WorldWideScience

Sample records for activated cell sorting

  1. Fluorescence activated cell sorting of plant protoplasts.

    Science.gov (United States)

    Bargmann, Bastiaan O R; Birnbaum, Kenneth D

    2010-02-18

    High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis. An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: P(SCR;)::GFP (endodermis and quiescent center) and P(WOX5;)::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution. FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time. This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce

  2. Buoyancy-activated cell sorting using targeted biotinylated albumin microbubbles.

    Directory of Open Access Journals (Sweden)

    Yu-Ren Liou

    Full Text Available Cell analysis often requires the isolation of certain cell types. Various isolation methods have been applied to cell sorting, including fluorescence-activated cell sorting and magnetic-activated cell sorting. However, these conventional approaches involve exerting mechanical forces on the cells, thus risking cell damage. In this study we applied a novel isolation method called buoyancy-activated cell sorting, which involves using biotinylated albumin microbubbles (biotin-MBs conjugated with antibodies (i.e., targeted biotin-MBs. Albumin MBs are widely used as contrast agents in ultrasound imaging due to their good biocompatibility and stability. For conjugating antibodies, biotin is conjugated onto the albumin MB shell via covalent bonds and the biotinylated antibodies are conjugated using an avidin-biotin system. The albumin microbubbles had a mean diameter of 2 μm with a polydispersity index of 0.16. For cell separation, the MDA-MB-231 cells are incubated with the targeted biotin-MBs conjugated with anti-CD44 for 10 min, centrifuged at 10 g for 1 min, and then allowed 1 hour at 4 °C for separation. The results indicate that targeted biotin-MBs conjugated with anti-CD44 antibodies can be used to separate MDA-MB-231 breast cancer cells; more than 90% of the cells were collected in the MB layer when the ratio of the MBs to cells was higher than 70:1. Furthermore, we found that the separating efficiency was higher for targeted biotin-MBs than for targeted avidin-incorporated albumin MBs (avidin-MBs, which is the most common way to make targeted albumin MBs. We also demonstrated that the recovery rate of targeted biotin-MBs was up to 88% and the sorting purity was higher than 84% for a a heterogenous cell population containing MDA-MB-231 cells (CD44(+ and MDA-MB-453 cells (CD44-, which are classified as basal-like breast cancer cells and luminal breast cancer cells, respectively. Knowing that the CD44(+ is a commonly used cancer-stem-cell

  3. Fluorescence-Activated Cell Sorting of Live Versus Dead Bacterial Cells and Spores

    Science.gov (United States)

    Bernardini, James N.; LaDuc, Myron T.; Diamond, Rochelle; Verceles, Josh

    2012-01-01

    This innovation is a coupled fluorescence-activated cell sorting (FACS) and fluorescent staining technology for purifying (removing cells from sampling matrices), separating (based on size, density, morphology, and live versus dead), and concentrating cells (spores, prokaryotic, eukaryotic) from an environmental sample.

  4. PCR-activated cell sorting for cultivation-free enrichment and sequencing of rare microbes.

    Science.gov (United States)

    Lim, Shaun W; Tran, Tuan M; Abate, Adam R

    2015-01-01

    Microbial systems often exhibit staggering diversity, making the study of rare, interesting species challenging. For example, metagenomic analyses of mixed-cell populations are often dominated by the sequences of the most abundant organisms, while those of rare microbes are detected only at low levels, if at all. To overcome this, selective cultivation or fluorescence-activated cell sorting (FACS) can be used to enrich for the target species prior to sequence analysis; however, since most microbes cannot be grown in the lab, cultivation strategies often fail, while cell sorting requires techniques to uniquely label the cell type of interest, which is often not possible with uncultivable microbes. Here, we introduce a culture-independent strategy for sorting microbial cells based on genomic content, which we term PCR-activated cell sorting (PACS). This technology, which utilizes the power of droplet-based microfluidics, is similar to FACS in that it uses a fluorescent signal to uniquely identify and sort target species. However, PACS differs importantly from FACS in that the signal is generated by performing PCR assays on the cells in microfluidic droplets, allowing target cells to be identified with high specificity with suitable design of PCR primers and TaqMan probes. The PACS assay is general, requires minimal optimization and, unlike antibody methods, can be developed without access to microbial antigens. Compared to non-specific methods in which cells are sorted based on size, granularity, or the ability to take up dye, PACS enables genetic sequence-specific sorting and recovery of the cell genomes. In addition to sorting microbes, PACS can be applied to eukaryotic cells, viruses, and naked nucleic acids. PMID:25629401

  5. Isolation of Cells Specialized in Anticancer Alkaloid Metabolism by Fluorescence-Activated Cell Sorting.

    Science.gov (United States)

    Carqueijeiro, Inês; Guimarães, Ana Luísa; Bettencourt, Sara; Martínez-Cortés, Teresa; Guedes, Joana G; Gardner, Rui; Lopes, Telma; Andrade, Cláudia; Bispo, Cláudia; Martins, Nuno Pimpão; Andrade, Paula; Valentão, Patrícia; Valente, Inês M; Rodrigues, José A; Duarte, Patrícia; Sottomayor, Mariana

    2016-08-01

    Plant specialized metabolism often presents a complex cell-specific compartmentation essential to accomplish the biosynthesis of valuable plant natural products. Hence, the disclosure and potential manipulation of such pathways may depend on the capacity to isolate and characterize specific cell types. Catharanthus roseus is the source of several medicinal terpenoid indole alkaloids, including the low-level anticancer vinblastine and vincristine, for which the late biosynthetic steps occur in specialized mesophyll cells called idioblasts. Here, the optical, fluorescence, and alkaloid-accumulating properties of C. roseus leaf idioblasts are characterized, and a methodology for the isolation of idioblast protoplasts by fluorescence-activated cell sorting is established, taking advantage of the distinctive autofluorescence of these cells. This achievement represents a crucial step for the development of differential omic strategies leading to the identification of candidate genes putatively involved in the biosynthesis, pathway regulation, and transmembrane transport leading to the anticancer alkaloids from C. roseus. PMID:27356972

  6. A monolithic glass chip for active single-cell sorting based on mechanical phenotyping

    OpenAIRE

    Faigle, C.; Lautenschläger, F.; Whyte, G; Homewood, P.; Martín Badosa, Estela; Guck, J.

    2014-01-01

    The mechanical properties of biological cells have long been considered as inherent markers of biological function and disease. However, the screening and active sorting of heterogeneous populations based on serial single-cell mechanical measurements has not been demonstrated. Here we present a novel monolithic glass chip for combined fluorescence detection and mechanical phenotyping using an optical stretcher. A new design and manufacturing process, involving the bonding of two asymmetricall...

  7. Characterization of Rat Hair Follicle Stem Cells Selected by Vario Magnetic Activated Cell Sorting System

    International Nuclear Information System (INIS)

    Hair follicle stem cells (HfSCs) play crucial roles in hair follicle morphogenesis and hair cycling. These stem cells are self-renewable and have the multi-lineage potential to generate epidermis, sebaceous glands, and hair follicle. The separation and identification of hair follicle stem cells are important for further research in stem cell biology. In this study, we report on the successful enrichment of rat hair follicle stem cells through vario magnetic activated cell sorting (Vario MACS) and the biological characteristics of the stem cells. We chose the HfSCs positive surface markers CD34, α6-integrin and the negative marker CD71 to design four isolation strategies: positive selection with single marker of CD34, positive selection with single marker of α6-integrin, CD71 depletion followed by CD34 positive selection, and CD71 depletion followed by α6-integrin positive selection. The results of flow cytometry analysis showed that all four strategies had ideal effects. Specifically, we conducted a series of researches on HfSCs characterized by their high level of CD34, termed CD34bri cells, and low to undetectable expression of CD34, termed CD34dim cells. CD34bri cells had greater proliferative potential and higher colony-forming ability than CD34dim cells. Furthermore, CD34bri cells had some typical characteristics as progenitor cells, such as large nucleus, obvious nucleolus, large nuclear:cytoplasmic ratio and few cytoplasmic organelles. Our findings clearly demonstrated that HfSCs with high purity and viability could be successfully enriched with Vario MACS

  8. Tracking heavy water (D2O) incorporation for identifying and sorting active microbial cells

    DEFF Research Database (Denmark)

    Berry, David; Mader, Esther; Lee, Tae Kwon;

    2015-01-01

    distinctive response patterns to amendments of mucin and sugars. By Ramanbased cell sorting of active (deuterated) cells with optical tweezers and subsequent multiple displacement amplification and DNA sequencing, novel cecal microbes stimulated by mucin and/ or glucosamine were identified, demonstrating...

  9. 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.

  10. Rapid isolation of antibody from a synthetic human antibody library by repeated fluorescence-activated cell sorting (FACS.

    Directory of Open Access Journals (Sweden)

    Sung Sun Yim

    Full Text Available Antibodies and their derivatives are the most important agents in therapeutics and diagnostics. Even after the significant progress in the technology for antibody screening from huge libraries, it takes a long time to isolate an antibody, which prevents a prompt action against the spread of a disease. Here, we report a new strategy for isolating desired antibodies from a combinatorial library in one day by repeated fluorescence-activated cell sorting (FACS. First, we constructed a library of synthetic human antibody in which single-chain variable fragment (scFv was expressed in the periplasm of Escherichia coli. After labeling the cells with fluorescent antigen probes, the highly fluorescent cells were sorted by using a high-speed cell sorter, and these cells were reused without regeneration in the next round of sorting. After repeating this sorting, the positive clones were completely enriched in several hours. Thus, we screened the library against three viral antigens, including the H1N1 influenza virus, Hepatitis B virus, and Foot-and-mouth disease virus. Finally, the potential antibody candidates, which show K(D values between 10 and 100 nM against the target antigens, could be successfully isolated even though the library was relatively small (∼ 10(6. These results show that repeated FACS screening without regeneration of the sorted cells can be a powerful method when a rapid response to a spreading disease is required.

  11. Efficient selective breeding of live oil-rich Euglena gracilis with fluorescence-activated cell sorting.

    Science.gov (United States)

    Yamada, Koji; Suzuki, Hideyuki; Takeuchi, Takuto; Kazama, Yusuke; Mitra, Sharbanee; Abe, Tomoko; Goda, Keisuke; Suzuki, Kengo; Iwata, Osamu

    2016-01-01

    Euglena gracilis, a microalgal species of unicellular flagellate protists, has attracted much attention in both the industrial and academic sectors due to recent advances in the mass cultivation of E. gracilis that have enabled the cost-effective production of nutritional food and cosmetic commodities. In addition, it is known to produce paramylon (β-1,3-glucan in a crystalline form) as reserve polysaccharide and convert it to wax ester in hypoxic and anaerobic conditions-a promising feedstock for biodiesel and aviation biofuel. However, there remain a number of technical challenges to be solved before it can be deployed in the competitive fuel market. Here we present a method for efficient selective breeding of live oil-rich E. gracilis with fluorescence-activated cell sorting (FACS). Specifically, the selective breeding method is a repetitive procedure for one-week heterotrophic cultivation, staining intracellular lipids with BODIPY(505/515), and FACS-based isolation of top 0.5% lipid-rich E. gracilis cells with high viability, after inducing mutation with Fe-ion irradiation to the wild type (WT). Consequently, we acquire a live, stable, lipid-rich E. gracilis mutant strain, named B1ZFeL, with 40% more lipid content on average than the WT. Our method paves the way for rapid, cost-effective, energy-efficient production of biofuel. PMID:27212384

  12. Cell sorting by deterministic cell rolling

    OpenAIRE

    Choi, Sungyoung; Karp, Jeffrey M.; Karnik, Rohit

    2011-01-01

    This communication presents the concept of “deterministic cell rolling”, which leverages transient cell-surface molecular interactions that mediate cell rolling to sort cells with high purity and efficiency in a single step.

  13. The ROCK inhibitor Y-27632 improves recovery of human embryonic stem cells after fluorescence-activated cell sorting with multiple cell surface markers.

    Directory of Open Access Journals (Sweden)

    Nil Emre

    Full Text Available BACKGROUND: Due to the inherent sensitivity of human embryonic stem cells (hESCs to manipulations, the recovery and survival of hESCs after fluorescence-activated cell sorting (FACS can be low. Additionally, a well characterized and robust methodology for performing FACS on hESCs using multiple-cell surface markers has not been described. The p160-Rho-associated coiled kinase (ROCK inhibitor, Y-27632, previously has been identified as enhancing survival of hESCs upon single-cell dissociation, as well as enhancing recovery from cryopreservation. Here we examined the application of Y-27632 to hESCs after FACS to improve survival in both feeder-dependent and feeder-independent growth conditions. METHODOLOGY/PRINCIPAL FINDINGS: HESCs were sorted using markers for SSEA-3, TRA-1-81, and SSEA-1. Cells were plated after sorting for 24 hours in either the presence or the absence of Y-27632. In both feeder-dependent and feeder-independent conditions, cell survival was greater when Y-27632 was applied to the hESCs after sort. Specifically, treatment of cells with Y-27632 improved post-sort recovery up to four fold. To determine the long-term effects of sorting with and without the application of Y-27632, hESCs were further analyzed. Specifically, hESCs sorted with and without the addition of Y-27632 retained normal morphology, expressed hESC-specific markers as measured by immunocytochemistry and flow cytometry, and maintained a stable karyotype. In addition, the hESCs could differentiate into three germ layers in vitro and in vivo in both feeder-dependent and feeder-independent growth conditions. CONCLUSIONS/SIGNIFICANCE: The application of Y-27632 to hESCs after cell sorting improves cell recovery with no observed effect on pluripotency, and enables the consistent recovery of hESCs by FACS using multiple surface markers. This improved methodology for cell sorting of hESCs will aid many applications such as removal of hESCs from secondary cell types

  14. Fluorescence-Activated Cell Sorting of EGFP-Labeled Neural Crest Cells From Murine Embryonic Craniofacial Tissue

    Directory of Open Access Journals (Sweden)

    Saurabh Singh

    2005-01-01

    Full Text Available During the early stages of embryogenesis, pluripotent neural crest cells (NCC are known to migrate from the neural folds to populate multiple target sites in the embryo where they differentiate into various derivatives, including cartilage, bone, connective tissue, melanocytes, glia, and neurons of the peripheral nervous system. The ability to obtain pure NCC populations is essential to enable molecular analyses of neural crest induction, migration, and/or differentiation. Crossing Wnt1-Cre and Z/EG transgenic mouse lines resulted in offspring in which the Wnt1-Cre transgene activated permanent EGFP expression only in NCC. The present report demonstrates a flow cytometric method to sort and isolate populations of EGFP-labeled NCC. The identity of the sorted neural crest cells was confirmed by assaying expression of known marker genes by TaqMan Quantitative Real-Time Polymerase Chain Reaction (QRT-PCR. The molecular strategy described in this report provides a means to extract intact RNA from a pure population of NCC thus enabling analysis of gene expression in a defined population of embryonic precursor cells critical to development.

  15. Toward label-free Raman-activated cell sorting of cardiomyocytes derived from human embryonic stem cells

    Science.gov (United States)

    Pascut, Flavius C.; Goh, Huey T.; George, Vinoj; Denning, Chris; Notingher, Ioan

    2011-04-01

    Raman micro-spectroscopy (RMS) has been recently proposed for label-free phenotypic identification of human embryonic stem cells (hESC)-derived cardiomyocytes. However, the methods used for measuring the Raman spectra led to acquisition times of minutes per cell, which is prohibitive for rapid cell sorting applications. In this study we evaluated two measurement strategies that could reduce the measurement time by a factor of more than 100. We show that sampling individual cells with a laser beam focused to a line could eliminate the need of cell raster scanning and achieve high prediction accuracies (>95% specificity and >96% sensitivity) with acquisition times ~5 seconds per cell. However, the use of commercially-available higher power lasers could potentially lead to sorting speeds of ~10 cells per s. This would start to progress RMS to the field of cell sorting for applications such as enrichment and purification of hESC-derived cardiomyocytes.

  16. Fluorescently activated cell sorting followed by microarray profiling of helper T cell subtypes from human peripheral blood.

    Directory of Open Access Journals (Sweden)

    Chiaki Ono

    Full Text Available BACKGROUND: Peripheral blood samples have been subjected to comprehensive gene expression profiling to identify biomarkers for a wide range of diseases. However, blood samples include red blood cells, white blood cells, and platelets. White blood cells comprise polymorphonuclear leukocytes, monocytes, and various types of lymphocytes. Blood is not distinguishable, irrespective of whether the expression profiles reflect alterations in (a gene expression patterns in each cell type or (b the proportion of cell types in blood. CD4+ Th cells are classified into two functionally distinct subclasses, namely Th1 and Th2 cells, on the basis of the unique characteristics of their secreted cytokines and their roles in the immune system. Th1 and Th2 cells play an important role not only in the pathogenesis of human inflammatory, allergic, and autoimmune diseases, but also in diseases that are not considered to be immune or inflammatory disorders. However, analyses of minor cellular components such as CD4+ cell subpopulations have not been performed, partly because of the limited number of these cells in collected samples. METHODOLOGY/PRINCIPAL FINDINGS: We describe fluorescently activated cell sorting followed by microarray (FACS-array technology as a useful experimental strategy for characterizing the expression profiles of specific immune cells in the circulation. We performed reproducible gene expression profiling of Th1 and Th2, respectively. Our data suggest that this procedure provides reliable information on the gene expression profiles of certain small immune cell populations. Moreover, our data suggest that GZMK, GZMH, EOMES, IGFBP3, and STOM may be novel markers for distinguishing Th1 cells from Th2 cells, whereas IL17RB and CNTNAP1 can be Th2-specific markers. CONCLUSIONS/SIGNIFICANCE: Our approach may help in identifying aberrations and novel therapeutic or diagnostic targets for diseases that affect Th1 or Th2 responses and elucidating the

  17. Fluorescence-Activated Nucleolus Sorting in Arabidopsis.

    Science.gov (United States)

    Pontvianne, Frédéric; Boyer-Clavel, Myriam; Sáez-Vásquez, Julio

    2016-01-01

    Nucleolar isolation allows exhaustive characterization of the nucleolar content. Centrifugation-based protocols are not adapted to isolation of nucleoli directly from a plant tissue because of copurification of cellular debris. We describe here a method that allows the purification of nucleoli using fluorescent-activated cell sorting from Arabidopsis thaliana leaves. This approach requires the expression of a specific nucleolar protein such as fibrillarin fused to green fluorescent protein in planta. PMID:27576720

  18. Polarized sorting and trafficking in epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Xinwang Cao; Michal A Surma; Kai Simons

    2012-01-01

    The polarized distribution of proteins and lipids at the surface membrane of epithelial cells results in the formation of an apical and a basolateral domain,which are separated by tight junctions.The generation and maintenance of epithelial polarity require elaborate mechanisms that guarantee correct sorting and vectorial delivery of cargo molecules.This dynamic process involves the interaction of sorting signals with sorting machineries and the formation of transport carriers.Here we review the recent advances in the field of polarized sorting in epithelial cells.We especially highlight the role of lipid rafts in apical sorting.

  19. Isolating Epithelial and Epithelial-to-Mesenchymal Transition Populations from Primary Tumors by Fluorescence-Activated Cell Sorting.

    Science.gov (United States)

    Aiello, Nicole M; Rhim, Andrew D; Stanger, Ben Z

    2016-01-01

    Transgenic mice that express conditional reporters allow for the isolation of specific cell lineages. These cells can be further stratified by gene expression and collected by fluorescence-activated cell sorting (FACS) for further analysis. Using Cre-recombinase (Cre) technology we have generated a transgenic mouse line termed PKCY in which all pancreatic epithelial cells and therefore all pancreatic cancer cells are constitutively labeled with yellow fluorescent protein (YFP). We have used immunofluorescent staining for E-cadherin to divide the YFP(+) tumor population into epithelial cells (E-cadherin positive) and cells that have undergone an epithelial-to-mesenchymal transition (EMT; E-cadherin negative). This protocol describes how to prepare a tumor sample for FACS, with an emphasis on separating epithelial and EMT populations. These cells can then be used for a number of applications including, but not limited to, the generation of cell lines, gene-expression analysis by quantitative polymerase chain reaction (qPCR) or RNA sequencing, DNA sequencing, chromatin immunoprecipitation, and western blots. PMID:26729901

  20. Identification of microbes from the surfaces of food-processing lines based on the flow cytometric evaluation of cellular metabolic activity combined with cell sorting.

    Science.gov (United States)

    Juzwa, W; Duber, A; Myszka, K; Białas, W; Czaczyk, K

    2016-09-01

    In this study the design of a flow cytometry-based procedure to facilitate the detection of adherent bacteria from food-processing surfaces was evaluated. The measurement of the cellular redox potential (CRP) of microbial cells was combined with cell sorting for the identification of microorganisms. The procedure enhanced live/dead cell discrimination owing to the measurement of the cell physiology. The microbial contamination of the surface of a stainless steel conveyor used to process button mushrooms was evaluated in three independent experiments. The flow cytometry procedure provided a step towards monitoring of contamination and enabled the assessment of microbial food safety hazards by the discrimination of active, mid-active and non-active bacterial sub-populations based on determination of their cellular vitality and subsequently single cell sorting to isolate microbial strains from discriminated sub-populations. There was a significant correlation (r = 0.97; p < 0.05) between the bacterial cell count estimated by the pour plate method and flow cytometry, despite there being differences in the absolute number of cells detected. The combined approach of flow cytometric CRP measurement and cell sorting allowed an in situ analysis of microbial cell vitality and the identification of species from defined sub-populations, although the identified microbes were limited to culturable cells. PMID:27406324

  1. Minimal residual disease surveillance in chronic lymphocytic leukemia by fluorescence-activated cell sorting.

    Science.gov (United States)

    Ringelstein-Harlev, Shimrit; Fineman, Riva

    2014-10-01

    Achievement of complete response (CR) to therapy in chronic lymphocytic leukemia (CLL) has become a feasible goal, directly correlating with prolonged survival. It has been established that the classic definition of CR actually encompasses a variety of disease loads, and more sensitive multiparameter flow cytometry and polymerase chain reaction methods can detect the disease burden with a much higher sensitivity. Detection of malignant cells with a sensitivity of 1 tumor cell in 10,000 cells (10(-4)), using the abovementioned sophisticated techniques, is the current cutoff for minimal residual disease (MRD). Tumor burdens lower than 10(-4) are defined as MRD-negative. Several studies in CLL have determined the achievement of MRD negativity as an independent favorable prognostic factor, leading to prolonged disease-free and overall survival, regardless of the treatment protocol or the presence of other pre-existing prognostic indicators. Minimal residual disease evaluation using flow cytometry is a sensitive and applicable approach which is expected to become an integral part of future prospective trials in CLL designed to assess the role of MRD surveillance in treatment tailoring.

  2. Minimal Residual Disease Surveillance in Chronic Lymphocytic Leukemia by Fluorescence-Activated Cell Sorting

    Directory of Open Access Journals (Sweden)

    Shimrit Ringelstein-Harlev

    2014-10-01

    Full Text Available Achievement of complete response (CR to therapy in chronic lymphocytic leukemia (CLL has become a feasible goal, directly correlating with prolonged survival. It has been established that the classic definition of CR actually encompasses a variety of disease loads, and more sensitive multiparameter flow cytometry and polymerase chain reaction methods can detect the disease burden with a much higher sensitivity. Detection of malignant cells with a sensitivity of 1 tumor cell in 10,000 cells (10–4, using the abovementioned sophisticated techniques, is the current cutoff for minimal residual disease (MRD. Tumor burdens lower than 10–4 are defined as MRD-negative. Several studies in CLL have determined the achievement of MRD negativity as an independent favorable prognostic factor, leading to prolonged disease-free and overall survival, regardless of the treatment protocol or the presence of other pre-existing prognostic indicators. Minimal residual disease evaluation using flow cytometry is a sensitive and applicable approach which is expected to become an integral part of future prospective trials in CLL designed to assess the role of MRD surveillance in treatment tailoring.

  3. Noninvasive prenatal diagnosis. Use of density gradient centrifugation, magnetically activated cell sorting and in situ hybridization

    DEFF Research Database (Denmark)

    Campagnoli, C; Multhaupt, H A; Ludomirski, A;

    1997-01-01

    centrifugation and dual antibody labeling methods. The protocol was designed to compare the efficacy of antitransferrin receptor (CD71)/antiglycophorin A (GPA) antibodies with antithrom-bospondin receptor (CD36)/anti-GPA antibodies in identifying nucleated erythrocytes in maternal blood. Cytospin preparations...... cells recovered did not differ. Seven of seven male pregnancies were correctly identified. One case of trisomy 21 was detected. CONCLUSION: The in situ hybridization analysis of fetal nucleated erythrocytes isolated from maternal blood using single density gradient centrifugation, anti-CD71/anti...

  4. Identification of microbes from the surfaces of food-processing lines based on the flow cytometric evaluation of cellular metabolic activity combined with cell sorting.

    Science.gov (United States)

    Juzwa, W; Duber, A; Myszka, K; Białas, W; Czaczyk, K

    2016-09-01

    In this study the design of a flow cytometry-based procedure to facilitate the detection of adherent bacteria from food-processing surfaces was evaluated. The measurement of the cellular redox potential (CRP) of microbial cells was combined with cell sorting for the identification of microorganisms. The procedure enhanced live/dead cell discrimination owing to the measurement of the cell physiology. The microbial contamination of the surface of a stainless steel conveyor used to process button mushrooms was evaluated in three independent experiments. The flow cytometry procedure provided a step towards monitoring of contamination and enabled the assessment of microbial food safety hazards by the discrimination of active, mid-active and non-active bacterial sub-populations based on determination of their cellular vitality and subsequently single cell sorting to isolate microbial strains from discriminated sub-populations. There was a significant correlation (r = 0.97; p method and flow cytometry, despite there being differences in the absolute number of cells detected. The combined approach of flow cytometric CRP measurement and cell sorting allowed an in situ analysis of microbial cell vitality and the identification of species from defined sub-populations, although the identified microbes were limited to culturable cells.

  5. Integration through a Card-Sort Activity

    Science.gov (United States)

    Green, Kris; Ricca, Bernard P.

    2015-01-01

    Learning to compute integrals via the various techniques of integration (e.g., integration by parts, partial fractions, etc.) is difficult for many students. Here, we look at how students in a college level Calculus II course develop the ability to categorize integrals and the difficulties they encounter using a card sort-resort activity. Analysis…

  6. Machine-vision based optofluidic cell sorting

    DEFF Research Database (Denmark)

    Glückstad, Jesper; Bañas, Andrew

    In contemporary life science there is an increasing emphasis on sorting rare disease-indicating cells within small dilute quantities such as in the confines of optofluidic lab-on-chip devices. Our approach to this is based on the use of optical forces to isolate red blood cells detected by advanced...... machine vision1. This approach is gentler, less invasive and more economical compared to conventional FACS-systems. As cells are less responsive to plastic or glass objects commonly used in the optical manipulation literature2, and since laser safety would be an issue in clinical use, we develop efficient...... the available light and creating 2D or 3D beam distributions aimed at the positions of the detected cells. Furthermore, the beam shaping freedom provided by GPC can allow optimizations in the beam’s propagation and its interaction with the laser catapulted and sorted cells....

  7. Comparative analysis of circulating endothelial progenitor cells in age-related macular degeneration patients using automated rare cell analysis (ARCA and fluorescence activated cell sorting (FACS.

    Directory of Open Access Journals (Sweden)

    Emil Anthony T Say

    Full Text Available BACKGROUND: Patients with age-related macular degeneration (ARMD begin with non-neovascular (NNV phenotypes usually associated with good vision. Approximately 20% of NNV-ARMD patients will convert to vision debilitating neovascular (NV ARMD, but precise timing of this event is unknown. Developing a clinical test predicting impending conversion to NV-ARMD is necessary to prevent vision loss. Endothelial progenitor cells (EPCs, defined as CD34(+VEGR2(+ using traditional fluorescence activated cell sorting (FACS, are rare cell populations known to be elevated in patients with NV-ARMD compared to NNV-ARMD. FACS has high inter-observer variability and subjectivity when measuring rare cell populations precluding development into a diagnostic test. We hypothesized that automated rare cell analysis (ARCA, a validated and FDA-approved technology for reproducible rare cell identification, can enumerate EPCs in ARMD patients more reliably. This pilot study serves as the first step in developing methods for reproducibly predicting ARMD phenotype conversion. METHODS: We obtained peripheral venous blood samples in 23 subjects with NNV-ARMD or treatment naïve NV-ARMD. Strict criteria were used to exclude subjects with known angiogenic diseases to minimize confounding results. Blood samples were analyzed in masked fashion in two separate laboratories. EPCs were independently enumerated using ARCA and FACS within 24 hours of blood sample collection, and p<0.2 was considered indicative of a trend for this proof of concept study, while statistical significance was established at 0.05. RESULTS: We measured levels of CD34(+VEGFR2(+ EPCs suggestive of a trend with higher values in patients with NV compared to NNV-ARMD (p = 0.17 using ARCA. Interestingly, CD34(+VEGR2(+ EPC analysis using FACS did not produce similar results (p = 0.94. CONCLUSIONS: CD34(+VEGR2(+ may have predictive value for EPC enumeration in future ARCA studies. EPC measurements in a small sample

  8. Cell sorting using efficient light shaping approaches

    DEFF Research Database (Denmark)

    Banas, Andrew; Palima, Darwin; Villangca, Mark Jayson;

    2016-01-01

    Early detection of diseases can save lives. Hence, there is emphasis in sorting rare disease-indicating cells within small dilute quantities such as in the confines of lab-on-a-chip devices. In our work, we use optical forces to isolate red blood cells detected by machine vision. This approach...... and light modulation devices. The Generalized Phase Contrast (GPC) method that can be used for efficiently illuminating spatial light modulators or creating well-defined contiguous optical traps is supplemented by diffractive techniques capable of integrating the available light and creating 2D or 3D beam...

  9. Separation of uncompromised whole blood mixtures for single source STR profiling using fluorescently-labeled human leukocyte antigen (HLA) probes and fluorescence activated cell sorting (FACS).

    Science.gov (United States)

    Dean, Lee; Kwon, Ye Jin; Philpott, M Katherine; Stanciu, Cristina E; Seashols-Williams, Sarah J; Dawson Cruz, Tracey; Sturgill, Jamie; Ehrhardt, Christopher J

    2015-07-01

    Analysis of biological mixtures is a significant problem for forensic laboratories, particularly when the mixture contains only one cell type. Contributions from multiple individuals to biologic evidence can complicate DNA profile interpretation and often lead to a reduction in the probative value of DNA evidence or worse, its total loss. To address this, we have utilized an analytical technique that exploits the intrinsic immunological variation among individuals to physically separate cells from different sources in a mixture prior to DNA profiling. Specifically, we applied a fluorescently labeled antibody probe to selectively bind to one contributor in a mixture through allele-specific interactions with human leukocyte antigen (HLA) proteins that are expressed on the surfaces of most nucleated cells. Once the contributor's cells were bound to the probe, they were isolated from the mixture using fluorescence activated cell sorting (FACS)-a high throughput technique for separating cell populations based on their optical properties-and then subjected to STR analysis. We tested this approach on two-person and four-person whole blood mixtures where one contributor possessed an HLA allele (A*02) that was not shared by other contributors to the mixture. Results showed that hybridization of the mixture with a fluorescently-labeled antibody probe complimentary to the A*02 allele's protein product created a cell population with a distinct optical profile that could be easily differentiated from other cells in the mixture. After sorting the cells with FACS, genetic analysis showed that the STR profile of this cell population was consistent with that of the contributor who possessed the A*02 allele. Minor peaks from the A*02 negative contributor(s) were observed but could be easily distinguished from the profile generated from A*02 positive cells. Overall, this indicates that HLA antibody probes coupled to FACS may be an effective approach for generating STR profiles of

  10. Safe sorting of GFP-transduced live cells for subsequent culture using a modified FACS vantage

    DEFF Research Database (Denmark)

    Sørensen, T U; Gram, G J; Nielsen, S D;

    1999-01-01

    culture. RESULTS: The bacteriophage sorting showed that the biologically active material was confined to the sorting chamber. A failure mode simulating a nozzle blockage resulted in detectable droplets inside the sorting chamber, but no droplets could be detected when an additional air suction from....... Safety tests with bacteriophages were performed to evaluate the potential spread of biologically active material during cell sorting. Cells transduced with a retroviral vector carrying the gene for GFP were sorted on the basis of their GFP fluorescence, and GFP expression was followed during subsequent...

  11. Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays

    OpenAIRE

    Saliba, Antoine-Emmanuel; Saias, Laure; Psychari, Eleni; Minc, Nicolas; Simon, Damien; Bidard, François-Clément; Mathiot, Claire; Pierga, Jean-Yves; Fraisier, Vincent; Salamero, Jean; Saada, Véronique; Farace, Françoise; Vielh, Philippe; Malaquin, Laurent; Viovy, Jean-Louis

    2010-01-01

    We propose a unique method for cell sorting, “Ephesia,” using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On c...

  12. Microfluidic-chip platform for cell sorting

    Science.gov (United States)

    Malik, Sarul; Balyan, Prerna; Akhtar, J.; Agarwal, Ajay

    2016-04-01

    Cell sorting and separation are considered to be very crucial preparatory steps for numerous clinical diagnostics and therapeutics applications in cell biology research arena. Label free cell separation techniques acceptance rate has been increased to multifold by various research groups. Size based cell separation method focuses on the intrinsic properties of the cell which not only avoids clogging issues associated with mechanical and centrifugation filtration methods but also reduces the overall cost for the process. Consequentially flow based cell separation method for continuous flow has attracted the attention of millions. Due to the realization of structures close to particle size in micro dimensions, the microfluidic devices offer precise and rapid particle manipulation which ultimately leads to an extraordinary cell separation results. The proposed microfluidic device is fabricated to separate polystyrene beads of size 1 µm, 5 µm, 10 µm and 20 µm. The actual dimensions of blood corpuscles were kept in mind while deciding the particle size of polystyrene beads which are used as a model particles for study.

  13. Cell sorting using efficient light shaping approaches

    Science.gov (United States)

    Bañas, Andrew; Palima, Darwin; Villangca, Mark; Glückstad, Jesper

    2016-03-01

    Early detection of diseases can save lives. Hence, there is emphasis in sorting rare disease-indicating cells within small dilute quantities such as in the confines of lab-on-a-chip devices. In our work, we use optical forces to isolate red blood cells detected by machine vision. This approach is gentler, less invasive and more economical compared to conventional FACS systems. As cells are less responsive to plastic or glass beads commonly used in the optical manipulation literature, and since laser safety would be an issue in clinical use, we develop efficient approaches in utilizing lasers and light modulation devices. The Generalized Phase Contrast (GPC) method that can be used for efficiently illuminating spatial light modulators or creating well-defined contiguous optical traps is supplemented by diffractive techniques capable of integrating the available light and creating 2D or 3D beam distributions aimed at the positions of the detected cells. Furthermore, the beam shaping freedom provided by GPC can allow optimizations in the beam's propagation and its interaction with the catapulted cells.

  14. Osteogenic potential of sorted equine mesenchymal stem cell subpopulations

    OpenAIRE

    Radtke, Catherine L.; Nino-Fong, Rodolfo; Rodriguez-Lecompte, Juan Carlos; Esparza Gonzalez, Blanca P.; Stryhn, Henrik; McDuffee, Laurie A.

    2015-01-01

    The objectives of this study were to use non-equilibrium gravitational field-flow fractionation (GrFFF), an immunotag-less method of sorting mesenchymal stem cells (MSCs), to sort equine muscle tissue-derived mesenchymal stem cells (MMSCs) and bone marrow-derived mesenchymal stem cells (BMSC) into subpopulations and to carry out assays in order to compare their osteogenic capabilities. Cells from 1 young adult horse were isolated from left semitendinosus muscle tissue and from bone marrow asp...

  15. Real-Time Fluorescence Lifetime Actuation for Cell Sorting using a CMOS SPAD Silicon Photomultiplier

    OpenAIRE

    Mattioli della Rocca, Francescopaolo; Nedbal, Jakub; Tyndall, David; Krstajic, Nikola; Li, David Day-Uei; Ameer-Beg, Simon; Henderson, Robert

    2016-01-01

    Time-correlated single photon counting (TCSPC) is a fundamental fluorescence lifetime measurement technique offering high signal to noise ratio (SNR). However, its requirement for complex software algorithms for histogram processing restricts throughput in flow cytometers and prevents on-the-fly sorting of cells. We present a single-point digital silicon photomultiplier (SiPM) detector accomplishing real-time fluorescence lifetime-activated actuation targeting cell sorting applications in flo...

  16. Cell wall sorting of lipoproteins in Staphylococcus aureus.

    OpenAIRE

    Navarre, W W; Daefler, S; Schneewind, O

    1996-01-01

    Many surface proteins are thought to be anchored to the cell wall of gram-positive organisms via their C termini, while the N-terminal domains of these molecules are displayed on the bacterial surface. Cell wall anchoring of surface proteins in Staphylococcus aureus requires both an N-terminal leader peptide and a C-terminal cell wall sorting signal. By fusing the cell wall sorting of protein A to the C terminus of staphylococcal beta-lactamase, we demonstrate here that lipoproteins can also ...

  17. Recombinant Escherichia coli produces tailor-made biopolyester granules for applications in fluorescence activated cell sorting: functional display of the mouse interleukin-2 and myelin oligodendrocyte glycoprotein

    Directory of Open Access Journals (Sweden)

    Brockelbank Jane A

    2007-01-01

    Full Text Available Abstract Background Fluorescence activated cell sorting (FACS is a powerful technique for the qualitative and quantitative detection of biomolecules used widely in both basic research and clinical diagnostic applications. Beads displaying a specific antigen are used to bind antibodies which are then fluorescently labelled using secondary antibodies. As the individual suspension bead passes through the sensing region of the FACS machine, fluorescent signals are acquired and analysed. Currently, antigens are tediously purified and chemically cross-linked to preformed beads. Purification and coupling of proteins often renders them inactive and they will not be displayed in its native configuration. As an alternative, we genetically engineered Escherichia coli to produce biopolyester (polyhdroxyalkanoate=PHA granules displaying diagnostically relevant antigens in their native conformation and suitable for FACS analysis. Results Hybrid genes were constructed, which encode either the mouse interleukin-2 (IL2 or the myelin oligodendrocyte glycoprotein (MOG fused via an enterokinase site providing linker region to the C terminus of the PHA granule associated protein PhaP, respectively. The hybrid genes were expressed in PHA-accumulating recombinant E. coli. MOG and IL2 fusion proteins were abundantly attached to PHA granules and were identified by MALDI-TOF/MS analysis and N terminal sequencing. A more abundant second fusion protein of either MOG or IL2 resulted from an additional N terminal fusion, which did surprisingly not interfere with attachment to PHA granule. PHA granules displaying either IL2 or MOG were used for FACS using monoclonal anti-IL2 or anti-MOG antibodies conjugated to a fluorescent dye. FACS analysis showed significant and specific binding of respective antibodies. Enterokinase treatment of IL2 displaying PHA granules enabled removal of IL2 as monitored by FACS analysis. Mice were immunized with either MOG or OVA (ovalbumin and the

  18. Development of the Adolescent and Young Adult Activity Card Sort.

    Science.gov (United States)

    Berg, Christine; McCollum, Mary; Cho, Esther; Jason, Dawn

    2015-10-01

    Emerging adulthood defines transition to employment, higher education, and domestic life. This study describes the development of an assessment of self-reported participation in a range of age-appropriate activities. Item selection was established from literature review, feedback from youth and professionals, the former Adolescent Activity Card Sort (AACS), and the original Activity Card Sort (ACS). Iterative item selection occurred with three separate samples of emerging adults and six professionals. Test-retest reliability was evaluated. The Adolescent and Young Adult Activity Card Sort (AYA-ACS) consists of chores (11 items), leisure (13), social (10), health and fitness (9), work (10), education (8), and parenting (9). Test-retest reliability showed significant moderate to substantial Kappa agreement (.48-.85) for all domains except parenting (κ = .15). This preliminary study describes the development of the AYA-ACS to be used with individuals who encounter challenges when transitioning to young adulthood. PMID:27505902

  19. Sphingolipid trafficking and protein sorting in epithelial cells

    NARCIS (Netherlands)

    Slimane, TA; Hoekstra, D

    2002-01-01

    Sphingolipids represent a minor, but highly dynamic subclass of lipids in all eukaryotic cells. They are involved in functions that range from structural protection to signal transduction and protein sorting, and participate in lipid raft assembly. In polarized epithelial cells, which display an asy

  20. An analytical comparison of the information in sorted and non-sorted cosine-tuned spike activity

    Science.gov (United States)

    Won, D. S.; Tiesinga, P. H. E.; Henriquez, C. S.; Wolf, P. D.

    2007-09-01

    Spike sorting is a technologically expensive component of the signal processing chain required to interpret population spike activity acquired in a neuromotor prosthesis. No systematic analysis of the value of spike sorting has been carried out, and little is known about the effects of spike sorting error on the ability of a brain-machine interface (BMI) to decode intended motor commands. We developed a theoretical framework to examine the effects of spike processing on the information available to a BMI decoder. We computed the mutual information in neural activity in a simplified model of directional cosine tuning to compare the effects of pooling activity from up to four neurons to the effects of sorting with varying amounts of spike error. The results showed that information in a small population of cosine-tuned neurons is maximized when the responses are sorted and there is diverse tuning of units, but information was affected little when pooling units with similar preferred directions. Spike error had adverse effects on information, such that non-sorted population activity had 79-92% of the information in its sorted counterpart for reasonable amounts of detection and sorting error and for units with moderate differences in preferred direction. This quantification of information loss associated with pooling units and with spike detection and sorting error will help to guide the engineering decisions in designing a BMI spike processing system.

