulin-releasing cell lines

    Directory of Open Access Journals (Sweden)

    Joffé Elisa

    2009-06-01

    Full Text Available Abstract Background The in vitro culture of insulinomas provides an attractive tool to study cell proliferation and insulin synthesis and secretion. However, only a few human beta cell lines have been described, with long-term passage resulting in loss of insulin secretion. Therefore, we set out to establish and characterize human insulin-releasing cell lines. Results We generated ex-vivo primary cultures from two independent human insulinomas and from a human nesidioblastosis, all of which were cultured up to passage number 20. All cell lines secreted human insulin and C-peptide. These cell lines expressed neuroendocrine and islets markers, confirming the expression profile found in the biopsies. Although all beta cell lineages survived an anchorage independent culture, none of them were able to invade an extracellular matrix substrate. Conclusion We have established three human insulin-releasing cell lines which maintain antigenic characteristics and insulin secretion profiles of the original tumors. These cell lines represent valuable tools for the study of molecular events underlying beta cell function and dysfunction.

  6. Generation and characterization of human insulin-releasing cell lines

    Science.gov (United States)

    Labriola, Leticia; Peters, Maria G; Krogh, Karin; Stigliano, Iván; Terra, Letícia F; Buchanan, Cecilia; Machado, Marcel CC; Joffé, Elisa Bal de Kier; Puricelli, Lydia; Sogayar, Mari C

    2009-01-01

    Background The in vitro culture of insulinomas provides an attractive tool to study cell proliferation and insulin synthesis and secretion. However, only a few human beta cell lines have been described, with long-term passage resulting in loss of insulin secretion. Therefore, we set out to establish and characterize human insulin-releasing cell lines. Results We generated ex-vivo primary cultures from two independent human insulinomas and from a human nesidioblastosis, all of which were cultured up to passage number 20. All cell lines secreted human insulin and C-peptide. These cell lines expressed neuroendocrine and islets markers, confirming the expression profile found in the biopsies. Although all beta cell lineages survived an anchorage independent culture, none of them were able to invade an extracellular matrix substrate. Conclusion We have established three human insulin-releasing cell lines which maintain antigenic characteristics and insulin secretion profiles of the original tumors. These cell lines represent valuable tools for the study of molecular events underlying beta cell function and dysfunction. PMID:19545371

  7. An Integrated Analysis of the Genome-Wide Profiles of DNA Methylation and mRNA Expression Defining the Side Population of a Human Malignant Mesothelioma Cell Line

    Science.gov (United States)

    Kim, Myung-Chul; Kim, Na-Yon; Seo, Yu-Ri; Kim, Yongbaek

    2016-01-01

    Intratumoral heterogeneity is a hallmark of all cancers and functions as the major barrier against effective cancer therapy. In contrast to genetic mutations, the role of epigenetic modifications in the generation and maintenance of heterogeneous cancer cells remains largely undetermined. This study was performed to evaluate the epigenetic mechanisms involved in the tumor cell heterogeneity using side population (SP) and non-SP cells isolated from a human malignant mesothelioma (HMM) cell line. The subpopulations of cancer cells were analyzed by methylated DNA immunoprecipitation combined with high-throughput sequencing (MeDIP-seq) and RNA-seq methodology. The RNA-seq data were analyzed with the MeDIP-seq data in an integrated way to identify the epigenetically modified genes that defined the SP. Concomitant changes in mRNA expression and DNA methylation were found in 122 genes, including 118 down-regulated genes with hypermethylation and 4 up-regulated genes with hypomethylation. Gene ontology revealed that a large portion of the genes belonged to the groups of biological processes such as stem cell maintenance, stem cell development, stem cell differentiation, and the negative regulation of the developmental process. Among these genes, BNC1, RPS6KA3, TWSG1 and DUSP15 contained aberrant methylation in the CpG islands of the promoter region, indicating that the genes regulated by DNA methylation characterized a distinct subpopulation of HMM cells. The present study provided valuable information to shed light on the epigenetic contributions to the generation and maintenance of tumor cell heterogeneity.

  8. Functional characterization of the human mariner transposon Hsmar2.

    Directory of Open Access Journals (Sweden)

    Estel Gil

    Full Text Available DNA transposons are mobile elements with the ability to mobilize and transport genetic information between different chromosomal loci. Unfortunately, most transposons copies are currently inactivated, little is known about mariner elements in humans despite their role in the evolution of the human genome, even though the Hsmar2 transposon is associated to hotspots for homologous recombination involved in human genetic disorders as Charcot-Marie-Tooth, Prader-Willi/Angelman, and Williams syndromes. This manuscript describes the functional characterization of the human HSMAR2 transposase generated from fossil sequences and shows that the native HSMAR2 is active in human cells, but also in bacteria, with an efficiency similar to other mariner elements. We observe that the sub-cellular localization of HSMAR2 is dependent on the host cell type, and is cytotoxic when overexpressed in HeLa cells. Finally, we also demonstrate that the binding of HSMAR2 to its own ITRs is specific, and that the excision reaction leaves non-canonical footprints both in bacteria and eukaryotic cells.

  9. Circulating Vitamin D3 and 25-hydroxyvitamin D in Humans: An Important Tool to Define Adequate Nutritional Vitamin D Status

    OpenAIRE

    Bruce W Hollis; Wagner, Carol L.; Drezner, Mark K.; Binkley, Neil C

    2007-01-01

    Circulating 25-hydroxyvitamin D [25(OH)D] is generally considered the means by which we define nutritional vitamin D status. There is much debate, however, with respect to what a healthy minimum level of circulation 25(OH)D should be. Recent data using various biomarkers such as intact parathyroid hormone (PTH), intestinal calcium absorption, and skeletal density measurements suggest this minimum level to be 80 nmol (32 ng/mL). Surprisingly, the relationship between circulating vitamin D3 and...

  10. Integrated proteogenomic characterization of human high grade serous ovarian cancer

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Hui; Liu, Tao; Zhang, Zhen; Payne, Samuel H.; Zhang, Bai; McDermott, Jason E.; Zhou, Jian-Ying; Petyuk, Vladislav A.; Chen, Li; Ray, Debjit; Sun, Shisheng; Yang, Feng; Chen, Lijun; Wang, Jing; Shah, Punut; Cha, Seong Won; Aiyetan, Paul; Woo, Sunghee; Tian, Yuan; Gritsenko, Marina A.; Clauss, Therese RW; Choi, Caitlin; Monroe, Matthew E.; Thomas, Stefani N.; Nie, Song; Wu, Chaochao; Moore, Ronald J.; Yu, Kun-Hsing; Tabb, David L.; Fenyo, David; Bafna, Vineet; Wang, Yue; Rodriguez, Henry; Boja, Emily; Hiltket, Tara; Rivers, Robert; Sokoll, Lori J.; Zhu, Heng; Shih, Ie-Ming; Cope, Leslie; Pandey, Akhilesh; Zhang, Bing; Snyder, Michael; Levine, Douglas; Smith, Richard D.; Chan, Daniel W.; Rodland, Karin D.

    2016-07-28

    Ovarian cancer remains the most lethal gynecological malignancy in the developed world, despite recent advances in genomic information and treatment. To better understand this disease, define an integrated proteogenomic landscape, and identify factors associated with homologous repair deficiency (HRD) and overall survival, we performed a comprehensive proteomic characterization of ovarian high-grade serous carcinomas (HGSC) previously characterized by The Cancer Genome Atlas (TCGA). We observed that messenger RNA transcript abundance did not reliably predict abundance for 10,030 proteins across 174 tumors. Clustering of tumors based on protein abundance identified five subtypes, two of which correlated robustly with mesenchymal and proliferative subtypes, while tumors characterized as immunoreactive or differentiated at the transcript level were intermixed at the protein level. At the genome level, HGSC is characterized by a complex landscape of somatic copy number alterations (CNA), which individually do not correlate significantly with survival. Correlation of CNAs with protein abundances identified loci with significant trans regulatory effects mapping to pathways associated with proliferation, cell motility/invasion, and immune regulation, three known hallmarks of cancer. Using the trans regulated proteins we also created models significantly correlated with patient survival by multivariate analysis. Integrating protein abundance with specific post-translational modification data identified subnetworks correlated with HRD status; specifically, acetylation of Lys12 and Lys16 on histone H4 was associated with HRD status. Using quantitative phosphoproteomics data covering 4,420 proteins as reflective of pathway activity, we identified the PDGFR and VEGFR signaling pathways as significantly up-regulated in patients with short overall survival, independent of PDGFR and VEGFR protein levels, potentially informing the use of anti-angiogenic therapies. Components of

  11. Defined Conditions for the Isolation and Expansion of Basal Prostate Progenitor Cells of Mouse and Human Origin

    OpenAIRE

    Thomas Höfner; Christian Eisen; Corinna Klein; Teresa Rigo-Watermeier; Stephan M. Goeppinger; Anna Jauch; Brigitte Schoell; Vanessa Vogel; Elisa Noll; Wilko Weichert; Irène Baccelli; Anja Schillert; Steve Wagner; Sascha Pahernik; Martin R. Sprick

    2015-01-01

    Summary Methods to isolate and culture primary prostate epithelial stem/progenitor cells (PESCs) have proven difficult and ineffective. Here, we present a method to grow and expand both murine and human basal PESCs long term in serum- and feeder-free conditions. The method enriches for adherent mouse basal PESCs with a Lin−SCA-1+CD49f+TROP2high phenotype. Progesterone and sodium selenite are additionally required for the growth of human Lin−CD49f+TROP2high PESCs. The gene-expression profiles ...

  12. The monitoring of gene functions on a cell-defined siRNA microarray in human bone marrow stromal and U2OS cells

    Directory of Open Access Journals (Sweden)

    Hi Chul Kim

    2016-06-01

    Full Text Available Here, we developed a cell defined siRNA microarray (CDSM for human bone marrow stromal cells (hBMSCs designed to control the culture of cells inside the spot area without reducing the efficiency of siRNA silencing, “Development of a cell-defined siRNA microarray for analysis of gene functionin human bone marrow stromal cells” (Kim et al., 2016 [1]. First, we confirmed that p65 protein inhibition efficiency was maintained when hBMSCs were culture for 7 days on the siRNA spot, and siRNA spot activity remained in spite of long term storage (10 days and 2 months. Additionally, we confirmed p65 protein inhibition in U2OS cells after 48 h reverse transfection.

  13. Characterization of NAADP-mediated calcium signaling in human spermatozoa

    Energy Technology Data Exchange (ETDEWEB)

    Sánchez-Tusie, A.A. [Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos (Mexico); Vasudevan, S.R.; Churchill, G.C. [Department of Pharmacology, University of Oxford, Oxford OX1 3QT, England (United Kingdom); Nishigaki, T. [Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos (Mexico); Treviño, C.L., E-mail: ctrevino@ibt.unam.mx [Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos (Mexico)

    2014-01-10

    Highlights: •Human sperm cells synthesize NAADP. •NAADP-AM mediates [Ca{sup 2+}]{sub i} increases in human sperm in the absence of [Ca{sup 2+}]{sub o}. •Human sperm have two acidic compartments located in the head and midpiece. -- Abstract: Ca{sup 2+} signaling in spermatozoa plays a crucial role during processes such as capacitation and release of the acrosome, but the underlying molecular mechanisms still remain unclear. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca{sup 2+}-releasing second messenger in a variety of cellular processes. The presence of a NAADP synthesizing enzyme in sea urchin sperm has been previously reported, suggesting a possible role of NAADP in sperm Ca{sup 2+} signaling. In this work we used in vitro enzyme assays to show the presence of a novel NAADP synthesizing enzyme in human sperm, and to characterize its sensitivity to Ca{sup 2+} and pH. Ca{sup 2+} fluorescence imaging studies demonstrated that the permeable form of NAADP (NAADP-AM) induces intracellular [Ca{sup 2+}] increases in human sperm even in the absence of extracellular Ca{sup 2+}. Using LysoTracker®, a fluorescent probe that selectively accumulates in acidic compartments, we identified two such stores in human sperm cells. Their acidic nature was further confirmed by the reduction in staining intensity observed upon inhibition of the endo-lysosomal proton pump with Bafilomycin, or after lysosomal bursting with glycyl-L-phenylalanine-2-naphthylamide. The selective fluorescent NAADP analog, Ned-19, stained the same subcellular regions as LysoTracker®, suggesting that these stores are the targets of NAADP action.

  14. Characterization of NAADP-mediated calcium signaling in human spermatozoa

    International Nuclear Information System (INIS)

    Highlights: •Human sperm cells synthesize NAADP. •NAADP-AM mediates [Ca2+]i increases in human sperm in the absence of [Ca2+]o. •Human sperm have two acidic compartments located in the head and midpiece. -- Abstract: Ca2+ signaling in spermatozoa plays a crucial role during processes such as capacitation and release of the acrosome, but the underlying molecular mechanisms still remain unclear. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+-releasing second messenger in a variety of cellular processes. The presence of a NAADP synthesizing enzyme in sea urchin sperm has been previously reported, suggesting a possible role of NAADP in sperm Ca2+ signaling. In this work we used in vitro enzyme assays to show the presence of a novel NAADP synthesizing enzyme in human sperm, and to characterize its sensitivity to Ca2+ and pH. Ca2+ fluorescence imaging studies demonstrated that the permeable form of NAADP (NAADP-AM) induces intracellular [Ca2+] increases in human sperm even in the absence of extracellular Ca2+. Using LysoTracker®, a fluorescent probe that selectively accumulates in acidic compartments, we identified two such stores in human sperm cells. Their acidic nature was further confirmed by the reduction in staining intensity observed upon inhibition of the endo-lysosomal proton pump with Bafilomycin, or after lysosomal bursting with glycyl-L-phenylalanine-2-naphthylamide. The selective fluorescent NAADP analog, Ned-19, stained the same subcellular regions as LysoTracker®, suggesting that these stores are the targets of NAADP action

  15. Characterization of the Phosphoproteome in Human Bronchoalveolar Lavage Fluid

    Directory of Open Access Journals (Sweden)

    Francesco Giorgianni

    2012-01-01

    Full Text Available Global-scale examination of protein phosphorylation in human biological fluids by phosphoproteomics approaches is an emerging area of research with potential for significant contributions towards discovery of novel biomarkers. In this pilot work, we analyzed the phosphoproteome in human bronchoalveolar lavage fluid (BAL from nondiseased subjects. The main objectives were to assess the feasibility to probe phosphorylated proteins in human BAL and to obtain the initial catalog of BAL phosphoproteins, including protein identities and exact description of their phosphorylation sites. We used a gel-free bioanalytical workflow that included whole-proteome digestion of depleted BAL proteins, enrichment of phosphopeptides by immobilized metal ion affinity chromatography (IMAC, LC-MS/MS analyses with a linear ion trap mass spectrometer, and searches of a protein sequence database to generate a panel of BAL phosphoproteins and their sites of phosphorylation. Based on sequence-diagnostic MS/MS fragmentation patterns, we identified a collection of 36 phosphopeptides that contained 26 different phosphorylation sites. These phosphopeptides mapped to 21 phosphoproteins including, for example, vimentin, plastin-2, ferritin heavy chain, kininogen-1, and others. The characterized phosphoproteins have diverse characteristics in terms of cellular origin and biological function. To the best of our knowledge, results of this study represent the first description of the human BAL phosphoproteome.

  16. Towards spatially smart abatement of human pharmaceuticals in surface waters: defining impact of sewage treatment plants on susceptible functions

    NARCIS (Netherlands)

    Gils, J.A.G.; Coppens, L.J.C.; Laak, ter T.L.; Raterman, B.W.; Wezel, van A.P.

    2015-01-01

    For human pharmaceuticals, sewage treatment plants (STPs) are a major point of entry to surface waters. The receiving waters provide vital functions. Modeling the impact of STPs on susceptible functions of the surface water system allows for a spatially smart implementation of abatement options at,

  17. Characterization of novel neutralizing monoclonal antibodies specific to human neurturin.

    Science.gov (United States)

    Hongo, J A; Tsai, S P; Moffat, B; Schroeder, K A; Jung, C; Chuntharapai, A; Lampe, P A; Johnson, E M; de Sauvage, F J; Armanini, M; Phillips, H; Devaux, B

    2000-08-01

    Neurturin (NTN) a structural and functional relative of glial cell line-derived neurotrophic factor, was originally identified based on its ability to support the survival of sympathetic neurons in culture. Similar to glial cell line-derived neurotrophic factor (GDNF), Neurturin has been shown to bind to a high affinity glycosylphosphatidylinositol (GPI)-linked receptor (GFRalpha2) and induce phosphorylation of the tyrosine kinase receptor Ret, resulting in the activation of the mitogen activated protein kinase (MAPK) signalling pathway. A panel of six novel murine monoclonal antibodies (MAbs) specific to human Neurturin has been developed and characterized. Four of the MAbs tested inhibit, to varying degrees, binding of NTN to the GPI-linked GFRalpha2 receptor. Three MAbs cross-react with the murine homolog. These antibodies have been shown to be useful reagents for Western blotting, immunohistochemistry, and also for the development of a sensitive, quantitative enzyme-linked immunosorbent assay (ELISA) for human NTN. Novel, specific MAbs with varying epitope specificities and blocking activity will be valuable tools for both the in vitro and in vivo characterization of NTN and its relationship to the GFRalpha2 and Ret receptors. PMID:11001403

  18. Universal power-law scaling of water diffusion in human brain defines what we see with MRI

    CERN Document Server

    Veraart, Jelle; Novikov, Dmitry S

    2016-01-01

    Development of successful therapies for neurological disorders depends on our ability to diagnose and monitor the progression of underlying pathologies at the cellular level. Physics and physiology limit the resolution of human MRI to millimeters, three orders of magnitude coarser than the cell dimensions of microns. A promising way to access cellular structure is provided by diffusion-weighted MRI (dMRI), a modality which exploits the sensitivity of the MRI signal to micron-level Brownian motion of water molecules strongly hindered by cell walls. By analyzing diffusion of water molecules in human subjects, here we demonstrate that biophysical modeling has the potential to break the intrinsic MRI resolution limits. The observation of a universal power-law scaling of the dMRI signal identifies the contribution from water specifically confined inside narrow impermeable axons, validating the overarching assumption behind models of diffusion in neuronal tissue. This scaling behavior establishes dMRI as an in vivo...

  19. Methods for defining distinct bioenergetic profiles in platelets, lymphocytes, monocytes, and neutrophils, and the oxidative burst from human blood

    OpenAIRE

    Chacko, Balu K; Kramer, Philip A.; Ravi, Saranya; Johnson, Michelle S.; Hardy, Robert W.; Ballinger, Scott W.; Darley-Usmar, Victor M.

    2013-01-01

    Peripheral blood mononuclear cells and platelets have long been recognized as having the potential to act as sensitive markers for mitochondrial dysfunction in a broad range of pathological conditions. However, the bioenergetic function of these cells has not been examined from the same donors, yet this is important for the selection of cell types for translational studies. Here, we demonstrate the measurement of cellular bioenergetics in isolated human monocytes, lymphocytes, and platelets, ...

  20. Metabolomic analysis of human fecal microbiota: a comparison of feces-derived communities and defined mixed communities.

    Science.gov (United States)

    Yen, Sandi; McDonald, Julie A K; Schroeter, Kathleen; Oliphant, Kaitlyn; Sokolenko, Stanislav; Blondeel, Eric J M; Allen-Vercoe, Emma; Aucoin, Marc G

    2015-03-01

    The extensive impact of the human gut microbiota on its human host calls for a need to understand the types of communication that occur among the bacteria and their host. A metabolomics approach can provide a snapshot of the microbe-microbe interactions occurring as well as variations in the microbes from different hosts. In this study, metabolite profiles from an anaerobic continuous stirred-tank reactors (CSTR) system supporting the growth of several consortia of bacteria representative of the human gut were established and compared. Cell-free supernatant samples were analyzed by 1D (1)H nuclear magnetic resonance (NMR) spectroscopy, producing spectra representative of the metabolic activity of a particular community at a given time. Using targeted profiling, specific metabolites were identified and quantified on the basis of NMR analyses. Metabolite profiles discriminated each bacterial community examined, demonstrating that there are significant differences in the microbiota metabolome between each cultured community. We also found unique compounds that were identifying features of individual bacterial consortia. These findings are important because they demonstrate that metabolite profiles of gut microbial ecosystems can be constructed by targeted profiling of NMR spectra. Moreover, examination of these profiles sheds light on the type of microbes present in the gut and their metabolic interactions.

  1. Molecular clocks and the human condition: approaching their characterization in human physiology and disease.

    Science.gov (United States)

    Fitzgerald, G A; Yang, G; Paschos, G K; Liang, X; Skarke, C

    2015-09-01

    Molecular clockworks knit together diverse biological networks and compelling evidence from model systems infers their importance in metabolism, immunological and cardiovascular function. Despite this and the diurnal variation in many aspects of human physiology and the phenotypic expression of disease, our understanding of the role and importance of clock function and dysfunction in humans is modest. There are tantalizing hints of connection across the translational divide and some correlative evidence of gene variation and human disease but most of what we know derives from forced desynchrony protocols in controlled environments. We now have the ability to monitor quantitatively ex vivo or in vivo the genome, metabolome, proteome and microbiome of humans in the wild. Combining this capability, with the power of mobile telephony and the evolution of remote sensing, affords a new opportunity for deep phenotyping, including the characterization of diurnal behaviour and the assessment of the impact of the clock on approved drug function. PMID:26332979

  2. Genetic Characterization and Classification of Human and Animal Sapoviruses.

    Directory of Open Access Journals (Sweden)

    Tomoichiro Oka

    Full Text Available Sapoviruses (SaVs are enteric caliciviruses that have been detected in multiple mammalian species, including humans, pigs, mink, dogs, sea lions, chimpanzees, and rats. They show a high level of diversity. A SaV genome commonly encodes seven nonstructural proteins (NSs, including the RNA polymerase protein NS7, and two structural proteins (VP1 and VP2. We classified human and animal SaVs into 15 genogroups (G based on available VP1 sequences, including three newly characterized genomes from this study. We sequenced the full length genomes of one new genogroup V (GV, one GVII and one GVIII porcine SaV using long range RT-PCR including newly designed forward primers located in the conserved motifs of the putative NS3, and also 5' RACE methods. We also determined the 5'- and 3'-ends of sea lion GV SaV and canine GXIII SaV. Although the complete genomic sequences of GIX-GXII, and GXV SaVs are unavailable, common features of SaV genomes include: 1 "GTG" at the 5'-end of the genome, and a short (9~14 nt 5'-untranslated region; and 2 the first five amino acids (M [A/V] S [K/R] P of the putative NS1 and the five amino acids (FEMEG surrounding the putative cleavage site between NS7 and VP1 were conserved among the chimpanzee, two of five genogroups of pig (GV and GVIII, sea lion, canine, and human SaVs. In contrast, these two amino acid motifs were clearly different in three genogroups of porcine (GIII, GVI and GVII, and bat SaVs. Our results suggest that several animal SaVs have genetic similarities to human SaVs. However, the ability of SaVs to be transmitted between humans and animals is uncertain.

  3. Genetic Characterization and Classification of Human and Animal Sapoviruses.

    Science.gov (United States)

    Oka, Tomoichiro; Lu, Zhongyan; Phan, Tung; Delwart, Eric L; Saif, Linda J; Wang, Qiuhong

    2016-01-01

    Sapoviruses (SaVs) are enteric caliciviruses that have been detected in multiple mammalian species, including humans, pigs, mink, dogs, sea lions, chimpanzees, and rats. They show a high level of diversity. A SaV genome commonly encodes seven nonstructural proteins (NSs), including the RNA polymerase protein NS7, and two structural proteins (VP1 and VP2). We classified human and animal SaVs into 15 genogroups (G) based on available VP1 sequences, including three newly characterized genomes from this study. We sequenced the full length genomes of one new genogroup V (GV), one GVII and one GVIII porcine SaV using long range RT-PCR including newly designed forward primers located in the conserved motifs of the putative NS3, and also 5' RACE methods. We also determined the 5'- and 3'-ends of sea lion GV SaV and canine GXIII SaV. Although the complete genomic sequences of GIX-GXII, and GXV SaVs are unavailable, common features of SaV genomes include: 1) "GTG" at the 5'-end of the genome, and a short (9~14 nt) 5'-untranslated region; and 2) the first five amino acids (M [A/V] S [K/R] P) of the putative NS1 and the five amino acids (FEMEG) surrounding the putative cleavage site between NS7 and VP1 were conserved among the chimpanzee, two of five genogroups of pig (GV and GVIII), sea lion, canine, and human SaVs. In contrast, these two amino acid motifs were clearly different in three genogroups of porcine (GIII, GVI and GVII), and bat SaVs. Our results suggest that several animal SaVs have genetic similarities to human SaVs. However, the ability of SaVs to be transmitted between humans and animals is uncertain.

  4. Genetic Characterization and Classification of Human and Animal Sapoviruses

    Science.gov (United States)

    Oka, Tomoichiro; Lu, Zhongyan; Phan, Tung; Delwart, Eric L.; Saif, Linda J.; Wang, Qiuhong

    2016-01-01

    Sapoviruses (SaVs) are enteric caliciviruses that have been detected in multiple mammalian species, including humans, pigs, mink, dogs, sea lions, chimpanzees, and rats. They show a high level of diversity. A SaV genome commonly encodes seven nonstructural proteins (NSs), including the RNA polymerase protein NS7, and two structural proteins (VP1 and VP2). We classified human and animal SaVs into 15 genogroups (G) based on available VP1 sequences, including three newly characterized genomes from this study. We sequenced the full length genomes of one new genogroup V (GV), one GVII and one GVIII porcine SaV using long range RT-PCR including newly designed forward primers located in the conserved motifs of the putative NS3, and also 5' RACE methods. We also determined the 5’- and 3’-ends of sea lion GV SaV and canine GXIII SaV. Although the complete genomic sequences of GIX-GXII, and GXV SaVs are unavailable, common features of SaV genomes include: 1) “GTG” at the 5′-end of the genome, and a short (9~14 nt) 5′-untranslated region; and 2) the first five amino acids (M [A/V] S [K/R] P) of the putative NS1 and the five amino acids (FEMEG) surrounding the putative cleavage site between NS7 and VP1 were conserved among the chimpanzee, two of five genogroups of pig (GV and GVIII), sea lion, canine, and human SaVs. In contrast, these two amino acid motifs were clearly different in three genogroups of porcine (GIII, GVI and GVII), and bat SaVs. Our results suggest that several animal SaVs have genetic similarities to human SaVs. However, the ability of SaVs to be transmitted between humans and animals is uncertain. PMID:27228126

  5. Mapping of Variable DNA Methylation Across Multiple Cell Types Defines a Dynamic Regulatory Landscape of the Human Genome

    Science.gov (United States)

    Gu, Junchen; Stevens, Michael; Xing, Xiaoyun; Li, Daofeng; Zhang, Bo; Payton, Jacqueline E.; Oltz, Eugene M.; Jarvis, James N.; Jiang, Kaiyu; Cicero, Theodore; Costello, Joseph F.; Wang, Ting

    2016-01-01

    DNA methylation is an important epigenetic modification involved in many biological processes and diseases. Many studies have mapped DNA methylation changes associated with embryogenesis, cell differentiation, and cancer at a genome-wide scale. Our understanding of genome-wide DNA methylation changes in a developmental or disease-related context has been steadily growing. However, the investigation of which CpGs are variably methylated in different normal cell or tissue types is still limited. Here, we present an in-depth analysis of 54 single-CpG-resolution DNA methylomes of normal human cell types by integrating high-throughput sequencing-based methylation data. We found that the ratio of methylated to unmethylated CpGs is relatively constant regardless of cell type. However, which CpGs made up the unmethylated complement was cell-type specific. We categorized the 26,000,000 human autosomal CpGs based on their methylation levels across multiple cell types to identify variably methylated CpGs and found that 22.6% exhibited variable DNA methylation. These variably methylated CpGs formed 660,000 variably methylated regions (VMRs), encompassing 11% of the genome. By integrating a multitude of genomic data, we found that VMRs enrich for histone modifications indicative of enhancers, suggesting their role as regulatory elements marking cell type specificity. VMRs enriched for transcription factor binding sites in a tissue-dependent manner. Importantly, they enriched for GWAS variants, suggesting that VMRs could potentially be implicated in disease and complex traits. Taken together, our results highlight the link between CpG methylation variation, genetic variation, and disease risk for many human cell types. PMID:26888867

  6. Differential DNA methylation profiles of coding and non-coding genes define hippocampal sclerosis in human temporal lobe epilepsy

    Science.gov (United States)

    Miller-Delaney, Suzanne F.C.; Bryan, Kenneth; Das, Sudipto; McKiernan, Ross C.; Bray, Isabella M.; Reynolds, James P.; Gwinn, Ryder; Stallings, Raymond L.

    2015-01-01

    Temporal lobe epilepsy is associated with large-scale, wide-ranging changes in gene expression in the hippocampus. Epigenetic changes to DNA are attractive mechanisms to explain the sustained hyperexcitability of chronic epilepsy. Here, through methylation analysis of all annotated C-phosphate-G islands and promoter regions in the human genome, we report a pilot study of the methylation profiles of temporal lobe epilepsy with or without hippocampal sclerosis. Furthermore, by comparative analysis of expression and promoter methylation, we identify methylation sensitive non-coding RNA in human temporal lobe epilepsy. A total of 146 protein-coding genes exhibited altered DNA methylation in temporal lobe epilepsy hippocampus (n = 9) when compared to control (n = 5), with 81.5% of the promoters of these genes displaying hypermethylation. Unique methylation profiles were evident in temporal lobe epilepsy with or without hippocampal sclerosis, in addition to a common methylation profile regardless of pathology grade. Gene ontology terms associated with development, neuron remodelling and neuron maturation were over-represented in the methylation profile of Watson Grade 1 samples (mild hippocampal sclerosis). In addition to genes associated with neuronal, neurotransmitter/synaptic transmission and cell death functions, differential hypermethylation of genes associated with transcriptional regulation was evident in temporal lobe epilepsy, but overall few genes previously associated with epilepsy were among the differentially methylated. Finally, a panel of 13, methylation-sensitive microRNA were identified in temporal lobe epilepsy including MIR27A, miR-193a-5p (MIR193A) and miR-876-3p (MIR876), and the differential methylation of long non-coding RNA documented for the first time. The present study therefore reports select, genome-wide DNA methylation changes in human temporal lobe epilepsy that may contribute to the molecular architecture of the epileptic brain. PMID

  7. Defining the Pathogenesis of the Human Atp12p W94R Mutation Using a Saccharomyces cerevisiae Yeast Model*

    OpenAIRE

    Meulemans, Ann; Seneca, Sara; Pribyl, Thomas; Smet, Joel; Alderweirldt, Valerie; Waeytens, Anouk; Lissens, Willy; Van Coster, Rudy; De Meirleir, Linda; di Rago, Jean-Paul; Gatti, Domenico L; Ackerman, Sharon H.

    2009-01-01

    Studies in yeast have shown that a deficiency in Atp12p prevents assembly of the extrinsic domain (F1) of complex V and renders cells unable to make ATP through oxidative phosphorylation. De Meirleir et al. (De Meirleir, L., Seneca, S., Lissens, W., De Clercq, I., Eyskens, F., Gerlo, E., Smet, J., and Van Coster, R. (2004) J. Med. Genet. 41, 120–124) have reported that a homozygous missense mutation in the gene for human Atp12p (HuAtp12p), which replaces Trp-94 with Arg, was linked to the dea...

  8. Unsolved Mysteries of the Human Mammary Gland: Defining and Redefining the Critical Questions from the Lactation Consultant's Perspective.

    Science.gov (United States)

    Marasco, Lisa Ann

    2014-12-01

    Despite advances in knowledge about human lactation, clinicians face many problems when advising mothers who are experiencing breastfeeding difficulties that do not respond to normal management strategies. Primary insufficient milk production is now being acknowledged, but incidence rates have not been well studied. Many women have known histories of infertility, polycystic ovary syndrome, obesity, hypertension, insulin resistance, thyroid dysfunction, hyperandrogenism or other hormonal imbalances, while others have no obvious risk factors. Some present with obviously abnormal breasts that are pubescent, tuberous/tubular or asymmetric in shape, raising the question of insufficient mammary gland tissue. Other women have breasts that appear within normal limits yet do not lactate normally. Endocrine disruptors may underlie some of these cases but their impact on human milk production has not been well explored. Similarly, any problem with prolactin such as a deficiency in serum prolactin or receptor number, receptor resistance, or poor bioavailability or bioactivity could underlie some cases of insufficient lactation, yet these possibilities are rarely investigated. A weak or suppressed milk ejection reflex, often assumed to be psychosomatic, could be related to thyroid dysfunction or caused by downstream post-receptor pathway problems. In the absence of sufficient data regarding these situations, desperate mothers may turn to non-evidence-based remedies, sometimes at considerable cost and unknown risk. Research targeted to these clinical dilemmas is critical in order to develop evidence-based strategies and increase breastfeeding duration and success rates. PMID:26084427

  9. Injectable hybrid hydrogels of hyaluronic Acid crosslinked by well-defined synthetic polycations: preparation and characterization in vitro and in vivo.

    Science.gov (United States)

    Cross, Daisy; Jiang, Xiaoze; Ji, Weihang; Han, Wenqing; Wang, Chun

    2015-05-01

    An injectable hybrid hydrogel system was developed consisting of hyaluronic acid (HA) crosslinked by well-defined block copolymers of the cationic poly(2-aminoethyl methacrylate) (PAEM) and polyethylene glycol (PEG). Robust, shear-thinning hybrid hydrogel was produced by mixing HA and 4-arm star PEG-PAEM block copolymer at 1:1 charge ratio. The encapsulation and release of highly viable human mesenchymal stem cells in physiological media was demonstrated. After subcutaneous injection of the hybrid gel in mice, mild but resolvable inflammatory response was observed. This hybrid gel could serve as a model system for studying structure-function relationship of polyelectrolyte hydrogels and as a practical injectable biomaterial for medical applications. PMID:25630277

  10. Xeno-Free and Defined Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells Functionally Integrate in a Large-Eyed Preclinical Model

    Directory of Open Access Journals (Sweden)

    Alvaro Plaza Reyes

    2016-01-01

    Full Text Available Human embryonic stem cell (hESC-derived retinal pigment epithelial (RPE cells could replace lost tissue in geographic atrophy (GA but efficacy has yet to be demonstrated in a large-eyed model. Also, production of hESC-RPE has not yet been achieved in a xeno-free and defined manner, which is critical for clinical compliance and reduced immunogenicity. Here we describe an effective differentiation methodology using human laminin-521 matrix with xeno-free and defined medium. Differentiated cells exhibited characteristics of native RPE including morphology, pigmentation, marker expression, monolayer integrity, and polarization together with phagocytic activity. Furthermore, we established a large-eyed GA model that allowed in vivo imaging of hESC-RPE and host retina. Cells transplanted in suspension showed long-term integration and formed polarized monolayers exhibiting phagocytic and photoreceptor rescue capacity. We have developed a xeno-free and defined hESC-RPE differentiation method and present evidence of functional integration of clinically compliant hESC-RPE in a large-eyed disease model.

  11. The meta-epigenomic structure of purified human stem cell populations is defined at cis-regulatory sequences

    Science.gov (United States)

    Zhao, Yong Mei; Golden, Aaron; Mar, Jessica C.; Einstein, Francine H.; Greally, John M.

    2014-01-01

    The mechanism and significance of epigenetic variability in the same cell type between healthy individuals are not clear. Here, we purify human CD34+ hematopoietic stem and progenitor cells (HSPCs) from different individuals and find that there is increased variability of DNA methylation at loci with properties of promoters and enhancers. The variability is especially enriched at candidate enhancers near genes transitioning between silent and expressed states, and encoding proteins with leukocyte differentiation properties. Our findings of increased variability at loci with intermediate DNA methylation values, at candidate “poised” enhancers, and at genes involved in HSPC lineage commitment suggest that CD34+ cell subtype heterogeneity between individuals is a major mechanism for the variability observed. Epigenomic studies performed on cell populations, even when purified, are testing collections of epigenomes, or meta-epigenomes. Our findings show that meta-epigenomic approaches to data analysis can provide insights into cell subpopulation structure. PMID:25327398

  12. Characterization of acid sphingomyelinase activity in human cerebrospinal fluid.

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    Christiane Mühle

    Full Text Available BACKGROUND: As a key enzyme in sphingolipid metabolism, acid sphingomyelinase (ASM is involved in the regulation of cell fate and signaling via hydrolysis of sphingomyelin to form ceramide. While increased activity of the lysosomal form has been associated with various pathological conditions, there are few studies on secretory ASM limited only to cell models, plasma or serum. METHODS: An optimized assay based on a fluorescent substrate was applied to measure the ASM activity in cerebrospinal fluid (CSF collected from mice and from 42 patients who were classified as controls based on normal routine CSF values. RESULTS: We have detected ASM activity in human CSF, established a sensitive quantitative assay and characterized the enzyme's properties. The enzyme resembles plasmatic ASM including protein stability and Zn(2+-dependence but the assays differ considerably in the optimal detergent concentration. Significantly increased activities in the CSF of ASM transgenic mice and undetectable levels in ASM knock-out mice prove that the measured ASM activity originates from the ASM-encoding gene SMPD1. CSF localized ASM activities were comparable to corresponding serum ASM levels at their respective optimal reaction conditions, but no correlation was observed. The large variance in ASM activity was independent of sex, age or analyzed routine CSF parameters. CONCLUSIONS: Human and mouse CSF contain detectable levels of secretory ASM, which are unrelated to serum ASM activities. Further investigations in humans and in animal models will help to elucidate the role of this enzyme in human disease and to assess its value as a potential biomarker for disease type, severity, progress or therapeutic success.

  13. Defining GERD.

    Science.gov (United States)

    Sontag, S J

    1999-01-01

    "It is not the death of GERD that I seek, but that it turns from its evil ways and follows the path of righteousness." The reflux world is fully aware of what GERD is and what GERD does. What the world does not know, however, is the answer to the most important yet least asked question surrounding GERD's raison-d'etre: Why is GERD here and why do we have it? What GERD is: abnormal gastric reflux into the esophagus that causes any type of mischief. What GERD does: causes discomfort and/or pain with or without destroying the mucosa; causes stricture or stenosis, preventing food from being swallowed; sets the stage for the development of esophageal adenocarcinoma; invades the surrounding lands to harass the peaceful oropharyngeal, laryngeal and broncho-pulmonary territories; reminds us that we are not only human, but that we are dust and ashes. Why GERD is here: We propose three separate and distinct etiologies of GERD, and we offer the following three hypotheses to explain why, after 1.5 million years of standing erect, we have evolved into a species (specifically Homosapiens sapiens) that is destined to live with the scourge of GERD. Hypothesis 1: congenital. The antireflux barrier, comprising the smooth-muscled lower esophageal sphincter, the skeletal-muscled right crural diaphragm and the phreno-esophageal ligament does not completely develop due to a developmental anomaly or incomplete gestation. Hypothesis 2: acute trauma: The antireflux barrier in adults suffering acute traumatic injury to the abdomen or chest is permanently disrupted by unexpected forces, such as motor vehicle accidents (with steering wheel crush impact), blows to the abdomen (from activities such as boxing, etc.), heavy lifting or moving (e.g., pianos, refrigerators) or stress positions (e.g., hand stands on parallel gym bars). The trauma creates a hiatal hernia that renders the antireflux mechanism useless and incapable of preventing GERD. Hypothesis 3: chronic trauma: The antireflux barrier

  14. Isolation and Characterization of Human Lung Lymphatic Endothelial Cells

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    Bruno Lorusso

    2015-01-01

    Full Text Available Characterization of lymphatic endothelial cells from the respiratory system may be crucial to investigate the role of the lymphatic system in the normal and diseased lung. We describe a simple and inexpensive method to harvest, isolate, and expand lymphatic endothelial cells from the human lung (HL-LECs. Fifty-five samples of healthy lung selected from patients undergoing lobectomy were studied. A two-step purification tool, based on paramagnetic sorting with monoclonal antibodies to CD31 and Podoplanin, was employed to select a pure population of HL-LECs. The purity of HL-LECs was assessed by morphologic criteria, immunocytochemistry, flow cytometry, and functional assays. Interestingly, these cells retain in vitro several receptor tyrosine kinases (RTKs implicated in cell survival and proliferation. HL-LECs represent a clinically relevant cellular substrate to study lymphatic biology, lymphoangiogenesis, interaction with microbial agents, wound healing, and anticancer therapy.

  15. Expression, purification and characterization of human Dopamine ß-monooxygenase

    DEFF Research Database (Denmark)

    Vendelboe, Trine Vammen

    others, one of the reasons why these proteins are considered to follow the same mechanism. DBM converts dopamine (DA) into Norepinphrine (NE). Both substrate and product functions as neurotransmitters and the levels of these are involved in many different disorders such as depression and hypertension...... and also the reason why DBM in the past decades has been considered an important therapeutic target. The ambition with this project was to develop a solid and efficient expression system for human DBM (hDBM) and subsequent to characterize it with mass spectrometry (MS) and X-ray crystallography. hDBM has...... that the tetramer was significantly more stable than the dimer and that to two tetramer forms were equally stable. The effect of ascorbic acid (Asc) and DA on the stability of the protein was also evaluated with DSF, and the conclusion was that the presence of both substrates had a significant effect...

  16. Defined MicroRNAs Induce Aspects of Maturation in Mouse and Human Embryonic-Stem-Cell-Derived Cardiomyocytes

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    Desy S. Lee

    2015-09-01

    Full Text Available Pluripotent-cell-derived cardiomyocytes have great potential for use in research and medicine, but limitations in their maturity currently constrain their usefulness. Here, we report a method for improving features of maturation in murine and human embryonic-stem-cell-derived cardiomyocytes (m/hESC-CMs. We found that coculturing m/hESC-CMs with endothelial cells improves their maturity and upregulates several microRNAs. Delivering four of these microRNAs, miR-125b-5p, miR-199a-5p, miR-221, and miR-222 (miR-combo, to m/hESC-CMs resulted in improved sarcomere alignment and calcium handling, a more negative resting membrane potential, and increased expression of cardiomyocyte maturation markers. Although this could not fully phenocopy all adult cardiomyocyte characteristics, these effects persisted for two months following delivery of miR-combo. A luciferase assay demonstrated that all four miRNAs target ErbB4, and siRNA knockdown of ErbB4 partially recapitulated the effects of miR-combo. In summary, a combination of miRNAs induced via endothelial coculture improved ESC-CM maturity, in part through suppression of ErbB4 signaling.

  17. Integrating mechanistic and polymorphism data to characterize human genetic susceptibility for environmental chemical risk assessment in the 21st century

    Energy Technology Data Exchange (ETDEWEB)

    Mortensen, Holly M., E-mail: mortensen.holly@epa.gov [Office of Research and Development, US Environmental Protection Agency, National Center for Computational Toxicology, US EPA, 109 TW Alexander Dr., Mailcode B205-01, Research Triangle Park, NC 27711 (United States); Euling, Susan Y. [Office of Research and Development, US Environmental Protection Agency, National Center for Environmental Assessment, US EPA, 1200 Pennsylvania Ave., NW, Mail Code 8623P, Washington, DC 20460 (United States)

    2013-09-15

    Response to environmental chemicals can vary widely among individuals and between population groups. In human health risk assessment, data on susceptibility can be utilized by deriving risk levels based on a study of a susceptible population and/or an uncertainty factor may be applied to account for the lack of information about susceptibility. Defining genetic susceptibility in response to environmental chemicals across human populations is an area of interest in the NAS' new paradigm of toxicity pathway-based risk assessment. Data from high-throughput/high content (HT/HC), including -omics (e.g., genomics, transcriptomics, proteomics, metabolomics) technologies, have been integral to the identification and characterization of drug target and disease loci, and have been successfully utilized to inform the mechanism of action for numerous environmental chemicals. Large-scale population genotyping studies may help to characterize levels of variability across human populations at identified target loci implicated in response to environmental chemicals. By combining mechanistic data for a given environmental chemical with next generation sequencing data that provides human population variation information, one can begin to characterize differential susceptibility due to genetic variability to environmental chemicals within and across genetically heterogeneous human populations. The integration of such data sources will be informative to human health risk assessment.

  18. Integrating mechanistic and polymorphism data to characterize human genetic susceptibility for environmental chemical risk assessment in the 21st century

    International Nuclear Information System (INIS)

    Response to environmental chemicals can vary widely among individuals and between population groups. In human health risk assessment, data on susceptibility can be utilized by deriving risk levels based on a study of a susceptible population and/or an uncertainty factor may be applied to account for the lack of information about susceptibility. Defining genetic susceptibility in response to environmental chemicals across human populations is an area of interest in the NAS' new paradigm of toxicity pathway-based risk assessment. Data from high-throughput/high content (HT/HC), including -omics (e.g., genomics, transcriptomics, proteomics, metabolomics) technologies, have been integral to the identification and characterization of drug target and disease loci, and have been successfully utilized to inform the mechanism of action for numerous environmental chemicals. Large-scale population genotyping studies may help to characterize levels of variability across human populations at identified target loci implicated in response to environmental chemicals. By combining mechanistic data for a given environmental chemical with next generation sequencing data that provides human population variation information, one can begin to characterize differential susceptibility due to genetic variability to environmental chemicals within and across genetically heterogeneous human populations. The integration of such data sources will be informative to human health risk assessment

  19. Transcriptional program induced by Wnt protein in human fibroblasts suggests mechanisms for cell cooperativity in defining tissue microenvironments.

    Directory of Open Access Journals (Sweden)

    Zach Klapholz-Brown

    Full Text Available BACKGROUND: The Wnt signaling system plays key roles in development, regulation of stem cell self-renewal and differentiation, cell polarity, morphogenesis and cancer. Given the multifaceted roles of Wnt signaling in these processes, its transcriptional effects on the stromal cells that make up the scaffold and infrastructure of epithelial tissues are of great interest. METHODS AND RESULTS: To begin to investigate these effects, we used DNA microarrays to identify transcriptional targets of the Wnt pathway in human lung fibroblasts. Cells were treated with active Wnt3a protein in culture, and RNA was harvested at 4 hours and 24 hours. Nuclear accumulation of ss-Catenin, as shown by immunofluorescence, and induction of AXIN2 demonstrate that fibroblasts are programmed to respond to extracellular Wnt signals. In addition to several known Wnt targets, we found many new Wnt induced genes, including many transcripts encoding regulatory proteins. Transcription factors with important developmental roles, including HOX genes, dominated the early transcriptional response. Furthermore, we found differential expression of several genes that play direct roles in the Wnt signaling pathway, as well as genes involved in other cell signaling pathways including fibroblast growth factor (FGF and bone morphogenetic protein (BMP signaling. The gene most highly induced by Wnt3a was GREMLIN2, which encodes a secreted BMP antagonist. CONCLUSIONS: Elevated expression of GREMLIN2 suggests a new role for Wnt signals in the maintenance of stem cell niches, whereby Wnt signals induce nearby fibroblasts to produce a BMP antagonist, inhibiting differentiation and promoting expansion of stem cells in their microenvironment. We suggest that Wnt-induced changes in the gene expression program of local stromal cells may play an important role in the establishment of specialized niches hospitable to the self-renewal of normal or malignant epithelial stem cells in vivo.

  20. Preliminary characterization of human skin microbiome in healthy Egyptian individuals.

    Science.gov (United States)

    Ramadan, M; Solyman, S; Taha, M; Hanora, A

    2016-01-01

    Human skin is a large, complex ecosystem that harbors diverse microbial communities. The rapid advances in molecular techniques facilitate the exploration of skin associated bacterial populations. The objective of this study was to perform a preliminary characterization of skin associated bacterial populations in Egyptian individuals. Samples were collected from five healthy subjects from two skin sites; Antecubital Fossa (AF) and Popliteal Fossa (PF). Genomic DNA was extracted and used to amplify bacterial 16S rRNA genes which were sequenced on Illumina MiSeq platform. The two sites showed distinct diversity where PF was more diverse than AF. Taxonomic analysis of sequences revealed four main phyla Proteobacteria, Firmicutes, Actinobacteria and Deinococcus-Thermus, with Proteobacteria presenting the highest diversity. Klebsiella, Bacillus, Pseudomonas and Escherichia were the most predominant genera. Our data suggest that environmental factors can shape the composition of the skin microbiome in certain geographical regions. This study presents a new insight for subsequent analyses of human microbiome in Egypt. PMID:27545210

  1. Characterization of Nanostructures in Acellular Cementum of Human Tooth Roots

    Institute of Scientific and Technical Information of China (English)

    GAO Yong-bo; LIU Ming-xia; SUN Zhi-qiang; ZHANG Kai; DONG Feng-xia; SUN Hong-chen; YANG Bai

    2012-01-01

    Determining the microstructure in human cementum might help us design new kinds of replacement materials for the treatment of teeth injury and disease.The authors characterized the nanostructures in the cementum of health human teeth via scanning electronic microscopy(SEM).It was found that the acellular cementum is mainly composed of two kinds of nanostructures——inorganic nanoparticles and organic nanofibers.And the inorganic nanoparticles show a tendency to arrange along the organic nanofibers.Based on the micro-molding in capillary strategy,the distribution of organic component in acellular cementum was copied with UV curable resin.After removing the inorganic nanoparticles by acid etching,many isolated spindle shape nanopores were left in polymer,which suggested that the inorganic nanoparticles should have been isolated by the organic component in cementum,and should be oval or nanosheet in shape.We hope the present work could provide reference for the biomimetic preparation of tissue engineering materials,and help us design new types of tooth implant.

  2. Characterization of Microvesicles Released from Human Red Blood Cells

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    Duc Bach Nguyen

    2016-03-01

    Full Text Available Background/Aims: Extracellular vesicles (EVs are spherical fragments of cell membrane released from various cell types under physiological as well as pathological conditions. Based on their size and origin, EVs are classified as exosome, microvesicles (MVs and apoptotic bodies. Recently, the release of MVs from human red blood cells (RBCs under different conditions has been reported. MVs are released by outward budding and fission of the plasma membrane. However, the outward budding process itself, the release of MVs and the physical properties of these MVs have not been well investigated. The aim of this study is to investigate the formation process, isolation and characterization of MVs released from RBCs under conditions of stimulating Ca2+ uptake and activation of protein kinase C. Methods: Experiments were performed based on single cell fluorescence imaging, fluorescence activated cell sorter/flow cytometer (FACS, scanning electron microscopy (SEM, atomic force microscopy (AFM and dynamic light scattering (DLS. The released MVs were collected by differential centrifugation and characterized in both their size and zeta potential. Results: Treatment of RBCs with 4-bromo-A23187 (positive control, lysophosphatidic acid (LPA, or phorbol-12 myristate-13 acetate (PMA in the presence of 2 mM extracellular Ca2+ led to an alteration of cell volume and cell morphology. In stimulated RBCs, exposure of phosphatidylserine (PS and formation of MVs were observed by using annexin V-FITC. The shedding of MVs was also observed in the case of PMA treatment in the absence of Ca2+, especially under the transmitted bright field illumination. By using SEM, AFM and DLS the morphology and size of stimulated RBCs, MVs were characterized. The sizes of the two populations of MVs were 205.8 ± 51.4 nm and 125.6 ± 31.4 nm, respectively. Adhesion of stimulated RBCs and MVs was observed. The zeta potential of MVs was determined in the range from - 40 mV to - 10 m

  3. A novel in vitro human microglia model: characterization of human monocyte-derived microglia.

    Science.gov (United States)

    Etemad, Samar; Zamin, Rasheeda Mohd; Ruitenberg, Marc J; Filgueira, Luis

    2012-07-30

    Microglia are the innate immune cells of the central nervous system. They help maintaining physiological homeostasis and contribute significantly to inflammatory responses in the course of infection, injury and degenerative processes. To date, there is no standardized simple model available to investigate the biology of human microglia. The aim of this study was to establish a new human microglia model. For that purpose, human peripheral blood monocytes were cultured in serum free medium in the presence of M-CSF, GM-CSF, NGF and CCL2 to generate monocyte-derived microglia (M-MG). M-MG were clearly different in morphology, phenotype and function from freshly isolated monocytes, cultured monocytes in the absence of the cytokines and monocyte-derived dendritic cells (M-DC) cultured in the presence of GM-CSF and IL-4. M-MG acquired a ramified morphology with primary and secondary processes. M-MG displayed a comparable phenotype to the human microglia cell line HMC3, expressing very low levels of CD45, CD14 and HLA-DR, CD11b and CD11c; and undetectable levels of CD40, CD80 and CD83, and a distinct pattern of chemokine receptors (positive for CCR1, CCR2, CCR4, CCR5, CXCR1, CXCR3, CX3CR1; negative for CCR6 and CCR7). In comparison with M-DC, M-MG displayed lower T-lymphocyte stimulatory capacity, as well as lower phagocytosis activity. The described protocol for the generation of human monocyte-derived microglia is feasible, well standardized and reliable, as it uses well defined culture medium and recombinant cytokines, but no serum or conditioned medium. This protocol will certainly be very helpful for future studies investigating the biology and pathology of human microglia. PMID:22659341

  4. The Development of Dynamic Human Reliability Analysis Simulations for Inclusion in Risk Informed Safety Margin Characterization Frameworks

    Energy Technology Data Exchange (ETDEWEB)

    Jeffrey C. Joe; Diego Mandelli; Ronald L. Boring; Curtis L. Smith; Rachel B. Shirley

    2015-07-01

    The United States Department of Energy is sponsoring the Light Water Reactor Sustainability program, which has the overall objective of supporting the near-term and the extended operation of commercial nuclear power plants. One key research and development (R&D) area in this program is the Risk-Informed Safety Margin Characterization pathway, which combines probabilistic risk simulation with thermohydraulic simulation codes to define and manage safety margins. The R&D efforts to date, however, have not included robust simulations of human operators, and how the reliability of human performance or lack thereof (i.e., human errors) can affect risk-margins and plant performance. This paper describes current and planned research efforts to address the absence of robust human reliability simulations and thereby increase the fidelity of simulated accident scenarios.

  5. Design of a Vitronectin-Based Recombinant Protein as a Defined Substrate for Differentiation of Human Pluripotent Stem Cells into Hepatocyte-Like Cells.

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    Masato Nagaoka

    Full Text Available Maintenance and differentiation of human pluripotent stem cells (hPSCs usually requires culture on a substrate for cell adhesion. A commonly used substratum is Matrigel purified from Engelbreth-Holm-Swarm sarcoma cells, and consists of a complex mixture of extracellular matrix proteins, proteoglycans, and growth factors. Several studies have successfully induced differentiation of hepatocyte-like cells from hPSCs. However, most of these studies have used Matrigel as a cell adhesion substrate, which is not a defined culture condition. In an attempt to generate a substratum that supports undifferentiated properties and differentiation into hepatic lineage cells, we designed novel substrates consisting of vitronectin fragments fused to the IgG Fc domain. hPSCs adhered to these substrates via interactions between integrins and the RGD (Arg-Gly-Asp motif, and the cells maintained their undifferentiated phenotypes. Using a previously established differentiation protocol, hPSCs were efficiently differentiated into mesendodermal and hepatic lineage cells on a vitronectin fragment-containing substrate. We found that full-length vitronectin did not support stable cell adhesion during the specification stage. Furthermore, the vitronectin fragment with the minimal RGD-containing domain was sufficient for differentiation of human induced pluripotent stem cells into hepatic lineage cells under completely defined conditions that facilitate the clinical application of cells differentiated from hPSCs.

  6. Viewing the human microbiome through three-dimensional glasses: integrating structural and functional studies to better define the properties of myriad carbohydrate-active enzymes

    International Nuclear Information System (INIS)

    Metagenomics has unleashed a deluge of sequencing data describing the organismal, genetic, and transcriptional diversity of the human microbiome. To better understand the precise functions of the myriad proteins encoded by the microbiome, including carbohydrate-active enzymes, it will be critical to combine structural studies with functional analyses. Recent studies have provided an unprecedented view of the trillions of microbes associated with the human body. The human microbiome harbors tremendous diversity at multiple levels: the species that colonize each individual and each body habitat; the genes that are found in each organism’s genome; the expression of these genes and the interactions and activities of their protein products. The sources of this diversity are wide-ranging and reflect both environmental and host factors. A major challenge moving forward is defining the precise functions of members of various families of proteins represented in our microbiomes, including the highly diverse carbohydrate-active enzymes (CAZymes) involved in numerous biologically important chemical transformations, such as the degradation of complex dietary polysaccharides. Coupling metagenomic analyses to structural genomics initiatives and to biochemical and other functional assays of CAZymes will be essential for determining how these as well as other microbiome-encoded proteins operate to shape the properties of microbial communities and their human hosts

  7. Characterizing Cognitive Aging in Humans with Links to Animal Models

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    Gene E Alexander

    2012-09-01

    Full Text Available With the population of older adults expected to grow rapidly over the next two decades, it has become increasingly important to advance research efforts to elucidate the mechanisms associated with cognitive aging, with the ultimate goal of developing effective interventions and prevention therapies. Although there has been a vast research literature on the use of cognitive tests to evaluate the effects of aging and age-related neurodegenerative disease, the need for a set of standardized measures to characterize the cognitive profiles specific to healthy aging has been widely recognized. Here we present a review of selected methods and approaches that have been applied in human research studies to evaluate the effects of aging on cognition, including executive function, memory, processing speed, language, and visuospatial function. The effects of healthy aging on each of these cognitive domains are discussed with examples from cognitive/experimental and clinical/neuropsychological approaches. Further, we consider those measures that have clear conceptual and methodological links to tasks currently in use for non-human animal studies of aging, as well as those that have the potential for translation to animal aging research. Having a complementary set of measures to assess the cognitive profiles of healthy aging across species provides a unique opportunity to enhance research efforts for cross-sectional, longitudinal, and intervention studies of cognitive aging. Taking a cross-species, translational approach will help to advance cognitive aging research, leading to a greater understanding of associated neurobiological mechanisms with the potential for developing effective interventions and prevention therapies for age-related cognitive decline.

  8. Characterization of side population cells from human airway epithelium.

    Science.gov (United States)

    Hackett, Tillie-Louise; Shaheen, Furquan; Johnson, Andrew; Wadsworth, Samuel; Pechkovsky, Dmitri V; Jacoby, David B; Kicic, Anthony; Stick, Stephen M; Knight, Darryl A

    2008-10-01

    The airway epithelium is the first line of contact with the inhaled external environment and is continuously exposed to and injured by pollutants, allergens, and viruses. However, little is known about epithelial repair and in particular the identity and role of tissue resident stem/progenitor cells that may contribute to epithelial regeneration. The aims of the present study were to identify, isolate, and characterize side population (SP) cells in human tracheobronchial epithelium. Epithelial cells were obtained from seven nontransplantable healthy lungs and four asthmatic lungs by pronase digestion. SP cells were identified by verapamil-sensitive efflux of the DNA-binding dye Hoechst 33342. Using flow cytometry, CD45(-) SP, CD45(+) SP, and non-SP cells were isolated and sorted. CD45(-) SP cells made up 0.12% +/- 0.01% of the total epithelial cell population in normal airway but 4.1% +/- 0.06% of the epithelium in asthmatic airways. All CD45(-) SP cells showed positive staining for epithelial-specific markers cytokeratin-5, E-cadherin, ZO-1, and p63. CD45(-) SP cells exhibited stable telomere length and increased colony-forming and proliferative potential, undergoing population expansion for at least 16 consecutive passages. In contrast with non-SP cells, fewer than 100 CD45(-) SP cells were able to generate a multilayered and differentiated epithelium in air-liquid interface culture. SP cells are present in human tracheobronchial epithelium, exhibit both short- and long-term proliferative potential, and are capable of generation of differentiated epithelium in vitro. The number of SP cells is significantly greater in asthmatic airways, providing evidence of dysregulated resident SP cells in the asthmatic epithelium. Disclosure of potential conflicts of interest is found at the end of this article. PMID:18653771

  9. A defined and xeno-free culture method enabling the establishment of clinical-grade human embryonic, induced pluripotent and adipose stem cells.

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    Kristiina Rajala

    Full Text Available BACKGROUND: The growth of stem cells in in vitro conditions requires optimal balance between signals mediating cell survival, proliferation, and self-renewal. For clinical application of stem cells, the use of completely defined conditions and elimination of all animal-derived materials from the establishment, culture, and differentiation processes is desirable. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report the development of a fully defined xeno-free medium (RegES, capable of supporting the expansion of human embryonic stem cells (hESC, induced pluripotent stem cells (iPSC and adipose stem cells (ASC. We describe the use of the xeno-free medium in the derivation and long-term (>80 passages culture of three pluripotent karyotypically normal hESC lines: Regea 06/015, Regea 07/046, and Regea 08/013. Cardiomyocytes and neural cells differentiated from these cells exhibit features characteristic to these cell types. The same formulation of the xeno-free medium is capable of supporting the undifferentiated growth of iPSCs on human feeder cells. The characteristics of the pluripotent hESC and iPSC lines are comparable to lines derived and cultured in standard undefined culture conditions. In the culture of ASCs, the xeno-free medium provided significantly higher proliferation rates than ASCs cultured in medium containing allogeneic human serum (HS, while maintaining the differentiation potential and characteristic surface marker expression profile of ASCs, although significant differences in the surface marker expression of ASCs cultured in HS and RegES media were revealed. CONCLUSION/SIGNIFICANCE: Our results demonstrate that human ESCs, iPSCs and ASCs can be maintained in the same defined xeno-free medium formulation for a prolonged period of time while maintaining their characteristics, demonstrating the applicability of the simplified xeno-free medium formulation for the production of clinical-grade stem cells. The basic xeno-free formulation

  10. Characterization of golimumab, a human monoclonal antibody specific for human tumor necrosis factor α.

    Science.gov (United States)

    Shealy, David J; Cai, Ann; Staquet, Kim; Baker, Audrey; Lacy, Eilyn R; Johns, Laura; Vafa, Omid; Gunn, George; Tam, Susan; Sague, Sarah; Wang, Dana; Brigham-Burke, Mike; Dalmonte, Paul; Emmell, Eva; Pikounis, Bill; Bugelski, Peter J; Zhou, Honghui; Scallon, Bernard J; Giles-Komar, Jill

    2010-01-01

    We prepared and characterized golimumab (CNTO148), a human IgG1 tumor necrosis factor alpha (TNFα) antagonist monoclonal antibody chosen for clinical development based on its molecular properties. Golimumab was compared with infliximab, adalimumab and etanercept for affinity and in vitro TNFα neutralization. The affinity of golimumab for soluble human TNFα, as determined by surface plasmon resonance, was similar to that of etanercept (18 pM versus 11 pM), greater than that of infliximab (44 pM) and significantly greater than that of adalimumab (127 pM, p=0.018).  The concentration of golimumab necessary to neutralize TNFα-induced E-selectin expression on human endothelial cells by 50% was significantly less than those for infliximab (3.2 fold; p=0.017) and adalimumab (3.3-fold; p=0.008) and comparable to that for etanercept. The conformational stability of golimumab was greater than that of infliximab (primary melting temperature [Tm] 74.8 °C vs. 69.5 °C) as assessed by differential scanning calorimetry.  In addition, golimumab showed minimal aggregation over the intended shelf life when formulated as a high concentration liquid product (100 mg/mL) for subcutaneous administration.  In vivo, golimumab at doses of 1 and 10 mg/kg significantly delayed disease progression in a mouse model of human TNFα-induced arthritis when compared with untreated mice, while infliximab was effective only at 10 mg/kg. Golimumab also significantly reduced histological scores for arthritis severity and cartilage damage, as well as serum levels of pro-inflammatory cytokines and chemokines associated with arthritis. Thus, we have demonstrated that golimumab is a highly stable human monoclonal antibody with high affinity and capacity to neutralize human TNFα in vitro and in vivo.

  11. Characterization and comparison of the tissue-related modules in human and mouse.

    Directory of Open Access Journals (Sweden)

    Ruolin Yang

    Full Text Available BACKGROUND: Due to the advances of high throughput technology and data-collection approaches, we are now in an unprecedented position to understand the evolution of organisms. Great efforts have characterized many individual genes responsible for the interspecies divergence, yet little is known about the genome-wide divergence at a higher level. Modules, serving as the building blocks and operational units of biological systems, provide more information than individual genes. Hence, the comparative analysis between species at the module level would shed more light on the mechanisms underlying the evolution of organisms than the traditional comparative genomics approaches. RESULTS: We systematically identified the tissue-related modules using the iterative signature algorithm (ISA, and we detected 52 and 65 modules in the human and mouse genomes, respectively. The gene expression patterns indicate that all of these predicted modules have a high possibility of serving as real biological modules. In addition, we defined a novel quantity, "total constraint intensity," a proxy of multiple constraints (of co-regulated genes and tissues where the co-regulation occurs on the evolution of genes in module context. We demonstrate that the evolutionary rate of a gene is negatively correlated with its total constraint intensity. Furthermore, there are modules coding the same essential biological processes, while their gene contents have diverged extensively between human and mouse. CONCLUSIONS: Our results suggest that unlike the composition of module, which exhibits a great difference between human and mouse, the functional organization of the corresponding modules may evolve in a more conservative manner. Most importantly, our findings imply that similar biological processes can be carried out by different sets of genes from human and mouse, therefore, the functional data of individual genes from mouse may not apply to human in certain occasions.

  12. Use of xenofree matrices and molecularly-defined media to control human embryonic stem cell pluripotency: effect of low physiological TGF-beta concentrations.

    Science.gov (United States)

    Peiffer, Isabelle; Barbet, Romain; Zhou, Yi-Ping; Li, Ma-Lin; Monier, Marie-Noëlle; Hatzfeld, Antoinette; Hatzfeld, Jacques A

    2008-06-01

    To monitor human embryonic stem cell (hESC) self-renewal without differentiation, we used quantitative RT-PCR to study a selection of hESC genes, including markers for self-renewal, commitment/differentiation, and members of the TGF-beta superfamily and DAN gene family. Indeed, low commitment/differentiation gene expression, together with a significant self-renewal gene expres sion, provides a better pluripotency index than self-renewal genes alone. We demonstrate that matrices derived from human mesenchymal stem cells (hMSCs) can advantageously replace murine embryonic fibroblasts (MEF) or hMSC feeders. Moreover, a xenofree molecularly-defined SBX medium, containing a synthetic lipid carrier instead of albumin, can replace SR medium. The number of selected differentiation genes expressed by hESCs in these culture conditions was significantly lower than those expressed on MEF feeders in SR medium. In SBX, the positive effect of a non-physiological concentration of activin A (10-30 ng/mL) to reduce differentiation during self-renewal could also be obtained by physiological concentrations of TGF-beta(100-300 pg/mL). In contrast, these TGF-beta concentrations added to activin favored differentiation as previously observed with TGF-beta concentrations of 1 ng/mL or more. Compared to SR-containing medium, SBX medium promoted down-regulation of CER1 and LEFTIES and up-regulation of GREM1. Thus these genes better control self-renewal and pluripotency and prevent differentiation. A strategy is proposed to analyze, in more physiological, xenofree, molecularly-defined media and matrices, the numerous genes with still unknown functions controlling hESCs or human-induced pluripotent stem cells (iPS). PMID:18513159

  13. Dynamic Propagation Channel Characterization and Modeling for Human Body Communication

    Directory of Open Access Journals (Sweden)

    Lei Wang

    2012-12-01

    Full Text Available This paper presents the first characterization and modeling of dynamic propagation channels for human body communication (HBC. In-situ experiments were performed using customized transceivers in an anechoic chamber. Three HBC propagation channels, i.e., from right leg to left leg, from right hand to left hand and from right hand to left leg, were investigated under thirty-three motion scenarios. Snapshots of data (2,800,000 were acquired from five volunteers. Various path gains caused by different locations and movements were quantified and the statistical distributions were estimated. In general, for a given reference threshold è = −10 dB, the maximum average level crossing rate of the HBC was approximately 1.99 Hz, the maximum average fade time was 59.4 ms, and the percentage of bad channel duration time was less than 4.16%. The HBC exhibited a fade depth of −4 dB at 90% complementary cumulative probability. The statistical parameters were observed to be centered for each propagation channel. Subsequently a Fritchman model was implemented to estimate the burst characteristics of the on-body fading. It was concluded that the HBC is motion-insensitive, which is sufficient for reliable communication link during motions, and therefore it has great potential for body sensor/area networks.

  14. Molecular Characterization of China Human Rabies Vaccine Strains

    Institute of Scientific and Technical Information of China (English)

    Xiaoyan Tao; Na Han; Zhenyang Guo; Qing Tang; Simon Rayner; Guodong Liang

    2013-01-01

    To understand the molecular characteristics of China human rabies vaccine strains,we report the full-length genome of the aG strain and present a comprehensive analysis of this strain and almost all available lyssavirus genomes (58 strains) from GenBank (as of Jan 6,2011).It is generally considered that the G protein plays a predominant role in determining the pathogenicity of the virus,to this end we predicted the tertiary structure of the G protein of aG strain,CTN 181 strain and wild type strain HN 10 based on the crystal structure of Vesicular stomatitis virus (VSV) G.The predicted RABV G structure has a similar topology to VSV G and the ectodomain can be divided into 4 distinct domains DI-DIV.By mapping the characterized mutations to this structure between China vaccine strains and their close street strains,we speculate that the G303(P-H) mutations of CTN181 and HN10 causing D Ⅱ 3D change may be associated with the attenuated virulence in both strains.Specifically,the two signature mutations (G165P and G231P) in the aG strain are withinβsheets,suggesting that both sites are of structural importance.

  15. Characterization of a Canine Homolog of Human Aichivirus▿

    Science.gov (United States)

    Kapoor, Amit; Simmonds, Peter; Dubovi, Edward J.; Qaisar, Natasha; Henriquez, Jose A.; Medina, Jan; Shields, Shelly; Lipkin, W. Ian

    2011-01-01

    Many of our fatal “civilization” infectious diseases have arisen from domesticated animals. Although picornaviruses infect most mammals, infection of a companion animal is not known. Here we describe the identification and genomic characterization of the first canine picornavirus. Canine kobuvirus (CKoV), identified in stool samples from dogs with diarrhea, has a genomic organization typical of a picornavirus and encodes a 2,469-amino-acid polyprotein flanked by 5′ and 3′ untranslated regions. Comparative phylogenetic analysis using various structural and nonstructural proteins of CKoV confirmed it as the animal virus homolog most closely related to human Aichivirus (AiV). Bayesian Markov chain Monte Carlo analysis suggests a mean recent divergence time of CKoV and AiV within the past 20 to 50 years, well after the domestication of canines. The discovery of CKoV provides new insights into the origin and evolution of AiV and the species specificity and pathogenesis of kobuviruses. PMID:21880761

  16. Characterization of a canine homolog of human Aichivirus.

    Science.gov (United States)

    Kapoor, Amit; Simmonds, Peter; Dubovi, Edward J; Qaisar, Natasha; Henriquez, Jose A; Medina, Jan; Shields, Shelly; Lipkin, W Ian

    2011-11-01

    Many of our fatal "civilization" infectious diseases have arisen from domesticated animals. Although picornaviruses infect most mammals, infection of a companion animal is not known. Here we describe the identification and genomic characterization of the first canine picornavirus. Canine kobuvirus (CKoV), identified in stool samples from dogs with diarrhea, has a genomic organization typical of a picornavirus and encodes a 2,469-amino-acid polyprotein flanked by 5' and 3' untranslated regions. Comparative phylogenetic analysis using various structural and nonstructural proteins of CKoV confirmed it as the animal virus homolog most closely related to human Aichivirus (AiV). Bayesian Markov chain Monte Carlo analysis suggests a mean recent divergence time of CKoV and AiV within the past 20 to 50 years, well after the domestication of canines. The discovery of CKoV provides new insights into the origin and evolution of AiV and the species specificity and pathogenesis of kobuviruses. PMID:21880761

  17. Production and Characterization of Monoclonal Antibody Against Recombinant Human Erythropoietin

    Institute of Scientific and Technical Information of China (English)

    JIE-BO MI; JIN YAN; XIAO-JIE DING; ZHEN-QUAN GUO; MEI-PING ZHAO; WEN-BAO CHANG

    2007-01-01

    Objective To produce specific monoclonal antibody(mAb)against recombinant human erythropoietin(rHuEPO)for development of higmy efficient methods for erythropoietin detection in biological fluids.Methods rHuEPO was covalently coupled with bovine serum albumin(BSA)and the conjugate was used to immunize mice to produce specific mAb against rHuEPO based on hybridoma technology.The obtained F3-mAb was characterized by enzyme-linked immunosorbent assay (ELISA),SDS-PAGE and Western blot.Results The isotype of F3-mAb Was found to be IgM with an affinity constant of 2.1x108 L/mol.The competitive ELISA using the obtained IgM showed a broader linear range and lower detection limit compared with previous work.Conclusions The modification of rHuEPO was proved to be successful in generating required specific mAb with high avidity to rHuEPO.

  18. A Newly Defined and Xeno-Free Culture Medium Supports Every-Other-Day Medium Replacement in the Generation and Long-Term Cultivation of Human Pluripotent Stem Cells.

    Science.gov (United States)

    Ahmadian Baghbaderani, Behnam; Tian, Xinghui; Scotty Cadet, Jean; Shah, Kevan; Walde, Amy; Tran, Huan; Kovarcik, Don Paul; Clarke, Diana; Fellner, Thomas

    2016-01-01

    Human pluripotent stem cells (hPSCs) present an unprecedented opportunity to advance human health by offering an alternative and renewable cell resource for cellular therapeutics and regenerative medicine. The present demand for high quality hPSCs for use in both research and clinical studies underscores the need to develop technologies that will simplify the cultivation process and control variability. Here we describe the development of a robust, defined and xeno-free hPSC medium that supports reliable propagation of hPSCs and generation of human induced pluripotent stem cells (hiPSCs) from multiple somatic cell types; long-term serial subculturing of hPSCs with every-other-day (EOD) medium replacement; and banking fully characterized hPSCs. The hPSCs cultured in this medium for over 40 passages are genetically stable, retain high expression levels of the pluripotency markers TRA-1-60, TRA-1-81, Oct-3/4 and SSEA-4, and readily differentiate into ectoderm, mesoderm and endoderm. Importantly, the medium plays an integral role in establishing a cGMP-compliant process for the manufacturing of hiPSCs that can be used for generation of clinically relevant cell types for cell replacement therapy applications. PMID:27606941

  19. Production, Characterization, and Biological Evaluation of Well-Defined IgG1 Fc Glycoforms as a Model System for Biosimilarity Analysis.

    Science.gov (United States)

    Okbazghi, Solomon Z; More, Apurva S; White, Derek R; Duan, Shaofeng; Shah, Ishan S; Joshi, Sangeeta B; Middaugh, C Russell; Volkin, David B; Tolbert, Thomas J

    2016-02-01

    Four different well-defined IgG1 Fc glycoforms are proposed as a model system to examine important biological and physicochemical features for protein drug biosimilar analyses. The IgG1 Fc glycoforms were produced by yeast expression combined with in vitro enzymatic synthesis as a series of sequentially truncated high-mannose IgG1 Fc glycoforms with an anticipated range of biological activity and structural stability. Initial characterization with mass spectrometry, SDS-PAGE, size exclusion HPLC, and capillary isoelectric focusing confirmed that the glycoproteins are overall highly similar with the only major difference being glycosylation state. Binding to the activating Fc receptor, FcγRIIIa was used to evaluate the potential biological activity of the IgG1 Fc glycoproteins. Two complementary methods using biolayer interferometry, 1 with protein G-immobilized IgG1 Fc and the other with streptavidin-immobilized FcγRIIIa, were developed to assess FcγRIIIa affinity in kinetic binding studies. The high-mannose IgG1 Fc and Man5-IgG1 Fc glycoforms were highly similar to one another with high affinity for FcγRIIIa, whereas GlcNAc-Fc had weak affinity, and the nonglycosylated N297Q-Fc had no measurable affinity for FcγRIIIa. These 4 IgG1 Fc glycoforms were also evaluated in terms of physical and chemical stability profiles and then used as a model system to mathematically assess overall biosimilarity, as described in a series of companion articles. PMID:26869419

  20. 235U isotopic characterization of natural and enriched uranium materials by using multigroup analysis (MGA method at a defined geometry using different absorbers and collimators

    Directory of Open Access Journals (Sweden)

    Yücel Haluk

    2015-09-01

    Full Text Available Characterization of nuclear materials is an important topic within the context of nuclear safeguards, homeland security and nuclear forensics. This paper deals with the performance of multigroup gamma-ray analysis (MGA method using the X- and γ-rays in the 80-130 keV region and enrichment meter principle (EMP based on the analysis of 185.7 keV peak for a certain geometry using different absorbers and collimators. The results from MGA and those of EMP are compared. In particular, the effect of aluminum/lead absorbers and lead collimator on the enrichment determination of 235U in natural and low enriched samples is investigated in a given source-detector geometry. The optimum diameter/height ratio for the Pb-collimator is found to be Dc/Hc = 1.4-1.6 in the chosen geometry. In order to simulate the container walls, ten different thicknesses of Al-absorbers of 141 to 840 mg·cm-2 and six different thicknesses of Pb-absorbers of 1120-7367 mg·cm-2 are interposed between sample and detector. The calibration coefficients (% enrichment/cps are calculated for each geometry. The comparison of the MGA and EMP methods shows that the enrichment meter principle provides more accurate and precise results for 235U abundance than those of MGA method at the chosen geometrical conditions. The present results suggest that a two-step procedure should be used in analyses of uranium enrichment. Firstly MGA method can be applied in situ and then EMP method can be used at a defined geometry in laboratory.

  1. Defining talent management components

    OpenAIRE

    Golchin Shafieian

    2014-01-01

    In today's world of global trade, it can guarantee success and excellence of organizations against competitors is talented human resources in an organization, especially at the managerial level. But the challenges that organizations face include: attracting, evaluating, exploring, and developing and keeping talent. The main objective of this study is to define the components of talent management in the University of Chaloos, in order to provide a way to reduce the challenges and short comings...

  2. Operationally defined species characterization and bioaccessibility evaluation of cobalt, copper and selenium in Cape gooseberry (Physalis Peruviana L.) by SEC-ICP MS.

    Science.gov (United States)

    Wojcieszek, Justyna; Ruzik, Lena

    2016-03-01

    Physalis peruviana could attract great interest because of its nutritional and industrial properties. It is an excellent source of vitamins, minerals, essential fatty acids and carotenoids. Physalis Peruviana is also known to have a positive impact on human health. Unfortunately, still little is known about trace elements present in Physalis Peruviana and their forms available for the human body. Thus, the aim of this study was to estimate bioaccessibility and characterization of species of cobalt, copper and selenium in Physalis Peruviana fruits. Total and extractable contents of elements were determined by mass spectrometer with inductively coupled plasma (ICP MS). In order to separate the different types of metal complexes Physalis peruviana fruits were treated with the following solvents: Tris-HCl (pH 7.4), sodium dodecyl sulfate (SDS) (pH 7.4) and ammonium acetate (pH 5.5). The best efficiency of extraction of: cobalt was obtained for ammonium acetate (56%) and Tris-HCl (60%); for copper was obtained for SDS (66%), for selenium the best extraction efficiency was obtained after extraction with SDS (48%). To obtain information about bioaccessibility of investigated elements, enzymatic extraction based on in vitro simulation of gastric (pepsin) and intestinal (pancreatin) digestion was performed. For copper and selenium the simulation of gastric digestion leads to the extraction yield above 90%, while both steps of digestion method were necessary to obtain satisfactory extraction yield in the case of cobalt. Size exclusion chromatography (SEC) coupled to on-line ICP MS detection was used to investigate collected metal species. The main fraction of metal compounds was found in the 17 kDa region. Cobalt and copper create complexes mostly with compounds extracted by means of ammonium acetate and SDS, respectively. Cobalt, copper and selenium were found to be highly bioaccessible from Physalis Peruviana. Investigation of available standards of cobalt and selenium

  3. Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions.

    Science.gov (United States)

    Slamecka, Jaroslav; Salimova, Lilia; McClellan, Steven; van Kelle, Mathieu; Kehl, Debora; Laurini, Javier; Cinelli, Paolo; Owen, Laurie; Hoerstrup, Simon P; Weber, Benedikt

    2016-01-01

    Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However, upgrading them to pluripotency confers refractoriness toward senescence, higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling, such as Down syndrome or β-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing, feeder-dependent culture. Here, we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium, a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4, Nanog, Sox2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 in a pattern typical for human primed PSC. Additionally, the cells formed teratomas, and were deemed pluripotent by PluriTest, a global expression microarray-based in-silico pluripotency assay. However, we found that the PluriTest scores were borderline, indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology, non-integrating reprogramming and chemically defined culture are more acceptable.

  4. An IgE epitope of Bet v 1 and fagales PR10 proteins as defined by a human monoclonal IgE

    DEFF Research Database (Denmark)

    Hecker, J.; Diethers, A.; Schulz, D.;

    2012-01-01

    formats including IgE. Using variants of Bet v 1, the epitope of the antibody was mapped and extrapolated to other PR10 proteins. RESULTS: The obtained antibodies exhibited pronounced reactivity with Bet v 1, but were not reactive with the homologous PR10 protein Mal d 1. The epitope as defined by the Ig......-reactivities predicted by primary structure analyses of different isoforms and PR10 proteins were verified by allergen chip-based analyses. CONCLUSIONS: The obtained results demonstrate that hybrid IgE repertoires represent a source for human antibodies with genuine paratopes. The IgE-derived information about the Ig......E epitope nature of Bet v 1 and homologues allows for detailed insights into molecular aspects of allergenicity and cross-reactivity within the PR10 protein family....

  5. Effects of defined mixtures of persistent organic pollutants (POPs) on multiple cellular responses in the human hepatocarcinoma cell line, HepG2, using high content analysis screening.

    Science.gov (United States)

    Wilson, Jodie; Berntsen, Hanne Friis; Zimmer, Karin Elisabeth; Frizzell, Caroline; Verhaegen, Steven; Ropstad, Erik; Connolly, Lisa

    2016-03-01

    Persistent organic pollutants (POPs) are toxic substances, highly resistant to environmental degradation, which can bio-accumulate and have long-range atmospheric transport potential. Most studies focus on single compound effects, however as humans are exposed to several POPs simultaneously, investigating exposure effects of real life POP mixtures on human health is necessary. A defined mixture of POPs was used, where the compound concentration reflected its contribution to the levels seen in Scandinavian human serum (total mix). Several sub mixtures representing different classes of POPs were also constructed. The perfluorinated (PFC) mixture contained six perfluorinated compounds, brominated (Br) mixture contained seven brominated compounds, chlorinated (Cl) mixture contained polychlorinated biphenyls and also p,p'-dichlorodiphenyldichloroethylene, hexachlorobenzene, three chlordanes, three hexachlorocyclohexanes and dieldrin. Human hepatocarcinoma (HepG2) cells were used for 2h and 48h exposures to the seven mixtures and analysis on a CellInsight™ NXT High Content Screening platform. Multiple cytotoxic endpoints were investigated: cell number, nuclear intensity and area, mitochondrial mass and membrane potential (MMP) and reactive oxygen species (ROS). Both the Br and Cl mixtures induced ROS production but did not lead to apoptosis. The PFC mixture induced ROS production and likely induced cell apoptosis accompanied by the dissipation of MMP. Synergistic effects were evident for ROS induction when cells were exposed to the PFC+Br mixture in comparison to the effects of the individual mixtures. No significant effects were detected in the Br+Cl, PFC+Cl or total mixtures, which contain the same concentrations of chlorinated compounds as the Cl mixture plus additional compounds; highlighting the need for further exploration of POP mixtures in risk assessment. PMID:26772051

  6. Defining the optimal window for cranial transplantation of human induced pluripotent stem cell-derived cells to ameliorate radiation-induced cognitive impairment.

    Science.gov (United States)

    Acharya, Munjal M; Martirosian, Vahan; Christie, Lori-Ann; Riparip, Lara; Strnadel, Jan; Parihar, Vipan K; Limoli, Charles L

    2015-01-01

    Past preclinical studies have demonstrated the capability of using human stem cell transplantation in the irradiated brain to ameliorate radiation-induced cognitive dysfunction. Intrahippocampal transplantation of human embryonic stem cells and human neural stem cells (hNSCs) was found to functionally restore cognition in rats 1 and 4 months after cranial irradiation. To optimize the potential therapeutic benefits of human stem cell transplantation, we have further defined optimal transplantation windows for maximizing cognitive benefits after irradiation and used induced pluripotent stem cell-derived hNSCs (iPSC-hNSCs) that may eventually help minimize graft rejection in the host brain. For these studies, animals given an acute head-only dose of 10 Gy were grafted with iPSC-hNSCs at 2 days, 2 weeks, or 4 weeks following irradiation. Animals receiving stem cell grafts showed improved hippocampal spatial memory and contextual fear-conditioning performance compared with irradiated sham-surgery controls when analyzed 1 month after transplantation surgery. Importantly, superior performance was evident when stem cell grafting was delayed by 4 weeks following irradiation compared with animals grafted at earlier times. Analysis of the 4-week cohort showed that the surviving grafted cells migrated throughout the CA1 and CA3 subfields of the host hippocampus and differentiated into neuronal (∼39%) and astroglial (∼14%) subtypes. Furthermore, radiation-induced inflammation was significantly attenuated across multiple hippocampal subfields in animals receiving iPSC-hNSCs at 4 weeks after irradiation. These studies expand our prior findings to demonstrate that protracted stem cell grafting provides improved cognitive benefits following irradiation that are associated with reduced neuroinflammation.

  7. Complete Genome Sequence of Germline Chromosomally Integrated Human Herpesvirus 6A and Analyses Integration Sites Define a New Human Endogenous Virus with Potential to Reactivate as an Emerging Infection

    OpenAIRE

    Tweedy, J.; Spyrou, MA; Pearson, M.; Lassner, D.; Kuhl, U.; Gompels, UA

    2016-01-01

    Human herpesvirus-6A and B (HHV-6A, HHV-6B) have recently defined endogenous genomes, resulting from integration into the germline: chromosomally-integrated "CiHHV-6A/B". These affect approximately 1.0% of human populations, giving potential for virus gene expression in every cell. We previously showed that CiHHV-6A was more divergent than CiHHV-6B by examining four genes in 44 European CiHHV-6A/B cardiac/haematology patients. There was evidence for gene expression/reactivation, implying func...

  8. Complete Genome Sequence of Germline Chromosomally Integrated Human Herpesvirus 6A and Analyses Integration Sites Define a New Human Endogenous Virus with Potential to Reactivate as an Emerging Infection

    OpenAIRE

    Joshua Tweedy; Maria Alexandra Spyrou; Max Pearson; Dirk Lassner; Uwe Kuhl; Gompels, Ursula A.

    2016-01-01

    Human herpesvirus-6A and B (HHV-6A, HHV-6B) have recently defined endogenous genomes, resulting from integration into the germline: chromosomally-integrated “CiHHV-6A/B”. These affect approximately 1.0% of human populations, giving potential for virus gene expression in every cell. We previously showed that CiHHV-6A was more divergent than CiHHV-6B by examining four genes in 44 European CiHHV-6A/B cardiac/haematology patients. There was evidence for gene expression/reactivation, implying func...

  9. Structural and Biochemical Characterization of the Human Cyclophilin Family of Peptidyl-Prolyl Isomerases

    Energy Technology Data Exchange (ETDEWEB)

    Davis, Tara L.; Walker, John R.; Campagna-Slater, Valérie; Finerty, Jr., Patrick J.; Paramanathan, Ragika; Bernstein, Galina; MacKenzie, Farrell; Tempel, Wolfram; Ouyang, Hui; Lee, Wen Hwa; Eisenmesser, Elan Z.; Dhe-Paganon, Sirano (Toronto); (Colorado)

    2011-12-14

    Peptidyl-prolyl isomerases catalyze the conversion between cis and trans isomers of proline. The cyclophilin family of peptidyl-prolyl isomerases is well known for being the target of the immunosuppressive drug cyclosporin, used to combat organ transplant rejection. There is great interest in both the substrate specificity of these enzymes and the design of isoform-selective ligands for them. However, the dearth of available data for individual family members inhibits attempts to design drug specificity; additionally, in order to define physiological functions for the cyclophilins, definitive isoform characterization is required. In the current study, enzymatic activity was assayed for 15 of the 17 human cyclophilin isomerase domains, and binding to the cyclosporin scaffold was tested. In order to rationalize the observed isoform diversity, the high-resolution crystallographic structures of seven cyclophilin domains were determined. These models, combined with seven previously solved cyclophilin isoforms, provide the basis for a family-wide structure:function analysis. Detailed structural analysis of the human cyclophilin isomerase explains why cyclophilin activity against short peptides is correlated with an ability to ligate cyclosporin and why certain isoforms are not competent for either activity. In addition, we find that regions of the isomerase domain outside the proline-binding surface impart isoform specificity for both in vivo substrates and drug design. We hypothesize that there is a well-defined molecular surface corresponding to the substrate-binding S2 position that is a site of diversity in the cyclophilin family. Computational simulations of substrate binding in this region support our observations. Our data indicate that unique isoform determinants exist that may be exploited for development of selective ligands and suggest that the currently available small-molecule and peptide-based ligands for this class of enzyme are insufficient for isoform

  10. Characterization of Evidence for Human System Risk Assessment

    Science.gov (United States)

    Steinberg, S. L.; Van Baalen, M.; Rossi, M.; Riccio, G.; Romero, E.; Francisco, D.

    2016-01-01

    Understanding the kinds of evidence available and using the best evidence to answer a question is critical to evidenced-based decision-making, and it requires synthesis of evidence from a variety of sources. Categorization of human system risks in spaceflight, in particular, focuses on how well the integration and interpretation of all available evidence informs the risk statement that describes the relationship between spaceflight hazards and an outcome of interest. A mature understanding and categorization of these risks requires: 1) sufficient characterization of risk, 2) sufficient knowledge to determine an acceptable level of risk (i.e., a standard), 3) development of mitigations to meet the acceptable level of risk, and 4) identification of factors affecting generalizability of the evidence to different design reference missions. In the medical research community, evidence is often ranked by increasing confidence in findings gleaned from observational and experimental research (e.g., "levels of evidence"). However, an approach based solely on aspects of experimental design is problematic in assessing human system risks for spaceflight. For spaceflight, the unique challenges and opportunities include: (1) The independent variables in most evidence are the hazards of spaceflight, such as space radiation or low gravity, which cannot be entirely duplicated in terrestrial (Earth-based) analogs, (2) Evidence is drawn from multiple sources including medical and mission operations, Lifetime Surveillance of Astronaut Health (LSAH), spaceflight research (LSDA), and relevant environmental & terrestrial databases, (3) Risk metrics based primarily on LSAH data are typically derived from available prevalence or incidence data, which may limit rigorous interpretation, (4) The timeframe for obtaining adequate spaceflight sample size (n) is very long, given the small population, (5) Randomized controlled trials are unattainable in spaceflight, (6) Collection of personal and

  11. "Human Health Impact Characterization of Toxic Chemicals for Sustainable Design and Manufacturing

    OpenAIRE

    Yuan, Chris; Dornfeld, David

    2009-01-01

    A schematic method to characterize the human health impact of toxic chemicals is presented. This schematic method uses a streamlined three-tiered hierarchy process which includes intake, toxicity and persistence of a chemical release for its impact characterization. The human health impact of a chemical is represented by its position in a two-dimensional characterization plot, which enables the benchmarking of chemicals to be easily made by comparing the relative positions of the chemicals in...

  12. Determination of human DNA polymerase utilization for the repair of a model ionizing radiation-induced DNA strand break lesion in a defined vector substrate

    Science.gov (United States)

    Winters, T. A.; Russell, P. S.; Kohli, M.; Dar, M. E.; Neumann, R. D.; Jorgensen, T. J.

    1999-01-01

    Human DNA polymerase and DNA ligase utilization for the repair of a major class of ionizing radiation-induced DNA lesion [DNA single-strand breaks containing 3'-phosphoglycolate (3'-PG)] was examined using a novel, chemically defined vector substrate containing a single, site-specific 3'-PG single-strand break lesion. In addition, the major human AP endonuclease, HAP1 (also known as APE1, APEX, Ref-1), was tested to determine if it was involved in initiating repair of 3'-PG-containing single-strand break lesions. DNA polymerase beta was found to be the primary polymerase responsible for nucleotide incorporation at the lesion site following excision of the 3'-PG blocking group. However, DNA polymerase delta/straightepsilon was also capable of nucleotide incorporation at the lesion site following 3'-PG excision. In addition, repair reactions catalyzed by DNA polymerase beta were found to be most effective in the presence of DNA ligase III, while those catalyzed by DNA polymerase delta/straightepsilon appeared to be more effective in the presence of DNA ligase I. Also, it was demonstrated that the repair initiating 3'-PG excision reaction was not dependent upon HAP1 activity, as judged by inhibition of HAP1 with neutralizing HAP1-specific polyclonal antibody.

  13. CXCR6, a newly defined biomarker of tissue-specific stem cell asymmetric self-renewal, identifies more aggressive human melanoma cancer stem cells.

    Directory of Open Access Journals (Sweden)

    Rouzbeh Taghizadeh

    Full Text Available A fundamental problem in cancer research is identifying the cell type that is capable of sustaining neoplastic growth and its origin from normal tissue cells. Recent investigations of a variety of tumor types have shown that phenotypically identifiable and isolable subfractions of cells possess the tumor-forming ability. In the present paper, using two lineage-related human melanoma cell lines, primary melanoma line IGR39 and its metastatic derivative line IGR37, two main observations are reported. The first one is the first phenotypic evidence to support the origin of melanoma cancer stem cells (CSCs from mutated tissue-specific stem cells; and the second one is the identification of a more aggressive subpopulation of CSCs in melanoma that are CXCR6+.We defined CXCR6 as a new biomarker for tissue-specific stem cell asymmetric self-renewal. Thus, the relationship between melanoma formation and ABCG2 and CXCR6 expression was investigated. Consistent with their non-metastatic character, unsorted IGR39 cells formed significantly smaller tumors than unsorted IGR37 cells. In addition, ABCG2+ cells produced tumors that had a 2-fold greater mass than tumors produced by unsorted cells or ABCG2- cells. CXCR6+ cells produced more aggressive tumors. CXCR6 identifies a more discrete subpopulation of cultured human melanoma cells with a more aggressive MCSC phenotype than cells selected on the basis of the ABCG2+ phenotype alone.The association of a more aggressive tumor phenotype with asymmetric self-renewal phenotype reveals a previously unrecognized aspect of tumor cell physiology. Namely, the retention of some tissue-specific stem cell attributes, like the ability to asymmetrically self-renew, impacts the natural history of human tumor development. Knowledge of this new aspect of tumor development and progression may provide new targets for cancer prevention and treatment.

  14. Characterization Analysis of Human Anti-Ferritin Autoantibodies

    OpenAIRE

    Shusaku Higashi; Kosei Nagasawa; Yasunaga Yoshikawa; Kiyotaka Watanabe; Koichi Orino

    2014-01-01

    Anti-ferritin autoantibodies are found in many animals. Human ferritin-binding proteins (FBPs) were partially purified from human serum by ion-exchange chromatography and immobilized metal affinity chromatography with Zn2+. Crude FBPs were immunocoprecipitated with canine liver ferritin followed by the addition of anti-ferritin antibodies. Immunoglobulins in the immunocoprecipitate were detected with antibodies specific for human IgG, IgM or IgA heavy chains, and immunoglobulins IgG, IgM and ...

  15. Plasmid characterization of Salmonella typhimurium transmitted from animals to humans.

    OpenAIRE

    Olsvik, O; Sørum, H; Birkness, K; Wachsmuth, K; Fjølstad, M; Lassen, J; Fossum, K; Feeley, J C

    1985-01-01

    The transmission of pathogenic bacteria from animals to humans is widely studied because of its public health importance. In this study, we show the transmission of Salmonella typhimurium from cattle which had received no growth-promoting antibiotics to humans who had direct contact with the ill animals. On one cattle farm, the veterinarian attending the sick animals became ill, and two other individuals living on the farm later developed salmonellosis. The strains isolated from both humans a...

  16. Define Digital Vernacular

    Institute of Scientific and Technical Information of China (English)

    刘佳; 李海英; James Stevens; Rough Nelson

    2014-01-01

    As science and technology developed, the tools of humans developed from humans’hands, to mechanical and digital technologies. The tools influ-ence almost everything in the humans’world, so does vernacular. The digital vernacular could be understood as using digital technology to vernacular; the digital means technologies. It also could be understood as doing vernacular in a digital way;the digital means data and information, in other words it can be seeking truth from facts. Define digital vernacular is not only what is digital vernacular, but also about how to do the digital vernacular and what kind of attitude we should hold to-ward the digital vernacular. Define digital vernacular as both thinking and doing.

  17. Isolation and functional characterization of the human 90K promoter

    DEFF Research Database (Denmark)

    Brakebusch, C; Sures, I; Jallal, B;

    1999-01-01

    that it is a TATA-less promoter, but neither GC-rich nor dependent on SP1 sites. RNase protection assays detected one major transcription start site (+1) and several minor transcription start sites upstream and downstream. Deletion studies defined a minimal promoter (-103 --> -49) and indirectly suggested positive...

  18. Molecular characterization of bacterial communities in the human gastrointestinal tract

    NARCIS (Netherlands)

    Zoetendal, E.G.

    2001-01-01

    The human gastrointestinal (GI) tract is a complex ecosystem in which host and microbial cells live in close contact with each other. The microbial community in the human GI tract has an important nutritional and protective function and mainly consists of anaerobic bacteria. After birth, the germ-fr

  19. Characterization of interleukin-8 receptors in non-human primates

    Energy Technology Data Exchange (ETDEWEB)

    Alvarez, V.; Coto, E.; Gonzalez-Roces, S.; Lopez-Larrea, C. [Hospital Central de Asturias, Oviedo (Spain)] [and others

    1996-09-01

    Interleukin-8 is a chemokine with a potent neutrophil chemoatractant activity. In humans, two different cDNAs encoding human IL8 receptors designated IL8RA and IL8RB have been cloned. IL8RA binds IL8, while IL8RB binds IL8 as well as other {alpha}-chemokines. Both human IL8Rs are encoded by two genes physically linked on chromosome 2. The IL8RA and IL8RB genes have open reading frames (ORF) lacking introns. By direct sequencing of the polymerase chain reaction products, we sequenced the IL8R genes of cell lines from four non-human primates: chimpanzee, gorilla, orangutan, and macaca. The IL8RB encodes an ORF in the four non-human primates, showing 95%-99% similarity to the human IL8RB sequence. The IL8RA homologue in gorilla and chimpanzee consisted of two ORF 98%-99% identical to the human sequence. The macaca and orangutan IL8RA homologues are pseudogenes: a 2 base pair insertion generated a sequence with several stop codons. In addition, we describe the physical linkage of these genes in the four non-human primates and discuss the evolutionary implications of these findings. 25 refs., 5 figs., 3 tabs.

  20. Isolation and characterization of the human Gsα gene

    International Nuclear Information System (INIS)

    The gene for Gsα (the α subunit of the guanine nucleotide-binding protein Gs) was isolated from human genomic libraries using rat Gsα cDNA as a probe. Comparison of the nucleotide sequence of the human gene with that of the rat cDNA revealed that the human Gsα gene spans ∼20 kilobases and is composed of 13 exons and 12 introns. Genomic Southern blot analysis suggests that the human haploid genome contains a single Gsα gene. Previous reports indicated the presence of multiple species of Gsα cDNA. The structure of the human Gsα gene suggests that four types of Gsα mRNAs may be generated from a single Gsα gene by alternate use of exon 3 and/or of two 3' splice sites of intron 3, where an unusual splice junction sequence (TG) instead of the consensus (AG) is used. S1 nuclease mapping analysis of human Gsα mRNA identified multiple transcriptional initiation sites. The promoter region of the human Gsα gene has extremely high G+C content (85%). It contains 4 GC boxes, but no typical TATA or CAAT box sequence. In the 5' flanking region, there are several blocks of sequences that are similar to the sequences of the 5' flanking region of the human cKi-ras2 gene

  1. CHARACTERIZATION OF THE OLFACTORY RECEPTORS EXPRESSED IN HUMAN SPERMATOZOA

    Directory of Open Access Journals (Sweden)

    Caroline eFlegel

    2016-01-01

    Full Text Available The detection of external cues is fundamental for human spermatozoa to locate the oocyte in the female reproductive tract. This task requires a specific chemoreceptor repertoire that is expressed on the surface of human spermatozoa, which is not fully identified to date. Olfactory receptors (ORs are candidate molecules and have been attributed to be involved in sperm chemotaxis and chemokinesis, indicating an important role in mammalian spermatozoa. An increasing importance has been suggested for spermatozoal RNA, which led us to investigate the expression of all 387 OR genes. This study provides the first comprehensive analysis of OR transcripts in human spermatozoa of several individuals by RNA-Seq. We detected 91 different transcripts in the spermatozoa samples that could be aligned to annotated OR genes. Using stranded mRNA-Seq, we detected a class of these putative OR transcripts in an antisense orientation, indicating a different function, rather than coding for a functional OR protein. Nevertheless, we were able to detect OR proteins in various compartments of human spermatozoa, indicating distinct functions in human sperm. A panel of various OR ligands induced Ca2+ signals in human spermatozoa, which could be inhibited by mibefradil. This study indicated that a variety of ORs are expressed at the mRNA and protein level in human spermatozoa and demonstrates that ORs are involved in the physiological processes.

  2. Defining the impact on yeast ATP synthase of two pathogenic human mitochondrial DNA mutations, T9185C and T9191C.

    Science.gov (United States)

    Kabala, Anna Magdalena; Lasserre, Jean-Paul; Ackerman, Sharon H; di Rago, Jean-Paul; Kucharczyk, Roza

    2014-05-01

    Mutations in the human mitochondrial ATP6 gene encoding ATP synthase subunit a/6 (referred to as Atp6p in yeast) are at the base of neurodegenerative disorders like Neurogenic Ataxia and Retinitis Pigmentosa (NARP), Leigh syndrome (LS), Charcot-Marie-Tooth (CMT), and ataxia telangiectasia. In previous studies, using the yeast Saccharomyces cerevisiae as a model we were able to better define how several of these mutations impact the ATP synthase. Here we report the construction of yeast models of two other ATP6 pathogenic mutations, T9185C and T9191C. The first one was reported as conferring a mild, sometimes reversible, CMT clinical phenotype; the second one has been described in a patient presenting with severe LS. We found that an equivalent of the T9185C mutation partially impaired the functioning of yeast ATP synthase, with only a 30% deficit in mitochondrial ATP production. An equivalent of the mutation T9191C had much more severe effects, with a nearly complete block in yeast Atp6p assembly and an >95% drop in the rate of ATP synthesis. These findings provide a molecular basis for the relative severities of the diseases induced by T9185C and T9191C.

  3. Pluripotent and Metabolic Features of Two Types of Porcine iPSCs Derived from Defined Mouse and Human ES Cell Culture Conditions.

    Directory of Open Access Journals (Sweden)

    Wei Zhang

    Full Text Available The domestic pig is an excellent animal model for stem cell research and clinical medicine. There is still no suitable culture condition to generate authentic porcine embryonic stem cells (pESCs and high quality porcine induced pluripotent stem cells (piPSCs. In this study, we found that culture conditions affected pluripotent and metabolic features of piPSCs. Using defined human embryonic stem cell (hESC and mouse ESC (mESC culture conditions, we generated two types of piPSCs, one of which was morphologically similar to hESCs (here called hpiPSCs, the other resembled mESCs (here called mpiPSCs. Transcriptome analysis and signaling pathway inhibition results suggested that mpiPSCs shared more of mESC signaling pathways, such as the BMP pathway and JAK/STAT pathway and hpiPSCs shared more hESC signaling pathways, such as the FGF pathway. Importantly, the mpiPSCs performed embryonic chimera incorporation more efficiently than the hpiPSCs did. In addition, the mpiPSCs showed mitochondrial features of naive ESCs and lipid droplets accumulation. These evidences may facilitate understanding of the gene regulation network and metabolism in piPSCs and promote derivation of bona fide pESCs for translational medicine.

  4. Technical advance: immunophenotypical characterization of human neutrophil differentiation

    DEFF Research Database (Denmark)

    Mora-Jensen, Helena Isabel; Jendholm, Johan; Fossum, Anna;

    2011-01-01

    The current study reports a flow cytometry-based protocol for the prospective purification of human BM populations representing six successive stages of terminal neutrophil differentiation, including early promyelocytes and late promyelocytes, myelocytes, metamyelocytes, band cells, and PMN neutr...

  5. Development and characterization of a new human hepatic cell line

    Science.gov (United States)

    Ramboer, Eva; De Craene, Bram; De Kock, Joey; Berx, Geert; Rogiers, Vera; Vanhaecke, Tamara; Vinken, Mathieu

    2015-01-01

    The increasing demand and hampered use of primary human hepatocytes for research purposes have urged scientists to search for alternative cell sources, such as immortalized hepatic cell lines. The aim of this study was to develop a human hepatic cell line using the combined overexpression of TERT and the cell cycle regulators cyclin D1 and mutant isoform CDK4R24C. Following transduction of adult human primary hepatocytes with the selected immortalization genes, cell growth was triggered and a cell line was established. When cultured under appropriate conditions, the cell line expressed several hepatocytic markers and liver-enriched transcription factors at the transcriptional and/or translational level, secreted liver-specific proteins and showed glycogen deposition. These results suggest that the immortalization strategy applied to primary human hepatocytes could generate a novel hepatic cell line that seems to retain some key hepatic characteristics. PMID:26869867

  6. Characterization of the human GARP (Golgi associated retrograde protein) complex

    International Nuclear Information System (INIS)

    The Golgi associated retrograde protein complex (GARP) or Vps fifty-three (VFT) complex is part of cellular inter-compartmental transport systems. Here we report the identification of the VFT tethering factor complex and its interactions in mammalian cells. Subcellular fractionation shows that human Vps proteins are found in the smooth membrane/Golgi fraction but not in the cytosol. Immunostaining of human Vps proteins displays a vesicular distribution most concentrated at the perinuclear envelope. Co-staining experiments with endosomal markers imply an endosomal origin of these vesicles. Significant accumulation of VFT complex positive endosomes is found in the vicinity of the Trans Golgi Network area. This is in accordance with a putative role in Golgi associated transport processes. In Saccharomyces cerevisiae, GARP is the main effector of the small GTPase Ypt6p and interacts with the SNARE Tlg1p to facilitate membrane fusion. Accordingly, the human homologue of Ypt6p, Rab6, specifically binds hVps52. In human cells, the 'orphan' SNARE Syntaxin 10 is the genuine binding partner of GARP mediated by hVps52. This reveals a previously unknown function of human Syntaxin 10 in membrane docking and fusion events at the Golgi. Taken together, GARP shows significant conservation between various species but diversification and specialization result in important differences in human cells

  7. Characterization of fasted human gastric fluid for relevant rheological parameters and gastric lipase activities

    DEFF Research Database (Denmark)

    Pedersen, Pernille Barbre; Vilmann, Peter; Bar-Shalom, Daniel;

    2013-01-01

    PURPOSE: To characterize human gastric fluid with regard to rheological properties and gastric lipase activity. In addition, traditional physicochemical properties were determined. METHODS: Fasted HGA were collected from 19 healthy volunteers during a gastroscopic examination. Rheological charact...

  8. Use of gene expression and pathway signatures to characterize the complexity of human melanoma.

    Science.gov (United States)

    Freedman, Jennifer A; Tyler, Douglas S; Nevins, Joseph R; Augustine, Christina K

    2011-06-01

    A defining characteristic of most human cancers is heterogeneity, resulting from the somatic acquisition of a complex array of genetic and genomic alterations. Dissecting this heterogeneity is critical to developing an understanding of the underlying mechanisms of disease and to paving the way toward personalized treatments of the disease. We used gene expression data sets from the analysis of primary and metastatic melanomas to develop a molecular description of the heterogeneity that characterizes this disease. Unsupervised hierarchical clustering, gene set enrichment analyses, and pathway activity analyses were used to describe the genetic heterogeneity of melanomas. Patterns of gene expression that revealed two distinct classes of primary melanoma, two distinct classes of in-transit melanoma, and at least three subgroups of metastatic melanoma were identified. Expression signatures developed to predict the status of oncogenic signaling pathways were used to explore the biological basis underlying these differential patterns of expression. This analysis of activities revealed unique pathways that distinguished the primary and metastatic subgroups of melanoma. Distinct patterns of gene expression across primary, in-transit, and metastatic melanomas underline the genetic heterogeneity of this disease. This heterogeneity can be described in terms of deregulation of signaling pathways, thus increasing the knowledge of the biological features underlying individual melanomas and potentially directing therapeutic opportunities to individual patients with melanoma.

  9. Comprehensive molecular characterization of human colon and rectal cancer

    NARCIS (Netherlands)

    Muzny, Donna M.; Bainbridge, Matthew N.; Chang, Kyle; Dinh, Huyen H.; Drummond, Jennifer A.; Fowler, Gerald; Kovar, Christie L.; Lewis, Lora R.; Morgan, Margaret B.; Newsham, Irene F.; Reid, Jeffrey G.; Santibanez, Jireh; Shinbrot, Eve; Trevino, Lisa R.; Wu, Yuan-Qing; Wang, Min; Gunaratne, Preethi; Donehower, Lawrence A.; Creighton, Chad J.; Wheeler, David A.; Gibbs, Richard A.; Lawrence, Michael S.; Voet, Douglas; Jing, Rui; Cibulskis, Kristian; Sivachenko, Andrey; Stojanov, Petar; McKenna, Aaron; Lander, Eric S.; Gabriel, Stacey; Getz, Gad; Ding, Li; Fulton, Robert S.; Koboldt, Daniel C.; Wylie, Todd; Walker, Jason; Dooling, David J.; Fulton, Lucinda; Delehaunty, Kim D.; Fronick, Catrina C.; Demeter, Ryan; Mardis, Elaine R.; Wilson, Richard K.; Chu, Andy; Chun, Hye-Jung E.; Mungall, Andrew J.; Pleasance, Erin; Robertson, A. Gordon; Stoll, Dominik; Balasundaram, Miruna; Birol, Inanc; Butterfield, Yaron S. N.; Chuah, Eric; Coope, Robin J. N.; Dhalla, Noreen; Guin, Ranabir; Hirst, Carrie; Hirst, Martin; Holt, Robert A.; Lee, Darlene; Li, Haiyan I.; Mayo, Michael; Moore, Richard A.; Schein, Jacqueline E.; Slobodan, Jared R.; Tam, Angela; Thiessen, Nina; Varhol, Richard; Zeng, Thomas; Zhao, Yongjun; Jones, Steven J. M.; Marra, Marco A.; Bass, Adam J.; Ramos, Alex H.; Saksena, Gordon; Cherniack, Andrew D.; Schumacher, Stephen E.; Tabak, Barbara; Carter, Scott L.; Pho, Nam H.; Nguyen, Huy; Onofrio, Robert C.; Crenshaw, Andrew; Ardlie, Kristin; Beroukhim, Rameen; Winckler, Wendy; Getz, Gad; Meyerson, Matthew; Protopopov, Alexei; Zhang, Juinhua; Hadjipanayis, Angela; Lee, Eunjung; Xi, Ruibin; Yang, Lixing; Ren, Xiaojia; Zhang, Hailei; Sathiamoorthy, Narayanan; Shukla, Sachet; Chen, Peng-Chieh; Haseley, Psalm; Xiao, Yonghong; Lee, Semin; Seidman, Jonathan; Chin, Lynda; Park, Peter J.; Kucherlapati, Raju; Auman, J. Todd; Hoadley, Katherine A.; Du, Ying; Wilkerson, Matthew D.; Shi, Yan; Liquori, Christina; Meng, Shaowu; Li, Ling; Turman, Yidi J.; Topal, Michael D.; Tan, Donghui; Waring, Scot; Buda, Elizabeth; Walsh, Jesse; Jones, Corbin D.; Mieczkowski, Piotr A.; Singh, Darshan; Wu, Junyuan; Gulabani, Anisha; Dolina, Peter; Bodenheimer, Tom; Hoyle, Alan P.; Simons, Janae V.; Soloway, Matthew; Mose, Lisle E.; Jefferys, Stuart R.; Balu, Saianand; O'Connor, Brian D.; Prins, Jan F.; Chiang, Derek Y.; Hayes, D. Neil; Perou, Charles M.; Hinoue, Toshinori; Weisenberger, Daniel J.; Maglinte, Dennis T.; Pan, Fei; Berman, Benjamin P.; Van den Berg, David J.; Shen, Hui; Jr, Timothy Triche; Baylin, Stephen B.; Laird, Peter W.; Getz, Gad; Noble, Michael; Voet, Doug; Saksena, Gordon; Gehlenborg, Nils; DiCara, Daniel; Zhang, Juinhua; Zhang, Hailei; Wu, Chang-Jiun; Liu, Spring Yingchun; Shukla, Sachet; Lawrence, Michael S.; Zhou, Lihua; Sivachenko, Andrey; Lin, Pei; Stojanov, Petar; Jing, Rui; Park, Richard W.; Nazaire, Marc-Danie; Robinson, Jim; Thorvaldsdottir, Helga; Mesirov, Jill; Park, Peter J.; Chin, Lynda; Thorsson, Vesteinn; Reynolds, Sheila M.; Bernard, Brady; Kreisberg, Richard; Lin, Jake; Iype, Lisa; Bressler, Ryan; Erkkilae, Timo; Gundapuneni, Madhumati; Liu, Yuexin; Norberg, Adam; Robinson, Tom; Da Yang, [No Value; Zhang, Wei; Shmulevich, Ilya; De Ronde, Jorma J.; Schultz, Nikolaus; Cerami, Ethan; Ciriello, Giovanni; Goldberg, Arthur P.; Gross, Benjamin; Jacobsen, Anders; Gao, Jianjiong; Kaczkowski, Bogumil; Sinha, Rileen; Aksoy, B. Arman; Antipin, Yevgeniy; Reva, Boris; Shen, Ronglai; Taylor, Barry S.; Chan, Timothy A.; Ladanyi, Marc; Sander, Chris; Akbani, Rehan; Zhang, Nianxiang; Broom, Bradley M.; Casasent, Tod; Unruh, Anna; Wakefield, Chris; Hamilton, Stanley R.; Cason, R. Craig; Baggerly, Keith A.; Weinstein, John N.; Haussler, David; Benz, Christopher C.; Stuart, Joshua M.; Benz, Stephen C.; Sanborn, J. Zachary; Vaske, Charles J.; Zhu, Jingchun; Szeto, Christopher; Scott, Gary K.; Yau, Christina; Ng, Sam; Goldstein, Ted; Ellrott, Kyle; Collisson, Eric; Cozen, Aaron E.; Zerbino, Daniel; Wilks, Christopher; Craft, Brian; Spellman, Paul; Penny, Robert; Shelton, Troy; Hatfield, Martha; Morris, Scott; Yena, Peggy; Shelton, Candace; Sherman, Mark; Paulauskis, Joseph; Gastier-Foster, Julie M.; Bowen, Jay; Ramirez, Nilsa C.; Black, Aaron; Pyatt, Robert; Wise, Lisa; White, Peter; Bertagnolli, Monica; Brown, Jen; Chan, Timothy A.; Chu, Gerald C.; Czerwinski, Christine; Denstman, Fred; Dhir, Rajiv; Doerner, Arnulf; Fuchs, Charles S.; Guillem, Jose G.; Iacocca, Mary; Juhl, Hartmut; Kaufman, Andrew; Kohl, Bernard; Van Le, Xuan; Mariano, Maria C.; Medina, Elizabeth N.; Meyers, Michael; Nash, Garrett M.; Paty, Phillip B.; Petrelli, Nicholas; Rabeno, Brenda; Richards, William G.; Solit, David; Swanson, Pat; Temple, Larissa; Tepper, Joel E.; Thorp, Richard; Vakiani, Efsevia; Weiser, Martin R.; Willis, Joseph E.; Witkin, Gary; Zeng, Zhaoshi; Zinner, Michael J.; Zornig, Carsten; Jensen, Mark A.; Sfeir, Robert; Kahn, Ari B.; Chu, Anna L.; Kothiyal, Prachi; Wang, Zhining; Snyder, Eric E.; Pontius, Joan; Pihl, Todd D.; Ayala, Brenda; Backus, Mark; Walton, Jessica; Whitmore, Jon; Baboud, Julien; Berton, Dominique L.; Nicholls, Matthew C.; Srinivasan, Deepak; Raman, Rohini; Girshik, Stanley; Kigonya, Peter A.; Alonso, Shelley; Sanbhadti, Rashmi N.; Barletta, Sean P.; Greene, John M.; Pot, David A.; Shaw, Kenna R. Mills; Dillon, Laura A. L.; Buetow, Ken; Davidsen, Tanja; Demchok, John A.; Eley, Greg; Ferguson, Martin; Fielding, Peter; Schaefer, Carl; Sheth, Margi; Yang, Liming; Guyer, Mark S.; Ozenberger, Bradley A.; Palchik, Jacqueline D.; Peterson, Jane; Sofia, Heidi J.; Thomson, Elizabeth

    2012-01-01

    To characterize somatic alterations in colorectal carcinoma, we conducted a genome-scale analysis of 276 samples, analysing exome sequence, DNA copy number, promoter methylation and messenger RNA and microRNA expression. A subset of these samples (97) underwent low-depth-of-coverage whole-genome seq

  10. Measurements and Characterizations of Mechanical Properties of Human Skins

    Science.gov (United States)

    Song, Han Wook; Park, Yon Kyu

    A skin is an indispensible organ for humans because it contributes to metabolism using its own biochemical functions and protects the human body from external stimuli. Recently, mechanical properties such as a thickness, a friction and an elastic coefficient have been used as a decision index in the skin physiology and in the skin care market due to the increased awareness of wellbeing issues. In addition, the use of mechanical properties is known to have good discrimination ability in the classification of human constitutions, which are used in the field of an alternative medicine. In this study, a system that measures mechanical properties such as a friction and an elastic coefficient is designed. The equipment consists of a load cell type (manufactured by the authors) for the measurements of a friction coefficient, a decompression tube for the measurement of an elastic coefficient. Using the proposed system, the mechanical properties of human skins from different constitutions were compared, and the relative repeatability error for measurements of mechanical properties was determined to be less than 2%. Combining the inspection results of medical doctors in the field of an alternative medicine, we could conclude that the proposed system might be applicable to a quantitative constitutional diagnosis between human constitutions within an acceptable level of uncertainty.