  1. Multiplexed labeling system for high-throughput cell sorting.

    Science.gov (United States)

    Shin, Seung Won; Park, Kyung Soo; Song, In Hyun; Shin, Woo Jung; Kim, Byung Woo; Kim, Dong-Ik; Um, Soong Ho

    2016-09-01

    Flow cytometry and fluorescence activated cell sorting techniques were designed to realize configurable classification and separation of target cells. A number of cell phenotypes with different functionalities have recently been revealed. Before simultaneous selective capture of cells, it is desirable to label different samples with the corresponding dyes in a multiplexing manner to allow for a single analysis. However, few methods to obtain multiple fluorescent colors for various cell types have been developed. Even when restricted laser sources are employed, a small number of color codes can be expressed simultaneously. In this study, we demonstrate the ability to manifest DNA nanostructure-based multifluorescent colors formed by a complex of dyes. Highly precise self-assembly of fluorescent dye-conjugated oligonucleotides gives anisotropic DNA nanostructures, Y- and tree-shaped DNA (Y-DNA and T-DNA, respectively), which may be used as platforms for fluorescent codes. As a proof of concept, we have demonstrated seven different fluorescent codes with only two different fluorescent dyes using T-DNA. This method provides maximum efficiency for current flow cytometry. We are confident that this system will provide highly efficient multiplexed fluorescent detection for bioanalysis compared with one-to-one fluorescent correspondence for specific marker detection. PMID:27181032

  2. New high-speed cell sorting methods for stem cell sorting and breast cancer cell purging

    Science.gov (United States)

    Leary, James F.; McLaughlin, Scott R.; Hokanson, James A.; Rosenblatt, Judah I.

    1998-04-01

    An important problem in clinical medicine is that of positively selecting hematopoietic stem cells or mobilized peripheral blood stem cells for autologous bone marrow transplantation while purging it of contaminating tumor cells. Since both the stem cells to be positively selected and the tumor cells to be purged are relatively rare cells, this poses special problems for their isolation in terms of purity and yield of stem cells, with a high penalty of misclassification for contaminating tumor cells. A model system of tumor cells spiked into bone marrow or blood cells was used to validate the system. Multiparameter data mixtures of human MCF-7 breast cancer cells and human peripheral blood or bone marrow cells were first analyzed by discriminant function analysis. Mathematical methods were developed to assess the relative probabilities of misclassification. Cell identification tags, implemented as additional correlated listmode parameters not used for these analyses, were used to uniquely identify each cell type and to compare classifier results. The performance of classifier systems was also assessed using ROC (`receiver operating characteristics') analysis. Then the classification system was implemented using lookup tables allowing for real-time (in this system approximately 625 microseconds) rapid separation of these cell types. Isolated cell types, purities and yields were assessed by single-cell PCR molecular characterizations.

  3. Lipid polarity and sorting in epithelial cells

    NARCIS (Netherlands)

    van Meer, G.; Simons, K.

    1988-01-01

    Apical and basolateral membrane domains of epithelial cell plasma membranes possess unique lipid compositions. The tight junction, the structure separating the two domains, forms a diffusion barrier for membrane components and thereby prevents intermixing of the two sets of lipids. The barrier appar

  4. Unravelling the pivotal role of Alix in MVB sorting and silencing of the activated EGFR.

    Science.gov (United States)

    Sun, Sheng; Zhou, Xi; Zhang, Wei; Gallick, Gary E; Kuang, Jian

    2015-03-15

    Endosomal sorting complex required for transport (ESCRT)-III-mediated membrane invagination and scission are a critical step in multivesicular body (MVB) sorting of ubiquitinated membrane receptors, and generally thought to be required for degradation of these receptors in lysosomes. The adaptor protein Alix is critically involved in multiple ESCRT-III-mediated, membrane-remodelling processes in mammalian cells. However, Alix knockdown does not inhibit degradation of the activated epidermal growth factor receptor (EGFR) in mammalian cell lines, leading to a widely held notion that Alix is not critically involved in MVB sorting of ubiquitinated membrane receptors in mammalian cells. In the present study, we demonstrate that, despite its non-essential role in degradation of the activated EGFR, Alix plays a critical role in its MVB sorting and silencing Epidermal growth factor (EGF) stimulation of mammalian cell lines induces Alix's interaction with the ubiquitinated EGFR via the Alix V domain, and increases Alix's association with membrane-bound charged multivesicular body protein 4 (CHMP4) via the Alix Bro1 domain. Under both continuous and pulse-chase EGF stimulation conditions, inhibition of Alix's interaction with membrane-bound CHMP4, inhibition of Alix dimerization through the V domain or Alix knockdown dramatically inhibits MVB sorting of the activated EGFR and promotes sustained activation of extracellular-signal regulated kinase (ERK)1/2. Under the continuous EGF stimulation conditions, these cell treatments also retard degradation of the activated EGFR. These findings indicate that Alix is critically involved in MVB sorting of ubiquitinated membrane receptors in mammalian cells.

  5. Large area magnetic micropallet arrays for cell colony sorting.

    Science.gov (United States)

    Cox-Muranami, Wesley A; Nelson, Edward L; Li, G P; Bachman, Mark

    2016-01-01

    A new micropallet array platform for adherent cell colony sorting has been developed. The platform consisted of thousands of square plastic pallets, 270 μm by 270 μm on each side, large enough to hold a single colony of cells. Each pallet included a magnetic core, allowing them to be collected with a magnet after being released using a microscope mounted laser system. The micropallets were patterned from 1002F epoxy resist and were fabricated on translucent, gold coated microscope slides. The gold layer was used as seed for electroplating the ferromagnetic cores within every individual pallet. The gold layer also facilitated the release of each micropallet during laser release. This array allows for individual observation, sorting and collection of isolated cell colonies for biological cell colony research. In addition to consistent release and recovery of individual colonies, we demonstrated stable biocompatibility and minimal loss in imaging quality compared to previously developed micropallet arrays.

  6. Large area magnetic micropallet arrays for cell colony sorting.

    Science.gov (United States)

    Cox-Muranami, Wesley A; Nelson, Edward L; Li, G P; Bachman, Mark

    2016-01-01

    A new micropallet array platform for adherent cell colony sorting has been developed. The platform consisted of thousands of square plastic pallets, 270 μm by 270 μm on each side, large enough to hold a single colony of cells. Each pallet included a magnetic core, allowing them to be collected with a magnet after being released using a microscope mounted laser system. The micropallets were patterned from 1002F epoxy resist and were fabricated on translucent, gold coated microscope slides. The gold layer was used as seed for electroplating the ferromagnetic cores within every individual pallet. The gold layer also facilitated the release of each micropallet during laser release. This array allows for individual observation, sorting and collection of isolated cell colonies for biological cell colony research. In addition to consistent release and recovery of individual colonies, we demonstrated stable biocompatibility and minimal loss in imaging quality compared to previously developed micropallet arrays. PMID:26606460

  7. Automated single cell sorting and deposition in submicroliter drops

    Science.gov (United States)

    Salánki, Rita; Gerecsei, Tamás; Orgovan, Norbert; Sándor, Noémi; Péter, Beatrix; Bajtay, Zsuzsa; Erdei, Anna; Horvath, Robert; Szabó, Bálint

    2014-08-01

    Automated manipulation and sorting of single cells are challenging, when intact cells are needed for further investigations, e.g., RNA or DNA sequencing. We applied a computer controlled micropipette on a microscope admitting 80 PCR (Polymerase Chain Reaction) tubes to be filled with single cells in a cycle. Due to the Laplace pressure, fluid starts to flow out from the micropipette only above a critical pressure preventing the precise control of drop volume in the submicroliter range. We found an anomalous pressure additive to the Laplace pressure that we attribute to the evaporation of the drop. We have overcome the problem of the critical dropping pressure with sequentially operated fast fluidic valves timed with a millisecond precision. Minimum drop volume was 0.4-0.7 μl with a sorting speed of 15-20 s per cell. After picking NE-4C neuroectodermal mouse stem cells and human primary monocytes from a standard plastic Petri dish we could gently deposit single cells inside tiny drops. 94 ± 3% and 54 ± 7% of the deposited drops contained single cells for NE-4C and monocytes, respectively. 7.5 ± 4% of the drops contained multiple cells in case of monocytes. Remaining drops were empty. Number of cells deposited in a drop could be documented by imaging the Petri dish before and after sorting. We tuned the adhesion force of cells to make the manipulation successful without the application of microstructures for trapping cells on the surface. We propose that our straightforward and flexible setup opens an avenue for single cell isolation, critically needed for the rapidly growing field of single cell biology.

  8. Psychometric properties of the Arab Heritage Activity Card Sort.

    Science.gov (United States)

    Hamed, Razan; Holm, Margo B

    2013-03-01

    The Activity Card Sort is a valid and reliable assessment tool that was created to assess Participation. It has been translated to several languages and adapted to different international cultures. The most recent version of this tool is the Arabic Heritage Activity Card Sort (A-ACS). The purpose of this study was to establish the psychometric properties of the new Arabic version in Jordanian adults. Forty three Jordanian patients with multiple sclerosis (MS) and 62 healthy adults were recruited to test the psychometric properties of the tool. The A-ACS correlated moderately with the participation index of the Mayo-Portland Adaptability Inventory (r = -0.458, p scores of the Mayo-Portland Adaptability Inventory (r = -0.458, p score on the Arabic version of the self-report Performance Assessment of Self-care Skills (r = 0.581, p Card Sort is a valid and reliable tool for Arabic-speaking occupational therapists to use when assessing participation in Jordanian patients with MS or healthy adults. Limitations of this study include using only one diagnostic group from Jordan and examining only the Recovery and Community Versions of the tool. Future studies are needed to examine further psychometric properties for patients with different diagnoses and from different countries in the Arabic region for all three versions of the A-ACS.

  9. Endogenous RNAs Modulate MicroRNA Sorting to Exosomes and Transfer to Acceptor Cells

    Directory of Open Access Journals (Sweden)

    Mario Leonardo Squadrito

    2014-09-01

    Full Text Available MicroRNA (miRNA transfer via exosomes may mediate cell-to-cell communication. Interestingly, specific miRNAs are enriched in exosomes in a cell-type-dependent fashion. However, the mechanisms whereby miRNAs are sorted to exosomes and the significance of miRNA transfer to acceptor cells are unclear. We used macrophages and endothelial cells (ECs as a model of heterotypic cell communication in order to investigate both processes. RNA profiling of macrophages and their exosomes shows that miRNA sorting to exosomes is modulated by cell-activation-dependent changes of miRNA target levels in the producer cells. Genetically perturbing the expression of individual miRNAs or their targeted transcripts promotes bidirectional miRNA relocation from the cell cytoplasm/P bodies (sites of miRNA activity to multivesicular bodies (sites of exosome biogenesis and controls miRNA sorting to exosomes. Furthermore, the use of Dicer-deficient cells and reporter lentiviral vectors (LVs for miRNA activity shows that exosomal miRNAs are transferred from macrophages to ECs to detectably repress targeted sequences.

  10. Fast polyhedral cell sorting for interactive rendering of unstructured grids

    Energy Technology Data Exchange (ETDEWEB)

    Combra, J; Klosowski, J T; Max, N; Silva, C T; Williams, P L

    1998-10-30

    Direct volume rendering based on projective methods works by projecting, in visibility order, the polyhedral cells of a mesh onto the image plane, and incrementally compositing the cell's color and opacity into the final image. Crucial to this method is the computation of a visibility ordering of the cells. If the mesh is ''well-behaved'' (acyclic and convex), then the MPVO method of Williams provides a very fast sorting algorithm; however, this method only computes an approximate ordering in general datasets, resulting in visual artifacts when rendered. A recent method of Silva et al. removed the assumption that the mesh is convex, by means of a sweep algorithm used in conjunction with the MPVO method; their algorithm is substantially faster than previous exact methods for general meshes. In this paper we propose a new technique, which we call BSP-XMPVO, which is based on a fast and simple way of using binary space partitions on the boundary elements of the mesh to augment the ordering produced by MPVO. Our results are shown to be orders of magnitude better than previous exact methods of sorting cells.

  11. Separation and sorting of cells in microsystems using physical principles

    International Nuclear Information System (INIS)

    In the last decade, microfabrication techniques have been combined with microfluidics and applied to cell biology. Utilizing such new techniques, various cell studies have been performed for the research of stem cells, immune cells, cancer, neurons, etc. Among the various biological applications of microtechnology-based platforms, cell separation technology has been highly regarded in biological and clinical fields for sorting different types of cells, finding circulating tumor cells (CTCs), and blood cell separation, amongst other things. Many cell separation methods have been created using various physical principles. Representatively, these include hydrodynamic, acoustic, dielectrophoretic, magnetic, optical, and filtering methods. In this review, each of these methods will be introduced, and their physical principles and sample applications described. Each physical principle has its own advantages and disadvantages. The engineers who design the systems and the biologists who use them should understand the pros and cons of each method or principle, to broaden the use of microsystems for cell separation. Continuous development of microsystems for cell separation will lead to new opportunities for diagnosing CTCs and cancer metastasis, as well as other elements in the bloodstream. (topical review)

  12. Separation and sorting of cells in microsystems using physical principles

    Science.gov (United States)

    Lee, Gi-Hun; Kim, Sung-Hwan; Ahn, Kihoon; Lee, Sang-Hoon; Park, Joong Yull

    2016-01-01

    In the last decade, microfabrication techniques have been combined with microfluidics and applied to cell biology. Utilizing such new techniques, various cell studies have been performed for the research of stem cells, immune cells, cancer, neurons, etc. Among the various biological applications of microtechnology-based platforms, cell separation technology has been highly regarded in biological and clinical fields for sorting different types of cells, finding circulating tumor cells (CTCs), and blood cell separation, amongst other things. Many cell separation methods have been created using various physical principles. Representatively, these include hydrodynamic, acoustic, dielectrophoretic, magnetic, optical, and filtering methods. In this review, each of these methods will be introduced, and their physical principles and sample applications described. Each physical principle has its own advantages and disadvantages. The engineers who design the systems and the biologists who use them should understand the pros and cons of each method or principle, to broaden the use of microsystems for cell separation. Continuous development of microsystems for cell separation will lead to new opportunities for diagnosing CTCs and cancer metastasis, as well as other elements in the bloodstream.

  13. Novel serial positive enrichment technology enables clinical multiparameter cell sorting.

    Directory of Open Access Journals (Sweden)

    Christian Stemberger

    Full Text Available A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve--especially for clinical cell products. Therefore, we have generated low-affinity antibody-derived Fab-fragments, which stain like parental antibodies when multimerized via Strep-tag and Strep-Tactin, but can subsequently be removed entirely from the target cell population. Such reagents can be generated for virtually any antigen and can be used for sequential positive enrichment steps via paramagnetic beads. First protocols for multiparameter enrichment of two clinically relevant cell populations, CD4(high/CD25(high/CD45RA(high 'regulatory T cells' and CD8(high/CD62L(high/CD45RA(neg 'central memory T cells', have been established to determine quality and efficacy parameters of this novel technology, which should have broad applicability for clinical cell sorting as well as basic research.

  14. Protein characterization of intracellular target-sorted, formalin-fixed cell subpopulations

    Science.gov (United States)

    Sadick, Jessica S.; Boutin, Molly E.; Hoffman-Kim, Diane; Darling, Eric M.

    2016-01-01

    Cellular heterogeneity is inherent in most human tissues, making the investigation of specific cell types challenging. Here, we describe a novel, fixation/intracellular target-based sorting and protein extraction method to provide accurate protein characterization for cell subpopulations. Validation and feasibility tests were conducted using homogeneous, neural cell lines and heterogeneous, rat brain cells, respectively. Intracellular proteins of interest were labeled with fluorescent antibodies for fluorescence-activated cell sorting. Reproducible protein extraction from fresh and fixed samples required lysis buffer with high concentrations of Tris-HCl and sodium dodecyl sulfate as well as exposure to high heat. No deterioration in protein amount or quality was observed for fixed, sorted samples. For the feasibility experiment, a primary rat subpopulation of neuronal cells was selected for based on high, intracellular β-III tubulin signal. These cells showed distinct protein expression differences from the unsorted population for specific (phosphorylated tau) and non-specific (total tau) protein targets. Our approach allows for determining more accurate protein profiles directly from cell types of interest and provides a platform technology in which any cell subpopulation can be biochemically investigated. PMID:27666089

  15. Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays

    Science.gov (United States)

    Saliba, Antoine-Emmanuel; Saias, Laure; Psychari, Eleni; Minc, Nicolas; Simon, Damien; Bidard, François-Clément; Mathiot, Claire; Pierga, Jean-Yves; Fraisier, Vincent; Salamero, Jean; Saada, Véronique; Farace, Françoise; Vielh, Philippe; Malaquin, Laurent; Viovy, Jean-Louis

    2010-01-01

    We propose a unique method for cell sorting, “Ephesia,” using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples—blood, pleural effusion, and fine needle aspirates— issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost. PMID:20679245

  16. Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays.

    Science.gov (United States)

    Saliba, Antoine-Emmanuel; Saias, Laure; Psychari, Eleni; Minc, Nicolas; Simon, Damien; Bidard, François-Clément; Mathiot, Claire; Pierga, Jean-Yves; Fraisier, Vincent; Salamero, Jean; Saada, Véronique; Farace, Françoise; Vielh, Philippe; Malaquin, Laurent; Viovy, Jean-Louis

    2010-08-17

    We propose a unique method for cell sorting, "Ephesia," using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples--blood, pleural effusion, and fine needle aspirates--issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost. PMID:20679245

  17. Novel Serial Positive Enrichment Technology Enables Clinical Multiparameter Cell Sorting

    Science.gov (United States)

    Tschulik, Claudia; Piossek, Christine; Bet, Jeannette; Yamamoto, Tori N.; Schiemann, Matthias; Neuenhahn, Michael; Martin, Klaus; Schlapschy, Martin; Skerra, Arne; Schmidt, Thomas; Edinger, Matthias; Riddell, Stanley R.; Germeroth, Lothar; Busch, Dirk H.

    2012-01-01

    A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve – especially for clinical cell products. Therefore, we have generated low-affinity antibody-derived Fab-fragments, which stain like parental antibodies when multimerized via Strep-tag and Strep-Tactin, but can subsequently be removed entirely from the target cell population. Such reagents can be generated for virtually any antigen and can be used for sequential positive enrichment steps via paramagnetic beads. First protocols for multiparameter enrichment of two clinically relevant cell populations, CD4high/CD25high/CD45RAhigh ‘regulatory T cells’ and CD8high/CD62Lhigh/CD45RAneg ‘central memory T cells’, have been established to determine quality and efficacy parameters of this novel technology, which should have broad applicability for clinical cell sorting as well as basic research. PMID:22545138

  18. Identification and characterization of EGF receptor in individual exosomes by fluorescence-activated vesicle sorting.

    Science.gov (United States)

    Higginbotham, James N; Zhang, Qin; Jeppesen, Dennis K; Scott, Andrew M; Manning, H Charles; Ochieng, Josiah; Franklin, Jeffrey L; Coffey, Robert J

    2016-01-01

    Exosomes are small, 40-130 nm secreted extracellular vesicles that recently have become the subject of intense focus as agents of intercellular communication, disease biomarkers and potential vehicles for drug delivery. It is currently unknown whether a cell produces different populations of exosomes with distinct cargo and separable functions. To address this question, high-resolution methods are needed. Using a commercial flow cytometer and directly labelled fluorescent antibodies, we show the feasibility of using fluorescence-activated vesicle sorting (FAVS) to analyse and sort individual exosomes isolated by sequential ultracentrifugation from the conditioned medium of DiFi cells, a human colorectal cancer cell line. EGFR and the exosomal marker, CD9, were detected on individual DiFi exosomes by FAVS; moreover, both markers were identified by high-resolution stochastic optical reconstruction microscopy on individual, approximately 100 nm vesicles from flow-sorted EGFR/CD9 double-positive exosomes. We present evidence that the activation state of EGFR can be assessed in DiFi-derived exosomes using a monoclonal antibody (mAb) that recognizes "conformationally active" EGFR (mAb 806). Using human antigen-specific antibodies, FAVS was able to detect human EGFR and CD9 on exosomes isolated from the plasma of athymic nude mice bearing DiFi tumour xenografts. Multicolour FAVS was used to simultaneously identify CD9, EGFR and an EGFR ligand, amphiregulin (AREG), on human plasma-derived exosomes from 3 normal individuals. These studies demonstrate the feasibility of FAVS to both analyse and sort individual exosomes based on specific cell-surface markers. We propose that FAVS may be a useful tool to monitor EGFR and AREG in circulating exosomes from individuals with colorectal cancer and possibly other solid tumours. PMID:27345057

  19. Identification and characterization of EGF receptor in individual exosomes by fluorescence-activated vesicle sorting

    Science.gov (United States)

    Higginbotham, James N.; Zhang, Qin; Jeppesen, Dennis K.; Scott, Andrew M.; Manning, H. Charles; Ochieng, Josiah; Franklin, Jeffrey L.; Coffey, Robert J.

    2016-01-01

    Exosomes are small, 40–130 nm secreted extracellular vesicles that recently have become the subject of intense focus as agents of intercellular communication, disease biomarkers and potential vehicles for drug delivery. It is currently unknown whether a cell produces different populations of exosomes with distinct cargo and separable functions. To address this question, high-resolution methods are needed. Using a commercial flow cytometer and directly labelled fluorescent antibodies, we show the feasibility of using fluorescence-activated vesicle sorting (FAVS) to analyse and sort individual exosomes isolated by sequential ultracentrifugation from the conditioned medium of DiFi cells, a human colorectal cancer cell line. EGFR and the exosomal marker, CD9, were detected on individual DiFi exosomes by FAVS; moreover, both markers were identified by high-resolution stochastic optical reconstruction microscopy on individual, approximately 100 nm vesicles from flow-sorted EGFR/CD9 double-positive exosomes. We present evidence that the activation state of EGFR can be assessed in DiFi-derived exosomes using a monoclonal antibody (mAb) that recognizes “conformationally active” EGFR (mAb 806). Using human antigen-specific antibodies, FAVS was able to detect human EGFR and CD9 on exosomes isolated from the plasma of athymic nude mice bearing DiFi tumour xenografts. Multicolour FAVS was used to simultaneously identify CD9, EGFR and an EGFR ligand, amphiregulin (AREG), on human plasma-derived exosomes from 3 normal individuals. These studies demonstrate the feasibility of FAVS to both analyse and sort individual exosomes based on specific cell-surface markers. We propose that FAVS may be a useful tool to monitor EGFR and AREG in circulating exosomes from individuals with colorectal cancer and possibly other solid tumours. PMID:27345057

  20. Viable cell sorting of dinoflagellates by multi-parametric flow cytometry.

    Science.gov (United States)

    Electronic cell sorting for isolation and culture of dinoflagellates and other marine eukaryotic phytoplankton was compared to the traditional method of manually picking of cells using a micropipette. Trauma to electronically sorted cells was not a limiting factor as fragile dinoflagellates, such a...

  1. Lgr5 positive stem cells sorted from small intestines of diabetic mice differentiate into higher proportion of absorptive cells and Paneth cells in vitro.

    Science.gov (United States)

    Zhong, Xian-Yang; Yu, Tao; Zhong, Wa; Li, Jie-Yao; Xia, Zhong-Sheng; Yuan, Yu-Hong; Yu, Zhong; Chen, Qi-Kui

    2015-08-01

    Intestinal epithelial stem cells (IESCs) can differentiate into all types of intestinal epithelial cells (IECs) and Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) is a marker for IESC. Previous studies reported enhanced proliferation of IECs in diabetic mice. In this study, the in vitro differentiation of Lgr5 positive IESCs sorted from diabetic mice was further investigated. The diabetic mouse model was induced by streptozotocin (STZ), and crypt IECs were isolated from small intestines. Subsequently, Lgr5 positive IESCs were detected by flow cytometry (FCM) and sorted by magnetic activated cell sorting (MACS). Differentiation of the sorted IESCs was investigated by detecting the IEC markers in the diabetic mice using immunostaining, quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR), and Western blot analysis, which was compared with normal mice. We found that the proportion of Lgr5 positive cells in the crypt IECs of diabetic mice was higher than that of control mice (P absorptive cell marker sucrase-isomaltase (SI) and the Paneth cell marker lysozyme 1 (Lyz1) were more highly expressed in the differentiated cells derived from Lgr5 positive IESCs of diabetic mice in vitro (P small intestines of STZ-induced diabetic mice. Lgr5 positive IESCs sorted from the diabetic mice can differentiate into a higher proportion of absorptive cells and Paneth cells in vitro. We characterized the expression of Lgr5 in the small intestine of diabetic mice, and sorted Lgr5 positive intestinal epithelial stem cells (IESCs) for investigating their differentiation in vitro. We proved that the quantity of Lgr5 positive IESCs was significantly increased in the small intestines of diabetic mice. IESCs sorted from the diabetic mice can differentiate into a higher proportion of absorptive cells and Paneth cells in vitro.

  2. Sorting live stem cells based on Sox2 mRNA expression.

    Directory of Open Access Journals (Sweden)

    Hans M Larsson

    Full Text Available While cell sorting usually relies on cell-surface protein markers, molecular beacons (MBs offer the potential to sort cells based on the presence of any expressed mRNA and in principle could be extremely useful to sort rare cell populations from primary isolates. We show here how stem cells can be purified from mixed cell populations by sorting based on MBs. Specifically, we designed molecular beacons targeting Sox2, a well-known stem cell marker for murine embryonic (mES and neural stem cells (NSC. One of our designed molecular beacons displayed an increase in fluorescence compared to a nonspecific molecular beacon both in vitro and in vivo when tested in mES and NSCs. We sorted Sox2-MB(+SSEA1(+ cells from a mixed population of 4-day retinoic acid-treated mES cells and effectively isolated live undifferentiated stem cells. Additionally, Sox2-MB(+ cells isolated from primary mouse brains were sorted and generated neurospheres with higher efficiency than Sox2-MB(- cells. These results demonstrate the utility of MBs for stem cell sorting in an mRNA-specific manner.

  3. Cell Specific eQTL Analysis without Sorting Cells

    NARCIS (Netherlands)

    Westra, Harm-Jan; Arends, Danny; Esko, Tonu; Peters, Marjolein J.; Schurmann, Claudia; Schramm, Katharina; Kettunen, Johannes; Yaghootkar, Hanieh; Fairfax, Benjamin P.; Andiappan, Anand Kumar; Li, Yang; Fu, Jingyuan; Karjalainen, Juha; Platteel, Mathieu; Visschedijk, Marijn; Weersma, Rinse K.; Kasela, Silva; Milani, Lili; Tserel, Liina; Peterson, Part; Reinmaa, Eva; Hofman, Albert; Uitterlinden, Andre G.; Rivadeneira, Fernando; Homuth, Georg; Petersmann, Astrid; Lorbeer, Roberto; Prokisch, Holger; Meitinger, Thomas; Herder, Christian; Roden, Michael; Grallert, Harald; Ripatti, Samuli; Perola, Markus; Wood, Andrew R.; Melzer, David; Ferrucci, Luigi; Singleton, Andrew B.; Hernandez, Dena G.; Knight, Julian C.; Melchiotti, Rossella; Lee, Bernett; Poidinger, Michael; Zolezzi, Francesca; Larbi, Anis; Wang, De Yun; van den Berg, Leonard H.; Veldink, Jan H.; Rotzschke, Olaf; Makino, Seiko; Salomaa, Veikko; Strauch, Konstantin; Voelker, Uwe; van Meurs, Joyce B. J.; Metspalu, Andres; Wijmenga, Cisca; Jansen, Ritsert C.; Franke, Lude

    2015-01-01

    The functional consequences of trait associated SNPs are often investigated using expression quantitative trait locus (eQTL) mapping. While trait-associated variants may operate in a cell-type specific manner, eQTL datasets for such cell-types may not always be available. We performed a genome-envir

  4. Cell Specific eQTL Analysis without Sorting Cells

    NARCIS (Netherlands)

    H.J. Westra (Harm-Jan); D. Arends (Danny); T. Esko (Tõnu); M.J. Peters (Marjolein); C. Schurmann (Claudia); K. Schramm (Katharina); J. Kettunen (Johannes); H. Yaghootkar (Hanieh); B.P. Fairfax (Benjamin); A.K. Andiappan (Anand Kumar); Y. Li (Yang); J. Fu (Jingyuan); J. Karjalainen (Juha); I. Platteel (Inge); M. Visschedijk (Marijn); R.K. Weersma (Rinse K.); S. Kasela (Silva); L. Milani (Lili); L. Tserel (Liina); P. Peterson (Pärt); E. Reinmaa (Eva); A. Hofman (Albert); A.G. Uitterlinden (André); F. Rivadeneira Ramirez (Fernando); G. Homuth (Georg); A. Petersmann (Astrid); R. Lorbeer (Roberto); H. Prokisch (Holger); T. Meitinger (Thomas); C. Herder (Christian); M. Roden (Michael); H. Grallert (Harald); S. Ripatti (Samuli); M. Perola (Markus); A.R. Wood (Andrew); D. Melzer (David); L. Ferrucci (Luigi); A. Singleton (Andrew); D.G. Hernandez (Dena); J.C. Knight (Julian); R. Melchiotti (Rossella); B. Lee (Bernett); M. Poidinger (Michael); F. Zolezzi (Francesca); A. Larbi (Anis); D.Y. Wang (De Yun); L.H. van den Berg (Leonard); J.H. Veldink (Jan); O. Rotzschke (Olaf); S. Makino (Seiko); V. Salomaa (Veikko); K. Strauch (Konstantin); U. Völker (Uwe); J.B.J. van Meurs (Joyce); A. Metspalu (Andres); C. Wijmenga (Cisca); R.C. Jansen (Ritsert); L. Franke (Lude)

    2015-01-01

    textabstractThe functional consequences of trait associated SNPs are often investigated using expression quantitative trait locus (eQTL) mapping. While trait-associated variants may operate in a cell-type specific manner, eQTL datasets for such cell-types may not always be available. We performed a

  5. Cell Specific eQTL Analysis without Sorting Cells.

    Directory of Open Access Journals (Sweden)

    Harm-Jan Westra

    2015-05-01

    Full Text Available The functional consequences of trait associated SNPs are often investigated using expression quantitative trait locus (eQTL mapping. While trait-associated variants may operate in a cell-type specific manner, eQTL datasets for such cell-types may not always be available. We performed a genome-environment interaction (GxE meta-analysis on data from 5,683 samples to infer the cell type specificity of whole blood cis-eQTLs. We demonstrate that this method is able to predict neutrophil and lymphocyte specific cis-eQTLs and replicate these predictions in independent cell-type specific datasets. Finally, we show that SNPs associated with Crohn's disease preferentially affect gene expression within neutrophils, including the archetypal NOD2 locus.

  6. Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

    OpenAIRE

    Bieberich, Erhard

    2011-01-01

    The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins bindin...

  7. Side population sorting separates subfractions of cycling and non-cycling intestinal stem cells

    Directory of Open Access Journals (Sweden)

    Richard J. von Furstenberg

    2014-03-01

    Full Text Available We report here that side population (SP sorting allows for the simultaneous isolation of two intestinal stem cell (ISC subsets from wild-type (WT mice which are phenotypically different and represent cycling and non-cycling pools of cells. Following 5-ethynyl-2′-deoxyuridine (EdU injection, in the upper side population (USP the percentage of EdU+ was 36% showing this fraction to be highly proliferative. In the lower side population (LSP, only 0.4% of cells were EdU+, indicating this fraction to be predominantly non-cycling. Using Lgr5-EGFP mice, we show that Lgr5-EGFPhi cells, representing actively cycling ISCs, are essentially exclusive to the USP. In contrast, using histone 2B-YFP mice, SP analysis revealed YFP label retaining cells (LRCs in both the USP and the LSP. Correspondingly, evaluation of the SP fractions for mRNA markers by qRT-PCR showed that the USP was enriched in transcripts associated with both quiescent and active ISCs. In contrast, the LSP expressed mRNA markers of quiescent ISCs while being de-enriched for those of the active ISC. Both the USP and LSP are capable of generating enteroids in culture which include the four intestinal lineages. We conclude that sorting of USP and LSP fractions represents a novel isolation of cycling and non-cycling ISCs from WT mice.

  8. Accuracy of the Fluorescence-Activated Cell Sorting Assay for the Aquaporin-4 Antibody (AQP4-Ab): Comparison with the Commercial AQP4-Ab Assay Kit

    Science.gov (United States)

    Kim, Yoo-Jin; Cheon, So Young; Kim, Boram; Jung, Kyeong Cheon; Park, Kyung Seok

    2016-01-01

    Background The aquaporin-4 antibody (AQP4-Ab) is a disease-specific autoantibody to neuromyelitis optica (NMO). We aimed to evaluate the accuracy of the FACS assay in detecting the AQP4-Ab compared with the commercial cell-based assay (C-CBA) kit. Methods Human embryonic kidney-293 cells were transfected with human aquaporin-4 (M23) cDNA. The optimal cut off values of FACS assay was tested using 1123 serum samples from patients with clinically definite NMO, those at high risk for NMO, patients with multiple sclerosis, patients with other idiopathic inflammatory demyelinating diseases, and negative controls. The accuracy of FACS assay and C-CBA were compared in consecutive 225 samples that were collected between January 2014 and June 2014. Results With a cut-off value of MFIi of 3.5 and MFIr of 2.0, the receiver operating characteristic curve for the FACS assay showed an area under the curve of 0.876. Among 225 consecutive sera, the FACS assay and C-CBA had a sensitivity of 77.3% and 69.7%, respectively, in differentiating the sera of definite NMO patients from sera of controls without IDD or of MS. Both assay had a good specificity of 100% in it. The overall positivity of the C-CBA among FACS-positive sera was 81.5%; moreover, its positivity was low as 50% among FACS-positive sera with relatively low MFIis. Conclusions Both the FACS assay and C-CBA are sensitive and highly specific assays in detecting AQP4-Ab. However, in some sera with relatively low antibody titer, FACS-assay can be a more sensitive assay option. In real practice, complementary use of FACS assay and C-CBA will benefit the diagnosis of NMO patients, because the former can be more sensitive among low titer sera and the latter are easier to use therefore can be widely used. PMID:27658059

  9. A kinetic mechanism for cell sorting based on local variations in cell motility

    Science.gov (United States)

    Strandkvist, Charlotte; Juul, Jeppe; Baum, Buzz; Kabla, Alexandre J.; Duke, Tom

    2014-01-01

    Our current understanding of cell sorting relies on physical difference, either in the interfacial properties or motile force, between cell types. But is such asymmetry a prerequisite for cell sorting? We test this using a minimal model in which the two cell populations are identical with respect to their physical properties and differences in motility arise solely from how cells interact with their surroundings. The model resembles the Schelling model used in social sciences to study segregation phenomena at the scale of societies. Our results demonstrate that segregation can emerge solely from cell motility being a dynamic property that changes in response to the local environment of the cell, but that additional mechanisms are necessary to reproduce the envelopment behaviour observed in vitro. The time course of segregation follows a power law, in agreement with the scaling reported from experiment and in other models of motility-driven segregation. PMID:25485079

  10. High-throughput sorting of the highest producing cell via a transiently protein-anchored system.

    Directory of Open Access Journals (Sweden)

    Kuo-Hsiang Chuang

    Full Text Available Developing a high-throughput method for the effecient selection of the highest producing cell is very important for the production of recombinant protein drugs. Here, we developed a novel transiently protein-anchored system coupled with fluorescence activated cell sorting (FACS for the efficient selection of the highest producing cell. A furin cleavage peptide (RAKR was used to join a human anti-epithelial growth factor antibody (αEGFR Ab and the extracellular-transmembrane-cytosolic domains of the mouse B7-1 antigen (B7. The furin inhibitor can transiently switch secreted αEGFR Ab into a membrane-anchored form. After cell sorting, the level of membrane αEGFR Ab-RAKR-B7 is proportional to the amount of secreted αEGFR Ab in the medium. We further selected 23 αEGFR Ab expressing cells and demonstrated a high correlation (R2 = 0.9165 between the secretion level and surface expression levels of αEGFR Ab. These results suggested that the novel transiently protein-anchored system can easily and efficiently select the highest producing cells, reducing the cost for the production of biopharmaceuticals.

  11. Real-time fluorescence lifetime actuation for cell sorting using a CMOS SPAD silicon photomultiplier.

    Science.gov (United States)

    Rocca, Francescopaolo Mattioli Della; Nedbal, Jakub; Tyndall, David; Krstajić, Nikola; Li, David Day-Uei; Ameer-Beg, Simon M; Henderson, Robert K

    2016-02-15

    Time-correlated single photon counting (TCSPC) is a fundamental fluorescence lifetime measurement technique offering high signal to noise ratio (SNR). However, its requirement for complex software algorithms for histogram processing restricts throughput in flow cytometers and prevents on-the-fly sorting of cells. We present a single-point digital silicon photomultiplier (SiPM) detector accomplishing real-time fluorescence lifetime-activated actuation targeting cell sorting applications in flow cytometry. The sensor also achieves burst-integrated fluorescence lifetime (BIFL) detection by TCSPC. The SiPM is a single-chip complementary metal-oxide-semiconductor (CMOS) sensor employing a 32×32 single-photon avalanche diode (SPAD) array and eight pairs of time-interleaved time to digital converters (TI-TDCs) with a 50 ps minimum timing resolution. The sensor's pile-up resistant embedded center of mass method (CMM) processor accomplishes low-latency measurement and thresholding of fluorescence lifetime. A digital control signal is generated with a 16.6 μs latency for cell sorter actuation allowing a maximum cell throughput of 60,000 cells per second and an error rate of 0.6%. PMID:26872160

  12. Cell wall sorting signals in surface proteins of gram-positive bacteria.

    OpenAIRE

    Schneewind, O; Mihaylova-Petkov, D; Model, P

    1993-01-01

    Staphylococcal protein A is anchored to the cell wall, a unique cellular compartment of Gram-positive bacteria. The sorting signal sufficient for cell wall anchoring consists of an LPXTG motif, a C-terminal hydrophobic domain and a charged tail. Homologous sequences are found in many surface proteins of Gram-positive bacteria and we explored the universality of these sequences to serve as cell wall sorting signals. We show that several signals are able to anchor fusion proteins to the staphyl...

  13. Analyzing the role of AP-1B in polarized sorting from recycling endosomes in epithelial cells.

    Science.gov (United States)

    Fölsch, Heike

    2015-01-01

    Epithelial cells polarize their plasma membrane into apical and basolateral domains where the apical membrane faces the luminal side of an organ and the basolateral membrane is in contact with neighboring cells and the basement membrane. To maintain this polarity, newly synthesized and internalized cargos must be sorted to their correct target domain. Over the last ten years, recycling endosomes have emerged as an important sorting station at which proteins destined for the apical membrane are segregated from those destined for the basolateral membrane. Essential for basolateral sorting from recycling endosomes is the tissue-specific adaptor complex AP-1B. This chapter describes experimental protocols to analyze the AP-1B function in epithelial cells including the analysis of protein sorting in LLC-PK1 cells lines, immunoprecipitation of cargo proteins after chemical crosslinking to AP-1B, and radioactive pulse-chase experiments in MDCK cells depleted of the AP-1B subunit μ1B.

  14. Dipeptidyl peptidase IV is sorted to the secretory granules in pancreatic islet A-cells

    DEFF Research Database (Denmark)

    Poulsen, Mona Dam; Hansen, Gert Helge; Dabelsteen, Erik;

    1993-01-01

    labeling using a monoclonal glucagon antibody as the second primary antibody. These results show that DP IV is sorted to secretory granules in the pig pancreatic islet A-cells. Furthermore, this secretory granule enzyme, as opposed to intestinal brush border DP IV, is suggested to be a soluble protein......Dipeptidyl peptidase IV (DP IV:EC 3.4.14.5) was localized in endocrine cells of pig pancreas by immunohistochemical and enzyme histochemical methods. Immunolight microscopy with both monoclonal and polyclonal antibodies demonstrated DP IV immunoreactivity in cells located in the peripheral part...... of the islets of Langerhans. The antigen is enzymatically active, as shown by enzyme histochemical analysis with a synthetic DP IV substrate. By immunoelectron microscopy (immunogold labeling), the labeling of DP IV in the islets was associated with the secretory granules of the A-cells, as identified by double...

  15. Enhanced cell sorting and manipulation with combined optical tweezer and microfluidic chip technologies.

    Science.gov (United States)

    Wang, Xiaolin; Chen, Shuxun; Kong, Marco; Wang, Zuankai; Costa, Kevin D; Li, Ronald A; Sun, Dong

    2011-11-01

    Sorting (or isolation) and manipulation of rare cells with high recovery rate and purity are of critical importance to a wide range of physiological applications. In the current paper, we report on a generic single cell manipulation tool that integrates optical tweezers and microfluidic chip technologies for handling small cell population sorting with high accuracy. The laminar flow nature of microfluidics enables the targeted cells to be focused on a desired area for cell isolation. To recognize the target cells, we develop an image processing methodology with a recognition capability of multiple features, e.g., cell size and fluorescence label. The target cells can be moved precisely by optical tweezers to the desired destination in a noninvasive manner. The unique advantages of this sorter are its high recovery rate and purity in small cell population sorting. The design is based on dynamic fluid and dynamic light pattern, in which single as well as multiple laser traps are employed for cell transportation, and a recognition capability of multiple cell features. Experiments of sorting yeast cells and human embryonic stem cells are performed to demonstrate the effectiveness of the proposed cell sorting approach. PMID:21918752

  16. Prenatal diagnosis from maternal blood: simultaneous immunophenotyping and FISH of fetal nucleated erythrocytes isolated by negative magnetic cell sorting.