  11. Characterization of human-dog social interaction using owner report.

    Science.gov (United States)

    Lit, Lisa; Schweitzer, Julie B; Oberbauer, Anita M

    2010-07-01

    Dog owners were surveyed for observations of social behaviors in their dogs, using questions adapted from the human Autism Diagnostic Observation Schedule (ADOS) pre-verbal module. Using 939 responses for purebred and mixed-breed dogs, three factors were identified: initiation of reciprocal social behaviors (INIT), response to social interactions (RSPNS), and communication (COMM). There were small or no effects of sex, age, breed group or training. For six breeds with more than 35 responses (Border Collie, Rough Collie, German Shepherd, Golden Retriever, Labrador Retriever, Standard Poodle), the behaviors eye contact with humans, enjoyment in interactions with human interaction, and name recognition demonstrated little variability across breeds, while asking for objects, giving/showing objects to humans, and attempts to direct humans' attention showed higher variability across these breeds. Breeds with genetically similar backgrounds had similar response distributions for owner reports of dog response to pointing. When considering these breeds according to the broad categories of "herders" and "retrievers," owners reported that the "herders" used more eye contact and vocalization, while the "retrievers" used more body contact. Information regarding social cognitive abilities in dogs provided by owner report suggest that there is variability across many social cognitive abilities in dogs and offers direction for further experimental investigations. PMID:20438815

  12. Characterization of a Novel Human-Specific STING Agonist that Elicits Antiviral Activity Against Emerging Alphaviruses.

    Directory of Open Access Journals (Sweden)

    Tina M Sali

    2015-12-01

    Full Text Available Pharmacologic stimulation of innate immune processes represents an attractive strategy to achieve multiple therapeutic outcomes including inhibition of virus replication, boosting antitumor immunity, and enhancing vaccine immunogenicity. In light of this we sought to identify small molecules capable of activating the type I interferon (IFN response by way of the transcription factor IFN regulatory factor 3 (IRF3. A high throughput in vitro screen yielded 4-(2-chloro-6-fluorobenzyl-N-(furan-2-ylmethyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide (referred to herein as G10, which was found to trigger IRF3/IFN-associated transcription in human fibroblasts. Further examination of the cellular response to this molecule revealed expression of multiple IRF3-dependent antiviral effector genes as well as type I and III IFN subtypes. This led to the establishment of a cellular state that prevented replication of emerging Alphavirus species including Chikungunya virus, Venezuelan Equine Encephalitis virus, and Sindbis virus. To define cellular proteins essential to elicitation of the antiviral activity by the compound we employed a reverse genetics approach that utilized genome editing via CRISPR/Cas9 technology. This allowed the identification of IRF3, the IRF3-activating adaptor molecule STING, and the IFN-associated transcription factor STAT1 as required for observed gene induction and antiviral effects. Biochemical analysis indicates that G10 does not bind to STING directly, however. Thus the compound may represent the first synthetic small molecule characterized as an indirect activator of human STING-dependent phenotypes. In vivo stimulation of STING-dependent activity by an unrelated small molecule in a mouse model of Chikungunya virus infection blocked viremia demonstrating that pharmacologic activation of this signaling pathway may represent a feasible strategy for combating emerging Alphaviruses.

  13. Purification, characterization and development of radioimmunoassay of human liver ribonuclease

    International Nuclear Information System (INIS)

    Human liver ribonuclease (RNase) was purified 3600-fold into an electrophoretically homogeneous state by column chromatography on phosphocellulose, gel filtration, poly(G) affinity chromatography, and heparin affinity chromatography. The molecular weight of the RNase estimated by SDS disc electrophoresis was 19500. RNase was a heat- and pH-stable protein, and optimum activity was obtained at pH 7.0. The radioimmunoassay (RIA) for human liver RNase has been developed and the assay was shown to be sensitive (20 ng/ml), reproducible and specific. A good parallel relationship was observed between the standard curve and the dilution curves for serum and urine. No cross-reactivity was demonstrated between human liver and pancreatic RNase (less than 1%). In 44 normal subjects, the mean serum concentration of liver RNase determined by the RIA was found to be 99.4 ng/ml (SD+-66.3). (Auth.)

  14. A Colorimetric Characterization of the Raw Digital Data of the Visible Human Dataset Images

    OpenAIRE

    Bonacina, Stefano; Masseroli, Marco; Menegoni, Francesco; Quattrone, Giorgio; Pinciroli, Francesco

    2003-01-01

    A colorimetric characterization of the all about 9 thousand Visible Human Dataset (VHD) cryosectioned color images of the male and female body is described here. Such characterization is performed keeping limited the computational time besides the high resolution of the considered VHD images. The about 27 thousand distinct histograms obtained are downloadable from the VHD Milano Mirror Site® ftp server.

  15. Isolation and characterization of the human prolactin gene.

    OpenAIRE

    Truong, A T; Duez, C.; Belayew, A.; Renard, A.; Pictet, R; Bell, G I; Martial, J A

    1984-01-01

    Prolactin (PRL) and growth hormone (GH) genes derive from a common ancestor and still share some sequence homologies. Their expression in the pituitary gland is regulated in opposite directions by most of the many hormones acting on them. This provides an interesting system to study sequences involved in gene expression. Using a human PRL cDNA clone as a probe, we screened a human genomic DNA library in lambda phage and isolated a single recombinant comprising the whole hPRL gene. It was char...

  16. Development of a cell-defined siRNA microarray for analysis of gene function in human bone marrow stromal cells.

    Science.gov (United States)

    Kim, Hi Chul; Kim, Gi-Hwan; Cho, Ssang-Goo; Lee, Eun Ju; Kwon, Yong-Jun

    2016-03-01

    Small interfering RNA (siRNA) screening approaches have provided useful tools for the validation of genetic functions; however, image-based siRNA screening using multiwell plates requires large numbers of cells and time, which could be the barrier in application for gene mechanisms study using human adult cells. Therefore, we developed the advanced method with the cell-defined siRNA microarray (CDSM), for functional analysis of genes in small scale within slide glass using human bone marrow stromal cells (hBMSCs). We designed cell spot system with biomaterials (sucrose, gelatin, poly-L-lysine and matrigel) to control the attachment of hBMSCs inside spot area on three-dimensional (3D) hydrogel-coated slides. The p65 expression was used as a validation standard which described our previous report. For the optimization of siRNA mixture, first, we detected five kinds of commercialized reagent (Lipofectamine 2000, RNAi-Max, Metafectine, Metafectine Pro, TurboFectin 8.0) via validation. Then, according to quantification of p65 expression, we selected 2 μl of RNAi-Max as the most effective reagent condition on our system. Using same validation standard, we optimized sucrose and gelatin concentration (80 mM and 0.13%), respectively. Next, we performed titration of siRNA quantity (2.66-5.55 μM) by reverse transfection time (24 h, 48 h, 72 h) and confirmed 3.75 μM siRNA concentration and 48 h as the best condition. To sum up the process for optimized CDSM, 3 μl of 20 μM siRNA (3.75 μM) was transferred to the 384-well V-bottom plate containing 2 μl of dH2O and 2 μl of 0.6M sucrose (80 mM). Then, 2 μl of RNAi-Max was added and incubated for 20 min at room temperature after mixing gently and centrifugation shortly. Five microliters of gelatin (0.26%) and 2 μl of growth factor reduced phenol red-free matrigel (12.5%) were added and mixed by pipetting gently. Finally, optimized siRNA mixture was printed on 3D hydrogel-coated slides and cell-defined attachment and si

  17. Purification and characterization of osteopontin from human milk

    DEFF Research Database (Denmark)

    Sørensen, Steen; Justesen, Steen Just; Johnsen, Anders H

    2003-01-01

    biological source is missing. A four-step procedure was used to purify OPN from human milk, based on its crystal growth inhibitory activity, including anion exchange chromatography, the elimination of casein, hydroxyapatite chromatography, and negative affinity chromatography. Purified OPN was further...

  18. Purification and characterization of a soluble calnexin from human placenta

    DEFF Research Database (Denmark)

    Olsen, Dorthe T; Peng, Li; Træholt, Sofie D;

    2013-01-01

    Calreticulin (Crt) and calnexin (Cnx) are homologous endoplasmic reticulum (ER) chaperones involved in protein folding and quality control. Crt is a soluble ER luminal Mr 46 kDa protein and Cnx is a Mr 67kDa ER membrane protein. During purification of Crt from human placenta a soluble form of Cnx...

  19. Outcomes of Interferon/Ribavirin Therapy in Patients with HCV Defined by Expression of Plasma Soluble Human Leukocyte Antigen-G but Not IL-37

    Science.gov (United States)

    Ding, Shi-xiong; Ma, Jian-bo; Hu, Yao-ren; Hu, Ai-rong; Shen, Qiang; Gao, Guo-sheng

    2016-01-01

    Background Chronic hepatitis C virus (HCV) infection leads to life-threatening complications worldwide. Immunomodulation signals the response to virus clearance. The immune-suppressive molecule human leukocyte antigen-G (HLA-G) has been shown to function in inhibiting both innate and adaptive immune responses. The objective of this study was to investigate the expression of HLA-G and IL-37 in sustained virological response (SVR) and non-SVR HCV-positive patients before and after complete treatment with a combination of pegylated interferon (IFN) and ribavirin (RBV). Material/Methods Our study included 132 chronic hepatitis C patents who received combined therapy with IFN-α and RBV. Both SVR and non-SVR patients were included. The end-of-treatment response was defined as undetectable HCV RNA at week 48. Patients with end-of-treatment response were detected by HCV RNA at 24 weeks after therapy. The expression levels of HLA-G and IL-37 at the end and 24 weeks after treatment were detected by ELISA. Results Plasma HLA-G and IL-37 were significantly increased in HCV-infected patients compared with healthy individuals before treatment. Furthermore, HLA-G in SVR patients was noticeably decreased after treatment, while HLA-G in non-SVR patients had no changes after treatment. Additionally, both in SVR and non-SVR patients, the expression of IL-37 was remarkably reduced compared with baseline after treatment. Conclusions These findings suggest that elevation of HLA-G and IL-37 in HCV may play an important role in response to combined therapy with IFN-α and RBV. Monitoring the expression of HLA-G during therapy could contribute to adjusting the treatment program of HCV-infected patients. PMID:27112970

  20. Characterization of a Canine Homolog of Human Aichivirus▿

    OpenAIRE

    Kapoor, A; Simmonds, P.; Dubovi, E J; Qaisar, N.; Henriquez, J. A.; J. Medina; S Shields; Lipkin, W. I.

    2011-01-01

    Many of our fatal "civilization" infectious diseases have arisen from domesticated animals. Although picornaviruses infect most mammals, infection of a companion animal is not known. Here we describe the identification and genomic characterization of the first canine picornavirus. Canine kobuvirus (CKoV), identified in stool samples from dogs with diarrhea, has a genomic organization typical of a picornavirus and encodes a 2,469-amino-acid polyprotein flanked by 5' and 3' untranslated regions...

  1. Advanced approaches to characterize the human intestinal microbiota by computational meta-analysis

    NARCIS (Netherlands)

    Nikkilä, J.; Vos, de W.M.

    2010-01-01

    GOALS: We describe advanced approaches for the computational meta-analysis of a collection of independent studies, including over 1000 phylogenetic array datasets, as a means to characterize the variability of human intestinal microbiota. BACKGROUND: The human intestinal microbiota is a complex micr

  2. Characterization of human serum spreading factor with monoclonal antibody.

    OpenAIRE

    Barnes, D W; Silnutzer, J; See, C; Shaffer, M

    1983-01-01

    Serum spreading factor is a glycoprotein isolated from human serum that promotes spreading of a variety of cell types on culture dishes. We developed mouse hybridoma lines secreting monoclonal antibody to serum spreading factor that markedly inhibited the rate of serum spreading factor-promoted spreading of both fibroblastic and epithelial cells in culture. Fibronectin-promoted cell spreading was unaffected by monoclonal antibody to serum spreading factor, and the factor appeared to be distin...

  3. Characterization of optimal resting tension in human pulmonary arteries

    Science.gov (United States)

    Hussain, Azar; Bennett, Robert T; Chaudhry, Mubarak A; Qadri, Syed S; Cowen, Mike; Morice, Alyn H; Loubani, Mahmoud

    2016-01-01

    AIM To determine the optimum resting tension (ORT) for in vitro human pulmonary artery (PA) ring preparations. METHODS Pulmonary arteries were dissected from disease free sections of the resected lung in the operating theatre and tissue samples were directly sent to the laboratory in Krebs-Henseleit solution (Krebs). The pulmonary arteries were then cut into 2 mm long rings. PA rings were mounted in 25 mL organ baths or 8 mL myograph chambers containing Krebs compound (37 °C, bubbled with 21% O2: 5% CO2) to measure changes in isometric tension. The resting tension was set at 1-gram force (gf) with vessels being left static to equilibrate for duration of one hour. Baseline contractile reactions to 40 mmol/L KCl were obtained from a resting tension of 1 gf. Contractile reactions to 40 mmol/L KCl were then obtained from stepwise increases in resting tension (1.2, 1.4, 1.6, 1.8 and 2.0 gf). RESULTS Twenty PA rings of internal diameter between 2-4 mm were prepared from 4 patients. In human PA rings incrementing the tension during rest stance by 0.6 gf, up to 1.6 gf significantly augmented the 40 mmol/L KCl stimulated tension. Further enhancement of active tension by 0.4 gf, up to 2.0 gf mitigate the 40 mmol/L KCl stimulated reaction. Both Myograph and the organ bath demonstrated identical conclusions, supporting that the radial optimal resting tension for human PA ring was 1.61 g. CONCLUSION The radial optimal resting tension in our experiment is 1.61 gf (15.78 mN) for human PA rings. PMID:27721938

  4. Radioimmunoassay of human growth hormone: characterization of a monoclonal antibody

    International Nuclear Information System (INIS)

    The introduction of cell-hybridization techniques by Koehler and Milstein in 1975 to produce monoclonal antibodies (MA) was a definite improvement in methodological tools of radioimmunoassay, quite apart from all other applications of this technique in immunohistochemistry, affinity chromatography, target-directed drug delivery systems etc. MAs would ideally be suited in RIAs, when the specificity is the crucial aspect of the determination. However, for reasons which are not completely understood, assays with MAs very often lack the attribute of being highly sensitive. Despite several reports in the literature on MAs against human growth hormone (HGH), none of these seem sensitive enough to be of use in clinical chemistry, where a too strongly marked specificity may even be unwanted. However, from the scientific point of view, MAs against polypeptide hormones are of great interest. An MA to HGH was developed with a sensitivity limit of 0.2 ng. The titre of the ascites fluid is higher than 1:106 and the specificity against human placental lactogen, human prolactin and rat growth hormone is nearly complete. A critical step of the RIA procedure is the separation of bound and free hormone. A combination of human immunoglobulin (Sandoglobulin) with polyethylene glycol gives optimal results. A Scatchard plot reveals an affinity constant of 4x10-11M-bar and a maximal binding capacity of 2x108cpm/μL. In conclusion, our monoclonal antibody represents an excellent investigational tool for endocrine research and it seems to be the most sensitive and specific MA to HGH described to date. However, for practical clinical applications, there seems to be little advantage of an MA over a conventional polyclonal antiserum. (author)

  5. Molecular Diagnostic Methods for Detection and Characterization of Human Noroviruses

    OpenAIRE

    Chen, HaiFeng; Hu, Yuan

    2016-01-01

    Human noroviruses are a group of viral agents that afflict people of all age groups. The viruses are now recognized as the most common causative agent of nonbacterial acute gastroenteritis and foodborne viral illness worldwide. However, they have been considered to play insignificant roles in the disease burden of acute gastroenteritis for the past decades until the recent advent of new and more sensitive molecular diagnostic methods. The availability and application of the molecular diagnost...

  6. Characterization of a monoclonal antibody to human erythropoietin.

    OpenAIRE

    Weiss, T L; Kavinsky, C J; Goldwasser, E

    1982-01-01

    Hybrid cells that synthesize a monospecific antibody directed toward erythropoietin have been produced by the fusion of mouse plasmacytoma cells with spleen cells from rats immunized against human erythropoietin. The antibody binds the alpha and beta forms and the asialo alpha form of erythropoietin to the same extent. It is an immunoglobulin of the IgG class and binds only erythropoietin in an impure preparation of the hormone. Biologically active unlabeled erythropoietin competes with biolo...

  7. Isolation and characterization of human membrane carboxypeptidase (HMCP)

    International Nuclear Information System (INIS)

    The authors detected a membrane-bound carboxypeptidase in human placenta and other tissues which cleaves C-terminal Lys or Arg of peptides such as Lys6-Met5-enkephalin. The enzyme was solubilized from placental microvilli with 0.8% CHAPS and purified 427-fold by ion-exchange chromatography, Sepharose-arginine affinity chromatography, chromatofocusing and gel filtration on HPLC. HMCP had a mol. wt. of 67,000 in SDS-PAGE and 65,300 in gel filtration and a pH optimum of 7.0. HMCP cleaved Bz-Gly-argininic acid the fastest (90 μmol/min/mg) followed by Bz-Ala-Lys (41), Bz-Phe-Lys (26), Bz-Gly-Arg (1.7) and Bz-Gly-Lys (1.6). Activity was stimulated by CoCl2 and inhibited by cadmium acet., o-phenanthroline and 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid but not by phenylmethylsulfonyl fluoride, aprotinin or p-chloromercuriphenylsulfonate. The enzyme was stable for 1 hr at room temp. at pH 4.25, but lost 31% activity at pH 4.0. HMCP did not react with antiserum to human plasma carboxypeptidase N in Western blotting. This study shows that human placental microvilli contain a membrane carboxypeptidase, that differs from other carboxypeptidases, and cleaves C-terminal basic amino acids from peptides. This enzyme could be involved in regulating the level of peptide hormones in the placenta and other tissues

  8. Cloning and characterization of human DNA repair genes

    International Nuclear Information System (INIS)

    The isolation of two addition human genes that give efficient restoration of the repair defects in other CHO mutant lines is reported. The gene designated ERCC2 (Excision Repair Complementing Chinese hamster) corrects mutant UV5 from complementation group 1. They recently cloned this gene by first constructing a secondary transformant in which the human gene was shown to have become physically linked to the bacterial gpt dominant-marker gene by cotransfer in calcium phosphate precipitates in the primary transfection. Transformants expressing both genes were recovered by selecting for resistance to both UV radiation and mycophenolic acid. Using similar methods, the human gene that corrects CHO mutant EM9 was isolated in cosmids and named XRCC1 (X-ray Repair Complementing Chinese hamster). In this case, transformants were recovered by selecting for resistance to CldUrd, which kills EM9 very efficiently. In both genomic and cosmid transformants, the XRCC1 gene restored resistance to the normal range. DNA repair was studied using the kinetics of strand-break rejoining, which was measured after exposure to 137Cs γ-rays

  9. Functional characterization of human cancer-derived TRKB mutations.

    Directory of Open Access Journals (Sweden)

    Thomas R Geiger

    Full Text Available Cancer originates from cells that have acquired mutations in genes critical for controlling cell proliferation, survival and differentiation. Often, tumors continue to depend on these so-called driver mutations, providing the rationale for targeted anticancer therapies. To date, large-scale sequencing analyses have revealed hundreds of mutations in human tumors. However, without their functional validation it remains unclear which mutations correspond to driver, or rather bystander, mutations and, therefore, whether the mutated gene represents a target for therapeutic intervention. In human colorectal tumors, the neurotrophic receptor TRKB has been found mutated on two different sites in its kinase domain (TRKB(T695I and TRKB(D751N. Another site, in the extracellular part of TRKB, is mutated in a human lung adenocarcinoma cell line (TRKB(L138F. Lastly, our own analysis has identified one additional TRKB point mutation proximal to the kinase domain (TRKB(P507L in a human melanoma cell line. The functional consequences of all these point mutations, however, have so far remained elusive. Previously, we have shown that TRKB is a potent suppressor of anoikis and that TRKB-expressing cells form highly invasive and metastatic tumors in nude mice. To assess the functional consequences of these four TRKB mutations, we determined their potential to suppress anoikis and to form tumors in nude mice. Unexpectedly, both colon cancer-derived mutants, TRKB(T695I and TRKB(D751N, displayed reduced activity compared to that of wild-type TRKB. Consistently, upon stimulation with the TRKB ligand BDNF, these mutants were impaired in activating TRKB and its downstream effectors AKT and ERK. The two mutants derived from human tumor cell lines (TRKB(L138F and TRKB(P507L were functionally indistinguishable from wild-type TRKB in both in-vitro and in-vivo assays. In conclusion, we fail to detect any gain-of-function of four cancer-derived TRKB point mutations.

  10. Functional characterization of serotonin receptor subtypes in human duodenal secretion

    DEFF Research Database (Denmark)

    Engelmann, Bodil Elisabeth; Bindslev, Niels; Poulsen, Steen Seier;

    2006-01-01

    Serotonin (5-HT) stimulates ion secretion in the gastrointestinal tract and the sensitivity for 5-HT might be altered in dyspeptic patients infected with Helicobacter pylori. The purpose of the present study was to characterize the 5-HT-induced electrogenic ion transport in the duodenum of dyspep......Serotonin (5-HT) stimulates ion secretion in the gastrointestinal tract and the sensitivity for 5-HT might be altered in dyspeptic patients infected with Helicobacter pylori. The purpose of the present study was to characterize the 5-HT-induced electrogenic ion transport in the duodenum...... of dyspeptic patients with or without Helicobacter pylori infection, and to determine the 5-HT receptor subtypes functionally involved. Biopsies from the second part of duodenum were obtained from 43 dyspeptic patients during routine endoscopy. Biopsies were mounted in modified Ussing chambers with air suction...... for measurements of short-circuit current by a previously validated technique. Short-circuit current was measured before and after application of graded cumulative doses of 5-HT and a single dose of bumetanide (an inhibitor of chloride/bicarbonate transport), or one of the selective 5-HT receptor antagonists...

  11. Defining talent management components

    Directory of Open Access Journals (Sweden)

    Golchin Shafieian

    2014-07-01

    Full Text Available In today's world of global trade, it can guarantee success and excellence of organizations against competitors is talented human resources in an organization, especially at the managerial level. But the challenges that organizations face include: attracting, evaluating, exploring, and developing and keeping talent. The main objective of this study is to define the components of talent management in the University of Chaloos, in order to provide a way to reduce the challenges and short comings and also to look for ways to improve employee performance is discussed. This study is based on data collected from all employees of the University of Chaloos in August 2013; through two questionnaires have been developed. The statistical population has been considered 407 persons in these universities and according the Morgan Table, 198 persons were selected as sample of among research society. More over, 28 questions are used for determining components of talent management. Finally, we concluded that the component of talent management have been properly defined in the Universities of Chaloos.

  12. Functional Characterization of Cholera Toxin Inhibitors Using Human Intestinal Organoids.

    Science.gov (United States)

    Zomer-van Ommen, Domenique D; Pukin, Aliaksei V; Fu, Ou; Quarles van Ufford, Linda H C; Janssens, Hettie M; Beekman, Jeffrey M; Pieters, Roland J

    2016-07-28

    Preclinical drug testing in primary human cell models that recapitulate disease can significantly reduce animal experimentation and time-to-the-clinic. We used intestinal organoids to quantitatively study the potency of multivalent cholera toxin inhibitors. The method enabled the determination of IC50 values over a wide range of potencies (15 pM to 9 mM). The results indicate for the first time that an organoid-based swelling assay is a useful preclinical method to evaluate inhibitor potencies of drugs that target pathogen-derived toxins. PMID:27347611

  13. Defining the Anthropocene

    Science.gov (United States)

    Lewis, Simon; Maslin, Mark

    2016-04-01

    Time is divided by geologists according to marked shifts in Earth's state. Recent global environmental changes suggest that Earth may have entered a new human-dominated geological epoch, the Anthropocene. Should the Anthropocene - the idea that human activity is a force acting upon the Earth system in ways that mean that Earth will be altered for millions of years - be defined as a geological time-unit at the level of an Epoch? Here we appraise the data to assess such claims, first in terms of changes to the Earth system, with particular focus on very long-lived impacts, as Epochs typically last millions of years. Can Earth really be said to be in transition from one state to another? Secondly, we then consider the formal criteria used to define geological time-units and move forward through time examining whether currently available evidence passes typical geological time-unit evidence thresholds. We suggest two time periods likely fit the criteria (1) the aftermath of the interlinking of the Old and New Worlds, which moved species across continents and ocean basins worldwide, a geologically unprecedented and permanent change, which is also the globally synchronous coolest part of the Little Ice Age (in Earth system terms), and the beginning of global trade and a new socio-economic "world system" (in historical terms), marked as a golden spike by a temporary drop in atmospheric CO2, centred on 1610 CE; and (2) the aftermath of the Second World War, when many global environmental changes accelerated and novel long-lived materials were increasingly manufactured, known as the Great Acceleration (in Earth system terms) and the beginning of the Cold War (in historical terms), marked as a golden spike by the peak in radionuclide fallout in 1964. We finish by noting that the Anthropocene debate is politically loaded, thus transparency in the presentation of evidence is essential if a formal definition of the Anthropocene is to avoid becoming a debate about bias. The

  14. Evaluation of CD24 as a marker to rapidly define the mesenchymal stem cell phenotype and its differentiation in human nucleus pulposus

    Institute of Scientific and Technical Information of China (English)

    Guan Xiaoming; Ma Xun; Zhang Li; Feng Haoyu; Ma Zhuo

    2014-01-01

    Background Recent studies have indicated that human nucleus pulposus contain mesenchymal stem cells (NP-MSCs).However,the immunophenotypic variation of NP-MSCs in vitro was unclear.The present study was conducted to address the immunophenotypic variation of mesenchymal stem cells in nucleus pulposus under continuous proliferation in vitro and show the difference between mesenchymal stem cells and nucleus pulposus cell.Methods Tissue samples were obtained from thoracolumbar burst fracture patients and degenerative disc disease patients who underwent discectomy and fusion procedures.Flow cytometric and laser scanning confocal microscope (LSCM) were used to detect the variation of mesenchymal stem cells in nucleus pulposus which were expressing CD105 and CD24 in condition with or without transforming growth factor β1 (TGF-β1).Results More than 90% of the analyzed primary cells of mesenchymal stem cells in nucleus pulposus fulfilled the general immunophenotyping criteria for MSCs,such as CD44,CD105 and CD29,but the marker of mature NP cells characterized as CD24 was negative.In continuous cultures,the proportion of mesenchymal stem cells which were expressing CD44,CD105 and CD29 in nucleus pulposus gradually decreased.The mesenchymal stem cells in nucleus pulposus cells were positive for CD105 and CD29,with slight positivity for CD44.The CD24 expression gradually increased in proliferation.Biparametric flow cytometry and laser scanning confocal microscopy confirmed the presence of cells which were expressing CD105 and CD24 independently,and only a small part of cells expressed both CD105 and CD24 simultaneously.TGF-β1 could stimulate mesenchymal stem cells in nucleus pulposus to express CD24.Conclusions Non-degenerative and degenerative NP contains mesechymal stem cells.The variation of CD24 can be used as a marker to identify the NP-MSCs differentiation into NP-like cells.

  15. Biochemical characterization of human peroxiredoxin 2, an antioxidative protein

    Institute of Scientific and Technical Information of China (English)

    Sheng Yan; Shaopei Chen; Zhendong Li; Haiying Wang; Tuxiong Huang; Xiaoning Wang; Jufang Wang

    2012-01-01

    Human peroxiredoxin 2 (Prx2),which is abundant in erythrocytes,has been shown to play a key role in protecting erythrocytes against oxidative stress by scavenging reactive oxygen species as well as participating in cell signal transduction.Here,human Prx2 gene was successfully cloned into Escherichia coli BL21 (DE3) for Prx2 expression.Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis suggested that the recombinant protein was expressed mainly in a soluble form.The recombinant protein was purified by one-step Ni-nitrilotriacetic acid chelating affinity chromatography to a purity of up to 91.5%.The peroxidase activity of Prx2 to scavenge H2O2was determined by a ferrithiocyanate assay.The ability of Prx2 to protect plasmid DNA was tested by using a mixed-function oxidation system,and results showed that Prx2 could prevent DNA from undergoing oxidative stress. Ultraviolet (UV)-induced cell apoptosis assay demonstrated that Prx2 is also able to protect NIH/3T3 cells from UV-induced damage,suggesting its possible applications in cosmetics and other areas.

  16. Proteogenomic characterization of human colon and rectal cancer

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Bing; Wang, Jing; Wang, Xiaojing; Zhu, Jing; Liu, Qi; Shi, Zhiao; Chambers, Matthew C.; Zimmerman, Lisa J.; Shaddox, Kent F.; Kim, Sangtae; Davies, Sherri; Wang, Sean; Wang, Pei; Kinsinger, Christopher; Rivers, Robert; Rodriguez, Henry; Townsend, Reid; Ellis, Matthew; Carr, Steven A.; Tabb, David L.; Coffey, Robert J.; Slebos, Robbert; Liebler, Daniel

    2014-09-18

    We analyzed proteomes of colon and rectal tumors previously characterized by the Cancer Genome Atlas (TCGA) and performed integrated proteogenomic analyses. Protein sequence variants encoded by somatic genomic variations displayed reduced expression compared to protein variants encoded by germline variations. mRNA transcript abundance did not reliably predict protein expression differences between tumors. Proteomics identified five protein expression subtypes, two of which were associated with the TCGA "MSI/CIMP" transcriptional subtype, but had distinct mutation and methylation patterns and associated with different clinical outcomes. Although CNAs showed strong cis- and trans-effects on mRNA expression, relatively few of these extend to the protein level. Thus, proteomics data enabled prioritization of candidate driver genes. Our analyses identified HNF4A, a novel candidate driver gene in tumors with chromosome 20q amplifications. Integrated proteogenomic analysis provides functional context to interpret genomic abnormalities and affords novel insights into cancer biology.

  17. Defining natural history: assessment of the ability of college students to aid in characterizing clinical progression of Niemann-Pick disease, type C.

    Directory of Open Access Journals (Sweden)

    Jenny Shin

    Full Text Available Niemann-Pick Disease, type C (NPC is a fatal, neurodegenerative, lysosomal storage disorder. It is a rare disease with broad phenotypic spectrum and variable age of onset. These issues make it difficult to develop a universally accepted clinical outcome measure to assess urgently needed therapies. To this end, clinical investigators have defined emerging, disease severity scales. The average time from initial symptom to diagnosis is approximately 4 years. Further, some patients may not travel to specialized clinical centers even after diagnosis. We were therefore interested in investigating whether appropriately trained, community-based assessment of patient records could assist in defining disease progression using clinical severity scores. In this study we evolved a secure, step wise process to show that pre-existing medical records may be correctly assessed by non-clinical practitioners trained to quantify disease progression. Sixty-four undergraduate students at the University of Notre Dame were expertly trained in clinical disease assessment and recognition of major and minor symptoms of NPC. Seven clinical records, randomly selected from a total of thirty seven used to establish a leading clinical severity scale, were correctly assessed to show expected characteristics of linear disease progression. Student assessment of two new records donated by NPC families to our study also revealed linear progression of disease, but both showed accelerated disease progression, relative to the current severity scale, especially at the later stages. Together, these data suggest that college students may be trained in assessment of patient records, and thus provide insight into the natural history of a disease.

  18. Characterizing synaptic protein development in human visual cortex enables alignment of synaptic age with rat visual cortex

    Directory of Open Access Journals (Sweden)

    Joshua G.A Pinto

    2015-02-01

    Full Text Available Although many potential neuroplasticity based therapies have been developed in the lab, few have translated into established clinical treatments for human neurologic or neuropsychiatric diseases. Animal models, especially of the visual system, have shaped our understanding of neuroplasticity by characterizing the mechanisms that promote neural changes and defining timing of the sensitive period. The lack of knowledge about development of synaptic plasticity mechanisms in human cortex, and about alignment of synaptic age between animals and humans, has limited translation of neuroplasticity therapies. In this study, we quantified expression of a set of highly conserved pre- and post-synaptic proteins (Synapsin, Synaptophysin, PSD-95, Gephyrin and found that synaptic development in human primary visual cortex continues into late childhood. Indeed, this is many years longer than suggested by neuroanatomical studies and points to a prolonged sensitive period for plasticity in human sensory cortex. In addition, during childhood we found waves of inter-individual variability that are different for the 4 proteins and include a stage during early development (<1 year when only Gephyrin has high inter-individual variability. We also found that pre- and post-synaptic protein balances develop quickly, suggesting that maturation of certain synaptic functions happens within the first year or two of life. A multidimensional analysis (principle component analysis showed that most of the variance was captured by the sum of the 4 synaptic proteins. We used that sum to compare development of human and rat visual cortex and identified a simple linear equation that provides robust alignment of synaptic age between humans and rats. Alignment of synaptic ages is important for age-appropriate targeting and effective translation of neuroplasticity therapies from the lab to the clinic.

  19. Structural characterization of human heparanase reveals insights into substrate recognition.

    Science.gov (United States)

    Wu, Liang; Viola, Cristina M; Brzozowski, Andrzej M; Davies, Gideon J

    2015-12-01

    Heparan sulfate (HS) is a glycosaminoglycan that forms a key component of the extracellular matrix (ECM). Breakdown of HS is carried out by heparanase (HPSE), an endo-β-glucuronidase of the glycoside hydrolase 79 (GH79) family. Overexpression of HPSE results in breakdown of extracellular HS and release of stored growth factors and hence is strongly linked to cancer metastasis. Here we present crystal structures of human HPSE at 1.6-Å to 1.9-Å resolution that reveal how an endo-acting binding cleft is exposed by proteolytic activation of latent proHPSE. We used oligosaccharide complexes to map the substrate-binding and sulfate-recognition motifs. These data shed light on the structure and interactions of a key enzyme involved in ECM maintenance and provide a starting point for the design of HPSE inhibitors for use as biochemical tools and anticancer therapeutics. PMID:26575439

  20. Characterization of monoclonal antibodies directed against human thyroid stimulating hormone

    International Nuclear Information System (INIS)

    Monoclonal antibodies directed against human thyroid stimulating hormone (TSH) were obtained from hybrid myelomas, following fusion of mouse NSI myeloma cells with mouse spleen cells. Ten different antibodies were obtained from 4 separate fusions. Eight antibodies were of the IgG1 subclass. Affinities of antibodies for TSH were in the range 2 x 108-5 x 1010 M-1. Five of the antibodies were specific for TSH and did not react with LH, FSH or hCG. The remaining antibodies reacted with all these hormones and were assumed to recognise their common (α) subunit. The 5 specific antibodies fell into 3 subgroups recognising distinct antigenic determinants, whereas the 5 non-specific antibodies recognised a single determinant or closely related set of sites. It is concluded that these antibodies should be valuable reagents for use in sensitive and specific two-site immunoradiometric assays. (Auth.)

  1. Characterizing the human postural control system using detrended fluctuation analysis

    Science.gov (United States)

    Teresa Blázquez, M.; Anguiano, Marta; de Saavedra, Fernando Arias; Lallena, Antonio M.; Carpena, Pedro

    2010-01-01

    Detrended fluctuation analysis is used to study the behaviour of the time series of the position of the center of pressure, output from the activity of a human postural control system. The results suggest that these trajectories present a crossover in their scaling properties from persistent (for high frequencies, short-range time scale) to anti-persistent (for low frequencies, long-range time scale) behaviours. The values of the scaling exponent found for the persistent parts of the trajectories are very similar for all the cases analysed. The similarity of the results obtained for the measurements done with both eyes open and both eyes closed indicate either that the visual system may be disregarded by the postural control system, while maintaining quiet standing, or that the control mechanisms associated with each type of information (visual, vestibular and somatosensory) cannot be disentangled with this technique.

  2. Cytogenetic and molecular characterization of human radio-induced tumours

    International Nuclear Information System (INIS)

    After a brief recall of some fundamentals regarding radiobiology, this research thesis discusses some epidemiological aspects of radio carcinogenesis, based on epidemiological studies performed on people having survived to Hiroshima, Nagasaki and Chernobyl, but also performed on people submitted to domestic or professional exposures to radon, or to medicine-related exposures. The author highlights some predispositions to radio-induced cancers. Then, she discusses the genetic mechanisms of radio-induced carcinogenesis and the genetic alterations observed in human radio-induced tumours. She discusses and comments the genomic instability, its mechanisms and some models observed on mice, and describes the various forms of radio-induced genomic instability. After a discussion of all these aspects, the author draws some perspectives for future research works

  3. Characterization of ionizing radiation effects on human skin allografts

    International Nuclear Information System (INIS)

    The skin has a fundamental role in the viability of the human body. In the cases of extensive wounds, allograft skin provides an alternative to cover temporarily the damaged areas. After donor screening and preservation in glycerol (above 85%), the skin can be stored in the Skin Banks. The glycerol at this concentration has a bacteriostatic effect after certain time of preservation. On the other hand, skin sterilization by ionizing radiation may reduces the quarantine period for transplantation in patients and its safety is considered excellent. The objectives of this work were to establish procedures using two sources of ionizing radiation for sterilization of human skin allograft, and to evaluate the skin after gamma and electron beam irradiation. The analysis of stress-strain intended to verify possible effects of the radiation on the structure of preserved grafts. Skin samples were submitted to doses of 25 kGy and 50 kGy in an irradiator of 60Co and in an electron beam accelerator. Morphology and ultra-structure studies were also accomplished. The samples irradiated with a dose of 25 kGy seemed to maintain the bio mechanic characteristics. The gamma irradiated samples with a dose of 50 kGy and submitted to an electron beam at doses of 25 kGy and 50 kGy presented significant differences in the values of the elasticity modulus, in relation to the control. The analysis of the ultramicrographies revealed modifications in the structure and alterations in the pattern of collagen fibrils periodicity of the irradiated samples. (author)

  4. Characterization of testis-specific isoenzyme of human pyruvate dehydrogenase.

    Science.gov (United States)

    Korotchkina, Lioubov G; Sidhu, Sukhdeep; Patel, Mulchand S

    2006-04-01

    Pyruvate dehydrogenase (PDH), the first component of the human pyruvate dehydrogenase complex, has two isoenzymes, somatic cell-specific PDH1 and testis-specific PDH2 with 87% sequence identity in the alpha subunit of alpha(2) beta(2) PDH. The presence of functional testis-specific PDH2 is important for sperm cells generating nearly all their energy from carbohydrates via pyruvate oxidation. Kinetic and regulatory properties of recombinant human PDH2 and PDH1 were compared in this study. Site-specific phosphorylation/dephosphorylation of the three phosphorylation sites by four PDH kinases (PDK1-4) and two PDH phosphatases (PDP1-2) were investigated by substituting serines with alanine or glutamate in PDHs. PDH2 was found to be very similar to PDH1 as follows: (i) in specific activities and kinetic parameters as determined by the pyruvate dehydrogenase complex assay; (ii) in thermostability at 37 degrees C; (iii) in the mechanism of inactivation by phosphorylation of three sites; and (iv) in the phosphorylation of sites 1 and 2 by PDK3. In contrast, the differences for PDH2 were indicated as follows: (i) by a 2.4-fold increase in binding affinity for the PDH-binding domain of dihydrolipoamide acetyltransferase as measured by surface plasmon resonance; (ii) by possible involvement of Ser-264 (site 1) of PDH2 in catalysis as evident by its kinetic behavior; and (iii) by the lower activities of PDK1, PDK2, and PDK4 as well as PDP1 and PDP2 toward PDH2. These differences between PDH2 and PDH1 are less than expected from substitution of 47 amino acids in each PDH2 alpha subunit. The multiple substitutions may have compensated for any drastic alterations in PDH2 structure thereby preserving its kinetic and regulatory characteristics largely similar to that of PDH1. PMID:16436377

  5. Chromosome Structural Alteration an Unusual Abnormality Characterizing Human Neoplasia

    Directory of Open Access Journals (Sweden)

    Abolfazl Movafagh

    2016-04-01

    Full Text Available Background and Aim: Ring chromosomes are rare cytogenetic abnormalities that occur in less than 10% of hematopoietic malignancies. They are rare in blood disorder. The present review has focused on the ring chromosome associated with oncology malignancies. Materials and Methods: By reviewing the web-based search for all English scientific peer review articles published, was initiated using Medline/PubMed, Mitelman database (http://cgap.nci.nih.gov/Chromosomes/Mitelman, and other pertinent references on websites about ring chromosomes in Oncology. The software program as End Note was used to handle the proper references for instruction to author. Karyotype descriptions were cited according to ISCN.Conclusion: Ring chromosomes are rare chromosomal aberrations, almost many times are of de novo origin, presenting a different phenotype regarding the loss of genetic material. The karyotype represents the main analysis for detection of ring chromosomes, but other molecular technics are necessary for complete characterization. The information of this review article adds to the spectrum of both morphology and genetic rearrangements in the field of oncology malignancies.

  6. MicroRNAs-Proteomic Networks Characterizing Human Medulloblastoma-SLCs.

    Science.gov (United States)

    Catanzaro, Giuseppina; Besharat, Zein Mersini; Garg, Neha; Ronci, Maurizio; Pieroni, Luisa; Miele, Evelina; Mastronuzzi, Angela; Carai, Andrea; Alfano, Vincenzo; Po, Agnese; Screpanti, Isabella; Locatelli, Franco; Urbani, Andrea; Ferretti, Elisabetta

    2016-01-01

    Medulloblastoma (MB) is the most common malignant brain tumor of pediatric age and is characterized by cells expressing stem, astroglial, and neuronal markers. Among them, stem-like cells (hMB-SLCs) represent a fraction of the tumor cell population with the potential of self-renewal and proliferation and have been associated with tumor poor prognosis. In this context, microRNAs have been described as playing a pivotal role in stem cells differentiation. In our paper, we analyze microRNAs profile and genes expression of hMB-SLCs before and after Retinoic Acid- (RA-) induced differentiation. We aimed to identify pivotal players of specific pathways sustaining stemness and/or tumor development and progression and integrate the results of our recent proteomic study. Our results uncovered 22 differentially expressed microRNAs that were used as input together with deregulated genes and proteins in the Genomatix Pathway System (GePS) analysis revealing 3 subnetworks that could be interestingly involved in the maintenance of hMB-SLCs proliferation. Taken together, our findings highlight microRNAs, genes, and proteins that are significantly modulated in hMB-SLCs with respect to their RA-differentiated counterparts and could open new perspectives for prognostic and therapeutic intervention on MB. PMID:26880947

  7. Human health risk characterization of petroleum coke calcining facility emissions.

    Science.gov (United States)

    Singh, Davinderjit; Johnson, Giffe T; Harbison, Raymond D

    2015-12-01

    Calcining processes including handling and storage of raw petroleum coke may result in Particulate Matter (PM) and gaseous emissions. Concerns have been raised over the potential association between particulate and aerosol pollution and adverse respiratory health effects including decrements in lung function. This risk characterization evaluated the exposure concentrations of ambient air pollutants including PM10 and gaseous pollutants from a petroleum coke calciner facility. The ambient air pollutant levels were collected through monitors installed at multiple locations in the vicinity of the facility. The measured and modeled particulate levels in ambient air from the calciner facility were compared to standards protective of public health. The results indicated that exposure levels were, on occasions at sites farther from the facility, higher than the public health limit of 150 μg/m(3) 24-h average for PM10. However, the carbon fraction demonstrated that the contribution from the calciner facility was de minimis. Exposure levels of the modeled SO2, CO, NOx and PM10 concentrations were also below public health air quality standards. These results demonstrate that emissions from calcining processes involving petroleum coke, at facilities that are well controlled, are below regulatory standards and are not expected to produce a public health risk. PMID:26520182

  8. Molecular cytogenetic characterization of a human thyroid cancercell line

    Energy Technology Data Exchange (ETDEWEB)

    Weier, Heinz-Ulrich G.; Tuton, Tiffany B.; Ito, Yuko; Chu, LisaW.; Lu, Chung-Mei; Baumgartner, Adolf; Zitzelsberger, Horst F.; Weier,Jingly F.

    2006-01-04

    The incidence of papillary thyroid carcinoma (PTC) increases significantly after exposure of the head and neck region to ionizing radiation, yet we know neither the steps involved in malignant transformation of thyroid epithelium nor the specific carcinogenic mode of action of radiation. Such increased tumor frequency became most evident in children after the 1986 nuclear accident in Chernobyl, Ukraine. In the twelve years following the accident, the average incidence of childhood PTCs (chPTC) increased over one hundred-fold compared to the rate of about 1 tumor incidence per 10{sup 6} children per year prior to 1986. To study the etiology of radiation-induced thyroid cancer, we formed an international consortium to investigate chromosomal changes and altered gene expression in cases of post-Chernobyl chPTC. Our approach is based on karyotyping of primary cultures established from chPTC specimens, establishment of cell lines and studies of genotype-phenotype relationships through high resolution chromosome analysis, DNA/cDNA micro-array studies, and mouse xenografts that test for tumorigenicity. Here, we report the application of fluorescence in situ hybridization (FISH)-based techniques for the molecular cytogenetic characterization of a highly tumorigenic chPTC cell line, S48TK, and its subclones. Using chromosome 9 rearrangements as an example, we describe a new approach termed ''BAC-FISH'' to rapidly delineate chromosomal breakpoints, an important step towards a better understanding of the formation of translocations and their functional consequences.

  9. Human health risk characterization of petroleum coke calcining facility emissions.

    Science.gov (United States)

    Singh, Davinderjit; Johnson, Giffe T; Harbison, Raymond D

    2015-12-01

    Calcining processes including handling and storage of raw petroleum coke may result in Particulate Matter (PM) and gaseous emissions. Concerns have been raised over the potential association between particulate and aerosol pollution and adverse respiratory health effects including decrements in lung function. This risk characterization evaluated the exposure concentrations of ambient air pollutants including PM10 and gaseous pollutants from a petroleum coke calciner facility. The ambient air pollutant levels were collected through monitors installed at multiple locations in the vicinity of the facility. The measured and modeled particulate levels in ambient air from the calciner facility were compared to standards protective of public health. The results indicated that exposure levels were, on occasions at sites farther from the facility, higher than the public health limit of 150 μg/m(3) 24-h average for PM10. However, the carbon fraction demonstrated that the contribution from the calciner facility was de minimis. Exposure levels of the modeled SO2, CO, NOx and PM10 concentrations were also below public health air quality standards. These results demonstrate that emissions from calcining processes involving petroleum coke, at facilities that are well controlled, are below regulatory standards and are not expected to produce a public health risk.

  10. Molecular characterization of Chlamydia pneumoniae in animals and humans from Argentina: Genetic characterization of Chlamydia pneumoniae.

    Science.gov (United States)

    Frutos, María C; Monetti, Marina S; Mosmann, Jessica; Kiguen, Ana X; Venezuela, Fernando R; Ré, Viviana E; Cuffini, Cecilia G

    2016-10-01

    In this study, genetic diversity of Chlamydia pneumoniae was investigated and the relationships between sequences amplified of different sources, clinical conditions and geographical regions of central Argentina were established. Samples amplified were similar to human C. pneumoniae patterns and show the high clonality of the population. PMID:27328126

  11. 3D Human cartilage surface characterization by optical coherence tomography

    Science.gov (United States)

    Brill, Nicolai; Riedel, Jörn; Schmitt, Robert; Tingart, Markus; Truhn, Daniel; Pufe, Thomas; Jahr, Holger; Nebelung, Sven

    2015-10-01

    Early diagnosis and treatment of cartilage degeneration is of high clinical interest. Loss of surface integrity is considered one of the earliest and most reliable signs of degeneration, but cannot currently be evaluated objectively. Optical Coherence Tomography (OCT) is an arthroscopically available light-based non-destructive real-time imaging technology that allows imaging at micrometre resolutions to millimetre depths. As OCT-based surface evaluation standards remain to be defined, the present study investigated the diagnostic potential of 3D surface profile parameters in the comprehensive evaluation of cartilage degeneration. To this end, 45 cartilage samples of different degenerative grades were obtained from total knee replacements (2 males, 10 females; mean age 63.8 years), cut to standard size and imaged using a spectral-domain OCT device (Thorlabs, Germany). 3D OCT datasets of 8  ×  8, 4  ×  4 and 1  ×  1 mm (width  ×  length) were obtained and pre-processed (image adjustments, morphological filtering). Subsequent automated surface identification algorithms were used to obtain the 3D primary profiles, which were then filtered and processed using established algorithms employing ISO standards. The 3D surface profile thus obtained was used to calculate a set of 21 3D surface profile parameters, i.e. height (e.g. Sa), functional (e.g. Sk), hybrid (e.g. Sdq) and segmentation-related parameters (e.g. Spd). Samples underwent reference histological assessment according to the Degenerative Joint Disease classification. Statistical analyses included calculation of Spearman’s rho and assessment of inter-group differences using the Kruskal Wallis test. Overall, the majority of 3D surface profile parameters revealed significant degeneration-dependent differences and correlations with the exception of severe end-stage degeneration and were of distinct diagnostic value in the assessment of surface integrity. None of the 3D

  12. Peptide Characterization of Mature Fluorotic and Control Human Enamel.

    Science.gov (United States)

    Lelis, Isabel Maria Porto; Molina, Gabriela F; Souza, Cláudia; Perez, Walter B; Laure, Helen J; Rosa, José C; Gerlach, Raquel F

    2016-01-01

    Exposure to high fluoride levels during amelogenesis causes enamel fluorosis. This study aimed to determine and compare the amino acid sequences in the enamel of fluorotic and control teeth. This investigation included enamel samples obtained from erupted and non-erupted third molars with either TF grade 4-6 (n=7) fluorosis or no sign of fluorosis (controls, n=7). The samples were kept frozen at -20 °C until protein extraction. Samples were etched and processed with a cocktail of proteinase inhibitors and immediately analyzed. Matrix Assisted Laser Desorption/Ionization-Time-Of-Flight/Time-of-Flight Mass Spectrometry (MALDI-TOF/TOF) followed by MASCOT search aided the peptides analysis. The more abundant peptides bore the N-terminal amelogenin sequences WYQSIRPPYP (which is specific for the X-encoded amelogenin) and MPLPPHPGHPGYINF (which does not show sexual dimorphism) were not different in control or fluorotic enamel. There was no missing proteolytic cleavage in the fluorotic samples, which suggested that the increased amount of protein described in fluorotic enamel did not stem from the decreased ability of proteinases to cleave the proteins in humans. This study showed how to successfully obtain peptide from superficial enamel. A relatively low number of teeth was sufficient to provide good data on the actual peptides found in mature enamel.

  13. Peptide Characterization of Mature Fluorotic and Control Human Enamel.

    Science.gov (United States)

    Lelis, Isabel Maria Porto; Molina, Gabriela F; Souza, Cláudia; Perez, Walter B; Laure, Helen J; Rosa, José C; Gerlach, Raquel F

    2016-01-01

    Exposure to high fluoride levels during amelogenesis causes enamel fluorosis. This study aimed to determine and compare the amino acid sequences in the enamel of fluorotic and control teeth. This investigation included enamel samples obtained from erupted and non-erupted third molars with either TF grade 4-6 (n=7) fluorosis or no sign of fluorosis (controls, n=7). The samples were kept frozen at -20 °C until protein extraction. Samples were etched and processed with a cocktail of proteinase inhibitors and immediately analyzed. Matrix Assisted Laser Desorption/Ionization-Time-Of-Flight/Time-of-Flight Mass Spectrometry (MALDI-TOF/TOF) followed by MASCOT search aided the peptides analysis. The more abundant peptides bore the N-terminal amelogenin sequences WYQSIRPPYP (which is specific for the X-encoded amelogenin) and MPLPPHPGHPGYINF (which does not show sexual dimorphism) were not different in control or fluorotic enamel. There was no missing proteolytic cleavage in the fluorotic samples, which suggested that the increased amount of protein described in fluorotic enamel did not stem from the decreased ability of proteinases to cleave the proteins in humans. This study showed how to successfully obtain peptide from superficial enamel. A relatively low number of teeth was sufficient to provide good data on the actual peptides found in mature enamel. PMID:27007349

  14. Structural and functional characterization of the human KIN17 protein

    International Nuclear Information System (INIS)

    Human KIN17 (hKIN17) is highly conserved, more abundant in proliferating cells than in quiescent cells and over expressed after UV irradiation and ionizing radiation. All theses features raised our interest for its structural and functional analysis at the molecular level in order to describe its functions at the cellular level. Several properties have been described for the hKIN17 protein in different cellular mechanisms such as DNA replication, DNA repair and RNA metabolism. During my project, I showed by combining data from CD, NMR and SAXS that hKIN17 has a structured domain probably exhibiting a zinc finger fold and a winged helix domain in its N-terminus region. Only the first domain binds to nucleic acids. The second domain binds to several RNA helicases involved in transcription and translation. Using X-ray crystallography, I solved the structure of the C-terminal domain which is found only in higher eukaryotes and folds into a double SH3-like domain. I showed using different biochemical methods (molecular filtration, non denaturing IEF) that this domain anchors the hKIN17 protein in a nuclear and acidic complex of high molecular weight, whose components are currently identified by mass spectrometry. Moreover, this domain binds to RNA and modified histones. Taken together, these results confirm the involvement of hKIN17 in RNA metabolism and clarify the role of each domain within the protein. (author)

  15. Characterization of Large Structural Genetic Mosaicism in Human Autosomes

    Science.gov (United States)

    Machiela, Mitchell J.; Zhou, Weiyin; Sampson, Joshua N.; Dean, Michael C.; Jacobs, Kevin B.; Black, Amanda; Brinton, Louise A.; Chang, I-Shou; Chen, Chu; Chen, Constance; Chen, Kexin; Cook, Linda S.; Crous Bou, Marta; De Vivo, Immaculata; Doherty, Jennifer; Friedenreich, Christine M.; Gaudet, Mia M.; Haiman, Christopher A.; Hankinson, Susan E.; Hartge, Patricia; Henderson, Brian E.; Hong, Yun-Chul; Hosgood, H. Dean; Hsiung, Chao A.; Hu, Wei; Hunter, David J.; Jessop, Lea; Kim, Hee Nam; Kim, Yeul Hong; Kim, Young Tae; Klein, Robert; Kraft, Peter; Lan, Qing; Lin, Dongxin; Liu, Jianjun; Le Marchand, Loic; Liang, Xiaolin; Lissowska, Jolanta; Lu, Lingeng; Magliocco, Anthony M.; Matsuo, Keitaro; Olson, Sara H.; Orlow, Irene; Park, Jae Yong; Pooler, Loreall; Prescott, Jennifer; Rastogi, Radhai; Risch, Harvey A.; Schumacher, Fredrick; Seow, Adeline; Setiawan, Veronica Wendy; Shen, Hongbing; Sheng, Xin; Shin, Min-Ho; Shu, Xiao-Ou; VanDen Berg, David; Wang, Jiu-Cun; Wentzensen, Nicolas; Wong, Maria Pik; Wu, Chen; Wu, Tangchun; Wu, Yi-Long; Xia, Lucy; Yang, Hannah P.; Yang, Pan-Chyr; Zheng, Wei; Zhou, Baosen; Abnet, Christian C.; Albanes, Demetrius; Aldrich, Melinda C.; Amos, Christopher; Amundadottir, Laufey T.; Berndt, Sonja I.; Blot, William J.; Bock, Cathryn H.; Bracci, Paige M.; Burdett, Laurie; Buring, Julie E.; Butler, Mary A.; Carreón, Tania; Chatterjee, Nilanjan; Chung, Charles C.; Cook, Michael B.; Cullen, Michael; Davis, Faith G.; Ding, Ti; Duell, Eric J.; Epstein, Caroline G.; Fan, Jin-Hu; Figueroa, Jonine D.; Fraumeni, Joseph F.; Freedman, Neal D.; Fuchs, Charles S.; Gao, Yu-Tang; Gapstur, Susan M.; Patiño-Garcia, Ana; Garcia-Closas, Montserrat; Gaziano, J. Michael; Giles, Graham G.; Gillanders, Elizabeth M.; Giovannucci, Edward L.; Goldin, Lynn; Goldstein, Alisa M.; Greene, Mark H.; Hallmans, Goran; Harris, Curtis C.; Henriksson, Roger; Holly, Elizabeth A.; Hoover, Robert N.; Hu, Nan; Hutchinson, Amy; Jenab, Mazda; Johansen, Christoffer; Khaw, Kay-Tee; Koh, Woon-Puay; Kolonel, Laurence N.; Kooperberg, Charles; Krogh, Vittorio; Kurtz, Robert C.; LaCroix, Andrea; Landgren, Annelie; Landi, Maria Teresa; Li, Donghui; Liao, Linda M.; Malats, Nuria; McGlynn, Katherine A.; McNeill, Lorna H.; McWilliams, Robert R.; Melin, Beatrice S.; Mirabello, Lisa; Peplonska, Beata; Peters, Ulrike; Petersen, Gloria M.; Prokunina-Olsson, Ludmila; Purdue, Mark; Qiao, You-Lin; Rabe, Kari G.; Rajaraman, Preetha; Real, Francisco X.; Riboli, Elio; Rodríguez-Santiago, Benjamín; Rothman, Nathaniel; Ruder, Avima M.; Savage, Sharon A.; Schwartz, Ann G.; Schwartz, Kendra L.; Sesso, Howard D.; Severi, Gianluca; Silverman, Debra T.; Spitz, Margaret R.; Stevens, Victoria L.; Stolzenberg-Solomon, Rachael; Stram, Daniel; Tang, Ze-Zhong; Taylor, Philip R.; Teras, Lauren R.; Tobias, Geoffrey S.; Viswanathan, Kala; Wacholder, Sholom; Wang, Zhaoming; Weinstein, Stephanie J.; Wheeler, William; White, Emily; Wiencke, John K.; Wolpin, Brian M.; Wu, Xifeng; Wunder, Jay S.; Yu, Kai; Zanetti, Krista A.; Zeleniuch-Jacquotte, Anne; Ziegler, Regina G.; de Andrade, Mariza; Barnes, Kathleen C.; Beaty, Terri H.; Bierut, Laura J.; Desch, Karl C.; Doheny, Kimberly F.; Feenstra, Bjarke; Ginsburg, David; Heit, John A.; Kang, Jae H.; Laurie, Cecilia A.; Li, Jun Z.; Lowe, William L.; Marazita, Mary L.; Melbye, Mads; Mirel, Daniel B.; Murray, Jeffrey C.; Nelson, Sarah C.; Pasquale, Louis R.; Rice, Kenneth; Wiggs, Janey L.; Wise, Anastasia; Tucker, Margaret; Pérez-Jurado, Luis A.; Laurie, Cathy C.; Caporaso, Neil E.; Yeager, Meredith; Chanock, Stephen J.