    OpenAIRE

    Zheng, Y.L.; Carter, N. P.; Price, C M; Colman, S. M.; Milton, P J; Hackett, G A; Greaves, M F; Ferguson-Smith, M A

    1993-01-01

    Fetal nucleated cells in the maternal circulation constitute a potential source of cells for the non-invasive prenatal diagnosis of fetal genetic abnormalities. We have investigated the use of the Magnetic Activated Cell Sorter (MACS) for enriching fetal nucleated erythrocytes. Mouse monoclonal antibodies specific for CD45 and CD32 were used to deplete leucocytes from maternal blood using MACS sorting, thus enriching for fetal nucleated erythrocytes which do not express either of these antige...

  17. A cell counting/sorting system incorporated with a microfabricated flow cytometer chip

    Science.gov (United States)

    Yang, Sung-Yi; Hsiung, Suz-Kai; Hung, Yung-Ching; Chang, Chen-Min; Liao, Teh-Lu; Lee, Gwo-Bin

    2006-07-01

    Flow cytometry is a popular technique for counting and sorting individual cells. This study presents and demonstrates a new cell counting/sorting system integrated with several essential components including a micromachined flow cytometer chip device, an optical detection system and a data analysis and control system to achieve the functions of cell sample injection, optical signal detection and cell collection. By using MEMS technology, we have integrated several microfluidic components such as micro pneumatic pumps/valves onto a polymer-based chip device. Three pneumatic micropumps are used to provide the hydrodynamic driving force for both sample and sheath flows such that hydrodynamic flow focusing can be achieved, and a micro flow switch device comprising three pneumatic microvalves located downstream of the micro sample flow channel is used for cell collection. Cell samples of human lung cancer cells labelled with commercially available fluorescent dyes have been detected and collected successfully utilizing the developed device. The real-time image of dye-labelled cell samples being excited and detected can be monitored and observed through the LCD panel by a custom designed CCD/APD holder and moving stage. Finally, micro flow switch devices were used to successfully sort the cells into the desired outlet channel, and the counting results of the specific cell samples were monitored through the counting panel. The current study focuses on the setup of the overall system. The proposed flow cytometer system has several advantages such as portability, low cost and easy operation process. The size of the system is 37 cm × 16 cm × 18 cm and the weight is 3.5 kg. The error rate of counting and sorting was 1.5% and 2%, respectively. The sorting frequency of the microvalve device is calculated to be 120 cells min-1. The developed microfluidic chip device could be a promising tool for cell-based application fields such as profiling, counting and sorting.

  18. Spectral representation: analyzing single-unit activity in extracellularly recorded neuronal data without spike sorting

    OpenAIRE

    Luczak, Artur; Narayanan, Nandakumar S.

    2005-01-01

    One step in the conventional analysis of extracellularly recorded neuronal data is spike sorting, which separates electrical signal into action potentials from different neurons. Because spike sorting involves human judgment, it can be subjective and time intensive, particularly for large sets of neurons. Here we propose a simple, automated way to construct alternative representations of neuronal activity, called spectral representation (SR). In this approach, neuronal spikes are mapped to a ...

  19. The viral spike protein is not involved in the polarized sorting of coronaviruses in epithelial cells

    NARCIS (Netherlands)

    Rossen, J W; de Beer, R; Godeke, G J; Raamsman, M J; Horzinek, M C; Vennema, H; Rottier, P J

    1998-01-01

    Coronaviruses are assembled by budding into a pre-Golgi compartment from which they are transported along the secretory pathway to leave the cell. In cultured epithelial cells, they are released in a polarized fashion; depending on the virus and cell type, they are sorted preferentially either to th

  20. Accurate determination of plasmid copy number of flow-sorted cells using droplet digital PCR.

    Science.gov (United States)

    Jahn, Michael; Vorpahl, Carsten; Türkowsky, Dominique; Lindmeyer, Martin; Bühler, Bruno; Harms, Hauke; Müller, Susann

    2014-06-17

    Many biotechnological processes rely on the expression of a plasmid-based target gene. A constant and sufficient number of plasmids per cell is desired for efficient protein production. To date, only a few methods for the determination of plasmid copy number (PCN) are available, and most of them average the PCN of total populations disregarding heterogeneous distributions. Here, we utilize the highly precise quantification of DNA molecules by droplet digital PCR (ddPCR) and combine it with cell sorting using flow cytometry. A duplex PCR assay was set up requiring only 1000 sorted cells for precise determination of PCN. The robustness of this method was proven by thorough optimization of cell sorting, cell disruption, and PCR conditions. When non plasmid-harboring cells of Pseudomonas putida KT2440 were spiked with different dilutions of the expression plasmid pA-EGFP_B, a PCN from 1 to 64 could be accurately detected. As a proof of principle, induced cultures of P. putida KT2440 producing an EGFP-fused model protein by means of the plasmid pA-EGFP_B were investigated by flow cytometry and showed two distinct subpopulations, fluorescent and nonfluorescent cells. These two subpopulations were sorted for PCN determination with ddPCR. A remarkably diverging plasmid distribution was found within the population, with nonfluorescent cells showing a much lower PCN (≤1) than fluorescent cells (PCN of up to 5) under standard conditions.

  1. Novel Serial Positive Enrichment Technology Enables Clinical Multiparameter Cell Sorting

    OpenAIRE

    Christian Stemberger; Stefan Dreher; Claudia Tschulik; Christine Piossek; Jeannette Bet; Yamamoto, Tori N.; Matthias Schiemann; Michael Neuenhahn; Klaus Martin; Martin Schlapschy; Arne Skerra; Thomas Schmidt; Matthias Edinger; Riddell, Stanley R.; Lothar Germeroth

    2012-01-01

    A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve--especially for clinical cell product...

  2. Computational modeling reveals that a combination of chemotaxis and differential adhesion leads to robust cell sorting during tissue patterning.

    Directory of Open Access Journals (Sweden)

    Rui Zhen Tan

    Full Text Available Robust tissue patterning is crucial to many processes during development. The "French Flag" model of patterning, whereby naïve cells in a gradient of diffusible morphogen signal adopt different fates due to exposure to different amounts of morphogen concentration, has been the most widely proposed model for tissue patterning. However, recently, using time-lapse experiments, cell sorting has been found to be an alternative model for tissue patterning in the zebrafish neural tube. But it remains unclear what the sorting mechanism is. In this article, we used computational modeling to show that two mechanisms, chemotaxis and differential adhesion, are needed for robust cell sorting. We assessed the performance of each of the two mechanisms by quantifying the fraction of correct sorting, the fraction of stable clusters formed after correct sorting, the time needed to achieve correct sorting, and the size variations of the cells having different fates. We found that chemotaxis and differential adhesion confer different advantages to the sorting process. Chemotaxis leads to high fraction of correct sorting as individual cells will either migrate towards or away from the source depending on its cell type. However after the cells have sorted correctly, there is no interaction among cells of the same type to stabilize the sorted boundaries, leading to cell clusters that are unstable. On the other hand, differential adhesion results in low fraction of correct clusters that are more stable. In the absence of morphogen gradient noise, a combination of both chemotaxis and differential adhesion yields cell sorting that is both accurate and robust. However, in the presence of gradient noise, the simple combination of chemotaxis and differential adhesion is insufficient for cell sorting; instead, chemotaxis coupled with delayed differential adhesion is required to yield optimal sorting.

  3. Identification, visualization, and sorting of translationally active microbial consortia from deep-sea methane seeps

    Science.gov (United States)

    Hatzenpichler, R.; Connon, S. A.; Goudeau, D.; Malmstrom, R.; Woyke, T.; Orphan, V. J.

    2015-12-01

    Within the past few years, great progress has been made in tapping the genomes of individual cells separated from environmental samples. Unfortunately, however, most often these efforts have been target blind, as they did not pre-select for taxa of interest or focus on metabolically active cells that could be considered key species of the system at the time. This problem is particularly pronounced in low-turnover systems such as deep sea sediments. In an effort to tap the genetic potential hidden within functionally active cells, we have recently developed an approach for the in situ fluorescent tracking of protein synthesis in uncultured cells via bioorthogonal non-canonical amino acid-tagging (BONCAT). This technique depends on the incorporation of synthetic amino acids that carry chemically modifiable tags into newly made proteins, which later can be visualized via click chemistry-mediated fluorescence-labeling. BONCAT is thus able to specifically target proteins that have been expressed in reaction to an experimental condition. We are particularly interested in using BONCAT to understand the functional potential of slow-growing syntrophic consortia of anaerobic methanotrophic archaea and sulfate-reducing bacteria which together catalyze the anaerobic oxidation of methane (AOM) in marine methane seeps. In order to specifically target consortia that are active under varying environmental regimes, we are studying different subpopulations of these inter-domain consortia via a combination of BONCAT with rRNA-targeted FISH. We then couple the BONCAT-enabled staining of active consortia with their separation from inactive members of the community via fluorescence-activated cell-sorting (FACS) and metagenomic sequencing of individual consortia. Using this approach, we were able to identify previously unrecognized AOM-partnerships. By comparing the mini-metagenomes obtained from individual consortia with each other we are starting to gain a more hollistic understanding

  4. Using pico-LCoS SLMs for high speed cell sorting

    DEFF Research Database (Denmark)

    Bañas, Andrew Rafael; Aabo, Thomas; Palima, Darwin;

    2012-01-01

    We propose the use of consumer pico projectors as cost effective spatial light modulators in cell sorting applications. The matched filtering Generalized Phase Contrast (mGPC) beam shaping method is used to produce high intensity optical spots for trapping and catapulting cells. A pico projector...

  5. Postendocytic sorting of constitutively internalized dopamine transporter in cell lines and dopaminergic neurons

    DEFF Research Database (Denmark)

    Eriksen, Jacob; Bjørn-Yoshimoto, Walden Emil; Jørgensen, Trine Nygaard;

    2010-01-01

    lines, we fused the one-transmembrane segment protein Tac to DAT, thereby generating a transporter (TacDAT) with an extracellular antibody epitope suited for trafficking studies. TacDAT was functional and endocytosed constitutively in HEK293 cells. According to an ELISA-based assay, TacDAT intracellular......The dopamine transporter (DAT) mediates reuptake of released dopamine and is the target for psychostimulants, such as cocaine and amphetamine. DAT undergoes marked constitutive endocytosis, but little is known about the fate and sorting of the endocytosed transporter. To study DAT sorting in cells...

  6. Participation in Occupational Performance: Reliability and Validity of the Activity Card Sort.

    Science.gov (United States)

    Katz, Noomi; Karpin, Hanah; Lak, Arit; Furman, Tania; Hartman-Maeir, Adina

    2003-01-01

    A study assessed the reliability and validity of the Activity Card Sort (ACS) within different adult groups (n=263): healthy adults, healthy older adults, Alzheimer's caregivers, multiple sclerosis patients, and stroke survivors. Found that the ACS had high internal consistency for daily living and social-cultural activities and a lower…

  7. Rare Cell Separation and Analysis by Magnetic Sorting

    OpenAIRE

    Zborowski, Maciej; Chalmers, Jeffrey J.

    2011-01-01

    The separation and or isolation of rare cells using magnetic forces is commonly used and growing in use ranging from simple sample prep for further studies to a FDA approved, clinical diagnostic test. This grown is the result of both the demand to obtain homogeneous rare cells for molecular analysis and the dramatic increases in the power of permanent magnets that even allow the separation of some unlabeled cells based on intrinsic magnetic moments, such as malaria parasite-infected red blood...

  8. Flow sorting in the study of teratocarcinoma cell differentiation

    NARCIS (Netherlands)

    G.H. Schaap (Gerard Hendrik)

    1984-01-01

    textabstractFlow cytometry is a technique by which particles (cells, subcellular fragments, bacteria) in aqueous suspension are passed one by one through a sensing region where optical (or electrical) signals are generated. These signals for each individual cell are collected and processed, and may

  9. Proliferation of sorted human and rat beta cells

    DEFF Research Database (Denmark)

    Parnaud, G; Bosco, D; Berney, T;

    2008-01-01

    The aim of the study was to determine whether purified beta cells can replicate in vitro and whether this is enhanced by extracellular matrix (ECM) and growth factors.......The aim of the study was to determine whether purified beta cells can replicate in vitro and whether this is enhanced by extracellular matrix (ECM) and growth factors....

  10. Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases

    Directory of Open Access Journals (Sweden)

    Arildo Nerys-Junior

    2014-01-01

    Full Text Available Engineered nucleases such as zinc finger nucleases (ZFN and transcription activator-like effector nucleases (TALEN are one of the most promising tools for modifying genomes. These site-specific enzymes cause double- strand breaks that allow gene disruption or gene insertion, thereby facilitating genetic manipulation. The major problem associated with this approach is the labor-intensive procedures required to screen and confirm the cellular modification by nucleases. In this work, we produced a TALEN that targets the human CCR5 gene and developed a heteroduplex mobility assay for HEK 293T cells to select positive colonies for sequencing. This approach provides a useful tool for the quick detection and easy assessment of nuclease activity.

  11. Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics.

    Science.gov (United States)

    Daynac, Mathieu; Morizur, Lise; Kortulewski, Thierry; Gauthier, Laurent R; Ruat, Martial; Mouthon, Marc-André; Boussin, François D

    2015-01-01

    Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies. PMID:26436641

  12. Below Regulatory Concern Owners Group: An evaluation of dry active waste sorting: Final report

    International Nuclear Information System (INIS)

    The objective of this research was to determine the accuracy of manual inspection of Dry Active Waste (DAW). Three studies were conducted at two nuclear power plants in which unmodified DAW waste streams of roughly 10,000 items each were inspected by technicians using pancake probes. Sorting performance was measured unobtrusively by intercepting the ''outflow'' from inspection stations. Verification of sorting accuracy was performed with a prototype, semi-automated sorting table employing a matrix of fixed plastic scintillation detectors. More than 30,000 items of trash were examined, classified, counted, and verified, and the composition of the ''inflow'' to the inspection stations was determined by reconstructing the ''outflow'' components, as determined during verification procedures. The results showed that between 1 and 19% of all items in each of the three DAW waste streams were contaminated at levels ≥100 ccpm. Sixty-two percent of the ''contaminated'' items in Study I, 87% of the contaminated items in Study II, and 97% of the contaminated items in Study III were detected. One-half to one percent of all items classified as <100 ccpm by technicians were actually ≥100 ccpm. False positive rates were very high in all three studies. The production rates and accuracy obtained on the semi-automated plastic scintillation sorting table used during the verification stages of this project greatly exceeded the rates for manual sorting. 9 figs., 13 tabs

  13. Sorting and biological characteristics analysis for side population cells in human primary hepatocellular carcinoma

    Science.gov (United States)

    Jiang, Yegui; Gao, Hucheng; Liu, Mingdong; Mao, Qing

    2016-01-01

    Hepatocellular carcinoma (HCC) is the fifth most common cause of the tumor worldwide, its incidence is increasing year by year. This study aims to investigate the sorting and biological characteristics of side population (SP) cells. Human HCC tissues used were obtained from patients undergoing surgical resection. SP cells were sorted using flow cytometry. Cell cycle assay, apoptosis assay and colony formation assay were performed to detect cell proliferation and apoptosis. Invasion assay was employed to examine SP cell invasion. Tumorigenicity assay was used to evaluate tumorigenicity. HCC related microRNAs (miRNA) were analyzed using Micro-array analysis. Target genes were predicted using miRNA database. GO analsis was employed to predict target gene function. Apoptosis percentage was lower and cell viability was higher in SP cells than non-SP (NSP) cells. Colony forming ability of SP cells was significantly higher than NSP cells. Transwell assay positive cells in SP cells were higher significantly than NSP cells. Tumorigenicity of SP cells was higher significantly than NSP cells. 107 differentially expression miRNA were discovered, including 45 up-expressed miRNAs and 62 down-expressed miRNAs in SP cells. Up-regulated hsa-miR-193b-3p and hsa-miR-505-3p predict 25 and 35 target genes, and correlated with 4 and 42 GO terms, respectively. Down-regulated hsa-miR-200a-3p, hsa-miR-194-5p, hsa-miR-130b-3p predict 133, 48 and 127 target genes, and correlate with 10, 7 and 109 GO terms, respectively. In conclusion, proliferation, colony formation, anti-apoptosis, self-renewal capavility, invasive characteristic and tumorigenicity in SP cells isolated from HCC tissues was higher compared to NSP cells. Therefore, sorted SP cells could characterize with biological functions of cancer stem cells.

  14. A simple add-on microfluidic appliance for accurately sorting small populations of cells with high fidelity

    International Nuclear Information System (INIS)

    Current advances in single cell sequencing, gene expression and proteomics require the isolation of single cells, frequently from a very small source population. In this work we describe the design and characterization of a manually operated microfluidic cell sorter that (1) can accurately sort single or small groups of cells from very small cell populations with minimal losses, (2) that is easy to operate and that can be used in any laboratory that has a basic fluorescent microscope and syringe pump, (3) that can be assembled within minutes, (4) that can sort cells in very short time (minutes) with minimum cell stress, (5) that is cheap and reusable. This microfluidic sorter is made from hard plastic material (PMMA) into which microchannels are directly milled with hydraulic diameter of 70 µm. Inlet and outlet reservoirs are drilled through the chip. Sorting occurs through hydrodynamic switching ensuring low hydrodynamic shear stresses, which were modeled and experimentally confirmed to be below the cell damage threshold. Manually operated, the maximum sorting frequencies were approximately 10 cells min−1. Experiments verified that cell sorting operations could be achieved in as little as 15 min, including the assembly and testing of the sorter. In only one out of ten sorting experiments the sorted cells were contaminated with another cell type. This microfluidic cell sorter represents an important capability for protocols requiring fast isolation of single cells from small number of rare cell populations. (technical note)

  15. Improved method and apparatus for electrostatically sorting biological cells. [DOE patent application

    Science.gov (United States)

    Merrill, J.T.

    An improved method of sorting biological cells in a conventional cell sorter apparatus includes generating a fluid jet containing cells to be sorted, measuring the distance between the centers of adjacent droplets in a zone thereof defined at the point where the fluid jet separates into descrete droplets, setting the distance between the center of a droplet in said separation zone and the position along said fluid jet at which the cell is optically sensed for specific characteristics to be an integral multiple of said center-to-center distance, and disabling a charger from electrically charging a specific droplet if a cell is detected by the optical sensor in a position wherein it will be in the neck area between droplets during droplet formation rather than within a predetermined distance from the droplet center.

  16. Computational cell model based on autonomous cell movement regulated by cell-cell signalling successfully recapitulates the "inside and outside" pattern of cell sorting

    Directory of Open Access Journals (Sweden)

    Ajioka Itsuki

    2007-09-01

    Full Text Available Abstract Background Development of multicellular organisms proceeds from a single fertilized egg as the combined effect of countless numbers of cellular interactions among highly dynamic cells. Since at least a reminiscent pattern of morphogenesis can be recapitulated in a reproducible manner in reaggregation cultures of dissociated embryonic cells, which is known as cell sorting, the cells themselves must possess some autonomous cell behaviors that assure specific and reproducible self-organization. Understanding of this self-organized dynamics of heterogeneous cell population seems to require some novel approaches so that the approaches bridge a gap between molecular events and morphogenesis in developmental and cell biology. A conceptual cell model in a computer may answer that purpose. We constructed a dynamical cell model based on autonomous cell behaviors, including cell shape, growth, division, adhesion, transformation, and motility as well as cell-cell signaling. The model gives some insights about what cellular behaviors make an appropriate global pattern of the cell population. Results We applied the model to "inside and outside" pattern of cell-sorting, in which two different embryonic cell types within a randomly mixed aggregate are sorted so that one cell type tends to gather in the central region of the aggregate and the other cell type surrounds the first cell type. Our model can modify the above cell behaviors by varying parameters related to them. We explored various parameter sets with which the "inside and outside" pattern could be achieved. The simulation results suggested that direction of cell movement responding to its neighborhood and the cell's mobility are important for this specific rearrangement. Conclusion We constructed an in silico cell model that mimics autonomous cell behaviors and applied it to cell sorting, which is a simple and appropriate phenomenon exhibiting self-organization of cell population. The model

  17. Specific rates of leucine incorporation by marine bacterioplantkon in the open Mediterranean Sea in summer using cell sorting

    Science.gov (United States)

    Talarmin, A.; van Wambeke, F.; Catala, P.; Courties, C.; Lebaron, P.

    2010-08-01

    Cell-specific leucine incorporation rates were determined in early summer across the open stratified Mediterranean Sea along vertical profiles from 0 to 200 m. During the period of our study, the bulk leucine incorporation rate was on average 5.0 ± 4.0 (n=31) pmol leu l-1 h-1. After 3H-radiolabeled leucine incorporation and SyBR Green I staining, populations were sorted using flow cytometry. Heterotrophic prokaryotes (Hprok) were divided in several clusters according to the cytometric properties of side scatter and green fluorescence of the cells: the low nucleic acid content cells (LNA) and the high nucleic acid content cells (HNA), with high size and low size (HNA-hs and HNA-ls, respectively). LNA cells represented 45 to 63% of the Hprok abundance between surface and 200 m, and significantly contributed to the bulk activity, from 17 to 55% all along the transect. The HNA/LNA ratio of cell-specific activities was on average 2.1 ± 0.7 (n=31). Among Hprok populations from surface samples (0 down to the deep chlorophyll depth, DCM), HNA-hs was mostly responsible for the leucine incorporation activity. Its cell-specific activity was up to 13.3 and 6.9-fold higher than that of HNA-ls and LNA, respectively, and it varied within a wide range of values (0.9-54.3×10-21 mol leu cell-1 h-1). At the opposite, ratios between the specific activities of the 3 populations tended to get closer to each other, below the DCM, implying a potentially higher homogeneity in activity of Hprok in the vicinity of nutriclines. Prochlorococcus cells were easily sorted near the DCM and displayed cell-specific activities equally high, sometimes higher than the HNA-hs group (2.5-55×10-21 mol leu cell-1 h-1). We then showed that all the sorted populations were key-players in leucine incorporation into proteins. The mixotrophic feature of certain photosynthetic prokaryotes and the non-negligible activity of LNA cells all over Mediterranean were reinforced.

  18. Specific rates of leucine incorporation by marine bacterioplantkon in the open Mediterranean Sea in summer using cell sorting

    Directory of Open Access Journals (Sweden)

    A. Talarmin

    2010-08-01

    Full Text Available Cell-specific leucine incorporation rates were determined in early summer across the open stratified Mediterranean Sea along vertical profiles from 0 to 200 m. During the period of our study, the bulk leucine incorporation rate was on average 5.0 ± 4.0 (n=31 pmol leu l−1 h−1. After 3H-radiolabeled leucine incorporation and SyBR Green I staining, populations were sorted using flow cytometry. Heterotrophic prokaryotes (Hprok were divided in several clusters according to the cytometric properties of side scatter and green fluorescence of the cells: the low nucleic acid content cells (LNA and the high nucleic acid content cells (HNA, with high size and low size (HNA-hs and HNA-ls, respectively. LNA cells represented 45 to 63% of the Hprok abundance between surface and 200 m, and significantly contributed to the bulk activity, from 17 to 55% all along the transect. The HNA/LNA ratio of cell-specific activities was on average 2.1 ± 0.7 (n=31. Among Hprok populations from surface samples (0 down to the deep chlorophyll depth, DCM, HNA-hs was mostly responsible for the leucine incorporation activity. Its cell-specific activity was up to 13.3 and 6.9-fold higher than that of HNA-ls and LNA, respectively, and it varied within a wide range of values (0.9–54.3×10−21 mol leu cell−1 h−1. At the opposite, ratios between the specific activities of the 3 populations tended to get closer to each other, below the DCM, implying a potentially higher homogeneity in activity of Hprok in the vicinity of nutriclines. Prochlorococcus cells were easily sorted near the DCM and displayed cell-specific activities equally high, sometimes higher than the HNA-hs group (2.5–55×10−21 mol leu cell−1 h−1. We then showed that all the sorted populations were key-players in leucine incorporation into proteins. The mixotrophic feature of

  19. Index sorting resolves heterogeneous murine hematopoietic stem cell populations

    Science.gov (United States)

    Schulte, Reiner; Wilson, Nicola K.; Prick, Janine C.M.; Cossetti, Chiara; Maj, Michal K.; Gottgens, Berthold; Kent, David G.

    2015-01-01

    Recent advances in the cellular and molecular biology of single stem cells have uncovered significant heterogeneity in the functional properties of stem cell populations. This has prompted the development of approaches to study single cells in isolation, often performed using multiparameter flow cytometry. However, many stem cell populations are too rare to test all possible cell surface marker combinations, and virtually nothing is known about functional differences associated with varying intensities of such markers. Here we describe the use of index sorting for further resolution of the flow cytometric isolation of single murine hematopoietic stem cells (HSCs). Specifically, we associate single-cell functional assay outcomes with distinct cell surface marker expression intensities. High levels of both CD150 and EPCR associate with delayed kinetics of cell division and low levels of differentiation. Moreover, cells that do not form single HSC-derived clones appear in the 7AADdim fraction, suggesting that even low levels of 7AAD staining are indicative of less healthy cell populations. These data indicate that when used in combination with single-cell functional assays, index sorting is a powerful tool for refining cell isolation strategies. This approach can be broadly applied to other single-cell systems, both to improve isolation and to acquire additional cell surface marker information. PMID:26051918

  20. Sorting Out Sorts

    OpenAIRE

    Jonathan B. Berk

    1998-01-01

    In this paper we analyze the theoretical implications of sorting data into groups and then running asset pricing tests within each group. We show that the way this procedure is implemented introduces a severe bias in favor of rejecting the model under consideration. By simply picking enough groups to sort into even the true asset pricing model can be shown to have no explanatory power within each group.

  1. Reduced graft-versus-host disease-inducing capacity of T cells after activation, culturing, and magnetic cell sorting selection in an allogeneic bone marrow transplantation model in rats

    NARCIS (Netherlands)

    Weijtens, M; van Spronsen, A; Hagenbeek, A; Braakman, E; Martens, A

    2002-01-01

    Graft-versus-host disease (GvHD), a major complication of allogeneic bone marrow transplantation, has been ascribed to mature T cells in the graft. Because T cells play an important role in engraftment of the bone marrow and decrease the probability of relapse of leukemia, a treatment strategy was d

  2. Polarized Trafficking of the Sorting Receptor SorLA in Neurons and MDCK Cells

    DEFF Research Database (Denmark)

    Klinger, Stine C; Højland, Anne; Jain, Shweta;

    2016-01-01

    The sorting receptor SorLA is highly expressed in neurons and is also found in other polarized cells. The receptor has been reported to participate in the trafficking of several ligands, some of which are linked to human diseases, including the amyloid precursor protein, TrkB and lipoprotein lipase...... (LpL). Despite this, only the trafficking in non-polarized cells has been described so far. Due to the many differences between polarized and non-polarized cells, we examined the localization and trafficking of SorLA in epithelial Madin-Darby canine kidney (MDCK) cells and rat hippocampal neurons. We...

  3. Vacuolar Sorting Receptor-Mediated Trafficking of Soluble Vacuolar Proteins in Plant Cells

    Directory of Open Access Journals (Sweden)

    Hyangju Kang

    2014-08-01

    Full Text Available Vacuoles are one of the most prominent organelles in plant cells, and they play various important roles, such as degradation of waste materials, storage of ions and metabolites, and maintaining turgor. During the past two decades, numerous advances have been made in understanding how proteins are specifically delivered to the vacuole. One of the most crucial steps in this process is specific sorting of soluble vacuolar proteins. Vacuolar sorting receptors (VSRs, which are type I membrane proteins, are involved in the sorting and packaging of soluble vacuolar proteins into transport vesicles with the help of various accessory proteins. To date, large amounts of data have led to the development of two different models describing VSR-mediated vacuolar trafficking that are radically different in multiple ways, particularly regarding the location of cargo binding to, and release from, the VSR and the types of carriers utilized. In this review, we summarize current literature aimed at elucidating VSR-mediated vacuolar trafficking and compare the two models with respect to the sorting signals of vacuolar proteins, as well as the molecular machinery involved in VSR-mediated vacuolar trafficking and its action mechanisms.

  4. An efficient method of sorting liver stem cells by using immuno-magnetic microbeads

    Institute of Scientific and Technical Information of China (English)

    Yu-Fei He; Yin-Kun Liu; Dong-Mei Gao; Jun Chen; Peng-Yuan Yang

    2006-01-01

    AIM: To develop a method to isolate liver stem cells fast and efficiently.METHODS: Fetal mouse liver cells were characterized by cell surface antigens (c-Kit and CD45/TER119) using flow cytometry. The candidate liver stem cells were sorted by using immuno-magnetic microbeads and identified by clone-forming culture, RT-PCR and immunofluorescence assays.RESULTS: The c-Kit-(CD45/TER119)-cell population with 97.9% of purity were purified by immuno-magnetic microbeads at one time. The yield of this separation was about 6% of the total sorting cells and the cell viability was above 98%. When cultured in vitro these cells had high clone-forming and self-renewing ability and expressed markers of hepatocytes and bile duct cells.Functionally mature hepatocytes were observed after 21 d of culture.CONCLUSION: This method offers an excellent tool for the enrichment of liver stem cells with high purity and viability, which could be used for further studies. It is fast, efficient, simple and not expensive.

  5. echinus, required for interommatidial cell sorting and cell death in the Drosophila pupal retina, encodes a protein with homology to ubiquitin-specific proteases

    Directory of Open Access Journals (Sweden)

    Gorski Sharon M

    2007-07-01

    Full Text Available Abstract Background Programmed cell death is used to remove excess cells between ommatidia in the Drosophila pupal retina. This death is required to establish the crystalline, hexagonal packing of ommatidia that characterizes the adult fly eye. In previously described echinus mutants, interommatidial cell sorting, which precedes cell death, occurred relatively normally. Interommatidial cell death was partially suppressed, resulting in adult eyes that contained excess pigment cells, and in which ommatidia were mildly disordered. These results have suggested that echinus functions in the pupal retina primarily to promote interommatidial cell death. Results We generated a number of new echinus alleles, some likely null mutants. Analysis of these alleles provides evidence that echinus has roles in cell sorting as well as cell death. echinus encodes a protein with homology to ubiquitin-specific proteases. These proteins cleave ubiquitin-conjugated proteins at the ubiquitin C-terminus. The echinus locus encodes multiple splice forms, including two proteins that lack residues thought to be critical for deubiquitination activity. Surprisingly, ubiquitous expression in the eye of versions of Echinus that lack residues critical for ubiquitin specific protease activity, as well as a version predicted to be functional, rescue the echinus loss-of-function phenotype. Finally, genetic interactions were not detected between echinus loss and gain-of-function and a number of known apoptotic regulators. These include Notch, EGFR, the caspases Dronc, Drice, Dcp-1, Dream, the caspase activators, Rpr, Hid, and Grim, the caspase inhibitor DIAP1, and Lozenge or Klumpfuss. Conclusion The echinus locus encodes multiple splice forms of a protein with homology to ubiquitin-specific proteases, but protease activity is unlikely to be required for echinus function, at least when echinus is overexpressed. Characterization of likely echinus null alleles and genetic interactions

  6. Measuring and sorting cell populations expressing isospectral fluorescent proteins with different fluorescence lifetimes.

    Directory of Open Access Journals (Sweden)

    Bryan Sands

    Full Text Available Study of signal transduction in live cells benefits from the ability to visualize and quantify light emitted by fluorescent proteins (XFPs fused to different signaling proteins. However, because cell signaling proteins are often present in small numbers, and because the XFPs themselves are poor fluorophores, the amount of emitted light, and the observable signal in these studies, is often small. An XFP's fluorescence lifetime contains additional information about the immediate environment of the fluorophore that can augment the information from its weak light signal. Here, we constructed and expressed in Saccharomyces cerevisiae variants of Teal Fluorescent Protein (TFP and Citrine that were isospectral but had shorter fluorescence lifetimes, ∼ 1.5 ns vs ∼ 3 ns. We modified microscopic and flow cytometric instruments to measure fluorescence lifetimes in live cells. We developed digital hardware and a measure of lifetime called a "pseudophasor" that we could compute quickly enough to permit sorting by lifetime in flow. We used these abilities to sort mixtures of cells expressing TFP and the short-lifetime TFP variant into subpopulations that were respectively 97% and 94% pure. This work demonstrates the feasibility of using information about fluorescence lifetime to help quantify cell signaling in living cells at the high throughput provided by flow cytometry. Moreover, it demonstrates the feasibility of isolating and recovering subpopulations of cells with different XFP lifetimes for subsequent experimentation.

  7. Polarized trafficking of the sorting receptor SorLA in neurons and MDCK cells.

    Science.gov (United States)

    Klinger, Stine C; Højland, Anne; Jain, Shweta; Kjolby, Mads; Madsen, Peder; Svendsen, Anna Dorst; Olivecrona, Gunilla; Bonifacino, Juan S; Nielsen, Morten S

    2016-07-01

    The sorting receptor SorLA is highly expressed in neurons and is also found in other polarized cells. The receptor has been reported to participate in the trafficking of several ligands, some of which are linked to human diseases, including the amyloid precursor protein, TrkB, and Lipoprotein Lipase (LpL). Despite this, only the trafficking in nonpolarized cells has been described so far. Due to the many differences between polarized and nonpolarized cells, we examined the localization and trafficking of SorLA in epithelial Madin-Darby canine kidney (MDCK) cells and rat hippocampal neurons. We show that SorLA is mainly found in sorting endosomes and on the basolateral surface of MDCK cells and in the somatodendritic domain of neurons. This polarized distribution of SorLA respectively depends on an acidic cluster and an extended version of this cluster and involves the cellular adaptor complex AP-1. Furthermore, we show that SorLA can mediate transcytosis across a tight cell layer. PMID:27192064

  8. Grain sorting in the morphological active layer of a braided river physical model

    Science.gov (United States)

    Leduc, P.; Ashmore, P.; Gardner, J. T.

    2015-07-01

    A physical scale model of a gravel-bed braided river was used to measure vertical grain size sorting in the morphological active layer aggregated over the width of the river. This vertical sorting is important for analyzing braided river sedimentology, for numerical modeling of braided river morpho-dynamics and for measuring and predicting bed load transport rate. We define the morphological active layer as the bed material between the maximum and minimum bed elevations at a point over extended time periods sufficient for braiding processes to re-work the river bed. The vertical extent of the active layer was measured using 40 hourly high-resolution DEMs of the model river bed. An image texture algorithm was used to map bed material grain size of each DEM. Analysis of the 40 DEMs and texture maps provides data on the geometry of the morphological active layer and variation in grain size in three-dimensions. Normalizing active layer thickness and dividing into 10 sub-layers we show that all grain sizes occur with almost equal frequency in all sub-layers. Occurrence of patches and strings of coarser (or finer) material relates to preservation of particular morpho-textural features within the active layer. For numerical modeling and bed load prediction a morphological active layer that is fully mixed with respect to grain size is a reliable approximation.

  9. Electronic Sorting of Immune Cell Subpopulations Based on Highly Plastic Genes.

    Science.gov (United States)

    Wang, Pingzhang; Han, Wenling; Ma, Dalong

    2016-07-15

    Immune cells are highly heterogeneous and plastic with regard to gene expression and cell phenotype. In this study, we categorized genes into those with low and high gene plasticity, and those categories revealed different functions and applications. We proposed that highly plastic genes could be suited for the labeling of immune cell subpopulations; thus, novel immune cell subpopulations could be identified by gene plasticity analysis. For this purpose, we systematically analyzed highly plastic genes in human and mouse immune cells. In total, 1,379 human and 883 mouse genes were identified as being extremely plastic. We also expanded our previous immunoinformatic method, electronic sorting, which surveys big data to perform virtual analysis. This approach used correlation analysis and took dosage changes into account, which allowed us to identify the differentially expressed genes. A test with human CD4(+) T cells supported the method's feasibility, effectiveness, and predictability. For example, with the use of human nonregulatory T cells, we found that FOXP3(hi)CD4(+) T cells were highly expressive of certain known molecules, such as CD25 and CTLA4, and that this process of investigation did not require isolating or inducing these immune cells in vitro. Therefore, the sorting process helped us to discover the potential signature genes or marker molecules and to conduct functional evaluations for immune cell subpopulations. Finally, in human CD4(+) T cells, 747 potential immune cell subpopulations and their candidate signature genes were identified, which provides a useful resource for big data-driven knowledge discoveries. PMID:27288532

  10. Cell cycle variation in x-ray survival for cells from spheroids measured by volume cell sorting

    International Nuclear Information System (INIS)

    Considerable work has been done studying the variation in cell survival as a function of cell cycle position for monolayers or single cells exposed to radiation. Little is known about the effects of multicellular growth on the relative radiation sensitivity of cells in different cell cycle stages. The authors have developed a new technique for measuring the response of cells, using volume cell sorting, which is rapid, non-toxic, and does not require cell synchronization. By combining this technique with selective spheroid dissociation,they have measured the age response of cells located at various depths in EMT6 and Colon 26 spheroids. Although cells in the inner region had mostly G1-phase DNA contents, 15-20% had S- and G2-phase DNA contents. Analysis of these cells using BrdU labeling and flow cytometric analysis with a monoclonal antibody to BrdU indicated that the inner region cells were not synthesizing DNA. Thus, the authors were able to measure the radiation response of cells arrested in G1, S and G2 cell cycle phases. Comparison of inner and outer spheroid regions, and monolayer cultures, indicates that it is improper to extrapolate age response data in standard culture conditions to the situation in spheroids

  11. One-step fabrication of 3D silver paste electrodes into microfluidic devices for enhanced droplet-based cell sorting

    Directory of Open Access Journals (Sweden)

    Lang Rao

    2015-05-01

    Full Text Available 3D microelectrodes are one-step fabricated into a microfluidic droplet separator by filling conductive silver paste into PDMS microchambers. The advantages of 3D silver paste electrodes in promoting droplet sorting accuracy are systematically demonstrated by theoretical calculation, numerical simulation and experimental validation. The employment of 3D electrodes also helps to decrease the droplet sorting voltage, guaranteeing that cells encapsulated in droplets undergo chip-based sorting processes are at better metabolic status for further potential cellular assays. At last, target droplet containing single cell are selectively sorted out from others by an appropriate electric pulse. This method provides a simple and inexpensive alternative to fabricate 3D electrodes, and it is expected our 3D electrode-integrated microfluidic droplet separator platform can be widely used in single cell operation and analysis.

  12. SorLA Controls Neurotrophic Activity by Sorting of GDNF and Its Receptors GFRα1 and RET

    DEFF Research Database (Denmark)

    Glerup, Simon; Lume, Maria; Olsen, Ditte;

    2013-01-01

    Glial cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor that has reached clinical trials for Parkinson's disease. GDNF binds to its coreceptor GFRα1 and signals through the transmembrane receptor tyrosine kinase RET, or RET independently through NCAM or syndecan-3....... Whereas the GDNF signaling cascades are well described, cellular turnover and trafficking of GDNF and its receptors remain poorly characterized. Here, we find that SorLA acts as sorting receptor for the GDNF/GFRα1 complex, directing it from the cell surface to endosomes. Through this mechanism, GDNF...... is targeted to lysosomes and degraded while GFRα1 recycles, creating an efficient GDNF clearance pathway. The SorLA/GFRα1 complex further targets RET for endocytosis but not for degradation, affecting GDNF-induced neurotrophic activities. SorLA-deficient mice display elevated GDNF levels, altered dopaminergic...

  13. Flow fraction in charged rectangular microchannel to optimally design hydrodynamic filtration chip for cell sorting

    Science.gov (United States)

    Chun, Myung-Suk; Jeong, Sohyun; Kim, Jae Hun; Lee, Tae Seok

    2015-11-01

    Among the passive separations, hydrodynamic filtration (HDF) can perform the fractionation of cells or particles by selective extraction of streamlines controlled by the flow fraction at each branch. Only the stream near the sidewall enters the branches as the focusing, with the amount of fluid leaving the main channel being determined by the flow distribution related to the hydraulic flow resistances. Its understanding is important, but in-depth consideration has not been treated until now. The virtual boundary of the fluid layer should be first specified, and the parabolic velocity profile starts to form from the steady state flow with high Péclet numbers. We computed the 3-dimensional flow profile at the rectangular cross-section with any aspect ratios, by considering electrokinetic transport coupled with the Poisson-Boltzmann and Navier-Stokes equations. The chip was designed with the parameters rigorously determined by the complete analysis of laminar flow for flow fraction and complicated networks of main and multi-branched channels for cell sorting into the finite number of subpopulations. For potential applications to the precise sorting, our designed microfluidic chip can be validated by applying model cells consisting of heterogeneous subpopulations. Supported by the KIST Institutional Program (No. 2E25382).