    2015-01-01

    Analyses of genome-wide association study (GWAS) data have revealed that detectable genetic mosaicism involving large (>2 Mb) structural autosomal alterations occurs in a fraction of individuals. We present results for a set of 24,849 genotyped individuals (total GWAS set II [TGSII]) in whom 341 large autosomal abnormalities were observed in 168 (0.68%) individuals. Merging data from the new TGSII set with data from two prior reports (the Gene-Environment Association Studies and the total GWAS set I) generated a large dataset of 127,179 individuals; we then conducted a meta-analysis to investigate the patterns of detectable autosomal mosaicism (n = 1,315 events in 925 [0.73%] individuals). Restricting to events >2 Mb in size, we observed an increase in event frequency as event size decreased. The combined results underscore that the rate of detectable mosaicism increases with age (p value = 5.5 × 10−31) and is higher in men (p value = 0.002) but lower in participants of African ancestry (p value = 0.003). In a subset of 47 individuals from whom serial samples were collected up to 6 years apart, complex changes were noted over time and showed an overall increase in the proportion of mosaic cells as age increased. Our large combined sample allowed for a unique ability to characterize detectable genetic mosaicism involving large structural events and strengthens the emerging evidence of non-random erosion of the genome in the aging population. PMID:25748358

  16. Cloning and characterization of a functional human ¿-aminobutyric acid (GABA) transporter, human GAT-2

    DEFF Research Database (Denmark)

    Christiansen, Bolette; Meinild, Anne-Kristine; Jensen, Anders A.;

    2007-01-01

    at the transporter. Expression analysis by reverse transcription-PCR showed that GAT-2 mRNA is present in human brain, kidney, lung, and testis. The finding of the human GAT-2 demonstrates for the first time that the four plasma membrane GABA transporters identified in several mammalian species are all conserved...

  17. Structural and Histone Binding Ability Characterizations of Human PWWP Domains

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Hong; Zeng, Hong; Lam, Robert; Tempel, Wolfram; Amaya, Maria F.; Xu, Chao; Dombrovski, Ludmila; Qiu, Wei; Wang, Yanming; Min, Jinrong (Toronto); (Penn)

    2013-09-25

    The PWWP domain was first identified as a structural motif of 100-130 amino acids in the WHSC1 protein and predicted to be a protein-protein interaction domain. It belongs to the Tudor domain 'Royal Family', which consists of Tudor, chromodomain, MBT and PWWP domains. While Tudor, chromodomain and MBT domains have long been known to bind methylated histones, PWWP was shown to exhibit histone binding ability only until recently. The PWWP domain has been shown to be a DNA binding domain, but sequence analysis and previous structural studies show that the PWWP domain exhibits significant similarity to other 'Royal Family' members, implying that the PWWP domain has the potential to bind histones. In order to further explore the function of the PWWP domain, we used the protein family approach to determine the crystal structures of the PWWP domains from seven different human proteins. Our fluorescence polarization binding studies show that PWWP domains have weak histone binding ability, which is also confirmed by our NMR titration experiments. Furthermore, we determined the crystal structures of the BRPF1 PWWP domain in complex with H3K36me3, and HDGF2 PWWP domain in complex with H3K79me3 and H4K20me3. PWWP proteins constitute a new family of methyl lysine histone binders. The PWWP domain consists of three motifs: a canonical {beta}-barrel core, an insertion motif between the second and third {beta}-strands and a C-terminal {alpha}-helix bundle. Both the canonical {beta}-barrel core and the insertion motif are directly involved in histone binding. The PWWP domain has been previously shown to be a DNA binding domain. Therefore, the PWWP domain exhibits dual functions: binding both DNA and methyllysine histones.

  18. Radiochemical Characterization of Algae Products Commercialized for Human Consumption.

    Science.gov (United States)

    Desideri, Donatella; Cantaluppi, Chiara; Ceccotto, Federica; Meli, Maria Assunta; Roselli, Carla; Feduzi, Laura

    2016-09-01

    Natural radionuclides and Cs were determined by alpha (U, U, Th,Po,Th, and Th) and gamma spectrometry (Cs, K, Ra, Pb, and Ra via Ac) in 14 dried seaweeds commercialized for human nutrition in Italy. The study was carried out in order to provide information on the concentrations of natural and artificial radionuclides. Cesium-137 (Cs) concentrations in all analyzed samples were always <2.0 Bq kg (dry weight), while the naturally occurring radionuclide concentrations were detectable in all the samples and significantly different in the analyzed seaweeds. Potassium-40 (K) showed a mean activity of 894 Bq kg with a range of 14.1-3,256 Bq kg. The mean of activity for Po was 5.1 Bq kg with a range of 1.5-13.6 Bq kg. The mean of activity for Pb was 8.9 Bq kg with a range of 2.9-25.7 Bq kg. The mean of U and Ra activity concentration was 4.7 and 8.4 Bq kg with a range of 0.1-27.7 and 3.2-24.6 Bq kg, respectively. The mean activity concentrations for Th, Th, and Ra were 0.69, 3.0, and 6.6 Bq kg with ranges of 0.05-3.39, 0.24-15.7, and 0.5-35.6 Bq kg, respectively. Radionuclide activity concentrations were within ranges reported in the scientific literature. The committed effective dose due to all the radionuclides analyzed, from ingestion of 1 kg y of seaweeds, accounts for 0.66% of the natural radiation exposure in Italy. PMID:27472751

  19. Genetic Characterization of Atypical Mansonella (Mansonella) ozzardi Microfilariae in Human Blood Samples from Northeastern Peru

    OpenAIRE

    Marcos, Luis A.; Arrospide, Nancy; Recuenco, Sergio; Cabezas, Cesar; Gary J Weil; Fischer, Peter U.

    2012-01-01

    DNA sequence comparisons are useful for characterizing proposed new parasite species or strains. Microfilariae with an atypical arrangement of nuclei behind the cephalic space have been recently described in human blood samples from the Amazon region of Peru. Three blood specimens containing atypical microfilariae were genetically characterized using three DNA markers (5S ribosomal DNA, 12S ribosomal DNA, and cytochrome oxidase I). All atypical microfilariae were clustered into the Mansonella...

  20. Characterization of a novel cutaneous human papillomavirus genotype HPV-125.

    Directory of Open Access Journals (Sweden)

    Anja Kovanda

    Full Text Available The DNA genome of a novel HPV genotype, HPV-125, isolated from a hand wart of an immuno-competent 19-year old male was fully cloned, sequenced and characterized. The full genome of HPV-125 is 7,809-bp in length with a GC content of 46.4%. By comparing the nucleotide sequence of the complete L1 gene, HPV-125 is phylogenetically placed within cutaneotrophic species 2 of Alphapapillomaviruses, and is most closely related to HPV-3 and HPV-28. HPV-125 has a typical genomic organization of Alphapapillomaviruses and contains genes coding for five early proteins, E6, E7, E1, E2 and E4 and two late capsid proteins, L1 and L2. The genome contains two non-coding regions: the first located between the L1 and E6 genes (nucleotide positions 7,137-7,809, length 673-bp and the second between genes E2 and L2 (nucleotide positions 3,757-4,216, length 460-bp. The E6 protein of HPV-125 contains two regular zinc-binding domains at amino acid positions 29 and 102, whereas the E7 protein exhibits one such domain at position 50. HPV-125 lacks the regular pRb-binding core sequence within its E7 protein. In order to assess the tissue predilection and clinical significance of HPV-125, a quantitative type-specific real-time PCR was developed. The 95% limit-of-detection of the assay was 2.5 copies per reaction (range 1.7-5.7 and the intra- and inter-assay coefficients of variation were 0.47 and 2.00 for 100 copies per reaction, and 1.15 and 2.15 for 10 copies per reaction, respectively. Testing of a representative collection of HPV-associated mucosal and cutaneous benign and malignant neoplasms and hair follicles (a total of 601 samples showed that HPV-125 is a relatively rare HPV genotype, with cutaneous tropism etiologically linked with sporadic cases of common warts.

  1. GEOPHYSICS AND SITE CHARACTERIZATION AT THE HANFORD SITE THE SUCCESSFUL USE OF ELECTRICAL RESISTIVITY TO POSITION BOREHOLES TO DEFINE DEEP VADOSE ZONE CONTAMINATION - 11509

    Energy Technology Data Exchange (ETDEWEB)

    GANDER MJ; LEARY KD; LEVITT MT; MILLER CW

    2011-01-14

    Historic boreholes confirmed the presence of nitrate and radionuclide contaminants at various intervals throughout a more than 60 m (200 ft) thick vadose zone, and a 2010 electrical resistivity survey mapped the known contamination and indicated areas of similar contaminants, both laterally and at depth; therefore, electrical resistivity mapping can be used to more accurately locate characterization boreholes. At the Hanford Nuclear Reservation in eastern Washington, production of uranium and plutonium resulted in the planned release of large quantities of contaminated wastewater to unlined excavations (cribs). From 1952 until 1960, the 216-U-8 Crib received approximately 379,000,000 L (100,000,000 gal) of wastewater containing 25,500 kg (56,218 lb) uranium; 1,029,000 kg (1,013 tons) of nitrate; 2.7 Ci of technetium-99; and other fission products including strontium-90 and cesium-137. The 216-U-8 Crib reportedly holds the largest inventory of waste uranium of any crib on the Hanford Site. Electrical resistivity is a geophysical technique capable of identifying contrasting physical properties; specifically, electrically conductive material, relative to resistive native soil, can be mapped in the subsurface. At the 216-U-8 Crib, high nitrate concentrations (from the release of nitric acid [HNO{sub 3}] and associated uranium and other fission products) were detected in 1994 and 2004 boreholes at various depths, such as at the base of the Crib at 9 m (30 ft) below ground surface (bgs) and sporadically to depths in excess of 60 m (200 ft) bgs. These contaminant concentrations were directly correlative with the presence of observed low electrical resistivity responses delineated during the summer 2010 geophysical survey. Based on this correlation and the recently completed mapping of the electrically conductive material, additional boreholes are planned for early 2011 to identify nitrate and radionuclide contamination: (a) throughout the entire vertical length of the

  2. Isolation and characterization of human liver guanine deaminase.

    Science.gov (United States)

    Gupta, N K; Glantz, M D

    1985-01-01

    Guanine deaminase (EC 3.5.4.3, guanine aminohydrolase [GAH]) was purified 3248-fold from human liver to homogeneity with a specific activity of 21.5. A combination of ammonium sulfate fractionation, and DEAE-cellulose, hydroxylapatite, and affinity chromatography with guanine triphosphate ligand were used to purify the enzyme. The enzyme was a dimer protein of a molecular weight of 120,000 with each subunit of 59,000 as determined by gel filtration and sodium dodecyl sulfate-gel electrophoresis. Isoelectric focusing gave a pI of 4.76. It was found to be an acidic protein, as evidenced by the amino acid analysis, enriched with glutamate, aspartate, alanine and glycine. It showed a sharp pH optimum of 8.0. The apparent Km for guanine was determined to be 1.53 X 10(-5) M at pH 6.0 and 2 X 10(-4) M for 8-azaguanine as a substrate at pH 6.0. The enzyme was found to be sensitive to p-hydroxymercuribenzoate inhibition with a Ki of 1.53 X 10(-5) M and a Ki of 5 X 10(-5) M with 5-aminoimidazole-4-carboxamide as an inhibitor. The inhibition with iodoacetic acid showed only a 7% loss in the activity at 1 X 10(-4) M and a 24% loss at 1 X 10(-3) M after 30 min of incubation, whereas p-hydroxymercuribenzoate incubation for 30 min resulted in a 91% loss of activity at a concentration of 1 X 10(-4) M. Guanine was the substrate for all of the inhibition studies. The enzyme was observed to be stable up to 40 degrees C, with a loss of almost all activity at 65 degrees C with 30 min incubation. Two pKa values were obtained at 5.85 and 8.0. Analysis of the N-terminal amino acid proved to be valine while the C-terminal residue was identified as alanine. PMID:3966794

  3. PXRF characterization of gold nanoparticles in human cancer cell

    International Nuclear Information System (INIS)

    Full Text: Nanomaterials, including nanoparticles and nanotubes, have been widely used in biotechnology and medicine. Applications include systems for controlled release of drugs, biomarkers, biosensors and devices for diagnosis of various diseases like cancer, tuberculosis and Chagas. In the case of cancer, the success of current anti-cancer therapy depends on the speed of diagnosis. Gold nanoparticles can be used to visualize lesions or to destroy cancer cells. In this study, was developed a methodology for identification and quantification of gold nanoparticles (AuNPs) deposited on polymer films and matrices that simulate the human skin with pH 7.4, as well as determining the radiation dose of the measures. The nanoparticles were chemically synthesized at the Laboratory of Nanomedicine and nanotoxicology the IFSC/USP and used in concentrations from 1 ppm to 1000 ppm. The measurements were performed with the portable system of the Laboratory of Applied Nuclear Physics (LFNA-UEL), composed of X-ray detector type S-PIN with a resolution of 149 eV for the line at 5.9 keV Mn (AMPTEK) and an X-ray detector type Si-Drift with a resolution of 139 eV for 5.9 keV line of Fe (AMPTEK) and standard electronics, the excitation of samples was performed with a mini X-ray tube with target Silver operated at 28 kV and 10μ A (MOXTEK). Filters were used for silver and aluminum in the output of X-ray tube. The positioner for the set of excitation-detection allows degrees of freedom of translation and rotation. The lower limit of quantification was 5 ppm of gold, using measurement times of 300 s to 1000 s, with R2 ranging from 0.92 to 0.96. X-ray fluorescence lines of gold were analyzed by 8.5 keV, 9.7 keV and 11.4 keV showing a statistical deviation of 0.24. (author)

  4. PXRF characterization of gold nanoparticles in human cancer cell

    Energy Technology Data Exchange (ETDEWEB)

    Lopes, Fabio; Estevam, Marcelo; Appoloni, Carlos R.; Panis, Carolina; Galvao, Tiago Dutra [Universidade Estadual de Londrina (UEL), PR (Brazil); Zucolotto, Valtencir [Universidade de Sao Paulo (USP), Sao Carlos, SP (Brazil). Inst. de Fisica

    2011-07-01

    Full Text: Nanomaterials, including nanoparticles and nanotubes, have been widely used in biotechnology and medicine. Applications include systems for controlled release of drugs, biomarkers, biosensors and devices for diagnosis of various diseases like cancer, tuberculosis and Chagas. In the case of cancer, the success of current anti-cancer therapy depends on the speed of diagnosis. Gold nanoparticles can be used to visualize lesions or to destroy cancer cells. In this study, was developed a methodology for identification and quantification of gold nanoparticles (AuNPs) deposited on polymer films and matrices that simulate the human skin with pH 7.4, as well as determining the radiation dose of the measures. The nanoparticles were chemically synthesized at the Laboratory of Nanomedicine and nanotoxicology the IFSC/USP and used in concentrations from 1 ppm to 1000 ppm. The measurements were performed with the portable system of the Laboratory of Applied Nuclear Physics (LFNA-UEL), composed of X-ray detector type S-PIN with a resolution of 149 eV for the line at 5.9 keV Mn (AMPTEK) and an X-ray detector type Si-Drift with a resolution of 139 eV for 5.9 keV line of Fe (AMPTEK) and standard electronics, the excitation of samples was performed with a mini X-ray tube with target Silver operated at 28 kV and 10{mu} A (MOXTEK). Filters were used for silver and aluminum in the output of X-ray tube. The positioner for the set of excitation-detection allows degrees of freedom of translation and rotation. The lower limit of quantification was 5 ppm of gold, using measurement times of 300 s to 1000 s, with R2 ranging from 0.92 to 0.96. X-ray fluorescence lines of gold were analyzed by 8.5 keV, 9.7 keV and 11.4 keV showing a statistical deviation of 0.24. (author)

  5. Structural and histone binding ability characterizations of human PWWP domains.

    Directory of Open Access Journals (Sweden)

    Hong Wu

    Full Text Available BACKGROUND: The PWWP domain was first identified as a structural motif of 100-130 amino acids in the WHSC1 protein and predicted to be a protein-protein interaction domain. It belongs to the Tudor domain 'Royal Family', which consists of Tudor, chromodomain, MBT and PWWP domains. While Tudor, chromodomain and MBT domains have long been known to bind methylated histones, PWWP was shown to exhibit histone binding ability only until recently. METHODOLOGY/PRINCIPAL FINDINGS: The PWWP domain has been shown to be a DNA binding domain, but sequence analysis and previous structural studies show that the PWWP domain exhibits significant similarity to other 'Royal Family' members, implying that the PWWP domain has the potential to bind histones. In order to further explore the function of the PWWP domain, we used the protein family approach to determine the crystal structures of the PWWP domains from seven different human proteins. Our fluorescence polarization binding studies show that PWWP domains have weak histone binding ability, which is also confirmed by our NMR titration experiments. Furthermore, we determined the crystal structures of the BRPF1 PWWP domain in complex with H3K36me3, and HDGF2 PWWP domain in complex with H3K79me3 and H4K20me3. CONCLUSIONS: PWWP proteins constitute a new family of methyl lysine histone binders. The PWWP domain consists of three motifs: a canonical β-barrel core, an insertion motif between the second and third β-strands and a C-terminal α-helix bundle. Both the canonical β-barrel core and the insertion motif are directly involved in histone binding. The PWWP domain has been previously shown to be a DNA binding domain. Therefore, the PWWP domain exhibits dual functions: binding both DNA and methyllysine histones. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web

  6. Controlled interactions between anhydrous keggin-type heteropolyacids and silica support: Preparation and characterization of well-defined silica-supported polyoxometalate species

    KAUST Repository

    Grinenval, Eva

    2010-11-11

    Anhydrous Keggin-type phosphorus heteropolyacids were deposited on partially dehydroxylated silica by using the surface organometallic chemistry (SOMC) strategy. The resulting solids were characterized by a combination of physicochemical methods including IR, Raman, 1D and 2D 1H, and 31P MAS NMR, electron microscopy experiments and density functional theory (DFT) calculations. It is shown that the main surface species is [ - Si(OH...H+)]2[H+]1[PM 12O403-] where the polyoxometalate is linked to the support by proton interaction with two silanols. Two other minor species (10% each) are formed by coordination of the polyoxometalate to the surface via the interaction between all three protons with three silanol groups or via three covalent bonds formed by dehydroxylation of the above species. Comparison of the reactivity of these solids and of compounds prepared by a classical way shows that the samples prepared by the SOMC approach contain ca. 7 times more acid sites. © 2010 American Chemical Society.

  7. Defining microbiota for developing new probiotics.

    Science.gov (United States)

    Collado, Maria Carmen; Bäuerl, Christine; Pérez-Martínez, Gaspar

    2012-01-01

    The human body harbors complex communities of microbes that play a prominent role in human health. Detailed characterization of the microbiota in the target population forms the basis of probiotic use. Probiotics are defined as live bacterial preparations with clinically documented health effects in humans, and independent of their genus and species, probiotic strains are unique and their beneficial properties on human health have to be assessed in a case-by-case manner. Understanding the mechanisms by which probiotics influence microbiota would facilitate the use of probiotics for both dietary management and reduction in risk of specific diseases. The development of high throughput sequencing methods has allowed metagenomic approaches to study the human microbiome. These efforts are starting to generate an inventory of bacterial taxons and functional features bound to particular health or disease status that allow inferring aspects of the microbiome's function. In the future, this information will allow the rational design of dietary interventions aimed to improve consumer's health via modulation of the microbiota.

  8. Defining microbiota for developing new probiotics

    Science.gov (United States)

    Collado, Maria Carmen; Bäuerl, Christine; Pérez-Martínez, Gaspar

    2012-01-01

    The human body harbors complex communities of microbes that play a prominent role in human health. Detailed characterization of the microbiota in the target population forms the basis of probiotic use. Probiotics are defined as live bacterial preparations with clinically documented health effects in humans, and independent of their genus and species, probiotic strains are unique and their beneficial properties on human health have to be assessed in a case-by-case manner. Understanding the mechanisms by which probiotics influence microbiota would facilitate the use of probiotics for both dietary management and reduction in risk of specific diseases. The development of high throughput sequencing methods has allowed metagenomic approaches to study the human microbiome. These efforts are starting to generate an inventory of bacterial taxons and functional features bound to particular health or disease status that allow inferring aspects of the microbiome's function. In the future, this information will allow the rational design of dietary interventions aimed to improve consumer's health via modulation of the microbiota. PMID:23990820

  9. Defining microbiota for developing new probiotics

    Directory of Open Access Journals (Sweden)

    Maria Carmen Collado

    2012-06-01

    Full Text Available The human body harbors complex communities of microbes that play a prominent role in human health. Detailed characterization of the microbiota in the target population forms the basis of probiotic use. Probiotics are defined as live bacterial preparations with clinically documented health effects in humans, and independent of their genus and species, probiotic strains are unique and their beneficial properties on human health have to be assessed in a case-by-case manner. Understanding the mechanisms by which probiotics influence microbiota would facilitate the use of probiotics for both dietary management and reduction in risk of specific diseases. The development of high throughput sequencing methods has allowed metagenomic approaches to study the human microbiome. These efforts are starting to generate an inventory of bacterial taxons and functional features bound to particular health or disease status that allow inferring aspects of the microbiome's function. In the future, this information will allow the rational design of dietary interventions aimed to improve consumer's health via modulation of the microbiota.

  10. Characterization of the oligosaccharide structure of human glycosylated prolactin (G-hPRL) native and recombinant

    International Nuclear Information System (INIS)

    Human prolactin (hPRL) is a polypeptide hormone secreted by the anterior pituitary under the regulation of the hypothalamus, involved in a variety of biological processes such as mammary gland development and lactation. The recombinant product is important in medical diagnosis and treatment of failure of lactation. This hormone may occur in the form of non-glycosylated protein (NGhPRL) and glycosylated (G-hPRL) with molecular weights of approximately 23 and 25 kilodalton (kDa), respectively; has a single N-glycosylation site located at asparagine (Asn) position 31, which is partially occupied, thus being a particularly interesting model of glycosylation. The biological activity of G-hPRL is lower compared to NG-hPRL (~4 times) and its physiological function is not well defined: the portion of carbohydrate appears to have an important role in the hormone biosynthesis, secretion, biological activity, and plasma survival of the hormone. The main objective of this study was to compare the structures of N-glycans present in glycosylated pituitary prolactin (G-hPRL-NHPP) with those present in the recombinant. To obtain the recombinant G-hPRL the production was performed in laboratory scale from Chinese hamster ovary cells (CHO), genetically modified and adapted to growth in suspension. Cycloheximide (CHX), whose main effect was to increase the ratio G-hPRL/NG-hPRL from 5% to 38% was added to the culture medium, thereby facilitating the purification of G-hPRL. The G-hPRL was purified in two steps, a cation exchanger followed by a purification by reversed-phase high performance liquid chromatography (RP-HPLC) which demonstrated the efficient separation of the two isoforms of hPRL. Recombinant G-hPRL-IPEN was well characterized by several techniques confirming its purity and biological activity, including comparisons with other reference preparation of pituitary origin purchased from the National Hormone & Peptide Program (NHPPU. S.). The composition of N-glycans present in

  11. Characterization of Phascolarctobacterium succinatutens sp. nov., an Asaccharolytic, Succinate-Utilizing Bacterium Isolated from Human Feces

    OpenAIRE

    Watanabe, Yohei; Nagai, Fumiko; Morotomi, Masami

    2012-01-01

    Isolation, cultivation, and characterization of the intestinal microorganisms are important for understanding the comprehensive physiology of the human gastrointestinal (GI) tract microbiota. Here, we isolated two novel bacterial strains, YIT 12067T and YIT 12068, from the feces of healthy human adults. Phylogenetic analysis indicated that they belonged to the same species and were most closely related to Phascolarctobacterium faecium ACM 3679T, with 91.4% to 91.5% 16S rRNA gene sequence simi...

  12. Not all sperm are equal: functional mitochondria characterize a subpopulation of human sperm with better fertilization potential.

    Directory of Open Access Journals (Sweden)

    Ana Paula Sousa

    Full Text Available Human sperm samples are very heterogeneous and include a low amount of truly functional gametes. Distinct strategies have been developed to characterize and isolate this specific subpopulation. In this study we have used fluorescence microscopy and fluorescence-activated cell sorting to determine if mitochondrial function, as assessed using mitochondrial-sensitive probes, could be employed as a criterion to obtain more functional sperm from a given ejaculate. We first determined that mitochondrial activity correlated with the quality of distinct human samples, from healthy donors to patients with decreased semen quality. Furthermore, using fluorescence-activated cell sorting to separate sperm with active and inactive mitochondria we found that this was also true within samples. Indeed, sperm with active mitochondria defined a more functional subpopulation, which contained more capacitated and acrosome intact cells, sperm with lower chromatin damage, and, crucially, sperm more able to decondense and participate in early development using both chemical induction and injection into mature bovine oocytes. Furthermore, cell sorting using mitochondrial activity produced a more functional sperm subpopulation than classic swim-up, both in terms of improvement in a variety of functional sperm parameters and in statistical significance. In conclusion, whatever the true biological role of sperm mitochondria in fertilization, mitochondrial activity is a clear hallmark of human sperm functionality.

  13. Not all sperm are equal: functional mitochondria characterize a subpopulation of human sperm with better fertilization potential.

    Science.gov (United States)

    Sousa, Ana Paula; Amaral, Alexandra; Baptista, Marta; Tavares, Renata; Caballero Campo, Pedro; Caballero Peregrín, Pedro; Freitas, Albertina; Paiva, Artur; Almeida-Santos, Teresa; Ramalho-Santos, João

    2011-03-23

    Human sperm samples are very heterogeneous and include a low amount of truly functional gametes. Distinct strategies have been developed to characterize and isolate this specific subpopulation. In this study we have used fluorescence microscopy and fluorescence-activated cell sorting to determine if mitochondrial function, as assessed using mitochondrial-sensitive probes, could be employed as a criterion to obtain more functional sperm from a given ejaculate. We first determined that mitochondrial activity correlated with the quality of distinct human samples, from healthy donors to patients with decreased semen quality. Furthermore, using fluorescence-activated cell sorting to separate sperm with active and inactive mitochondria we found that this was also true within samples. Indeed, sperm with active mitochondria defined a more functional subpopulation, which contained more capacitated and acrosome intact cells, sperm with lower chromatin damage, and, crucially, sperm more able to decondense and participate in early development using both chemical induction and injection into mature bovine oocytes. Furthermore, cell sorting using mitochondrial activity produced a more functional sperm subpopulation than classic swim-up, both in terms of improvement in a variety of functional sperm parameters and in statistical significance. In conclusion, whatever the true biological role of sperm mitochondria in fertilization, mitochondrial activity is a clear hallmark of human sperm functionality.

  14. Selection and characterization of a human neutralizing antibody to human fibroblast growth factor-2

    Energy Technology Data Exchange (ETDEWEB)

    Tao, Jun [Department of Immunology, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Xiang, Jun-Jian, E-mail: txjj@jnu.edu.cn [Laboratory of Antibody Engineering, College of Life Sciences and Technologies, Jinan University, Guangzhou 510632 (China); Department of Immunology, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Li, Dan [Department of Immunology, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Deng, Ning; Wang, Hong; Gong, Yi-Ping [Laboratory of Antibody Engineering, College of Life Sciences and Technologies, Jinan University, Guangzhou 510632 (China)

    2010-04-09

    Compelling evidences suggest that fibroblast growth factor-2 (FGF-2) plays important roles in tumor growth, angiogenesis and metastasis. Molecules blocking the FGF-2 signaling have been proposed as anticancer agents. Through screening of a human scFv phage display library, we have isolated several human single-chain Fv fragments (scFvs) that bind to human FGF-2. After expression and purification in bacteria, one scFv, named 1A2, binds to FGF-2 with a high affinity and specificity, and completes with FGF-2 binding to its receptor. This 1A2 scFv was then cloned into the pIgG1 vector and expressed in 293T cells. The purified hIgG1-1A2 antibody showed a high binding affinity of 8 x 10{sup -9} M to rhFGF-2. In a set of vitro assays, it inhibited various biological activities of FGF-2 such as the proliferation, migration and tube formation of human umbilical vein endothelial cells. More importantly, hIgG1-1A2 antibody also efficiently blocked the growth while inducing apoptosis of glioma cells. For the first time, we generated a human anti-FGF-2 antibody with proven in vitro anti-tumor activity. It may therefore present a new therapeutic candidate for the treatment of cancers that are dependent on FGF-2 signaling for growth and survival.

  15. MOLECULAR CHARACTERIZATION OF A RECURRING COMPLEX CHROMOSOMAL TRANSLOCATION IN 2 HUMAN EXTRAGONADAL GERM-CELL TUMORS

    NARCIS (Netherlands)

    SINKE, RJ; WEGHUIS, DO; SUIJKERBUIJK, RF; TANIGAMI, A; NAKAMURA, Y; LARSSON, C; WEBER, G; DEJONG, B; OOSTERHUIS, JW; MOLENAAR, WM; VANKESSEL, AG

    1994-01-01

    The molecular characterization of a recurring complex chromosomal translocation involving 6p21, 6p22, 6p23, and 11q13 in two independent bur similar extragonadal human germ cell rumors was initiated using fluorescence in situ hybridization (FISH) and pulse field gel electrophoresis (PFGE) techniques

  16. Molecular characterization of a recurring complex chromosomal translocation in two human extragonadal germ cell tumors.

    NARCIS (Netherlands)

    Sinke, R J; Weghuis, D O; Suijkerbuijk, R F; Tanigami, A; Nakamura, Y; Larsson, C; Weber, G; Jong, B de; Oosterhuis, J W; Molenaar, W M

    1994-01-01

    The molecular characterization of a recurring complex chromosomal translocation involving 6p21, 6p22, 6q23, and 11q13 in two independent but similar extragonadal human germ cell tumors was initiated using fluorescence in situ hybridization (FISH) and pulse field gel electrophoresis (PFGE) techniques

  17. Characterization of human placental glycosaminoglycans and regional binding to VAR2CSA in malaria infected erythrocytes

    DEFF Research Database (Denmark)

    Beaudet, Julie M; Mansur, Leandra; Joo, Eun Ji;

    2014-01-01

    and their VAR2CSA binding were characterized. All portions of human placenta contained sufficient amounts of the appropriate low-sulfated chondroitin sulfate-A to display high-affinity binding to a recombinant truncated VAR2CSA construct, as determined using surface plasmon resonance. The cotyledon is the only...

  18. ADHESION AND SPREADING OF HUMAN SKIN FIBROBLASTS ON PHYSICOCHEMICALLY CHARACTERIZED GRADIENT SURFACES

    NARCIS (Netherlands)

    RUARDY, TG; SCHAKENRAAD, JM; VANDERMEI, HC; BUSSCHER, HJ

    1995-01-01

    In this study, adhesion and spreading of human skin fibroblasts on gradient surfaces of dichlorodimethylsilane (DDS) coupled to glass was investigated. Gradient surfaces were prepared by the diffusion technique and characterized by the Wilhelmy plate technique for their wettability and by scanning x

  19. Human Tick-Borne Encephalitis and Characterization of Virus from Biting Tick.

    Science.gov (United States)

    Henningsson, Anna J; Lindqvist, Richard; Norberg, Peter; Lindblom, Pontus; Roth, Anette; Forsberg, Pia; Bergström, Tomas; Överby, Anna K; Lindgren, Per-Eric

    2016-08-01

    We report a case of human tick-borne encephalitis (TBE) in which the TBE virus was isolated from the biting tick. Viral growth and sequence were characterized and compared with those of a reference strain. Virus isolation from ticks from patients with TBE may offer a new approach for studies of epidemiology and pathogenicity. PMID:27434395

  20. Construction and characterization of human chromosome 2-specific cosmid, fosmid, and PAC clone libraries

    Energy Technology Data Exchange (ETDEWEB)

    Gingrich, J.C.; Boehrer, D.M.; Garnes, J.A. [Lawrence Livermore National Lab., CA (United States)] [and others

    1996-02-15

    This article discusses the construction and characterization of three human chromosome 2-specific clone libraries. A chromosome 2-specific PAC library was also constructed from a hybrid cell line. The chromosome 2 coverage of each of the three libraries was further determined by PCR screening clone pools with 82 chromosome 2-specific STSs. 47 refs., 3 figs., 1 tab.

  1. Human Tick-Borne Encephalitis and Characterization of Virus from Biting Tick

    Science.gov (United States)

    Lindqvist, Richard; Norberg, Peter; Lindblom, Pontus; Roth, Anette; Forsberg, Pia; Bergström, Tomas; Överby, Anna K.; Lindgren, Per-Eric

    2016-01-01

    We report a case of human tick-borne encephalitis (TBE) in which the TBE virus was isolated from the biting tick. Viral growth and sequence were characterized and compared with those of a reference strain. Virus isolation from ticks from patients with TBE may offer a new approach for studies of epidemiology and pathogenicity. PMID:27434395

  2. Characterization of human chromosomal DNA sequences which replicate autonomously in Saccharomyces cerevisiae.

    OpenAIRE

    Montiel, J F; Norbury, C. J.; Tuite, M F; Dobson, M J; Mills, J S; Kingsman, A J; Kingsman, S M

    1984-01-01

    We have characterised two restriction fragments, isolated from a "shotgun" collection of human DNA, which function as autonomously replicating sequences (ARSs) in Saccharomyces cerevisiae. Functional domains of these fragments have been defined by subcloning and exonuclease (BAL 31) deletion analysis. Both fragments contain two spatially distinct domains. One is essential for high frequency transformation and is termed the Replication Sequence (RS) domain, the other, termed the Replication En...

  3. Definable deduction relation

    Institute of Scientific and Technical Information of China (English)

    张玉平

    1999-01-01

    The nonmonotonic deduction relation in default reasoning is defined with fixed point style, which has the many-extension property that classical logic is not possessed of. These two kinds of deductions both have boolean definability property, that is, their extensions or deductive closures can be defined by boolean formulas. A generalized form of fixed point method is employed to define a class of deduction relations, which all have the above property. Theorems on definability and atomless boolean algebras in model theory are essential in dealing with this assertion.

  4. Robust estimation of fractal measures for characterizing the structural complexity of the human brain: optimization and reproducibility.

    Science.gov (United States)

    Goñi, Joaquín; Sporns, Olaf; Cheng, Hu; Aznárez-Sanado, Maite; Wang, Yang; Josa, Santiago; Arrondo, Gonzalo; Mathews, Vincent P; Hummer, Tom A; Kronenberger, William G; Avena-Koenigsberger, Andrea; Saykin, Andrew J; Pastor, María A

    2013-12-01

    High-resolution isotropic three-dimensional reconstructions of human brain gray and white matter structures can be characterized to quantify aspects of their shape, volume and topological complexity. In particular, methods based on fractal analysis have been applied in neuroimaging studies to quantify the structural complexity of the brain in both healthy and impaired conditions. The usefulness of such measures for characterizing individual differences in brain structure critically depends on their within-subject reproducibility in order to allow the robust detection of between-subject differences. This study analyzes key analytic parameters of three fractal-based methods that rely on the box-counting algorithm with the aim to maximize within-subject reproducibility of the fractal characterizations of different brain objects, including the pial surface, the cortical ribbon volume, the white matter volume and the gray matter/white matter boundary. Two separate datasets originating from different imaging centers were analyzed, comprising 50 subjects with three and 24 subjects with four successive scanning sessions per subject, respectively. The reproducibility of fractal measures was statistically assessed by computing their intra-class correlations. Results reveal differences between different fractal estimators and allow the identification of several parameters that are critical for high reproducibility. Highest reproducibility with intra-class correlations in the range of 0.9-0.95 is achieved with the correlation dimension. Further analyses of the fractal dimensions of parcellated cortical and subcortical gray matter regions suggest robustly estimated and region-specific patterns of individual variability. These results are valuable for defining appropriate parameter configurations when studying changes in fractal descriptors of human brain structure, for instance in studies of neurological diseases that do not allow repeated measurements or for disease

  5. PK/PD Modelling of the QT Interval: a Step Towards Defining the Translational Relationship Between In Vitro, Awake Beagle Dogs, and Humans.

    Science.gov (United States)

    Marostica, Eleonora; Van Ammel, Karel; Teisman, Ard; Gallacher, David; Van Bocxlaer, Jan; De Ridder, Filip; Boussery, Koen; Vermeulen, An

    2016-07-01

    Inhibiting the human ether-a-go-go-related gene (hERG)-encoded potassium ion channel is positively correlated with QT-interval prolongation in vivo, which is considered a risk factor for the occurrence of Torsades de Pointes (TdP). A pharmacokinetic/pharmacodynamic model was developed for four compounds that reached the clinic, to relate drug-induced QT-interval change in awake dogs and humans and to derive a translational scaling factor a 1. Overall, dogs were more sensitive than humans to QT-interval change, an a 1 of 1.5 was found, and a 10% current inhibition in vitro produced a higher percent QT-interval change in dogs as compared to humans. The QT-interval changes in dogs were predictive for humans. In vitro and in vivo information could reliably describe the effects in humans. Robust translational knowledge is likely to reduce the need for expensive thorough QT studies; therefore, expanding this work to more compounds is recommended.

  6. Genetic Characterization of Human Populations: From ABO to a Genetic Map of the British People

    Science.gov (United States)

    Bodmer, Walter

    2015-01-01

    From 1900, when Landsteiner first described the ABO blood groups, to the present, the methods used to characterize the genetics of human populations have undergone a remarkable development. Concomitantly, our understanding of the history and spread of human populations across the earth has become much more detailed. As has often been said, a better understanding of the genetic relationships among the peoples of the world is one of the best antidotes to racial prejudices. Such an understanding provides us with a fascinating, improved insight into our origins as well as with valuable information about population differences that are of medical relevance. The study of genetic polymorphisms has been essential to the analysis of the relationships between human populations. The evolution of methods used to study human polymorphisms and the resulting contributions to our understanding of human health and history is the subject of this Perspectives. PMID:25657345

  7. Molecular characterization of Echinococcus granulosus sensu stricto and Echinococcus canadensis in humans and livestock from Algeria.

    Science.gov (United States)

    Zait, Houria; Kouidri, Mokhtaria; Grenouillet, Florence Elisabeth; Umhang, Gérald; Millon, Laurence; Hamrioui, Boussad; Grenouillet, Frédéric

    2016-06-01

    In Algeria, previous studies investigated genotypes of Echinococcus granulosus sensu lato in animals and identified E. granulosus sensu stricto (s.s.) genotypes G1 and G3 whereas Echinococcus canadensis genotype G6 was only reported from dromedary cysts. Molecular data on human cystic echinococcosis (CE) were limited. We implemented a large genotyping study of hydatid cysts from humans and livestock animals to specify CE's molecular epidemiology and the genetic diversity in Algeria. Fifty-four human CE cysts from patients predominantly admitted in surgical units from Mustapha Hospital, Algiers, and 16 cysts from livestock animals gathered in two geographically distinct slaughterhouses, Tiaret and Tamanrasset, were collected. Molecular characterization was performed using sequencing of two mitochondrial genes, cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit I (NDI). In humans, G1 of E. granulosus s.s. was the main genotype (90.7 %); four samples (7.4 %) were characterized as E. granulosus s.s. G3 and one cyst as E. canadensis G6 (1.8 %). This molecular confirmation of E. canadensis G6 human infection in Algeria was observed in a Tuareg female living in a desertic area in Tamanrasset. All cysts from sheep, cattle, and goat were identified as E. granulosus s.s. G1 and the two cysts originating from dromedary as E. canadensis G6. Twenty concatenated haplotypes (COI + NDI) were characterized. Among E. granulosus s.s., one haplotype (HL1) was highly predominant in both humans and animals cysts (71.6 %). This study revealed main occurrence of E. granulosus s.s. in humans and livestock animals, with description of a predominant shared haplotype corresponding to the main worldwide observed haplotype E.granulosus s.s. G1. E. canadensis G6 was limited to South Algeria, in dromedary as well as in human.

  8. Molecular characterization of Echinococcus granulosus sensu stricto and Echinococcus canadensis in humans and livestock from Algeria.

    Science.gov (United States)

    Zait, Houria; Kouidri, Mokhtaria; Grenouillet, Florence Elisabeth; Umhang, Gérald; Millon, Laurence; Hamrioui, Boussad; Grenouillet, Frédéric

    2016-06-01

    In Algeria, previous studies investigated genotypes of Echinococcus granulosus sensu lato in animals and identified E. granulosus sensu stricto (s.s.) genotypes G1 and G3 whereas Echinococcus canadensis genotype G6 was only reported from dromedary cysts. Molecular data on human cystic echinococcosis (CE) were limited. We implemented a large genotyping study of hydatid cysts from humans and livestock animals to specify CE's molecular epidemiology and the genetic diversity in Algeria. Fifty-four human CE cysts from patients predominantly admitted in surgical units from Mustapha Hospital, Algiers, and 16 cysts from livestock animals gathered in two geographically distinct slaughterhouses, Tiaret and Tamanrasset, were collected. Molecular characterization was performed using sequencing of two mitochondrial genes, cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit I (NDI). In humans, G1 of E. granulosus s.s. was the main genotype (90.7 %); four samples (7.4 %) were characterized as E. granulosus s.s. G3 and one cyst as E. canadensis G6 (1.8 %). This molecular confirmation of E. canadensis G6 human infection in Algeria was observed in a Tuareg female living in a desertic area in Tamanrasset. All cysts from sheep, cattle, and goat were identified as E. granulosus s.s. G1 and the two cysts originating from dromedary as E. canadensis G6. Twenty concatenated haplotypes (COI + NDI) were characterized. Among E. granulosus s.s., one haplotype (HL1) was highly predominant in both humans and animals cysts (71.6 %). This study revealed main occurrence of E. granulosus s.s. in humans and livestock animals, with description of a predominant shared haplotype corresponding to the main worldwide observed haplotype E.granulosus s.s. G1. E. canadensis G6 was limited to South Algeria, in dromedary as well as in human. PMID:27021186

  9. Α-galactosylceramide analogs with weak agonist activity for human iNKT cells define new candidate anti-inflammatory agents.

    Science.gov (United States)

    Bricard, Gabriel; Venkataswamy, Manjunatha M; Yu, Karl O A; Im, Jin S; Ndonye, Rachel M; Howell, Amy R; Veerapen, Natacha; Illarionov, Petr A; Besra, Gurdyal S; Li, Qian; Chang, Young-Tae; Porcelli, Steven A

    2010-12-17

    CD1d-restricted natural killer T cells with invariant T cell receptor α chains (iNKT cells) are a unique lymphocyte subset that responds to recognition of specific lipid and glycolipid antigens. They are conserved between mice and humans and exert various immunoregulatory functions through their rapid secretion of a variety of cytokines and secondary activation of dendritic cells, B cells and NK cells. In the current study, we analyzed the range of functional activation states of human iNKT cells using a library of novel analogs of α-galactosylceramide (αGalCer), the prototypical iNKT cell antigen. Measurement of cytokines secreted by human iNKT cell clones over a wide range of glycolipid concentrations revealed that iNKT cell ligands could be classified into functional groups, correlating with weak versus strong agonistic activity. The findings established a hierarchy for induction of different cytokines, with thresholds for secretion being consistently lowest for IL-13, higher for interferon-γ (IFNγ), and even higher for IL-4. These findings suggested that human iNKT cells can be intrinsically polarized to selective production of IL-13 by maintaining a low level of activation using weak agonists, whereas selective polarization to IL-4 production cannot be achieved through modulating the strength of the activating ligand. In addition, using a newly designed in vitro system to assess the ability of human iNKT cells to transactivate NK cells, we found that robust secondary induction of interferon-γ secretion by NK cells was associated with strong but not weak agonist ligands of iNKT cells. These results indicate that polarization of human iNKT cell responses to Th2-like or anti-inflammatory effects may best be achieved through selective induction of IL-13 and suggest potential discrepancies with findings from mouse models that may be important in designing iNKT cell-based therapies in humans.

  10. Α-galactosylceramide analogs with weak agonist activity for human iNKT cells define new candidate anti-inflammatory agents.

    Directory of Open Access Journals (Sweden)

    Gabriel Bricard

    Full Text Available CD1d-restricted natural killer T cells with invariant T cell receptor α chains (iNKT cells are a unique lymphocyte subset that responds to recognition of specific lipid and glycolipid antigens. They are conserved between mice and humans and exert various immunoregulatory functions through their rapid secretion of a variety of cytokines and secondary activation of dendritic cells, B cells and NK cells. In the current study, we analyzed the range of functional activation states of human iNKT cells using a library of novel analogs of α-galactosylceramide (αGalCer, the prototypical iNKT cell antigen. Measurement of cytokines secreted by human iNKT cell clones over a wide range of glycolipid concentrations revealed that iNKT cell ligands could be classified into functional groups, correlating with weak versus strong agonistic activity. The findings established a hierarchy for induction of different cytokines, with thresholds for secretion being consistently lowest for IL-13, higher for interferon-γ (IFNγ, and even higher for IL-4. These findings suggested that human iNKT cells can be intrinsically polarized to selective production of IL-13 by maintaining a low level of activation using weak agonists, whereas selective polarization to IL-4 production cannot be achieved through modulating the strength of the activating ligand. In addition, using a newly designed in vitro system to assess the ability of human iNKT cells to transactivate NK cells, we found that robust secondary induction of interferon-γ secretion by NK cells was associated with strong but not weak agonist ligands of iNKT cells. These results indicate that polarization of human iNKT cell responses to Th2-like or anti-inflammatory effects may best be achieved through selective induction of IL-13 and suggest potential discrepancies with findings from mouse models that may be important in designing iNKT cell-based therapies in humans.

  11. Characterization of Glass Fibre – Coconut Coir– Human Hair Hybrid Composites

    Directory of Open Access Journals (Sweden)

    D. Senthilnathan

    2014-03-01

    Full Text Available A composite material is a combination of two or more materials arranged in the form of layer one on the other layer using binding material through some prescribed methods. In the Glass fibre coconut fibre human hair hybrid composite method, the epoxy resin is used as binding material, in which one layer is formed of glass fibre, followed by coconut fibre and then by human hair. By using hand layup method and by changing the above arrangement of layers, six types of laminates are prepared by using the resin of LY556 and hardness HY951.The six types of laminates are Glass fibre reinforced plastic (GFRPCoconut coir reinforced plastic(CCRP,Human hair reinforced plastic(HHRP,Glass – Coconut coir - human hair- Glass hybrid composite (GCHGRP,Coconut coir-glass- human hair-coconut coir hybrid composite (CGHCRP and human hair-coconut coir-glass-human hair hybrid composite (HCGHRP . As per ASTM standard, specimens are prepared in the above said six conduct various tests such as tensile, flexural, shear, impact and hardness tests. From the characterization of Glass-coconut coir-Human hair hybrid composite, it is observed that Eco-friendly coconut coir and human hair inclusion in the Glass fibre laminates have significantly influenced the mechanical properties.

  12. The human granzyme A (HFSP, CTLA3) gene maps to 5q11-q12 and defines a new locus of the serine protease superfamily

    Energy Technology Data Exchange (ETDEWEB)

    Fink, T.M.; Lichter, P. (Institut fuer angewandte Tumorvirologie, Heidelberg (Germany)); Wekerle, H.; Zimmer, M.; Jenne, D.E. (Max-Planck-Institut fuer Psychiatrie, Planegg-Martinsried (Germany))

    1993-11-01

    Human granzyme A (HFSP, Hanukah factor serine protease; CTLA3, cytotoxic T-lymphocyte-associated serine esterase-3), a homodimeric, trypsin-like serine protease of 60 kDa found in granules of cytolytic T cells and natural killer cells, is implicated in lymphocyte-mediated target cell lysis. It contributes to DNA fragmentation in perforin (PRF1)-lysed target cells through an unknown mechanism. The authors have isolated a cosmid clone for the functional gene of human granzyme A and established its complete exon-intron map of 10 kb. Using an 11-kb subfragment of the cloned genomic DNA as a probe, they have identified the chromosomal position of human granzyme A on 5q11-q12. Thus, the human granzyme A gene falls into a region of homology between human chromosome 5 and mouse chromosome 13, band D, where the mouse granzyme A gene has been located previously. The granzyme A gene is not linked to known members of the large superfamily of serine proteases. 20 refs., 2 figs.

  13. Judicial activism, the Biotech Directive and its institutional implications – Is the Court acting as a legislator or a court when defining the ‘human embryo’?

    DEFF Research Database (Denmark)

    Faeh, Andrea Beata

    2015-01-01

    The Court of Justice of the European Union (Court) delivered a preliminary ruling in 2011 in the case of Oliver Brüstle v Greenpeace on the interpretation of Article 6(2) of the Biotech Directive and thereby established an autonomous concept of the term ‘human embryo’. The Brüstle decision raises...... for it to be regarded as a “human embryo”.’ This contribution discusses the two judgments both in relation to the role of the EU institutions and the potential effects of the judgments on biotech inventions, in particular on medical innovation...

  14. Defining and measuring ecological specialization

    OpenAIRE

    Devictor, Vincent; Clavel, Joanne; Julliard, Romain; Lavergne, Sébastien; Mouillot, David; Thuiller, Wilfried; Venail, Patrick; Villéger, Sébastien; Mouquet, Nicolas

    2010-01-01

    1.  Ecological specialization is one of the main concepts in ecology and conservation. However, this concept has become highly context-dependent and is now obscured by the great variability of existing definitions and methods used to characterize ecological specialization. 2.  In this study, we clarify this concept by reviewing the strengths and limitations of different approaches commonly used to define and measure ecological specialization. We first show that ecological specialization can e...

  15. Characterization of human PGD blastocysts with unbalanced chromosomal translocations and human embryonic stem cell line derivation?

    Science.gov (United States)

    Frydman, N; Féraud, O; Bas, C; Amit, M; Frydman, R; Bennaceur-Griscelli, A; Tachdjian, G

    2009-01-01

    Novel embryonic stem cell lines derived from embryos carrying structural chromosomal abnormalities obtained after preimplantation genetic diagnosis (PGD) are of interest to study in terms of the influence of abnormalities on further development. A total of 22 unbalanced blastocysts obtained after PGD were analysed for structural chromosomal defects. Morphological description and chromosomal status of these blastocysts was established and they were used to derive human embryonic stem cell (ESC) lines. An outgrowth of cells was observed for six blastocysts (6/22; 27%). For two blastocysts, the exact morphology was unknown since they were at early stage, and for four blastocysts, the inner cell mass was clearly visible. Fifteen blastocysts carried an unbalanced chromosomal defect linked to a reciprocal translocation, resulting in a positive outgrowth of cells for five blastocysts. One human ESC line was obtained from a blastocyst carrying a partial chromosome-21 monosomy and a partial chromosome-1 trisomy. Six blastocysts carried an unbalanced chromosomal defect linked to a Robertsonian translocation, and one showed a positive outgrowth of cells. One blastocyst carried an unbalanced chromosomal defect linked to an insertion and no outgrowth was observed. The efficiency of deriving human ESC lines with constitutional chromosomal disorders was low and probably depends on the initial morphological aspect of the blastocysts and/or the type of the chromosomal disorders.

  16. Defining Quantum Control Flow

    OpenAIRE

    Ying, Mingsheng; Yu, Nengkun; Feng, Yuan

    2012-01-01

    A remarkable difference between quantum and classical programs is that the control flow of the former can be either classical or quantum. One of the key issues in the theory of quantum programming languages is defining and understanding quantum control flow. A functional language with quantum control flow was defined by Altenkirch and Grattage [\\textit{Proc. LICS'05}, pp. 249-258]. This paper extends their work, and we introduce a general quantum control structure by defining three new quantu...

  17. When mothering goes awry: Challenges and opportunities for utilizing evidence across rodent, nonhuman primate and human studies to better define the biological consequences of negative early caregiving.

    Science.gov (United States)

    Drury, Stacy S; Sánchez, Mar M; Gonzalez, Andrea

    2016-01-01

    This article is part of a Special Issue "Parental Care".Across mammalian species, mothers shape socio-emotional development and serve as essential external regulators of infant physiology, brain development, behavior patterns, and emotional regulation. Caregiving quality, consistency and predictability shape the infant's underlying neurobiological processes. Although the requirements for "optimal" caregiving differ across species, the negative long-term consequences of the absence of needed caregiving (e.g. neglect) or the presence of harmful/aversive caregiving (e.g. physical abuse), are translatable across species. Recognizing the significant potential of cross species comparisons in terms of defining underlying mechanisms, effective translation requires consideration of the evolutionary, ecological, and fundamental biological and developmental differences between and among species. This review provides both an overview of several success stories of cross-species translations in relation to negative caregiving and a template for future studies seeking to most effectively define the underlying biological processes and advance research dedicated to mitigating the lasting negative health consequences of child maltreatment. PMID:26506032

  18. Generation of eye field/optic vesicle-like structures from human embryonic stem cells under two-dimensional and chemically defined conditions.

    Science.gov (United States)

    Parvini, Maryam; Parivar, Kazem; Safari, Fatemeh; Tondar, Mahdi

    2015-03-01

    Despite the enormous progress in studying retinal cell differentiation from human embryonic stem cells (hESCs), none of the reported protocols have produced a cost-effective eye field cells with the capability to further differentiate into retinal derivatives. In this study, by drawing chemicals on our four-step differentiation strategy, we demonstrated the ability of hESCs in assembling such qualifications to follow human retinogenesis in a serum- and feeder-free adherent condition. Two-dimensional (2D) populations of eye field cells arose within early forebrain progeny upon hESCs differentiation. Gene expression analysis showed that the treatment of hESCs with a combination of selected small molecules (SMs) gave rise to the higher expressions of eye field-specific genes, PAX6, RX, and SIX3. Thereafter, a subset of cells gained the transient features of advancing retinal differentiation, including optic vesicle (OV)-like structures, which expressed MITF and CHX10 in a manner imitated in vivo human retinal development. The competency of derived cells in differentiation to retinal derivatives was further investigated. The gene analysis of the cells showed more propensity for generating retinal pigment epithelial (RPE) than neural retina (NR). The generation of OV-like structures in 2D cultures can shed light on molecular events governing retinal specification. It can also facilitate the study of human retinal development.

  19. Defining an emerging disease.

    Science.gov (United States)

    Moutou, F; Pastoret, P-P

    2015-04-01

    Defining an emerging disease is not straightforward, as there are several different types of disease emergence. For example, there can be a 'real' emergence of a brand new disease, such as the emergence of bovine spongiform encephalopathy in the 1980s, or a geographic emergence in an area not previously affected, such as the emergence of bluetongue in northern Europe in 2006. In addition, disease can emerge in species formerly not considered affected, e.g. the emergence of bovine tuberculosis in wildlife species since 2000 in France. There can also be an unexpected increase of disease incidence in a known area and a known species, or there may simply be an increase in our knowledge or awareness of a particular disease. What all these emerging diseases have in common is that human activity frequently has a role to play in their emergence. For example, bovine spongiform encephalopathy very probably emerged as a result of changes in the manufacturing of meat-and-bone meal, bluetongue was able to spread to cooler climes as a result of uncontrolled trade in animals, and a relaxation of screening and surveillance for bovine tuberculosis enabled the disease to re-emerge in areas that had been able to drastically reduce the number of cases. Globalisation and population growth will continue to affect the epidemiology of diseases in years to come and ecosystems will continue to evolve. Furthermore, new technologies such as metagenomics and high-throughput sequencing are identifying new microorganisms all the time. Change is the one constant, and diseases will continue to emerge, and we must consider the causes and different types of emergence as we deal with these diseases in the future. PMID:26470448

  20. Multifractal characterization of morphology of human red blood cells membrane skeleton.

    Science.gov (United States)

    Ţălu, Ş; Stach, S; Kaczmarska, M; Fornal, M; Grodzicki, T; Pohorecki, W; Burda, K

    2016-04-01

    The purpose of this paper is to show applicability of multifractal analysis in investigations of the morphological changes of ultra-structures of red blood cells (RBCs) membrane skeleton measured using atomic force microscopy (AFM). Human RBCs obtained from healthy and hypertensive donors as well as healthy erythrocytes irradiated with neutrons (45 μGy) were studied. The membrane skeleton of the cells was imaged using AFM in a contact mode. Morphological characterization of the three-dimensional RBC surfaces was realized by a multifractal method. The nanometre scale study of human RBCs surface morphology revealed a multifractal geometry. The generalized dimensions Dq and the singularity spectrum f(α) provided quantitative values that characterize the local scale properties of their membrane skeleton organization. Surface characterization was made using areal ISO 25178-2: 2012 topography parameters in combination with AFM topography measurement. The surface structure of human RBCs is complex with hierarchical substructures resulting from the organization of the erythrocyte membrane skeleton. The analysed AFM images confirm a multifractal nature of the surface that could be useful in histology to quantify human RBC architectural changes associated with different disease states. In case of very precise measurements when the red cell surface is not wrinkled even very fine differences can be uncovered as was shown for the erythrocytes treated with a very low dose of ionizing radiation.

  1. Characterization of the Human Pancreatic Islet Proteome by Two-Dimensional LC/MS/MS

    Energy Technology Data Exchange (ETDEWEB)

    Metz, Thomas O.; Jacobs, Jon M.; Gritsenko, Marina A.; Fontes, Ghislaine; Qian, Weijun; Camp, David G.; Poitout, Vincent J.; Smith, Richard D.

    2006-12-01

    Research to elucidate the pathogenesis of type 1 diabetes mellitus has traditionally focused on the genetic and immunological factors associated with the disease, and, until recently, has not considered the target cell. While there have been reports detailing proteomic analyses of established islet cell lines or isolated rodent islets, the information gained is not always easily extrapolated to humans. Therefore, extensive characterization of the human islet proteome could result in better understanding of islet biology and lead to more effective treatment strategies. We have applied a two-dimensional LC-MS/MS-based analysis to the characterization of the human islet proteome, resulting in the detection of 29,021 unique peptides corresponding to 4,925 proteins. As expected, major islet hormones (insulin, glucagon, somatostatin), beta-cell enriched secretory products (IAPP), ion channels (K-ATP channel), and transcription factors (PDX-1, Nkx 6.1, HNF-1 beta) were detected. In addition, significant proteome coverage of metabolic enzymes and cellular pathways was obtained, including the insulin signaling cascade and the MAP kinase, NF-κβ, and JAK/STAT pathways. This work represents the most extensive characterization of the human islet proteome to date and provides a peptide reference library that may be utilized in future studies of islet biology and type 1 diabetes.

  2. Discovery and Characterization of Chromatin States for Systematic Annotation of the Human Genome

    Science.gov (United States)

    Ernst, Jason; Kellis, Manolis

    A plethora of epigenetic modifications have been described in the human genome and shown to play diverse roles in gene regulation, cellular differentiation and the onset of disease. Although individual modifications have been linked to the activity levels of various genetic functional elements, their combinatorial patterns are still unresolved and their potential for systematic de novo genome annotation remains untapped. Here, we use a multivariate Hidden Markov Model to reveal chromatin states in human T cells, based on recurrent and spatially coherent combinations of chromatin marks.We define 51 distinct chromatin states, including promoter-associated, transcription-associated, active intergenic, largescale repressed and repeat-associated states. Each chromatin state shows specific enrichments in functional annotations, sequence motifs and specific experimentally observed characteristics, suggesting distinct biological roles. This approach provides a complementary functional annotation of the human genome that reveals the genome-wide locations of diverse classes of epigenetic function.

  3. Characterization of receptors for recombinant human tumor necrosis factor-alpha from human placental membranes

    International Nuclear Information System (INIS)

    High affinity receptors for recombinant human tumor necrosis factor-alpha (rhTNF-alpha) were identified on membranes prepared from full term human placenta. Highly purified rhTNF-alpha iodinated by the iodogen method was found to bind placental membranes in a displaceable manner with an approximate dissociation constant (KD) of 1.9 nM. The membrane bound TNF-alpha receptor could be solubilized by several detergents with optimum extraction being obtained with 1% Triton X-100. The binding of 125I-rhTNF-alpha to the solubilized receptor was found to be time and temperature dependent, yielding maximum binding within 1 h, 24 h and 48 h at 37 degrees C, 24 degrees C and 4 degrees C, respectively. However, the maximum binding obtainable at 4 degrees C was only 40% of that at 37 degrees C. The binding 125I-rhTNF-alpha to solubilized placental membrane extracts was displaceable by unlabeled rhTNF-alpha, but not by a related protein recombinant human tumor necrosis factor-beta (rhTNF-beta; previously called lymphotoxin). This is similar to the behavior of TNF-alpha receptors derived from detergent-solubilized cell extracts, although on intact cells, both rhTNF-alpha and rhTNF-beta bind with equal affinity to TNF receptors. The Scatchard analysis of the binding data of the solubilized receptor revealed high affinity binding sites with a KD of approximately 0.5 nM and a receptor concentration of about 1 pmole/mg protein. Gel filtration of the solubilized receptor-ligand complexes on Sephacryl S-300 revealed two different peaks of radioactivity at approximate molecular masses of 50,000 Da and 400,000 Da. The 400,000 dalton peak corresponded to the receptor-ligand complex. Overall, our results suggest that high affinity receptors for TNF-alpha are present on human placental membranes and provide evidence that these receptors may be different from that of rhTNF-beta

  4. Genetic characterization of atypical Mansonella (Mansonella) ozzardi microfilariae in human blood samples from northeastern Peru.

    Science.gov (United States)

    Marcos, Luis A; Arrospide, Nancy; Recuenco, Sergio; Cabezas, Cesar; Weil, Gary J; Fischer, Peter U

    2012-09-01

    DNA sequence comparisons are useful for characterizing proposed new parasite species or strains. Microfilariae with an atypical arrangement of nuclei behind the cephalic space have been recently described in human blood samples from the Amazon region of Peru. Three blood specimens containing atypical microfilariae were genetically characterized using three DNA markers (5S ribosomal DNA, 12S ribosomal DNA, and cytochrome oxidase I). All atypical microfilariae were clustered into the Mansonella group and indistinguishable from M. ozzardi based on these DNA markers. PMID:22826497

  5. Serological characterization of guinea pigs infected with H3N2 human influenza or immunized with hemagglutinin protein

    Directory of Open Access Journals (Sweden)

    Bushnell Ruth V

    2010-08-01

    Full Text Available Abstract Background Recent and previous studies have shown that guinea pigs can be infected with, and transmit, human influenza viruses. Therefore guinea pig may be a useful animal model for better understanding influenza infection and assessing vaccine strategies. To more fully characterize the model, antibody responses following either infection/re-infection with human influenza A/Wyoming/03/2003 H3N2 or immunization with its homologous recombinant hemagglutinin (HA protein were studied. Results Serological samples were collected and tested for anti-HA immunoglobulin by ELISA, antiviral antibodies by hemagglutination inhibition (HI, and recognition of linear epitopes by peptide scanning (PepScan. Animals inoculated with infectious virus demonstrated pronounced viral replication and subsequent serological conversion. Animals either immunized with the homologous HA antigen or infected, showed a relatively rapid rise in antibody titers to the HA glycoprotein in ELISA assays. Antiviral antibodies, measured by HI assay, were detectable after the second inoculation. PepScan data identified both previously recognized and newly defined linear epitopes. Conclusions Infection and/or recombinant HA immunization of guinea pigs with H3N2 Wyoming influenza virus resulted in a relatively rapid production of viral-specific antibody thus demonstrating the strong immunogenicity of the major viral structural proteins in this animal model for influenza infection. The sensitivity of the immune response supports the utility of the guinea pig as a useful animal model of influenza infection and immunization.

  6. Historically defined autobiographical periods

    DEFF Research Database (Denmark)

    Brown, Norman R.; Hansen, Tia G. B.; Lee, Peter J.;

    2012-01-01

    The chapter reviews a research programme that has demonstrated the existence of historically defined autobiographical periods and identified the conditions that bring them about. Data from four samples of World War II-generation adults show that historically defined autobiographical periods endure...

  7. Derivation and characterization of human embryonic stem cells on human amnion epithelial cells.

    Science.gov (United States)

    Lai, Dongmei; Wang, Yongwei; Sun, Jian; Chen, Yifei; Li, Ting; Wu, Yi; Guo, Lihe; Wei, Chunsheng

    2015-05-07

    Culture conditions that support the growth of undifferentiated human embryonic stem cells (hESCs) have already been established using primary human amnion epithelial cells (hAECs) as an alternative to traditional mitotically inactivated mouse embryonic fibroblasts (MEFs). In the present work, inner cell masses (ICM) were isolated from frozen embryos obtained as donations from couples undergoing in vitro fertilization (IVF) treatment and four new hESC lines were derived using hAECs as feeder cells. This feeder system was able to support continuous growth of what were, according to their domed shape and markers, undifferentiated naïve-like hESCs. Their pluripotent potential were also demonstrated by embryoid bodies developing to the expected three germ layers in vitro and the productions of teratoma in vivo. The cell lines retained their karyotypic integrity for over 35 passages. Transmission electron microscopy (TEM) indicated that these newly derived hESCs consisted mostly of undifferentiated cells with large nuclei and scanty cytoplasm. The new hESCs cultured on hAECs showed distinct undifferentiated characteristics in comparison to hESCs of the same passage maintained on MEFs. This type of optimized culture system may provide a useful platform for establishing clinical-grade hESCs and assessing the undifferentiated potential of hESCs.

  8. Selection, affinity maturation, and characterization of a human scFv antibody against CEA protein

    International Nuclear Information System (INIS)

    CEA is a tumor-associated antigen abundantly expressed on several cancer types, including those naturally refractory to chemotherapy. The selection and characterization of human anti-CEA single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer monoclonal antibodies designed for optimal blood clearance and tumor penetration. The human MA39 scFv, selected for its ability to recognize a CEA epitope expressed on human colon carcinomas, was first isolated from a large semi-synthetic ETH-2 antibody phage library, panned on human purified CEA protein. Subsequently, by in vitro mutagenesis of a gene encoding for the scFv MA39, a new library was established, and new scFv antibodies with improved affinity towards the CEA cognate epitope were selected and characterized. The scFv MA39 antibody was affinity-maturated by in vitro mutagenesis and the new scFv clone, E8, was isolated, typed for CEA family member recognition and its CEACAM1, 3 and 5 shared epitope characterized for expression in a large panel of human normal and tumor tissues and cells. The binding affinity of the scFv E8 is in a range for efficient, in vivo, antigen capture in tumor cells expressing a shared epitope of the CEACAM1, 3 and 5 proteins. This new immunoreagent meets all criteria for a potential anticancer compound: it is human, hence poorly or not at all immunogenic, and it binds selectively and with good affinity to the CEA epitope expressed by metastatic melanoma and colon and lung carcinomas. Furthermore, its small molecular size should provide for efficient tissue penetration, yet give rapid plasma clearance

  9. Molecular cloning and biological characterization of the human excision repair gene ERCC-3.

    OpenAIRE

    Weeda, G; van Ham, R C; Masurel, R; Westerveld, A; Odijk, H; Wit, J.; Bootsma, D; van der Eb, A J; Hoeijmakers, J. H.

    1990-01-01

    In this report we present the cloning, partial characterization, and preliminary studies of the biological activity of a human gene, designated ERCC-3, involved in early steps of the nucleotide excision repair pathway. The gene was cloned after genomic DNA transfection of human (HeLa) chromosomal DNA together with dominant marker pSV3gptH to the UV-sensitive, incision-defective Chinese hamster ovary (CHO) mutant 27-1. This mutant belongs to complementation group 3 of repair-deficient rodent m...

  10. Experimental characterization and damage model of the human skin response to dynamic loadings

    OpenAIRE

    JACQUEMOUD,C; Coret, M; BRUYERE-GARNIER,K; Brunet, M.

    2007-01-01

    The objective of this work is to characterize and to model the damage of planar and fibrous soft tissues at high strain rate. As first step, we choose to study the human skin. A dynamic tensile test up to failure is performed on 10x30mm human skin samples. The test is based on the drop test principle and allows loading of samples at a strain rate close to 40 s-1. Classical measurement techniques give global strains whereas a full local strain field is measured on the sample surface by an Imag...

  11. Characterization of Adherent Nonhematopoietic Cells Derived from Human Umbilical Cord Blood

    Institute of Scientific and Technical Information of China (English)

    安小惠; 蔡国平

    2003-01-01

    To confirm and characterize the adherent fibroblast-like progenitors in human umbilical cord blood, we isolated mononuclear cells from human umbilical cord blood by Ficoll-Hypaque.Two main morphologically different kinds of cells were formed by culturing the cells in collagen-coated 24-well plastic dishes and flasks.One type was the adherent fibroblast-like cells, while the other was loosely adherent clonally expanded round cells.Our experiments demonstrate that the adherent fibroblast-like cells possess multilineage potential, including the ability to differentiate into endothelial-like cells and to express the mesenchymal cell marker.

  12. Molecular cloning and characterization of the human RNase κ, an ortholog of Cc RNase

    OpenAIRE

    Economopoulou, Marie-angela I.; Fragoulis, Emmanouel G.; Sideris, Diamantis C.

    2007-01-01

    A novel protein family, designated hereafter as RNase κ (kappa) family, has been recently introduced with the characterization of the specific Cc RNase, isolated from the insect Ceratitis capitata. The human ortholog of this family consists of 98 amino acids and shares > 98% identity with its mammalian counterparts. This RNase is encoded by a single-copy gene found to be expressed in a wide spectrum of normal and cancer tissues. The cDNA of the human ribonuclease has been isolated and subclon...

  13. AIDS defining disease: Disseminated cryptococcosis

    Directory of Open Access Journals (Sweden)

    Roshan Anupama

    2006-01-01

    Full Text Available Disseminated cryptococcosis is one of the acquired immune deficiency syndrome defining criteria and the most common cause of life threatening meningitis. Disseminated lesions in the skin manifest as papules or nodules that mimic molluscum contagiosum (MC. We report here a human immunodeficiency virus positive patient who presented with MC like lesions. Disseminated cryptococcosis was confirmed by India ink preparation and histopathology. The condition of the patient improved with amphotercin B.

  14. Characterization of biomechanical properties of aged human and ovine mitral valve chordae tendineae.

    Science.gov (United States)

    Zuo, Keping; Pham, Thuy; Li, Kewei; Martin, Caitlin; He, Zhaoming; Sun, Wei

    2016-09-01

    The mitral valve (MV) is a highly complex cardiac valve consisting of an annulus, anterior and posterior leaflets, chordae tendineae (chords) and two papillary muscles. The chordae tendineae mechanics play a pivotal role in proper MV function: the chords help maintain proper leaflet coaptation and rupture of the chordae tendineae due to disease or aging can lead to mitral valve insufficiency. Therefore, the aim of this study was to characterize the mechanical properties of aged human and ovine mitral chordae tendineae. The human and ovine chordal specimens were categorized by insertion location (i.e., marginal, basal and strut) and leaflet type (i.e., anterior and posterior). The results show that human and ovine chords of differing types vary largely in size but do not have significantly different elastic and failure properties. The excess fibrous tissue layers surrounding the central core of human chords added thickness to the chords but did not contribute to the overall strength of the chords. In general, the thinner marginal chords were stiffer than the thicker basal and strut chords, and the anterior chords were stiffer and weaker than the posterior chords. The human chords of all types were significantly stiffer than the corresponding ovine chords and exhibited much lower failure strains. These findings can be explained by the diminished crimp pattern of collagen fibers of the human mitral chords observed histologically. Moreover, the mechanical testing data was modeled with the nonlinear hyperelastic Ogden strain energy function to facilitate accurate computational modeling of the human MV. PMID:27315372

  15. Human exonuclease 1 (EXO1) activity characterization and its function on FLAP structures

    DEFF Research Database (Denmark)

    Keijzers, Guido; Bohr, Vilhelm A; Juel Rasmussen, Lene

    2015-01-01

    Human exonuclease 1 (EXO1) is involved in multiple DNA metabolism processes, including DNA repair and replication. Most of the fundamental roles of EXO1 have been described in yeast. Here, we report a biochemical characterization of human full-length EXO1. Prior to assay EXO1 on different DNA flap...... structures, we determined factors essential for the thermodynamic stability of EXO1. We show that enzymatic activity and stability of EXO1 on DNA is modulated by temperature. By characterization of EXO1 flap activity using various DNA flap substrates, we show that EXO1 has a strong capacity for degrading...... double stranded DNA and has a modest endonuclease or 5' flap activity. Furthermore, we report novel mechanistic insights into the processing of flap structures, showing that EXO1 preferentially cleaves one nucleotide inwards in a double stranded region of a forked and nicked DNA flap substrates...

  16. Isolation & characterization of Brucella melitensis isolated from patients suspected for human brucellosis in India

    Directory of Open Access Journals (Sweden)

    Anita Barua

    2016-01-01

    Interpretation & conclusions: The present study showed an overall isolation rate of 17.64 per cent for B. melitensis. There is a need to establish facilities for isolation and characterization of Brucella species for effective clinical management of the disease among patients as well as surveillance and control of infection in domestic animals. Further studies are needed from different geographical areas of the country with different level of endemicity to plan and execute control strategies against human brucellosis.

  17. Identification and characterization of a novel repressor site in the human tumor necrosis factor alpha gene.

    OpenAIRE

    Fong, C L; Siddiqui, A H; Mark, D F

    1994-01-01

    In human monocytic cell lines, tumor necrosis factor alpha (TNF alpha) expression is induced by phorbol myristate acetate (PMA). We have identified positive and negative cis-acting elements in the TNF alpha promoter by deletion analysis. Here we present the initial characterization of the repressor element. The repressor element was shown to function in either orientation and at various distances upstream from the positive element of the TNF alpha promoter. The TNF alpha repressor site (TRS) ...

  18. Towards the Maturation and Characterization of Smooth Muscle Cells Derived from Human Embryonic Stem Cells

    OpenAIRE

    Helena Vazão; Ricardo Pires das Neves; Mário Grãos; Lino Ferreira

    2011-01-01

    In this study we demonstrate that CD34(+) cells derived from human embryonic stem cells (hESCs) have higher smooth muscle cell (SMC) potential than CD34(-) cells. We report that from all inductive signals tested, retinoic acid (RA) and platelet derived growth factor (PDGF(BB)) are the most effective agents in guiding the differentiation of CD34(+) cells into smooth muscle progenitor cells (SMPCs) characterized by the expression of SMC genes and proteins, secretion of SMC-related cytokines, co...

  19. Bioinformatic characterization and analysis of polymorphic inversions in the human genome

    OpenAIRE

    Alexander Mart\\xednez Fundichelly

    2013-01-01

    Within the great interest in the characterization of genomic structural variants (SVs) in the human genome, inversions present unique challenges and have been little studied. This thesis has developed "GRIAL", a new algorithm focused specifically in detect and map accurately inversions from paired-end mapping (PEM) data, which is the most widely used method to detect SVs. GRIAL is based on geometrical rules to cluster, merge and refine both breakpoints of putative inversions...

  20. Bioluminescence Reporter Gene Imaging Characterize Human Embryonic Stem Cell-Derived Teratoma Formation

    OpenAIRE

    Su, Weijun; Zhou, Manqian; Zheng, Yizhou; Fan, Yan; HAN, ZHONGCHAO; Kong, Deling; Wu, Joseph C.; Xiang, Rong; Li, Zongjin

    2011-01-01

    Human embryonic stem (hES) cells are capable of differentiation into virtually all cell types and hold tremendous potential as cell sources for regenerative therapies. However, teratoma formation can be the main obstacle for hES cells therapy. In order to understand the biology and physiology of hES cells teratoma formation, we investigated the angiogenic process within teratomas and characterized teratoma cells. In this study, hES cells transduced with double fusion reporter gene that consis...

  1. Isolation, culture and characterization of postnatal human umbilical vein-derived mesenchymal stem cells

    OpenAIRE

    Mehdi Kadivar; Shohreh Khatami; Yousef Mortazavi; Masoud Soleimani; Mohammad Taghikhani; Mohammad Ali Shokrgozar

    2005-01-01

    On the basis of reports that mesenchymal stem cells (MSCs) can be isolated from the placenta/umbilical cord stroma, the present study was undertaken to isolate and characterize MSCs from the human umbilical cord veins. In this investigation, a cell population was isolated which was derived from the endothelium/subendothelium layers of 20 umbilical cord veins obtained from term deliveries using a solution of 0.1% collagenase type IV. Results suggest that these cells possess morphological, immu...

  2. Characterization of scale-free properties of human electrocorticography in awake and slow wave sleep states

    Directory of Open Access Journals (Sweden)

    John M Zempel

    2012-06-01

    Full Text Available Like many complex dynamic systems, the brain exhibits scale-free dynamics that follow power law scaling. Broadband power spectral density (PSD of brain electrical activity exhibits state-dependent power law scaling with a log frequency exponent that varies across frequency ranges. Widely divergent naturally occurring neural states, awake and slow wave sleep (SWS periods, were used evaluate the nature of changes in scale-free indices. We demonstrate two analytic approaches to characterizing electrocorticographic (ECoG data obtained during Awake and SWS states. A data driven approach was used, characterizing all available frequency ranges. Using an Equal Error State Discriminator (EESD, a single frequency range did not best characterize state across data from all six subjects, though the ability to distinguish awake and SWS states in individual subjects was excellent. Multisegment piecewise linear fits were used to characterize scale-free slopes across the entire frequency range (0.2-200 Hz. These scale-free slopes differed between Awake and SWS states across subjects, particularly at frequencies below 10 Hz and showed little difference at frequencies above 70 Hz. A Multivariate Maximum Likelihood Analysis (MMLA method using the multisegment slope indices successfully categorized ECoG data in most subjects, though individual variation was seen. The ECoG spectrum is not well characterized by a single linear fit across a defined set of frequencies, but is best described by a set of discrete linear fits across the full range of available frequencies. With increasing computational tractability, the use of scale-free slope values to characterize EEG data will have practical value in clinical and research EEG studies.

  3. Characterization of human chromosomal DNA sequences which replicate autonomously in Saccharomyces cerevisiae.

    Science.gov (United States)

    Montiel, J F; Norbury, C J; Tuite, M F; Dobson, M J; Mills, J S; Kingsman, A J; Kingsman, S M

    1984-01-01

    We have characterised two restriction fragments, isolated from a "shotgun" collection of human DNA, which function as autonomously replicating sequences (ARSs) in Saccharomyces cerevisiae. Functional domains of these fragments have been defined by subcloning and exonuclease (BAL 31) deletion analysis. Both fragments contain two spatially distinct domains. One is essential for high frequency transformation and is termed the Replication Sequence (RS) domain, the other, termed the Replication Enhancer (RE) domain, has no inherent replication competence but is essential for ensuring maximum function of the RS domain. The nucleotide sequence of these domains reveals several conserved sequences one of which is strikingly similar to the yeast ARS consensus sequence. PMID:6320114

  4. How to define surveillance?

    Directory of Open Access Journals (Sweden)

    Christian Fuchs

    2011-12-01

    Full Text Available The task of this paper is to explore and compare ways of defining surveillance. In order to give meaning to concepts that describe the realities of society, social theory is needed. Therefore social theory is employed in this paper for discussing ways of defining surveillance. “Living in ‘surveillance societies’ may throw up challenges of a fundamental – ontological – kind” (Lyon, 1994, p.19. Social theory is a way of clarifying such ontological questions that concern the basic nature and reality of surveillance. A distinction between neutral and negative concepts of surveillance is drawn. Some potential disadvantages of neutral concepts of surveillance are outlined. This paper wants to contribute to the discussion of how to best define surveillance and wants to show that one of the main theoretical differences and questions in surveillance theory is if surveillance should be defined as a negative or a neutral concept.

  5. Adaptation of a Commonly Used, Chemically Defined Medium for Human Embryonic Stem Cells to Stable Isotope Labeling with Amino Acids in Cell Culture

    DEFF Research Database (Denmark)

    Liberski, A. R.; Al-Noubi, M. N.; Rahman, Z. H.;

    2013-01-01

    rarely employed in the context of complex culturing conditions as those required for human embryonic stem cells (hESCs). Classic hESC culture is based on the use of mouse embryonic fibroblasts (MEFs) as a feeder layer, and as a result, possible xenogeneic contamination, contribution of unlabeled amino......Metabolic labeling with stable isotopes is a prominent technique for comparative quantitative proteomics, and stable isotope labeling with amino acids in cell culture (SILAC) is the most commonly used approach. SILAC is, however, traditionally limited to simple tissue culture regimens and only...... developed by Ludwig et al. and commercially available as mTeSR1 [mTeSR1 is a trade mark of WiCell (Madison, WI) licensed to STEMCELL Technologies (Vancouver, Canada)]. This medium, together with adjustments to the culturing protocol, facilitates reproducible labeling that is easily scalable to the protein...

  6. Defining Documentary Film

    DEFF Research Database (Denmark)

    Juel, Henrik

    2006-01-01

    A discussion of various attemts at defining documentary film regarding form, content, truth, stile, genre or reception - and a propoposal of a positive list of essential, but non-exclusive characteristica of documentary film......A discussion of various attemts at defining documentary film regarding form, content, truth, stile, genre or reception - and a propoposal of a positive list of essential, but non-exclusive characteristica of documentary film...

  7. Advanced flow cytometric analysis of nanoparticle targeting to rare leukemic stem cells in peripheral human blood in a defined model system

    Science.gov (United States)

    Cooper, Christy L.; Leary, James F.

    2015-03-01

    Leukemia stem cells are both stem-like and leukemic-like. This complicates their detection as rare circulating tumor cells in the peripheral blood of leukemia patients. Since leukemic stem cells are also resistant to standard chemotherapeutic regimens, new therapeutic strategies need to be designed to kill the leukemic stem cells without killing normal stem cells. In these initial targeting studies we utilized a bioinformatics approach to design an antibodyfluorescent nanoparticle conjugate for targeting to these leukemic stem cells and to minimize targeting to normal stemprogenitor cells. Multicolor flow cytometric analyses were performed on a BD FACS Aria III. Human leukemic stem cell-like cell RS4;11 (with putative immunophenotype CD133+/CD24+/-, CD34+/-, CD38+, CD10-/Flt3+) was spiked into normal hematopoietic stem-progenitor cells obtained from a "buffy coat" prep (with putative immunophenotype CD133- /CD34+/CD38-/CD10-/Flt-3-) to be used as a model human leukemia patient. To analyze the model system, digital data mixtures of the two cell types were first created and assigned classifiers in order to create truth sets. ROC (Receiver Operating Characteristic) and multidimensional cluster analyses were used to evaluate the specificity and sensitivity of the immunophenotyping panel and for automated cell population identification, respectively. Costs of misclassification (false targeting) were also accounted for by this analysis scheme. Ultimately, this analysis scheme will be applied to use of nanoparticle-antibody conjugates at therapeutic doses for targeted killing of leukemia stem cells preferentially to normal stem -progenitor cells.

  8. Functional characterization in Caenorhabditis elegans of transmembrane worm-human orthologs

    Directory of Open Access Journals (Sweden)

    Baillie David L

    2004-11-01

    Full Text Available Abstract Background The complete genome sequences for human and the nematode Caenorhabditis elegans offer an opportunity to learn more about human gene function through functional characterization of orthologs in the worm. Based on a previous genome-wide analysis of worm-human orthologous transmembrane proteins, we selected seventeen genes to explore experimentally in C. elegans. These genes were selected on the basis that they all have high confidence candidate human orthologs and that their function is unknown. We first analyzed their phylogeny, membrane topology and domain organization. Then gene functions were studied experimentally in the worm by using RNA interference and transcriptional gfp reporter gene fusions. Results The experiments gave functional insights for twelve of the genes studied. For example, C36B1.12, the worm ortholog of three presenilin-like genes, was almost exclusively expressed in head neurons, suggesting an ancient conserved role important to neuronal function. We propose a new transmembrane topology for the presenilin-like protein family. sft-4, the worm ortholog of surfeit locus gene Surf-4, proved to be an essential gene required for development during the larval stages of the worm. R155.1, whose human ortholog is entirely uncharacterized, was implicated in body size control and other developmental processes. Conclusions By combining bioinformatics and C. elegans experiments on orthologs, we provide functional insights on twelve previously uncharacterized human genes.

  9. Establishment and characterization of two new human embryonic stem cell lines, SYSU-1 and SYSU-2

    Institute of Scientific and Technical Information of China (English)

    HUANG Guo; Andy Peng Xiang; LI Wei-qiang; CHEN Rui; CHEN Zhen-guang; ZHANG Xiu-ming; MAO Fu-xiang; HUANG Shao-liang; LI Shu-nong; Bruce T Lahn

    2007-01-01

    Background Human embryonic stem cells can propagate indefinitely in vitro and are able to differentiate into derivatives of all three embryonic germ layers. The excitement surrounding human embryonic stem cells lies largely in their potential to produce specialized cells that can be used for transplant therapies. However, further investigation requires additional cell lines with varying genetic background. Therefore, efforts to derive and establish more human embryonic stem cell lines are highly warranted.Methods Surplus embryos (blastocysts) from donors were used to isolate the inner cell mass by immunosurgery. All cells were cultured continuously on irradiated murine embryonic fibroblasts feed layer and likely human embryonic stem cell colonies were subsequently characterized by cell surface marker staining, karyotyping and teratoma formation.Results Two human embryonic stem cell lines (SYSU-1 and SYSU-2) were established from surplus embryos. The two lines express several pluripotency markers including alkaline phosphatase, SSEA- 4, Tra-1-60, Oct-4, Nanog and Rex-1.They remain in undifferentiated state with normal karyotype after prolonged passages and can form embryoid bodies in vitro and teratoma in vivo.Conclusion Two new human embryonic stem cell lines have been established from surplus embryos. They can be used to understand selfrenewal and differentiating mechanisms and provide more choices for regenerative medicine.

  10. Through-wall bio-radio location and characterization of human activities

    Science.gov (United States)

    Fu, Lei; Gui, Yong-Sheng; Hu, Can-Ming

    2016-06-01

    In this paper, a through-the-wall life detection system has been developed by using a broadband microwave technique. This system can not only determine and characterize human movement behind an obstacle but also determine the person’s position by employing the Fourier transform technique. The effectiveness of this system is shown by the experimental results where the presence of stationary and moving person behind an obstacle can be identified upto a distance of 17 and 30 m respectively. Since the movement of a human body is continuous, an averaged background subtraction technique has been developed which allows real time detection of human activities without requiring any prior knowledge of the environment, thus making the system suitable for practical applications.

  11. Molecular profiling of multiple human cancers defines an inflammatory cancer-associated molecular pattern and uncovers KPNA2 as a uniform poor prognostic cancer marker.

    Directory of Open Access Journals (Sweden)

    Saleh M Rachidi

    Full Text Available BACKGROUND: Immune evasion is one of the recognized hallmarks of cancer. Inflammatory responses to cancer can also contribute directly to oncogenesis. Since the immune system is hardwired to protect the host, there is a possibility that cancers, regardless of their histological origins, endow themselves with a common and shared inflammatory cancer-associated molecular pattern (iCAMP to promote oncoinflammation. However, the definition of iCAMP has not been conceptually and experimentally investigated. METHODS AND FINDINGS: Genome-wide cDNA expression data was analyzed for 221 normal and 324 cancer specimens from 7 cancer types: breast, prostate, lung, colon, gastric, oral and pancreatic. A total of 96 inflammatory genes with consistent dysregulation were identified, including 44 up-regulated and 52 down-regulated genes. Protein expression was confirmed by immunohistochemistry for some of these genes. The iCAMP contains proteins whose roles in cancer have been implicated and others which are yet to be appreciated. The clinical significance of many iCAMP genes was confirmed in multiple independent cohorts of colon and ovarian cancer patients. In both cases, better prognosis correlated strongly with high CXCL13 and low level of GREM1, LOX, TNFAIP6, CD36, and EDNRA. An "Inflammatory Gene Integrated Score" was further developed from the combination of 18 iCAMP genes in ovarian cancer, which predicted overall survival. Noticeably, as a selective nuclear import protein whose immuno-regulatory function just begins to emerge, karyopherin alpha 2 (KPNA2 is uniformly up-regulated across cancer types. For the first time, the cancer-specific up-regulation of KPNA2 and its clinical significance were verified by tissue microarray analysis in colon and head-neck cancers. CONCLUSION: This work defines an inflammatory signature shared by seven epithelial cancer types and KPNA2 as a consistently up-regulated protein in cancer. Identification of iCAMP may not only

  12. Affinity-purified antibodies of defined specificity for use in a solid-phase microplate radioimmunoassay of human Tamm-Horsfall glycoprotein in urine.

    Science.gov (United States)

    Hunt, J S; McGiven, A R; Groufsky, A; Lynn, K L; Taylor, M C

    1985-05-01

    Rabbit antibodies to human Tamm-Horsfall glycoprotein (prepared by salt precipitation from normal urine) were purified by affinity chromatography using columns containing Tamm-Horsfall glycoprotein linked to CNBr-activated Sepharose 4B. The specificity of these antibodies was determined by analysis of their binding characteristics on Western blots of Tamm-Horsfall protein from sodium dodecyl sulphate/polyacrylamide gradient gels and comparison with the reactivity of monoclonal antibodies to this glycoprotein. Optimal conditions of adsorption to poly(vinyl chloride) microtitre plates were established such that these purified antibodies could be used in a solid-phase radioimmunoassay for the determination of urinary Tamm-Horsfall-glycoprotein concentration. The specificity of the immunoassay was confirmed by competitive inhibition of the urinary Tamm-Horsfall glycoprotein by purified freeze-dried material in solution. A standard curve obtained with this material showed the radioimmunoassay to have a sensitivity of at least 5 ng/ml, with linearity between 30 and 600 ng/ml. The mean coefficient of variation over the linear section of the curve was 11.3 +/- 2.2% (n = 13). The effects of dialysis and freezing of urine samples before determination of Tamm-Horsfall-glycoprotein concentrations were investigated and the mean 24 h urinary excretion rate in 60 normal donors was shown to be 84.9 +/- 44.1 mg.

  13. A novel system for simultaneous or sequential integration of multiple gene-loading vectors into a defined site of a human artificial chromosome.

    Directory of Open Access Journals (Sweden)

    Teruhiko Suzuki

    Full Text Available Human artificial chromosomes (HACs are gene-delivery vectors suitable for introducing large DNA fragments into mammalian cells. Although a HAC theoretically incorporates multiple gene expression cassettes of unlimited DNA size, its application has been limited because the conventional gene-loading system accepts only one gene-loading vector (GLV into a HAC. We report a novel method for the simultaneous or sequential integration of multiple GLVs into a HAC vector (designated as the SIM system via combined usage of Cre, FLP, Bxb1, and φC31 recombinase/integrase. As a proof of principle, we first attempted simultaneous integration of three GLVs encoding EGFP, Venus, and TdTomato into a gene-loading site of a HAC in CHO cells. These cells successfully expressed all three fluorescent proteins. Furthermore, microcell-mediated transfer of HACs enabled the expression of those fluorescent proteins in recipient cells. We next demonstrated that GLVs could be introduced into a HAC one-by-one via reciprocal usage of recombinase/integrase. Lastly, we introduced a fourth GLV into a HAC after simultaneous integration of three GLVs by FLP-mediated DNA recombination. The SIM system expands the applicability of HAC vectors and is useful for various biomedical studies, including cell reprogramming.

  14. Characterization of temperate phages infecting Clostridium difficile isolates of human and animal origins.

    Science.gov (United States)

    Sekulovic, Ognjen; Garneau, Julian R; Néron, Audrey; Fortier, Louis-Charles

    2014-04-01

    Clostridium difficile is a Gram-positive pathogen infecting humans and animals. Recent studies suggest that animals could represent potential reservoirs of C. difficile that could then transfer to humans. Temperate phages contribute to the evolution of most bacteria, for example, by promoting the transduction of virulence, fitness, and antibiotic resistance genes. In C. difficile, little is known about their role, mainly because suitable propagating hosts and conditions are lacking. Here we report the isolation, propagation, and preliminary characterization of nine temperate phages from animal and human C. difficile isolates. Prophages were induced by UV light from 58 C. difficile isolates of animal and human origins. Using soft agar overlays with 27 different C. difficile test strains, we isolated and further propagated nine temperate phages: two from horse isolates (ΦCD481-1 and ΦCD481-2), three from dog isolates (ΦCD505, ΦCD506, and ΦCD508), and four from human isolates (ΦCD24-2, ΦCD111, ΦCD146, and ΦCD526). Two phages are members of the Siphoviridae family (ΦCD111 and ΦCD146), while the others are Myoviridae phages. Pulsed-field gel electrophoresis and restriction enzyme analyses showed that all of the phages had unique double-stranded DNA genomes of 30 to 60 kb. Phages induced from human C. difficile isolates, especially the members of the Siphoviridae family, had a broader host range than phages from animal C. difficile isolates. Nevertheless, most of the phages could infect both human and animal strains. Phage transduction of antibiotic resistance was recently reported in C. difficile. Our findings therefore call for further investigation of the potential risk of transduction between animal and human C. difficile isolates.

  15. Computational Characterization of Exogenous MicroRNAs that Can Be Transferred into Human Circulation.

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    Jiang Shu

    Full Text Available MicroRNAs have been long considered synthesized endogenously until very recent discoveries showing that human can absorb dietary microRNAs from animal and plant origins while the mechanism remains unknown. Compelling evidences of microRNAs from rice, milk, and honeysuckle transported to human blood and tissues have created a high volume of interests in the fundamental questions that which and how exogenous microRNAs can be transferred into human circulation and possibly exert functions in humans. Here we present an integrated genomics and computational analysis to study the potential deciding features of transportable microRNAs. Specifically, we analyzed all publicly available microRNAs, a total of 34,612 from 194 species, with 1,102 features derived from the microRNA sequence and structure. Through in-depth bioinformatics analysis, 8 groups of discriminative features have been used to characterize human circulating microRNAs and infer the likelihood that a microRNA will get transferred into human circulation. For example, 345 dietary microRNAs have been predicted as highly transportable candidates where 117 of them have identical sequences with their homologs in human and 73 are known to be associated with exosomes. Through a milk feeding experiment, we have validated 9 cow-milk microRNAs in human plasma using microRNA-sequencing analysis, including the top ranked microRNAs such as bta-miR-487b, miR-181b, and miR-421. The implications in health-related processes have been illustrated in the functional analysis. This work demonstrates the data-driven computational analysis is highly promising to study novel molecular characteristics of transportable microRNAs while bypassing the complex mechanistic details.

  16. Identification and characterization of Ca2+-activated K+ channels in granulosa cells of the human ovary

    Directory of Open Access Journals (Sweden)

    Berg Ulrike

    2009-04-01

    Full Text Available Abstract Background Granulosa cells (GCs represent a major endocrine compartment of the ovary producing sex steroid hormones. Recently, we identified in human GCs a Ca2+-activated K+ channel (KCa of big conductance (BKCa, which is involved in steroidogenesis. This channel is activated by intraovarian signalling molecules (e.g. acetylcholine via raised intracellular Ca2+ levels. In this study, we aimed at characterizing 1. expression and functions of KCa channels (including BKCa beta-subunits, and 2. biophysical properties of BKCa channels. Methods GCs were obtained from in vitro-fertilization patients and cultured. Expression of mRNA was determined by standard RT-PCR and protein expression in human ovarian slices was detected by immunohistochemistry. Progesterone production was measured in cell culture supernatants using ELISAs. Single channels were recorded in the inside-out configuration of the patch-clamp technique. Results We identified two KCa types in human GCs, the intermediate- (IK and the small-conductance KCa (SK. Their functionality was concluded from attenuation of human chorionic gonadotropin-stimulated progesterone production by KCa blockers (TRAM-34, apamin. Functional IK channels were also demonstrated by electrophysiological recording of single KCa channels with distinctive features. Both, IK and BKCa channels were found to be simultaneously active in individual GCs. In agreement with functional data, we identified mRNAs encoding IK, SK1, SK2 and SK3 in human GCs and proteins of IK and SK2 in corresponding human ovarian cells. Molecular characterization of the BKCa channel revealed the presence of mRNAs encoding several BKCa beta-subunits (beta2, beta3, beta4 in human GCs. The multitude of beta-subunits detected might contribute to variations in Ca2+ dependence of individual BKCa channels which we observed in electrophysiological recordings. Conclusion Functional and molecular studies indicate the presence of active IK and SK

  17. Biomaterial arrays with defined adhesion ligand densities and matrix stiffness identify distinct phenotypes for tumorigenic and nontumorigenic human mesenchymal cell types.

    Science.gov (United States)

    Hansen, Tyler D; Koepsel, Justin T; Le, Ngoc Nhi; Nguyen, Eric H; Zorn, Stefan; Parlato, Matthew; Loveland, Samuel G; Schwartz, Michael P; Murphy, William L

    2014-05-01

    Here, we aimed to investigate migration of a model tumor cell line (HT-1080 fibrosarcoma cells, HT-1080s) using synthetic biomaterials to systematically vary peptide ligand density and substrate stiffness. A range of substrate elastic moduli were investigated by using poly(ethylene glycol) (PEG) hydrogel arrays (0.34 - 17 kPa) and self-assembled monolayer (SAM) arrays (~0.1-1 GPa), while cell adhesion was tuned by varying the presentation of Arg-Gly-Asp (RGD)-containing peptides. HT-1080 motility was insensitive to cell adhesion ligand density on RGD-SAMs, as they migrated with similar speed and directionality for a wide range of RGD densities (0.2-5% mol fraction RGD). Similarly, HT-1080 migration speed was weakly dependent on adhesion on 0.34 kPa PEG surfaces. On 13 kPa surfaces, a sharp initial increase in cell speed was observed at low RGD concentration, with no further changes observed as RGD concentration was increased further. An increase in cell speed ~ two-fold for the 13 kPa relative to the 0.34 kPa PEG surface suggested an important role for substrate stiffness in mediating motility, which was confirmed for HT-1080s migrating on variable modulus PEG hydrogels with constant RGD concentration. Notably, despite ~ two-fold changes in cell speed over a wide range of moduli, HT-1080s adopted rounded morphologies on all surfaces investigated, which contrasted with well spread primary human mesenchymal stem cells (hMSCs). Taken together, our results demonstrate that HT-1080s are morphologically distinct from primary mesenchymal cells (hMSCs) and migrate with minimal dependence on cell adhesion for surfaces within a wide range of moduli, whereas motility is strongly influenced by matrix mechanical properties.

  18. Unique gene expression and MR T2 relaxometry patterns define chronic murine dextran sodium sulphate colitis as a model for connective tissue changes in human Crohn's disease.

    Directory of Open Access Journals (Sweden)

    Christine Breynaert

    Full Text Available INTRODUCTION: Chronically relapsing inflammation, tissue remodeling and fibrosis are hallmarks of inflammatory bowel diseases. The aim of this study was to investigate changes in connective tissue in a chronic murine model resulting from repeated cycles of dextran sodium sulphate (DSS ingestion, to mimic the relapsing nature of the human disease. MATERIALS AND METHODS: C57BL/6 mice were exposed to DSS in drinking water for 1 week, followed by a recovery phase of 2 weeks. This cycle of exposure was repeated for up to 3 times (9 weeks in total. Colonic inflammation, fibrosis, extracellular matrix proteins and colonic gene expression were studied. In vivo MRI T 2 relaxometry was studied as a potential non-invasive imaging tool to evaluate bowel wall inflammation and fibrosis. RESULTS: Repeated cycles of DSS resulted in a relapsing and remitting disease course, which induced a chronic segmental, transmural colitis after 2 and 3 cycles of DSS with clear induction of fibrosis and remodeling of the muscular layer. Tenascin expression mirrored its expression in Crohn's colitis. Microarray data identified a gene expression profile different in chronic colitis from that in acute colitis. Additional recovery was associated with upregulation of unique genes, in particular keratins, pointing to activation of molecular pathways for healing and repair. In vivo MRI T2 relaxometry of the colon showed a clear shift towards higher T2 values in the acute stage and a gradual regression of T2 values with increasing cycles of DSS. CONCLUSIONS: Repeated cycles of DSS exposure induce fibrosis and connective tissue changes with typical features, as occurring in Crohn's disease. Colonic gene expression analysis revealed unique expression profiles in chronic colitis compared to acute colitis and after additional recovery, pointing to potential new targets to intervene with the induction of fibrosis. In vivo T2 relaxometry is a promising non-invasive assessment of

  19. Distribution and characterization of progenitor cells within the human filum terminale.

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    Lisa Arvidsson

    Full Text Available BACKGROUND: Filum terminale (FT is a structure that is intimately associated with conus medullaris, the most caudal part of the spinal cord. It is well documented that certain regions of the adult human central nervous system contains undifferentiated, progenitor cells or multipotent precursors. The primary objective of this study was to describe the distribution and progenitor features of this cell population in humans, and to confirm their ability to differentiate within the neuroectodermal lineage. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that neural stem/progenitor cells are present in FT obtained from patients treated for tethered cord. When human or rat FT-derived cells were cultured in defined medium, they proliferated and formed neurospheres in 13 out of 21 individuals. Cells expressing Sox2 and Musashi-1 were found to outline the central canal, and also to be distributed in islets throughout the whole FT. Following plating, the cells developed antigen profiles characteristic of astrocytes (GFAP and neurons (β-III-tubulin. Addition of PDGF-BB directed the cells towards a neuronal fate. Moreover, the cells obtained from young donors shows higher capacity for proliferation and are easier to expand than cells derived from older donors. CONCLUSION/SIGNIFICANCE: The identification of bona fide neural progenitor cells in FT suggests a possible role for progenitor cells in this extension of conus medullaris and may provide an additional source of such cells for possible therapeutic purposes. Filum terminale, human, progenitor cells, neuron, astrocytes, spinal cord.

  20. Nouns to Define Homophobia

    Directory of Open Access Journals (Sweden)

    Adalberto Campo Arias

    2013-09-01

    Full Text Available Background. The term ‘homophobia’ was introduced in the academic context more than 40 years ago. However, its meaning has changed over time. Objective. To review the nouns used in the last twelve years to define homophobia. Methodology. The authors conducted a systematic search in Medline through Pubmed that included editorials, letters to editors, comments and narrative reviews, in English and Spanish. A qualitative analysis (Grounded theory was applied to analyze nouns used to define homophobia since 2001 through 2012. Results. Authors reviewed three papers including ten nouns to define homophobia, the most common noun was fear. The terms were grouped into two domains: negative attitude and discomfort with homosexuality. Conclusion. Fear is the most used word to describe homophobia. The terms were grouped into two domains: negative attitude and discomfort toward homosexuality.

  1. Characterizing primary human microglia: A comparative study with myeloid subsets and culture models.

    Science.gov (United States)

    Melief, J; Sneeboer, M A M; Litjens, M; Ormel, P R; Palmen, S J M C; Huitinga, I; Kahn, R S; Hol, E M; de Witte, L D

    2016-11-01

    The biology of microglia has become subject to intense study, as they are widely recognized as crucial determinants of normal and pathologic brain functioning. While they are well studied in animal models, it is still strongly debated what specifies most accurately the phenotype and functioning of microglia in the human brain. In this study, we therefore isolated microglia from postmortem human brain tissue of corpus callosum (CC) and frontal cortex (CTX). The cells were phenotyped for a panel of typical microglia markers and genes involved in myeloid cell biology. Furthermore, their response to pro- and anti-inflammatory stimuli was assessed. The microglia were compared to key human myeloid cell subsets, including monocytes, monocyte-derived macrophages and monocyte-derived dendritic cells, and several commonly used microglial cell models. Protein and mRNA expression profiles partly differed between microglia isolated from CC and frontal cortex and were clearly distinct from other myeloid subsets. Microglia responded to both pro- (LPS or poly I:C) and anti-inflammatory (IL-4 or dexamethasone) stimuli. Interestingly, pro-inflammatory responses differed between microglia and monocyte-derived macrophages, as the former responded more strongly to poly I:C and the latter more strongly to LPS. Furthermore, we defined a large phenotypic discrepancy between primary human microglia and currently used microglial cell models and cell lines. In conclusion, we further delineated the unique and specific features that discriminate human microglia from other myeloid subsets, and we show that currently used cellular models only partly reflect the phenotype of primary human microglia. GLIA 2016;64:1857-1868.

  2. Molecular characterization of various trichomonad species isolated from humans and related mammals in Indonesia.

    Science.gov (United States)

    Kamaruddin, Mudyawati; Tokoro, Masaharu; Rahman, Md Moshiur; Arayama, Shunsuke; Hidayati, Anggi P N; Syafruddin, Din; Asih, Puji B S; Yoshikawa, Hisao; Kawahara, Ei

    2014-10-01

    Trichomonad species inhabit a variety of vertebrate hosts; however, their potential zoonotic transmission has not been clearly addressed, especially with regard to human infection. Twenty-one strains of trichomonads isolated from humans (5 isolates), pigs (6 isolates), rodents (6 isolates), a water buffalo (1 isolate), a cow (1 isolate), a goat (1 isolate), and a dog (1 isolate) were collected in Indonesia and molecularly characterized. The DNA sequences of the partial 18S small subunit ribosomal RNA (rRNA) gene or 5.8S rRNA gene locus with its flanking regions (internal transcribed spacer region, ITS1 and ITS2) were identified in various trichomonads; Simplicimonas sp., Hexamastix mitis, and Hypotrichomonas sp. from rodents, and Tetratrichomonas sp. and Trichomonas sp. from pigs. All of these species were not detected in humans, whereas Pentatrichomonas hominis was identified in humans, pigs, the dog, the water buffalo, the cow, and the goat. Even when using the high-resolution gene locus of the ITS regions, all P. hominis strains were genetically identical; thus zoonotic transmission between humans and these closely related mammals may be occurring in the area investigated. The detection of Simplicimonas sp. in rodents (Rattus exulans) and P. hominis in water buffalo in this study revealed newly recognized host adaptations and suggested the existence of remaining unrevealed ranges of hosts in the trichomonad species. PMID:25352694

  3. Characterization of galactosyl glycerolipids in the HT29 human colon carcinoma cell line.

    Science.gov (United States)

    Påhlsson, P; Spitalnik, S L; Spitalnik, P F; Fantini, J; Rakotonirainy, O; Ghardashkhani, S; Lindberg, J; Konradsson, P; Larson, G

    2001-12-15

    Glycoglycerolipids constitute a family of glycolipids with apparently very restricted expression in human tissues. They have previously been detected only in the testis and the nervous system. In the present study, two glycoglycerolipids were isolated from the HT29 human colon carcinoma cell line. The glycoglycerolipids were structurally characterized as a monogalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(beta-galactosyl)-sn-glycerol) and a digalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(beta-galactosyl(1-4)alpha-galactosyl)-sn-glycerol) using NMR and mass spectrometry. This digalactosylglycerolipid has not previously been structurally characterized. When HT29 cells were allowed to differentiate into more enterocyte-like cells by culture in glucose-free medium, expression of both of these glycoglycerolipids was greatly diminished. The presence of glycoglycerolipids in a human colon carcinoma cell line indicates that expression of this family of glycolipids may not be as restricted as previously thought. Instead this class of glycolipids may serve as differentiation antigens in various normal tissues and in tumor development. The Galalpha1-4Gal epitope was previously identified as a receptor for bacterial adhesins and toxins. The finding that this epitope is also linked to a glycerolipid moiety opens up new possible roles for this carbohydrate receptor in intracellular signaling.

  4. Nanotribological and nanomechanical characterization of human hair using a nanoscratch technique

    Energy Technology Data Exchange (ETDEWEB)

    Wei Guohua [Nanotribology Laboratory for Information Storage and MEMS/NEMS, Ohio State University, 650 Ackerman Road, Suite 255, Columbus, OH 43202 (United States); Bhushan, Bharat [Nanotribology Laboratory for Information Storage and MEMS/NEMS, Ohio State University, 650 Ackerman Road, Suite 255, Columbus, OH 43202 (United States)]. E-mail: bhushan.2@osu.edu

    2006-06-15

    Human hair ({approx}50-100 {mu}m in diameter) is a nanocomposite biological fiber with well-characterized microstructures, and is of great interest for both cosmetic science and materials science. Characterization of nanotribological and nanomechanical properties of human hair including the coefficient of friction and scratch resistance is essential to develop better shampoo and conditioner products and advance biological and cosmetic science. In this paper, the coefficient of friction and scratch resistance of Caucasian and Asian hair at virgin, chemo-mechanically damaged, and conditioner-treated conditions are measured using a nanoscratch technique with a Nano Indenter II system. The scratch tests were performed on both the single cuticle cell and multiple cuticle cells of each hair sample, and the scratch wear tracks were studied using scanning electron microscopy (SEM) after the scratch tests. The effect of soaking on the coefficient of friction, scratch resistance, hardness and Young's modulus of hair surface were also studied by performing experiments on hair samples which had been soaked in de-ionized water for 5 min. The nanotribological and nanomechanical properties of human hair as a function of hair structure (hair of different ethnicity), damage, treatment and soaking are discussed.

  5. Identification and characterization of receptors for granulocyte colony-stimulating factor on human placenta and trophoblastic cells.

    OpenAIRE

    Uzumaki, H; Okabe, T.; Sasaki, N; Hagiwara, K.; Takaku, F; Tobita, M.; Yasukawa, K; Ito, S.; Umezawa, Y.

    1989-01-01

    Since radioiodination of human granulocyte colony-stimulating factor (G-CSF) is difficult, we synthesized a mutein of human G-CSF that retains full biological activity and receptor-binding capacity for at least 2 weeks after radioiodination. Receptors for human G-CSF were characterized in the plasma membrane fraction from the human term placenta (human placental membranes) and trophoblastic cells by using the 125I-labeled mutein of human G-CSF (KW-2228). The specific binding of 125I-labeled K...

  6. Characterization of the Human Insulin-induced Gene 2 (INSIG2) Promoter

    Science.gov (United States)

    Fernández-Alvarez, Ana; Soledad Alvarez, María; Cucarella, Carme; Casado, Marta

    2010-01-01

    Insulin-induced gene 2 (INSIG2) and its homolog INSIG1 encode closely related endoplasmic reticulum proteins that regulate the proteolytic activation of sterol regulatory element-binding proteins, transcription factors that activate the synthesis of cholesterol and fatty acids in animal cells. Several studies have been carried out to identify INSIG2 genetic variants associated with metabolic diseases. However, few data have been published regarding the regulation of INSIG2 gene expression. Two Insig2 transcripts have been described in rodents through the use of different promoters that produce different noncoding first exons that splice into a common second exon. Herein we report the cloning and characterization of the human INSIG2 promoter and the detection of an INSIG2-specific transcript homologous to the Insig2b mouse variant in human liver. Deletion analyses on 3 kb of 5′-flanking DNA of the human INSIG2 gene revealed the functional importance of a 350-bp region upstream of the transcription start site. Mutated analyses, chromatin immunoprecipitation assays, and RNA interference analyses unveiled the significance of an Ets-consensus motif in the proximal region and the interaction of the Ets family member SAP1a (serum response factor (SRF) accessory protein-1a) with this region of the human INSIG2 promoter. Moreover, our findings suggest that insulin activated the human INSIG2 promoter in a process mediated by phosphorylated SAP1a. Overall, these results map the functional elements in the human INSIG2 promoter sequence and suggest an unexpected regulation of INSIG2 gene expression in human liver. PMID:20145255

  7. On Defining Mass

    Science.gov (United States)

    Hecht, Eugene

    2011-01-01

    Though central to any pedagogical development of physics, the concept of mass is still not well understood. Properly defining mass has proven to be far more daunting than contemporary textbooks would have us believe. And yet today the origin of mass is one of the most aggressively pursued areas of research in all of physics. Much of the excitement…

  8. Defining Game Mechanics

    DEFF Research Database (Denmark)

    Sicart (Vila), Miguel Angel

    2008-01-01

    This article defins game mechanics in relation to rules and challenges. Game mechanics are methods invoked by agents for interacting with the game world. I apply this definition to a comparative analysis of the games Rez, Every Extend Extra and Shadow of the Colossus that will show the relevance...... of a formal definition of game mechanics. Udgivelsesdato: Dec 2008...

  9. Defining Data Science

    OpenAIRE

    Zhu, Yangyong; Xiong, Yun

    2015-01-01

    Data science is gaining more and more and widespread attention, but no consensus viewpoint on what data science is has emerged. As a new science, its objects of study and scientific issues should not be covered by established sciences. Data in cyberspace have formed what we call datanature. In the present paper, data science is defined as the science of exploring datanature.