  14. Evaluation of cell sorting aerosols and containment by an optical airborne particle counter.

    Science.gov (United States)

    Xie, Mike; Waring, Michael T

    2015-08-01

    Understanding aerosols produced by cell sorting is critical to biosafety risk assessment and validation of containment efficiency. In this study an Optical Airborne Particle Counter was used to analyze aerosols produced by the BD FACSAria and to assess the effectiveness of its aerosol containment. The suitability of using this device to validate containment was directly compared to the Glo-Germ method put forth by the International Society for Advancement of Cytometry (ISAC) as a standard for testing. It was found that high concentrations of aerosols ranging from 0.3 µm to 10 µm can be detected in failure mode, with most less than 5 µm. In most cases, while numerous aerosols smaller than 5 µm were detected by the Optical Airborne Particle Counter, no Glo-Germ particles were detected, indicating that small aerosols are under-evaluated by the Glo-Germ method. The results demonstrate that the Optical Airborne Particle Counter offers a rapid, economic, and quantitative analysis of cell sorter aerosols and represents an improved method over Glo-Germ for the task of routine validation and monitoring of aerosol containment for cell sorting. PMID:26012776

  15. Efficient Parallel Sorting for Migrating Birds Optimization When Solving Machine-Part Cell Formation Problems

    Directory of Open Access Journals (Sweden)

    Ricardo Soto

    2016-01-01

    Full Text Available The Machine-Part Cell Formation Problem (MPCFP is a NP-Hard optimization problem that consists in grouping machines and parts in a set of cells, so that each cell can operate independently and the intercell movements are minimized. This problem has largely been tackled in the literature by using different techniques ranging from classic methods such as linear programming to more modern nature-inspired metaheuristics. In this paper, we present an efficient parallel version of the Migrating Birds Optimization metaheuristic for solving the MPCFP. Migrating Birds Optimization is a population metaheuristic based on the V-Flight formation of the migrating birds, which is proven to be an effective formation in energy saving. This approach is enhanced by the smart incorporation of parallel procedures that notably improve performance of the several sorting processes performed by the metaheuristic. We perform computational experiments on 1080 benchmarks resulting from the combination of 90 well-known MPCFP instances with 12 sorting configurations with and without threads. We illustrate promising results where the proposal is able to reach the global optimum in all instances, while the solving time with respect to a nonparallel approach is notably reduced.

  16. The functional architecture of the human body: assessing body representation by sorting body parts and activities.

    Science.gov (United States)

    Bläsing, Bettina; Schack, Thomas; Brugger, Peter

    2010-05-01

    We investigated mental representations of body parts and body-related activities in two subjects with congenitally absent limbs (one with, the other without phantom sensations), a wheelchair sports group of paraplegic participants, and two groups of participants with intact limbs. To analyse mental representation structures, we applied Structure Dimensional Analysis. Verbal labels indicating body parts and related activities were presented in randomized lists that had to be sorted according to a hierarchical splitting paradigm. Participants were required to group the items according to whether or not they were considered related, based on their own body perception. Results of the groups of physically intact and paraplegic participants revealed separate clusters for the lower body, upper body, fingers and head. The participant with congenital phantom limbs also showed a clear separation between upper and lower body (but not between fingers and hands). In the participant without phantom sensations of the absent arms, no such modularity emerged, but the specific practice of his right foot in communication and daily routines was reflected. Sorting verbal labels of body parts and activities appears a useful method to assess body representation in individuals with special body anatomy or function and leads to conclusions largely compatible with other assessment procedures.

  17. Sorting of cells of the same size, shape, and cell cycle stage for a single cell level assay without staining

    Directory of Open Access Journals (Sweden)

    Yomo Tetsuya

    2006-06-01

    Full Text Available Abstract Background Single-cell level studies are being used increasingly to measure cell properties not directly observable in a cell population. High-performance data acquisition systems for such studies have, by necessity, developed in synchrony. However, improvements in sample purification techniques are also required to reveal new phenomena. Here we assessed a cell sorter as a sample-pretreatment tool for a single-cell level assay. A cell sorter is routinely used for selecting one type of cells from a heterogeneous mixture of cells using specific fluorescence labels. In this case, we wanted to select cells of exactly the same size, shape, and cell-cycle stage from a population, without using a specific fluorescence label. Results We used four light scatter parameters: the peak height and area of the forward scatter (FSheight and FSarea and side scatter (SSheight and SSarea. The rat pheochromocytoma PC12 cell line, a neuronal cell line, was used for all experiments. The living cells concentrated in the high FSarea and middle SSheight/SSarea fractions. Single cells without cell clumps were concentrated in the low SS and middle FS fractions, and in the higher FSheight/FSarea and SSheight/SSarea fractions. The cell populations from these viable, single-cell-rich fractions were divided into twelve subfractions based on their FSarea-SSarea profiles, for more detailed analysis. We found that SSarea was proportional to the cell volume and the FSarea correlated with cell roundness and elongation, as well as with the level of DNA in the cell. To test the method and to characterize the basic properties of the isolated single cells, sorted cells were cultured in separate wells. The cells in all subfractions survived, proliferated and differentiated normally, suggesting that there was no serious damage. The smallest, roundest, and smoothest cells had the highest viability. There was no correlation between proliferation and differentiation. NGF increases

  18. Red blood cell sorting with a multi-bed microfabricated filter

    International Nuclear Information System (INIS)

    A microfabricated fluidic chip for sorting red blood cells (RBCs) by size has been designed, fabricated and tested. The performance of the chip has been compared against a flow cytometer using samples from identical populations of cells, and statistically significant (p < 0.0005) differences in the measured cell size distributions were observed. The measurement paradigm reported here differs from previously demonstrated devices such as microfabricated Coulter counters or flow cytometers, in that the analysis is inherently parallel and is thus suitable for high throughput, point-of-care analysis. This study is empirical and semi-quantitative. However, important features of RBC trapping are characterized and indications for improved device design are described. (paper)

  19. Multiparametric analysis, sorting, and transcriptional profiling of plant protoplasts and nuclei according to cell type.

    Science.gov (United States)

    Galbraith, David W; Janda, Jaroslav; Lambert, Georgina M

    2011-01-01

    Flow cytometry has been employed for the analysis of higher plants for approximately the last 30 years. For the angiosperms, ∼500,000 species, itself a daunting number, parametric measurements enabled through the use of flow cytometers started with basic descriptors of the individual cells and their contents, and have both inspired the development of novel cytometric methods that subsequently have been applied to organisms within other kingdoms of life, and adopted cytometric methods devised for other species, particularly mammals. Higher plants offer unique challenges in terms of flow cytometric analysis, notably the facts that their organs and tissues are complex three-dimensional assemblies of different cell types, and that their individual cells are, in general, larger than those of mammals.This chapter provides an overview of the general types of parametric measurement that have been applied to plants, and provides detailed methods for selected examples based on the plant model Arabidopsis thaliana. These illustrate the use of flow cytometry for the analysis of protoplasts and nuclear DNA contents (genome size and the cell cycle). These are further integrated with measurements focusing on specific cell types, based on transgenic expression of Fluorescent Proteins (FPs), and on analysis of the spectrum of transcripts found within protoplasts and nuclei. These measurements were chosen in particular to illustrate, respectively, the issues encountered in the flow analysis and sorting of large biological cells, typified by protoplasts; how to handle flow analyses under conditions that require processing of large numbers of samples in which the individual samples contain only a very small minority of objects of interest; and how to deal with exceptionally small amounts of RNA within the sorted samples.

  20. Microfabrication of Bubbular Cavities in PDMS for Cell Sorting and Microcell Culture Applications

    Institute of Scientific and Technical Information of China (English)

    Ut-Binh T.Giang; Michael R.King; Lisa A.DeLouise

    2008-01-01

    We describe a novel technique, low surface energy Gas Expansion Molding (GEM), to fabricate microbubble arrays in polydimethylsiloxane (PDMS) which are incorporated into parallel plate flow chambers and tested in cell sorting and microcell culture applications. This architecture confers several operational advantages that distinguish this technology approach from currently used methods. Herein we describe the GEM process and the parameters that are used to control microbubble formation and a Vacuum-Assisted Coating (VAC) process developed to selectively and spatially alter the PDMS surface chemistry in the wells and on the microchannel surface. We describe results from microflow image visualization studies conducted to investigate fluid streams above and within microbubble wells and conclude with a discussion of cell culture studies in PDMS.

  1. Growth and airborne transmission of cell-sorted life cycle stages of Pneumocystis carinii.

    Science.gov (United States)

    Martinez, Anna; Halliez, Marie C M; Aliouat, El Moukhtar; Chabé, Magali; Standaert-Vitse, Annie; Fréalle, Emilie; Gantois, Nausicaa; Pottier, Muriel; Pinon, Anthony; Dei-Cas, Eduardo; Aliouat-Denis, Cécile-Marie

    2013-01-01

    Pneumocystis organisms are airborne opportunistic pathogens that cannot be continuously grown in culture. Consequently, the follow-up of Pneumocystis stage-to-stage differentiation, the sequence of their multiplication processes as well as formal identification of the transmitted form have remained elusive. The successful high-speed cell sorting of trophic and cystic forms is paving the way for the elucidation of the complex Pneumocystis life cycle. The growth of each sorted Pneumocystis stage population was followed up independently both in nude rats and in vitro. In addition, by setting up a novel nude rat model, we attempted to delineate which cystic and/or trophic forms can be naturally aerially transmitted from host to host. The results showed that in axenic culture, cystic forms can differentiate into trophic forms, whereas trophic forms are unable to evolve into cystic forms. In contrast, nude rats inoculated with pure trophic forms are able to produce cystic forms and vice versa. Transmission experiments indicated that 12 h of contact between seeder and recipient nude rats was sufficient for cystic forms to be aerially transmitted. In conclusion, trophic- to cystic-form transition is a key step in the proliferation of Pneumocystis microfungi because the cystic forms (but not the trophic forms) can be transmitted by aerial route from host to host. PMID:24223207

  2. Growth and airborne transmission of cell-sorted life cycle stages of Pneumocystis carinii.

    Directory of Open Access Journals (Sweden)

    Anna Martinez

    Full Text Available Pneumocystis organisms are airborne opportunistic pathogens that cannot be continuously grown in culture. Consequently, the follow-up of Pneumocystis stage-to-stage differentiation, the sequence of their multiplication processes as well as formal identification of the transmitted form have remained elusive. The successful high-speed cell sorting of trophic and cystic forms is paving the way for the elucidation of the complex Pneumocystis life cycle. The growth of each sorted Pneumocystis stage population was followed up independently both in nude rats and in vitro. In addition, by setting up a novel nude rat model, we attempted to delineate which cystic and/or trophic forms can be naturally aerially transmitted from host to host. The results showed that in axenic culture, cystic forms can differentiate into trophic forms, whereas trophic forms are unable to evolve into cystic forms. In contrast, nude rats inoculated with pure trophic forms are able to produce cystic forms and vice versa. Transmission experiments indicated that 12 h of contact between seeder and recipient nude rats was sufficient for cystic forms to be aerially transmitted. In conclusion, trophic- to cystic-form transition is a key step in the proliferation of Pneumocystis microfungi because the cystic forms (but not the trophic forms can be transmitted by aerial route from host to host.

  3. Endocytic Sorting of CFTR variants Monitored by Single Cell Fluorescence Ratio Image Analysis (FRIA) in Living Cells

    Science.gov (United States)

    Barriere, H.; Apaja, P.; Okiyoneda, T.; Lukacs, G. L.

    2016-01-01

    Summary The wild-type CFTR channel undergoes constitutive internalization and recycling at the plasma membrane. This process is initiated by the recognition of the Tyr- and di-Leu-based endocytic motifs of CFTR by the AP-2 adaptor complex, leading to the formation of clathrin-coated vesicles and the channel delivery to sorting/recycling endosomes. Accumulating evidence suggests that conformationally defective mutant CFTRs (e.g. rescued ΔF508 and glycosylation-deficient channel) are unstable at the plasma membrane and undergo augmented ubiquitination in post-Golgi compartments. Ubiquitination conceivably accounts for the metabolic instability at cell surface by provoking accelerated internalization, as well as rerouting the channel from recycling towards lysosomal degradation. We developed an in vivo fluorescence ratio imaging assay (FRIA) that in concert with genetic manipulation can be utilized to establish the post-endocytic fate and sorting determinants of mutant CFTRs. PMID:21594793

  4. High-speed real-time data classification and cell sorting using discriminant functions and probabilities of misclassification for stem cell enrichment and tumor purging

    Science.gov (United States)

    Leary, James F.; McLaughlin, Scott R.; Hokanson, James A.; Rosenblatt, Judah I.

    1998-04-01

    Data analysis and cell sorting are both fundamentally the same except in terms of the time available to make classification decisions. In the case of cell sorting the cell classification decisions must be made in real-time (in the case of cell sorting, real-time means in about 625 microseconds on this system). This dictates an approach to classification which can be implemented at memory speeds or in pre-programmed hardware. We have been developing new high-speed lookup table transformation methods, suitable for real-time data classification or cell sorting based on statistical classifiers. Multiparameter data mixtures of human MCF-7 breast cancer cells and human bone marrow were analyzed by discriminant function analysis. Cell identification tags, implemented as additional correlated listmode parameters not used for these analyses, were used to uniquely identify each cell type and to compare classifier results. The performance of classifier systems was also assessed using ROC ('receiver operating characteristics') analysis. The effectiveness of the classification system for cell sorting can be evaluated using molecular characterizations of sorted cells, either in small numbers or at single-cell level.

  5. Sorting choanoflagellates

    Science.gov (United States)

    Marconi, Veronica I.; Miño, Gaston L.; Sparacino, Javier; Banchio, Adolfo J.; Condat, Carlos A.; Koehl, Mimi A. R.; King, Nicole; Stocker, Roman

    2015-03-01

    In freshwater environments, as well as in oceans, environmental conditions are in constant fluctuation. Some heterotrophic plankton must adapt their swimming behavior in order to survive under these conditions. In the case of the choanoflagellate, the closest animal ancestor, the ability to forage for food is given not only by its single flagellum, but also by its differentiation between fast and slow swimmers. The understanding of how these cells with different strategies to swim search for food can give us a better insight into how eukaryotes respond to different stimuli. In this work, we have designed a microfluidic device that sorts choanoflagellates by their speed. The optimal geometry was found by a numerical model using the experimentally determined motilities of each swimmer type.

  6. SorLA Controls Neurotrophic Activity by Sorting of GDNF and Its Receptors GFRα1 and RET

    Directory of Open Access Journals (Sweden)

    Simon Glerup

    2013-01-01

    Full Text Available Glial cell-line-derived neurotrophic factor (GDNF is a potent neurotrophic factor that has reached clinical trials for Parkinson’s disease. GDNF binds to its coreceptor GFRα1 and signals through the transmembrane receptor tyrosine kinase RET, or RET independently through NCAM or syndecan-3. Whereas the GDNF signaling cascades are well described, cellular turnover and trafficking of GDNF and its receptors remain poorly characterized. Here, we find that SorLA acts as sorting receptor for the GDNF/GFRα1 complex, directing it from the cell surface to endosomes. Through this mechanism, GDNF is targeted to lysosomes and degraded while GFRα1 recycles, creating an efficient GDNF clearance pathway. The SorLA/GFRα1 complex further targets RET for endocytosis but not for degradation, affecting GDNF-induced neurotrophic activities. SorLA-deficient mice display elevated GDNF levels, altered dopaminergic function, marked hyperactivity, and reduced anxiety, all of which are phenotypes related to abnormal GDNF activity. Taken together, these findings establish SorLA as a critical regulator of GDNF activity in the CNS.

  7. Automated Chemotactic Sorting and Single-cell Cultivation of Microbes using Droplet Microfluidics

    Science.gov (United States)

    Dong, Libing; Chen, Dong-Wei; Liu, Shuang-Jiang; Du, Wenbin

    2016-04-01

    We report a microfluidic device for automated sorting and cultivation of chemotactic microbes from pure cultures or mixtures. The device consists of two parts: in the first part, a concentration gradient of the chemoeffector was built across the channel for inducing chemotaxis of motile cells; in the second part, chemotactic cells from the sample were separated, and mixed with culture media to form nanoliter droplets for encapsulation, cultivation, enumeration, and recovery of single cells. Chemotactic responses were assessed by imaging and statistical analysis of droplets based on Poisson distribution. An automated procedure was developed for rapid enumeration of droplets with cell growth, following with scale-up cultivation on agar plates. The performance of the device was evaluated by the chemotaxis assays of Escherichia coli (E. coli) RP437 and E. coli RP1616. Moreover, enrichment and isolation of non-labelled Comamonas testosteroni CNB-1 from its 1:10 mixture with E. coli RP437 was demonstrated. The enrichment factor reached 36.7 for CNB-1, based on its distinctive chemotaxis toward 4-hydroxybenzoic acid. We believe that this device can be widely used in chemotaxis studies without necessarily relying on fluorescent labelling, and isolation of functional microbial species from various environments.

  8. Screening of promoters from rhizosphere metagenomic DNA using a promoter-trap vector and flow cytometric cell sorting.

    Science.gov (United States)

    Lee, Se Hee; Kim, Jeong Myeong; Lee, Hyo Jung; Jeon, Che Ok

    2011-02-01

    We constructed a facilitative and efficient promoter-trap vector, pCM-EGFP, for capturing and analyzing functional promoters from environmental DNA. The pCM-EGFP vector showed good chloramphenicol sensitivity and no enhanced green fluorescent protein (EGFP) gene expression. Promoter libraries were constructed for screening promoters responding to naringenin, a key molecule released from plant roots. After electroporation, E. coli transformants were incubated in LB broth containing chloramphenicol (10 μg/ml) to select against transformants with no cloned promoter. E. coli cells were sorted using flow cytometry without naringenin, and then sorted again with high fluorescence after incubation in LB broth with naringenin (1 mM) at 28 °C for 12 h. The inducible properties of approximately 400 sorted cells were evaluated, with most cells showing only strong EGFP gene expression without inducible properties. Two clones (5-4E and 15-3D) displayed naringenin inducibility, and both contained a promoter bounded by a TetR-family regulator. The regulator knock-out mutant of the 5-4E clone lost its ability to be induced by naringenin. In conclusion, the pCM-EGFP vector may be used as an efficient promoter-trap vector and a combination of the vector with flow cytometric cell sorting was demonstrated to be an useful method for screening promoters responding to specific conditions or inducers. PMID:21259288

  9. Isolation of αL I domain mutants mediating firm cell adhesion using a novel flow-based sorting method.

    Science.gov (United States)

    Pepper, Lauren R; Parthasarathy, Ranganath; Robbins, Gregory P; Dang, Nicholas N; Hammer, Daniel A; Boder, Eric T

    2013-08-01

    The inserted (I) domain of αLβ2 integrin (LFA-1) contains the entire binding site of the molecule. It mediates both rolling and firm adhesion of leukocytes at sites of inflammation depending on the activation state of the integrin. The affinity change of the entire integrin can be mimicked by the I domain alone through mutations that affect the conformation of the molecule. High-affinity mutants of the I domain have been discovered previously using both rational design and directed evolution. We have found that binding affinity fails to dictate the behavior of I domain adhesion under shear flow. In order to better understand I domain adhesion, we have developed a novel panning method to separate yeast expressing a library of I domain variants on the surface by adhesion under flow. Using conditions analogous to those experienced by cells interacting with the post-capillary vascular endothelium, we have identified mutations supporting firm adhesion that are not found using typical directed evolution techniques that select for tight binding to soluble ligands. Mutants isolated using this method do not cluster with those found by sorting with soluble ligand. Furthermore, these mutants mediate shear-driven cell rolling dynamics decorrelated from binding affinity, as previously observed for I domains bearing engineered disulfide bridges to stabilize activated conformational states. Characterization of these mutants supports a greater understanding of the structure-function relationship of the αL I domain, and of the relationship between applied force and bioadhesion in a broader context.

  10. FACS-sorted putative oogonial stem cells from the ovary are neither DDX4-positive nor germ cells.

    Science.gov (United States)

    Zarate-Garcia, Larissa; Lane, Simon I R; Merriman, Julie A; Jones, Keith T

    2016-01-01

    Whether the adult mammalian ovary contains oogonial stem cells (OSCs) is controversial. They have been isolated by a live-cell sorting method using the germ cell marker DDX4, which has previously been assumed to be cytoplasmic, not surface-bound. Furthermore their stem cell and germ cell characteristics remain disputed. Here we show that although OSC-like cells can be isolated from the ovary using an antibody to DDX4, there is no good in silico modelling to support the existence of a surface-bound DDX4. Furthermore these cells when isolated were not expressing DDX4, and did not initially possess germline identity. Despite these unremarkable beginnings, they acquired some pre-meiotic markers in culture, including DDX4, but critically never expressed oocyte-specific markers, and furthermore were not immortal but died after a few months. Our results suggest that freshly isolated OSCs are not germ stem cells, and are not being isolated by their DDX4 expression. However it may be that culture induces some pre-meiotic markers. In summary the present study offers weight to the dogma that the adult ovary is populated by a fixed number of oocytes and that adult de novo production is a rare or insignificant event. PMID:27301892

  11. Efficient laboratory evolution of computationally designed enzymes with low starting activities using fluorescence-activated droplet sorting.

    Science.gov (United States)

    Obexer, Richard; Pott, Moritz; Zeymer, Cathleen; Griffiths, Andrew D; Hilvert, Donald

    2016-09-01

    De novo biocatalysts with non-natural functionality are accessible by computational enzyme design. The catalytic activities obtained for the initial designs are usually low, but can be optimized significantly by directed evolution. Nevertheless, rate accelerations approaching the level of natural enzymes can only be achieved over many rounds of tedious and time-consuming laboratory evolution. In this work, we show that microfluidic-based screening using fluorescence-activated droplet sorting (FADS) is ideally suited for efficient optimization of designed enzymes with low starting activity, essentially straight out of the computer. We chose the designed retro-aldolase RA95.0, which had been previously evolved by conventional microtiter plate screening, as an example and reoptimized it using the microfluidic-based assay. Our results show that FADS is sufficiently sensitive to detect enzyme activities as low as kcat/Km = 0.5 M(-1)s(-1) The ultra-high throughput of this system makes screening of large mutant libraries possible in which clusters of up to five residues are randomized simultaneously. Thus, combinations of beneficial mutations can be identified directly, leading to large jumps in catalytic activity of up to 80-fold within a single round of evolution. By exploring several evolutionary trajectories in parallel, we identify alternative active site arrangements that exhibit comparably enhanced efficiency but opposite enantioselectivity. PMID:27542390

  12. Generation of Recombinant Monoclonal Antibodies from Immunised Mice and Rabbits via Flow Cytometry and Sorting of Antigen-Specific IgG+ Memory B Cells.

    Directory of Open Access Journals (Sweden)

    Dale O Starkie

    Full Text Available Single B cell screening strategies, which avoid both hybridoma fusion and combinatorial display, have emerged as important technologies for efficiently sampling the natural antibody repertoire of immunized animals and humans. Having access to a range of methods to interrogate different B cell subsets provides an attractive option to ensure large and diverse panels of high quality antibody are produced. The generation of multiple antibodies and having the ability to find rare B cell clones producing IgG with unique and desirable characteristics facilitates the identification of fit-for-purpose molecules that can be developed into therapeutic agents or research reagents. Here, we describe a multi-parameter flow cytometry single-cell sorting technique for the generation of antigen-specific recombinant monoclonal antibodies from single IgG+ memory B cells. Both mouse splenocytes and rabbit PBMC from immunised animals were used as a source of B cells. Reagents staining both B cells and other unwanted cell types enabled efficient identification of class-switched IgG+ memory B cells. Concurrent staining with antigen labelled separately with two spectrally-distinct fluorophores enabled antigen-specific B cells to be identified, i.e. those which bind to both antigen conjugates (double-positive. These cells were then typically sorted at one cell per well using FACS directly into a 96-well plate containing reverse transcriptase reaction mix. Following production of cDNA, PCR was performed to amplify cognate heavy and light chain variable region genes and generate transcriptionally-active PCR (TAP fragments. These linear expression cassettes were then used directly in a mammalian cell transfection to generate recombinant antibody for further testing. We were able to successfully generate antigen-specific recombinant antibodies from both the rabbit and mouse IgG+ memory B cell subset within one week. This included the generation of an anti-TNFR2 blocking

  13. Driving gradual endogenous c-myc overexpression by flow-sorting: intracellular signaling and tumor cell phenotype correlate with oncogene expression

    DEFF Research Database (Denmark)

    Knudsen, Kasper Jermiin; Holm, G.M.N.; Krabbe, J.S.;

    2009-01-01

    Insulin-exposed rat mammary cancer cells were flow sorted based on a c-myc reporter plasmid encoding a destabilized green fluorescent protein. Sorted cells exhibited gradual increases in c-myc levels. Cells overexpressing c-myc by only 10% exhibited phenotypic changes attributable to c-myc overex...... an alternative modeling of the receptor-mediated carcinogenic process, compared to the currently used approaches of recombinant constitutive or conditional overexpression of oncogenic transmembrane receptor tyrosine kinases or oncogenic transcription factors....

  14. Skeletal stem cell isolation: A review on the state-of-the-art microfluidic label-free sorting techniques.

    Science.gov (United States)

    Xavier, Miguel; Oreffo, Richard O C; Morgan, Hywel

    2016-01-01

    Skeletal stem cells (SSC) are a sub-population of bone marrow stromal cells that reside in postnatal bone marrow with osteogenic, chondrogenic and adipogenic differentiation potential. SSCs reside only in the bone marrow and have organisational and regulatory functions in the bone marrow microenvironment and give rise to the haematopoiesis-supportive stroma. Their differentiation capacity is restricted to skeletal lineages and therefore the term SSC should be clearly distinguished from mesenchymal stem cells which are reported to exist in extra-skeletal tissues and, critically, do not contribute to skeletal development. SSCs are responsible for the unique regeneration capacity of bone and offer unlimited potential for application in bone regenerative therapies. A current unmet challenge is the isolation of homogeneous populations of SSCs, in vitro, with homogeneous regeneration and differentiation capacities. Challenges that limit SSC isolation include a) the scarcity of SSCs in bone marrow aspirates, estimated at between 1 in 10-100,000 mononuclear cells; b) the absence of specific markers and thus the phenotypic ambiguity of the SSC and c) the complexity of bone marrow tissue. Microfluidics provides innovative approaches for cell separation based on bio-physical features of single cells. Here we review the physical principles underlying label-free microfluidic sorting techniques and review their capacity for stem cell selection/sorting from complex (heterogeneous) samples. PMID:27236022

  15. A specific sorting signal is not required for the polarized secretion of newly synthesized proteins from cultured intestinal epithelial cells.

    Science.gov (United States)

    Rindler, M J; Traber, M G

    1988-08-01

    Caco-2 cells, derived from human colon, have the morphological, functional, and biochemical properties of small intestinal epithelial cells. After infection with enveloped viruses, influenza virions assembled at the apical plasma membrane while vesicular stomatitis virus (VSV) particles appeared exclusively at the basolateral membrane, similar to the pattern observed in virus-infected Madin-Darby canine kidney (MDCK). When grown in Millicell filter chamber devices and labeled with [35S]methionine, Caco-2 monolayers released all of their radiolabeled secretory products preferentially into the basal chamber. Among the proteins identified were apolipoproteins AI and E, transferrin, and alpha-fetoprotein. No proteins were observed to be secreted preferentially from the apical cell surface. The lysosomal enzyme beta-hexosaminidase was also secreted primarily from the basolateral surface of the cells in the presence or absence of lysosomotropic drugs or tunicamycin, which inhibit the targetting of lysosomal enzymes to lysosomes. Neither of these drug treatments significantly affected the polarized secretion of other nonlysosomal proteins. In addition, growth hormone (GH), which is released in a nonpolar fashion from MDCK cells, was secreted exclusively from the basolateral membrane after transfection of Caco-2 cells with GH cDNA in a pSV2-based expression vector. Similar results were obtained in transient expression experiments and after selection of permanently transformed Caco-2 cells expressing GH. Since both beta-hexosaminidase and GH would be expected to lack sorting signals for polarized exocytosis in epithelial cells, these results indicate that in intestinal cells, proteins transported via the basolateral secretory pathway need not have specific sorting signals.

  16. Microfluidic sorting of microtissues.

    Science.gov (United States)

    Buschke, D G; Resto, P; Schumacher, N; Cox, B; Tallavajhula, A; Vivekanandan, A; Eliceiri, K W; Williams, J C; Ogle, B M

    2012-03-01

    Increasingly, invitro culture of adherent cell types utilizes three-dimensional (3D) scaffolds or aggregate culture strategies to mimic tissue-like, microenvironmental conditions. In parallel, new flow cytometry-based technologies are emerging to accurately analyze the composition and function of these microtissues (i.e., large particles) in a non-invasive and high-throughput way. Lacking, however, is an accessible platform that can be used to effectively sort or purify large particles based on analysis parameters. Here we describe a microfluidic-based, electromechanical approach to sort large particles. Specifically, sheath-less asymmetric curving channels were employed to separate and hydrodynamically focus particles to be analyzed and subsequently sorted. This design was developed and characterized based on wall shear stress, tortuosity of the flow path, vorticity of the fluid in the channel, sorting efficiency and enrichment ratio. The large particle sorting device was capable of purifying fluorescently labelled embryoid bodies (EBs) from unlabelled EBs with an efficiency of 87.3% ± 13.5%, and enrichment ratio of 12.2 ± 8.4 (n = 8), while preserving cell viability, differentiation potential, and long-term function. PMID:22505992

  17. Biofunctionalized magnetic nanospheres-based cell sorting strategy for efficient isolation, detection and subtype analyses of heterogeneous circulating hepatocellular carcinoma cells.

    Science.gov (United States)

    Chen, Lan; Wu, Ling-Ling; Zhang, Zhi-Ling; Hu, Jiao; Tang, Man; Qi, Chu-Bo; Li, Na; Pang, Dai-Wen

    2016-11-15

    Hepatocellular carcinoma (HCC) is an awful threat to human health. Early-stage HCC may be detected by isolation of circulating tumor cells (CTCs) from peripheral blood samples, which is beneficial to the diagnosis and therapy. However, the extreme rarity and high heterogeneity of HCC CTCs have been restricting the relevant research. To achieve an efficient isolation, reliable detection and subtype analyses of heterogeneous HCC CTCs, herein, we present a cell sorting strategy based on anti-CD45 antibody-modified magnetic nanospheres. By this strategy, leukocyte depletion efficiency was up to 99.9% within 30min in mimic clinical samples, and the purity of the spiked HCC cells was improved 265-317-fold. Besides, the isolated HCC cells remained viable at 92.3% and could be directly recultured. Moreover, coupling the convenient, fast and effective cell sorting strategy with specific ICC identification via biomarkers AFP and GPC3, HCC CTCs were detectable in peripheral blood samples, showing the potential for HCC CTC detection in clinic. Notably, this immunomagnetic cell sorting strategy enabled isolating more heterogeneous HCC cells compared with the established EpCAM-based methods, and further achieved characterization of three different CTC subtypes from one clinical HCC blood sample, which may assist clinical HCC analyses such as prognosis or personalized treatment. PMID:27240010

  18. Quantitative and qualitative analysis of small RNAs in human endothelial cells and exosomes provides insights into localized RNA processing, degradation and sorting

    NARCIS (Netherlands)

    van Balkom, Bas W M; Eisele, Almut S; Pegtel, D Michiel; Bervoets, Sander; Verhaar, Marianne C

    2015-01-01

    Exosomes are small vesicles that mediate cell-cell communication. They contain proteins, lipids and RNA, and evidence is accumulating that these molecules are specifically sorted for release via exosomes. We recently showed that endothelial-cell-produced exosomes promote angiogenesis in vivo in a sm

  19. Resolving sorting mechanisms into exosomes

    NARCIS (Netherlands)

    Stoorvogel, Willem

    2015-01-01

    The complexity of mechanisms driving protein sorting into exosomes is only beginning to emerge. In a paper recently published in Cell Research, Roucourt et al. report that trimming of heparan sulfate side chains of syndecans by endosomal heparanase facilitates sorting into exosomes by the formation

  20. Antibody-free magnetic cell sorting of genetically modified primary human CD4+ T cells by one-step streptavidin affinity purification.

    Directory of Open Access Journals (Sweden)

    Nicholas J Matheson

    Full Text Available Existing methods for phenotypic selection of genetically modified mammalian cells suffer disadvantages of time, cost and scalability and, where antibodies are used to bind exogenous cell surface markers for magnetic selection, typically yield cells coated with antibody-antigen complexes and beads. To overcome these limitations we have developed a method termed Antibody-Free Magnetic Cell Sorting in which the 38 amino acid Streptavidin Binding Peptide (SBP is displayed at the cell surface by the truncated Low Affinity Nerve Growth Receptor (LNGFRF and used as an affinity tag for one-step selection with streptavidin-conjugated magnetic beads. Cells are released through competition with the naturally occurring vitamin biotin, free of either beads or antibody-antigen complexes and ready for culture or use in downstream applications. Antibody-Free Magnetic Cell Sorting is a rapid, cost-effective, scalable method of magnetic selection applicable to either viral transduction or transient transfection of cell lines or primary cells. We have optimised the system for enrichment of primary human CD4+ T cells expressing shRNAs and exogenous genes of interest to purities of >99%, and used it to isolate cells following Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9 genome editing.

  1. 3种花色苷对DF-1细胞的影响%Effect of Three Sorts of Anthocyanin on DF-1 Cells

    Institute of Scientific and Technical Information of China (English)

    盖丽丽; 张莉; 姜世金; 雷用东

    2012-01-01

    Objective: To investigate the impact of three sorts of anthocyanin including heart radish anthocyanin, purple potato anthocyanin and purple corn anthocyanin on DF-1 cell line. Methods: The impact of the three sorts of anthocyanin on DF-1 cells growth was explored and the final concentration was measured respectively by intuitive observation. The impact of the three sorts of anthocyanin in improving the role of cell activity was explored by MTT assay. Results: The different concentrations of anthocyanins were affected on DF-1 monolayer cells growth, and the optimal final concentration of heart radish anthocyanin, purple potato anthocyanin and purple corn anthocyanin was got for 100, 75 and 50 μg/mL respectively. At the same time, we mapped the chart based on the data measured by MTT assay. Conclusion: Different varieties of anthocyanins as different concentrations were affected on DF-1 monolayer cells growth significantly. These will provide data support for the research of anthocyanins antivirus in the future.%目的:探讨来自心里美萝卜、紫甘薯和紫玉米的3种不同的花色苷对DF-1细胞单层生长的影响.方法:直观观察3种花色苷对DF-1细胞生长的影响,初步摸索其最适终浓度;用MTT法检测花色苷在提高细胞活性方面的作用.结果:不同浓度的3种花色苷均对细胞单层生长有不同的影响,心里美萝卜花色苷、紫甘薯花色苷和紫玉米花色苷对细胞生长的最适终浓度分别为100、75和50 μg/mL;用MTT法获得相应数据并制成细胞活性图表.结论:不同品种来源及不同浓度的花色苷对DF-1细胞单层生长均有明显影响,为今后开展花色苷促细胞生长,提高细胞抗病毒活性研究提供了数据基础.

  2. Slashing the timelines: Opting to generate high-titer clonal lines faster via viability-based single cell sorting.

    Science.gov (United States)

    Misaghi, Shahram; Shaw, David; Louie, Salina; Nava, Adrian; Simmons, Laura; Snedecor, Brad; Poon, Chungkee; Paw, Jonathan S; Gilmour-Appling, Laurie; Cupp, James E

    2016-01-01

    Chinese hamster ovary (CHO) cell line development (CLD) is a long and laborious process, which requires up to 5 - 6 months in order to generate and bank CHO lines capable of stably expressing therapeutic molecules. Additionally, single cell cloning of these production lines is also necessary to confirm clonality of the production lines. Here we introduce the utilization of viability staining dye in combination with flow cytometer to isolate high titer clones from a pool of selected cells and single cell deposit them into the wells of culture plates. Our data suggests that a stringent selection procedure along with viability dye staining and flow cytometry-based sorting can be used to isolate high expressing clones with titers comparable to that of traditional CLD methods. This approach not only requires less labor and consumables, but it also shortens CLD timelines by at least 3 weeks. Furthermore, single cell deposition of selected cells by a flow sorter can be regarded as an additional clonality assurance factor that in combination with Day 0 imaging can ensure clonality of the production lines. PMID:26587808

  3. New Approach to Selective Stem Cell Sorting: Separation of Undifferentiated Stem Cells from Differentiated Stem Cells by Using Iron Oxide Core Shell Nanoparticles

    Science.gov (United States)

    Kisa, Fikrullah

    An alternative approach to stem cell enrichment in another words sorting methods without changing the microenvironment of the cells to avoid the detrimental effects of present cell sorting methods by adopting iron-oxide gold (cFeAu) core-shell nanoparticles (NPs) is the focus of this thesis. Each chapter of this thesis focuses on different preliminary research in order to engender the adoption of cFeAu NPs for the selective killing of the mouse embryonic stem cells that are immunolabeled with the nanoparticles. The first part of the research focuses on the synthesis of superparamagnetic iron-oxide nanoparticles with the co-precipitation method and coating the nanoparticles with colloidal gold (cAu) to stabilize the characteristics of the nanoparticles. Detailed information regarding the chemistry of iron-oxide nanoparticles, common synthesis methods, and some of the factors that affect nanoparticle growth and synthesis have been investigated. The heating ability of the nanoparticles under an oscillating magnetic field (OMF) and the size distribution of the particles under a transmission electron microscope (TEM) are shown. The second part of the research focuses on selectively killing the RAW 264.7 macrophages which have internalized the synthesized nanoparticles in order to prove the biocompatibility and effectiveness of the nanoparticles. The particles' effect on the cells, the mechanism of killing, and the effectiveness of nanoparticles coated with colloidal gold and bovine serum albumin are investigated. The last part of the research focuses on effectively labeling the mESCs with a specific antibody conjugated to cFeAu nanoparticles that has an affinity to stage specific embryonic antigen 1 (SSEA-1). The influence of the OMF and the effects of immunolabeling on cell growth were investigated. The successful conjugation of the nanoparticles onto the cell surface is shown under scanning electron microscope. The damage inflicted by the nanoparticles on the cells

  4. Activation cross-section measurement of a sort of nuclide produced with a target including two isotopes

    Institute of Scientific and Technical Information of China (English)

    ZHOU Feng-Qun; TIAN Ming-Li; SONG Yue-Li; LAN Chang-Lin; KONG Xiang-Zhong

    2013-01-01

    Based on a formula used to calculate the activation cross-section sum of two reactions producing a sort of nuclide with a target including two isotopes,the related problems in some references have been analyzed and discussed.It is pointed out that the calculation methods of the cross-section sum of two reactions producing the same radioactive nuclide for two isotopes in some references are improper and usually it is impossible to obtain the correct cross-section sum of two reactions producing the same radioactive nuclide for two isotopes in the case of using natural samples.At the same time,the related concepts are clarified and the correct processing method and representation are given.The comparison with the experimental results show that the theoretical analysis results are right.

  5. Ploidy of cell-sorted trophic and cystic forms of Pneumocystis carinii.

    Directory of Open Access Journals (Sweden)

    Anna Martinez

    Full Text Available Once regarded as an AIDS-defining illness, Pneumocystis pneumonia (PcP is nowadays prevailing in immunocompromised HIV-negative individuals such as patients receiving immunosuppressive therapies or affected by primary immunodeficiency. Moreover, Pneumocystis clinical spectrum is broadening to non-severely-immunocompromised subjects who could be colonized by the fungus while remaining asymptomatic for PcP, thus being able to transmit the infection by airborne route to susceptible hosts. Although the taxonomical position of the Pneumocystis genus has been clarified, several aspects of its life cycle remain elusive such as its mode of proliferation within the alveolus or its ploidy level. As no long-term culture model exists to grow Pneumocystis organisms in vitro, an option was to use a model of immunosuppressed rat infected with Pneumocystis carinii and sort life cycle stage fractions using a high-through-put cytometer. Subsequently, ploidy levels of the P. carinii trophic and cystic form fractions were measured by flow cytometry. In the cystic form, eight contents of DNA were measured thus strengthening the fact that each mature cyst contains eight haploid spores. Following release, each spore evolves into a trophic form. The majority of the trophic form fraction was haploid in our study. Some less abundant trophic forms displayed two contents of DNA indicating that they could undergo (i mating/fusion leading to a diploid status or (ii asexual mitotic division or (iii both. Even less abundant trophic forms with four contents of DNA were suggestive of mitotic divisions occurring following mating in diploid trophic forms. Of interest, was the presence of trophic forms with three contents of DNA, an unusual finding that could be related to asymmetrical mitotic divisions occurring in other fungal species to create genetic diversity at lower energetic expenses than mating. Overall, ploidy data of P. carinii life cycle stages shed new light on the

  6. High purity microfluidic sorting and analysis of circulating tumor cells: towards routine mutation detection.

    Science.gov (United States)

    Autebert, Julien; Coudert, Benoit; Champ, Jérôme; Saias, Laure; Guneri, Ezgi Tulukcuoglu; Lebofsky, Ronald; Bidard, François-Clément; Pierga, Jean-Yves; Farace, Françoise; Descroix, Stéphanie; Malaquin, Laurent; Viovy, Jean-Louis

    2015-05-01

    A new generation of the Ephesia cell capture technology optimized for CTC capture and genetic analysis is presented, characterized in depth and compared with the CellSearch system as a reference. This technology uses magnetic particles bearing tumour-cell specific EpCAM antibodies, self-assembled in a regular array in a microfluidic flow cell. 48,000 high aspect-ratio columns are generated using a magnetic field in a high throughput (>3 ml h(-1)) device and act as sieves to specifically capture the cells of interest through antibody-antigen interactions. Using this device optimized for CTC capture and analysis, we demonstrated the capture of epithelial cells with capture efficiency above 90% for concentrations as low as a few cells per ml. We showed the high specificity of capture with only 0.26% of non-epithelial cells captured for concentrations above 10 million cells per ml. We investigated the capture behavior of cells in the device, and correlated the cell attachment rate with the EpCAM expression on the cell membranes for six different cell lines. We developed and characterized a two-step blood processing method to allow for rapid processing of 10 ml blood tubes in less than 4 hours, and showed a capture rate of 70% for as low as 25 cells spiked in 10 ml blood tubes, with less than 100 contaminating hematopoietic cells. Using this device and procedure, we validated our system on patient samples using an automated cell immunostaining procedure and a semi-automated cell counting method. Our device captured CTCs in 75% of metastatic prostate cancer patients and 80% of metastatic breast cancer patients, and showed similar or better results than the CellSearch device in 10 out of 13 samples. Finally, we demonstrated the possibility of detecting cancer-related PIK3CA gene mutation in 20 cells captured in the chip with a good correlation between the cell count and the quantitation value Cq of the post-capture qPCR. PMID:25815443

  7. [Physical arrangement of membrane lipids susceptible to being used in the process of cell sorting of proteins].