  10. POC1A truncation mutation causes a ciliopathy in humans characterized by primordial dwarfism.

    Science.gov (United States)

    Shaheen, Ranad; Faqeih, Eissa; Shamseldin, Hanan E; Noche, Ramil R; Sunker, Asma; Alshammari, Muneera J; Al-Sheddi, Tarfa; Adly, Nouran; Al-Dosari, Mohammed S; Megason, Sean G; Al-Husain, Muneera; Al-Mohanna, Futwan; Alkuraya, Fowzan S

    2012-08-10

    Primordial dwarfism (PD) is a phenotype characterized by profound growth retardation that is prenatal in onset. Significant strides have been made in the last few years toward improved understanding of the molecular underpinning of the limited growth that characterizes the embryonic and postnatal development of PD individuals. These include impaired mitotic mechanics, abnormal IGF2 expression, perturbed DNA-damage response, defective spliceosomal machinery, and abnormal replication licensing. In three families affected by a distinct form of PD, we identified a founder truncating mutation in POC1A. This gene is one of two vertebrate paralogs of POC1, which encodes one of the most abundant proteins in the Chlamydomonas centriole proteome. Cells derived from the index individual have abnormal mitotic mechanics with multipolar spindles, in addition to clearly impaired ciliogenesis. siRNA knockdown of POC1A in fibroblast cells recapitulates this ciliogenesis defect. Our findings highlight a human ciliopathy syndrome caused by deficiency of a major centriolar protein.

  11. Detection and genetic characterization of a novel parvovirus distantly related to human bufavirus in domestic pigs.

    Science.gov (United States)

    Hargitai, Renáta; Pankovics, Péter; Kertész, Attila Mihály; Bíró, Hunor; Boros, Ákos; Phan, Tung Gia; Delwart, Eric; Reuter, Gábor

    2016-04-01

    In this study, a novel parvovirus (strain swine/Zsana3/2013/HUN, KT965075) was detected in domestic pigs and genetically characterized by viral metagenomics and PCR methods. The novel parvovirus was distantly related to the human bufaviruses and was detected in 19 (90.5 %) of the 21 and five (33.3 %) of the 15 faecal samples collected from animals with and without cases of posterior paraplegia of unknown etiology from five affected farms and one control farm in Hungary, respectively. Swine/Zsana3/2013/HUN is highly prevalent in domestic pigs and potentially represents a novel parvovirus species in the subfamily Parvovirinae.

  12. Characterization of humoral and cellular immune responses in patients with human papilloma virus

    International Nuclear Information System (INIS)

    A descriptive and cross-sectional study was carried out in 30 females infected with the human papilloma virus, attended in the office of Immunology of the Specialty Polyclinic belonging to 'Saturnino Lora' Provincial Clinical Surgical Teaching Hospital in Santiago de Cuba, from June 2009 to June 2010, in order to characterize them according to immune response. To evaluate the humoral and cellular immune response rosetting assay and quantification of immunoglobulins were used respectively. Women between 25-36 years of age (40 %) infected with this virus, especially those coming from urban areas, prevailed in the series, and a significant decrease of the cellular response as compared to the humoral response was evidenced

  13. Characterization of smooth muscle differentiation of purified human skeletal muscle-derived cells

    OpenAIRE

    Lu, Shing-Hwa; Lin, Alex TL; Chen, Kuang-Kuo; Chiang, Han Sun; Chang, Luke S.

    2010-01-01

    Abstract The purpose of this study is to characterize the smooth muscle differentiation of purified human muscle-derived cells (hMDCs). The isolation and purification of hMDCs were conducted by modified preplate technique and Dynal CD34 cell selection. Smooth muscle cell differentiation was induced by the use of smooth muscle induction medium (SMIM) and low-serum medium. The gene expressions at the mRNA and protein levels of undifferentiated and differentiated hMDCs were tested by RT-PCR, Wes...

  14. Study and structural and chemical characterization of human dental smalt by electron microscopy

    International Nuclear Information System (INIS)

    The study of human dental smalt has been subject to investigation for this methods with electron microscopy, electron diffraction, X-ray diffraction and image simulation programs have been used with the purpose to determine its chemical and structural characteristics of the organic and inorganic materials. This work has been held mainly for the characterization of hydroxyapatite (Ca)10 (PO4)6 (OH4)2, inorganic material which conforms the dental smalt in 97%, so observing its structural unity which is composed by the prisms and these by crystals and atoms. It was subsequently initiated the study of the organic material, with is precursor of itself. (Author)

  15. Characterization of two cysteine proteases secreted by Blastocystis ST7, a human intestinal parasite.

    Science.gov (United States)

    Wawrzyniak, Ivan; Texier, Catherine; Poirier, Philippe; Viscogliosi, Eric; Tan, Kevin S W; Delbac, Frédéric; El Alaoui, Hicham

    2012-09-01

    Blastocystis spp. are unicellular anaerobic intestinal parasites of both humans and animals and the most prevalent ones found in human stool samples. Their association with various gastrointestinal disorders raises the questions of its pathogenicity and of the molecular mechanisms involved. Since secreted proteases are well-known to be implicated in intestinal parasite virulence, we intended to determine whether Blastocystis spp. possess such pathogenic factors. In silico analysis of the Blastocystis subtype 7 (ST7) genome sequence highlighted 22 genes coding proteases which were predicted to be secreted. We characterized the proteolytic activities in the secretory products of Blastocystis ST7 using specific protease inhibitors. Two cysteine proteases, a cathepsin B and a legumain, were identified in the parasite culture supernatant by gelatin zymographic SDS-PAGE gel and MS/MS analysis. These proteases might act on intestinal cells and disturb gut function. This work provides serious molecular candidates to link Blastocystis spp. and intestinal disorders.

  16. Characterizing noise in nonhuman vocalizations: Acoustic analysis and human perception of barks by coyotes and dogs

    Science.gov (United States)

    Riede, Tobias; Mitchell, Brian R.; Tokuda, Isao; Owren, Michael J.

    2005-07-01

    Measuring noise as a component of mammalian vocalizations is of interest because of its potential relevance to the communicative function. However, methods for characterizing and quantifying noise are less well established than methods applicable to harmonically structured aspects of signals. Using barks of coyotes and domestic dogs, we compared six acoustic measures and studied how they are related to human perception of noisiness. Measures of harmonic-to-noise-ratio (HNR), percent voicing, and shimmer were found to be the best predictors of perceptual rating by human listeners. Both acoustics and perception indicated that noisiness was similar across coyote and dog barks, but within each species there was significant variation among the individual vocalizers. The advantages and disadvantages of the various measures are discussed.

  17. Discovery and Characterization of piRNAs in the Human Fetal Ovary

    Directory of Open Access Journals (Sweden)

    Zev Williams

    2015-10-01

    Full Text Available Piwi-interacting RNAs (piRNAs, a class of 26- to 32-nt non-coding RNAs (ncRNAs, function in germline development, transposon silencing, and epigenetic regulation. We performed deep sequencing and annotation of untreated and periodate-treated small RNA cDNA libraries from human fetal and adult germline and reference somatic tissues. This revealed abundant piRNAs originating from 150 piRNA-encoding genes, including some exhibiting gender-specific expression, in fetal ovary and adult testis—developmental periods coinciding with mitotic cell divisions expanding fetal germ cells prior to meiotic divisions. The absence of reads mapping uniquely to annotated piRNA genes demonstrated their paucity in fetal testis and adult ovary and absence in somatic tissues. We curated human piRNA-expressing regions and defined their precise borders and observed piRNA-guided cleavage of transcripts antisense to some piRNA-producing genes. This study provides insights into sex-specific mammalian piRNA expression and function and serves as a reference for human piRNA analysis and annotation.

  18. Molecular characterization of H1N1 influenza A viruses from human cases in North America

    Institute of Scientific and Technical Information of China (English)

    WU Bin; WANG ChengMin; DONG GuoYing; LUO Jing; ZHAO BaoHua; HE HongXuan

    2009-01-01

    Subtypes of H1N1 influenza virus can be found in humans in North America,while they are also associated with the infection of swine.Characterization of the genotypes of viral strains in human populations is important to understand the source and distribution of viral strains.Genomic and protein sequences of 10 isolates of the 2009 outbreak of influenza A (H1N1) virus in North America were obtained from GenBank database.To characterize the genotypes of these viruses,phylogenetic trees of genes PB2,PB1,PA,HA,NP,NA,NS and M were constructed by Phylip3.67 program and N-Linked glycosylation sites of HA,NA,PB2,NS1 and M2 proteins were analyzed online by NetNGIyc1.0 program.Phylogenetic analysis indicated that these isolates are virtually identical but may be recombinant viruses because their genomic fragments come from different viruses.The isolates also contain a characteristic lowly pathogenic amino acid motif at their HA cleavage sites (IPSIQSR↓GL),and an E residue at position 627 of the PB2 protein which shows its high affinity to humans.The homologous model of M proteins showed that the viruses had obtained the ability of anti-amantadine due to the mutation at the drug-sensitive site,while sequence analysis of NA proteins indicated that the viruses are still susceptible to the neuraminidase inhibitor drug (i.e.oseltamivir and zanamivir) because no mutations have been observed.Our results strongly suggested that the viruses responsible for the 2009 outbreaks of influenza A (H1N1) virus have the ability to cross species barriers to infect human and mammalian animals based on molecular analysis.These findings may further facilitate the therapy and prevention of possible transmission from North America to other countries.

  19. Molecular cloning and biological characterization of the human excision repair gene ERCC-3

    International Nuclear Information System (INIS)

    In this report we present the cloning, partial characterization, and preliminary studies of the biological activity of a human gene, designated ERCC-3, involved in early steps of the nucleotide excision repair pathway. The gene was cloned after genomic DNA transfection of human (HeLa) chromosomal DNA together with dominant marker pSV3gptH to the UV-sensitive, incision-defective Chinese hamster ovary (CHO) mutant 27-1. This mutant belongs to complementation group 3 of repair-deficient rodent mutants. After selection of UV-resistant primary and secondary 27-1 transformants, human sequences associated with the induced UV resistance were rescued in cosmids from the DNA of a secondary transformant by using a linked dominant marker copy and human repetitive DNA as probes. From coinheritance analysis of the ERCC-3 region in independent transformants, we deduce that the gene has a size of 35 to 45 kilobases, of which one essential segment has so far been refractory to cloning. Conserved unique human sequences hybridizing to a 3.0-kilobase mRNA were used to isolate apparently full-length cDNA clones. Upon transfection to 27-1 cells, the ERCC-3 cDNA, inserted in a mammalian expression vector, induced specific and (virtually) complete correction of the UV sensitivity and unscheduled DNA synthesis of mutants of complementation group 3 with very high efficiency. Mutant 27-1 is, unlike other mutants of complementation group 3, also very sensitive toward small alkylating agents. This unique property of the mutant is not corrected by introduction of the ERCC-3 cDNA, indicating that it may be caused by an independent second mutation in another repair function. By hybridization to DNA of a human x rodent hybrid cell panel, the ERCC-3 gene was assigned to chromosome 2, in agreement with data based on cell fusion

  20. Mechanical Characterization and Constitutive Modeling of Human Trachea: Age and Gender Dependency

    Directory of Open Access Journals (Sweden)

    Farzaneh Safshekan

    2016-06-01

    Full Text Available Tracheal disorders can usually reduce the free lumen diameter or wall stiffness, and hence limit airflow. Trachea tissue engineering seems a promising treatment for such disorders. The required mechanical compatibility of the prepared scaffold with native trachea necessitates investigation of the mechanical behavior of the human trachea. This study aimed at mechanical characterization of human tracheas and comparing the results based on age and gender. After isolating 30 human tracheas, samples of tracheal cartilage, smooth muscle, and connective tissue were subjected to uniaxial tension to obtain force-displacement curves and calculate stress-stretch data. Among several models, the Yeoh and Mooney-Rivlin hyperelastic functions were best able to describe hyperelastic behavior of all three tracheal components. The mean value of the elastic modulus of human tracheal cartilage was calculated to be 16.92 ± 8.76 MPa. An overall tracheal stiffening with age was observed, with the most considerable difference in the case of cartilage. Consistently, we noticed some histological alterations in cartilage and connective tissue with aging, which may play a role in age-related tracheal stiffening. No considerable effect of gender on the mechanical behavior of tracheal components was observed. The results of this study can be applied in the design and fabrication of trachea tissue engineering scaffolds.

  1. Molecular Characterization of Human Rotavirus from Children with Diarrhoeal Disease in Sokoto State, Nigeria

    Science.gov (United States)

    Alkali, B. R.; Daneji, A. I.; Magaji, A. A.; Bilbis, L. S.

    2016-01-01

    This study was conducted to detect and characterize prevalent human group A rotavirus strains from 200 diarrheic children in Sokoto, Nigeria, by ELISA, monoclonal antibody (Mab) serotyping and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) techniques. Rotavirus was detected in 25.5% of the children. The G-serotypes observed in circulation were G4: 16 (59.3%), G1: 4 (14.8%), G2: 3 (11.1%), G3: 3 (11.1%), and G12: 1 (3.7%). The monoclonal antibody (Mab) serotyping detected G1 and G3 but did not detect G4 and G2 serotypes. The Mab typing of the G1 and G3 serotypes was consistent with the result of the RT-PCR. The VP4 genotypes detected were P[6] 3 (13%), P[8] 11 (47.8%), and the rare human P genotype (P[9]), found in 9 patients (39.1%). Nine strains identified with the common G and P combinations were G4 P[8] 5 (56%), G4 P[6] 1 (11%), G1 P[8] 2 (22%), and G3 P[8] 1 (11%), while seven strains with unusual combinations or rare G or P genotypes identified were G12 P[8] 1 (14%), G2 P[8] 2 (29%), and G4 P[9] 4 (57%). To our knowledge this is the first molecular study of human rotavirus and report of rare human G and P serotypes in Sokoto State. PMID:27051531

  2. Characterization of the Inflammasome in Human Kupffer Cells in Response to Synthetic Agonists and Pathogens.

    Science.gov (United States)

    Zannetti, Claudia; Roblot, Guillaume; Charrier, Emily; Ainouze, Michelle; Tout, Issam; Briat, François; Isorce, Nathalie; Faure-Dupuy, Suzanne; Michelet, Maud; Marotel, Marie; Kati, Semra; Schulz, Thomas F; Rivoire, Michel; Traverse-Glehen, Alexandra; Luangsay, Souphalone; Alatiff, Omran; Henry, Thomas; Walzer, Thierry; Durantel, David; Hasan, Uzma

    2016-07-01

    The liver is the largest gland in the human body and functions as an innate immune organ. Liver macrophages called Kupffer cells (KC) constitute the largest group of macrophages in the human body. Innate immune responses involving KC represent the first line of defense against pathogens in the liver. Human monocyte-derived macrophages have been used to characterize inflammasome responses that lead to the release of the proinflammatory cytokines IL-1β and IL-18, but it has not yet been determined whether human KC contain functional inflammasomes. We show, to our knowledge for the first time, that KC express genes and proteins that make up several different inflammasome complexes. Moreover, activation of KC in response to the absent in melanoma 2 (AIM2) inflammasome led to the production of IL-1β and IL-18, which activated IL-8 transcription and hepatic NK cell activity, respectively. Other inflammasome responses were also activated in response to selected bacteria and viruses. However, hepatitis B virus inhibited the AIM2 inflammasome by reducing the mRNA stability of IFN regulatory factor 7, which regulated AIM2 transcription. These data demonstrate the production of IL-1β and IL-18 in KC, suggesting that KC contain functional inflammasomes that could be important players in the innate immune response following certain infections of the liver. We think our findings could potentially aid therapeutic approaches against chronic liver diseases that activate the inflammasome. PMID:27226092

  3. Derivation and characterization of human embryonic stem cell lines from the Chinese population

    Institute of Scientific and Technical Information of China (English)

    Zhao Wu; Huimin Dai; Lei Qian; Qing Tian; Lei Xiao; Xiaojun Tan; Hui Li; Lingjun Rao; Lixiazi He; Lei Bao; Jing Liao; Chun Cui; Zhenyu Zuo; Qiao Li

    2011-01-01

    Human embryonic stem cells (hESCs) can self-renew indefinitely and differentiate into all cell types in the human body. Therefore, they are valuable in regenerative medicine, human developmental biology and drug discovery. A number of hESC lines have been derived from the Chinese population,but limited of them are available for research purposes. Here we report the derivation and characterization of two hESC lines derived from human blastocysts of Chinese origin. These hESCs express alkaline phosphatase and hESC-specific markers, including Oct4, Nanog, SSEA-3, SSEA-4,TRA-1-60 and TRA-1-81. They also have high levels of telomerase activity and normal karyotypes. These cells can form embryoid body in vitro and can be differentiated into all three germ layers in vivo by teratoma formation. The newly established hESCs will be distributed for research purposes.The availability of hESC lines from the Chinese population will facilitate studies on the differences in hESCs from different ethnic groups.

  4. Texture characterization for joint compression and classification based on human perception in the wavelet domain.

    Science.gov (United States)

    Fahmy, Gamal; Black, John; Panchanathan, Sethuraman

    2006-06-01

    Today's multimedia applications demand sophisticated compression and classification techniques in order to store, transmit, and retrieve audio-visual information efficiently. Over the last decade, perceptually based image compression methods have been gaining importance. These methods take into account the abilities (and the limitations) of human visual perception (HVP) when performing compression. The upcoming MPEG 7 standard also addresses the need for succinct classification and indexing of visual content for efficient retrieval. However, there has been no research that has attempted to exploit the characteristics of the human visual system to perform both compression and classification jointly. One area of HVP that has unexplored potential for joint compression and classification is spatial frequency perception. Spatial frequency content that is perceived by humans can be characterized in terms of three parameters, which are: 1) magnitude; 2) phase; and 3) orientation. While the magnitude of spatial frequency content has been exploited in several existing image compression techniques, the novel contribution of this paper is its focus on the use of phase coherence for joint compression and classification in the wavelet domain. Specifically, this paper describes a human visual system-based method for measuring the degree to which an image contains coherent (perceptible) phase information, and then exploits that information to provide joint compression and classification. Simulation results that demonstrate the efficiency of this method are presented. PMID:16764265

  5. Defining Legal Moralism

    DEFF Research Database (Denmark)

    Thaysen, Jens Damgaard

    2015-01-01

    This paper discusses how legal moralism should be defined. It is argued that legal moralism should be defined as the position that “For any X, it is always a pro tanto reason for justifiably imposing legal regulation on X that X is morally wrong (where “morally wrong” is not conceptually equivalent...... to “harmful”)”. Furthermore, a distinction between six types of legal moralism is made. The six types are grouped according to whether they are concerned with the enforcement of positive or critical morality, and whether they are concerned with criminalising, legally restricting, or refraining from legally...... protecting morally wrong behaviour. This is interesting because not all types of legal moralism are equally vulnerable to the different critiques of legal moralism that have been put forth. Indeed, I show that some interesting types of legal moralism have not been criticised at all....

  6. DEFINING SUCCESS IN PROJECTS

    Directory of Open Access Journals (Sweden)

    José Rodrigues de Farias Filho

    2011-05-01

    Full Text Available The Project Management Discipline has been widely used in the last years for companies around the world and these companies have been investing large amounts on surveys, training and consulting in order to get benefits for the organizations that need to create competitive advantage in the high competitive Market and to achieve their business strategy goals. Studies from the major world authors show many ways to define success at the organizations. In Brazil, the Benchmarking Study in GP from the PMI Chapters in 2009, show most studied companies don’t have a process do assess whether they are achieving the expected business goals with their investments in Project Management. This article goal is to demonstrate many ways to define success in project, which will facilitate the process to assess whether the companies are achieving these expected goals. The methodology used was a literature review, collecting publications, textbooks and documents from subject-matter-experts.

  7. Software Defined Networking

    OpenAIRE

    Roncero Hervás, Óscar

    2014-01-01

    Software Defined Networks (SDN) is a paradigm in which routing decisions are taken by a control layer. In contrast to conventional network structures, the control plane and forwarding plane are separated and communicate through standard protocols like OpenFlow. Historically, network management was based on a layered approach, each one isolated from the others. SDN proposes a radically different approach by bringing together the management of all these layers into a single controller. It is th...

  8. SDN : Software defined networks

    OpenAIRE

    Wiklund, Petter

    2014-01-01

    This report is a specialization in Software defined networking. SDN really comes to revolutionize the industry and it’s under constant development. But is the technology ready to be launched into operation yet? The report would initially involve a number of problems that today's network technology is facing. It then follows a deeper description of what this software-based networking technology really is and how it works. Further, the technique is being tested in a lab assignment, using a prog...

  9. Defining local food

    DEFF Research Database (Denmark)

    Eriksen, Safania Normann

    2013-01-01

    Despite evolving local food research, there is no consistent definition of “local food.” Various understandings are utilized, which have resulted in a diverse landscape of meaning. The main purpose of this paper is to examine how researchers within the local food systems literature define local...... food, and how these definitions can be used as a starting point to identify a new taxonomy of local food based on three domains of proximity....

  10. Solid-state NMR, electrophysiology and molecular dynamics characterization of human VDAC2

    Energy Technology Data Exchange (ETDEWEB)

    Gattin, Zrinka; Schneider, Robert; Laukat, Yvonne; Giller, Karin [Max Planck Institute for Biophysical Chemistry (Germany); Maier, Elke [Theodor-Boveri-Institut (Biozentrum) der Universität Würzburg, Lehrstuhl für Biotechnologie (Germany); Zweckstetter, Markus; Griesinger, Christian [Max Planck Institute for Biophysical Chemistry (Germany); Benz, Roland [Theodor-Boveri-Institut (Biozentrum) der Universität Würzburg, Lehrstuhl für Biotechnologie (Germany); Becker, Stefan; Lange, Adam, E-mail: alange@fmp-berlin.de [Max Planck Institute for Biophysical Chemistry (Germany)

    2015-04-15

    The voltage-dependent anion channel (VDAC) is the most abundant protein of the outer mitochondrial membrane and constitutes the major pathway for the transport of ADP, ATP, and other metabolites. In this multidisciplinary study we combined solid-state NMR, electrophysiology, and molecular dynamics simulations, to study the structure of the human VDAC isoform 2 in a lipid bilayer environment. We find that the structure of hVDAC2 is similar to the structure of hVDAC1, in line with recent investigations on zfVDAC2. However, hVDAC2 appears to exhibit an increased conformational heterogeneity compared to hVDAC1 which is reflected in broader solid-state NMR spectra and less defined electrophysiological profiles.

  11. Characterizing ncRNAs in human pathogenic protists using high-throughput sequencing technology

    Directory of Open Access Journals (Sweden)

    Lesley Joan Collins

    2011-12-01

    Full Text Available ncRNAs are key genes in many human diseases including cancer and viral infection, as well as providing critical functions in pathogenic organisms such as fungi, bacteria, viruses and protists. Until now the identification and characterization of ncRNAs associated with disease has been slow or inaccurate requiring many years of testing to understand complicated RNA and protein gene relationships. High-throughput sequencing now offers the opportunity to characterize miRNAs, siRNAs, snoRNAs and long ncRNAs on a genomic scale making it faster and easier to clarify how these ncRNAs contribute to the disease state. However, this technology is still relatively new, and ncRNA discovery is not an application of high priority for streamlined bioinformatics. Here we summarize background concepts and practical approaches for ncRNA analysis using high-throughput sequencing, and how it relates to understanding human disease. As a case study, we focus on the parasitic protists Giardia lamblia and Trichomonas vaginalis, where large evolutionary distance has meant difficulties in comparing ncRNAs with those from model eukaryotes. A combination of biological, computational and sequencing approaches has enabled easier classification of ncRNA classes such as snoRNAs, but has also aided the identification of novel classes. It is hoped that a higher level of understanding of ncRNA expression and interaction may aid in the development of less harsh treatment for protist-based diseases.

  12. Phenotypic and functional characterization of human memory T cell responses to Burkholderia pseudomallei.

    Directory of Open Access Journals (Sweden)

    Patcharaporn Tippayawat

    Full Text Available BACKGROUND: Infection with the Gram-negative bacterium Burkholderia pseudomallei is an important cause of community-acquired lethal sepsis in endemic regions in southeast Asia and northern Australia and is increasingly reported in other tropical areas. In animal models, production of interferon-gamma (IFN-gamma is critical for resistance, but in humans the characteristics of IFN-gamma production and the bacterial antigens that are recognized by the cell-mediated immune response have not been defined. METHODS: Peripheral blood from 133 healthy individuals who lived in the endemic area and had no history of melioidosis, 60 patients who had recovered from melioidosis, and 31 other patient control subjects were stimulated by whole bacteria or purified bacterial proteins in vitro, and IFN-gamma responses were analyzed by ELISPOT and flow cytometry. FINDINGS: B. pseudomallei was a potent activator of human peripheral blood NK cells for innate production of IFN-gamma. In addition, healthy individuals with serological evidence of exposure to B. pseudomallei and patients recovered from active melioidosis developed CD4(+ (and CD8(+ T cells that recognized whole bacteria and purified proteins LolC, OppA, and PotF, members of the B. pseudomallei ABC transporter family. This response was primarily mediated by terminally differentiated T cells of the effector-memory (T(EMRA phenotype and correlated with the titer of anti-B. pseudomallei antibodies in the serum. CONCLUSIONS: Individuals living in a melioidosis-endemic region show clear evidence of T cell priming for the ability to make IFN-gamma that correlates with their serological status. The ability to detect T cell responses to defined B. pseudomallei proteins in large numbers of individuals now provides the opportunity to screen candidate antigens for inclusion in protein or polysaccharide-conjugate subunit vaccines against this important but neglected disease.

  13. Defining the medical sphere

    NARCIS (Netherlands)

    Trappenburg, M.J.

    1997-01-01

    Part of the debate on cost containment in health care systems can be characterized as applied political philosophy. Three philosophical directions can be traced. (1) Norman Daniels and Ronald Dworkin advocate a health care distributional system based on a Rawls' A Theory of Justice. (2) Tristram Eng

  14. Biophysical characterization of G protein ectodomain of group B human respiratory syncytial virus from E. coli.

    Science.gov (United States)

    Khan, Wajihul Hasan; Srungaram, V L N Raghuram; Islam, Asimul; Beg, Ilyas; Haider, Md Shakir H; Ahmad, Faizan; Broor, Shobha; Parveen, Shama

    2016-07-01

    Human respiratory syncytial virus (hRSV) is an important pathogen of acute respiratory tract infection. The G protein of hRSV is a transmembrane glycoprotein that is a neutralizing antigen and is thus a vaccine candidate. In this study, synthetic codon optimized ectodomain G protein [G(ΔTM)] of BA genotype of group B hRSV was cloned, expressed, and characterized using biophysical techniques. The molar absorption coefficient and mean residue ellipticity at 222 nm ([θ]222) of G (ΔTM) was found to be 7950 M(-1) cm(-1) and -19701.7 deg cm(2) dmol(-1) respectively. It was concluded that G(ΔTM) mainly consist of α-helix (74.9%) with some amount of β-sheet (4%). The protein was stable up to 85°C without any transition curve. However, heat-induced denaturation of G(ΔTM) resulted in total loss of β-sheet whereas not much change was observed in the α-helix part of the secondary structure. It was concluded that G(ΔTM) is an α-helical protein and it is highly stable at high temperature, but could be easily denatured using high concentrations of GdmCl/urea or acidic condition. This is the first investigation of cloning, expression, and characterization of G(ΔTM) of BA viruses from India. Structural characterization of G protein will assist in drug designing and vaccine development for hRSV.

  15. Characterization of human arterial tissue affected by atherosclerosis using multimodal nonlinear optical microscopy

    Science.gov (United States)

    Baria, Enrico; Cicchi, Riccardo; Rotellini, Matteo; Nesi, Gabriella; Massi, Daniela; Pavone, Francesco S.

    2016-03-01

    Atherosclerosis is a widespread cardiovascular disease caused by the deposition of lipids (such as cholesterol and triglycerides) on the inner arterial wall. The rupture of an atherosclerotic plaque, resulting in a thrombus, is one of the leading causes of death in the Western World. Preventive assessment of plaque vulnerability is therefore extremely important and can be performed by studying collagen organization and lipid composition in atherosclerotic arterial tissues. Routinely used diagnostic methods, such as histopathological examination, are limited to morphological analysis of the examined tissues, whereas an exhaustive characterization requires immune-histochemical examination and a morpho-functional approach. Instead, a label-free and non-invasive alternative is provided by nonlinear microscopy. In this study, we combined SHG and FLIM microscopy in order to characterize collagen organization and lipids in human carotid ex vivo tissues affected by atherosclerosis. SHG and TPF images, acquired from different regions within atherosclerotic plaques, were processed through image pattern analysis methods (FFT, GLCM). The resulting information on collagen and cholesterol distribution and anisotropy, combined with collagen and lipids fluorescence lifetime measured from FLIM images, allowed characterization of carotid samples and discrimination of different tissue regions. The presented method can be applied for automated classification of atherosclerotic lesions and plaque vulnerability. Moreover, it lays the foundation for a potential in vivo diagnostic tool to be used in clinical setting.

  16. Defining Business Network

    Directory of Open Access Journals (Sweden)

    Raffaele Trequattrini

    2012-02-01

    Full Text Available The purpose of the present scientific contribution is to investigate from the business economics standpoing the emerging phenomenon of company networks. In particular, through the analysis of the theory of networks will be proposed the principal categories of business networks, and even before this the concept of the network will be defined. The proposed research, qualitatively, represents the point of departure for the study of the network phenomenon in light of the current economic phase termed “economy of knowledge”. Moreover, the research questions are the following: From where does the theory of networks arise? Do company networks consider themselves equal to knowledge networks?

  17. Defining Business Network

    Directory of Open Access Journals (Sweden)

    Raffaele Trequattrini

    2012-02-01

    Full Text Available The purpose of the present scientific contribution is to investigate from the businesseconomics standpoing the emerging phenomenon of company networks. In particular,through the analysis of the theory of networks will be proposed the principal categories ofbusiness networks, and even before this the concept of the network will be defined. Theproposed research, qualitatively, represents the point of departure for the study of thenetwork phenomenon in light of the current economic phase termed “economy of knowledge”.Moreover, the research questions are the following: From where does the theory of networksarise? Do company networks consider themselves equal to knowledge networks?

  18. Comprehensive transcriptomic and proteomic characterization of human mesenchymal stem cells reveals source specific cellular markers.

    Science.gov (United States)

    Billing, Anja M; Ben Hamidane, Hisham; Dib, Shaima S; Cotton, Richard J; Bhagwat, Aditya M; Kumar, Pankaj; Hayat, Shahina; Yousri, Noha A; Goswami, Neha; Suhre, Karsten; Rafii, Arash; Graumann, Johannes

    2016-01-01

    Mesenchymal stem cells (MSC) are multipotent cells with great potential in therapy, reflected by more than 500 MSC-based clinical trials registered with the NIH. MSC are derived from multiple tissues but require invasive harvesting and imply donor-to-donor variability. Embryonic stem cell-derived MSC (ESC-MSC) may provide an alternative, but how similar they are to ex vivo MSC is unknown. Here we performed an in depth characterization of human ESC-MSC, comparing them to human bone marrow-derived MSC (BM-MSC) as well as human embryonic stem cells (hESC) by transcriptomics (RNA-seq) and quantitative proteomics (nanoLC-MS/MS using SILAC). Data integration highlighted and validated a central role of vesicle-mediated transport and exosomes in MSC biology and also demonstrated, through enrichment analysis, their versatility and broad application potential. Particular emphasis was placed on comparing profiles between ESC-MSC and BM-MSC and assessing their equivalency. Data presented here shows that differences between ESC-MSC and BM-MSC are similar in magnitude to those reported for MSC of different origin and the former may thus represent an alternative source for therapeutic applications. Finally, we report an unprecedented coverage of MSC CD markers, as well as membrane associated proteins which may benefit immunofluorescence-based applications and contribute to a refined molecular description of MSC. PMID:26857143

  19. Characterization and Quantification of Innate Lymphoid Cell Subsets in Human Lung.

    Directory of Open Access Journals (Sweden)

    Katrien C De Grove

    Full Text Available Innate lymphoid cells (ILC are a new family of innate immune cells that have emerged as important regulators of tissue homeostasis and inflammation. However, limited data are available concerning the relative abundance and characteristics of ILC in the human lung.The aim of this study was to characterize and enumerate the different ILC subsets in human lung by multi-color flow cytometry.Within the CD45+ Lin- CD127+ pulmonary ILC population, we identified group 1 (ILC1, group 2 (ILC2 and group 3 (ILC3 innate lymphoid cells using specific surface markers (i.e. IL12Rβ2, CRTH2 and CD117 respectively and key transcription factors (i.e. T-bet, GATA-3 and RORγT respectively. Based on the presence of NKp44, ILC3 were further subdivided in natural cytotoxicity receptor (NCR+ and NCR- ILC3. In addition, we demonstrated the production of signature cytokines IFN-γ, IL-5, IL-17A, IL-22 and GM-CSF in the pulmonary ILC population. Interestingly, we observed a tendency to a higher frequency of NCR- ILC3 in lungs of patients with chronic obstructive pulmonary disease (COPD compared with controls.We show that the three main ILC subsets are present in human lung. Importantly, the relative abundance of ILC subsets tended to change in COPD patients in comparison to control individuals.

  20. Characterization of human thermal comfort in urban areas of brazilian semiarid

    Directory of Open Access Journals (Sweden)

    Pedro Vieira de Azevedo

    2015-12-01

    Full Text Available ABSTRACT Experimental studies were conducted aiming to characterize the thermal conditions in urban areas of the Brazilian semiarid for identifying the level of human thermal comfort in urban areas of the municipalities of Mossoró-RN, Serra Talhada-PE, Petrolina-PE and Juazeiro-BA. The results showed that the constituent elements of urban areas contribute to human thermal discomfort index (DIT. Both the wet and dry seasons showed up an uncomfortable condition in Mossoró on May 15th and on 23rd, 2008 (rural area and on May 25rd, 2008 (urban area. By applying Thom's equation for obtaining the DIT, it was observed that the urban areas reported thermal comfort conditions always inferior to those observed in the rural areas. The results indicate conditions of partial comfort for the all areas surveyed. However, the incidence of human thermal discomfort related to the minimum, average and maximum air temperatures are, probably associated to the formation of urban heat islands. In addition, the afforestation is an effective mechanism of mitigating the adverse effects of these structures with better quality of life to the population. Therefore, it can be stated that the Brazilian semiarid region has specific urban climate, which is influenced by the design characteristics of the urban structure and buildings.

  1. Production and Characterization of Monoclonal Antibodies against Human Nuclear Protein FAM76B.

    Directory of Open Access Journals (Sweden)

    Xiaojing Zheng

    Full Text Available Human FAM76B (hFAM76B is a 39 kDa protein that contains homopolymeric histidine tracts, a targeting signal for nuclear speckles. FAM76B is highly conserved among different species, suggesting that it may play an important physiological role in normal cellular functions. However, a lack of appropriate tools has hampered study of this potentially important protein. To facilitate research into the biological function(s of FAM76B, murine monoclonal antibodies (MAbs against hFAM76B were generated by using purified, prokaryotically expressed hFAM76B protein. Six strains of MAbs specific for hFAM76B were obtained and characterized. The specificity of MAbs was validated by using FAM76B-/- HEK 293 cell line. Double immunofluorescence followed by laser confocal microscopy confirmed the nuclear speckle localization of hFAM76B, and the specific domains recognized by different MAbs were further elucidated by Western blot. Due to the high conservation of protein sequences between mouse and human FAM76B, MAbs against hFAM76B were shown to react with mouse FAM76B (mFAM76B specifically. Lastly, FAM76B was found to be expressed in the normal tissues of most human organs, though to different extents. The MAbs produced in this study should provide a useful tool for investigating the biological function(s of FAM76B.

  2. Isolation and characterization of the human parathyroid hormone-like peptide gene

    International Nuclear Information System (INIS)

    A parathyroid hormone-like peptide (PTH-LP) has recently been identified in human tumors associated with the syndrome of humoral hypercalcemia of malignancy. The peptide appears to be encoded by a single-copy gene that gives rise to multiple mRNAs that are heterogeneous at both their 5' and their 3' ends. Alternative RNA splicing is responsible for the 3' heterogeneity and results in mRNAs encoding three different peptides, each with a unique C terminus. The authors have isolated and characterized the human PTHLP gene. The gene is a complex transcriptional unit spanning more than 12 kilobases of DNA and containing six exons. Two 5' exons encode distinct 5' untranslated regions and are separated by a putative promoter element, indicating that the gene either has two promoters or is alternatively spliced from a single promoter upstream of the first exon. The middle portion of the PTHLP gene, comprising exons 2-4, has an organizational pattern of introns and exons identical to that of the parathyroid hormone gene, consistent with a common ancestral origin of these two genes. Exon 4 of the PTHLP gene encodes the region common to all three peptides and the C terminus of the shortest peptide, and exons 5 and 6 encode the unique C termini of the other two peptides. Northern analysis of mRNAs from four human tumors of different histological types reveals the preferential use of 3' splicing patterns of individual tumors

  3. Establishment of a novel human medulloblastoma cell line characterized by highly aggressive stem-like cells.

    Science.gov (United States)

    Silva, Patrícia Benites Gonçalves da; Rodini, Carolina Oliveira; Kaid, Carolini; Nakahata, Adriana Miti; Pereira, Márcia Cristina Leite; Matushita, Hamilton; Costa, Silvia Souza da; Okamoto, Oswaldo Keith

    2016-08-01

    Medulloblastoma is a highly aggressive brain tumor and one of the leading causes of morbidity and mortality related to childhood cancer. These tumors display differential ability to metastasize and respond to treatment, which reflects their high degree of heterogeneity at the genetic and molecular levels. Such heterogeneity of medulloblastoma brings an additional challenge to the understanding of its physiopathology and impacts the development of new therapeutic strategies. This translational effort has been the focus of most pre-clinical studies which invariably employ experimental models using human tumor cell lines. Nonetheless, compared to other cancers, relatively few cell lines of human medulloblastoma are available in central repositories, partly due to the rarity of these tumors and to the intrinsic difficulties in establishing continuous cell lines from pediatric brain tumors. Here, we report the establishment of a new human medulloblastoma cell line which, in comparison with the commonly used and well-established cell line Daoy, is characterized by enhanced proliferation and invasion capabilities, stem cell properties, increased chemoresistance, tumorigenicity in an orthotopic metastatic model, replication of original medulloblastoma behavior in vivo, strong chromosome structural instability and deregulation of genes involved in neural development. These features are advantageous for designing biologically relevant experimental models in clinically oriented studies, making this novel cell line, named USP-13-Med, instrumental for the study of medulloblastoma biology and treatment. PMID:26358937

  4. Software-Defined Cluster

    Institute of Scientific and Technical Information of China (English)

    聂华; 杨晓君; 刘淘英

    2015-01-01

    The cluster architecture has played an important role in high-end computing for the past 20 years. With the advent of Internet services, big data, and cloud computing, traditional clusters face three challenges: 1) providing flexible system balance among computing, memory, and I/O capabilities;2) reducing resource pooling overheads;and 3) addressing low performance-power efficiency. This position paper proposes a software-defined cluster (SDC) architecture to deal with these challenges. The SDC architecture inherits two features of traditional cluster: its architecture is multicomputer and it has loosely-coupled interconnect. SDC provides two new mechanisms: global I/O space (GIO) and hardware-supported native access (HNA) to remote devices. Application software can define a virtual cluster best suited to its needs from resources pools provided by a physical cluster, and traditional cluster ecosystems need no modification. We also discuss a prototype design and implementation of a 32-processor cloud server utilizing the SDC architecture.

  5. Defining Communication Competence

    Science.gov (United States)

    Zascerinska, Jelena

    2008-01-01

    In order to become "the most competitive and dynamic knowledge-based economy in the world capable of sustainable economic growth with more and better jobs and greater social cohesion" the European Union realizes its people as the most important asset. Moreover, every human becomes a value for the whole society. It makes significant to take into…

  6. Defining the medical sphere

    OpenAIRE

    Trappenburg, M.J.

    1997-01-01

    Part of the debate on cost containment in health care systems can be characterized as applied political philosophy. Three philosophical directions can be traced. (1) Norman Daniels and Ronald Dworkin advocate a health care distributional system based on a Rawls' A Theory of Justice. (2) Tristram Engelhardt defends a market based approach, reminiscent of Nozick's Anarchy, State, and Utopia. (3) Daniel Callahan advocates a communitarian strategy which resembles the work of Christopher Lasch and...

  7. Production and characterization of antibodies against irradiated human erythrocytes membrane proteins

    International Nuclear Information System (INIS)

    Gamma irradiation affects people in several situations, with few if any sensitive biological assay of its action. Nucleic acids and proteins are affected by radiation, but only the former was used in most dosimetric techniques. The irradiation of proteins promotes structural modifications attributed to free radicals from water radiolysis. Theoretically, antibodies induced by irradiated proteins could recognize these radical-related new epitopes, allowing their use as a probe. Human erythrocyte membrane proteins (HEMP), few and well defined molecules, are certainly exposed to radiation, being the ideal target. With this rationale, we study the production of antibodies in mice immunized with 60 Co irradiated HEMPs. Menbranes from hypotonic lysis with differential centrifugation of A+ erythrocytes, were irradiated in a Gammacell 220 with 400, 800 and 1600 Gy, and used as immunogen for Balb/c mice, after SDS-PAGE. Irradiated HEMP induced antibodies recognize only irradiated human erthrocytes in an intact cell indirect immunofluorescence assay (ICIIFA). When used in Wester-blot against non-irradiated HEMPs, those sera recognize most proteins, suggesting a pool of abs directed both to native, as detected by Western Blot, or irradiated, as detected by ICIFA, HEMPs. Those data confirmed our assumptions, allowing the use of those abs in the search for a method of biological dosimetry. (author). 18 refs., 3 figs

  8. Isolation and characterization of cytotoxic effector cells and antibody producing cells from human intestine.

    Science.gov (United States)

    MacDermott, R P

    1985-01-01

    elevated spontaneous secretion of immunoglobulins in general and IgA in particular. Pure human bone marrow MNC exhibited high spontaneous secretion of IgA, and modest amounts of IgG and normal IgM secreting. The addition of PWM to cultures exhibiting high spontaneous synthesis and secretion of immunoglobulins resulted not in further enhancement but in suppression of antibody secretion. The characterization of types of IgA secreted by human IMC revealed that normal human bone marrow secretes almost exclusively monomeric IgA, while control human intestine secretes predominantly dimeric IgA. IMC from patients with CD and non-involved UC specimens also secreted predominantly dimeric IgA.(ABSTRACT TRUNCATED AT 400 WORDS)

  9. Human papillomaviruses associated with epidermodysplasia verruciformis. II. Molecular cloning and biochemical characterization of human papillomavirus 3a, 8, 10, and 12 genomes.

    OpenAIRE

    Kremsdorf, D; Jablonska, S; Favre, M; Orth, G

    1983-01-01

    The DNAs of four human papillomaviruses (HPVs) that were found in the benign lesions of three patients suffering from epidermodysplasia verruciformis have been characterized. The flat wart-like lesions and the macular lesions of patient 1 contained two viruses, HPV-3a and HPV-8, respectively, whose genomes had previously been only partially characterized. The flat wart-like lesions of patient 2 and the macular lesions of patient 3 each contained a virus previously considered as belonging to t...

  10. CHARACTERIZATION AND CHROMOSOMAL ASSIGNMENT OF YEAST ARTIFICIAL CHROMOSOMES CONTAINING HUMAN 3P13-P21-SPECIFIC SEQUENCE-TAGGED SITES

    NARCIS (Netherlands)

    MICHAELIS, SC; BARDENHEUER, W; LUX, A; SCHRAMM, A; GOCKEL, A; SIEBERT, R; WILLERS, C; SCHMIDTKE, K; TODT, B; VANDERHOUT, AH; BUYS, CHCM; HEPPELLPARTON, AC; RABBITTS, PH; UNGAR, S; SMITH, D; LEPASLIER, D; COHEN, D; OPALKA, B; SCHUTTE, J

    1995-01-01

    Human chromosomal region 3p12-p23 is proposed to harbor at least three tumor suppressor genes involved in the development of lung cancer, renal cell carcinoma, and other neoplasias. In order to identify one of these genes we defined sequence tagged sites (STSs) specific for 3p13-p24.2 by analyzing a

  11. Hematopoietic stem cell cytokines and fibroblast growth factor-2 stimulate human endothelial cell-pericyte tube co-assembly in 3D fibrin matrices under serum-free defined conditions.

    Directory of Open Access Journals (Sweden)

    Annie O Smith

    Full Text Available We describe a novel 3D fibrin matrix model using recombinant hematopoietic stem cell cytokines under serum-free defined conditions which promotes the assembly of human endothelial cell (EC tubes with co-associated pericytes. Individual ECs and pericytes are randomly mixed together and EC tubes form that is accompanied by pericyte recruitment to the EC tube abluminal surface over a 3-5 day period. These morphogenic processes are stimulated by a combination of the hematopoietic stem cell cytokines, stem cell factor, interleukin-3, stromal derived factor-1α, and Flt-3 ligand which are added in conjunction with fibroblast growth factor (FGF-2 into the fibrin matrix. In contrast, this tube morphogenic response does not occur under serum-free defined conditions when VEGF and FGF-2 are added together in the fibrin matrices. We recently demonstrated that VEGF and FGF-2 are able to prime EC tube morphogenic responses (i.e. added overnight prior to the morphogenic assay to hematopoietic stem cell cytokines in collagen matrices and, interestingly, they also prime EC tube morphogenesis in 3D fibrin matrices. EC-pericyte interactions in 3D fibrin matrices leads to marked vascular basement membrane assembly as demonstrated using immunofluorescence and transmission electron microscopy. Furthermore, we show that hematopoietic stem cell cytokines and pericytes stimulate EC sprouting in fibrin matrices in a manner dependent on the α5β1 integrin. This novel co-culture system, under serum-free defined conditions, allows for a molecular analysis of EC tube assembly, pericyte recruitment and maturation events in a critical ECM environment (i.e. fibrin matrices that regulates angiogenic events in postnatal life.

  12. Synthesis and characterization of Pt-Pd nanoparticles with core-shell morphology: Nucleation and overgrowth of the Pd shells on the as-prepared and defined Pt seeds

    Energy Technology Data Exchange (ETDEWEB)

    Long, Nguyen Viet, E-mail: nguyenvietlong@yahoo.com [Department of Materials Scienceand Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555 (Japan); Posts and Telecommunications Institute of Technology, km 10 Nguyen Trai, Hanoi (Viet Nam); Laboratory for Nanotechnology, Vietnam National University at Ho Chi Minh, Linh Trung, Thu Duc, Ho Chi Minh (Viet Nam); Department of Molecular and Material Sciences, Interdisciplinary Graduate School of Engineering Sciences, 6-1 Kasugakouen, Kasuga, Fukuoka 861-8580 (Japan); Hien, Tong Duy [Laboratory for Nanotechnology, Vietnam National University at Ho Chi Minh, Linh Trung, Thu Duc, Ho Chi Minh (Viet Nam); Asaka, Toru [Department of Materials Scienceand Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555 (Japan); Ohtaki, Michitaka [Department of Molecular and Material Sciences, Interdisciplinary Graduate School of Engineering Sciences, 6-1 Kasugakouen, Kasuga, Fukuoka 861-8580 (Japan); Nogami, Masayuki, E-mail: nogami@nitech.ac.jp [Department of Materials Scienceand Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555 (Japan)

    2011-07-21

    Highlights: > The Pt-Pd core-shell nanoparticles based on the as-prepared Pt cores are synthesized. > Not only the Pt-Pd core-shell nanoparticles are formed, but also the separate formation of Pd nanoparticles as well. > The Pt cores without the morphological changes are protected by the Pd-shell overgrowths. > There are the co-existence of the layer-by-layer and island-on-wetting-layer growth modes of the Pd shells and the latter becomes the favorable overgrowth in the formation of core-shell structures. - Abstract: In the present research, Pt-Pd core-shell nanoparticles based on the as-prepared and defined Pt-seed cores with well-controlled size and morphology were synthesized. Their characterizations were investigated by using UV-vis spectroscopy, transmission electron microscopy (TEM), and high resolution (HR)TEM measurements. The high resolution elemental mappings were performed in the operation of high angle annular dark field (HAADF) in conjunction with scanning (S)TEM mode and X-ray energy dispersive spectroscopy (XEDS). It is found that not only the Pt-Pd core-shell nanoparticles were formed, but also the nucleation, growth, and the separate formation of single Pd nanoparticles as well. Interestingly, the as-prepared Pt cores without the morphological changes were protected by the overgrowths of the Pd shells during the successive reduction of sodium tetrachloropalladate (II) hydrate. There were the co-existence of the Frank-van der Merwe (FM) layer-by-layer and Stranski-Krastanov (SK) island-on-wetting-layer growth modes of the Pd shells on the as-prepared Pt cores. It is predicted that the SK growth became the favorable growth mode in the formation of the Pd shells in the formation Pt-Pd core-shell nanoparticles.

  13. Defining critical thoughts.

    Science.gov (United States)

    Lovatt, Abbie

    2014-05-01

    Nursing education has long struggled to define critical thinking and explain how the process of critical thinking fits into the context of nursing. Despite this long time struggle, nurses and nurse educators continue to strive to foster critical thinking skills in nursing students as intuitively most nurses believe that critical thinking is necessary to function competently in the workplace. This article explores the most recent work of Dr. Stephen Brookfield and ties the concepts which are explored in Brookfield's work to nursing practice. Brookfield identifies that learners understand the meaning of critical thinking the best when the process is first demonstrated. Role modeling is a method educators can use to demonstrate critical thinking and is a strategy which nurses often use in the clinical area to train and mentor new nursing staff. Although it is not a new strategy in nursing education, it is a valuable strategy to engage learners in critical thinking activities. PMID:24418065

  14. Implementing Software Defined Radio

    CERN Document Server

    Grayver, Eugene

    2013-01-01

    Software Defined Radio makes wireless communications easier, more efficient, and more reliable. This book bridges the gap between academic research and practical implementation. When beginning a project, practicing engineers, technical managers, and graduate students can save countless hours by considering the concepts presented in these pages. The author covers the myriad options and trade-offs available when selecting an appropriate hardware architecture. As demonstrated here, the choice between hardware- and software-centric architecture can mean the difference between meeting an aggressive schedule and bogging down in endless design iterations. Because of the author’s experience overseeing dozens of failed and successful developments, he is able to present many real-life examples. Some of the key concepts covered are: Choosing the right architecture for the market – laboratory, military, or commercial Hardware platforms – FPGAs, GPPs, specialized and hybrid devices Standardization efforts to ens...

  15. Defining cyber warfare

    Directory of Open Access Journals (Sweden)

    Dragan D. Mladenović

    2012-04-01

    Full Text Available Cyber conflicts represent a new kind of warfare that is technologically developing very rapidly. Such development results in more frequent and more intensive cyber attacks undertaken by states against adversary targets, with a wide range of diverse operations, from information operations to physical destruction of targets. Nevertheless, cyber warfare is waged through the application of the same means, techniques and methods as those used in cyber criminal, terrorism and intelligence activities. Moreover, it has a very specific nature that enables states to covertly initiate attacks against their adversaries. The starting point in defining doctrines, procedures and standards in the area of cyber warfare is determining its true nature. In this paper, a contribution to this effort was made through the analysis of the existing state doctrines and international practice in the area of cyber warfare towards the determination of its nationally acceptable definition.

  16. Reusing information for high-level fusion: characterizing bias and uncertainty in human-generated intelligence

    Science.gov (United States)

    Burke, Dustin; Carlin, Alan; Picciano, Paul; Levchuk, Georgiy; Riordan, Brian

    2013-05-01

    To expedite the intelligence collection process, analysts reuse previously collected data. This poses the risk of analysis failure, because these data are biased in ways that the analyst may not know. Thus, these data may be incomplete, inconsistent or incorrect, have structural gaps and limitations, or simply be too old to accurately represent the current state of the world. Incorporating human-generated intelligence within the high-level fusion process enables the integration of hard (physical sensors) and soft information (human observations) to extend the ability of algorithms to associate and merge disparate pieces of information for a more holistic situational awareness picture. However, in order for high-level fusion systems to manage the uncertainty in soft information, a process needs to be developed for characterizing the sources of error and bias specific to human-generated intelligence and assessing the quality of this data. This paper outlines an approach Towards Integration of Data for unBiased Intelligence and Trust (TID-BIT) that implements a novel Hierarchical Bayesian Model for high-level situation modeling that allows the analyst to accurately reuse existing data collected for different intelligence requirements. TID-BIT constructs situational, semantic knowledge graphs that links the information extracted from unstructured sources to intelligence requirements and performs pattern matching over these attributed-network graphs for integrating information. By quantifying the reliability and credibility of human sources, TID-BIT enables the ability to estimate and account for uncertainty and bias that impact the high-level fusion process, resulting in improved situational awareness.

  17. Identification and characterization of microRNAs in Clonorchis sinensis of human health significance

    Directory of Open Access Journals (Sweden)

    Yuan Zi-Guo

    2010-09-01

    Full Text Available Abstract Background Clonorchis sinensis is a zoonotic parasite causing clonorchiasis-associated human disease such as biliary calculi, cholecystitis, liver cirrhosis, and it is currently classified as carcinogenic to humans for cholangiocarcinoma. MicroRNAs (miRNAs are non-coding, regulating small RNA molecules which are essential for the complex life cycles of parasites and are involved in parasitic infections. To identify and characterize miRNAs expressed in adult C. sinensis residing chronically in the biliary tract, we developed an integrative approach combining deep sequencing and bioinformatic predictions with stem-loop real-time PCR analysis. Results Here we report the use of this approach to identify and clone 6 new and 62,512 conserved C. sinensis miRNAs which belonged to 284 families. There was strong bias on families, family members and sequence nucleotides in C. sinensis. Uracil was the dominant nucleotide, particularly at positions 1, 14 and 22, which were located approximately at the beginning, middle and end of conserved miRNAs. There was no significant "seed region" at the first and ninth positions which were commonly found in human, animals and plants. Categorization of conserved miRNAs indicated that miRNAs of C. sinensis were still innovated and concentrated along three branches of the phylogenetic tree leading to bilaterians, insects and coelomates. There were two miRNA strategies in C. sinensis for its parasitic life: keeping a large category of miRNA families of different animals and keeping stringent conserved seed regions with high active innovation in other places of miRNAs mainly in the middle and the end, which were perfect for the parasite to perform its complex life style and for host changes. Conclusions The present study represented the first large scale characterization of C. sinensis miRNAs, which have implications for understanding the complex biology of this zoonotic parasite, as well as miRNA studies of other

  18. Characterization of cytoskeletal and junctional proteins expressed by cells cultured from human arachnoid granulation tissue

    Directory of Open Access Journals (Sweden)

    Mehta Bhavya C

    2005-10-01

    Full Text Available Abstract Background The arachnoid granulations (AGs are projections of the arachnoid membrane into the dural venous sinuses. They function, along with the extracranial lymphatics, to circulate the cerebrospinal fluid (CSF to the systemic venous circulation. Disruption of normal CSF dynamics may result in increased intracranial pressures causing many problems including headaches and visual loss, as in idiopathic intracranial hypertension and hydrocephalus. To study the role of AGs in CSF egress, we have grown cells from human AG tissue in vitro and have characterized their expression of those cytoskeletal and junctional proteins that may function in the regulation of CSF outflow. Methods Human AG tissue was obtained at autopsy, and explanted to cell culture dishes coated with fibronectin. Typically, cells migrated from the explanted tissue after 7–10 days in vitro. Second or third passage cells were seeded onto fibronectin-coated coverslips at confluent densities and grown to confluency for 7–10 days. Arachnoidal cells were tested using immunocytochemical methods for the expression of several common cytoskeletal and junctional proteins. Second and third passage cultures were also labeled with the common endothelial markers CD-31 or VE-cadherin (CD144 and their expression was quantified using flow cytometry analysis. Results Confluent cultures of arachnoidal cells expressed the intermediate filament protein vimentin. Cytokeratin intermediate filaments were expressed variably in a subpopulation of cells. The cultures also expressed the junctional proteins connexin43, desmoplakin 1 and 2, E-cadherin, and zonula occludens-1. Flow cytometry analysis indicated that second and third passage cultures failed to express the endothelial cell markers CD31 or VE-cadherin in significant quantities, thereby showing that these cultures did not consist of endothelial cells from the venous sinus wall. Conclusion To our knowledge, this is the first report of

  19. Characterizing Active Pharmaceutical Ingredient Binding to Human Serum Albumin by Spin-Labeling and EPR Spectroscopy.

    Science.gov (United States)

    Hauenschild, Till; Reichenwallner, Jörg; Enkelmann, Volker; Hinderberger, Dariush

    2016-08-26

    Drug binding to human serum albumin (HSA) has been characterized by a spin-labeling and continuous-wave (CW) EPR spectroscopic approach. Specifically, the contribution of functional groups (FGs) in a compound on its albumin-binding capabilities is quantitatively described. Molecules from different drug classes are labeled with EPR-active nitroxide radicals (spin-labeled pharmaceuticals (SLPs)) and in a screening approach CW-EPR spectroscopy is used to investigate HSA binding under physiological conditions and at varying ratios of SLP to protein. Spectral simulations of the CW-EPR spectra allow extraction of association constants (KA ) and the maximum number (n) of binding sites per protein. By comparison of data from 23 SLPs, the mechanisms of drug-protein association and the impact of chemical modifications at individual positions on drug uptake can be rationalized. Furthermore, new drug modifications with predictable protein binding tendency may be envisaged. PMID:27460503

  20. Characterization of a gene from the EDM1-PSACH region of human chromosome 19p

    Energy Technology Data Exchange (ETDEWEB)

    Lennon, G.G.; Giorgi, D.; Martin, J.R. [Lawrence Livermore National Lab., CA (United States)] [and others

    1994-09-01

    Genetic linkage mapping has indicated that both multiple epiphyseal dysplasia (EDM1), a dominantly inherited chondrodysplasia, and pseudoachondroplasia (PSACH), a skeletal disorder associated with dwarfism, map to a 2-3 Mb region of human chromosome 19p. We have isolated a partial cDNA from this region using hybrid selection, and report on progress towards the characterization of the genomic structure and transcription of the corresponding gene. Sequence analysis of the cDNA to date indicates that this gene is likely to be expressed within extracellular matrix tissues. Defects in this gene or neighboring gene family members may therefore lead to EDM1, PSACH, or other connective tissue and skeletal disorders.