    Science.gov (United States)

    Wolf, C; Quinn, P; Koumanov, K; Chachaty, C; Tenchov, B

    1999-01-01

    Detection of immiscible lipid domains in biological membranes offers an alternative support to protein sorting. Liquid ordered domains ("rafts") comprising cholesterol and saturated sphingolipids incorporate saturated glycosyl-phosphatidylinositol (GPI)-anchored or acylated (palmitoyl- and myristoyl-) proteins or particular transmembrane protein sequences. These lipid domains can be isolated in the form of Detergent resistant membranes (DRM) from biological plasma membrane preparations. Caveolae appear to be a differentiated fraction of plasma membranes comprising such numerous cross-linked microdomains associated with caveolin in different cell types. While the biological relevance of such membrane domains is evidenced in vivo by co-patching of proteins sharing the identical affinity for sphingolipids and by the disruption of co-patching following cell cholesterol depletion, only a few physical studies confort the principle of membrane heterogeneity. Results are now presented where cholesterol addition in a tertiary lipid mixture forces outphase-separation, as a realistic model where the lipid segregation can promote protein sorting to the segregated Lo phase. A lipid mixture comprising phosphatidylserine, phosphatidylethanolamine and sphingomyelin of natural origin in the ratio (1/4/3: mole/mole) has been rendered neatly heterogeneous after the addition of cholesterol (27 mole%). Xray diffraction (Small angle Xray scattering) showed the splitting of two neatly resolved lamellar diffractions in the presence of cholesterol. Above 37 degrees C the heterogeneity was traceable by a broadened diffraction spot up to the complete get-to-liquid transition of sphingomyelin at temperatures > 40 degrees C where the spot became again symmetrical and narrow. The large temperature range where the immiscible lamellar phases are detected, the specific requirement for cholesterol association with sphingomyelin, the positive influence of calcium and the reversibility of domain

  8. [Physical arrangement of membrane lipids susceptible to being used in the process of cell sorting of proteins].

    Science.gov (United States)

    Wolf, C; Quinn, P; Koumanov, K; Chachaty, C; Tenchov, B

    1999-01-01

    Detection of immiscible lipid domains in biological membranes offers an alternative support to protein sorting. Liquid ordered domains ("rafts") comprising cholesterol and saturated sphingolipids incorporate saturated glycosyl-phosphatidylinositol (GPI)-anchored or acylated (palmitoyl- and myristoyl-) proteins or particular transmembrane protein sequences. These lipid domains can be isolated in the form of Detergent resistant membranes (DRM) from biological plasma membrane preparations. Caveolae appear to be a differentiated fraction of plasma membranes comprising such numerous cross-linked microdomains associated with caveolin in different cell types. While the biological relevance of such membrane domains is evidenced in vivo by co-patching of proteins sharing the identical affinity for sphingolipids and by the disruption of co-patching following cell cholesterol depletion, only a few physical studies confort the principle of membrane heterogeneity. Results are now presented where cholesterol addition in a tertiary lipid mixture forces outphase-separation, as a realistic model where the lipid segregation can promote protein sorting to the segregated Lo phase. A lipid mixture comprising phosphatidylserine, phosphatidylethanolamine and sphingomyelin of natural origin in the ratio (1/4/3: mole/mole) has been rendered neatly heterogeneous after the addition of cholesterol (27 mole%). Xray diffraction (Small angle Xray scattering) showed the splitting of two neatly resolved lamellar diffractions in the presence of cholesterol. Above 37 degrees C the heterogeneity was traceable by a broadened diffraction spot up to the complete get-to-liquid transition of sphingomyelin at temperatures > 40 degrees C where the spot became again symmetrical and narrow. The large temperature range where the immiscible lamellar phases are detected, the specific requirement for cholesterol association with sphingomyelin, the positive influence of calcium and the reversibility of domain

  9. Isolation, cultivation and identification of brain glioma stem cells by magnetic bead sorting

    Institute of Scientific and Technical Information of China (English)

    Xiuping Zhou; Chao Zheng; Qiong Shi; Xiang Li; Zhigang Shen; Rutong Yu

    2012-01-01

    This study describes a detailed process for obtaining brain glioma stem cells from freshly dissected human brain glioma samples using an immunomagnetic bead technique combined with serum-free media pressure screening. Furthermore, the proliferation, differentiation and self-renewal biological features of brain glioma stem cells were identified. Results showed that a small number of CD133 positive tumor cells isolated from brain glioma samples survived as a cell suspension in serum-free media and proliferated. Subcultured CD133 positive cells maintained a potent self-renewal and proliferative ability, and expressed the stem cell-specific markers CD133 and nestin. After incubation with fetal bovine serum, the number of glial fibrillary acidic protein and microtubule associated protein 2 positive cells increased significantly, indicating that the cultured brain glioma stem cells can differentiate into astrocytes and neurons. Western blot analysis showed that tumor suppressor phosphatase and tensin homolog was highly expressed in tumor spheres compared with the differentiated tumor cells. These experimental findings indicate that the immunomagnetic beads technique is a useful method to obtain brain glioma stem cells from human brain tumors.

  10. Sorting through subsets: Which T cell populations mediate highly effective adoptive immunotherapy?

    Science.gov (United States)

    Klebanoff, Christopher A.; Gattinoni, Luca; Restifo, Nicholas P.

    2012-01-01

    CD8+ T cells have been described as being naïve (TN) or one of four antigen-experienced subtypes representing a continuum of differentiation and maturation: stem cell memory (TSCM), central memory (TCM), effector memory (TEM), and terminally differentiated effector T cells (TEFF). In mice, adoptive cell transfer (ACT) of less differentiated TN, TSCM and TCM subsets have consistently demonstrated superior in vivo expansion, persistence, and antitumor capacities relative to the more differentiated TEM and TEFF cells. Retrospective analyses from human ACT trials have confirmed that transfer of less differentiated T cell subsets is highly correlated with objective clinical responses. These findings, combined with the recent ability to convey de novo antigen reactivity with high efficiency through genetic engineering of exogenous T cell or chimeric antigen receptors, now challenge the field with three important questions: 1) how should less differentiated T cell subsets be isolated for human clinical trials?; 2) what is the best means of expanding T cells ex vivo in such a way as to not corrupt the beneficial traits of the younger subsets?; and 3) is it necessary to physically separate younger subsets from their more differentiated counterparts? Answering these questions will allow for the rational development of the next generation of highly effective and potentially curative T cell therapies for the treatment of cancer. PMID:23090074

  11. Induction and repair of DNA damage measured by the comet assay in human T lymphocytes separated by immunomagnetic cell sorting.

    Science.gov (United States)

    Bausinger, Julia; Speit, Günter

    2014-11-01

    The comet assay is widely used in human biomonitoring to measure DNA damage in whole blood or isolated peripheral blood mononuclear cells (PBMC) as a marker of exposure to genotoxic agents. Cytogenetic assays with phytohemagglutinin (PHA)-stimulated cultured T lymphocytes are also frequently performed in human biomonitoring. Cytogenetic effects (micronuclei, chromosome aberrations, sister chromatid exchanges) may be induced in vivo but also occur ex vivo during the cultivation of lymphocytes as a consequence of DNA damage present in lymphocytes at the time of sampling. To better understand whether DNA damage measured by the comet assay in PBMC is representative for DNA damage in T cells, we comparatively investigated DNA damage and its repair in PBMC and T cells obtained by immunomagnetic cell sorting. PBMC cultures and T cell cultures were exposed to mutagens with different modes of genotoxic action and DNA damage was measured by the comet assay after the end of a 2h exposure and after 18h post-incubation. The mutagens tested were methyl methanesulfonate (MMS), (±)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), 4-nitroquinoline-1-oxide (4NQO), styrene oxide and potassium bromate. MMS and potassium bromate were also tested by the modified comet assay with formamido pyrimidine glycosylase (FPG) protein. The results indicate that the mutagens tested induce DNA damage in PBMC and T cells in the same range of concentrations and removal of induced DNA lesions occurs to a comparable extent. Based on these results, we conclude that the comet assay with PBMC is suited to predict DNA damage and its removal in T cells.

  12. Parallel optical sorting of biological cells using the generalized phase contrast method

    DEFF Research Database (Denmark)

    Rindorf, Lars; Bu, Minqiang; Glückstad, Jesper

    2014-01-01

    Optical forces are used to fixate biological cells with optical tweezers where numerous biological parameters and phenomena can be studied. Optical beams carry a small momentum which generates a weak optical force, but on a cellular level this force is strong enough to allow for manipulation...... of biological cells in microfluidic systems exclusively using light. We demonstrate an optical cell sorter that uses simultaneous manipulation by multiple laser beams using the Generalized Phase Contrast method (GPC). The basic principle in an optical sorter is that the radiation force of the optical beam can...

  13. Lipoprotein sorting in bacteria.

    Science.gov (United States)

    Okuda, Suguru; Tokuda, Hajime

    2011-01-01

    Bacterial lipoproteins are synthesized as precursors in the cytoplasm and processed into mature forms on the cytoplasmic membrane. A lipid moiety attached to the N terminus anchors these proteins to the membrane surface. Many bacteria are predicted to express more than 100 lipoproteins, which play diverse functions on the cell surface. The Lol system, composed of five proteins, catalyzes the localization of Escherichia coli lipoproteins to the outer membrane. Some lipoproteins play vital roles in the sorting of other lipoproteins, lipopolysaccharides, and β-barrel proteins to the outer membrane. On the basis of results from biochemical, genetic, and structural studies, we discuss the biogenesis of lipoproteins in bacteria, their importance in cellular functions, and the molecular mechanisms underlying efficient sorting of hydrophobic lipoproteins to the outer membrane through the hydrophilic periplasm. PMID:21663440

  14. Palmitoylation of protease-activated receptor-1 regulates adaptor protein complex-2 and -3 interaction with tyrosine-based motifs and endocytic sorting.

    Science.gov (United States)

    Canto, Isabel; Trejo, JoAnn

    2013-05-31

    Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor for the coagulant protease thrombin. Thrombin binds to and cleaves the N terminus of PAR1, generating a new N terminus that functions as a tethered ligand that cannot diffuse away. In addition to rapid desensitization, PAR1 trafficking is critical for the regulation of cellular responses. PAR1 displays constitutive and agonist-induced internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), which binds to a distal tyrosine-based motif localized within the C-terminal tail (C-tail) domain. Once internalized, PAR1 is sorted from endosomes to lysosomes via AP-3 interaction with a second C-tail tyrosine motif proximal to the transmembrane domain. However, the regulatory processes that control adaptor protein recognition of PAR1 C-tail tyrosine-based motifs are not known. Here, we report that palmitoylation of PAR1 is critical for regulating proper utilization of tyrosine-based motifs and endocytic sorting. We show that PAR1 is basally palmitoylated at highly conserved C-tail cysteines. A palmitoylation-deficient PAR1 mutant is competent to signal and exhibits a marked increase in constitutive internalization and lysosomal degradation compared with wild type receptor. Intriguingly, enhanced constitutive internalization of PAR1 is mediated by AP-2 and requires the proximal tyrosine-based motif rather than the distal tyrosine motif used by wild type receptor. Moreover, palmitoylation-deficient PAR1 displays increased degradation that is mediated by AP-3. These findings suggest that palmitoylation of PAR1 regulates appropriate utilization of tyrosine-based motifs by adaptor proteins and endocytic trafficking, processes that are critical for maintaining appropriate expression of PAR1 at the cell surface. PMID:23580642

  15. Towards microfluidic sperm refinement : impedance-based analysis and sorting of sperm cells

    NARCIS (Netherlands)

    Wagenaar, de B.; Dekker, S.; Boer, de H.L.; Bomer, J.G.; Olthuis, W.; Berg, van den A.; Segerink, L.I.

    2016-01-01

    The use of high quality semen for artificial insemination in the livestock industry is essential for successful outcome. Insemination using semen with a high number of sperm cells containing morphological defects has a negative impact on fertilization outcome. Therefore, semen with a high number of

  16. COATED ENDOSOMAL VESICLES - SORTING AND RECYCLING COMPARTMENT FOR TRANSFERRIN IN BHK CELLS

    NARCIS (Netherlands)

    ESKELINEN, S; KOK, JW; SORMUNEN, R; HOEKSTRA, D

    1991-01-01

    We have investigated receptor-mediated endocytosis of transferrin (Tf) in baby hamster kidney (BHK) cells, using fluorescence and electron microscopy, and by carrying out colocalization experiments with clathrin antibodies and a fluorescently tagged glycolipid. Early during internalization, Tf was f

  17. Sorting for storage in myeloid cells of nonmyeloid proteins and chimeras with the propeptide of myeloperoxidase precursor.

    Science.gov (United States)

    Bülow, E; Nauseef, W M; Goedken, M; McCormick, S; Calafat, J; Gullberg, U; Olsson, I

    2002-02-01

    During formation of polymorphonuclear neutrophils, proteins are synthesized for storage in granules. Whereas sorting of proteins into distinct subtypes of cytoplasmic granules may reflect the coordinated expression of the proteins contained in them, still the mechanism(s) for the retrieval of proteins from the constitutive secretion is unknown. To investigate the mechanisms of retrieval, nonmyeloid secretory proteins were expressed in myeloid cell lines, and their subcellular fate was assessed. The contribution of the propeptide (MPOpro) of the myeloperoxidase (MPO) precursor was investigated by determining the fate of chimeras containing MPOpro. The nonmyeloid protein alpha(1)-microglobulin (alpha(1)-m) was targeted to storage organelles in 32D cells and colocalized with the lysosomal marker LAMP-1, whereas soluble TNF receptor 1 (sTNFR1) was secreted without granule targeting. Fusion of MPOpro to alpha(1)-m delayed exit from endoplasmic reticulum (ER), but subsequent targeting to dense organelles was indistinguishable from that of alpha(1)-m alone. Fusion proteins between MPOpro and sTNFR1 or green fluorescent protein expressed in myeloid 32D, K562, or PLB-985 cells did not associate stably with calreticulin or calnexin, molecular chaperones that normally interact transiently with the MPO precursor, but were still efficiently retained in the ER followed by degradation. We conclude that normally secreted, nonmyeloid proteins can be targeted efficiently to storage organelles in myeloid cells, that myeloid cells selectively target some proteins for storage but not others, and that MPOpro may contribute to the prolonged ER retention of the MPO precursor independent of the ER-molecular chaperones calreticulin and calnexin.

  18. Word Sorts for General Music Classes

    Science.gov (United States)

    Cardany, Audrey Berger

    2015-01-01

    Word sorts are standard practice for aiding children in acquiring skills in English language arts. When included in the general music classroom, word sorts may aid students in acquiring a working knowledge of music vocabulary. The author shares a word sort activity drawn from vocabulary in John Lithgow's children's book "Never Play…

  19. Optical force on diseased blood cells: Towards the optical sorting of biological matter

    KAUST Repository

    Gongora, Juan Sebastian Totero

    2015-05-01

    By employing a series of massively parallel ab-initio simulations, we study how optical forces act on biological matter subject to morphological disease. As a representative case study, we here consider the case of Plasmodium falciparum on red blood cells (RBC) illuminated by a monochromatic plane wave. Realistic parameters for the geometry and the refractive index are then taken from published experiments. In our theoretical campaign, we study the dependence of the optical force on the disease stage for different incident wavelengths. We show that optical forces change significantly with the disease, with amplitude variation in the hundreds of pN range. Our results open up new avenues for the design of new optical systems for the treatment of human disease. © 2015 Elsevier Ltd.

  20. Optical force on diseased blood cells: towards the optical sorting of biological matter

    CERN Document Server

    Gongora, Juan Sebastian Totero

    2016-01-01

    By employing a series of massively parallel ab-initio simulations, we study how optical forces act on biological matter subject to morphological disease. As a representative case study, we here consider the case of Plasmodium Falciparum on red blood cells (RBC) illuminated by a monochromatic plane wave. Realistic parameters for the geometry and the refractive index are then taken from published experiments. In our theoretical campaign, we study the dependence of the optical force on the disease stage for different incident wavelengths. We show that optical forces change significantly with the disease, with amplitude variation in the hundreds of pN range. Our results open up new avenues for the design of new optical systems for the treatment of human disease.

  1. k -Bitonic sort

    Institute of Scientific and Technical Information of China (English)

    高庆狮; 胡玥; 刘志勇

    1999-01-01

    A k-bitonic sort which generalizes the bitonic sort is proposed. The theorem of the bitonic sort, which merges two monotonic sequences into one order sequence, is extended into the theorem of k-bitonic sort. The k-bitonic sort merges K (=2k or 2k-1) monotonic sequences into one order sequence in steps, where k=[K/2] is an integer and k≥1. The k-bitonic sort is the Batcher’s bitonic sort when k=1.

  2. Rapid method to screen and sort lipid accumulating microalgae

    NARCIS (Netherlands)

    Dominguez Teles, I.; Zwart, van der M.; Kleinegris, D.M.M.; Barbosa, M.J.; Wijffels, R.H.

    2015-01-01

    The present work established an efficient staining method for fluorescence activated cell sorting (FACS) with Chlorococcum littorale maintaining cellular viability. The method was designed to detect high-lipid cells and to guarantee cellular viability. BODIPY505/515 (BP) was more suitable to FACS wh

  3. Phenotypic heterogeneity in metabolic traits among single cells of a rare bacterial species in its natural environment quantified with a combination of flow cell sorting and NanoSIMS

    Directory of Open Access Journals (Sweden)

    Matthias eZimmermann

    2015-04-01

    Full Text Available Populations of genetically identical microorganisms residing in the same environment can display marked variability in their phenotypic traits; this phenomenon is termed phenotypic heterogeneity. The relevance of such heterogeneity in natural habitats is unknown, because phenotypic characterization of a sufficient number of single cells of the same species in complex microbial communities is technically difficult. We report a procedure that allows to measure phenotypic heterogeneity in bacterial populations from natural environments, and use it to analyze N2 and CO2 fixation of single cells of the green sulfur bacterium Chlorobium phaeobacteroides from the meromictic lake Lago di Cadagno. We incubated lake water with 15N2 and 13CO2 under in situ conditions with and without NH4+. Subsequently, we used flow cell sorting with auto-fluorescence gating based on a pure culture isolate to concentrate C. phaeobacteroides from its natural abundance of 0.2 % to 26.5 % of total bacteria. C. phaeobacteroides cells were identified using catalyzed-reporter deposition fluorescence in situ hybridization (CARD-FISH targeting the 16S rRNA in the sorted population with a species-specific probe. In a last step, we used nanometer-scale secondary-ion mass spectrometry (NanoSIMS to measure the incorporation 15N and 13C stable isotopes in more than 252 cells. We found that C. phaeobacteroides fixes N2 in the absence of NH4+, but not in the presence of NH4+ as has previously been suggested. N2 and CO2 fixation were heterogeneous among cells and positively correlated indicating that N2 and CO2 fixation activity interact and positively facilitate each other in individual cells. However, because CARD-FISH identification cannot detect genetic variability among cells of the same species, we cannot exclude genetic variability as a source for phenotypic heterogeneity in this natural population. Our study demonstrates the technical feasibility of measuring phenotypic

  4. Apposition of iroquois expressing and non-expressing cells leads to cell sorting and fold formation in the Drosophila imaginal wing disc

    Directory of Open Access Journals (Sweden)

    González-Pérez Esther

    2007-09-01

    Full Text Available Abstract Background The organization of the different tissues of an animal requires mechanisms that regulate cell-cell adhesion to promote and maintain the physical separation of adjacent cell populations. In the Drosophila imaginal wing disc the iroquois homeobox genes are expressed in the notum anlage and contribute to the specification of notum identity. These genes are not expressed in the adjacent wing hinge territory. These territories are separated by an approximately straight boundary that in the mature disc is associated with an epithelial fold. The mechanism by which these two cell populations are kept separate is unclear. Results Here we show that the Iro-C genes participate in keeping the notum and wing cell populations separate. Indeed, within the notum anlage, cells not expressing Iro-C tend to join together and sort out from their Iro-C expressing neighbours. We also show that apposition of Iro-C expressing and non-expressing cells induces invagination and apico-basal shortening of the Iro-C- cells. This effect probably underlies formation of the fold that separates the notum and wing hinge territories. In addition, cells overexpressing a member of the Iro-C contact one another and become organized in a network of thin strings that surrounds and isolates large groups of non-overexpressing cells. The strings appear to exert a pulling force along their longitudinal axis. Conclusion Apposition of cells expressing and non-expressing the Iro-C, as it occurs in the notum-wing hinge border of the Drosophila wing disc, influences cell behaviour. It leads to cell sorting, and cellular invagination and apical-basal shortening. These effects probably account for keeping the prospective notum and wing hinge cell populations separate and underlie epithelial fold formation. Cells that overexpress a member of the Iro-C and that confront non-expressing cells establish contacts between themselves and become organized in a network of thin strings

  5. Preoperative sorting of circulating T lymphocytes in patients with esophageal squamous cell carcinoma: Its prognostic significance

    Institute of Scientific and Technical Information of China (English)

    Tadahiro Nozoe; Yoshihiko Maehara; Keizo Sugimachi

    2005-01-01

    AIM: To elucidate the immunologic parameters for the outcome of patients with malignant tumors, especially esophageal squamous cell carcinoma (ESCC) associated with high malignant potential.METHODS: Clinicopathologic features were compared between patients with lower and higher CD4 and CD8values as well as CD4/CD8 ratio in peripheral blood.RESULTS: The survival rate of patients with higher CD4 value was significantly better than that in patients with lower CD4 value (P = 0.039). The survival rate of patients with higher CD8 value was significantly worse than that of patients with lower CD8 value (P = 0.026).Similarly, the survival rate of patients with higher CD4/CD8 ratio was significantly better than that of patients with lower CD4/CD8 ratio (P = 0.042). Additionally,multivariate analysis demonstrated that lower CD8and lower CD4/CD8 ratio were factors independently associated with worse prognosis of patients.CONCLUSION: All the immunologic parameters can predict the outcome of patients with ESCC.

  6. What is a Sorting Function?

    DEFF Research Database (Denmark)

    Henglein, Fritz

    2009-01-01

    What is a sorting function—not a sorting function for a given ordering relation, but a sorting function with nothing given? Formulating four basic properties of sorting algorithms as defining requirements, we arrive at intrinsic notions of sorting and stable sorting: A function is a sorting funct...

  7. Parallel sorting algorithms

    CERN Document Server

    Akl, Selim G

    1985-01-01

    Parallel Sorting Algorithms explains how to use parallel algorithms to sort a sequence of items on a variety of parallel computers. The book reviews the sorting problem, the parallel models of computation, parallel algorithms, and the lower bounds on the parallel sorting problems. The text also presents twenty different algorithms, such as linear arrays, mesh-connected computers, cube-connected computers. Another example where algorithm can be applied is on the shared-memory SIMD (single instruction stream multiple data stream) computers in which the whole sequence to be sorted can fit in the

  8. Femtosecond laser fabricated microfluorescence-activated cell sorter for single cell recovery

    Science.gov (United States)

    Bragheri, F.; Paiè, P.; Nava, G.; Yang, T.; Minzioni, P.; Martinez Vazquez, R.; Bellini, N.; Ramponi, R.; Cristiani, I.; Osellame, R.

    2014-03-01

    Manipulation, sorting and recovering of specific live cells from samples containing less than a few thousand cells is becoming a major hurdle in rare cell exploration such as stem cell research or cell based diagnostics. Moreover the possibility of recovering single specific cells for culturing and further analysis would be of great impact in many biological fields ranging from regenerative medicine to cancer therapy. In recent years considerable effort has been devoted to the development of integrated and low-cost optofluidic devices able to handle single cells, which usually rely on microfluidic circuits that guarantee a controlled flow of the cells. Among the different microfabrication technologies, femtosecond laser micromachining (FLM) is ideally suited for this purpose as it provides the integration of both microfluidic and optical functions on the same glass chip leading to monolithic, robust and portable devices. Here a new optofluidic device is presented, which is capable of sorting and recovering of single cells, through optical forces, on the basis of their fluorescence and. Both fluorescence detection and single cell sorting functions are integrated in the microfluidic chip by FLM. The device, which is specifically designed to operate with a limited amount of cells but with a very high selectivity, is fabricated by a two-step process that includes femtosecond laser irradiation followed by chemical etching. The capability of the device to act as a micro fluorescence-activated cell sorter has been tested on polystyrene beads and on tumor cells and the results on the single live cell recovery are reported.

  9. Development of a cell sorting procedure to increase the sensitivity of detection of protein-protein interactions in plant protoplasts.

    Science.gov (United States)

    Zhang, Xin; Wong, Sek Man

    2011-05-01

    To visualize subcellular localization of viral proteins and interactions between viral proteins and host proteins in vivo, transfection of plasmids into protoplasts to over-express transiently fusion proteins with a fluorescent tag is a common method. However, due to the low efficiency (0.1-3.0%) of plasmid transfection into protoplasts, it is difficult to identify protoplasts that emit fluorescence using confocal microscopy. A flow cytometry sorting protocol was developed for separating kenaf protoplasts that emit yellow fluorescence. The sorted protoplasts showed strong fluorescence and the protoplasts were intact. This will improve the use of confocal microscopy for studying subcellular localization and protein interactions in protoplasts isolated from plants with low transfection efficiency.

  10. Sorting a distribution theory

    CERN Document Server

    Mahmoud, Hosam M

    2011-01-01

    A cutting-edge look at the emerging distributional theory of sorting Research on distributions associated with sorting algorithms has grown dramatically over the last few decades, spawning many exact and limiting distributions of complexity measures for many sorting algorithms. Yet much of this information has been scattered in disparate and highly specialized sources throughout the literature. In Sorting: A Distribution Theory, leading authority Hosam Mahmoud compiles, consolidates, and clarifies the large volume of available research, providing a much-needed, comprehensive treatment of the

  11. Designing sorting networks

    CERN Document Server

    Baddar, Sherenaz W Al-Haj

    2012-01-01

    Designing Sorting Networks: A New Paradigm provides an in-depth guide to maximizing the efficiency of sorting networks, and uses 0/1 cases, partially ordered sets and Haase diagrams to closely analyze their behavior in an easy, intuitive manner. This book also outlines new ideas and techniques for designing faster sorting networks using Sortnet, and illustrates how these techniques were used to design faster 12-key and 18-key sorting networks through a series of case studies. Finally, it examines and explains the mysterious behavior exhibited by the fastest-known 9-step 16-key network. Designi

  12. Coupling Binding to Catalysis – Using Yeast Cell Surface Display to Select Enzymatic Activities

    OpenAIRE

    Zhang, Keya; Bhuripanyo, Karan; Wang, Yiyang; Yin, Jun

    2015-01-01

    We find yeast cell surface display can be used to engineer enzymes by selecting the enzyme library for high affinity binding to reaction intermediates. Here we cover key steps of enzyme engineering on the yeast cell surface including library design, construction, and selection based on magnetic and fluorescence activated cell sorting.

  13. Coupling Binding to Catalysis: Using Yeast Cell Surface Display to Select Enzymatic Activities.

    Science.gov (United States)

    Zhang, Keya; Bhuripanyo, Karan; Wang, Yiyang; Yin, Jun

    2015-01-01

    We find yeast cell surface display can be used to engineer enzymes by selecting the enzyme library for high affinity binding to reaction intermediates. Here we cover key steps of enzyme engineering on the yeast cell surface including library design, construction, and selection based on magnetic and fluorescence-activated cell sorting. PMID:26060080

  14. Three Sorts of Naturalism

    DEFF Research Database (Denmark)

    Fink, Hans

    2006-01-01

    In "Two sorts of Naturalism" John McDowell is sketching his own sort of naturalism in ethics as an alternative to "bald naturalism". In this paper I distinguish materialist, idealist and absolute conceptions of nature and of naturalism in order to provide a framework for a clearer understanding o...

  15. Automated Sorting of Transuranic Waste

    Energy Technology Data Exchange (ETDEWEB)

    Shurtliff, Rodney Marvin

    2001-03-01

    The HANDSS-55 Transuranic Waste Sorting Module is designed to sort out items found in 55-gallon drums of waste as determined by an operator. Innovative imaging techniques coupled with fast linear motor-based motion systems and a flexible end-effector system allow the operator to remove items from the waste stream by a touch of the finger. When all desired items are removed from the waste stream, the remaining objects are automatically moved to a repackaging port for removal from the glovebox/cell. The Transuranic Waste Sorting Module consists of 1) a high accuracy XYZ Stereo Measurement and Imaging system, 2) a vibrating/tilting sorting table, 3) an XY Deployment System, 4) a ZR Deployment System, 5) several user-selectable end-effectors, 6) a waste bag opening system, 7) control and instrumentation, 8) a noncompliant waste load-out area, and 9) a Human/Machine Interface (HMI). The system is modular in design to accommodate database management tools, additional load-out ports, and other enhancements. Manually sorting the contents of a 55-gallon drum takes about one day per drum. The HANDSS-55 Waste Sorting Module is designed to significantly increase the throughput of this sorting process by automating those functions that are strenuous and tiresome for an operator to perform. The Waste Sorting Module uses the inherent ability of an operator to identify the items that need to be segregated from the waste stream and then, under computer control, picks that item out of the waste and deposits it in the appropriate location. The operator identifies the object by locating the visual image on a large color display and touches the image on the display with his finger. The computer then determines the location of the object, and performing a highspeed image analysis determines its size and orientation, so that a robotic gripper can be deployed to pick it up. Following operator verification by voice or function key, the object is deposited into a specified location.

  16. Deformability-based capsule sorting

    Science.gov (United States)

    Le Goff, Anne; Munier, Nadege; Maire, Pauline; Edwards-Levy, Florence; Salsac, Anne-Virginie

    2015-11-01

    Many microfluidic devices have been developed for cancer diagnosis applications, most of which relying on costly antibodies. Since some cancer cells display abnormal mechanical properties, new sorting tools based on mechanical sensing are of particular interest. We present a simple, passive pinched flow microfluidic system for capsule sorting. The device consists of a straight microchannel containing a cylindrical obstacle. Thanks to a flow-focusing module placed at the channel entrance, capsules arrive well-centered in the vicinity of the obstacle. Pure size-sorting can be achieved at low shear rate. When increasing the shear rate, capsules are deformed in the narrow space between the pillar and the wall. The softer the capsule, the more tightly it wraps around the obstacle. After the obstacle, streamlines diverge, allowing for the separation between soft capsules, that follow central streamlines, and stiff capsules, that drift away from the obstacle with a wider angle. This proves that we have developed a flexible multipurpose sorting microsystem based on a simple design.

  17. Increased basolateral sorting of carcinoembryonic antigen in a polarized colon carcinoma cell line after cholesterol depletion-Implications for treatment of inflammatory bowel disease

    Institute of Scientific and Technical Information of China (English)

    Robert Ehehalt; Markus Krautter; Martin Zorn; Richard Sparla; Joachim Fūllekrug; Hasan Kulaksiz; Wolfgang Stremmel

    2008-01-01

    AIM:To investigate a possible increase of basolateral expression of carcinoembryonic antigen(CEA)by interfering with the apical transport machinery,we studied the effect of cholesterol depletion on CEA sorting and secretion.METHODS:Cholesterol depletion was performed in polarized Caco-2 cells using Iovastatin and methyl-βcyclodextrin.RESULTS:We show that CEA is predominantly expressed and secreted at the apical surface.Reduction of the cholesterol level of the cell by 40%-50% with Iovastatin and methyl-β-cyclodextrin led to a significant change of the apical-to-basolateral transport ratio towards the basolateral membrane.CONCLUSION:As basolateral expression of CEA has been suggested to have anti-inflamatory properties,Cholesterol depletion of enterocytes might be a potential approach to influence the course of inflammatory bowel disease.

  18. Hash sort: A linear time complexity multiple-dimensional sort algorithm

    OpenAIRE

    Gilreath, William F.

    2004-01-01

    Sorting and hashing are two completely different concepts in computer science, and appear mutually exclusive to one another. Hashing is a search method using the data as a key to map to the location within memory, and is used for rapid storage and retrieval. Sorting is a process of organizing data from a random permutation into an ordered arrangement, and is a common activity performed frequently in a variety of applications. Almost all conventional sorting algorithms work by comparison, and ...

  19. Guest Editorial: Card sort methodology: An objective measure in rehabilitation research

    OpenAIRE

    Haitham Jahrami, PhD

    2012-01-01

    Card sort clinical tests such as the Wisconsin Card Sort Test and Activity Card Sort are well known in several clinical practices, including psychiatry, neurology, neuropsychology, and learning disabilities. However, card sort methodology is less famous as a research methodology. This editorial attempts to shed light on the novelty of the card sort methodology and its relevance to rehabilitation research.

  20. A Framework for the Comparative Assessment of Neuronal Spike Sorting Algorithms towards More Accurate Off-Line and On-Line Microelectrode Arrays Data Analysis

    OpenAIRE

    Giulia Regalia; Stefania Coelli; Emilia Biffi; Giancarlo Ferrigno; Alessandra Pedrocchi

    2016-01-01

    Neuronal spike sorting algorithms are designed to retrieve neuronal network activity on a single-cell level from extracellular multiunit recordings with Microelectrode Arrays (MEAs). In typical analysis of MEA data, one spike sorting algorithm is applied indiscriminately to all electrode signals. However, this approach neglects the dependency of algorithms’ performances on the neuronal signals properties at each channel, which require data-centric methods. Moreover, sorting is commonly perfor...

  1. LazySorted: A Lazily, Partially Sorted Python List

    Directory of Open Access Journals (Sweden)

    Naftali Harris

    2015-06-01

    Full Text Available LazySorted is a Python C extension implementing a partially and lazily sorted list data structure. It solves a common problem faced by programmers, in which they need just part of a sorted list, like its middle element (the median, but sort the entire list to get it. LazySorted presents them with the abstraction that they are working with a fully sorted list, while actually only sorting the list partially with quicksort partitions to return the requested sub-elements. This enables programmers to use naive "sort first" algorithms but nonetheless attain linear run-times when possible. LazySorted may serve as a drop-in replacement for the built-in sorted function in most cases, and can sometimes achieve run-times more than 7 times faster.

  2. Exosome and Exosomal MicroRNA:Trafficking, Sorting, and Function

    Institute of Scientific and Technical Information of China (English)

    Jian Zhang; Sha Li; Lu Li; Meng Li; Chongye Guo; Jun Yao; Shuangli Mi

    2015-01-01

    Exosomes are 40–100 nm nano-sized vesicles that are released from many cell types into the extracellular space. Such vesicles are widely distributed in various body fluids. Recently, mRNAs and microRNAs (miRNAs) have been identified in exosomes, which can be taken up by neighboring or distant cells and subsequently modulate recipient cells. This suggests an active sort-ing mechanism of exosomal miRNAs, since the miRNA profiles of exosomes may differ from those of the parent cells. Exosomal miRNAs play an important role in disease progression, and can stimu-late angiogenesis and facilitate metastasis in cancers. In this review, we will introduce the origin and the trafficking of exosomes between cells, display current research on the sorting mechanism of exo-somal miRNAs, and briefly describe how exosomes and their miRNAs function in recipient cells. Finally, we will discuss the potential applications of these miRNA-containing vesicles in clinical settings.

  3. A non-destructive culturing and cell sorting method for cardiomyocytes and neurons using a double alginate layer.

    Directory of Open Access Journals (Sweden)

    Hideyuki Terazono

    Full Text Available A non-destructive method of collecting cultured cells after identifying their in situ functional characteristics is proposed. In this method, cells are cultivated on an alginate layer in a culture dish and released by spot application of a calcium chelate buffer that locally melts the alginate layer and enables the collection of cultured cells at the single-cell level. Primary hippocampal neurons, beating human embryonic stem (hES cell-derived cardiomyocytes, and beating hES cell-derived cardiomyocyte clusters cultivated on an alginate layer were successfully released and collected with a micropipette. The collected cells were recultured while maintaining their physiological function, including beating, and elongated neurites. These results suggest that the proposed method may eventually facilitate the transplantation of ES- or iPS-derived cardiomyocytes and neurons differentiated in culture.

  4. Ready, steady, SORT!

    CERN Multimedia

    Laëtitia Pedroso

    2010-01-01

    The selective or ecological sorting of waste is already second nature to many of us and concerns us all. As the GS Department's new awareness-raising campaign reminds us, everything we do to sort waste contributes to preserving the environment.    Placemats printed on recycled paper using vegetable-based ink will soon be distributed in Restaurant No.1.   Environmental protection is never far from the headlines, and CERN has a responsibility to ensure that the 3000 tonnes and more of waste it produces every year are correctly and selectively sorted. Materials can be given a second life through recycling and re-use, thereby avoiding pollution from landfill sites and incineration plants and saving on processing costs. The GS Department is launching a new poster campaign designed to raise awareness of the importance of waste sorting and recycling. "After conducting a survey to find out whether members of the personnel were prepared to make an effort to sort a...

  5. The Proper Criteria for Identification and Sorting of Very Small Embryonic-Like Stem Cells, and Some Nomenclature Issues

    Science.gov (United States)

    Suszynska, Malwina; Zuba-Surma, Ewa K.; Maj, Magdalena; Mierzejewska, Kasia; Ratajczak, Janina; Kucia, Magda

    2014-01-01

    Evidence has accumulated that both murine and human adult tissues contain early-development stem cells with a broader differentiation potential than other adult monopotent stem cells. These cells, being pluripotent or multipotent, exist at different levels of specification and most likely represent overlapping populations of cells that, depending on the isolation strategy, ex vivo expansion protocol, and markers employed for their identification, have been given different names. In this review, we will discuss a population of very small embryonic-like stem cells (VSELs) in the context of other stem cells that express pluripotent/multipotent markers isolated from adult tissues as well as review the most current, validated working criteria on how to properly identify and isolate these very rare cells. VSELs have been successfully purified in several laboratories; however, a few have failed to isolate them, which has raised some unnecessary controversy in the field. Therefore, in this short review, we will address the most important reasons that some investigators have experienced problems in isolating these very rare cells and discuss some still unresolved challenges which should be overcome before these cells can be widely employed in the clinic. PMID:24299281

  6. Sorting and sustaining cooperation

    DEFF Research Database (Denmark)

    Vikander, Nick

    2013-01-01

    This paper looks at cooperation in teams where some people are selfish and others are conditional cooperators, and where lay-offs will occur at a fixed future date. I show that the best way to sustain cooperation prior to the lay-offs is often in a sorting equilibrium, where conditional cooperators...... can identify and then work with one another. Changes to parameters that would seem to make cooperation more attractive, such as an increase in the discount factor or the fraction of conditional cooperators, can reduce equilibrium cooperation if they decrease a selfish player's incentive to sort....

  7. Wage Sorting Trends

    DEFF Research Database (Denmark)

    Bagger, Jesper; Vejlin, Rune Majlund; Sørensen, Kenneth Lykke

    Using a population-wide Danish Matched Employer-Employee panel from 1980-2006, we document a strong trend towards more positive assortative wage sorting. The correlation between worker and firm fixed effects estimated from a log wage regression increases from -0.07 in 1981 to .14 in 2001. The non......Using a population-wide Danish Matched Employer-Employee panel from 1980-2006, we document a strong trend towards more positive assortative wage sorting. The correlation between worker and firm fixed effects estimated from a log wage regression increases from -0.07 in 1981 to .14 in 2001...

  8. Characterization and localization of side population cells in the lens

    OpenAIRE

    Oka, Mikako; Toyoda, Chizuko; Kaneko, Yuka; Nakazawa, Yosuke; Aizu-Yokota, Eriko; Takehana, Makoto

    2010-01-01

    Purpose Side population (SP) cells were isolated and the possibility whether lens epithelial cells contain stem cells was investigated. Methods Mouse lens epithelial cells were stained by Hoechst 33342 and then sorted by fluorescence-activated cell sorting (FACS). The expression of stem cell markers in sorted SP cells and the main population of epithelial cells were analyzed by quantitative real-time PCR. Localization of SP cells in the mouse lens was studied by fluorescence microscopy. Resul...

  9. Efficient selective breeding of live oil-rich Euglena gracilis with fluorescence-activated cell sorting

    OpenAIRE

    Koji Yamada; Hideyuki Suzuki; Takuto Takeuchi; Yusuke Kazama; Sharbanee Mitra; Tomoko Abe; Keisuke Goda; Kengo Suzuki; Osamu Iwata

    2016-01-01

    Euglena gracilis, a microalgal species of unicellular flagellate protists, has attracted much attention in both the industrial and academic sectors due to recent advances in the mass cultivation of E. gracilis that have enabled the cost-effective production of nutritional food and cosmetic commodities. In addition, it is known to produce paramylon (β-1,3-glucan in a crystalline form) as reserve polysaccharide and convert it to wax ester in hypoxic and anaerobic conditions–a promising feedstoc...

  10. Online Sorted Range Reporting

    DEFF Research Database (Denmark)

    Brodal, Gerth Stølting; Fagerberg, Rolf; Greve, Mark;

    2009-01-01

    We study the following one-dimensional range reporting problem: On an arrayA of n elements, support queries that given two indices i ≤ j and an integerk report the k smallest elements in the subarray A[i..j] in sorted order. We present a data structure in the RAM model supporting such queries in ...

  11. Gender Differences in Sorting

    DEFF Research Database (Denmark)

    Merlino, Luca Paolo; Parrotta, Pierpaolo; Pozzoli, Dario

    In this paper, we investigate the sorting of workers in firms to understand gender gaps in labor market outcomes. Using Danish employer-employee matched data, we fiend strong evidence of glass ceilings in certain firms, especially after motherhood, preventing women from climbing the career ladder...

  12. Det sorte USA

    DEFF Research Database (Denmark)

    Brøndal, Jørn

    Bogen gennemgår det sorte USAs historie fra 1776 til 2016, idet grundtemaet er spændingsforholdet mellem USAs grundlæggelsesidealer og den racemæssige praksis, et spændingsforhold som Gunnar Myrdal kaldte "det amerikanske dilemma." Bogen, der er opbygget som politisk, social og racemæssig historie...

  13. Sorting of ligand-activated epidermal growth factor receptor to lysosomes requires its actin-binding domain

    NARCIS (Netherlands)

    Stoorvogel, W; Kerstens, S; Fritzsche, I; den Hartigh, JC; Oud, R; van der Heyden, MAG; Henegouwen, PMPVE

    2004-01-01

    Ligand-induced down-regulation of the epidermal growth factor receptor (EGFR) comprises activation of two sequential transport steps. The first involves endocytic uptake by clathrin-coated vesicles, the second transfer of endocytosed EGFR from endosomes to lysosomes. Here we demonstrate that the sec

  14. Visual ergonomics interventions in mail sorting facilities.

    Science.gov (United States)

    Hemphälä, H; Hansson, G-Å; Dahlqvist, C; Eklund, J

    2012-01-01

    This study was performed between 2004 and 2011 at mail sorting facilities in Sweden. During this time, different interventions were performed. The first was a lighting intervention that had a positive impact on the postal workers, especially those with eyestrain. A new lighting system also improved the illuminance and gave better light distribution. The second intervention involved new personal spectacles for the postal workers who needed them and this had a positive effect on eyestrain. The third intervention involved a specific type of sorting spectacles for the postal workers who already used progressive lenses privately. The reading distances that the postal workers had while sorting the mail was inverted to the distances in their regular progressive lenses. The new sorting spectacles had a positive effect on head postures and on muscular activity. PMID:22317243

  15. Selective isolation of ammonia-oxidizing bacteria from autotrophic nitrifying granules by applying cell-sorting and sub-culturing of microcolonies

    Directory of Open Access Journals (Sweden)

    Hirotsugu eFujitani

    2015-10-01

    Full Text Available Nitrification is a key process in the biogeochemical nitrogen cycle and biological wastewater treatment that consists of two stepwise reactions, ammonia oxidation by ammonia-oxidizing bacteria (AOB or archaea followed by nitrite oxidation by nitrite-oxidizing bacteria. One of the representative of the AOB group is Nitrosomonas mobilis species. Although a few pure strains of this species have been isolated so far, approaches to their preservation in pure culture have not been established. Here, we report isolation of novel members of the N. mobilis species from autotrophic nitrifying granules used for ammonia-rich wastewater treatment. We developed an isolation method focusing on microcolonies formation of nitrifying bacteria. Two kinds of distinctive light scattering signatures in a cell-sorting system enabled to separate microcolonies from single cells and heterogeneous aggregates within granule samples. Inoculation of a pure microcolony into 96-well microtiter plates led to successful sub-culturing and increased probability of isolation. Obtained strain Ms1 is cultivated in the liquid culture with relatively high ammonia or nitrite concentration, not extremely slow growing. Considering environmental clones that were closely related to N. mobilis and detected in various environments, the availability of this novel strain would facilitate to reveal this member’s ecophysiology in a variety of habitats.

  16. Selective isolation of ammonia-oxidizing bacteria from autotrophic nitrifying granules by applying cell-sorting and sub-culturing of microcolonies.

    Science.gov (United States)

    Fujitani, Hirotsugu; Kumagai, Asami; Ushiki, Norisuke; Momiuchi, Kengo; Tsuneda, Satoshi

    2015-01-01

    Nitrification is a key process in the biogeochemical nitrogen cycle and biological wastewater treatment that consists of two stepwise reactions, ammonia oxidation by ammonia-oxidizing bacteria (AOB) or archaea followed by nitrite oxidation by nitrite-oxidizing bacteria. One of the representatives of the AOB group is Nitrosomonas mobilis species. Although a few pure strains of this species have been isolated so far, approaches to their preservation in pure culture have not been established. Here, we report isolation of novel members of the N. mobilis species from autotrophic nitrifying granules used for ammonia-rich wastewater treatment. We developed an isolation method focusing on microcolonies formation of nitrifying bacteria. Two kinds of distinctive light scattering signatures in a cell-sorting system enabled to separate microcolonies from single cells and heterogeneous aggregates within granule samples. Inoculation of a pure microcolony into 96-well microtiter plates led to successful sub-culturing and increased probability of isolation. Obtained strain Ms1 is cultivated in the liquid culture with relatively high ammonia or nitrite concentration, not extremely slow growing. Considering environmental clones that were closely related to N. mobilis and detected in various environments, the availability of this novel strain would facilitate to reveal this member's ecophysiology in a variety of habitats. PMID:26528282

  17. Sorting quantum systems efficiently

    Science.gov (United States)

    Ionicioiu, Radu

    2016-05-01

    Measuring the state of a quantum system is a fundamental process in quantum mechanics and plays an essential role in quantum information and quantum technologies. One method to measure a quantum observable is to sort the system in different spatial modes according to the measured value, followed by single-particle detectors on each mode. Examples of quantum sorters are polarizing beam-splitters (PBS) – which direct photons according to their polarization – and Stern-Gerlach devices. Here we propose a general scheme to sort a quantum system according to the value of any d-dimensional degree of freedom, such as spin, orbital angular momentum (OAM), wavelength etc. Our scheme is universal, works at the single-particle level and has a theoretical efficiency of 100%. As an application we design an efficient OAM sorter consisting of a single multi-path interferometer which is suitable for a photonic chip implementation.

  18. Teaching Sorting in ICT

    OpenAIRE

    Szlávi, Péter; Törley, Gábor

    2009-01-01

    This article is aimed at considering how an algorithmic problem - more precisely a sorting problem - can be used in an informatics class in primary and secondary education to make students mobilize the largest possible amount of their intellectual skills in the problem solving process. We will be outlining a method which essentially forces students to utilize their mathematical knowledge besides algorithmization in order to provide an efficient solution. What is more, they are expected to ...

  19. K-sort: A new sorting algorithm that beats Heap sort for n <= 70 lakhs!

    CERN Document Server

    Sundararajan, Kiran Kumar; Chakraborty, Soubhik; Mahanti, N C

    2011-01-01

    Sundararajan and Chakraborty (2007) introduced a new version of Quick sort removing the interchanges. Khreisat (2007) found this algorithm to be competing well with some other versions of Quick sort. However, it uses an auxiliary array thereby increasing the space complexity. Here, we provide a second version of our new sort where we have removed the auxiliary array. This second improved version of the algorithm, which we call K-sort, is found to sort elements faster than Heap sort for an appreciably large array size (n <= 70,00,000) for uniform U[0, 1] inputs.

  20. Chip-based droplet sorting

    Energy Technology Data Exchange (ETDEWEB)

    Beer, Neil Reginald; Lee, Abraham; Hatch, Andrew

    2014-07-01

    A non-contact system for sorting monodisperse water-in-oil emulsion droplets in a microfluidic device based on the droplet's contents and their interaction with an applied electromagnetic field or by identification and sorting.

  1. Chip-based droplet sorting

    Science.gov (United States)

    Beer, Neil Reginald; Lee, Abraham; Hatch, Andrew

    2014-07-01

    A non-contact system for sorting monodisperse water-in-oil emulsion droplets in a microfluidic device based on the droplet's contents and their interaction with an applied electromagnetic field or by identification and sorting.

  2. Immobilization of cells via activated cell walls

    Energy Technology Data Exchange (ETDEWEB)

    Markt, M.; Kas, J.; Valentova, O.; Demnerova, K.; Vodrazka, Z.

    1986-10-01

    Cell walls of Saccharomyces cerevisiae and S. uvarum were activated by periodate oxidation of vicinal diol groups in cell wall polysaccharides. The aldehyde groups thus generated allow the yeast cells to be covalently bound to modified bead cellulose or macroporous glycidyl methacrylate supports, or to enzymes such as glucose oxidase and catalase. 6 references.

  3. Pair Wise Sorting: A New Way of Sorting

    Directory of Open Access Journals (Sweden)

    Md. Jahangir Alam

    2010-12-01

    Full Text Available This paper presents a technique for sorting numerical data in an efficient way. The numbers of comparisons i.e. the running time of this technique is dependent on distribution or diversity of the value of data items as like as other efficient algorithms. When the total number of data is even, this method groups that data into a collection of pairs and therefore establishes the sorting constraints on each of the pairs. The control is traversed through the list of elements by changing the position of each pair which is the major principle of this technique. On the other hand, when the total number of elements is odd, this method sorts all elements except the last one in the same was as mentioned earlier and the last element is sorted using the general Insertion Sort. This algorithm is therefore a hybrid sorting method that sorts elementary numeric data in a faster and efficient manner.

  4. Spike sorting in the frequency domain with overlap detection

    CERN Document Server

    Rinberg, D; Davidowitz, H; Tishby, N; Rinberg, Dima; Bialek, William; Davidowitz, Hanan; Tishby, Naftali

    2003-01-01

    This paper deals with the problem of extracting the activity of individual neurons from multi-electrode recordings. Important aspects of this work are: 1) the sorting is done in two stages - a statistical model of the spikes from different cells is built and only then are occurrences of these spikes in the data detected by scanning through the original data, 2) the spike sorting is done in the frequency domain, 3) strict statistical tests are applied to determine if and how a spike should be classiffed, 4) the statistical model for detecting overlaping spike events is proposed, 5) slow dynamics of spike shapes are tracked during long experiments. Results from the application of these techniques to data collected from the escape response system of the American cockroach, Periplaneta americana, are presented.

  5. Assessment of Preschool Classroom Practices: Application of Q-Sort Methodology

    Science.gov (United States)

    Bracken, Stacey Storch; Fischel, Janet E.

    2006-01-01

    This paper reports on the application of Q-sort methodology to the development of the Preschool Classroom Practices (PCP) Q-sort. The PCP Q-sort was tested in a sample of 66 preschool teachers and assistants. Results demonstrated the existence of a 2-cluster structure within the Q-sort, comprised of Cognitive Development Activities and…

  6. Selective sorting of waste

    CERN Multimedia

    2007-01-01

    Not much effort needed, just willpower In order to keep the cost of disposing of waste materials as low as possible, CERN provides two types of recipient at the entrance to each building: a green plastic one for paper/cardboard and a metal one for general refuse. For some time now we have noticed, to our great regret, a growing negligence as far as selective sorting is concerned, with, for example, the green recipients being filled with a mixture of cardboard boxes full of polystyrene or protective wrappers, plastic bottles, empty yogurts pots, etc. …We have been able to ascertain, after careful checking, that this haphazard mixing of waste cannot be attributed to the cleaning staff but rather to members of the personnel who unscrupulously throw away their rubbish in a completely random manner. Non-sorted waste entails heavy costs for CERN. For information, once a non-compliant item is found in a green recipient, the entire contents are sent off for incineration rather than recycling… We are all concerned...

  7. A two-channel detection method for autofluorescence correction and efficient on-bead screening of one-bead one-compound combinatorial libraries using the COPAS fluorescence activated bead sorting system

    International Nuclear Information System (INIS)

    One-bead one-compound combinatorial library beads exhibit varying levels of autofluorescence after solid phase combinatorial synthesis. Very often this causes significant problems for automated on-bead screening using TentaGel beads and fluorescently labeled target proteins. Herein, we present a method to overcome this limitation when fluorescence activated bead sorting is used as the screening method. We have equipped the COPAS bead sorting instrument with a high-speed profiling unit and developed a spectral autofluorescence correction method. The correction method is based on a simple algebraic operation using the fluorescence data from two detection channels and is applied on-the-fly in order to reliably identify hit beads by COPAS bead sorting. Our method provides a practical tool for the fast and efficient isolation of hit beads from one-bead one-compound library screens using either fluorescently labeled target proteins or biotinylated target proteins. This method makes hit bead identification easier and more reliable. It reduces false positives and eliminates the need for time-consuming pre-sorting of library beads in order to remove autofluorescent beads. (technical note)

  8. Spin-the-bottle Sort and Annealing Sort: Oblivious Sorting via Round-robin Random Comparisons

    CERN Document Server

    Goodrich, Michael T

    2010-01-01

    We study sorting algorithms based on randomized round-robin comparisons. Specifically, we study Spin-the-bottle sort, where comparisons are unrestricted, and Annealing sort, where comparisons are restricted to a distance bounded by a \\emph{temperature} parameter. Both algorithms are simple, randomized, data-oblivious sorting algorithms, which are useful in privacy-preserving computations, but, as we show, Annealing sort is much more efficient. We show that there is an input permutation that causes Spin-the-bottle sort to require $\\Omega(n^2\\log n)$ expected time in order to succeed, and that in $O(n^2\\log n)$ time this algorithm succeeds with high probability for any input. We also show there is an implementation of Annealing sort that runs in $O(n\\log n)$ time and succeeds with very high probability.

  9. Determination of telomerase activity in stem cells and non-stem cells of breast cancer

    Institute of Scientific and Technical Information of China (English)

    LI Zhi; HE Yanli; ZHANG Jiahua; ZHANG Jinghui; HUANG Tao

    2007-01-01

    Although all normal tissue cells,including stem cells,are genetically homologous,variation in gene expression patterns has already determined the distinct roles for individual cells in the physiological process due to the occurrence of epigenetic modification.This is of special importance for the existenee of tissue stem cells because they are exclusively immortal within the body,capable of selfreplicating and differentiating by which tissues renew and repair itself and the total tissue cell population maintains a steady-state.Impairment of tissue stem cells is usually accompanied by a reduction in cell number,slows down the repair process and causes hypofunction.For instance,chemotherapy usually leads to depression of bone marrow and hair loss.Cellular aging is closely associated with the continuous erosion of the telomere while activation of telomerase repairs and maintains telomeres,thus slowing the aging process and prolonging cell life.In normal adults,telomerase activation mainly presents in tissue stem cells and progenitor cells giving them unlimited growth potential.Despite the extensive demonstration of telomerase activation in malignancy(>80%),scientists found that heterogeneity also exists among the tumor cells and only minorities of cells,designated as cancer stem cells,andergo processes analogous to the self-renewal and differentiation of normal stem ceils while the rest have limited lifespans.In this study,telomerase activity was measured and compared in breast cancer stem cells and non-stem cells that were phenotypically sorted by examining surface marker expression.The results indicated that cancer stem cells show a higher level of enzyme activity than non-stem cells.In addition,associated with the repair of cancer tissue(or relapse)after chemotherapy,telomerase activity in stem cells was markedly increased.

  10. Phenotypic and functional characterization of earthworm coelomocyte subsets: Linking light scatter-based cell typing and imaging of the sorted populations

    DEFF Research Database (Denmark)

    Engelmann, Péter; Hayashi, Yuya; Bodo, Kornélia;

    2016-01-01

    of lectin binding capacity indicated wheat germ agglutinin (WGA) as the strongest reactor to amoebocytes. This is further evidenced by WGA inhibition assays that suggest high abundance of N-acetyl-d-glucosamine in amoebocytes. Post-sort phagocytosis assays confirmed the functional differences between...

  11. T cell receptor zeta allows stable expression of receptors containing the CD3gamma leucine-based receptor-sorting motif

    DEFF Research Database (Denmark)

    Dietrich, J; Geisler, C

    1998-01-01

    that the leucine-based motif in these complexes was inactive. In contrast, the CD4/CD3gamma chimeras did not associate with TCRzeta, and the leucine-based motif in these chimeras was constitutively active resulting in a high spontaneous internalization rate and low expression of the chimeras at the cell surface...

  12. Cell sorting enables interphase fluorescence in situ hybridization detection of low BCR-ABL1 producing stem cells in chronic myeloid leukaemia patients beyond deep molecular remission

    DEFF Research Database (Denmark)

    van Kooten Niekerk, Peter Buur; Petersen, Charlotte Christie; Nyvold, Charlotte Guldborg;

    2014-01-01

    The exact disease state of chronic myeloid leukaemia (CML) patients in deep molecular remission is unknown, because even the most sensitive quantitative reverse transcription polymerase chain reaction (qPCR) methods cannot identify patients prone to relapse after treatment withdrawal. To elucidate......) ), n = 11) using both sensitive qPCR and interphase fluorescence in situ hybridization (iFISH). Despite evaluating fewer cells, iFISH proved superior to mRNA-based qPCR in detecting residual Ph(+) stem cells (P = 0·005), and detected Ph(+) stem- and progenitor cells in 9/10 patients at frequencies of 2......-14%. Moreover, while all qPCR(+) samples also were iFISH(+) , 9/33 samples were qPCR-/iFISH(+) , including all positive samples from MR(4) patients. Our findings show that residual Ph(+) cells are low BCR-ABL1 producers, and that DNA-based methods are required to assess the content of persisting Ph(+) stem...

  13. A Micro Fluorescent Activated Cell Sorter for Astrobiology Applications

    Science.gov (United States)

    Platt, Donald W.; Hoover, Richard B.

    2009-01-01

    A micro-scale Fluorescent Activated Cell Sorter (microFACS) for astrobiology applications is under development. This device is designed to have a footprint of 7 cm x 7 cm x 4 cm and allow live-dead counts and sorting of cells that have fluorescent characteristics from staining. The FACS system takes advantage of microfluidics to create a cell sorter that can fit in the palm of the hand. A micron-scale channel allows cells to pass by a blue diode which causes emission of marker-expressed cells which are detected by a filtered photodetector. A small microcontroller then counts cells and operates high speed valves to select which chamber the cell is collected in (a collection chamber or a waste chamber). Cells with the expressed characteristic will be collected in the collection chamber. This system has been built and is currently being tested. We are also designing a system with integrated MEMS-based pumps and valves for a small and compact unit to fly on small satellite-based biology experiments.

  14. Parallel Algorithms for Neuronal Spike Sorting

    OpenAIRE

    Bergheim, Thomas Stian; Skogvold, Arve Aleksander Nymo

    2011-01-01

    Neurons communicate through electrophysiological signals, which may be recorded using electrodes inserted into living tissue.When a neuron emits a signal, it is referred to as a spike, and an electrode can detect these from multiple neurons.Neuronal spike sorting is the process of classifying the spike activity based on which neuron each spike signal is emitted from.Advances in technology have introduced better recording equipment, which allows the recording of many neurons at the same time.H...

  15. Natural Selection Is a Sorting Process: What Does that Mean?

    Science.gov (United States)

    Price, Rebecca M.

    2013-01-01

    To learn why natural selection acts only on existing variation, students categorize processes as either creative or sorting. This activity helps students confront the misconception that adaptations evolve because species need them.

  16. Interplay of Endosomal pH and Ligand Occupancy in Integrin α5β1 Ubiquitination, Endocytic Sorting, and Cell Migration

    Directory of Open Access Journals (Sweden)

    Dmitri Kharitidi

    2015-10-01

    Full Text Available Membrane trafficking of integrins plays a pivotal role in cell proliferation and migration. How endocytosed integrins are targeted either for recycling or lysosomal delivery is not fully understood. Here, we show that fibronectin (FN binding to α5β1 integrin triggers ubiquitination and internalization of the receptor complex. Acidification facilitates FN dissociation from integrin α5β1 in vitro and in early endosomes, promoting receptor complex deubiquitination by the USP9x and recycling to the cell surface. Depending on residual ligand occupancy of receptors, some α5β1 integrins remain ubiquitinated and are captured by ESCRT-0/I, containing histidine domain-containing protein tyrosine phosphatase (HD-PTP and ubiquitin-associated protein 1 (UBAP1, and are directed for lysosomal proteolysis, limiting receptor downstream signaling and cell migration. Thus, HD-PTP or UBAP1 depletion confers a pro-invasive phenotype. Thus, pH-dependent FN-integrin dissociation and deubiquitination of the activated integrin α5β1 are required for receptor resensitization and cell migration, representing potential targets to modulate tumor invasiveness.

  17. An activated set point of T-cell and monocyte inflammatory networks in recent-onset schizophrenia patients involves both pro- and anti-inflammatory forces

    NARCIS (Netherlands)

    Drexhage, Roosmarijn C.; Hoogenboezem, Thomas A.; Cohen, Dan; Versnel, Marjan A.; Nolen, Willem A.; van Beveren, Nico J. M.; Drexhage, Hemmo A.

    2011-01-01

    We recently described a pro-inflammatory gene expression signature in the monocytes of 60% of patients with recent-onset schizophrenia (SCZ). Here we investigated whether the T-cell system is also in a pro-inflammatory state. A detailed fluorescence-activated cell sorting (FACS) analysis, e. g. of C

  18. Review on Sorting Algorithms A Comparative Study

    Directory of Open Access Journals (Sweden)

    Khalid Suleiman Al-Kharabsheh

    2013-09-01

    Full Text Available There are many popular problems in different practical fields of computer sciences, database applications, Networks and Artificial intelligence. One of these basic operations and problems is sorting algorithm; the sorting problem has attracted a great deal of research. A lot of sorting algorithms has been developed to enhance the performance in terms of computational complexity. there are several factors that must be taken in consideration; time complexity, stability, memory space. Information growth rapidly in our world leads to increase developing sort algorithms .a stable sorting algorithms maintain the relative order of records with equal keys This paper makes a comparison between the Grouping Comparison Sort (GCS and conventional algorithm such as Selection sort, Quick sort, Insertion sort , Merge sort and Bubble sort with respect execution time to show how this algorithm perform reduce execution time.

  19. Hybrid optical and acoustic force based sorting

    Science.gov (United States)

    O'Mahoney, Paul; Brodie, Graham W.; Wang, Han; Demore, Christine E. M.; Cochran, Sandy; Spalding, Gabriel C.; MacDonald, Michael P.

    2014-09-01

    We report the combined use of optical sorting and acoustic levitation to give particle sorting. Differing sizes of microparticles are sorted optically both with and without the aid of acoustic levitation, and the results compared to show that the use of acoustic trapping can increase sorting efficiency. The use of a transparent ultrasonic transducer is also shown to streamline the integration of optics and acoustics. We also demonstrate the balance of optical radiation pressure and acoustic levitation to achieve vertical sorting.

  20. A Heapify Based Parallel Sorting Algorithm

    Directory of Open Access Journals (Sweden)

    M. A.A.A. Hija

    2008-01-01

    Full Text Available Quick sort is a sorting algorithm whose worst case running time is θ(n2 on an input array of n numbers. It is the best practical for sorting because it has the advantage of sorting in place. Problem statement: Behavior of quick sort is complex, we proposed in-place 2m threads parallel heap sort algorithm which had advantage in sorting in place and had better performance than classical sequential quick sort in running time. Approach: The algorithm consisted of several stages, in first stage; it splits input data into two partitions, next stages it did the same partitioning for prior stage which had been spitted until 2 m partitions was reached equal to the number of available processors, finally it used heap sort to sort respectively ordered of non internally sorted partitions in parallel. Results: Results showed the speed of algorithm about double speed of classical Quick sort for a large input size. The number of comparisons needed was reduced significantly. Conclusion: In this study we had been proposed a sorting algorithm that uses less number of comparisons with respect to original quick sort that in turn requires less running time to sort the same input data.

  1. Sorting and selection in posets

    DEFF Research Database (Denmark)

    Daskalakis, Constantinos; Karp, Richard M.; Mossel, Elchanan;

    2011-01-01

    Classical problems of sorting and searching assume an underlying linear ordering of the objects being compared. In this paper, we study these problems in the context of partially ordered sets, in which some pairs of objects are incomparable. This generalization is interesting from a combinatorial...

  2. Molecular characterization of flow-sorted mammalian centromeres

    Energy Technology Data Exchange (ETDEWEB)

    Hamkalo, B.A.; Henschen, A.; Parseghian, M.H. [Univ. of Calfornia, Irvine, CA (United States). Dept. of Molecular Biology and Biochemistry] [and others

    1998-12-31

    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The project involved experiments directed towards developing a molecular characterization of the centromere region of mammalian chromosomes. Attempts to purify this essential chromosomal locus by conventional methods have thus far been unsuccessful. However, preliminary data obtained in collaboration with the National Flow Cytometry Resource (NFCR) showed that it is possible to purify a chromosome fragment that is present in certain cultured mouse cell lines and has all the properties expected of an intact centromere region. To begin sorting this minichromosome for the identification of proteins preferentially associated with centromere regions, standard buffers utilized in chromosome sorting were evaluated for potential effects on maintenance of chromosomal proteins during sorting. The data indicate that the presence of several buffer constituents results in the extraction of all but a few chromosomal proteins. The subsequent use of a magnesium sulfate buffer resulted in the sorting of mouse chromosomes that do not suffer a significant loss of proteins. Several DNA stains were also evaluated for causing protein dissociation, but no significant losses were observed. Although flow-sorted chromosomes have been used extensively for DNA analysis and cloning, this is a pioneering effort by the NFCR, and its collaborators, to exploit chromosome sorting capabilities for the analysis of chromosomal proteins.

  3. Swarm-Based Spatial Sorting

    CERN Document Server

    Amos, Martyn

    2008-01-01

    Purpose: To present an algorithm for spatially sorting objects into an annular structure. Design/Methodology/Approach: A swarm-based model that requires only stochastic agent behaviour coupled with a pheromone-inspired "attraction-repulsion" mechanism. Findings: The algorithm consistently generates high-quality annular structures, and is particularly powerful in situations where the initial configuration of objects is similar to those observed in nature. Research limitations/implications: Experimental evidence supports previous theoretical arguments about the nature and mechanism of spatial sorting by insects. Practical implications: The algorithm may find applications in distributed robotics. Originality/value: The model offers a powerful minimal algorithmic framework, and also sheds further light on the nature of attraction-repulsion algorithms and underlying natural processes.

  4. Development of innovative PCR and fluorescence activated cell sorting methodologies for the detection of a range of foodborne pathogens.

    OpenAIRE

    Martinon, Alice Marie Amelie

    2011-01-01

    peer-reviewed The early and rapid detection of the Enterobacteriaceae, Staphylococcus aureus and Listeria monocytogenes is desirable to prevent foodborne outbreaks. In this thesis, novel molecular and flow cytometric based methods were developed for their qualitative and quantitative detection. For S. aureus and L. monocytogenes, two highly specific primer sets were selected for application in simplex and duplex SYBR Green-based real-time PCR assays. Melting curve analysis confirm...

  5. Single beam atom sorting machine

    International Nuclear Information System (INIS)

    We create two overlapping one-dimensional optical lattices using a single laser beam, a spatial light modulator and a high numerical aperture lens. These lattices have the potential to trap single atoms, and using the dynamic capabilities of the spatial light modulator may shift and sort atoms to a minimum atom-atom separation of 1.52 μm. We show how a simple feedback circuit can compensate for the spatial light modulator's intensity modulation

  6. Swarm-Based Spatial Sorting

    OpenAIRE

    Amos, Martyn; Don, Oliver

    2008-01-01

    Purpose: To present an algorithm for spatially sorting objects into an annular structure. Design/Methodology/Approach: A swarm-based model that requires only stochastic agent behaviour coupled with a pheromone-inspired "attraction-repulsion" mechanism. Findings: The algorithm consistently generates high-quality annular structures, and is particularly powerful in situations where the initial configuration of objects is similar to those observed in nature. Research limitations/implications: Exp...

  7. Sorting Techniques for Plastics Recycling

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    This paper presents the basic principles of three different types of separating methods and a general guideline for choosing the most effective method for sorting plastic mixtures. It also presents the results of the tests carried out for separation of PVC, ABS and PET from different kinds of plastic mixtures in order to improve the grade of the raw input used in mechanical or feedstock recycling.

  8. Mechanisms of cell propulsion by active stresses

    Energy Technology Data Exchange (ETDEWEB)

    Carlsson, A E, E-mail: aec@wustl.edu [Department of Physics, Washington University, Campus Box 1105, One Brookings Drive, St. Louis, MO 63130 (United States)

    2011-07-15

    The mechanisms by which cytoskeletal flows and cell-substrate interactions interact to generate cell motion are explored by using a simplified model of the cytoskeleton as a viscous gel containing active stresses. This model yields explicit general results relating cell speed and traction forces to the distributions of active stress and cell-substrate friction. It is found that (i) the cell velocity is given by a function that quantifies the asymmetry of the active-stress distribution, (ii) gradients in cell-substrate friction can induce motion even when the active stresses are symmetrically distributed, (iii) the traction-force dipole is enhanced by protrusive stresses near the cell edges or contractile stresses near the center of the cell and (iv) the cell velocity depends biphasically on the cell-substrate adhesion strength if active stress is enhanced by adhesion. Specific experimental tests of the calculated dependences are proposed.

  9. The cell biology of T-dependent B cell activation

    DEFF Research Database (Denmark)

    Owens, T; Zeine, R

    1989-01-01

    The requirement that CD4+ helper T cells recognize antigen in association with class II Major Histocompatibility Complex (MHC) encoded molecules constrains T cells to activation through intercellular interaction. The cell biology of the interactions between CD4+ T cells and antigen-presenting cells...

  10. Design of Garbage Sorting Machine

    Directory of Open Access Journals (Sweden)

    Stephen K. Adzimah

    2009-01-01

    Full Text Available Problem statement: Domestic waste collection, sorting and disposal are major problems in many developing countries such as Ghana. It is an undeniable fact that the environment has been engulfed in filth. This filth comprises of the garbage and waste generated in homes, workplace and industrial setups. Most of this waste has found its way into the streets, gutters, in and around the homes, dung hills and worst of all, water bodies, many of which are sources of the drinking water treated at high costs or not treated at all. Approach: Garbage needs to be sorted into various components and each of such components like textile materials, polythene, foodstuffs, metals and glassware would then have to be handled separately at the disposal or recycling site. Such a process required a certain degree of literacy, discipline and certain basic equipment, for example separate collector bins or sorting bags. In the developed world this is not much a problem because every home has different polythene bags into which the various constituents of domestic waste are put right at the generation point. Separate collection bins were also provided at vantage points for the various types of domestic garbage collection. In the developing countries these arrangements have not been feasible because of the level of literacy, lack of appreciation of the problem, non-availability of the different types of polythene bags and poverty. Currently, most garbage collection in the developing countries is done by depositing every thing into a single container from where they are hauled to be dumped in landfills or burned in incinerators. Refuse disposal by land filling requires a sizeable land for the sole purpose of refuse disposal. This may lead to (1: Encumbering large tracks of prime land, which could not be put to other uses (2: Pollution of ground water by the leachate from the landfills (3: Breeding of leaches, rodents, mosquitoes and (4: Generation of strong stench coming

  11. Importance of glycolipid synthesis for butyric acid-induced sensitization to Shiga toxin and intracellular sorting of toxin in A431 cells

    NARCIS (Netherlands)

    Sandvig, K.; Garred, Ø.; van Helvoort, A.; van Meer, G.; van Deurs, B.

    1996-01-01

    The human epidermoid carcinoma cell line A431 becomes highly sensitive to Shiga toxin upon treatment with butyric acid. This strong sensitization (>1000-fold) is accompanied by an increase in the fraction of cell-associated toxin transported to the Golgi apparatus and to the endoplasmic reticulum (E

  12. On the Construction of Sorted Reactive Systems

    DEFF Research Database (Denmark)

    Birkedal, Lars; Debois, Søren; Hildebrandt, Thomas

    2008-01-01

    . Here we present a general construction of sortings. The constructed sortings always sustain the behavioural theory of pure bigraphs (in a precise sense), thus obviating the need to redevelop that theory for each new application. As an example, we recover Milner's local bigraphs as a sorting on pure...

  13. "A Shock of Electricity Just Sort of Goes through My Body": Physical Activity and Embodied Reflexive Practices in Young Female Ballet Dancers

    Science.gov (United States)

    Wellard, Ian; Pickard, Angela; Bailey, Richard

    2007-01-01

    Participation in physical activities, in and out of school, remains heavily influenced by social constructions of gendered behaviour. In addition, the body plays a significant part in the presentation of legitimate performances of physical practice and the construction of a physical "identity". The consequence is that in formalized activities many…

  14. MiR-95 induces proliferation and chemo- or radioresistance through directly targeting sorting nexin1 (SNX1) in non-small cell lung cancer.

    Science.gov (United States)

    Chen, Xiaochun; Chen, Shaomu; Hang, Weijie; Huang, Haitao; Ma, Haitao

    2014-06-01

    MicroRNAs are emerging as a class of small regulatory RNAs whose specific roles and significant functions in the majority of carcinomas have yet to be entirely illustrated. The aim of this study is to explore the effect of miR-95 and determine whether miR-95 could be a potential therapeutic target for human non-small cell lung cancer. First of all, our study showed that miR-95 was highly expressed in both NSCLC cell lines (compared with normal cells) and tumor tissues (compared with corresponding normal tissues), whereas the protein level of SNX1 was downregulated in NSCLC cell lines. Next, we found that ectopic overexpression of miR-95 in A549 or H226 contributed to tumor growth in xenograft mouse models. In addition, the results also indicated that upregulation of miR-95 could significantly enhance the susceptibilities of NSCLC cells to chemo- or radiotherapy. Furthermore, using the luciferase reporter, we demonstrated that SNX1 is a direct target of miR-95. Meanwhile, overexpression of SNX1 could abrogate the growth of NSCLC cells induced by miR-95. Taken together, these results suggest that miR-95 functions as an oncogene role in NSCLC cells by directly targeting SNX1. PMID:24835695

  15. Differentiation of immortal cells inhibits telomerase activity.

    OpenAIRE

    Sharma, H W; Sokoloski, J A; Perez, J.R.; Maltese, J Y; Sartorelli, A C; Stein, C A; Nichols, G; Khaled, Z.; Telang, N T; Narayanan, R.

    1995-01-01

    Telomerase, a ribonucleic acid-protein complex, adds hexameric repeats of 5'-TTAGGG-3' to the ends of mammalian chromosomal DNA (telomeres) to compensate for the progressive loss that occurs with successive rounds of DNA replication. Although somatic cells do not express telomerase, germ cells and immortalized cells, including neoplastic cells, express this activity. To determine whether the phenotypic differentiation of immortalized cells is linked to the regulation of telomerase activity, t...

  16. How does the Shift-insertion sort behave when the sorting elements follow a Normal distribution?

    CERN Document Server

    Pal, Mita; Mahanti, N C

    2012-01-01

    The present paper examines the behavior of Shift-insertion sort (insertion sort with shifting) for normal distribution inputs and is in continuation of our earlier work on this new algorithm for discrete distribution inputs, namely, negative binomial. Shift insertion sort is found more sensitive for main effects but not for all interaction effects compared to conventional insertion sort.

  17. Telomerase activity in plasma cell dyscrasias

    OpenAIRE

    Xu, D; Zheng, C.; Bergenbrant, S; Holm, G; Björkholm, M.; Yi, Q; Gruber, A

    2001-01-01

    Activation of telomerase is essential for in vitro cellular immortalization and tumorigenesis. In the present study, we investigated telomerase activation and its implications in plasma cell dyscrasias including monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma (MM) and plasma cell leukaemia (PCL). All 5 patients with MGUS exhibited normal levels of telomerase activity in their plasma cells. Elevated telomerase activity was found in the samples from 21/27 patients wi...

  18. Proteins interacting with Membranes: Protein Sorting and Membrane Shaping

    Science.gov (United States)

    Callan-Jones, Andrew

    2015-03-01

    Membrane-bound transport in cells requires generating membrane curvature. In addition, transport is selective, in order to establish spatial gradients of membrane components in the cell. The mechanisms underlying cell membrane shaping by proteins and the influence of curvature on membrane composition are active areas of study in cell biophysics. In vitro approaches using Giant Unilamellar Vesicles (GUVs) are a useful tool to identify the physical mechanisms that drive sorting of membrane components and membrane shape change by proteins. I will present recent work on the curvature sensing and generation of IRSp53, a protein belonging to the BAR family, whose members, sharing a banana-shaped backbone, are involved in endocytosis. Pulling membrane tubes with 10-100 nm radii from GUVs containing encapsulated IRSp53 have, unexpectedly, revealed a non-monotonic dependence of the protein concentration on the tube as a function of curvature. Experiments also show that bound proteins alter the tube mechanics and that protein phase separation along the tube occurs at low tensions. I will present accompanying theoretical work that can explain these findings based on the competition between the protein's intrinsic curvature and the effective rigidity of a membrane-protein patch.

  19. Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting: Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4{sup +} T cells compared

    Energy Technology Data Exchange (ETDEWEB)

    Ford, A.L.; Goodsall, A.L.; Sedgwick, J.D. [Centenary Institute of Cancer Medicine and Cell Biology, Sydney (Australia)] [and others

    1995-05-01

    Ramified microglia in the adult central nervous system (CNS) are the principal glial element up-regulating MHC class I and II expression in response to inflammatory events or neuronal damage. A proportion of these cells also express MHC class II constitutively in the normal CNS. The role of microglia as APCs for CD4{sup +} cells extravasating into the CNS remains undefined. In this study, using irradiation bone marrow chimeras in CD45-congenic rats, the phenotype CD45{sup low}CD11b/c{sup +} is shown to identify microglial cells specifically within the CNS. Highly purified populations of microglia and nonmicroglial but CNS-associated macrophages (CD45{sup high}CD11b/c{sup +}) have been obtained directly from the adult CNS, by using flow cytometric sorting. Morphologically, freshly isolated microglia vs other CNS macrophages are quite distinct. Of the two populations recovered from the normal CNS, it is the minority CD45{sup high}CD11 b/c{sup +} transitional macrophage population, and not microglia, that is the effective APC for experimental autoimmune encephalomyelitis-inducing CD4{sup +} myelin basic protein (MBP)-reactive T cells. CD45{sup high}CD11b/c{sup +} CNS macrophages also stimulate MBP-reactive T cells without addition of MBP to culture suggesting presentation of endogenous Ag. This is the first study in which microglia vs other CNS macrophages have been analyzed for APC ability directly from the CNS, with substantial cross-contamination between the two populations eliminated. The heterogeneity of these populations in terms of APC function is clearly demonstrated. Evidence is still lacking that adult CNS microglia have the capacity to interact with and stimulate CD4{sup +} T cells to proliferate or secrete IL-2. 60 refs., 6 figs., 1 tab.

  20. Cell division activity during apical hook development

    NARCIS (Netherlands)

    Raz, V.; Koornneef, M.

    2001-01-01

    Growth during plant development is predominantly governed by the combined activities of cell division and cell elongation. The relative contribution of both activities controls the growth of a tissue. A fast change in growth is exhibited at the apical hypocotyl of etiolated seedlings where cells gro

  1. Marital Sorting and Parental Wealth

    OpenAIRE

    Kerwin Kofi Charles; Erik Hurst; Alexandra Killewald

    2011-01-01

    Using data from the Panel Study of Income Dynamics (PSID), this paper studies the degree to which spouses sort in the marriage market on the basis of parental wealth. We estimate a variety of models, including transition matrices, OLS and TSLS models to deal with measurement error in wealth reports. Our various results show that men and women in the U.S. marry spouses whose parents have wealth similar to that of their own parents; and are very unlikely to marry persons from very different par...

  2. Rapid Evaluation of Mutant Exon-11 in c-kit in a Recurrent MCT Case Using CD117 Immunocytofluorescence, FACS-Cell Sorting, and PCR

    Directory of Open Access Journals (Sweden)

    Dettachai Ketpun

    2013-01-01

    Full Text Available A 13-year-old, poodle-mixed, male dog was referred to the oncology unit in our faculty’s small animal teaching hospital with the problem of rapid recurrent MCT. The owner and the veterinarian would like to use a tyrosine kinase inhibitor (TKI for the dog. Therefore, fine-needle aspiration (FNA was performed to collect the MCT cells and these cells were submitted to our laboratory for the detection of internal-tandem-duplicated (ITD mutation of exon-11 in c-kit, prior to the treatment. The aim of this paper is to demonstrate the use of combinatorial protocol for the rapid evaluation of ITD mutation in MCT cells harvested by FNA. However, there was no ITD-mutant exon-11 that had been observed in this case.

  3. A Framework for the Comparative Assessment of Neuronal Spike Sorting Algorithms towards More Accurate Off-Line and On-Line Microelectrode Arrays Data Analysis.