  1. Characterization of Dielectric Responses of Human Cancer Cells in the Terahertz Region

    Science.gov (United States)

    Shiraga, Keiichiro; Ogawa, Yuichi; Suzuki, Tetsuhito; Kondo, Naoshi; Irisawa, Akiyoshi; Imamura, Motoki

    2014-05-01

    Terahertz time-domain attenuated total reflection spectroscopy, in combination with a two-interface model, is used to determine the complex dielectric constants of cultured human cancer cells (DLD-1, HEK293 and HeLa). Picosecond and sub-picosecond water dynamics are dominant in the measured complex dielectric constants of these cells. We demonstrate that dielectric responses below 1.0 THz best characterize the particular water dynamics of cancer cells when compared with extracellular water. Debye-Lorentz fitting revealed that this is due to a significantly attenuated slow relaxation mode and enhanced fast relaxation mode of the water in these cancer cells. These findings could lead to a new procedure to digitally evaluate cellular activities or functions, in terms of intracellular water dynamics, and remove the veil from the mysterious intracellular milieu.

  2. Characterization of human interleukin-3 receptors on a multi-factor-dependent cell line

    International Nuclear Information System (INIS)

    Recombinant human interleukin-3 (hIL-3) was radioiodinated by Bolton-Hunter method with maintenance of biological activity. Using 125I-hIL-3, hIL-3 receptors were characterized on a multi-factor-dependent cell line TF-1. Equilibrium binding studies revealed the existence of a single class of binding sites (667 +/- 306 sites/cell) with a Kd of 173 +/- 25 pM. Affinity labeling of TF-1 cells with 125I-IL-3 yielded two bands of 150 kDa and 85 kDa, implying molecular weights of 135 kDa and 70 kDa for the hIL-3 receptors

  3. Characterization of the Usage of the Serine Metabolic Network in Human Cancer

    Directory of Open Access Journals (Sweden)

    Mahya Mehrmohamadi

    2014-11-01

    Full Text Available The serine, glycine, one-carbon (SGOC metabolic network is implicated in cancer pathogenesis, but its general functions are unknown. We carried out a computational reconstruction of the SGOC network and then characterized its expression across thousands of cancer tissues. Pathways including methylation and redox metabolism exhibited heterogeneous expression indicating a strong context dependency of their usage in tumors. From an analysis of coexpression, simultaneous up- or downregulation of nucleotide synthesis, NADPH, and glutathione synthesis was found to be a common occurrence in all cancers. Finally, we developed a method to trace the metabolic fate of serine using stable isotopes, high-resolution mass spectrometry, and a mathematical model. Although the expression of single genes didn’t appear indicative of flux, the collective expression of several genes in a given pathway allowed for successful flux prediction. Altogether, these findings identify expansive and heterogeneous functions for the SGOC metabolic network in human cancer.

  4. Characterization of black raspberry functional food products for cancer prevention human clinical trials.

    Science.gov (United States)

    Gu, Junnan; Ahn-Jarvis, Jennifer H; Riedl, Kenneth M; Schwartz, Steven J; Clinton, Steven K; Vodovotz, Yael

    2014-05-01

    Our team is designing and fully characterizing black raspberry (BRB) food products suitable for long-term cancer prevention studies. The processing, scale-up, and storage effects on the consistency, quality, bioactive stability, and sensory acceptability of two BRB delivery systems of various matrices are presented. BRB dosage, pH, water activity, and texture were consistent in the scale-up production. Confections retained >90% of anthocyanins and ellagitannin after processing. Nectars had >69% of anthocyanins and >66% of ellagitannin retention, which varied with BRB dosage due to the processing difference. Texture remained unchanged during storage. BRB products consumed in a prostate cancer clinical trial were well accepted in sensory tests. Thus, this study demonstrates that two different BRB foods can be formulated to meet quality standards with a consistent bioactive pattern and successfully scaled up for a large human clinical trial focusing on cancer risk and other health outcomes.

  5. Prevalence and molecular characterization of human noroviruses and sapoviruses in Ethiopia.

    Science.gov (United States)

    Sisay, Zufan; Djikeng, Appolinaire; Berhe, Nega; Belay, Gurja; Gebreyes, Wondwossen; Abegaz, Woldaregay Erku; Njahira, Moses N; Wang, Q H; Saif, Linda J

    2016-08-01

    Viral gastroenteritis is a major public health problem worldwide. In Ethiopia, very limited studies have been done on the epidemiology of enteropathogenic viruses. The aim of this study was to detect and characterize noroviruses (NoVs) and sapoviruses (SaVs) from acute gastroenteritis patients of all ages. Fecal samples were collected from diarrheic patients (n = 213) in five different health centers in Addis Ababa during June-September 2013. The samples were screened for caliciviruses by reverse transcription polymerase chain reaction (RT-PCR) using universal and genogroup-specific primer pairs. Phylogenetic analyses were conducted using the sequences of the PCR products. Of the clinical samples, 25.3 % and 4.2 % were positive for NoV and SaV RNA, respectively. Among the norovirus positives, 22 were sequenced further, and diverse norovirus strains were identified: GI (n = 4), GII (n = 17) and GIV (n = 1). Most strains were GII (n = 17/22: 77.2 %), which were further divided into three different genotypes (GII.4, GII.12/GII.g recombinant-like and GII.17), with GII.17 being the dominant (7/17) strain detected. GI noroviruses, in particular GI.4 (n = 1), GI.5 (n = 2) and GI.8 (n = 1), were also detected and characterized. The GIV strain detected is the first from East Africa. The sapoviruses sequenced were also the first reported from Ethiopia. Collectively, this study showed the high burden and diversity of noroviruses and circulation of sapoviruses in diarrheic patients in Ethiopia. Continued surveillance to assess their association with diarrhea is needed to define their epidemiology, disease burden, and impact on public health. PMID:27193022

  6. Characterization of plasmids in a human clinical strain of Lactococcus garvieae.

    Directory of Open Access Journals (Sweden)

    Mónica Aguado-Urda

    Full Text Available The present work describes the molecular characterization of five circular plasmids found in the human clinical strain Lactococcus garvieae 21881. The plasmids were designated pGL1-pGL5, with molecular sizes of 4,536 bp, 4,572 bp, 12,948 bp, 14,006 bp and 68,798 bp, respectively. Based on detailed sequence analysis, some of these plasmids appear to be mosaics composed of DNA obtained by modular exchange between different species of lactic acid bacteria. Based on sequence data and the derived presence of certain genes and proteins, the plasmid pGL2 appears to replicate via a rolling-circle mechanism, while the other four plasmids appear to belong to the group of lactococcal theta-type replicons. The plasmids pGL1, pGL2 and pGL5 encode putative proteins related with bacteriocin synthesis and bacteriocin secretion and immunity. The plasmid pGL5 harbors genes (txn, orf5 and orf25 encoding proteins that could be considered putative virulence factors. The gene txn encodes a protein with an enzymatic domain corresponding to the family actin-ADP-ribosyltransferases toxins, which are known to play a key role in pathogenesis of a variety of bacterial pathogens. The genes orf5 and orf25 encode two putative surface proteins containing the cell wall-sorting motif LPXTG, with mucin-binding and collagen-binding protein domains, respectively. These proteins could be involved in the adherence of L. garvieae to mucus from the intestine, facilitating further interaction with intestinal epithelial cells and to collagenous tissues such as the collagen-rich heart valves. To our knowledge, this is the first report on the characterization of plasmids in a human clinical strain of this pathogen.

  7. Development, characterization, and photocytotoxicity assessment on human melanoma of chloroaluminum phthalocyanine nanocapsules

    International Nuclear Information System (INIS)

    In this work we have developed nanocapsules containing chloroaluminum phthalocyanine (ClAlPc) and assessed their phototoxic action on WM1552C, WM278, and WM1617 human melanoma cell lines. The ClAlPc-loaded nanocapsules were prepared by the nanoprecipitation method and optimized by means of a 23 full factorial design. The ClAlPc nanocapsules were characterized by particle size and distribution, zeta potential, morphology, encapsulation efficiency, singlet oxygen production, stability, and phototoxic action on melanoma cells. Both the development and optimization studies revealed that stable colloidal formulations could be obtained by using 1.75% (w/v) soybean lecithin, 1.25% (w/v) Poloxamer 188, 2.5% (v/v) soybean oil, and 0.75% (w/v) poly(D,L-lactide-co-glycolide). The nanocapsules had a mean diameter of 230 nm, homogeneous size distribution (polydispersity index −1) under light irradiation at 20 mJ cm−2. On the other hand, the cell survival percentage for all the melanoma cell lines treated with the highest light dose (150 mJ cm−2) was lower than 10%. In summary, ClAlPc nanoencapsulation could enable application of this hydrophobic photosensitizer in the treatment of malignant melanoma with the use of both low sensitizer drug concentration and light dose. - Highlights: ► Nanocapsules containing a hydrophobic metallophthalocyanine (ClAlPc) were developed. ► The colloidal formulations were characterized by their physicochemical parameters. ► ClAlPc nanocapsules were used for the photosensitization of human melanoma cell lines. ► Phototoxicity was achieved with low ClAlPc nanocapsules concentration and light dose

  8. Development and characterization of human-induced pluripotent stem cell-derived cholangiocytes.

    Science.gov (United States)

    De Assuncao, Thiago M; Sun, Yan; Jalan-Sakrikar, Nidhi; Drinane, Mary C; Huang, Bing Q; Li, Ying; Davila, Jaime I; Wang, Ruisi; O'Hara, Steven P; Lomberk, Gwen A; Urrutia, Raul A; Ikeda, Yasuhiro; Huebert, Robert C

    2015-06-01

    Cholangiocytes are the target of a heterogeneous group of liver diseases known as the cholangiopathies. An evolving understanding of the mechanisms driving biliary development provides the theoretical underpinnings for rational development of induced pluripotent stem cell (iPSC)-derived cholangiocytes (iDCs). Therefore, the aims of this study were to develop an approach to generate iDCs and to fully characterize the cells in vitro and in vivo. Human iPSC lines were generated by forced expression of the Yamanaka pluripotency factors. We then pursued a stepwise differentiation strategy toward iDCs, using precise temporal exposure to key biliary morphogens, and we characterized the cells, using a variety of morphologic, molecular, cell biologic, functional, and in vivo approaches. Morphology shows a stepwise phenotypic change toward an epithelial monolayer. Molecular analysis during differentiation shows appropriate enrichment in markers of iPSC, definitive endoderm, hepatic specification, hepatic progenitors, and ultimately cholangiocytes. Immunostaining, western blotting, and flow cytometry demonstrate enrichment of multiple functionally relevant biliary proteins. RNA sequencing reveals that the transcriptome moves progressively toward that of human cholangiocytes. iDCs generate intracellular calcium signaling in response to ATP, form intact primary cilia, and self-assemble into duct-like structures in three-dimensional culture. In vivo, the cells engraft within mouse liver, following retrograde intrabiliary infusion. In summary, we have developed a novel approach to generate mature cholangiocytes from iPSCs. In addition to providing a model of biliary differentiation, iDCs represent a platform for in vitro disease modeling, pharmacologic testing, and individualized, cell-based, regenerative therapies for the cholangiopathies. PMID:25867762

  9. Characterization of the replication timing program of 6 human model cell lines.

    Science.gov (United States)

    Hadjadj, Djihad; Denecker, Thomas; Maric, Chrystelle; Fauchereau, Fabien; Baldacci, Giuseppe; Cadoret, Jean-Charles

    2016-09-01

    During the S-phase, the DNA replication process is finely orchestrated and regulated by two programs: the spatial program that determines where replication will start in the genome (Cadoret et al. (2008 Oct 14), Cayrou et al. (2011 Sep), Picard et al. (2014 May 1) [1], [2], [3]), and the temporal program that determines when during the S phase different parts of the genome are replicated and when origins are activated. The temporal program is so well conserved for each cell type from independent individuals [4] that it is possible to identify a cell type from an unknown sample just by determining its replication timing program. Moreover, replicative domains are strongly correlated with the partition of the genome into topological domains (determined by the Hi-C method, Lieberman-Aiden et al. (2009 Oct 9), Pope et al. (2014 Nov 20) [5], [6]). On the one hand, replicative areas are well defined and participate in shaping the spatial organization of the genome for a given cell type. On the other hand, studies on the timing program during cell differentiation showed a certain plasticity of this program according to the stage of cell differentiation Hiratani et al. (2008 Oct 7, 2010 Feb) [7], [8]. Domains where a replication timing change was observed went through a nuclear re-localization. Thus the temporal program of replication can be considered as an epigenetic mark Hiratani and Gilbert (2009 Feb 16) [9]. We present the genomic data of replication timing in 6 human model cell lines: U2OS (GSM2111308), RKO (GSM2111309), HEK 293T (GSM2111310), HeLa (GSM2111311), MRC5-SV (GSM2111312) and K562 (GSM2111313). A short comparative analysis was performed that allowed us to define regions common to the 6 cell lines. These replication timing data can be taken into account when performing studies that use these model cell lines. PMID:27508120

  10. Characterization of the replication timing program of 6 human model cell lines

    Directory of Open Access Journals (Sweden)

    Djihad Hadjadj

    2016-09-01

    Full Text Available During the S-phase, the DNA replication process is finely orchestrated and regulated by two programs: the spatial program that determines where replication will start in the genome (Cadoret et al. (2008 Oct 14, Cayrou et al. (2011 Sep, Picard et al. (2014 May 1 [1–3], and the temporal program that determines when during the S phase different parts of the genome are replicated and when origins are activated. The temporal program is so well conserved for each cell type from independent individuals [4] that it is possible to identify a cell type from an unknown sample just by determining its replication timing program. Moreover, replicative domains are strongly correlated with the partition of the genome into topological domains (determined by the Hi-C method, Lieberman-Aiden et al. (2009 Oct 9, Pope et al. (2014 Nov 20 [5,6]. On the one hand, replicative areas are well defined and participate in shaping the spatial organization of the genome for a given cell type. On the other hand, studies on the timing program during cell differentiation showed a certain plasticity of this program according to the stage of cell differentiation Hiratani et al. (2008 Oct 7, 2010 Feb [7,8]. Domains where a replication timing change was observed went through a nuclear re-localization. Thus the temporal program of replication can be considered as an epigenetic mark Hiratani and Gilbert (2009 Feb 16 [9]. We present the genomic data of replication timing in 6 human model cell lines: U2OS (GSM2111308, RKO (GSM2111309, HEK 293T (GSM2111310, HeLa (GSM2111311, MRC5-SV (GSM2111312 and K562 (GSM2111313. A short comparative analysis was performed that allowed us to define regions common to the 6 cell lines. These replication timing data can be taken into account when performing studies that use these model cell lines.

  11. Biochemical and Structural Characterization of the Human TL1A Ectodomain

    Energy Technology Data Exchange (ETDEWEB)

    Zhan, C.; Yan, Q; Patskovsky, Y; Li, Z; Toro, R; Meyer, A; Cheng, H; Brenowitz, M; Nathenson, S; Almo, S

    2009-01-01

    TNF-like 1A (TL1A) is a newly described member of the TNF superfamily that is directly implicated in the pathogenesis of autoimmune diseases, including inflammatory bowel disease, atherosclerosis, and rheumatoid arthritis. We report the crystal structure of the human TL1A extracellular domain at a resolution of 2.5 {angstrom}, which reveals a jelly-roll fold typical of the TNF superfamily. This structural information, in combination with complementary mutagenesis and biochemical characterization, provides insights into the binding interface and the specificity of the interactions between TL1A and the DcR3 and DR3 receptors. These studies suggest that the mode of interaction between TL1A and DcR3 differs from other characterized TNF ligand/receptor complexes. In addition, we have generated functional TL1A mutants with altered disulfide bonding capability that exhibit enhanced solution properties, which will facilitate the production of materials for future cell-based and whole animal studies. In summary, these studies provide insights into the structure and function of TL1A and provide the basis for the rational manipulation of its interactions with cognate receptors.

  12. Human-factors-based implementation of the remote characterization system high-level control station

    International Nuclear Information System (INIS)

    The detection and characterization of buried objects and materials is an important first step in the restoration of the numerous US Department of Energy (DOE) and US Department of Defense waste disposal sites. DOE, through its Environmental Restoration and Waste Management Robotics and Technology Development Program, has developed the Remote Characterization System (RCS) to address the needs of remote subsurfacecharacterization. The RCS consists of a low-metal-content (low-metallic-signature) remotely piloted vehicle, a high-level control station (HLCS) where operators can remotely control the vehicle and analyze real-time data from sensors, and an array of sensors that can be chosen to meet the survey task at hand. Communication between the vehicle and the base station is handled by a radio link. Site mapping is made possible through the use of geopositioning satellite data. The primary mode of vehicle operation is teleoperation, but provision has been made for semiautonomous or supervisory control that allows for automated sitesurvey on simple sites. Data analysis and display is supported for both real-time observation and postprocessing of data. The particular emphasis of this paper documents the human-factors-based design influences on the HLCS and describes the design in detail

  13. Partial Characterization of Bacteriocins Produced by Two New Enterococcus faecium Isolated from Human Intestine.

    Science.gov (United States)

    Turgis, Mélanie; Vu, Khanh Dang; Lacroix, Monique

    2013-06-01

    This study aimed at characterizing two novel bacteriocin-producing enterococcal strains isolated from human intestine. A total of 200 lactic acid bacteria were isolated from a woman stool sample. Two of them were selected for characterization due to their high antimicrobial activity against five strains of Listeria monocytogenes. The selected bacteria were identified as two different strains of Enterococcus faecium and designated MT 104 and MT 162. The bacteriocins produced by MT 104 and MT 162 were stable at different pH ranging from 2 to 11 and were active after different treatments such as heat, enzymes, detergents, and γ-irradiation. The two isolated strains exhibited some probiotic properties such as survival in simulated gastric fluid and intestinal fluid, lack of expression of bile salt hydrolase or hemolytic activity, adhesion to Caco-2 cells efficiently, and sensitivity to clinical antimicrobial agents. Thus, the two isolated strains of E. faecium could become new probiotic bacteria and their bacteriocins could be used for controlling L. monocytogenes in combination with irradiation for food preservation. PMID:26782736

  14. Synchrotron X-ray diffraction characterization of healthy and fluorotic human dental enamel

    Science.gov (United States)

    Colaço, M. V.; Barroso, R. C.; Porto, I. M.; Gerlach, R. F.; Costa, F. N.; Braz, D.; Droppa, R.; de Sousa, F. B.

    2012-10-01

    With the introduction of fluoride as the main anticaries agent used in preventive dentistry, and perhaps an increase in fluoride in our food chain, dental fluorosis has become an increasing world-wide problem. Visible signs of fluorosis begin to become obvious on the enamel surface as opacities, implying some porosity in the tissue. The mechanisms that conduct the formation of fluorotic enamel are unknown, but should involve modifications in the basic physical-chemistry reactions of demineralization and remineralisation of the enamel of the teeth, which is the same reaction of formation of the enamel's hydroxyapatite (HAp) in the maturation phase. The increase of the amount of fluoride inside of the apatite will result in gradual increase of the lattice parameters. The aim of this work is to characterize the healthy and fluorotic enamel in human tooth using Synchrotron X-ray diffraction. All the scattering profile measurements were carried out at the X-ray diffraction beamline (XRD1) at the Brazilian Synchrotron Light Laboratory—LNLS, Campinas, Brazil. X-ray diffraction experiments were performed both in powder samples and polished surfaces. The powder samples were analyzed to obtain the characterization of a typical healthy enamel pattern. The polished surfaces were analyzed in specific areas that have been identified as fluorotic ones. X-ray diffraction data were obtained for all samples and these data were compared with the control samples and also with the literature data.

  15. Characterization of nanobodies binding human fibrinogen selected by E. coli display.

    Science.gov (United States)

    Salema, Valencio; López-Guajardo, Ana; Gutierrez, Carlos; Mencía, Mario; Fernández, Luis Ángel

    2016-09-20

    Abnormal levels of fibrinogen (Fib) in blood plasma are associated with several pathological conditions and hence methods for its detection in blood and body fluids are essential. Nanobodies (Nbs) or (VHHs) are single domain antibodies derived from camelids with excellent biophysical and antigen-binding properties, showing great promise in diagnostics and therapy. In this work, we select and characterize high affinity Nbs binding human Fib employing an E. coli cell surface display system based on the fusion of an immune library of VHH domains with the β-domain of Intimin. Bacteria displaying high-affinity Nbs against Fib were selected using magnetic cell sorting (MACS). Specific binding of the selected clones to Fib was confirmed by flow cytometry of E. coli bacteria, as well as by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) with the purified Nbs. E. coli display also provided an excellent estimation of the affinity of the selected Nbs by flow cytometry analysis under equilibrium conditions, with equilibrium constant (KD) values very similar to those obtained by SPR analysis. Finally, pairwise epitope-scouting studies revealed that the selected Nbs bound distinct epitopes on Fib. The selected Nbs are promising diagnostic tools for determination of human Fib levels. PMID:27485813

  16. Cell surface and transcriptional characterization of human adipose-derived adherent stromal (hADAS) cells.

    Science.gov (United States)

    Katz, Adam J; Tholpady, Ashok; Tholpady, Sunil S; Shang, Hulan; Ogle, Roy C

    2005-03-01

    Adult human subcutaneous adipose tissue contains cells with intriguing multilineage developmental plasticity, much like marrow-derived mesenchymal stem cells. Putative stem or progenitor cells from fat have been given many different names in the literature, reflecting an early and evolving consensus regarding their phenotypic characterization. The study reported here used microarrays to evaluate over 170 genes relating to angiogenesis and extracellular matrix in undifferentiated, early-passage human adipose-derived adherent stromal (hADAS) cells isolated from three separate donors. The hADAS populations unanimously transcribed 66% of the screened genes, and 83% were transcribed by at least two of the three populations. The most highly transcribed genes relate to functional groupings such as cell adhesion, matrix proteins, growth factors and receptors, and proteases. The transcriptome of hADAS cells demonstrated by this work reveals many similarities to published profiles of bone marrow mesenchymal stem cells (MSCs). In addition, flow analysis of over 24 hADAS cell surface proteins (n = 7 donors) both confirms and expands on the existing literature and reveals strong intergroup correlation, despite an inconsistent nomenclature and the lack of standardized protocols for cell isolation and culture. Finally, based on flow analysis and reverse transcription polymerase chain reaction studies, our results suggest that hADAS cells do not express several proteins that are implicated as markers of "stemness" in other stem cell populations, including telomerase, CD133, and the membrane transporter ABCG2.

  17. Recombinant methionine aminopeptidase protein of Babesia microti: immunobiochemical characterization as a vaccine candidate against human babesiosis.

    Science.gov (United States)

    Munkhjargal, Tserendorj; Yokoyama, Naoaki; Igarashi, Ikuo

    2016-09-01

    Human babesiosis is the most important zoonotic protozoan infection in the world. This is the first report of the cloning, expression, purification, and immunobiochemical characterization of a methionine aminopeptidase 1 (MetAP1) protein from Babesia microti (B. microti). The gene encodes a MetAP1 protein of B. microti (BmMetAP1) of approximately 66.8 kDa that includes glutathione S-transferase (GST) tag and shows MetAP activity. BmMetAP1 was detected in a lysate of B. microti and further localized in cytoplasm of the B. microti merozoite. rBmMetAP1 was found to be immunogenic, eliciting a high antibody titer in mice. Moreover, rBmMetAP1 stimulated the production of IFN-γ and IL-12 but not IL-4. Finally, rBmMetAP1 was able to provide considerable protection to mice against a B. microti challenge infection based on a reduction in peak parasitemia levels and earlier clearance of the parasite as compared with control mice. Taken together, these results suggest that rBmMetAP1 confers significant protection against experimental B. microti infection and might be considered a potential vaccine target against human babesiosis. PMID:27306898

  18. Persea americana Mill. Seed: Fractionation, Characterization, and Effects on Human Keratinocytes and Fibroblasts.

    Science.gov (United States)

    Ramos-Jerz, Maria Del R; Villanueva, Socorro; Jerz, Gerold; Winterhalter, Peter; Deters, Alexandra M

    2013-01-01

    Methanolic avocado (Persea americana Mill., Lauraceae) seed extracts were separated by preparative HSCCC. Partition and HSCCC fractions were principally characterized by LC-ESI-MS/MS analysis. Their in vitro influence was investigated on proliferation, differentiation, cell viability, and gene expression on HaCaT and normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF). The methanol-water partition (M) from avocado seeds and HSCCC fraction 3 (M.3) were mostly composed of chlorogenic acid and its isomers. Both reduced NHDF but enhanced HaCaT keratinocytes proliferation. HSCCC fraction M.2 composed of quinic acid among chlorogenic acid and its isomers inhibited proliferation and directly induced differentiation of keratinocytes as observed on gene and protein level. Furthermore, M.2 increased NHDF proliferation via upregulation of growth factor receptors. Salidrosides and ABA derivatives present in HSCCC fraction M.6 increased NHDF and keratinocyte proliferation that resulted in differentiation. The residual solvent fraction M.7 contained among low concentrations of ABA derivatives high amounts of proanthocyanidins B1 and B2 as well as an A-type trimer and stimulated proliferation of normal cells and inhibited the proliferation of immortalized HaCaT keratinocytes.

  19. Persea americana Mill. Seed: Fractionation, Characterization, and Effects on Human Keratinocytes and Fibroblasts

    Directory of Open Access Journals (Sweden)

    Maria del R. Ramos-Jerz

    2013-01-01

    Full Text Available Methanolic avocado (Persea americana Mill., Lauraceae seed extracts were separated by preparative HSCCC. Partition and HSCCC fractions were principally characterized by LC-ESI-MS/MS analysis. Their in vitro influence was investigated on proliferation, differentiation, cell viability, and gene expression on HaCaT and normal human epidermal keratinocytes (NHEK and normal human dermal fibroblasts (NHDF. The methanol-water partition (M from avocado seeds and HSCCC fraction 3 (M.3 were mostly composed of chlorogenic acid and its isomers. Both reduced NHDF but enhanced HaCaT keratinocytes proliferation. HSCCC fraction M.2 composed of quinic acid among chlorogenic acid and its isomers inhibited proliferation and directly induced differentiation of keratinocytes as observed on gene and protein level. Furthermore, M.2 increased NHDF proliferation via upregulation of growth factor receptors. Salidrosides and ABA derivatives present in HSCCC fraction M.6 increased NHDF and keratinocyte proliferation that resulted in differentiation. The residual solvent fraction M.7 contained among low concentrations of ABA derivatives high amounts of proanthocyanidins B1 and B2 as well as an A-type trimer and stimulated proliferation of normal cells and inhibited the proliferation of immortalized HaCaT keratinocytes.

  20. Characterization of Human Dental Pulp Tissue Under Oscillatory Shear and Compression.

    Science.gov (United States)

    Ozcan, Burak; Bayrak, Ece; Erisken, Cevat

    2016-06-01

    Availability of material as well as biological properties of native tissues is critical for biomaterial design and synthesis for regenerative engineering. Until recently, selection of biomaterials and biomolecule carriers for dental pulp regeneration has been done randomly or based on experience mainly due to the absence of benchmark data for dental pulp tissue. This study, for the first time, characterizes the linear viscoelastic material functions and compressive properties of human dental pulp tissue harvested from wisdom teeth, under oscillatory shear and compression. The results revealed a gel-like behavior of the pulp tissue over the frequency range of 0.1-100 rps. Uniaxial compression tests generated peak normal stress and compressive modulus values of 39.1 ± 20.4 kPa and 5.5 ± 2.8 kPa, respectively. Taken collectively, the linear viscoelastic and uniaxial compressive properties of the human dental pulp tissue reported here should enable the better tailoring of biomaterials or biomolecule carriers to be employed in dental pulp regeneration. PMID:27093446

  1. Purification and characterization of α-L-fucosidase from human primary hepatocarcinoma tissue

    Institute of Scientific and Technical Information of China (English)

    Chao Li; Jie Qian; Ju-Sheng Lin

    2006-01-01

    AIM: To purify and characterize α-L-fucosidase fromhuman liver cancer tissue and to detect the localizationof α-L-fucosidase in tumor tissue.METHODS: Cation exchange chromatography onCM-52 and ultrafiltration were used to separate α-L-fucosidase (AFU) from crude extract of liver cancertissue. 4-methylumbelliferyl-α-L-fucopyranoside wasused as a fluorescent substrate to quantify the purifiedAFU activity in each step. A polyclonal antibody (pAb)against the purified AFU was obtained by anion exchangechromatography on DEAE-52 after ammonium sulfatefractionation and ultrafiltration. Immuohistochemicalstaining was used to observe the expression of AFU inmalignant and adjacent liver tissues.RESULTS: Human α-L-fucosidase was purified 74-fold to apparent homogeneity with 15% yield. SDS-PAGE indicated the presence of one subunit of molecularweight of 55 Ku. The specific activity of AFU in pooledfraction by chromatography was 10085 IU/mg. Westernblot analysis indicated that the pAb could recognizeone protein band of molecular weight of 55 Ku. Theexpression of AFU was observed in cytoplasm membraneof liver cancer tissue but not in that of adjacent tissue.CONCLUSION: The purified α-L-fucosidase from primaryhepatocarcinoma (PHC) is different in its properties fromα-L-fucosidase in human other organs. The polyclonalantibody prepared in this experiment can be applied tothe diagnosis of PHC.

  2. Generation and characterization of the human neutralizing antibody fragment Fab091 against rabies virus

    Institute of Scientific and Technical Information of China (English)

    Chen LI; Feng ZHANG; Hong LIN; Zhong-can WANG; Xin-jian LIU; Zhen-qing FENG; Jin ZHU; Xiao-hong GUAN

    2011-01-01

    Aim: To transform the human anti-rabies virus glycoprotein (anti-RABVG) single-chain variable fragment (scFv) into a Fab fragment and to analyze its immunological activity.Methods: The Fab gene was amplified using overlap PCR and inserted into the vector pComb3XSS. The recombinant vector was then transformed into E coli Top10F' for expression and purification. The purified Fab was characterized using SDS-PAGE, Western blotting,indirect ELISA, competitive ELISA, and the fluorescent antibody virus neutralization test (FAVN), respectively, and examined in a Kunming mouse challenge model in vivo.Results: A recombinant vector was constructed. The Fab was expressed in soluble form In E coll Top10F'. Specific binding of the Fab to rabies virus was confirmed by indirect ELISA and immunoprecipitation (IP). The neutralizing antibody titer of Fab was 10.26 IU/mL.The mouse group treated with both vaccine and human rabies immunoglobulin (HRIG)/Fab091 (32 IU/kg) showed protection against rabies, compared with the control group (P<0.05, Logrank test).Conclusion: The antibody fragment Fab was shown to be a neutralizing antibody against RABVG. It can be used together with other monoclonal antibodies for post-exposure prophylaxis of rabies virus in future studies.

  3. EEG-fMRI based information theoretic characterization of the human perceptual decision system.

    Directory of Open Access Journals (Sweden)

    Dirk Ostwald

    Full Text Available The modern metaphor of the brain is that of a dynamic information processing device. In the current study we investigate how a core cognitive network of the human brain, the perceptual decision system, can be characterized regarding its spatiotemporal representation of task-relevant information. We capitalize on a recently developed information theoretic framework for the analysis of simultaneously acquired electroencephalography (EEG and functional magnetic resonance imaging data (fMRI (Ostwald et al. (2010, NeuroImage 49: 498-516. We show how this framework naturally extends from previous validations in the sensory to the cognitive domain and how it enables the economic description of neural spatiotemporal information encoding. Specifically, based on simultaneous EEG-fMRI data features from n = 13 observers performing a visual perceptual decision task, we demonstrate how the information theoretic framework is able to reproduce earlier findings on the neurobiological underpinnings of perceptual decisions from the response signal features' marginal distributions. Furthermore, using the joint EEG-fMRI feature distribution, we provide novel evidence for a highly distributed and dynamic encoding of task-relevant information in the human brain.

  4. A Comparative Study of Theoretical Graph Models for Characterizing Structural Networks of Human Brain

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    Xiaojin Li

    2013-01-01

    Full Text Available Previous studies have investigated both structural and functional brain networks via graph-theoretical methods. However, there is an important issue that has not been adequately discussed before: what is the optimal theoretical graph model for describing the structural networks of human brain? In this paper, we perform a comparative study to address this problem. Firstly, large-scale cortical regions of interest (ROIs are localized by recently developed and validated brain reference system named Dense Individualized Common Connectivity-based Cortical Landmarks (DICCCOL to address the limitations in the identification of the brain network ROIs in previous studies. Then, we construct structural brain networks based on diffusion tensor imaging (DTI data. Afterwards, the global and local graph properties of the constructed structural brain networks are measured using the state-of-the-art graph analysis algorithms and tools and are further compared with seven popular theoretical graph models. In addition, we compare the topological properties between two graph models, namely, stickiness-index-based model (STICKY and scale-free gene duplication model (SF-GD, that have higher similarity with the real structural brain networks in terms of global and local graph properties. Our experimental results suggest that among the seven theoretical graph models compared in this study, STICKY and SF-GD models have better performances in characterizing the structural human brain network.

  5. Discovery and Characterization of Phage Display-Derived Human Monoclonal Antibodies against RSV F Glycoprotein.

    Science.gov (United States)

    Chen, Zhifeng; Zhang, Lan; Tang, Aimin; Callahan, Cheryl; Pristatsky, Pavlo; Swoyer, Ryan; Cejas, Pedro; Nahas, Debbie; Galli, Jennifer; Cosmi, Scott; DiStefano, Daniel; Hoang, Van M; Bett, Andrew; Casimiro, Danilo; Vora, Kalpit A

    2016-01-01

    Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants, the elderly and in immunosuppressed populations. The vast majority of neutralizing antibodies isolated from human subjects target the RSV fusion (F) glycoprotein, making it an attractive target for the development of vaccines and therapeutic antibodies. Currently, Synagis® (palivizumab) is the only FDA approved antibody drug for the prevention of RSV infection, and there is a great need for more effective vaccines and therapeutics. Phage display is a powerful tool in antibody discovery with the advantage that it does not require samples from immunized subjects. In this study, Morphosys HuCAL GOLD® phage libraries were used for panning against RSV prefusion and postfusion F proteins. Panels of human monoclonal antibodies (mAbs) against RSV F protein were discovered following phage library panning and characterized. Antibodies binding specifically to prefusion or postfusion F proteins and those binding both conformations were identified. 3B1 is a prototypic postfusion F specific antibody while 2E1 is a prototypic prefusion F specific antibody. 2E1 is a potent broadly neutralizing antibody against both RSV A and B strains. Epitope mapping experiments identified a conformational epitope spanning across three discontinuous sections of the RSV F protein, as well as critical residues for antibody interaction. PMID:27258388

  6. Discovery and Characterization of Phage Display-Derived Human Monoclonal Antibodies against RSV F Glycoprotein.

    Directory of Open Access Journals (Sweden)

    Zhifeng Chen

    Full Text Available Respiratory syncytial virus (RSV is a leading cause of lower respiratory tract infection in infants, the elderly and in immunosuppressed populations. The vast majority of neutralizing antibodies isolated from human subjects target the RSV fusion (F glycoprotein, making it an attractive target for the development of vaccines and therapeutic antibodies. Currently, Synagis® (palivizumab is the only FDA approved antibody drug for the prevention of RSV infection, and there is a great need for more effective vaccines and therapeutics. Phage display is a powerful tool in antibody discovery with the advantage that it does not require samples from immunized subjects. In this study, Morphosys HuCAL GOLD® phage libraries were used for panning against RSV prefusion and postfusion F proteins. Panels of human monoclonal antibodies (mAbs against RSV F protein were discovered following phage library panning and characterized. Antibodies binding specifically to prefusion or postfusion F proteins and those binding both conformations were identified. 3B1 is a prototypic postfusion F specific antibody while 2E1 is a prototypic prefusion F specific antibody. 2E1 is a potent broadly neutralizing antibody against both RSV A and B strains. Epitope mapping experiments identified a conformational epitope spanning across three discontinuous sections of the RSV F protein, as well as critical residues for antibody interaction.

  7. Persea americana Mill. Seed: Fractionation, Characterization, and Effects on Human Keratinocytes and Fibroblasts.

    Science.gov (United States)

    Ramos-Jerz, Maria Del R; Villanueva, Socorro; Jerz, Gerold; Winterhalter, Peter; Deters, Alexandra M

    2013-01-01

    Methanolic avocado (Persea americana Mill., Lauraceae) seed extracts were separated by preparative HSCCC. Partition and HSCCC fractions were principally characterized by LC-ESI-MS/MS analysis. Their in vitro influence was investigated on proliferation, differentiation, cell viability, and gene expression on HaCaT and normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF). The methanol-water partition (M) from avocado seeds and HSCCC fraction 3 (M.3) were mostly composed of chlorogenic acid and its isomers. Both reduced NHDF but enhanced HaCaT keratinocytes proliferation. HSCCC fraction M.2 composed of quinic acid among chlorogenic acid and its isomers inhibited proliferation and directly induced differentiation of keratinocytes as observed on gene and protein level. Furthermore, M.2 increased NHDF proliferation via upregulation of growth factor receptors. Salidrosides and ABA derivatives present in HSCCC fraction M.6 increased NHDF and keratinocyte proliferation that resulted in differentiation. The residual solvent fraction M.7 contained among low concentrations of ABA derivatives high amounts of proanthocyanidins B1 and B2 as well as an A-type trimer and stimulated proliferation of normal cells and inhibited the proliferation of immortalized HaCaT keratinocytes. PMID:24371457

  8. Characterization of Diaphanous-related formin FMNL2 in human tissues

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    Kampf Caroline

    2010-07-01

    Full Text Available Abstract Background Diaphanous-related formins govern actin-based processes involved in many cellular functions, such as cell movement and invasion. Possible connections to developmental processes and cellular changes associated with malignant phenotype make them interesting study targets. In spite of this, very little is known of the tissue distribution and cellular location of any mammalian formin. Here we have carried out a comprehensive analysis of the formin family member formin -like 2 (FMNL2 in human tissues. Results An FMNL2 antibody was raised and characterized. The affinity-purified FMNL2 antibody was validated by Western blotting, Northern blotting, a peptide competition assay and siRNA experiments. Bioinformatics-based mRNA profiling indicated that FMNL2 is widely expressed in human tissues. The highest mRNA levels were seen in central and peripheral nervous systems. Immunohistochemical analysis of 26 different human tissues showed that FMNL2 is widely expressed, in agreement with the mRNA profile. The widest expression was detected in the central nervous system, since both neurons and glial cells expressed FMNL2. Strong expression was also seen in many epithelia. However, the expression in different cell types was not ubiquitous. Many mesenchymal cell types showed weak immunoreactivity and cells lacking expression were seen in many tissues. The subcellular location of FMNL2 was cytoplasmic, and in some tissues a strong perinuclear dot was detected. In cultured cells FMNL2 showed mostly a cytoplasmic localization with perinuclear accumulation consistent with the Golgi apparatus. Furthermore, FMNL2 co-localized with F-actin to the tips of cellular protrusions in WM164 human melanoma cells. This finding is in line with FMNL2's proposed function in the formation of actin filaments in cellular protrusions, during amoeboid cellular migration. Conclusion FMNL2 is expressed in multiple human tissues, not only in the central nervous system

  9. Epidemiological Studies on Echinococcosis and Characterization of Human and Livestock Hydatid Cysts in Mauritania

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    CB Ould Ahmed Salem

    2011-03-01

    Full Text Available Background: Echinococcosis/hydatidosis is considered endemic in Mauritania. The aim of this study is to present an epidemiological study on the echinococcosis in man and animals in the Nouakchott region. Methods: The internal organs from livestock carcasses were inspected for research of hydatid cysts. The hydatid fluid was examined for research of the protoscoleces. Dogs were necropsied for the collect of Echinococcus granulosus.Results: In the Nouakchott Hospital, 24 surgical operation of human hydatid cysts have been per­formed, out of which 50% were localised in the lung, 33% in the liver and 17% elsewhere. Then, the incidence rate would be of 1.2% per 100 000 inhabitants in Mauritania. In the dog, the prevalence rate is 14%. The average number of E. granulosus on the whole dogs is 172 and 1227 on the positive dogs. Concerning the livestock, hydatid cysts found in 30.1% of the dromedary, 5.5% of the cattle and 6.5 of the sheep. The fertility rate of hydatid cysts in humans (75% and camels (76% was significantly higher than that of sheep (24% and cattle (23% (P<0.0001. Hydatid infestation is characterized globally by the dominance of pulmonary localiza­tions in hu­mans (50% and camels (72.7% and in the liver in sheep (76.1% and cattle (82.3%.Conclusion: The differences between prevalence rates, the fertility of hydatid cysts and diversity sites localization observed in humans and camels of one hand and the sheep and cattle on the other hand, depends possibly the strain(s diversity of E. granulosus.

  10. Characterization of pancreatic stem cells derived from adult human pancreas ducts by fluorescence activated cell sorting

    Institute of Scientific and Technical Information of China (English)

    Han-Tso Lin; Shih-Hwa Chiou; Chung-Lan Kao; Yi-Ming Shyr; Chien-Jen Hsu; Yih-Wen Tarng; Larry L-T Ho; Ching-Fai Kwok; Hung-Hai Ku

    2006-01-01

    AIM: To isolate putative pancreatic stem cells (PSCs)from human adult tissues of pancreas duct using serumfree, conditioned medium. The characterization of surface phenotype of these PSCs was analyzed by flow cytometry. The potential for pancreatic lineage and the capability of β-cell differentiation in these PSCs were evaluated as well.METHODS: By using serum-free medium supplemented with essential growth factors, we attempted to isolate the putative PSCs which has been reported to express nestin and pdx-1. The MatrigelTM was employed to evaluate the differential capacity of isolated cells. Dithizone staining, insulin content/secretion measurement, and immunohistochemistry staining were used to monitor the differentiation. Fluorescence activated cell sorting (FACS)was used to detect the phenotypic markers of putative PSCs.RESULTS: A monolayer of spindle-like cells was cultivated. The putative PSCs expressed pdx-1 and nestin.They were also able to differentiate into insulin-, glucagon-, and somatostatin-positive cells. The spectrum of phenotypic markers in PSCs was investigated; a similarity was revealed when using human bone marrow-derived stem cells as the comparative experiment, such as CD29,CD44, CD49, CD50, CD51, CD62E, PDGFR-α, CD73 (SH2),CD81, CD105(SH3).CONCLUSION: In this study, we successfully isolated PSCs from adult human pancreatic duct by using serumfree medium. These PSCs not only expressed nestin and pdx-1 but also exhibited markers attributable to mesenchymal stem cells. Although work is needed to elucidate the role of these cells, the application of these PSCs might be therapeutic strategies for diabetes mellitus.

  11. Characterizing Properties of Biochar Produced from Simulated Human Feces and Its Potential Applications.

    Science.gov (United States)

    Ilango, Ajaykannan; Lefebvre, Olivier

    2016-03-01

    This study presents a comprehensive characterization of biochar obtained from simulated human feces (SHF) with a view to improve human waste sanitization and stabilization before usage as a resource. The possible applications of SHF are as a fuel, as a soil amendment, or for emerging applications (e.g., activated carbon precursor and odor control), depending on the charring conditions. Simulated human feces were charred under different conditions of peak temperature (200-800°C), heating rate (2-50°C min), and holding time (0.5-6.0 h); these parameters have been shown to have the largest influence on the thermal and physicochemical characteristics of the final product. The peak temperature was shown to have a higher impact than the heating rate or the holding time. At 200°C, the very mild structural changes of the product were characteristic of dry torrefaction, a process useful to remove moisture and sterilize the product. At 400°C the carbon content (76.2 ± 0.4) and the calorific heat value (30.6 ± 0.4 MJ kg) of the product increased by 60%. From 600°C onward, the improved degree of aromatization verified by Fourier transform infrared spectrometry (alkene [C=C] stretching around 1680-1450 cm) and C nuclear magnetic resonance (C=C stretching at 140-110 ppm) made the biochar increasingly suitable for carbon sequestration or commercial fabrication of briquettes of charcoal. In conclusion, SHF proved to be a suitable feedstock to produce a biochar whose characteristics depended mostly on the peak charring temperature. Ultimately, the selection of a suitable application may depend on local and sociological considerations.

  12. Characterization of adipocyte differentiation from human mesenchymal stem cells in bone marrow

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    Huang Hai-Yan

    2010-05-01

    Full Text Available Abstract Background Adipocyte hyperplasia is associated with obesity and arises due to adipogenic differentiation of resident multipotent stem cells in the vascular stroma of adipose tissue and remote stem cells of other organs. The mechanistic characterization of adipocyte differentiation has been researched in murine pre-adipocyte models (i.e. 3T3-L1 and 3T3-F442A, revealing that growth-arrest pre-adipocytes undergo mitotic clonal expansion and that regulation of the differentiation process relies on the sequential expression of three key transcription factors (C/EBPβ, C/EBPα and PPARγ. However, the mechanisms underlying adipocyte differentiation from multipotent stem cells, particularly human mesenchymal stem cells (hBMSCs, remain poorly understood. This study investigated cell cycle regulation and the roles of C/EBPβ, C/EBPα and PPARγ during adipocyte differentiation from hBMSCs. Results Utilising a BrdU incorporation assay and manual cell counting it was demonstrated that induction of adipocyte differentiation in culture resulted in 3T3-L1 pre-adipocytes but not hBMSCs undergoing mitotic clonal expansion. Knock-down and over-expression assays revealed that C/EBPβ, C/EBPα and PPARγ were required for adipocyte differentiation from hBMSCs. C/EBPβ and C/EBPα individually induced adipocyte differentiation in the presence of inducers; PPARγ alone initiated adipocyte differentiation but the cells failed to differentiate fully. Therefore, the roles of these transcription factors during human adipocyte differentiation are different from their respective roles in mouse. Conclusions The characteristics of hBMSCs during adipogenic differentiation are different from those of murine cells. These findings could be important in elucidating the mechanisms underlying human obesity further.

  13. Genomic characterization of large heterochromatic gaps in the human genome assembly.

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    Nicolas Altemose

    2014-05-01

    Full Text Available The largest gaps in the human genome assembly correspond to multi-megabase heterochromatic regions composed primarily of two related families of tandem repeats, Human Satellites 2 and 3 (HSat2,3. The abundance of repetitive DNA in these regions challenges standard mapping and assembly algorithms, and as a result, the sequence composition and potential biological functions of these regions remain largely unexplored. Furthermore, existing genomic tools designed to predict consensus-based descriptions of repeat families cannot be readily applied to complex satellite repeats such as HSat2,3, which lack a consistent repeat unit reference sequence. Here we present an alignment-free method to characterize complex satellites using whole-genome shotgun read datasets. Utilizing this approach, we classify HSat2,3 sequences into fourteen subfamilies and predict their chromosomal distributions, resulting in a comprehensive satellite reference database to further enable genomic studies of heterochromatic regions. We also identify 1.3 Mb of non-repetitive sequence interspersed with HSat2,3 across 17 unmapped assembly scaffolds, including eight annotated gene predictions. Finally, we apply our satellite reference database to high-throughput sequence data from 396 males to estimate array size variation of the predominant HSat3 array on the Y chromosome, confirming that satellite array sizes can vary between individuals over an order of magnitude (7 to 98 Mb and further demonstrating that array sizes are distributed differently within distinct Y haplogroups. In summary, we present a novel framework for generating initial reference databases for unassembled genomic regions enriched with complex satellite DNA, and we further demonstrate the utility of these reference databases for studying patterns of sequence variation within human populations.

  14. Hanford Defined Wastes model

    International Nuclear Information System (INIS)

    This paper describes the methodology behind the Hanford Defined Wastes (HDW) model for estimating the contents of Hanford high level waste (HLW) tanks. The HDW model is based on historical process and transaction histories for each tank and has four major components: Waste Status and Transaction Record Summary (WSTRS), Tank Layer Model (TLM), Supernatant Mixing Model (SMM), and HDW Compositions. Three examples of the application of HDW model estimates are described, including comparisons with global site inventories, comparisons with per tank assays, and comparisons of HDW TOC (Total Organic Carbon) estimates with existing hydrogen watch list tanks. The HDW model provides a cross check on existing assumptions for the global site inventory of wastes. Note that existing inventories for Hanford are based on much the same source information as the HDW model, chemicals used and process flowsheet data. Despite that, the HDW model predicts that the sodium inventory for Hanford tanks is 40,300 MT (metric tonnes), which is only 58% of the previous baseline estimate of 69,000 MT. There are other significant differences for inventories of chromium, iron, and nitrate as well. There are two causes for these differences; (1) previous neglect of chemical inventory placed into the ground at Hanford; (2) double counting attributed to tank inventory that was retrieved, reprocessed, and returned to the tanks. This double-counted inventory was counted once when it first went into the tanks and then again after it was reprocessed. The HDW model estimates also can provide a basis for targeting tanks for organic safety issues. In particular, the HDW model has shown that 88% of flammable gas watch list tanks have HDW estimated organic concentrations in excess of 0.64 wt% TOC. Derivation of variabilities for the HDW model estimates and other potential uses will also be outlined

  15. Expression of human DNA polymerase β in Escherichia coli and characterization of the recombinant enzyme

    International Nuclear Information System (INIS)

    The coding region of a human β-polymerase cDNA, predicting a 335 amino acid protein, was subcloned in the Escherichia coli expression plasmid pRC23. After induction of transformed cells, the crude soluble extract was found to contain a new protein immunoreactive with β-polymerase antibody and corresponding in size to the protein deduced from the cDNA. This protein was purified in a yield of 1-2 mg/50 g of cells. The recombinant protein had about the same DNA polymerase specific activity as β-polymerase purified from mammalian tissues, and template-primer specificity and immunological properties of the recombinant polymerase were similar to those of natural β-polymerases. The purified enzyme was free of nuclease activity. The authors studied detailed catalytic properties of the recombinant β-polymerase using defined template-primer systems. The results indicate that this β-polymerase is essentially identical with natural β-polymerases. The recombinant enzyme is distributive in mode of synthesis and is capable of detecting changes in the integrity of the single-stranded template, such as methylated bases and a double-stranded region. The enzyme recognizes a template region four to seven bases downstream of the primer 3' end and utilizes alternative primers if this downstream template region is double stranded. The enzyme is unable to synthesize past methylated bases N3-methyl-dT or O6-methyl-dG

  16. Mapping and characterization of non-HLA multigene assemblages in the human MHC class I region

    Energy Technology Data Exchange (ETDEWEB)

    Venditti, C.P.; Harris, J.M.; Geraghty, D.E. [Pennsylvania State Univ. College of Medicine, Hershey, PA (United States)] [and others

    1994-07-15

    The major histocompatibility complex (MHC) class I region has been shown to be associated with a variety of immune and nonimmune disorders. In an effort to initiate steps designed to identify the idiopathic hemochromatosis disease gene (HFE), the authors have cloned and mapped two expressed messages using probes from the HLA-H subregion that lie immediately distal to the HLA-A9 breakpoint. Although the cDNA clones identify distinct multifragment families that are dispersed throughout the MHC, the gene sequences from which the two cDNA clones derive map centromeric to the HLA-B locus and are absent from the genomes of higher nonhuman primates. This suggests that a syntenic coding segment arose within a highly polymorphic region (TNF to HLA-B interval) as the result of an insertion event following the emergence of Homo sapiens. An additional syntenic cluster exists within a peak of linkage disequilibrium with the HFE gene and may define coding sequences that underlie the defect in genetic iron overload. These data generally support the concept that the class I region is potentially gene-rich and further highlight the possibility that these new coding sequences may play a role in the development of a variety of HLA-linked diseases. The observations presented suggest that interlocus exchanges have played a structural role in the genesis of the human class I region. 46 refs., 6 refs.

  17. Human RNASET2 derivatives as potential anti-angiogenic agents: actin binding sequence identification and characterization

    Science.gov (United States)

    Nesiel-Nuttman, Liron; Doron, Shani; Schwartz, Betty; Shoseyov, Oded

    2015-01-01

    Human RNASET2 (hRNASET2) has been demonstrated to exert antiangiogenic and antitumorigenic effects independent of its ribonuclease capacity. We suggested that RNASET2 exerts its antiangiogenic and antitumorigenic activities via binding to actin and consequently inhibits cell motility. We focused herein on the identification of the actin binding site of hRNASET2 using defined sequences encountered within the whole hRNASET2 protein. For that purpose we designed 29 different hRNASET2-derived peptides. The 29 peptides were examined for their ability to bind immobilized actin. Two selected peptides-A103-Q159 consisting of 57 amino acids and peptide K108-K133 consisting of 26 amino acids were demonstrated to have the highest actin binding ability and concomitantly the most potent anti-angiogenic activity. Further analyses on the putative mechanisms associated with angiogenesis inhibition exerted by peptide K108-K133 involved its location during treatment within the HUVE cells. Peptide K108-K133 readily penetrates the cell membrane within 10 min of incubation. In addition, supplementation with angiogenin delays the entrance of peptide K108-K133 to the cell suggesting competition on the same cell internalization route. The peptide was demonstrated to co-localize with angiogenin, suggesting that both molecules bind analogous cellular epitopes, similar to our previously reported data for ACTIBIND and trT2-50. PMID:25815360

  18. Characterization of the virulence, growth temperature and antibiotic resistance of the Campylobacter jejuni IAL 2383 strain isolated from humans

    Directory of Open Access Journals (Sweden)

    B.B. Fonseca

    2014-01-01

    Full Text Available The objective of this study was to characterize the C. jejuni IAL2383 strain isolated from humans in Brazil. Transcripts for the racR, dnaJ and ciaB genes were found and flaA, plda and cadF genes were present in the genome and bacteria was sensitive to most of the important antimicrobials used to treat humans. C. jejuni IAL2383 is a good experimental model to analyze the interactions with cells.

  19. Phenotypic and functional characterization of human mammary stem/progenitor cells in long term culture.

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    Devaveena Dey

    Full Text Available BACKGROUND: Cancer stem cells exhibit close resemblance to normal stem cells in phenotype as well as function. Hence, studying normal stem cell behavior is important in understanding cancer pathogenesis. It has recently been shown that human breast stem cells can be enriched in suspension cultures as mammospheres. However, little is known about the behavior of these cells in long-term cultures. Since extensive self-renewal potential is the hallmark of stem cells, we undertook a detailed phenotypic and functional characterization of human mammospheres over long-term passages. METHODOLOGY: Single cell suspensions derived from human breast 'organoids' were seeded in ultra low attachment plates in serum free media. Resulting primary mammospheres after a week (termed T1 mammospheres were subjected to passaging every 7th day leading to the generation of T2, T3, and T4 mammospheres. PRINCIPAL FINDINGS: We show that primary mammospheres contain a distinct side-population (SP that displays a CD24(low/CD44(low phenotype, but fails to generate mammospheres. Instead, the mammosphere-initiating potential rests within the CD44(high/CD24(low cells, in keeping with the phenotype of breast cancer-initiating cells. In serial sphere formation assays we find that even though primary (T1 mammospheres show telomerase activity and fourth passage T4 spheres contain label-retaining cells, they fail to initiate new mammospheres beyond T5. With increasing passages, mammospheres showed an increase in smaller sized spheres, reduction in proliferation potential and sphere forming efficiency, and increased differentiation towards the myoepithelial lineage. Significantly, staining for senescence-associated beta-galactosidase activity revealed a dramatic increase in the number of senescent cells with passage, which might in part explain the inability to continuously generate mammospheres in culture. CONCLUSIONS: Thus, the self-renewal potential of human breast stem cells is

  20. Characterization of functional properties of Enterococcus faecium strains isolated from human gut.

    Science.gov (United States)

    İspirli, Hümeyra; Demirbaş, Fatmanur; Dertli, Enes

    2015-11-01

    The aim of this work was to characterize the functional properties of Enterococcus faecium strains identified after isolation from human faeces. Of these isolates, strain R13 showed the best resistance to low pH, bile salts, and survival in the simulated in vitro digestion assay, and demonstrated an important level of adhesion to hexadecane as a potential probiotic candidate. Analysis of the antibiotic resistance of E. faecium strains indicated that in general these isolates were sensitive to the tested antibiotics and no strain appeared to be resistant to vancomycin. Examination of the virulence determinants for E. faecium strains demonstrated that all strains contained the virulence genes common in gut- and food-originated enterococci, and strain R13 harboured the lowest number of virulence genes. Additionally, no strain contained the genes related to cytolysin metabolism and showed hemolytic activity. The antimicrobial role of E. faecium strains was tested against several pathogens, in which different levels of inhibitory effects were observed, and strain R13 was inhibitory to all tested pathogens. PCR screening of genes encoding enterocin A and B indicated the presence of these genes in E. faecium strains. Preliminary characterization of bacteriocins revealed that their activity was lost after proteolytic enzyme treatments, but no alteration in antimicrobial activity was observed at different pHs (3.5 to 9.5) and after heat treatments. In conclusion, this study revealed the functional characteristics of E. faecium R13 as a gut isolate, and this strain could be developed as a new probiotic after further tests.

  1. The characterization of Helicobacter pylori DNA associated with ancient human remains recovered from a Canadian glacier.

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    Treena Swanston

    Full Text Available Helicobacter pylori is a gram-negative bacterium that colonizes the stomach of nearly half of the world's population. Genotypic characterization of H. pylori strains involves the analysis of virulence-associated genes, such as vacA, which has multiple alleles. Previous phylogenetic analyses have revealed a connection between modern H. pylori strains and the movement of ancient human populations. In this study, H. pylori DNA was amplified from the stomach tissue of the Kwäday Dän Ts'ìnchi individual. This ancient individual was recovered from the Samuel Glacier in Tatshenshini-Alsek Park, British Columbia, Canada on the traditional territory of the Champagne and Aishihik First Nations and radiocarbon dated to a timeframe of approximately AD 1670 to 1850. This is the first ancient H. pylori strain to be characterized with vacA sequence data. The Tatshenshini H. pylori strain has a potential hybrid vacA m2a/m1d middle (m region allele and a vacA s2 signal (s region allele. A vacA s2 allele is more commonly identified with Western strains, and this suggests that European strains were present in northwestern Canada during the ancient individual's time. Phylogenetic analysis indicated that the vacA m1d region of the ancient strain clusters with previously published novel Native American strains that are closely related to Asian strains. This indicates a past connection between the Kwäday Dän Ts'ìnchi individual and the ancestors who arrived in the New World thousands of years ago.