    Science.gov (United States)

    Regalia, Giulia; Coelli, Stefania; Biffi, Emilia; Ferrigno, Giancarlo; Pedrocchi, Alessandra

    2016-01-01

    Neuronal spike sorting algorithms are designed to retrieve neuronal network activity on a single-cell level from extracellular multiunit recordings with Microelectrode Arrays (MEAs). In typical analysis of MEA data, one spike sorting algorithm is applied indiscriminately to all electrode signals. However, this approach neglects the dependency of algorithms' performances on the neuronal signals properties at each channel, which require data-centric methods. Moreover, sorting is commonly performed off-line, which is time and memory consuming and prevents researchers from having an immediate glance at ongoing experiments. The aim of this work is to provide a versatile framework to support the evaluation and comparison of different spike classification algorithms suitable for both off-line and on-line analysis. We incorporated different spike sorting "building blocks" into a Matlab-based software, including 4 feature extraction methods, 3 feature clustering methods, and 1 template matching classifier. The framework was validated by applying different algorithms on simulated and real signals from neuronal cultures coupled to MEAs. Moreover, the system has been proven effective in running on-line analysis on a standard desktop computer, after the selection of the most suitable sorting methods. This work provides a useful and versatile instrument for a supported comparison of different options for spike sorting towards more accurate off-line and on-line MEA data analysis. PMID:27239191

  4. Lhcb transcription is coordinated with cell size and chlorophyll accumulation. Studies on fluorescence-activated, cell-sorter-purified single cells from wild-type and immutans Arabidopsis thaliana

    Energy Technology Data Exchange (ETDEWEB)

    Meehan, L.; Harkins, K.; Rodermel, S. [Iowa State Univ., Ames, IA (United States)] [and others

    1996-11-01

    To study the mechanisms that integrate pigment and chlorophyll a/b-binding apoprotein biosynthesis during light-harvesting complex II assembly, we have examined {beta}-glucuronidase (GUS) enzyme activities, cell-sorting-separated single cells sizes in fluorescence activated, cell-sorting-separated single cells from transgenic Arabidopsis thaliana wild-type and immutans variegation mutant plants that express an Lhcb (photosystem II chlorophyll a/b-binding polypeptide gene)/GUS promoter fusion. We found that GUS activities are positively correlated with chlorophyll content and cell size in green cells from the control and immutans plants, indicating that Lhcb gene transcription is coordinated with cell size in this species. Compared with the control plants, however, chlorophyll production is enhanced in the green cells of immutans; this may represent part of a strategy to maximize photosynthesis in the white sectors of the mutant. Lhcb transcription is significantly higher in pure-white cells of the transgenic immutans plants than in pure-white cells from norflurazon-treated, photooxidized A. thaliana leaves. This suggests that immutans partially uncouples Lhcb transcription from its normal dependence on chlorophyll accumulation and chloroplast development. We conclude that immutans may play a role in regulating Lhcb transcription, and may be a key component in the signal transduction pathways that control chloroplast biogenesis. 58 refs., 5 figs., 2 tabs.

  5. Sorting and Selection in Posets

    CERN Document Server

    Daskalakis, Constantinos; Mossel, Elchanan; Riesenfeld, Samantha; Verbin, Elad

    2007-01-01

    Classical problems of sorting and searching assume an underlying linear ordering of the objects being compared. In this paper, we study a more general setting, in which some pairs of objects are incomparable. This generalization is relevant in applications related to rankings in sports, college admissions, or conference submissions. It also has potential applications in biology, such as comparing the evolutionary fitness of different strains of bacteria, or understanding input-output relations among a set of metabolic reactions or the causal influences among a set of interacting genes or proteins. Our results improve and extend results from two decades ago of Faigle and Tur\\'{a}n. A measure of complexity of a partially ordered set (poset) is its width. Our algorithms obtain information about a poset by queries that compare two elements. We present an algorithm that sorts, i.e. completely identifies, a width w poset of size n and has query complexity O(wn + nlog(n)), which is within a constant factor of the in...

  6. The method of sorting out perivascular stem cells from human adipose tissue through flow cytometry%流式分析人脂肪组织中血管周围干细胞含量的方法探究

    Institute of Scientific and Technical Information of China (English)

    徐峰; 刘舒云; 王鑫; 彭江; 卢世璧; 袁玫; 许文静; 郭全义

    2015-01-01

    目的建立人脂肪组织中分离血管周围干细胞(PSCs)的方法,并研究其在脂肪组织细胞中所占的比例,为血管周围干细胞作为骨和软骨组织工程新的种子细胞奠定基础。  方法取人的脂肪组织分别用 I 型胶原酶和 II 型胶原酶消化得到血管基质成分(SVF),用细胞计数仪及流式细胞仪检测 SVF 中细胞密度、活细胞比例和 PSCs 细胞所占的比例。  结果用细胞计数仪分析得出用 II 型胶原酶消化脂肪组织所得到的 SVF 中活细胞比例更高,且差异具有统计学意义(P  结论使用 II 型胶原酶消化脂肪组织可以得到更多的血管周围干细胞 PSCs,其在脂肪组织中的含量可以满足骨和软骨损伤后自体细胞移植修复的需要。%Objective To establish the method of sorting out perivascular stem cells (PSCs) from human adipose tissue and study the proportion of these cells in adipose tissue cells. This research is to explore new seed cells for the bone and cartilage tissue engineering. Methods Stromal vascular fraction (SVF) was got from human adipose tissue that was digested by collagenase type I or collagenase type II. The cell density, proportion of living cells and proportion of PSCs in SVF were tested by the cell count and flow cytometry (FCM). Results The proportion of living cells in SVF digested by collagenase type II was much higher through analyzing by the cell count and the difference was statistically significant (P Conclusion A higher amount of PSCs can be got from human adipose digested by collagenase type II, and the content of PSCs in the adipose tissue can satisfy the needs of autologous cell transplantation for the bone and cartilage repair.

  7. Digital Sorting of Pure Cell Populations Enables Unambiguous Genetic Analysis of Heterogeneous Formalin-Fixed Paraffin-Embedded Tumors by Next Generation Sequencing

    OpenAIRE

    Chiara Bolognesi; Claudio Forcato; Genny Buson; Francesca Fontana; Chiara Mangano; Anna Doffini; Valeria Sero; Rossana Lanzellotto; Giulio Signorini; Alex Calanca; Maximilian Sergio; Rita Romano; Stefano Gianni; Gianni Medoro; Giuseppe Giorgini

    2016-01-01

    Precision medicine in oncology requires an accurate characterization of a tumor molecular profile for patient stratification. Though targeted deep sequencing is an effective tool to detect the presence of somatic sequence variants, a significant number of patient specimens do not meet the requirements needed for routine clinical application. Analysis is hindered by contamination of normal cells and inherent tumor heterogeneity, compounded with challenges of dealing with minute amounts of tiss...

  8. Digital Sorting of Pure Cell Populations Enables Unambiguous Genetic Analysis of Heterogeneous Formalin-Fixed Paraffin-Embedded Tumors by Next Generation Sequencing.

    Science.gov (United States)

    Bolognesi, Chiara; Forcato, Claudio; Buson, Genny; Fontana, Francesca; Mangano, Chiara; Doffini, Anna; Sero, Valeria; Lanzellotto, Rossana; Signorini, Giulio; Calanca, Alex; Sergio, Maximilian; Romano, Rita; Gianni, Stefano; Medoro, Gianni; Giorgini, Giuseppe; Morreau, Hans; Barberis, Massimo; Corver, Willem E; Manaresi, Nicolò

    2016-01-01

    Precision medicine in oncology requires an accurate characterization of a tumor molecular profile for patient stratification. Though targeted deep sequencing is an effective tool to detect the presence of somatic sequence variants, a significant number of patient specimens do not meet the requirements needed for routine clinical application. Analysis is hindered by contamination of normal cells and inherent tumor heterogeneity, compounded with challenges of dealing with minute amounts of tissue and DNA damages common in formalin-fixed paraffin-embedded (FFPE) specimens. Here we present an innovative workflow using DEPArray™ system, a microchip-based digital sorter to achieve 100%-pure, homogenous subpopulations of cells from FFPE samples. Cells are distinguished by fluorescently labeled antibodies and DNA content. The ability to address tumor heterogeneity enables unambiguous determination of true-positive sequence variants, loss-of-heterozygosity as well as copy number variants. The proposed strategy overcomes the inherent trade-offs made between sensitivity and specificity in detecting genetic variants from a mixed population, thus rescuing for analysis even the smaller clinical samples with low tumor cellularity. PMID:26864208

  9. Digital Sorting of Pure Cell Populations Enables Unambiguous Genetic Analysis of Heterogeneous Formalin-Fixed Paraffin-Embedded Tumors by Next Generation Sequencing

    Science.gov (United States)

    Bolognesi, Chiara; Forcato, Claudio; Buson, Genny; Fontana, Francesca; Mangano, Chiara; Doffini, Anna; Sero, Valeria; Lanzellotto, Rossana; Signorini, Giulio; Calanca, Alex; Sergio, Maximilian; Romano, Rita; Gianni, Stefano; Medoro, Gianni; Giorgini, Giuseppe; Morreau, Hans; Barberis, Massimo; Corver, Willem E.; Manaresi, Nicolò

    2016-01-01

    Precision medicine in oncology requires an accurate characterization of a tumor molecular profile for patient stratification. Though targeted deep sequencing is an effective tool to detect the presence of somatic sequence variants, a significant number of patient specimens do not meet the requirements needed for routine clinical application. Analysis is hindered by contamination of normal cells and inherent tumor heterogeneity, compounded with challenges of dealing with minute amounts of tissue and DNA damages common in formalin-fixed paraffin-embedded (FFPE) specimens. Here we present an innovative workflow using DEPArray™ system, a microchip-based digital sorter to achieve 100%-pure, homogenous subpopulations of cells from FFPE samples. Cells are distinguished by fluorescently labeled antibodies and DNA content. The ability to address tumor heterogeneity enables unambiguous determination of true-positive sequence variants, loss-of-heterozygosity as well as copy number variants. The proposed strategy overcomes the inherent trade-offs made between sensitivity and specificity in detecting genetic variants from a mixed population, thus rescuing for analysis even the smaller clinical samples with low tumor cellularity. PMID:26864208

  10. Energy efficient data sorting using standard sorting algorithms

    KAUST Repository

    Bunse, Christian

    2011-01-01

    Protecting the environment by saving energy and thus reducing carbon dioxide emissions is one of todays hottest and most challenging topics. Although the perspective for reducing energy consumption, from ecological and business perspectives is clear, from a technological point of view, the realization especially for mobile systems still falls behind expectations. Novel strategies that allow (software) systems to dynamically adapt themselves at runtime can be effectively used to reduce energy consumption. This paper presents a case study that examines the impact of using an energy management component that dynamically selects and applies the "optimal" sorting algorithm, from an energy perspective, during multi-party mobile communication. Interestingly, the results indicate that algorithmic performance is not key and that dynamically switching algorithms at runtime does have a significant impact on energy consumption. © Springer-Verlag Berlin Heidelberg 2011.

  11. Layers in sorting practices: Sorting out patients with potential cancer

    DEFF Research Database (Denmark)

    Møller, Naja Holten; Bjørn, Pernille

    2011-01-01

    are planned and pre-booked in order to manage patient trajectories. They are different from typical medical guidelines because they combine both administrative and clinical prescriptions. A key issue related to the enactment of a standardized cancer pathway concerns the decision to initiate a pathway...... in the pre-diagnostic work as being structured in layers of the interrelated, iterative practices of constructing, organizing, re-organizing, and merging the multiple queues within which each patient is simultaneously situated. We find that the ordering of patients in queues is guided by the formal sorting...... is a collaborative process of merging multiple queues while continuously deciding whether or not a patient’s symptoms point to potential cancer....

  12. Mechanically robust microfluidics and bulk wave acoustics to sort microparticles

    Science.gov (United States)

    Dauson, Erin R.; Gregory, Kelvin B.; Greve, David W.; Healy, Gregory P.; Oppenheim, Irving J.

    2016-04-01

    Sorting microparticles (or cells, or bacteria) is significant for scientific, medical and industrial purposes. Research groups have used lithium niobate SAW devices to produce standing waves, and then to align microparticles at the node lines in polydimethylsiloxane (PDMS, silicone) microfluidic channels. The "tilted angle" (skewed) configuration is a recent breakthrough producing particle trajectories that cross multiple node lines, making it practical to sort particles. However, lithium niobate wafers and PDMS microfluidic channels are not mechanically robust. We demonstrate "tilted angle" microparticle sorting in novel devices that are robust, rapidly prototyped, and manufacturable. We form our microfluidic system in a rigid polymethyl methacrylate (PMMA, acrylic) prism, sandwiched by lead-zirconium-titanate (PZT) wafers, operating in through-thickness mode with inertial backing, that produce standing bulk waves. The overall configuration is compact and mechanically robust, and actuating PZT wafers in through-thickness mode is highly efficient. Moving to this novel configuration introduced new acoustics questions involving internal reflections, but we show experimental images confirming the intended nodal geometry. Microparticles in "tilted angle" devices display undulating trajectories, where deviation from the straight path increases with particle diameter and with excitation voltage to create the mechanism by which particles are sorted. We show a simplified analytical model by which a "phase space" is constructed to characterize effective particle sorting, and we compare our experimental data to the predictions from that simplified model; precise correlation is not expected and is not observed, but the important physical trends from the model are paralleled in the measured particle trajectories.

  13. Fast Parallel Sorting Algorithms on GPUs

    Directory of Open Access Journals (Sweden)

    Bilal Jan

    2012-12-01

    Full Text Available This paper presents a comparative analysis of the three widely used parallel sorting algorithms: Odd-Even sort, Rank sort and Bitonic sort in terms of sorting rate, sorting time and speed-up on CPU anddifferent GPU architectures. Alongside we have implemented novel parallel algorithm: min-max butterflynetwork, for finding minimum and maximum in large data sets. All algorithms have been implementedexploiting data parallelism model, for achieving high performance, as available on multi-core GPUsusing the OpenCL specification. Our results depicts minimum speed-up19x of bitonic sort against oddevensorting technique for small queue sizes on CPU and maximum of 2300x speed-up for very largequeue sizes on Nvidia Quadro 6000 GPU architecture. Our implementation of full-butterfly networksorting results in relatively better performance than all of the three sorting techniques: bitonic, odd-evenand rank sort. For min-max butterfly network, our findings report high speed-up of Nvidia quadro 6000GPU for high data set size reaching 224 with much lower sorting time.

  14. Recyclable Waste Paper Sorting Using Template Matching

    Science.gov (United States)

    Osiur Rahman, Mohammad; Hussain, Aini; Scavino, Edgar; Hannan, M. A.; Basri, Hassan

    This paper explores the application of image processing techniques in recyclable waste paper sorting. In recycling, waste papers are segregated into various grades as they are subjected to different recycling processes. Highly sorted paper streams will facilitate high quality end products, and save processing chemicals and energy. Since 1932 to 2009, different mechanical and optical paper sorting methods have been developed to fill the demand of paper sorting. Still, in many countries including Malaysia, waste papers are sorted into different grades using manual sorting system. Due to inadequate throughput and some major drawbacks of mechanical paper sorting systems, the popularity of optical paper sorting systems is increased. Automated paper sorting systems offer significant advantages over human inspection in terms of fatigue, throughput, speed, and accuracy. This research attempts to develop a smart vision sensing system that able to separate the different grades of paper using Template Matching. For constructing template database, the RGB components of the pixel values are used to construct RGBString for template images. Finally, paper object grade is identified based on the maximum occurrence of a specific template image in the search image. The outcomes from the experiment in classification for White Paper, Old Newsprint Paper and Old Corrugated Cardboard are 96%, 92% and 96%, respectively. The remarkable achievement obtained with the method is the accurate identification and dynamic sorting of all grades of papers using simple image processing techniques.

  15. Receptorligand sorting along the endocytic pathway

    CERN Document Server

    Linderman, Jennifer J

    1989-01-01

    This research monograph focuses on a biomolecular separation process that occurs within most cells. Two types of molecules, receptors and ligands, are separated and routed along different intracellular pathways; this is a critical step in the process of receptor-mediated endocytosis. The development of an understanding of the basic mechanisms of this separation process is presented, with an emphasis on discovering the fundamental and measurable parameters that influence the event. Mathematical models of sorting are evaluated to predict the range of possible outcomes. These are compared with a variety of experimental data on different receptor/ligand systems. In addition, the influence of the separation on overall receptor/ligand processing dynamics is discussed. The book is intended for both biomathematicians and biologists. It is not necessary to understand the details of the model equations and their solution in order to test the models experimentally. The analysis suggests experiments that might be done to...

  16. Active cell mechanics: Measurement and theory.

    Science.gov (United States)

    Ahmed, Wylie W; Fodor, Étienne; Betz, Timo

    2015-11-01

    Living cells are active mechanical systems that are able to generate forces. Their structure and shape are primarily determined by biopolymer filaments and molecular motors that form the cytoskeleton. Active force generation requires constant consumption of energy to maintain the nonequilibrium activity to drive organization and transport processes necessary for their function. To understand this activity it is necessary to develop new approaches to probe the underlying physical processes. Active cell mechanics incorporates active molecular-scale force generation into the traditional framework of mechanics of materials. This review highlights recent experimental and theoretical developments towards understanding active cell mechanics. We focus primarily on intracellular mechanical measurements and theoretical advances utilizing the Langevin framework. These developing approaches allow a quantitative understanding of nonequilibrium mechanical activity in living cells. This article is part of a Special Issue entitled: Mechanobiology.

  17. Fixing the Sorting Algorithm for Android, Java and Python

    NARCIS (Netherlands)

    Gouw, C.P.T. de; Boer, F.S. de

    2015-01-01

    Tim Peters developed the Timsort hybrid sorting algorithm in 2002. TimSort was first developed for Python, a popular programming language, but later ported to Java (where it appears as java.util.Collections.sort and java.util.Arrays.sort). TimSort is today used as the default sorting algorithm in Ja

  18. Learning sorting algorithms through visualization construction

    Science.gov (United States)

    Cetin, Ibrahim; Andrews-Larson, Christine

    2016-01-01

    Recent increased interest in computational thinking poses an important question to researchers: What are the best ways to teach fundamental computing concepts to students? Visualization is suggested as one way of supporting student learning. This mixed-method study aimed to (i) examine the effect of instruction in which students constructed visualizations on students' programming achievement and students' attitudes toward computer programming, and (ii) explore how this kind of instruction supports students' learning according to their self-reported experiences in the course. The study was conducted with 58 pre-service teachers who were enrolled in their second programming class. They expect to teach information technology and computing-related courses at the primary and secondary levels. An embedded experimental model was utilized as a research design. Students in the experimental group were given instruction that required students to construct visualizations related to sorting, whereas students in the control group viewed pre-made visualizations. After the instructional intervention, eight students from each group were selected for semi-structured interviews. The results showed that the intervention based on visualization construction resulted in significantly better acquisition of sorting concepts. However, there was no significant difference between the groups with respect to students' attitudes toward computer programming. Qualitative data analysis indicated that students in the experimental group constructed necessary abstractions through their engagement in visualization construction activities. The authors of this study argue that the students' active engagement in the visualization construction activities explains only one side of students' success. The other side can be explained through the instructional approach, constructionism in this case, used to design instruction. The conclusions and implications of this study can be used by researchers and

  19. Cell death sensitization of leukemia cells by opioid receptor activation

    Science.gov (United States)

    Friesen, Claudia; Roscher, Mareike; Hormann, Inis; Fichtner, Iduna; Alt, Andreas; Hilger, Ralf A.; Debatin, Klaus-Michael; Miltner, Erich

    2013-01-01

    Cyclic AMP (cAMP) regulates a number of cellular processes and modulates cell death induction. cAMP levels are altered upon stimulation of specific G-protein-coupled receptors inhibiting or activating adenylyl cyclases. Opioid receptor stimulation can activate inhibitory Gi-proteins which in turn block adenylyl cyclase activity reducing cAMP. Opioids such as D,L-methadone induce cell death in leukemia cells. However, the mechanism how opioids trigger apoptosis and activate caspases in leukemia cells is not understood. In this study, we demonstrate that downregulation of cAMP induced by opioid receptor activation using the opioid D,L-methadone kills and sensitizes leukemia cells for doxorubicin treatment. Enhancing cAMP levels by blocking opioid-receptor signaling strongly reduced D,L-methadone-induced apoptosis, caspase activation and doxorubicin-sensitivity. Induction of cell death in leukemia cells by activation of opioid receptors using the opioid D,L-methadone depends on critical levels of opioid receptor expression on the cell surface. Doxorubicin increased opioid receptor expression in leukemia cells. In addition, the opioid D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux in leukemia cells, suggesting that the opioid D,L-methadone as well as doxorubicin mutually increase their cytotoxic potential. Furthermore, we found that opioid receptor activation using D,L-methadone alone or in addition to doxorubicin inhibits tumor growth significantly in vivo. These results demonstrate that opioid receptor activation via triggering the downregulation of cAMP induces apoptosis, activates caspases and sensitizes leukemia cells for doxorubicin treatment. Hence, opioid receptor activation seems to be a promising strategy to improve anticancer therapies. PMID:23633472

  20. Lactobacilli Differentially Activate Natural Killer Cells

    DEFF Research Database (Denmark)

    Fink, Lisbeth Nielsen; Christensen, Hanne Risager; Frøkiær, Hanne

    bacteria on regulatory functions of NK-cells. Here, we have investigated how human gut flora-derived non-pathogenic lactobacilli affect NK cells in vitro, by measuring proliferation and IFN-gamma production of human peripheral blood NK cells upon bacterial stimulation. CD3-CD56+ NK cells were isolated from...... having engulfed bacteria, stimulated the growth of the NK cells. In contrast, a Lactobacillus paracasei strain caused the NK cells to proliferate only in the presence of monocytes. These results demonstrate that various lactobacilli have the capacity to activate NK cells in vitro, in a monocyte dependent...

  1. Measurement of myeloid cell immune suppressive activity.

    Science.gov (United States)

    Dolcetti, Luigi; Peranzoni, Elisa; Bronte, Vincenzo

    2010-11-01

    This unit presents simple methods to assess the immunosuppressive properties of immunoregulatory cells of myeloid origin, such as myeloid-derived suppressor cells (MDSCs), both in vitro and in vivo. These methods are general and could be adapted to test the impact of different suppressive populations on T cell activation, proliferation, and cytotoxic activity; moreover they could be useful to assess the influence exerted on immune suppressive pathways by genetic modifications, chemical inhibitors, and drugs.

  2. Activated protein C modulates the proinflammatory activity of dendritic cells

    Directory of Open Access Journals (Sweden)

    Matsumoto T

    2015-05-01

    Full Text Available Takahiro Matsumoto,1,2* Yuki Matsushima,1* Masaaki Toda,1 Ziaurahman Roeen,1 Corina N D'Alessandro-Gabazza,1,5 Josephine A Hinneh,1 Etsuko Harada,1,3 Taro Yasuma,4 Yutaka Yano,4 Masahito Urawa,1,5 Tetsu Kobayashi,5 Osamu Taguchi,5 Esteban C Gabazza1 1Department of Immunology, Mie University Graduate School of Medicine, Tsu, Mie Prefecture, 2BONAC Corporation, BIO Factory 4F, Fukuoka, 3Iwade Research Institute of Mycology, 4Department of Endocrinology, Diabetes and Metabolism, 5Department of Pulmonary and Critical Care Medicine, Mie University Graduate School of Medicine, Tsu, Mie Prefecture, Japan *These authors contributed equally to this work Background: Previous studies have demonstrated the beneficial activity of activated protein C in allergic diseases including bronchial asthma and rhinitis. However, the exact mechanism of action of activated protein C in allergies is unclear. In this study, we hypothesized that pharmacological doses of activated protein C can modulate allergic inflammation by inhibiting dendritic cells. Materials and methods: Dendritic cells were prepared using murine bone marrow progenitor cells and human peripheral monocytes. Bronchial asthma was induced in mice that received intratracheal instillation of ovalbumin-pulsed dendritic cells. Results: Activated protein C significantly increased the differentiation of tolerogenic plasmacytoid dendritic cells and the secretion of type I interferons, but it significantly reduced lipopolysaccharide-mediated maturation and the secretion of inflammatory cytokines in myeloid dendritic cells. Activated protein C also inhibited maturation and the secretion of inflammatory cytokines in monocyte-derived dendritic cells. Activated protein C-treated dendritic cells were less effective when differentiating naïve CD4 T-cells from Th1 or Th2 cells, and the cellular effect of activated protein C was mediated by its receptors. Mice that received adoptive transfer of activated protein C

  3. Active Gel Model of Amoeboid Cell Motility

    CERN Document Server

    Callan-Jones, A C

    2013-01-01

    We develop a model of amoeboid cell motility based on active gel theory. Modeling the motile apparatus of a eukaryotic cell as a confined layer of finite length of poroelastic active gel permeated by a solvent, we first show that, due to active stress and gel turnover, an initially static and homogeneous layer can undergo a contractile-type instability to a polarized moving state in which the rear is enriched in gel polymer. This agrees qualitatively with motile cells containing an actomyosin-rich uropod at their rear. We find that the gel layer settles into a steadily moving, inhomogeneous state at long times, sustained by a balance between contractility and filament turnover. In addition, our model predicts an optimal value of the gel-susbstrate adhesion leading to maximum layer speed, in agreement with cell motility assays. The model may be relevant to motility of cells translocating in complex, confining environments that can be mimicked experimentally by cell migration through microchannels.

  4. Engineering a Cache-Oblivious Sorting Algorithm

    DEFF Research Database (Denmark)

    Brodal, Gerth Stølting; Fagerberg, Rolf; Vinther, Kristoffer

    2007-01-01

    This paper is an algorithmic engineering study of cache-oblivious sorting. We investigate by empirical methods a number of implementation issues and parameter choices for the cache-oblivious sorting algorithm Lazy Funnelsort, and compare the final algorithm with Quicksort, the established standard...

  5. Data Sorting Using Graphics Processing Units

    Directory of Open Access Journals (Sweden)

    M. J. Mišić

    2012-06-01

    Full Text Available Graphics processing units (GPUs have been increasingly used for general-purpose computation in recent years. The GPU accelerated applications are found in both scientific and commercial domains. Sorting is considered as one of the very important operations in many applications, so its efficient implementation is essential for the overall application performance. This paper represents an effort to analyze and evaluate the implementations of the representative sorting algorithms on the graphics processing units. Three sorting algorithms (Quicksort, Merge sort, and Radix sort were evaluated on the Compute Unified Device Architecture (CUDA platform that is used to execute applications on NVIDIA graphics processing units. Algorithms were tested and evaluated using an automated test environment with input datasets of different characteristics. Finally, the results of this analysis are briefly discussed.

  6. Activity driven fluctuations in living cells

    CERN Document Server

    Fodor, É; Gov, N S; Visco, P; Weitz, D A; van Wijland, F

    2015-01-01

    We propose a model for the dynamics of a probe embedded in a living cell, where both thermal fluctuations and nonequilibrium activity coexist. The model is based on a confining harmonic potential describing the elastic cytoskeletal matrix, which undergoes random active hops as a result of the nonequilibrium rearrangements within the cell. We describe the probe's statistics and we bring forth quantities affected by the nonequilibrium activity. We find an excellent agreement between the predictions of our model and experimental results for tracers inside living cells. Finally, we exploit our model to arrive at quantitative predictions for the parameters characterizing nonequilibrium activity, such as the typical time scale of the activity and the amplitude of the active fluctuations.

  7. Encapsulation of Biocatalysts (Cell/Enzyme) with High Retaining Activity

    OpenAIRE

    Liu, Tao

    2015-01-01

    Enzymes are always considered as great gifts from nature since they are holding brilliant properties, including high activity, selectivity and specificity. Nowadays, a variety of enzymes have been applied to many industry processes. However, challenges are still needed to be addressed while applying enzymes. It is worth to point out that enzymes are sensitive to the change of ambient conditions. Most of enzymes are unstable and work under certain sort of temperature and pH conditions. Since e...

  8. 考虑任务排序策略的舰船建造车间虚拟制造单元动态调度%Virtual manufacturing cell dynamic scheduling of ship construction workshop considering the task sorting strategy

    Institute of Scientific and Technical Information of China (English)

    韩文民; 孙晓梅; 孔鹏; 吕洁

    2014-01-01

    为提高舰船制造系统作业调度的柔性和效率,从车间层的作业计划角度出发,研究周期驱动条件下的虚拟制造单元多阶段动态调度问题并构建了动态调度数学模型。模型中考虑了加工任务动态需求、设备加工能力、负荷平衡、同类设备有多台的情况且提出共享资源协调排序策略,以实现最大完工时间和总物料运输距离之和最小化的目标。运用改进蚁群算法与启发式规则的混合算法进行求解。通过某船厂的实际生产数据验证了虚拟单元动态调度方法的可行性和有效性。%In order to improve the flexibility and efficiency of shipbuilding production scheduling system and develop shop floor short - term plans, virtual manufacturing cell multi-period dynamic scheduling model was established in condition of cycle driving mechanism. The model incorporated parameters of the processing task dynamic demand, equipment processing capability, load balance, and similar equipments have multiple; and put forward sharing resources coordination sorting strategy. The objective is to minimize completion time and the total materials and components travelling distance incurred. A hybrid algorithm, based on the improved ant colony algorithm and heuristic rules was proposed to solve the complex scheduling problem. Actual production data proved that the proposed approach was feasible and effective.

  9. Enhancement of Selection, Bubble and Insertion Sorting Algorithm

    Directory of Open Access Journals (Sweden)

    Muhammad Farooq Umar

    2014-07-01

    Full Text Available In everyday life there is a large amount of data to arrange because sorting removes any ambiguities and make the data analysis and data processing very easy, efficient and provides with cost less effort. In this study a set of improved sorting algorithms are proposed which gives better performance and design idea. In this study five new sorting algorithms (Bi-directional Selection Sort, Bi-directional bubble sort, MIDBiDirectional Selection Sort, MIDBidirectional bubble sort and linear insertion sort are presented. Bi-directional Selection Sort and MIDBiDirectional Selection Sort are the enhancement on basic selection sort while Bidirectional bubble sort and MIDBidirectional bubble sort are the enhancement on basic bubble sort by changing the selection and swapping mechanism of data for sorting. Enhanced sorting algorithms reduced the iteration by half and quarter times respectively. Asymptotically complexities of these algorithms are reduced to O (n2/2 and O (n2/4 from O (n2. Linear insertion sort is the enhancement of insertion sort by changing the design of algorithm (convert two loops to one loop. So asymptotically this algorithm is converted to linear time complexity from quadratic complexity. These sorting algorithms are described using C. The proposed algorithms are analyzed using asymptotic analysis and also using machine-running time and compared with their basic sorting algorithms. In this study we also discuss how the performance and complexity can be improved by optimizing the code and design.

  10. Measuring enzyme activity in single cells

    OpenAIRE

    Kovarik, Michelle L.; Allbritton, Nancy L.

    2011-01-01

    Seemingly identical cells can differ in their biochemical state, function and fate, and this variability plays an increasingly recognized role in organism-level outcomes. Cellular heterogeneity arises in part from variation in enzyme activity, which results from interplay between biological noise and multiple cellular processes. As a result, single-cell assays of enzyme activity, particularly those that measure product formation directly, are crucial. Recent innovations have yielded a range o...

  11. Syndecans: synergistic activators of cell adhesion

    DEFF Research Database (Denmark)

    Woods, A; Couchman, J R

    1998-01-01

    Cell-surface proteoglycans participate in cell adhesion, growth-factor signalling, lipase activity and anticoagulation. Until recently, only the roles of the glycosaminoglycan chains were investigated. Now, with molecular characterization of several core proteins, the roles of each individual...... molecules modulating integrin-based adhesion....

  12. Lipolytic activity in adipocyte cell fractions.

    Science.gov (United States)

    Oschry, Y; Shapiro, B

    1980-05-28

    Adipocytes release only negligible amounts of free fatty acids unless stimulated, but reveal considerable lipolytic activity when homogenized. Epinephrine treatment of the cells caused only a 20-40% increase in the activity of infranatants of homogenates while raising the activity associated with the fat layer up to 10-fold. Full activity (i.e. that of intact-activated cells) could be revealed by epinephrine treatment of the homogenate as well as by sonication of the fat layer in buffer. The combination of both treatments did not yield higher activities. The fat cake contains the bulk of the potential activities which are only realized when dispersed in the aqueous phase by sonication, or upon hormone activation of the whole homogenate. Increase in activity could also be obtained by removal of most of the lipid from the fat layer by extraction with petroleum ether. Re-introduction of extracted lipid inhibited lipolysis. The active enzyme could be separated by flotation at 1.12 specific gravity. The data suggest that the lack of activity in the intact non-stimulated cell may be due to the lack of availability of the aqueous phase to the enzyme. PMID:7378439

  13. An improved infrared technique for sorting pecans

    Science.gov (United States)

    Graeve, Thorsten; Dereniak, Eustace L.; Lamonica, John A., Jr.

    1991-10-01

    This paper presents the results of a study of pecan spectral reflectances. It describes an experiment for measuring the contrast between several components of raw pecan product to be sorted. An analysis of the experimental data reveals high contrast ratios in the infrared spectrum, suggesting a potential improvement in sorting efficiency when separating pecan meat from shells. It is believed that this technique has the potential to dramatically improve the efficiency of current sorting machinery, and to reduce the cost of processing pecans for the consumer market.

  14. Minimal Model Semantics for Sorted Constraint Representation

    Institute of Scientific and Technical Information of China (English)

    廖乐健; 史忠植

    1995-01-01

    Sorted constraint representation is a very useful representation in AI which combines class hierarchies and constraint networks.For such sorted constraint representation,a problem is how to generalize the idea of default inheritance to constraint network,where the attributes in a class or between different classes interact with each other via the network.To give a formal account for the defeasible reasoning in such representation,a general sorted constraint logic is proposed,and a minimal-model semantics for the logic is presented.

  15. Active oxygen and cell death in cereal aleurone cells.

    Science.gov (United States)

    Fath, Angelika; Bethke, Paul; Beligni, Veronica; Jones, Russell

    2002-05-01

    The cereal aleurone layer is a secretory tissue whose function is regulated by gibberellic acid (GA) and abscisic acid (ABA). Aleurone cells lack functional chloroplasts, thus excluding photosynthesis as a source of active oxygen species (AOS) in cell death. Incubation of barley aleurone layers or protoplasts in GA initiated the cell death programme, but incubation in ABA delays programmed cell death (PCD). Light, especially blue and UV-A light, and H(2)O(2) accelerate PCD of GA-treated aleurone cells, but ABA-treated aleurone cells are refractory to light and H(2)O(2) and are not killed. It was shown that light elevated intracellular H(2)O(2), and that the rise in H(2)O(2) was greater in GA-treated cells compared to cells in ABA. Experiments with antioxidants show that PCD in aleurone is probably regulated by AOS. The sensitivity of GA-treated aleurone to light and H(2)O(2) is a result of lowered amounts of enzymes that metabolize AOS. mRNAs encoding catalase, ascorbate peroxidase and superoxide dismutase are all reduced during 6-18 h of incubation in GA, but these mRNAs were present in higher amounts in cells incubated in ABA. The amounts of protein and enzyme activities encoded by these mRNAs were also dramatically reduced in GA-treated cells. Aleurone cells store and metabolize neutral lipids via the glyoxylate cycle in response to GA, and glyoxysomes are one potential source of AOS in the GA-treated cells. Mitochondria are another potential source of AOS in GA-treated cells. AOS generated by these organelles bring about membrane rupture and cell death.

  16. Bursts of activity in collective cell migration

    CERN Document Server

    Chepizhko, Oleksandr; Mastrapasqua, Eleonora; Nourazar, Mehdi; Ascagni, Miriam; Sugni, Michela; Fascio, Umberto; Leggio, Livio; Malinverno, Chiara; Scita, Giorgio; Santucci, Stephane; Alava, Mikko J; Zapperi, Stefano; La Porta, Caterina A M

    2016-01-01

    Dense monolayers of living cells display intriguing relaxation dynamics, reminiscent of soft and glassy materials close to the jamming transition, and migrate collectively when space is available, as in wound healing or in cancer invasion. Here we show that collective cell migration occurs in bursts that are similar to those recorded in the propagation of cracks, fluid fronts in porous media and ferromagnetic domain walls. In analogy with these systems, the distribution of activity bursts displays scaling laws that are universal in different cell types and for cells moving on different substrates. The main features of the invasion dynamics are quantitatively captured by a model of interacting active particles moving in a disordered landscape. Our results illustrate that collective motion of living cells is analogous to the corresponding dynamics in driven, but inanimate, systems.

  17. >Effect of progesterone hormon on cell viability and stem cell activation in dental pulp cells

    OpenAIRE

    Segah Altuntaş; Muhammed Ali Kara; Deniz Selin Aksoy; Zehra Dilşad Çoban; Şefik Güran

    2016-01-01

    Objective: The dental pulp is the part in the center of a tooth made up of living connective tissue and cells called odontoblasts. The vitality of the dentin structure, both during health and after injury, depends on pulp cell activity and the signaling processes that regulate the cell’s behavior. Dental pulp tissue has condensed stem cell activity. Dental pulp stem cells are multipotent stem cells that have the potential to differentiate into a variety of cell types. Several publications hav...

  18. Quantum Database Search can do without Sorting

    CERN Document Server

    Patel, A

    2001-01-01

    Sorting is a fundamental computational process, which facilitates subsequent searching of a database. It can be thought of as factorisation of the search process. The location of a desired item in a sorted database can be found by classical queries that inspect one letter of the label at a time. For an unsorted database, no such classical quick search algorithm is available. If the database permits quantum queries, however, then mere digitisation is sufficient for efficient search. Sorting becomes redundant with the quantum superposition of states. A quantum algorithm is written down which locates the desired item in an unsorted database a factor of two faster than the best classical algorithm can in a sorted database. This algorithm has close resemblance to the assembly process in DNA replication.

  19. Selection sorting Algorithm Visualization Using Flash

    Directory of Open Access Journals (Sweden)

    Hadi Sutopo

    2011-02-01

    Full Text Available This paper is intended to develop an algorithm visualization, particularly selection sorting for an Algorithm and Programming course. Algorithm visualization technology graphically illustrates howalgorithms work. This visualization can be used to explain how all data move to the proper position in order to be sorted in a display computer for education. This research consists of 6 steps which areconcept, design, obtaining content material, assembly, testing, and distribution. During the testing step, the application is run and checked to confirm that it performs exactly what the author has intended and the students can learn selection sorting algorithm by studying the visualization. Subjects of the research were students at Department of Informatics Universitas Persada Indonesia YAI for implementation of the learning. The data were analysed using the analytic descriptive method and interpreted in a narrativeway based on the research findings. The algorithm visualization indicates that students increase their motivation and ability to program variety of sorting in programming language they learn.

  20. Another Definition of Order—Sorted Algebra

    Institute of Scientific and Technical Information of China (English)

    何自强

    1998-01-01

    In this paper the definition of order-sorted algebra is generalized by introducing transformation functions between subtypes and supertypes.According to our definition,a type needn't be a subset of its supertype and a record model may form an order-sorted algebra.A new definition of equation is given.It has also been proved that equational theories and describing single inheritance have the initial model.

  1. Automatic spike sorting using tuning information.

    Science.gov (United States)

    Ventura, Valérie

    2009-09-01

    Current spike sorting methods focus on clustering neurons' characteristic spike waveforms. The resulting spike-sorted data are typically used to estimate how covariates of interest modulate the firing rates of neurons. However, when these covariates do modulate the firing rates, they provide information about spikes' identities, which thus far have been ignored for the purpose of spike sorting. This letter describes a novel approach to spike sorting, which incorporates both waveform information and tuning information obtained from the modulation of firing rates. Because it efficiently uses all the available information, this spike sorter yields lower spike misclassification rates than traditional automatic spike sorters. This theoretical result is verified empirically on several examples. The proposed method does not require additional assumptions; only its implementation is different. It essentially consists of performing spike sorting and tuning estimation simultaneously rather than sequentially, as is currently done. We used an expectation-maximization maximum likelihood algorithm to implement the new spike sorter. We present the general form of this algorithm and provide a detailed implementable version under the assumptions that neurons are independent and spike according to Poisson processes. Finally, we uncover a systematic flaw of spike sorting based on waveform information only.

  2. Specified neural progenitors sort to form sharp domains after noisy Shh signaling.

    Science.gov (United States)

    Xiong, Fengzhu; Tentner, Andrea R; Huang, Peng; Gelas, Arnaud; Mosaliganti, Kishore R; Souhait, Lydie; Rannou, Nicolas; Swinburne, Ian A; Obholzer, Nikolaus D; Cowgill, Paul D; Schier, Alexander F; Megason, Sean G

    2013-04-25

    Sharply delineated domains of cell types arise in developing tissues under instruction of inductive signal (morphogen) gradients, which specify distinct cell fates at different signal levels. The translation of a morphogen gradient into discrete spatial domains relies on precise signal responses at stable cell positions. However, cells in developing tissues undergoing morphogenesis and proliferation often experience complex movements, which may affect their morphogen exposure, specification, and positioning. How is a clear pattern achieved with cells moving around? Using in toto imaging of the zebrafish neural tube, we analyzed specification patterns and movement trajectories of neural progenitors. We found that specified progenitors of different fates are spatially mixed following heterogeneous Sonic Hedgehog signaling responses. Cell sorting then rearranges them into sharply bordered domains. Ectopically induced motor neuron progenitors also robustly sort to correct locations. Our results reveal that cell sorting acts to correct imprecision of spatial patterning by noisy inductive signals.