  2. Differential oxidative status and immune characterization of the early and advanced stages of human breast cancer.

    Science.gov (United States)

    Panis, C; Victorino, V J; Herrera, A C S A; Freitas, L F; De Rossi, T; Campos, F C; Simão, A N Colado; Barbosa, D S; Pinge-Filho, P; Cecchini, R; Cecchini, A L

    2012-06-01

    Breast cancer is the malignant neoplasia with the highest incidence in women worldwide. Chronic oxidative stress and inflammation have been indicated as major mediators during carcinogenesis and cancer progression. Human studies have not considered the complexity of tumor biology during the stages of cancer advance, limiting their clinical application. The purpose of this study was to characterize systemic oxidative stress and immune response parameters in early (ED; TNM I and II) and advanced disease (AD; TNM III and IV) of patients diagnosed with infiltrative ductal carcinoma breast cancer. Oxidative stress parameters were evaluated by plasmatic lipoperoxidation, carbonyl content, thiobarbituric reactive substances (TBARS), nitric oxide levels (NO), total radical antioxidant parameter (TRAP), superoxide dismutase, and catalase activities and GSH levels. Immune evaluation was determined by TNF-α, IL-1β, IL-12, and IL-10 levels and leukocytes oxidative burst evaluation by chemiluminescence. Tissue damage analysis included heart (total CK and CKMB), liver (AST, ALT, GGT), and renal (creatinine, urea, and uric acid) plasmatic markers. C-reactive protein (CRP) and iron metabolism were also evaluated. Analysis of the results verified different oxidative stress statuses occur at distinct cancer stages. ED was characterized by reduction in catalase, 8-isoprostanes, and GSH levels, with enhanced lipid peroxidation and TBARS levels. AD exhibited more pronounced oxidative status, with reduction in catalase activity and TRAP, intense lipid peroxidation and high levels of NO, TBARs, and carbonyl content. ED patients presented a Th2 immune pattern, while AD exhibited Th1 status. CRP levels and ferritin were increased in both stages of disease. Leukocytes burst impairment was observed in both the groups. Plasma iron levels were significantly elevated in AD. The data obtained indicated that oxidative stress enhancement and immune response impairment may be necessary to ensure

  3. Generation and Characterization of Novel Local and Metastatic Human Neuroblastoma Variants

    Directory of Open Access Journals (Sweden)

    Ido Nevo

    2008-08-01

    Full Text Available Neuroblastoma (NB is the most commonly occurring solid tumor in children. The disease usually arises in the adrenal medulla, and it is characterized by a remarkable heterogeneity in its progression. Most NB patients with an advanced disease have massive bone marrow infiltration at diagnosis. Lung metastasis represents a widely disseminated stage and is typically considered to be a terminal event. Much like other malignancies, NB progression is a complex, multistep process. The expression, function, and significance of the various factors involved in NB progression must be studied in relevant in vivo and in vitro models. Currently, models consisting of metastatic and nonmetastatic cell variants of the same genetic background exist for several types of cancer; however, none exists for NB. In the present study, we describe the generation of a NB metastasis model. SH-SY5Y and MHH-NB-11 NB cells were inoculated orthotopically into the adrenal glands of athymic nude mice. Neuroblastoma cells metastasizing to the lungs were isolated from mice bearing adrenal tumors. Lung metastatic variants were generated by repeated cycles of in vivo passage. Characterization of these variants included cellular morphology and immunophenotyping in vitro, aggressiveness in vivo, and various biologic parameters in vitro. The NB metastatic variant in each model displayed unique properties, and both metastatic variants demonstrated a metastatic phenotype in vivo. These reproducible models of human NB metastasis will serve as an unlimited source of transcriptomic and proteomic material. Such models can facilitate future studies on NB metastasis and the identification of novel NB biomarkers and targets for therapy.

  4. Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment.

    Science.gov (United States)

    Kirley, Terence L; Norman, Andrew B

    2015-01-01

    Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region.

  5. Preparation, Characterization, and Determination of Immunological Activities of Transfer Factor Specific to Human Sperm Antigen

    Directory of Open Access Journals (Sweden)

    Jianwei Zhou

    2013-01-01

    Full Text Available Objective. The objective of this study was to prepare, characterize, and determine immunological activities of specific transfer factor (STF specific to human sperm antigen (HSA for the preparation of antisperm contraceptive vaccine that can be used as an immunocontraceptive. Methods. HSA-STF was prepared using the spleens of rabbits vaccinated with HSA. The specific immunological activities were examined by lymphocyte proliferation test (LPT, leukocyte adhesion inhibition test (LAIT, and by determining the concentrations of IL-4, γ-IFN, and IL-21. HSA-STF was a helveolous substance, having a pH value of 7.0±0.4 and UV absorption maxima at 258 ± 6 nm. It contained seventeen amino acids; glycine and glutamic acids were the highest in terms of concentrations (38.8 μg/mL and 36.3 μg/mL, resp.. Results. The concentration of polypeptide was 2.34±0.31 mg/mL, and ribose was 0.717±0.043 mg/mL. The stimulation index for lymphocyte proliferation test was 1.84, and the leukocyte adhesion inhibition rate was 37.7%. There was a statistically significant difference between the cultural lymphocytes with HSA-STF and non-HSA-STF for γ-IFN and IL-21 (P0.05. Conclusion. HSA-STF was prepared and characterized successfully. It had immunological activity which could transfer the immune response specific to HSA and prove to be a potential candidate for the development of male immunocontraceptive agents.

  6. Characterization of human mesenchymal stem cell secretome at early steps of adipocyte and osteoblast differentiation

    Directory of Open Access Journals (Sweden)

    Alessi Marie-Christine

    2008-02-01

    Full Text Available Abstract Background It is well established that adipose tissue plays a key role in energy storage and release but is also a secretory organ and a source of stem cells. Among different lineages, stem cells are able to differentiate into adipocytes and osteoblasts. As secreted proteins could regulate the balance between both lineages, we aimed at characterizing the secretome of human multipotent adipose-derived stem cell (hMADS at an early step of commitment to adipocytes and osteoblasts. Results A proteomic approach, using mono-dimensional electrophoresis and tandem mass spectrometry, allowed us to identify a total of 73 proteins at day 0 and day 3 of adipocyte and osteoblast differentiation. Analysis of identified proteins showed that 52 % corresponded to classical secreted proteins characterized by a signal peptide, that 37 % previously described in the extracellular compartment were devoid of signal peptide and that 11 % neither exhibited a signal peptide nor had been previously described extracellularly. These proteins were classified into 8 clusters according to their function. Quantitative analysis has been performed for 8 candidates: PAI-1, PEDF, BIGH3, PTX3, SPARC, ENO1, GRP78 and MMP2. Among them, PAI-1 was detected at day 0 and day 3 of osteoblast differentiation but never in adipocyte secretome. Furthermore we showed that PAI-1 mRNA was down-regulated in the bone of ovariectomized mice. Conclusion Given its regulation during the early events of hMADS cell differentiation and its status in ovariectomized mice, PAI-1 could play a role in the adipocyte/osteoblast balance and thus in bone diseases such as osteoporosis.

  7. Development, characterization, and photocytotoxicity assessment on human melanoma of chloroaluminum phthalocyanine nanocapsules

    Energy Technology Data Exchange (ETDEWEB)

    Siqueira-Moura, Marigilson P. [Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP), Universidade de São Paulo, Ribeirão Preto-SP (Brazil); Departamento de Química, Laboratório de Fotobiologia e Fotomedicina, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto (FFCLRP), Universidade de São Paulo, Ribeirão Preto-SP (Brazil); Primo, Fernando L. [Departamento de Química, Laboratório de Fotobiologia e Fotomedicina, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto (FFCLRP), Universidade de São Paulo, Ribeirão Preto-SP (Brazil); Espreafico, Enilza M. [Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto (FMRP), Universidade de São Paulo, Ribeirão Preto-SP (Brazil); Tedesco, Antonio C., E-mail: atedesco@usp.br [Departamento de Química, Laboratório de Fotobiologia e Fotomedicina, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto (FFCLRP), Universidade de São Paulo, Ribeirão Preto-SP (Brazil)

    2013-04-01

    In this work we have developed nanocapsules containing chloroaluminum phthalocyanine (ClAlPc) and assessed their phototoxic action on WM1552C, WM278, and WM1617 human melanoma cell lines. The ClAlPc-loaded nanocapsules were prepared by the nanoprecipitation method and optimized by means of a 2{sup 3} full factorial design. The ClAlPc nanocapsules were characterized by particle size and distribution, zeta potential, morphology, encapsulation efficiency, singlet oxygen production, stability, and phototoxic action on melanoma cells. Both the development and optimization studies revealed that stable colloidal formulations could be obtained by using 1.75% (w/v) soybean lecithin, 1.25% (w/v) Poloxamer 188, 2.5% (v/v) soybean oil, and 0.75% (w/v) poly(D,L-lactide-co-glycolide). The nanocapsules had a mean diameter of 230 nm, homogeneous size distribution (polydispersity index < 0.3), and negative zeta potential (about − 30 mV). Their morphology was spherical, with evident polymer membrane coating droplet. The encapsulation efficiency was 70%, as expected for hydrophobic drugs, and the nanoencapsulated ClAlPc was able to produce high singlet oxygen quantum yield. ClAlPc nanocapsules exhibited good physical stability over a 12-month period. WM1552C primary melanoma cells were more sensitive (p < 0.05) to the phototoxic effect elicited by ClAlPc nanocapsules (0.3 μg ml{sup −1}) under light irradiation at 20 mJ cm{sup −2}. On the other hand, the cell survival percentage for all the melanoma cell lines treated with the highest light dose (150 mJ cm{sup −2}) was lower than 10%. In summary, ClAlPc nanoencapsulation could enable application of this hydrophobic photosensitizer in the treatment of malignant melanoma with the use of both low sensitizer drug concentration and light dose. - Highlights: ► Nanocapsules containing a hydrophobic metallophthalocyanine (ClAlPc) were developed. ► The colloidal formulations were characterized by their physicochemical parameters

  8. Establishment and characterization of a novel human cholangiocarcinoma cell line with high metastatic activity.

    Science.gov (United States)

    Uthaisar, Kwuntida; Vaeteewoottacharn, Kulthida; Seubwai, Wunchana; Talabnin, Chutima; Sawanyawisuth, Kanlayanee; Obchoei, Sumalee; Kraiklang, Ratthaphol; Okada, Seiji; Wongkham, Sopit

    2016-09-01

    Cholangiocarcinoma (CCA) is a highly metastatic tumor, and the lung is a common site of metastasis. A greater understanding of the biology of metastases is needed to improve treatment outcomes. Herein, a highly metastatic human CCA subline, KKU-213L5 from an original cell line, KKU-213 that has marginally metastatic ability, was established and characterized. KKU-213L5 was selected in vivo through the fifth serial passage of pulmonary metastasized tissues via tail-vein injection in NOD/scid/Jak3 mice. The metastatic abilities of the KKU-213L5 cells were compared with the parental line in vitro and in vivo. The expression profile of this metastatic cell line was determined using real-time PCR. KKU-213L5 cells were found to possess higher metastatic phenotypes, i.e., growth rates, stem cell surface markers (CD133), migration and invasion characteristics when compared with the parental cells. Compared to the KKU-213 cells, KKU-213L5 cells formed larger tumors in subcutaneous xenografted mice and had a >10-fold increase in lung metastases in the tail-vein injected metastatic mouse model. Mice injected intravenously with KKU-213L5 cells had a significantly shorter survival. Analysis of the expressed genes related to progression of cancer revealed significant upregulation of anterior gradient protein-2 (AGR2) and suppression of KiSS-1 in the KKU-213L5 cells. The association of these two genes with metastasis was affirmed in CCA patient tissues since increased AGR2 expression and decreased KiSS-1 expression were found in higher stage patient tumors. In conclusion, a highly metastatic human CCA cell line was established and characterized. It is plausible that the differential expression between the parental KKU-213 and highly metastatic KKU-213L5 cells may be beneficial to classify novel genes associated with metastasis. The KKU-213L5 cell line should serve as a valued device for discovering the molecular mechanisms of CCA metastasis and enabling the search for an

  9. Characterization of the Drosophila ortholog of the human Usher Syndrome type 1G protein sans.

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    Fabio Demontis

    Full Text Available BACKGROUND: The Usher syndrome (USH is the most frequent deaf-blindness hereditary disease in humans. Deafness is attributed to the disorganization of stereocilia in the inner ear. USH1, the most severe subtype, is associated with mutations in genes encoding myosin VIIa, harmonin, cadherin 23, protocadherin 15, and sans. Myosin VIIa, harmonin, cadherin 23, and protocadherin 15 physically interact in vitro and localize to stereocilia tips in vivo, indicating that they form functional complexes. Sans, in contrast, localizes to vesicle-like structures beneath the apical membrane of stereocilia-displaying hair cells. How mutations in sans result in deafness and blindness is not well understood. Orthologs of myosin VIIa and protocadherin 15 have been identified in Drosophila melanogaster and their genetic analysis has identified essential roles in auditory perception and microvilli morphogenesis, respectively. PRINCIPAL FINDINGS: Here, we have identified and characterized the Drosophila ortholog of human sans. Drosophila Sans is expressed in tubular organs of the embryo, in lens-secreting cone cells of the adult eye, and in microvilli-displaying follicle cells during oogenesis. Sans mutants are viable, fertile, and mutant follicle cells appear to form microvilli, indicating that Sans is dispensable for fly development and microvilli morphogenesis in the follicle epithelium. In follicle cells, Sans protein localizes, similar to its vertebrate ortholog, to intracellular punctate structures, which we have identified as early endosomes associated with the syntaxin Avalanche. CONCLUSIONS: Our work is consistent with an evolutionary conserved function of Sans in vesicle trafficking. Furthermore it provides a significant basis for further understanding of the role of this Usher syndrome ortholog in development and disease.

  10. Molecular and functional characterization of a human ATM gene analogue at Arabidopsis thaliana

    International Nuclear Information System (INIS)

    The human ATM gene, whose inactivation is responsible for the human disease ataxia telangiectasia is conserved throughout the Eukaryotes and plays an important role in the cellular responses to DNA damage, in particular to DNA double-strand breaks (DSBs). Here we describe the identification of an Arabidopsis thaliana homologue of ATM (AtATM), and the molecular and cytological characterization of plants, hereafter called atm, carrying a disrupting T-DNA insertion in this gene. AtATM covers a 32 kb region on chromosome 3. The AtATM transcript has a complex structure, is 12 kb long and formed by 79 exons. The transcriptional level of AtATM is very low in all the tissues tested, and does not vary after exposure to ionizing radiations (IR). In atm plants, the protein is not detected suggesting the mutants are null. The atm mutants are partially sterile. Aberrant segregation of chromosomes during meiosis I on both male and female sides account for this sterility. However, meiotic recombination frequency is normal. Mutant plants are also hypersensitive to gamma rays and methyl methane sulfonate, but not to UV-B, pointing to a specific defect of atm mutants in the response to DNA DSBs. In plants, ionizing radiations induce a strong, rapid and transient transcriptional activation of genes involved in the cellular response to or the repair of DSBs. This transcriptional regulation of AtRAD51, AtPARP1, atGR1 and AtL1G4 is lost in the atm mutants . The absence of AtRAD51 induction associated with ionizing radiation sensitivity suggest that AtAtm play an important function in DSB repair by homologous recombination. In addition we show that homologous intra-chromosomal recombination frequency is elevated in the mutant comparing to wild-type, with or without gamma irradiation. These results show the implication of AtAtm in the genomic stability. (author)

  11. Assessment and characterization of biofilm formation among human isolates of Streptococcus dysgalactiae subsp. equisimilis.

    Science.gov (United States)

    Genteluci, Gabrielle Limeira; Silva, Ligia Guedes; Souza, Maria Clara; Glatthardt, Thaís; de Mattos, Marcos Corrêa; Ejzemberg, Regina; Alviano, Celuta Sales; Figueiredo, Agnes Marie Sá; Ferreira-Carvalho, Bernadete Teixeira

    2015-12-01

    The capacity to form biofilm is considered a protective mechanism that allows the bacteria to survive and proliferate in hostile environments, facilitating the maintenance of the infectious process. Recently, biofilm has become a topic of interest in the study of the human pathogen group A Streptococcus (GAS). Although GAS has not been associated with infection on medical implants, the presence of microcolonies embedded in an extracellular matrix on infected tissues has been reported. Despite the similarity between GAS and Streptococcus dysgalactiae subspecies equisimilis (SDSE), there are no studies in the literature describing the production of biofilm by SDSE. In this work, we assessed and characterized biofilm development among SDSE human isolates of group C. The in vitro data showed that 59.3% of the 118 isolates tested were able to form acid-induced biofilm on glass, and 28% formed it on polystyrene surfaces. More importantly, biofilm was also formed in a foreign body model in mice. The biofilm structure was analyzed by confocal laser scanning microscopy, transmission electron microscopy, and scanning electron microscopy. Long fibrillar-like structures were observed by scanning electron microscopy. Additionally, the expression of a pilus associated gene of SDSE was increased for in vitro sessile cells compared with planktonics, and when sessile cells were collected from biofilms formed in the animal model compared with that of in vitro model. Results obtained from the immunofluorescence microscopy indicated the biofilm was immunogenic. Our data also suggested a role for proteins, exopolysaccharide and extracellular DNA in the formation and accumulation of biofilm by SDSE.

  12. Diffuse Optical Characterization of the Healthy Human Thyroid Tissue and Two Pathological Case Studies.

    Directory of Open Access Journals (Sweden)

    Claus Lindner

    Full Text Available The in vivo optical and hemodynamic properties of the healthy (n = 22 and pathological (n = 2 human thyroid tissue were measured non-invasively using a custom time-resolved spectroscopy (TRS and diffuse correlation spectroscopy (DCS system. Medical ultrasound was used to guide the placement of the hand-held hybrid optical probe. TRS measured the absorption and reduced scattering coefficients (μa, μs' at three wavelengths (690, 785 and 830 nm to derive total hemoglobin concentration (THC and oxygen saturation (StO2. DCS measured the microvascular blood flow index (BFI. Their dependencies on physiological and clinical parameters and positions along the thyroid were investigated and compared to the surrounding sternocleidomastoid muscle. The THC in the thyroid ranged from 131.9 μM to 144.8 μM, showing a 25-44% increase compared to the surrounding sternocleidomastoid muscle tissue. The blood flow was significantly higher in the thyroid (BFIthyroid = 16.0 × 10-9 cm2/s compared to the muscle (BFImuscle = 7.8 × 10-9 cm2/s, while StO2 showed a small (StO2, muscle = 63.8% to StO2, thyroid = 68.4%, yet significant difference. Two case studies with thyroid nodules underwent the same measurement protocol prior to thyroidectomy. Their THC and BFI reached values around 226.5 μM and 62.8 × 10-9 cm2/s respectively showing a clear contrast to the nodule-free thyroid tissue as well as the general population. The initial characterization of the healthy and pathologic human thyroid tissue lays the ground work for the future investigation on the use of diffuse optics in thyroid cancer screening.

  13. Diffuse Optical Characterization of the Healthy Human Thyroid Tissue and Two Pathological Case Studies.

    Science.gov (United States)

    Lindner, Claus; Mora, Mireia; Farzam, Parisa; Squarcia, Mattia; Johansson, Johannes; Weigel, Udo M; Halperin, Irene; Hanzu, Felicia A; Durduran, Turgut

    2016-01-01

    The in vivo optical and hemodynamic properties of the healthy (n = 22) and pathological (n = 2) human thyroid tissue were measured non-invasively using a custom time-resolved spectroscopy (TRS) and diffuse correlation spectroscopy (DCS) system. Medical ultrasound was used to guide the placement of the hand-held hybrid optical probe. TRS measured the absorption and reduced scattering coefficients (μa, μs') at three wavelengths (690, 785 and 830 nm) to derive total hemoglobin concentration (THC) and oxygen saturation (StO2). DCS measured the microvascular blood flow index (BFI). Their dependencies on physiological and clinical parameters and positions along the thyroid were investigated and compared to the surrounding sternocleidomastoid muscle. The THC in the thyroid ranged from 131.9 μM to 144.8 μM, showing a 25-44% increase compared to the surrounding sternocleidomastoid muscle tissue. The blood flow was significantly higher in the thyroid (BFIthyroid = 16.0 × 10-9 cm2/s) compared to the muscle (BFImuscle = 7.8 × 10-9 cm2/s), while StO2 showed a small (StO2, muscle = 63.8% to StO2, thyroid = 68.4%), yet significant difference. Two case studies with thyroid nodules underwent the same measurement protocol prior to thyroidectomy. Their THC and BFI reached values around 226.5 μM and 62.8 × 10-9 cm2/s respectively showing a clear contrast to the nodule-free thyroid tissue as well as the general population. The initial characterization of the healthy and pathologic human thyroid tissue lays the ground work for the future investigation on the use of diffuse optics in thyroid cancer screening. PMID:26815533

  14. Cytogenetic characterization of low-dose hyper-radiosensitivity in Cobalt-60 irradiated human lymphoblastoid cells

    Energy Technology Data Exchange (ETDEWEB)

    Joshi, Gnanada S. [Department of Biological Sciences, Wayne State University, Detroit, MI 48202 (United States); Joiner, Michael C. [Department of Radiation Oncology, Wayne State University, Detroit, MI 48201 (United States); Tucker, James D., E-mail: jtucker@biology.biosci.wayne.edu [Department of Biological Sciences, Wayne State University, Detroit, MI 48202 (United States)

    2014-12-15

    Highlights: • Human cells were irradiated in G1 or G2 and evaluated for micronuclei and bridges. • Cells irradiated in G2 but not in G1 exhibit low dose hyper-radiosensitivity. • Response curves of cells irradiated in G2 do not fit a linear-no-threshold model. • Response curves of cells irradiated in G1 fit a linear-no-threshold model. - Abstract: The dose-effect relationships of cells exposed to ionizing radiation are frequently described by linear quadratic (LQ) models over an extended dose range. However, many mammalian cell lines, when acutely irradiated in G2 at doses ≤0.3 Gy, show hyper-radiosensitivity (HRS) as measured by reduced clonogenic cell survival, thereby indicating greater cell lethality than is predicted by extrapolation from high-dose responses. We therefore hypothesized that the cytogenetic response in G2 cells to low doses would also be steeper than predicted by LQ extrapolation from high doses. We tested our hypothesis by exposing four normal human lymphoblastoid cell lines to 0–400 cGy of Cobalt-60 gamma radiation. The cytokinesis block micronucleus assay was used to determine the frequencies of micronuclei and nucleoplasmic bridges. To characterize the dependence of the cytogenetic damage on dose, univariate and multivariate regression analyses were used to compare the responses in the low- (HRS) and high-dose response regions. Our data indicate that the slope of the response for all four cell lines at ≤20 cGy during G2 is greater than predicted by an LQ extrapolation from the high-dose responses for both micronuclei and bridges. These results suggest that the biological consequences of low-dose exposures could be underestimated and may not provide accurate risk assessments following such exposures.

  15. PREPARATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODY AGAINST HUMAN TELOMERASE REVERSE TRANSCRIPTASE

    Institute of Scientific and Technical Information of China (English)

    王俊梅; 张波; 杨邵敏; 韩继生; 李冰思; 侯琳

    2003-01-01

    Objective. To develop monoclonal antibodies against the catalytic subunit of human telomerase reverse transcriptase (hTERT) for its expression detection of human tumors. Methods. A dominant epitope in hTERT (peptide hTERT7)was automatically synthesized based on Fmoc method, and was used to immunize Balb/c mice. Hybridomas were generated and screened by ELISA for specific monoclonal antibodies, and the characterization was performed by Western blotting and immunohistochemical staining. The heavy chain variable region of antibody was cloned by RT-PCR and sequenced. Results. Antigenic peptide hTERT7 was synthesized and confirmed by MALDI-TOF-MS and HPLC analysis. One hybridoma cell line secreting anti-hTERT7 antibodies designated as M2 was established after primary screening and consequent 3 rounds of limited dilution. M2 was IgG1 in isotyping. The competi tive assay showed that the M2 antibody was hTERT7 -specific, and the affinity constant was about 1×106 mol-1. The antibody reacted with cell extracts from HeLa cancer cells but not with those from normal 2BS cells in ELISA assay. For in situ staining of immunohistochemistry, the positive staining presented in the nuclear compartment of HeLa, while 2BS was negative. The heavy chain variable region from M2 re vealed that the monoclonal antibody was mouse origin. Conclusions. The developed mouse monoclonal antibody is hTERT-specific and able to recognize native cellular hTERT in ELISA and immunohistochemistry, which makes the immuno-detection of telom erase hTERT expression in cancer cells or tissues possible.

  16. Structural analysis and promoter characterization of the human collagenase-3 gene (MMP13)

    Energy Technology Data Exchange (ETDEWEB)

    Pendas, A.M.; Balbin, M.; Llano, E. [Universidad de Oviedo (Spain)] [and others

    1997-03-01

    Human collagenase-3 (MMP13) is a recently identified member of the matrix metalloproteinase (MMP) family that is expressed in breast carcinomas and in articular cartilage from arthritic patients. In this work we have isolated and characterized genomic clones coding for human collagenase-3. This gene is composed of 10 exons and 9 introns and spans over 12.5 kb. The overall organization of the collagenase-3 gene is similar to that of other MMP genes clustered at chromosome 11q22, including fibroblast collagenase (MMP-1), matrilysin (MMP-7), and macrophage metalloelastase (MMP-12), but is more distantly related to genes coding for stromelysin-3 (MMP-11), gelatinase-A (MMP-2), and gelatinase-B (MMP-9), which map outside of this gene cluster. Nucleotide sequence analysis of about 1 kb of the 5{prime}-flanking region of the collagenase-3 gene revealed the presence of a TATA box, an AP-1 motif, a PEA-3 consensus sequence, an osteoblast specific element (OSE-2), and a TGF-{beta} inhibitory element. Transient transfection experiments in HeLa and COS-1 cells with chloramphenicol acetyltransferase (CAT)-containing constructs showed that the AP-1 site is functional and responsible for the observed inducibility of the reporter gene by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). However, and in contrast to other MMP genes, no significative synergistic effect on CAT activity between the AP-1 and PEA-3 elements found in the collagenase-3 gene promoter was found. DNA binding analysis with nuclear extracts from HeLa cells revealed the formation of specific complexes between collagenase-3 promoter sequences containing the AP-1 site and nuclear proteins. The presence of this AP-1 functional site, which is able to confer responsiveness to a variety of tumor promoters and oncogene products, may contribute to explaining the high-level expression of collagenase-3 in breast carcinomas and degenerative joint diseases. 48 refs., 5 figs., 2 tabs.

  17. Purification of full-length human Pregnane and Xenobiotic Receptor: polyclonal antibody preparation for immunological characterization

    Institute of Scientific and Technical Information of China (English)

    Mallampati SARADHI; Biji KRISHNA; Gauranga MUKHOPADHYAY; Rakesh K TYAGI

    2005-01-01

    Pregnane and Xenobiotic Receptor (PXR; or Steroid and Xenobiotic Receptor, SXR), a new member of the nuclear receptor superfamily, is thought to modulate a network of genes that are involved in xenobiotic metabolism and elimination. To further explore the role of PXR in body's homeostatic mechanisms, we for the first time, report successful prokaryotic expression and purification of full-length PXR and preparation of polyclonal antibody against the whole protein. Thefull-length cDNA encoding a 434 amino acids protein was sub-cloned into prokaryotic expression vector, pET-30b and transformed into E. coli BL21 (DE3) cells for efficient over expression. The inclusion body fraction, containing the expressed recombinant protein, was purified first by solubilizing in sarcosine extraction buffer and then by affinity column chromatography using Ni-NTA His-Bind matrix. The efficacy of anti-PXR antibody was confirmed by immunocytology, Western blot analysis, EMSA and immunohistochemistry. The antibody obtained was capable of detecting human and mouse PXR with high specificity and sensitivity. Immunofluorescence staining of COS-1 cells transfected with human or mouse PXR showed a clear nuclear localization. Results from immunohistochemistry showed that level of PXR in liver sections is immunologically detectable in the nuclei. Similar to exogenously transfected PXR, Western blot analysis of cell extract from HepG2 and COLO320DM cells revealed a major protein band for endogenous PXR having the expected molecular weight of 50 kDa. Relevance of other immunodetectable bands with reference to PXR isoforms and current testimony are evaluated. Advantages of antibody raised against full-length PXR protein for functional characterization of receptor is discussed and its application for clinical purposes is envisaged.

  18. Reporter Cell Lines for the Characterization of the Interactions between Human Nuclear Receptors and Endocrine Disruptors.

    Science.gov (United States)

    Grimaldi, Marina; Boulahtouf, Abdelhay; Delfosse, Vanessa; Thouennon, Erwan; Bourguet, William; Balaguer, Patrick

    2015-01-01

    Endocrine-disrupting chemicals (EDCs) are exogenous substances interfering with hormone biosynthesis, metabolism, or action, and consequently causing disturbances in the endocrine system. Various pathways are activated by EDCs, including interactions with nuclear receptors (NRs), which are primary targets of numerous environmental contaminants. The main NRs targeted by environmental contaminants are the estrogen (ER α, β) and the androgen (AR) receptors. ERs and AR have pleiotropic regulatory roles in a diverse range of tissues, notably in the mammary gland, the uterus, and the prostate. Thus, dysfunctional ERs and AR signaling due to inappropriate exposure to environmental pollutants may lead to hormonal cancers and infertility. The pregnane X receptor (PXR) is also recognized by many environmental molecules. PXR has a protective role of the body through its ability to regulate proteins involved in the metabolism, the conjugation, and the transport of many exogenous and endogenous compounds. However, the permanent activation of this receptor by xenobiotics may lead to premature drug metabolism, the formation, and accumulation of toxic metabolites and defects in hormones homeostasis. The activity of other NRs can also be affected by environmental molecules. Compounds capable of inhibiting or activating the estrogen related (ERRγ), the thyroid hormone (TRα, β), the retinoid X receptors (RXRα, β, γ), and peroxisome proliferator-activated (PPAR α, γ) receptors have been identified and are highly suspected to promote developmental, reproductive, neurological, or metabolic diseases in humans and wildlife. In this review, we provide an overview of reporter cell lines established to characterize the human NR activities of a large panel of EDCs including natural as well as industrial compounds such as pesticides, plasticizers, surfactants, flame retardants, and cosmetics. PMID:26029163

  19. Characterization of the human myocardial proteome in inflammatory dilated cardiomyopathy by label-free quantitative shotgun proteomics of heart biopsies

    NARCIS (Netherlands)

    Hammer, E.; Goritzka, M.; Ameling, S.; Darm, K.; Steil, L.; Klingel, K.; Trimpert, C.; Herda, L.R.; Dorr, M.; Kroemer, H.K.; Kandolf, R.; Staudt, A.; Felix, S.B.; Volker, U.

    2011-01-01

    Dilated cardiomyopathy (DCM) is characterized by contractile dysfunction leading to heart failure. The molecular changes in the human heart associated with this disease have so far mostly been addressed at the gene expression level and only a few studies have analyzed global changes in the myocardia

  20. Characterization of connective tissue growth factor expression in primary cultures of human tubular epithelial cells: modulation by hypoxia

    NARCIS (Netherlands)

    S. Kroening; E. Neubauer; B. Wullich; J. Aten; M. Goppelt-Struebe

    2010-01-01

    Kroening S, Neubauer E, Wullich B, Aten J, Goppelt-Struebe M. Characterization of connective tissue growth factor expression in primary cultures of human tubular epithelial cells: modulation by hypoxia. Am J Physiol Renal Physiol 298:F796-F806, 2010. First published December 23, 2009; doi:10.1152/aj

  1. Human neutrophil elastase peptide sensors conjugated to cellulosic and nanocellulosic materials: part I, synthesis and characterization of fluorescent analogs

    Science.gov (United States)

    Here we describe the synthesis and characterization of peptide conjugated cellulose and nanocellulose materials as sensors for fluorescent detection of human neutrophil elastase (HNE). The cellulose sensor surfaces selected are filter paper (FP) and print cloth (PC) fabric, which are composed of pro...

  2. Characterization of human pseudogene-derived non-coding RNAs for functional potential.

    Directory of Open Access Journals (Sweden)

    Xingyi Guo

    Full Text Available Thousands of pseudogenes exist in the human genome and many are transcribed, but their functional potential remains elusive and understudied. To explore these issues systematically, we first developed a computational pipeline to identify transcribed pseudogenes from RNA-Seq data. Applying the pipeline to datasets from 16 distinct normal human tissues identified ∼ 3,000 pseudogenes that could produce non-coding RNAs in a manner of low abundance but high tissue specificity under normal physiological conditions. Cross-tissue comparison revealed that the transcriptional profiles of pseudogenes and their parent genes showed mostly positive correlations, suggesting that pseudogene transcription could have a positive effect on the expression of their parent genes, perhaps by functioning as competing endogenous RNAs (ceRNAs, as previously suggested and demonstrated with the PTEN pseudogene, PTENP1. Our analysis of the ENCODE project data also found many transcriptionally active pseudogenes in the GM12878 and K562 cell lines; moreover, it showed that many human pseudogenes produced small RNAs (sRNAs and some pseudogene-derived sRNAs, especially those from antisense strands, exhibited evidence of interfering with gene expression. Further integrated analysis of transcriptomics and epigenomics data, however, demonstrated that trimethylation of histone 3 at lysine 9 (H3K9me3, a posttranslational modification typically associated with gene repression and heterochromatin, was enriched at many transcribed pseudogenes in a transcription-level dependent manner in the two cell lines. The H3K9me3 enrichment was more prominent in pseudogenes that produced sRNAs at pseudogene loci and their adjacent regions, an observation further supported by the co-enrichment of SETDB1 (a H3K9 methyltransferase, suggesting that pseudogene sRNAs may have a role in regional chromatin repression. Taken together, our comprehensive and systematic characterization of pseudogene

  3. Biochemical characterization of the nucleic acids of some human and animal viruses

    International Nuclear Information System (INIS)

    The isolation and partial characterization of human polyomaviruses from a number of renal transplant patients is described. These isolates proved refractory to cell culture propagation by the methods used, and were thus extracted directly from large volumes of patient's urine. This approach has the advantage that the virus cannot undergo any possible genomic modification. The quantity of DNA obtained directly from urine was asually very limited. In order to produce adequate DNA for complete analysis, viral DNA was recombined with a bacterial vector and then cloned. This clone was used to prepare radioactively-labelled DNA probes for the detection of BK-specific sequences in urine isolates and in subsequent recombinants with patient material. The genomes of four rotaviruses were also studied. Experiments were performed to confirm the double-strainded RNA (dsRNA) nature of the Simian agents II genome. The difficulties in obtaining precise molecular weight values for rotavirus genome segments are also discussed. Gel systems were developed to improve on the resolution obtained in co-electrophoresis experiments. During attempts to culture Simian agents II and offal agent viruses in cell culture, it was observed that treatment of the cells and/or virus with versene-trypsin solution during infection gave a marked increase in virus yield

  4. X-ray characterization of mesophases of human telomeric G-quadruplexes and other DNA analogues

    Science.gov (United States)

    Yasar, Selcuk; Schimelman, Jacob B.; Aksoyoglu, M. Alphan; Steinmetz, Nicole F.; French, Roger H.; Parsegian, V. Adrian; Podgornik, Rudolf

    2016-06-01

    Observed in the folds of guanine-rich oligonucleotides, non-canonical G-quadruplex structures are based on G-quartets formed by hydrogen bonding and cation-coordination of guanosines. In dilute 5‧-guanosine monophosphate (GMP) solutions, G-quartets form by the self-assembly of four GMP nucleotides. We use x-ray diffraction to characterize the columnar liquid-crystalline mesophases in concentrated solutions of various model G-quadruplexes. We then probe the transitions between mesophases by varying the PEG solution osmotic pressure, thus mimicking in vivo molecular crowding conditions. Using the GMP-quadruplex, built by the stacking of G-quartets with no covalent linking between them, as the baseline, we report the liquid-crystalline phase behaviors of two other related G-quadruplexes: (i) the intramolecular parallel-stranded G-quadruplex formed by the 22-mer four-repeat human telomeric sequence AG3(TTAG3)3 and (ii) the intermolecular parallel-stranded G-quadruplex formed by the TG4T oligonucleotides. Finally, we compare the mesophases of the G-quadruplexes, under PEG-induced crowding conditions, with the corresponding mesophases of the canonical duplex and triplex DNA analogues.

  5. Y chromosome and mitochondrial DNA characterization of Pasiegos, a human isolate from Cantabria (Spain).

    Science.gov (United States)

    Maca-Meyer, N; Sánchez-Velasco, P; Flores, C; Larruga, J-M; González, A-M; Oterino, A; Leyva-Cobián, F

    2003-07-01

    Mitochondrial DNA sequences and Y chromosome haplotypes were characterized in Pasiegos, a human isolate from Cantabria, and compared with those of other Cantabrian and neighbouring Northern Spain populations. Cantabria appears to be a genetically heterogeneous community. Whereas Lebaniegos do not differ from their eastern Basque and western Asturian and Galician neighbours, Pasiegos and other non-Lebaniego Cantabrians show significant differences with all of them. Pasiegos are peculiar for their high frequencies of Y chromosomal markers (E-M81) with North African assignation, and Y chromosomal (R-SRY2627) and mtDNA (V, I, U5) markers related to northern European populations. This dual geographic contribution is more in agreement with the complex demographic history of this isolate, as opposed to recent drift effects. The high incidence in Cantabrians with pre-V and V mtDNA haplotypes, considered as a signal of Postglacial recolonization in Europe from south-western refugees, points to such refugees as a better candidate population than Basques for this expansion. However, this does not discount a conjoint recolonization. PMID:12914567

  6. Isolation of human salivary extracellular vesicles by iodixanol density gradient ultracentrifugation and their characterizations

    Directory of Open Access Journals (Sweden)

    Kazuya Iwai

    2016-05-01

    Full Text Available Diagnostic methods that focus on the extracellular vesicles (EVs present in saliva have been attracting great attention because of their non-invasiveness. EVs contain biomolecules such as proteins, messenger RNA (mRNA and microRNA (miRNA, which originate from cells that release EVs, making them an ideal source for liquid biopsy. Although there have been many reports on density-based fractionation of EVs from blood and urine, the number of reports on EVs from saliva has been limited, most probably because of the difficulties in separating EVs from viscous saliva using density gradient centrifugation. This article establishes a protocol for the isolation of EVs from human saliva using density gradient centrifugation. The fractionated salivary EVs were characterized by atomic force microscopy, western blot and reverse transcription polymerase chain reaction. The results indicate that salivary EVs have a smaller diameter (47.8±12.3 nm and higher density (1.11 g/ml than EVs isolated from conditioned cell media (74.0±23.5 nm and 1.06 g/ml, respectively. Additionally, to improve the throughput of density-based fractionation of EVs, the original protocol was further modified by using a fixed angle rotor instead of a swinging rotor. It was also confirmed that several miRNAs were expressed strongly in the EV-marker-expressing fractions.

  7. Characterization of dimeric forms of human pituitary growth hormone by bioassay, radioreceptor assay, and radioimmunoassay

    International Nuclear Information System (INIS)

    Seven highly purified dimeric forms of human pituitary growth hormone, composed of the monomeric forms 20 K hGH, 22K hGH and 24 k hBH linked together by noncovalent or covalent bounds, have been characterized by an in vitro bioassay (the Nb2 assay), a radioreceptor assay and a radioimmunoassay. Considerable differences in the ability to displace labelled recombinant hGH were observed in the radioreceptor assay. The seven dimeric forms varied over a range between 22 K hGH (most effective) and 20 K hGH. The three covalently-linked dimeric forms had nearly identical affinity constants. The mitogenic action of all but one of the hGH dimers in the Nb2 assay was in the same mutual order as the receptor binding activity in the radioreceptor assay. In the RIA, all dose-response curves were parallel except for those obtained with 20 K hGH and with the dimeric form (20 K-20 K)hGH. In this assay, dimeric variants of the constituents 22 K hGH and 24 K hGH were approximately twice as active as 22 K hGH on a molar basis, suggesting about the same affinity between the the antibodies and each of the monomeric forms. Determination of the amino acid compositions of the dimeric forms provided support for their content of monomeric constituents as established earlier by electrophoretic analysis. (author)

  8. Refolding and Characterization of Recombinant Human GST-PD-1 Fusion Protein Expressed in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    Da-Wei LI; Jian-Feng YU; Yong-Jing CHEN; Hong-Bing MA; Zheng-Fei WANG; Yi-Bei ZHU; Xue-Guang ZHANG

    2004-01-01

    Programmed death-1 (PD-1) is a costimulatory molecule of CD28 family expressed onactivated T, B and myeloid cells. The engagement of PD-1 with its two ligands, PD-L1 and PD-L2, inhibitsproliferation of T cell and production of a series of its cytokines. The blockade of PD-1 pathway is involvedin antiviral and antitumoral immunity. In this study, human PD-1 cDNA encoding extracellular domain wasamplified and cloned into expression plasmid pGEX-5x-3. The fusion protein GST-PD-1 was effectivelyexpressed in E. coli BL21 (DE3) as inclusion bodies and a denaturation and refolding procedure was performed to obtain bioactive soluble GST-PD-1. Fusion protein of above 95% purity was acquired by a convenient two-step purification using GST affinity and size exclusion columns. Furthermore, a PD-L1-dependentin vitro bioassay method was set up to characterize GST-PD-1 bioactivity. The results suggested that GSTPD-1 could competently block the interaction between PD-L1 and PD-1 and increase the production of IL2 and IFN-γ of phytohemagglutinin-activated T cells.

  9. Refoiding and Characterization of Recombinant Human GST-PD-1 Fusion Protein Expressed in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    Da-WeiLI; Jian-FengYU; Yong-JingCHEN; Hong-BingMA; Zheng-FeiWANG; Yi-BeiZHU; Xue-GuangZHANG

    2004-01-01

    Programmed death-1 (PD-1) is a costimulatory molecule of CD28 family expressed onactivated T, B and myeloid cells. The engagement of PD-1 with its two ligands, PD-L1 and PD-L2, inhibitsproliferation of T cell and production of a series of its cytokines. The blockade of PD-1 pathway is involvedin antiviral and antitumoral immunity. In this study, human PD-1 cDNA encoding extracellular domain wasamplified and cloned into expression plasmid pGEX-Sx-3. The fusion protein GST-PD-1 was effectivelyexpressed in E. coli BL21 (DE3) as inclusion bodies and a denaturation and refolding procedure was per-formed to obtain bioactive soluble GST-PD-I. Fusion protein of above 95% purity was acquired by a conve-nient two-step purification using GST affinity and size exclusion columns. Furthermore, a PD-L1-dependentin vitro bioassay method was set up to characterize GST-PD-1 bioactivity. The results suggested that GST-PD-1 could competently block the interaction between PD-Ll and PD-l and increase the production of IL-2 and IFN-γ of phytohemagglutinin-activated T cells.

  10. Characterizations of individual human red blood cells from patients with diabetes mellitus (Conference Presentation)

    Science.gov (United States)

    Lee, SangYun; Jang, Seongsoo; Park, HyunJoo; Park, YongKeun

    2016-03-01

    We systematically measure the morphological, biochemical, and biomechanical properties of individual human red blood cells (RBCs) from patients with diabetes mellitus using quantitative phase imaging technique to characterize the diabetic red cells with respect to those of the healthy. The 3-D refractive index tomograms and 2-D dynamic membrane fluctuation maps of individual RBCs are reconstructed from a set of the retrieved complex optical fields at various laser incidence angles using the Common-path diffraction optical tomography, from which volume, surface area, sphericity, hemoglobin (Hb) concentration, Hb content, and membrane fluctuation are obtained simultaneously. The correlative relations among the retrieved red cell indices of diabetic and healthy RBCs are also investigated with capabilities of individual cell measurement. As expected, there are no significant alterations in morphologies (cellular volumes, surface area, and sphericity) between diabetic and healthy RBCs. However, despite the minute mean corpuscular Hb differences in cell blood count datasheet, the measured Hb concentrations and Hb contents of diabetic RBCs are statistically higher than those of healthy RBCs, which might be related to the glycation of Hb molecules by hyperglycemia. Meanwhile, the membrane fluctuations of diabetic RBCs are clearly diminished compared to healthy red cells, implying the significantly decreased RBC deformability. In particular, it seems that the membrane fluctuations have mild negative relationships with the reported HbA1c levels.

  11. Genetic characterization of St. Louis encephalitis virus isolated from human in São Paulo, Brazil

    Directory of Open Access Journals (Sweden)

    Cecília Luiza Simões dos Santos

    2006-02-01

    Full Text Available The molecular characterization of SPH253157, a new strain of St. Louis encephalitis virus (SLEV, isolated in 2004 from the first case of human infection recognized in the state of São Paulo, Brazil, is reported. The patient, presenting a febrile illness without neurological involvement, was hospitalized as a probable case of dengue fever. Genomic RNA was isolated from the supernatant of C6/36 cells infected with acute phase-serum specimen of the patient and the envelope gene was amplified by reverse-transcription-polymerase chain reaction. The complete nucleotide sequence of the envelope gene of this isolate was directly sequenced from the amplified products and compared with other Brazilian and American SLEV strains. Phylogenetic analyses were carried out under maximum likelihood criterion with outgroups both included and excluded. Outgroups comprised four flavivirus of the Japanese encephalitis group. Phylogeny also included Bayesian analysis. The results indicated that the new SLEV isolate belongs to lineage III, being closely related to an Argentinean strain recovered from Culex sp. in 1979. It is concluded that there are at least 3 lineages of SLEV in Brazil.

  12. Immunoaffinity purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from human erythrocytes

    Institute of Scientific and Technical Information of China (English)

    Driss Mountassif; Tarik Baibai; Latifa Fourrat; Adnane Moutaouakkil; Abdelghani Iddar; M'Hammed Sa(i)d El Kebbaj; Abdelaziz Soukri

    2009-01-01

    A new procedure utilizing immunoaffinity column chromatography has been used for the purification of glyceraldehyde-3-phosphate dehydrogenase(GAPDH,EC 1.2.1.12)from human erythrocytes.The comparison between this rapid method(one step)and the traditional procedure including ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography shows that the new method gives a highest specific activity with a highest yield in a short time.The characterization of the purified GAPDH reveals that the native enzyme is a homotetramer of ~150 kDa with an absolute specificity for the oxidized form of nicotinamide adenine dinucleotide(NAD+).Western blot analysis using purified monospecific poly clonal antibodies raised against the purified GAPDH showed a singie 36 kDa band corresponding to the enzyme subunit.Studies on the effect of temperature and pH on enzyme activity revealed optimal values of about 43℃ and 8.5, respectively.The kinetic par ameters were also calculated:the Vmax was 4.3 U/mg and the Km values against G3P and NAD+ were 20.7and 17.8μM,respectively.The new protocol described represents a simple,economic,and reproducible tool for the purification Of GAPDH and can be used for other proteins.

  13. Characterization of Human Gingival Fibroblasts on Zirconia Surfaces Containing Niobium Oxide

    Directory of Open Access Journals (Sweden)

    Young-Dan Cho

    2015-09-01

    Full Text Available It was indicated that tetragonal zirconia polycrystal (TZP containing yttria (Y2O3 and niobium oxide (Nb2O5 ((Y,Nb-TZP could be an adequate dental material to be used at esthetically important sites. The (Y,Nb-TZP was also proved to possess its osteogenic potential comparable with those conventional dental implant material, titanium (Ti. The objective of the current study was to characterize cellular response of human gingival fibroblasts (HGFs to smooth and rough surfaces of the (Y,Nb-TZP disc, which were obtained by polishing and sandblasting, respectively. Various microscopic, biochemical, and molecular techniques were used to investigate the disc surfaces and cellular responses for the experimental (Y,Nb-TZP and the comparing Ti groups. Sandblasted rough (Y,Nb-TZP (Zir-R discs had the highest surface roughness. HGFs cultured on polished (Y,Nb-TZP (Zir showed a rounded cell morphology and light spreading at 6 h after seeding and its proliferation rate significantly increased during seven days of culture compared to other surfaces. The mRNA expressions of type I collagen, integrin α2 and β1 were significantly stimulated for the Zir group at 24 h after seeding. The current findings, combined with the previous results, indicate that (Y,Nb-TZP provides appropriate surface condition for osseointegration at the fixture level and for peri-implant mucosal sealing at the abutment level producing a suitable candidate for dental implantation with an expected favorable clinical outcome.

  14. The Human p73 Promoter: Characterization and Identification of Functional E2F Binding Sites

    Directory of Open Access Journals (Sweden)

    Ratnam S. Seelan

    2002-01-01

    Full Text Available p73, a member of the p53 family, is overexpressed in many cancers. To understand the mechanism(s underlying this overexpression, we have undertaken a detailed characterization of the human p73 promoter. The promoter is strongly activated in cells expressing exogenous E2F1 and suppressed by exogenous Rb. At least three functional E2F binding sites, located immediately upstream of exon 1 (at-284,-155 and-132 mediate this induction. 5' serially deleted promoter constructs and constructs harboring mutated E2F sites were analyzed for their response to exogenously expressed E2F1 or Rb to establish functionality of these sites. Authenticity of E2F sites was further confirmed by electrophoretic mobility shift assay (EMSA using E2F1 /DP1 heterodimers synthesized in vitro, followed by competition assays with unlabeled wild-type or mutant oligonucleotides and supershift analysis using anti-E2F1 antibodies. In vivo binding of E2F1 to the p73 promoter was demonstrated using nuclear extracts prepared from E2F1-inducible Saos2 cells. The region conferring the highest promoter activity was found to reside between-113 to-217 of the p73 gene. Two of the three functional E2F sites (at-155 and-132 reside within this region. Our results suggest that regulation of p73 expression is primarily mediated through binding of E2 F1 to target sites at-155 and-132.

  15. Identification and characterization of an alternative promoter of the human PGC-1{alpha} gene

    Energy Technology Data Exchange (ETDEWEB)

    Yoshioka, Toyo; Inagaki, Kenjiro [Division of Diabetes, Metabolism, and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Noguchi, Tetsuya, E-mail: noguchi@med.kobe-u.ac.jp [Division of Diabetes, Metabolism, and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Sakai, Mashito; Ogawa, Wataru; Hosooka, Tetsuya [Division of Diabetes, Metabolism, and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Iguchi, Haruhisa; Watanabe, Eijiro; Matsuki, Yasushi; Hiramatsu, Ryuji [Genomic Science Laboratories, DainipponSumitomo Pharma Co. Ltd., 4-2-1 Takatsukasa, Takarazuka 665-8555 (Japan); Kasuga, Masato [Division of Diabetes, Metabolism, and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655 (Japan)

    2009-04-17

    The transcriptional regulator peroxisome proliferator-activated receptor-{gamma} coactivator-1{alpha} (PGC-1{alpha}) controls mitochondrial biogenesis and energy homeostasis. Although physical exercise induces PGC-1{alpha} expression in muscle, the underlying mechanism of this effect has remained incompletely understood. We recently identified a novel muscle-enriched isoform of PGC-1{alpha} transcript (designated PGC-1{alpha}-b) that is derived from a previously unidentified first exon. We have now cloned and characterized the human PGC-1{alpha}-b promoter. The muscle-specific transcription factors MyoD and MRF4 transactivated this promoter through interaction with a proximal E-box motif. Furthermore, either forced expression of Ca{sup 2+}- and calmodulin-dependent protein kinase IV (CaMKIV), calcineurin A, or the p38 mitogen-activated protein kinase (p38 MAPK) kinase MKK6 or the intracellular accumulation of cAMP activated the PGC-1{alpha}-b promoter in cultured myoblasts through recruitment of cAMP response element (CRE)-binding protein (CREB) to a putative CRE located downstream of the E-box. Our results thus reveal a potential molecular basis for isoform-specific regulation of PGC-1{alpha} expression in contracting muscle.

  16. Characterization of GLP-1 effects on beta-cell function after meal ingestion in humans

    DEFF Research Database (Denmark)

    Ahrén, Bo; Holst, Jens Juul; Mari, Andrea

    2003-01-01

    have been characterized in humans during graded intravenous infusions of glucose, whereas its effects after more physiological stimuli, like meal intake, are not known. RESEARCH DESIGN AND METHODS: Eight women (aged 69 years, fasting glucose 3.7-10.3 mmol/l, BMI 22.4-43.9 kg/m(2)) who had fasted......OBJECTIVE: Glucagon-like peptide 1 (GLP-1) is an incretin that augments insulin secretion after meal intake and is developed for treatment of type 2 diabetes. As a novel therapeutic agent, characteristics of its beta-cell effects are important to establish. Previously, beta-cell effects of GLP-1......: -GLP-1 markedly augmented insulin secretion, despite lower glucose. Total insulin secretion was 29.7 +/- 4.2 nmol/m(2) with GLP-1 versus 21.0 +/- 1.6 nmol/m(2) with saline (P = 0.048). GLP-1 increased the dose-response relationship between glucose concentration and insulin secretion (70 +/- 26 with GLP...

  17. Identification and characterization of an alternative promoter of the human PGC-1α gene

    International Nuclear Information System (INIS)

    The transcriptional regulator peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) controls mitochondrial biogenesis and energy homeostasis. Although physical exercise induces PGC-1α expression in muscle, the underlying mechanism of this effect has remained incompletely understood. We recently identified a novel muscle-enriched isoform of PGC-1α transcript (designated PGC-1α-b) that is derived from a previously unidentified first exon. We have now cloned and characterized the human PGC-1α-b promoter. The muscle-specific transcription factors MyoD and MRF4 transactivated this promoter through interaction with a proximal E-box motif. Furthermore, either forced expression of Ca2+- and calmodulin-dependent protein kinase IV (CaMKIV), calcineurin A, or the p38 mitogen-activated protein kinase (p38 MAPK) kinase MKK6 or the intracellular accumulation of cAMP activated the PGC-1α-b promoter in cultured myoblasts through recruitment of cAMP response element (CRE)-binding protein (CREB) to a putative CRE located downstream of the E-box. Our results thus reveal a potential molecular basis for isoform-specific regulation of PGC-1α expression in contracting muscle.

  18. Expression Characterization and Preparation of Human Amyloid Precursor Protein in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    XU Guang-wei; WANG Jia-peng; HUANG Xue-mei; ZHANG Ying-jiu

    2009-01-01

    To analyze whether expressed amyloid precursor protein(APP) existed in hydrophilic(cytoplasmid) or hy-drophobic(lipid bilayer) environment in E. coli and to obtain intact APP for study on its function, we investigated the expression characterization and preparation of the three intact isoforms APP770, APP751, and APP695 in E. coli. The results show that these expressed APPs existed both in hydrophilic cytoplasm region as inclusion bodies and hy-drophobic membrane region as membrane-bound state in E. coll. APPs in inclusion bodies were purified on an NTA-Ni2. agarose column after dissolving in the urea buffer and APPs in membrane-bound state were obtained by ultracentrifugation. The activity analysis indicates that APP770 and APP751 exhibited strong trypsin-inhibitory activity like the natural ones. These results indicate that E. coil cells can be used as host cells for the expression of human integral membrane protein like APP in either soluble or membrane-bound state unless the interest protein undergone post-translational modification is required.

  19. Characterization of visual percepts evoked by noninvasive stimulation of the human posterior parietal cortex.

    Directory of Open Access Journals (Sweden)

    Peter J Fried

    Full Text Available Phosphenes are commonly evoked by transcranial magnetic stimulation (TMS to study the functional organization, connectivity, and excitability of the human visual brain. For years, phosphenes have been documented only from stimulating early visual areas (V1-V3 and a handful of specialized visual regions (V4, V5/MT+ in occipital cortex. Recently, phosphenes were reported after applying TMS to a region of posterior parietal cortex involved in the top-down modulation of visuo-spatial processing. In the present study, we systematically characterized parietal phosphenes to determine if they are generated directly by local mechanisms or emerge through indirect activation of other visual areas. Using technology developed in-house to record the subjective features of phosphenes, we found no systematic differences in the size, shape, location, or frame-of-reference of parietal phosphenes when compared to their occipital counterparts. In a second experiment, discrete deactivation by 1 Hz repetitive TMS yielded a double dissociation: phosphene thresholds increased at the deactivated site without producing a corresponding change at the non-deactivated location. Overall, the commonalities of parietal and occipital phosphenes, and our ability to independently modulate their excitability thresholds, lead us to conclude that they share a common neural basis that is separate from either of the stimulated regions.

  20. Biophysical characterization and modeling of human Ecdysoneless (ECD protein supports a scaffolding function

    Directory of Open Access Journals (Sweden)

    Nicholas P. Schafer

    2016-03-01

    Full Text Available The human homolog of Drosophila ecdysoneless protein (ECD is a p53 binding protein that stabilizes and enhances p53 functions. Homozygous deletion of mouse Ecd is early embryonic lethal and Ecd deletion delays G1-S cell cycle progression. Importantly, ECD directly interacts with the Rb tumor suppressor and competes with the E2F transcription factor for binding to Rb. Further studies demonstrated ECD is overexpressed in breast and pancreatic cancers and its overexpression correlates with poor patient survival. ECD overexpression together with Ras induces cellular transformation through upregulation of autophagy. Recently we demonstrated that CK2 mediated phosphorylation of ECD and interaction with R2TP complex are important for its cell cycle regulatory function. Considering that ECD is a component of multiprotein complexes and its crystal structure is unknown, we characterized ECD structure by circular dichroism measurements and sequence analysis software. These analyses suggest that the majority of ECD is composed of α-helices. Furthermore, small angle X-ray scattering (SAXS analysis showed that deletion fragments, ECD(1–432 and ECD(1–534, are both well-folded and reveals that the first 400 residues are globular and the next 100 residues are in an extended cylindrical structure. Taking all these results together, we speculate that ECD acts like a structural hub or scaffolding protein in its association with its protein partners. In the future, the hypothetical model presented here for ECD will need to be tested experimentally.