  3. Sorting and Manipulation of Magnetic Droplets in Continuous Flow

    Science.gov (United States)

    Al-Hetlani, Entesar; Hatt, Oliver J.; Vojtíšek, Martin; Tarn, Mark D.; Iles, Alexander; Pamme, Nicole

    2010-12-01

    We report the rapid on-chip generation and subsequent manipulation of magnetic droplets in continuous flow. Magnetic droplets were formed using aqueous-based ferrofluid as the dispersed phase and fluorocarbon oil as the continuous phase. Droplet manipulation was demonstrated with simple permanent magnets using two microfluidic platforms: (i) flow focusing droplet generation followed by their splitting into daughter droplets containing different amounts of magnetic nanoparticles, and (ii) droplet generation at a T-junction and their downstream deflection across a chamber for sorting based on the applied magnetic field and magnetite loading of the droplet. Both systems show great potential for performing a wide range of high throughput continuous flow processes including sample dilution, cell sorting and screening, and microparticle fabrication.

  4. Chemokines: a new dendritic cell signal for T cell activation

    Directory of Open Access Journals (Sweden)

    Christoph A Thaiss

    2011-08-01

    Full Text Available Dendritic cells (DCs are the main inducers and regulators of cytotoxic T lymphocyte (CTL responses against viruses and tumors. One checkpoint to avoid misguided CTL activation, which might damage healthy cells of the body, is the necessity for multiple activation signals, involving both antigenic as well as additional signals that reflect the presence of pathogens. DCs provide both signals when activated by ligands of pattern recognition receptors and licensed by helper lymphocytes. Recently, it has been established that such T cell licensing can be facilitated by CD4+ T helper cells (classical licensing or by NKT cells (alternative licensing. Licensing regulates the DC/CTL cross-talk at multiple layers. Direct recruitment of CTLs through chemokines released by licensed DCs has recently emerged as a common theme and has a crucial impact on the efficiency of CTL responses. Here, we discuss recent advances in our understanding of DC licensing for cross-priming and implications for the temporal and spatial regulation underlying this process. Future vaccination strategies will benefit from a deeper insight into the mechanisms that govern CTL activation.

  5. Dielectrophoresis microsystem with integrated flow cytometers for on-line monitoring of sorting efficiency

    DEFF Research Database (Denmark)

    Wang, Zhenyu; Hansen, Ole; Petersen, Peter Kalsen;

    2006-01-01

    Dielectrophoresis (DEP) and flow cytometry are powerful technologies and widely applied in microfluidic systems for handling and measuring cells and particles. Here, we present a novel microchip with a DEP selective filter integrated with two microchip flow cytometers (FCs) for on-line monitoring...... of cell sorting processes. On the microchip, the DEP filter is integrated in a microfluidic channel network to sort yeast cells by positive DER The two FCs detection windows are set upstream and downstream of the DEP filter. When a cell passes through the detection windows, the light scattered by the cell...

  6. Entangled active matter: From cells to ants

    Science.gov (United States)

    Hu, D. L.; Phonekeo, S.; Altshuler, E.; Brochard-Wyart, F.

    2016-07-01

    Both cells and ants belong to the broad field of active matter, a novel class of non-equilibrium materials composed of many interacting units that individually consume energy and collectively generate motion or mechanical stresses. However cells and ants differ from fish and birds in that they can support static loads. This is because cells and ants can be entangled, so that individual units are bound by transient links. Entanglement gives cells and ants a set of remarkable properties usually not found together, such as the ability to flow like a fluid, spring back like an elastic solid, and self-heal. In this review, we present the biology, mechanics and dynamics of both entangled cells and ants. We apply concepts from soft matter physics and wetting to characterize these systems as well as to point out their differences, which arise from their differences in size. We hope that our viewpoints will spur further investigations into cells and ants as active materials, and inspire the fabrication of synthetic active matter.

  7. Roles of specific membrane lipid domains in EGF receptor activation and cell adhesion molecule stabilization in a developing olfactory system.

    Directory of Open Access Journals (Sweden)

    Nicholas J Gibson

    Full Text Available BACKGROUND: Reciprocal interactions between glial cells and olfactory receptor neurons (ORNs cause ORN axons entering the brain to sort, to fasciculate into bundles destined for specific glomeruli, and to form stable protoglomeruli in the developing olfactory system of an experimentally advantageous animal species, the moth Manduca sexta. Epidermal growth factor receptors (EGFRs and the cell adhesion molecules (IgCAMs neuroglian and fasciclin II are known to be important players in these processes. METHODOLOGY/PRINCIPAL FINDINGS: We report in situ and cell-culture studies that suggest a role for glycosphingolipid-rich membrane subdomains in neuron-glia interactions. Disruption of these subdomains by the use of methyl-beta-cyclodextrin results in loss of EGFR activation, depletion of fasciclin II in ORN axons, and loss of neuroglian stabilization in the membrane. At the cellular level, disruption leads to aberrant ORN axon trajectories, small antennal lobes, abnormal arrays of olfactory glomerul, and loss of normal glial cell migration. CONCLUSIONS/SIGNIFICANCE: We propose that glycosphingolipid-rich membrane subdomains (possible membrane rafts or platforms are essential for IgCAM-mediated EGFR activation and for anchoring of neuroglian to the cytoskeleton, both required for normal extension and sorting of ORN axons.

  8. Critical telomerase activity for uncontrolled cell growth.

    Science.gov (United States)

    Wesch, Neil L; Burlock, Laura J; Gooding, Robert J

    2016-01-01

    The lengths of the telomere regions of chromosomes in a population of cells are modelled using a chemical master equation formalism, from which the evolution of the average number of cells of each telomere length is extracted. In particular, the role of the telomere-elongating enzyme telomerase on these dynamics is investigated. We show that for biologically relevant rates of cell birth and death, one finds a critical rate, R crit, of telomerase activity such that the total number of cells diverges. Further, R crit is similar in magnitude to the rates of mitosis and cell death. The possible relationship of this result to replicative immortality and its associated hallmark of cancer is discussed. PMID:27500377

  9. Critical telomerase activity for uncontrolled cell growth

    Science.gov (United States)

    Wesch, Neil L.; Burlock, Laura J.; Gooding, Robert J.

    2016-08-01

    The lengths of the telomere regions of chromosomes in a population of cells are modelled using a chemical master equation formalism, from which the evolution of the average number of cells of each telomere length is extracted. In particular, the role of the telomere-elongating enzyme telomerase on these dynamics is investigated. We show that for biologically relevant rates of cell birth and death, one finds a critical rate, R crit, of telomerase activity such that the total number of cells diverges. Further, R crit is similar in magnitude to the rates of mitosis and cell death. The possible relationship of this result to replicative immortality and its associated hallmark of cancer is discussed.

  10. Wave shape classification of spontaneous neuronal activity in cortical cultures on micro-electrode arrays

    OpenAIRE

    Staveren, van, R.; Buitenweg, J.R.; Heida, T.; Rutten, W.L.C.

    2002-01-01

    Dissociated embryonal or postnatal rat cortical cells were cultured onto multi electrode arrays (MEA's) with 61 electrode sites. They developed into networks and became spontaneously active after about one week in vitro. About 180,000 recorded action potential waveforms were sorted using several spike features and classified with a Mahalanobis distance sorting procedure. They were classified into six basic wave shapes.

  11. Oxide inspecting/sorting concepts. Revision 2

    International Nuclear Information System (INIS)

    The purpose of this document is to summarize the preferred methods for inspecting and sorting plutonium and uranium oxides in preparation for future processing. A limited number of commercially available systems were investigated in preparation for selecting the preferred candidate(s). A complete listing and description of all the oxides to be processed can be located in ''Materials Disposition Acceptance Specifications for the Plutonium Immobilization Project'', document number PIP-98-047. For the purposes of this document, they will be referred to simply as oxides, unless there is a specific characteristic requiring further explanation. The physical transfer of the oxides from a convenience can into a standard oxide can will occur in the High Contamination section of the Unpackaging/Sorting glovebox. The degree of oxide inspecting and sorting performed will depend on the processing that will occur after Unpackaging/Sorting, on the known condition of the oxide when it is first received and the degree of confidence in its condition. The order in which the steps are performed and what occurs in the individual steps may vary depending on the method selected. For the purpose of organization and to help clarify what may occur in the individual steps, the following guidelines are proposed for the Unpackaging/Sorting glovebox. Sorting will involve the capture and removal of foreign particles and/or to detain hard lumps for additional processing in Crush and Grind. Part of the sorting stage will involve reducing workable lumps (de-lumping) into a smaller particle size in preparation for the sizing stage. Sizing will involve classifying/grading the oxide powder into a particle size that is acceptable for the next processing stage. It could involve some mild form of processing (screening, eTc.), but is not expected to include any sophisticated type of processing. Inspection refers to the inspection of the final product to ensure it meets the particle size requirements for

  12. Three-Step Model for Polarized Sorting of KIF17 into Dendrites.

    Science.gov (United States)

    Franker, Mariella A; Esteves da Silva, Marta; Tas, Roderick P; Tortosa, Elena; Cao, Yujie; Frias, Cátia P; Janssen, Anne F J; Wulf, Phebe S; Kapitein, Lukas C; Hoogenraad, Casper C

    2016-07-11

    Kinesin and dynein motors drive bidirectional cargo transport along microtubules and have a critical role in polarized cargo trafficking in neurons [1, 2]. The kinesin-2 family protein KIF17 is a dendrite-specific motor protein and has been shown to interact with several dendritic cargoes [3-7]. However, the mechanism underlying the dendritic targeting of KIF17 remains poorly understood [8-11]. Using live-cell imaging combined with inducible trafficking assays to directly probe KIF17 motor activity in living neurons, we found that the polarized sorting of KIF17 to dendrites is regulated in multiple steps. First, cargo binding of KIF17 relieves autoinhibition and initiates microtubule-based cargo transport. Second, KIF17 does not autonomously target dendrites, but enters the axon where the actin cytoskeleton at the axon initial segment (AIS) prevents KIF17 vesicles from moving further into the axon. Third, dynein-based motor activity is able to redirect KIF17-coupled cargoes into dendrites. We propose a three-step model for polarized targeting of KIF17, in which the collective function of multiple motor teams is required for proper dendritic sorting. PMID:27265394

  13. PX domain and CD domain play different roles in localization and vacuolation of Sorting Nexin 10

    Institute of Scientific and Technical Information of China (English)

    YAO Dong; WU Bin; QIN BaoMing; PEI DuanQing

    2009-01-01

    Sorting nexins (SNXs) are PX domain containing proteins and essential for intracellular protein sorting,trafficking and signal transduction.The PX domains of SNXs can bind to various phosphorelated phosphoinositides (Pls) and target the host proteins to endosomes.Recently,we have reported that overexpression of SNX10 in mammalian cells could induce giant vacuoles.In this study,we aimed to identify regions in SNX10 critical for the vacuolation activity.We found that both the PX domain and the CD1 region were essential for vacuolation.We provided evidence that the PX domain was able to specifically bind to Ptdlns(3)P and target SNX10 to endosomes.A mutation in the β1 region of the PX domain (V15A) disrupted the Ptdlns(3)P binding ability and the endosomal localization of SNX10.However,correct subcellular localization alone was not sufficient for SNX10 to induce vacuoles.We found that the CD1 region,which was not required for the localization,was indispensable for the vacuolation activity of SNX10.In summary,both the PX domain and the CD1 region are necessary for SNX10 to induce vacuoles but they play different roles in this process.

  14. Sorting by Restricted-Length-Weighted Reversals

    Institute of Scientific and Technical Information of China (English)

    Thach Cam Nguyen; Hieu Trung Ngo; Nguyen Bao Nguyen

    2005-01-01

    Classical sorting by reversals uses the unit-cost model, that is, each reversal consumes an equal cost. This model limits the biological meaning of sorting by reversal.Bender and his colleagues extended it by assigning a cost function f(l) = lα for all α≥ 0, where l is the length of the reversed subsequence. In this paper, we extend their results by considering a model in which long reversals are prohibited. Using the same cost function above for permitted reversals, we present tight or nearly tight bounds for the worst-case cost of sorting by reversals. Then we develop algorithms to approximate the optimal cost to sort a given 0/1 sequence as well as a given permutation. Our proposed problems are more biologically meaningful and more algorithmically general and challenging than the problem considered by Bender et al. Furthermore, our bounds are tight and nearly tight, whereas our algorithms provide good approximation ratios compared to the optimal cost to sort 0/1 sequences or permutations by reversals.

  15. Mechanically activated artificial cell by using microfluidics.

    Science.gov (United States)

    Ho, Kenneth K Y; Lee, Lap Man; Liu, Allen P

    2016-01-01

    All living organisms sense mechanical forces. Engineering mechanosensitive artificial cell through bottom-up in vitro reconstitution offers a way to understand how mixtures of macromolecules assemble and organize into a complex system that responds to forces. We use stable double emulsion droplets (aqueous/oil/aqueous) to prototype mechanosensitive artificial cells. In order to demonstrate mechanosensation in artificial cells, we develop a novel microfluidic device that is capable of trapping double emulsions into designated chambers, followed by compression and aspiration in a parallel manner. The microfluidic device is fabricated using multilayer soft lithography technology, and consists of a control layer and a deformable flow channel. Deflections of the PDMS membrane above the main microfluidic flow channels and trapping chamber array are independently regulated pneumatically by two sets of integrated microfluidic valves. We successfully compress and aspirate the double emulsions, which result in transient increase and permanent decrease in oil thickness, respectively. Finally, we demonstrate the influx of calcium ions as a response of our mechanically activated artificial cell through thinning of oil. The development of a microfluidic device to mechanically activate artificial cells creates new opportunities in force-activated synthetic biology.

  16. Mechanically activated artificial cell by using microfluidics

    Science.gov (United States)

    Ho, Kenneth K. Y.; Lee, Lap Man; Liu, Allen P.

    2016-01-01

    All living organisms sense mechanical forces. Engineering mechanosensitive artificial cell through bottom-up in vitro reconstitution offers a way to understand how mixtures of macromolecules assemble and organize into a complex system that responds to forces. We use stable double emulsion droplets (aqueous/oil/aqueous) to prototype mechanosensitive artificial cells. In order to demonstrate mechanosensation in artificial cells, we develop a novel microfluidic device that is capable of trapping double emulsions into designated chambers, followed by compression and aspiration in a parallel manner. The microfluidic device is fabricated using multilayer soft lithography technology, and consists of a control layer and a deformable flow channel. Deflections of the PDMS membrane above the main microfluidic flow channels and trapping chamber array are independently regulated pneumatically by two sets of integrated microfluidic valves. We successfully compress and aspirate the double emulsions, which result in transient increase and permanent decrease in oil thickness, respectively. Finally, we demonstrate the influx of calcium ions as a response of our mechanically activated artificial cell through thinning of oil. The development of a microfluidic device to mechanically activate artificial cells creates new opportunities in force-activated synthetic biology. PMID:27610921

  17. 采用多功能流式细胞术分析分选造血干细胞和髓系定向分化祖细胞%Sorting and analysis of hematopoietic stem cells and myeloid lineage-committed progenitors using flow cytometry

    Institute of Scientific and Technical Information of China (English)

    崔巍; 许晓东; 许勇钢; 汪玄

    2008-01-01

    目的 探讨富集纯化造血干细胞(HSC)和髓系定向分化祖细胞的新实验方案.方法 根据造血干细胞和定向分化祖细胞在发育过程中表达某些特异性分化抗原的特性,通过免疫磁珠分选技术结合四色和六色流式细胞术分析14只健康小鼠的骨髓造血干细胞、造血祖细胞及定向分化祖细胞系列的表达,并对其进行分选,以进一步通过集落细胞培养和传代试验对分选后细胞的活性进行检测.结果 经上述实验方案分析,14只健康小鼠骨髓造血祖细胞(HPC)的表达率约为HSC的10倍;但其牛成活性远不如造血干细胞,共同髓系祖细胞(CMP)的传代能力仅为HSC的1/2,且次级分化的粒系单核系祖细胞(GMP)和红系巨核系祖细胞(MEP)的生成活性更弱,其传代次数为零.结论 通过多色流式细胞术实验方案可以分析纯化HSC和髓系定向分化祖细胞的表达,并精确计数HSC和祖细胞.%Objective To study the experimental protocol for purification and analysis of hematopoietic stem cells(HSC)and myeloid lineage-committed progenitors.Methods According to differentiation antigen expression pattern on hematopoietic stem cells(HSC) and progenitors during hematopoietic development,HSC and progenitors from bone marrow of 14 healthy mice were analyzed and sorted by magnetic nanoparticles and 4-color or 6-color flow cytometry using multiple antibody panels.Sorted HSC and progenitors were further tested by methylcellulose colony forming unit(CFU)and serial replatingassays.Results The expression of hematopoietic progenitor cells(HPC)was 10-fold higher expression than that of HSC.However,replating activity of common myeloid rogenitors(CMP)was only half of that of HSC.And there was almost 120 replating activity observed in granulocyte/macrophage lineage-restricted progenitors(GMP)and megakaryocyte/erythroeyte lineage-restricted progenitors(MEP).Conclusion Multiparametric flow cytometry could be used to isolate and

  18. A Success of Some Sort

    NARCIS (Netherlands)

    Venot, J.P.

    2016-01-01

    This paper explains the processes behind the framing of drip irrigation as a promising technology to address current poverty and environmental challenges in the developing world. I draw from critical development and science and technology studies and highlight that this imagery has been actively

  19. Identification of residual leukemic cells by flow cytometry in childhood B-cell precursor acute lymphoblastic leukemia: verification of leukemic state by flow-sorting and molecular/cytogenetic methods

    DEFF Research Database (Denmark)

    Obro, Nina F; Ryder, Lars P; Madsen, Hans O;

    2012-01-01

    Reduction in minimal residual disease, measured by real-time quantitative PCR or flow cytometry, predicts prognosis in childhood B-cell precursor acute lymphoblastic leukemia. We explored whether cells reported as minimal residual disease by flow cytometry represent the malignant clone harboring...... immunophenotype and antigen modulation) that highlight important methodological pitfalls. These findings demonstrate that with sufficient experience, flow cytometry is reliable for minimal residual disease monitoring in B-cell precursor acute lymphoblastic leukemia, although rare cases require supplementary PCR...

  20. The Role of the Clathrin Adaptor AP-1: Polarized Sorting and Beyond

    Directory of Open Access Journals (Sweden)

    Fubito Nakatsu

    2014-11-01

    Full Text Available The selective transport of proteins or lipids by vesicular transport is a fundamental process supporting cellular physiology. The budding process involves cargo sorting and vesicle formation at the donor membrane and constitutes an important process in vesicular transport. This process is particularly important for the polarized sorting in epithelial cells, in which the cargo molecules need to be selectively sorted and transported to two distinct destinations, the apical or basolateral plasma membrane. Adaptor protein (AP-1, a member of the AP complex family, which includes the ubiquitously expressed AP-1A and the epithelium-specific AP-1B, regulates polarized sorting at the trans-Golgi network and/or at the recycling endosomes. A growing body of evidence, especially from studies using model organisms and animals, demonstrates that the AP-1-mediated polarized sorting supports the development and physiology of multi-cellular units as functional organs and tissues (e.g., cell fate determination, inflammation and gut immune homeostasis. Furthermore, a possible involvement of AP-1B in the pathogenesis of human diseases, such as Crohn’s disease and cancer, is now becoming evident. These data highlight the significant contribution of AP-1 complexes to the physiology of multicellular organisms, as master regulators of polarized sorting in epithelial cells.

  1. Application of a microfluidic sperm sorter to in vitro production of dairy cattle sex-sorted embryos.

    Science.gov (United States)

    Li, Jingchun; Zhu, Sibing; He, Xianjing; Sun, Rui; He, Qianyu; Gan, Yi; Liu, Shengjun; Funahashi, Hiroaki; Li, Yanbing

    2016-04-15

    Viable sperm from sex-sorted semen without centrifugal treatment was separated by a microfluidic sperm sorter (MFSS) for IVF to improve in vitro embryo production of dairy cattle. The MFSS was originally developed to isolate motile human sperm by two laminar flows in the micro-channel (there are four chambers in an MFSS. Chamber A is the inlet for semen, chamber B is the inlet for the medium, chamber C is the exit chamber for motile sperm, and chamber D is the outlet for nonmotile sperm). Sex-sorted sperm were adjusted to 1 × 10(7) spermatozoa/mL (2 million cells/dose, sperm motility was 30% above after thawing). In a first experiment, diluted sex-sorted semen was mixed with modified Medium199(mM199) containing 5-mM caffeine for 5 minutes, resulting in variations in sperm concentration and quality parameters at chambers A, C, and D. In a second experiment, medium containing sperm from three MFSS chambers was collected and mitochondrial activity of the sperm was determined by flow cytometry, the relative activity of sperm mitochondria in chamber C (1.56 ± 0.03) was the highest in three observation areas (P competence of fertilized oocytes to the blastocyst stage was also higher in the MFSS-IVF system (40.12% ± 2.61%) than the modified standard IVF technique (24.55% ± 4.54%). These results demonstrate that a short coculture of dairy cattle oocytes with isolated motile sex-sorted spermatozoa gradually accumulated in the MFSS device improves the efficiencies of normally produced fertilized embryos and blastocyst formation. PMID:26768540

  2. Rac1+ cells distributed in accordance with CD 133+ cells in glioblastomas and the elevated invasiveness of CD 133+ glioma cells with higher Rac1 activity

    Institute of Scientific and Technical Information of China (English)

    ZHANG Bin; SUN Jian; YU Sheng-ping; CHEN Cong; LIU Bin; LIU Zhi-feng; REN Bing-cheng; MING Hao-lang; YANG Xue-jun

    2012-01-01

    Background Recent studies have suggested that cancer stem cells are one of the major causes for tumor recurrence due to their resistance to radiotherapy and chemotherapy.Although the highly invasive nature of glioblastoma (GBM)cells is also implicated in the failure of current therapies,it is not clear how glioma stem cells (GSCs) are involved in invasiveness.Rac1 activity is necessary for inducing reorganization of actin cytoskeleton and cell movement.In this study,we aimed to investigate the distribution characteristics of CD133+ cells and Rac1+ cells in GBM as well as Rac1 activity in CD133+ GBM cells,and analyze the migration and invasion potential of these cells.Methods A series of 21 patients with GBM were admitted consecutively and received tumor resection in Tianjin Medical University General Hospital during the first half of the year 2011.Tissue specimens were collected both from the peripheral and the central parts for each tumor under magnetic resonance imaging (MRI) navigation guidance.Immunohistochemical staining was used to detect the CD133+ cells and Rac1+ cells distribution in GBM specimens.Double-labeling immunofluorescence was further used to analyze CD133 and Rac1 co-expression and the relationship between CD133+ cells distribution and Rac1 expression.Serum-free medium culture and magnetic sorting were used to isolate CD133+ cells from U87 cell line.Rac1 activation assay was conducted to assess the activation of Rac1 in CD133+ and CD133-U87 cells.The migration and invasive ability of CD133+ and CD133-U87 cells were determined by cell migration and invasion assays in vitro.Student's t-test and one-way analysis of variance (ANOVA) test were used to determine statistical significance in this study.Results In the central parts of GBMs,CD133+ cells were found to cluster around necrosis and occasionally cluster around the vessels under the microscope by immunohistological staining.In the peripheral parts of the tumors,CD133+ cells were lined up along

  3. Sorting of CD133+ positive cells from nasopharyngeal carcinoma cell line%人鼻咽癌细胞株分选CD133+细胞实验研究

    Institute of Scientific and Technical Information of China (English)

    林国彪; 姚平; 张锡流

    2012-01-01

    目的 观察CD133在人鼻咽癌细胞株中的表达,探讨CD133+肿瘤细胞的体外增殖及分化能力从而确定鼻咽癌肿瘤干细胞的标志.方法 使用免疫细胞化学及流式细胞技术检测鼻咽癌CNE2Z细胞株中的表达,免疫磁珠分选技术纯化肿瘤细胞,体外培养,观察其增殖及分化能力.结果 鼻咽癌CNE2Z细胞株中有0.168%呈微量阳性表达,利用免疫磁珠分选技术纯化细胞的百分比为87.2%,免疫磁珠富集的CD133+肿瘤细胞在无血清培养基中1、3、5、7天的吸光度均高于相同条件下未分选细胞和CD133-细胞;CD133+在培养体系中的比例逐日下降,至培养的第12天,由第1天的87.2%下降至0.1%.结论 鼻咽癌CNE2Z细胞株中,CD133+癌细胞有比其他细胞亚群强的体外分化和增殖能力,具有自我更新、生成其他表型肿瘤细胞等干细胞样特性,CD133可能是肿瘤起始细胞的标志之一.%Purpose To detect the expression of CD133 in human nasopharyngeal carcinoma cell line, CNE2Z cell line and to observe the proliferation and differentiation ability of CD133 + groups in vitro. Methods Immunocytochemical staining and flow cytometry were used to detect the expression of putative tumor initiating cell marker CD133 in CNE2Z cell line, and the selective technique of inmu-nomagnetic beads was applied to purify CD133 positive cells. CD133+ tumor cells were cultured and their ability of proliferation and differentiation were observed in vitro. Results Only 0. 168% of cells in CNE2Z cell line expressed CD133. In senun-free RPMI1640, on days 1,3,5 and 7, their UV absorption was higher in CD133 cells than control CNE2Z cells. CD133 + cells demonstrated increased proliferating capacity. The proportion of CD133 + cells decreased in culture as days passed. In twelve days of culture, the percentage of CD133 + cells decreased from 87. 2% to 0. 1%. Conclusions CD133 + cells process stronger motility than CD133 - , CNE2Z cells in vitro and CD133

  4. THE ROLE OF VCAM-1/VLA-4 IN THE ACTIVATION OF ALLOGENIC T CELLS BY MURINE MACROPHAGES

    Institute of Scientific and Technical Information of China (English)

    He Long; Cao Xuetao; Zhang Weiping; Chen Guoyou; Zhu Xuejun; Yu Yizhi

    1998-01-01

    Vascular cell adhesion molecule 1 (VCAM-1) is a member of immunoglobulin superfamily. The principal ligand for VCAM-1 is integrin α4β1/VLA-4 (very late antigen 4). It was reported that VCAM-1 was expressed on macrophages and dendritic cells, but little is known about its function on these professional antigen presenting cells (APC). The present study was performed to investigate the expression of VCAM-1 on macrophages and the role of VCAM-1/VLA-4 in the activation of allogenic T cells by murine macrophages. We analyzed VCAM-1 expression on peritoneal macrophages and macrophage cell line J774A.1 by fluorescence-activated cell sorting (FACS). Using neutralizing antibodies, we further analyzed the role of VCAM-1/VLA-4 interaction in macrophage and allogenic T cell mixed lymphocyte reaction (MLR). We found that VCAM-1 was constitutively expressed on macrophages and its expression level was upregulated by soluble tumor associated antigen (freeze-thaw lysates of FBL-3 leukemia cells) and TNF-α.In MLR assays, we observed that blocking VCAM-1/VLA-4 interaction with anti-VCAM-1 or anti-VLA-4mAbs caused significant inhibition of the proliferative response and IL-2 production. These results suggest that VCAM-1on macrophages not only facilitates the cell-tocell contact through adhesive interaction but also plays a role in the costimulation of T cells via its interaction with VLA-4 on the T cells.

  5. Efficient sorting using registers and caches

    DEFF Research Database (Denmark)

    Wickremesinghe, Rajiv; Arge, Lars Allan; Chase, Jeffrey S.;

    2002-01-01

    Modern computer systems have increasingly complex memory systems. Common machine models for algorithm analysis do not reflect many of the features of these systems, e.g., large register sets, lockup-free caches, cache hierarchies, associativity, cache line fetching, and streaming behavior...... on sorting performance. We introduce a new cache-conscious sorting algorithm, R-MERGE, which achieves better performance in practice over algorithms that are superior in the theoretical models. R-MERGE is designed to minimize memory stall cycles rather than cache misses by considering features common to many...

  6. A Functionalized Sphingolipid Analogue for Studying Redistribution during Activation in Living T Cells.

    Science.gov (United States)

    Collenburg, Lena; Walter, Tim; Burgert, Anne; Müller, Nora; Seibel, Jürgen; Japtok, Lukasz; Kleuser, Burkhard; Sauer, Markus; Schneider-Schaulies, Sibylle

    2016-05-01

    Sphingolipids are major components of the plasma membrane. In particular, ceramide serves as an essential building hub for complex sphingolipids, but also as an organizer of membrane domains segregating receptors and signalosomes. Sphingomyelin breakdown as a result of sphingomyelinase activation after ligation of a variety of receptors is the predominant source of ceramides released at the plasma membrane. This especially applies to T lymphocytes where formation of ceramide-enriched membrane microdomains modulates TCR signaling. Because ceramide release and redistribution occur very rapidly in response to receptor ligation, novel tools to further study these processes in living T cells are urgently needed. To meet this demand, we synthesized nontoxic, azido-functionalized ceramides allowing for bio-orthogonal click-reactions to fluorescently label incorporated ceramides, and thus investigate formation of ceramide-enriched domains. Azido-functionalized C6-ceramides were incorporated into and localized within plasma membrane microdomains and proximal vesicles in T cells. They segregated into clusters after TCR, and especially CD28 ligation, indicating efficient sorting into plasma membrane domains associated with T cell activation; this was abolished upon sphingomyelinase inhibition. Importantly, T cell activation was not abrogated upon incorporation of the compound, which was efficiently excluded from the immune synapse center as has previously been seen in Ab-based studies using fixed cells. Therefore, the functionalized ceramides are novel, highly potent tools to study the subcellular redistribution of ceramides in the course of T cell activation. Moreover, they will certainly also be generally applicable to studies addressing rapid stimulation-mediated ceramide release in living cells. PMID:27036914

  7. Development of a Prototype Automated Sorting System for Plastic Recycling

    OpenAIRE

    D. A. Wahab; Hussain, A.; Scavino, E.; Mustafa, M.M.; Basri, H.

    2006-01-01

    Automated sorting for plastic recyclables has been seen as the way forward in the plastic recycling industry. Automated sorting provides significant improvements in terms of efficiency and consistency in the sorting process. In the case of macro sorting, which is the most common type of automated sorting, efficiency is determined by the mechanical details of the material handling system as well as the detection system. This paper provides a review on the state of-the-art technologies that hav...

  8. Enhancing and Optimization Sorting Algorithms: An Empirical Study

    OpenAIRE

    KARIMIZADEH, Mohammad Mehdi; RAFEAZADEH, Ehsan; AMIRI, Pouran; KHOLGHNIK, Dariush

    2015-01-01

    Abstract. Sorting algorithms are used to sort a list of data. Also sorting is used in other computer operations such as searching, merging, and normalization. Since the sorting is considered as a one of the key operation in computer science, recognition of an optimization approaches can develop this science considerably. Optimization in the sorting algorithms, even in small scale, can cause saving a lot of time.  The main discussion of the paper is on those algorithms which present optimized ...

  9. Activation-Induced Cell Death in T Cells and Autoimmunity

    Institute of Scientific and Technical Information of China (English)

    Jian Zhang; Xuemei Xu; Yong Liu

    2004-01-01

    Activation-induced cell death (AICD), which results from the interaction between Fas and Fas ligand, is responsible for maintaining tolerance to self-antigen. A defect in AICD may lead to development of autoimmunity. During the last several years, much progress has been made in understanding the mechanism(s) of AICD and its potential role in the pathogenesis of autoimmune diseases. In this review, we summarize the most recent progress on the regulation of the susceptibility of T cells to AICD and its possible involvement in autoimmune diseases.

  10. Decidual cell polyploidization necessitates mitochondrial activity.

    Directory of Open Access Journals (Sweden)

    Xinghong Ma

    Full Text Available Cellular polyploidy has been widely reported in nature, yet its developmental mechanism and function remain poorly understood. In the present study, to better define the aspects of decidual cell polyploidy, we isolated pure polyploid and non-polyploid decidual cell populations from the in vivo decidual bed. Three independent RNA pools prepared for each population were then subjected to the Affymetrix gene chip analysis for the whole mouse genome transcripts. Our data revealed up-regulation of 1015 genes and down-regulation of 1207 genes in the polyploid populations, as compared to the non-polyploid group. Comparative RT-PCR and in situ hybridization results indeed confirmed differential expressional regulation of several genes between the two populations. Based on functional enrichment analyses, up-regulated polyploidy genes appeared to implicate several functions, which primarily include cell/nuclear division, ATP binding, metabolic process, and mitochondrial activity, whereas that of down-regulated genes primarily included apoptosis and immune processes. Further analyses of genes that are related to mitochondria and bi-nucleation showed differential and regional expression within the decidual bed, consistent with the pattern of polyploidy. Consistently, studies revealed a marked induction of mitochondrial mass and ATP production in polyploid cells. The inhibition of mitochondrial activity by various pharmacological inhibitors, as well as by gene-specific targeting using siRNA-mediated technology showed a dramatic attenuation of polyploidy and bi-nucleation development during in vitro stromal cell decidualization, suggesting mitochondria play a major role in positive regulation of decidual cell polyploidization. Collectively, analyses of unique polyploidy markers and molecular signaling networks may be useful to further characterize functional aspects of decidual cell polyploidy at the site of implantation.

  11. Electrodynamic activity of healthy and cancer cells

    International Nuclear Information System (INIS)

    Microtubules in the cell form a structure capable of generating electrodynamic field and mitochondria form their supporting system for physical processes including energy supply. Mitochondria transfer protons from their matrix space into cytosol, create strong static field around them that causes ordering of water and altering it into quasi-elastic medium with reduced viscous damping. Microtubules are composed of heterodimers that are electric dipoles. Microtubule oscillations generate an electrodynamic field. The greatest energy supply may be provided by liberation of non-utilized energy from mitochondria. Microtubules and mitochondria form a unique cooperating system in the cell. Mitochondria form a boundary element whose function depends on chemical-genetic control but their output is essential for physical processes in the cell. Mitochondrial dysfunction in cancer cells results in diminished intensity of the static electric field, disturbed water ordering, increased damping of microtubule oscillations and their shift towards linear region, and decreased energy supply. Power and coherence of oscillations and generated electrodynamic field is weakened. Malignant properties of cancer cell, in particular local invasion and metastasis, may depend on disturbed electrodynamic field. Nanotechnology is promising for investigation of electrodynamic activity in living cells.

  12. FACS-sorted particles reduce the data variance in optical tweezers-assisted dynamic force spectroscopy measurements

    Science.gov (United States)

    Stangner, T.; Singer, D.; Wagner, C.; Gutsche, C.; Ueberschär, O.; Hoffmann, R.; Kremer, F.

    2013-08-01

    By combining optical tweezers-assisted dynamic force spectroscopy experiments with fluorescence activated cell sorting (FACS), we demonstrate a new approach to reducing the data variance in measuring receptor-ligand interactions on a single molecule level by ensuring similar coating densities. Therefore, the carboxyfluorescein-labelled monophosphorylated peptide tau226-240[pThr231] is anchored on melamine resin beads and these beads are sorted by FACS to achieve a homogeneous surface coverage. To quantify the impact of the fluorescence dye on the bond parameters between the phosphorylated peptide and the corresponding phosphorylation specific anti-human tau monoclonal antibody HPT-104, we perform dynamic force spectroscopy and compare the results to data using unsorted beads covered with the non-fluorescence peptide analogue. Finally, we demonstrate that the data variance of the relative binding frequency is significantly decreased by a factor of 3.4 using pre-sorted colloids with a homogeneous ligand coating compared to using unsorted colloids.

  13. System for optical sorting of microscopic objects

    DEFF Research Database (Denmark)

    2014-01-01

    The present invention relates to a system for optical sorting of microscopic objects and corresponding method. An optical detection system (52) is capable of determining the positions of said first and/or said second objects. One or more force transfer units (200, 205, 210, 215) are placed in a...

  14. Credit Scores, Race, and Residential Sorting

    Science.gov (United States)

    Nelson, Ashlyn Aiko

    2010-01-01

    Credit scores have a profound impact on home purchasing power and mortgage pricing, yet little is known about how credit scores influence households' residential location decisions. This study estimates the effects of credit scores on residential sorting behavior using a novel mortgage industry data set combining household demographic, credit, and…

  15. SELECTION SORTING ALGORITHM VISUALIZATION USING FLASH

    Directory of Open Access Journals (Sweden)

    Hadi Sutopo

    2011-02-01

    Full Text Available This paper is intended to develop an algorithm visualization, particularly selection sorting for an Algorithm and Programming course. Algorithm visualization technology graphically illustrates how algorithms work. This visualization can be used to explain how all data move to the proper position in order to be sorted in a display computer for education. This research consists of 6 steps which are concept, design, obtaining content material, assembly, testing, and distribution. During the testing step, the application is run and checked to confirm that it performs exactly what the author has intended and the students can learn selection sorting algorithm by studying the visualization. Subjects of the research were students at Department of Informatics Universitas Persada Indonesia YAI for implementation of the learning. The data were analysed using the analytic descriptive method and interpreted in a narrative way based on the research findings. The algorithm visualization indicates that students increase their motivation and ability to program variety of sorting in programming language they learn.

  16. Routes to the tonoplast: the sorting of tonoplast transporters in Arabidopsis mesophyll protoplasts.

    Science.gov (United States)

    Wolfenstetter, Susanne; Wirsching, Petra; Dotzauer, Dorina; Schneider, Sabine; Sauer, Norbert

    2012-01-01

    Vacuoles perform a multitude of functions in plant cells, including the storage of amino acids and sugars. Tonoplast-localized transporters catalyze the import and release of these molecules. The mechanisms determining the targeting of these transporters to the tonoplast are largely unknown. Using the paralogous Arabidopsis thaliana inositol transporters INT1 (tonoplast) and INT4 (plasma membrane), we performed domain swapping and mutational analyses and identified a C-terminal di-leucine motif responsible for the sorting of higher plant INT1-type transporters to the tonoplast in Arabidopsis mesophyll protoplasts. We demonstrate that this motif can reroute other proteins, such as INT4, SUCROSE TRANSPORTER2 (SUC2), or SWEET1, to the tonoplast and that the position of the motif relative to the transmembrane helix is critical. Rerouted INT4 is functionally active in the tonoplast and complements the growth phenotype of an int1 mutant. In Arabidopsis plants defective in the β-subunit of the AP-3 adaptor complex, INT1 is correctly localized to the tonoplast, while sorting of the vacuolar sucrose transporter SUC4 is blocked in cis-Golgi stacks. Moreover, we demonstrate that both INT1 and SUC4 trafficking to the tonoplast is sensitive to brefeldin A. Our data show that plants possess at least two different Golgi-dependent targeting mechanisms for newly synthesized transporters to the tonoplast.

  17. A cell penetrating peptide-integrated and enediyne-energized fusion protein shows potent antitumor activity.

    Science.gov (United States)

    Ru, Qin; Shang, Bo-Yang; Miao, Qing-Fang; Li, Liang; Wu, Shu-Ying; Gao, Rui-Juan; Zhen, Yong-Su

    2012-11-20

    Arginine-rich peptides belong to a subclass of cell penetrating peptides that are taken up by living cells and can be detected freely diffusing inside the cytoplasm and nucleoplasm. This phenomenon has been attributed to either an endocytotic mode of uptake and a subsequent release from vesicles or a direct membrane penetration. Lidamycin is an antitumor antibiotic, which consists of an active enediyne chromophore (AE) and a noncovalently bound apoprotein (LDP). In the present study, a fusion protein (Arg)(9)-LDP composed of cell penetrating peptide (Arg)(9) and LDP was prepared by DNA recombination, and the enediyne-energized fusion protein (Arg)(9)-LDP-AE was prepared by molecular reconstitution. The data in fixed cells demonstrated that (Arg)(9)-LDP could rapidly enter cells, and the results based on fluorescence activated cell sorting indicated that the major route for (Arg)(9)-mediated cellular uptake of protein molecules was endocytosis. (Arg)(9)-LDP-AE demonstrated more potent cytotoxicity against different carcinoma cell lines than lidamycin in vitro. In the mouse hepatoma 22 model, (Arg)(9)-LDP-AE (0.3mg/kg) suppressed the tumor growth by 89.2%, whereas lidamycin (0.05 mg/kg) by 74.6%. Furthermore, in the glioma U87 xenograft model in nude mice, (Arg)(9)-LDP-AE at 0.2mg/kg suppressed tumor growth by 88.8%, compared with that of lidamycin by 62.9% at 0.05 mg/kg. No obvious toxic effects were observed in all groups during treatments. The results showed that energized fusion protein (Arg)(9)-LDP-AE was more effective than lidamycin and would be a promising candidate for glioma therapy. In addition, this approach to manufacturing fusion proteins might serve as a technology platform for the development of new cell penetrating peptides-based drugs. PMID:22982402

  18. Development and validation of a simple cell-based fluorescence assay for dipeptidyl peptidase 1 (DPP1) activity.

    Science.gov (United States)

    Thong, Bob; Pilling, James; Ainscow, Edward; Beri, Raj; Unitt, John

    2011-01-01

    Dipeptidyl peptidase 1 (DPP1) (EC 3.4.14.1; also known as cathepsin C, cathepsin J, dipeptidyl aminopeptidase, and dipeptidyl aminotransferase) is a lysosomal cysteinyl protease of the papain family involved in the intracellular degradation of proteins. Isolated enzyme assays for DPP1 activity using a variety of synthetic substrates such as dipeptide or peptide linked to amino-methyl-coumarin (AMC) or other fluorophores are well established. There is, however, no report of a simple whole-cell-based assay for measuring lysosomal DPP1 activity other than the use of flow cytometry (fluorescence-activated cell sorting) or the use of invasive activity-based probes or the production of physiological products such as neutrophil elastase. The authors investigated a number of DPP1 fluorogenic substrates that have the potential to access the lysosome and enable the measurement of DPP1 enzyme activity in situ. They describe the development and evaluation of a simple noninvasive fluorescence assay for measuring DPP1 activity in fresh or cryopreserved human THP-1 cells using the substrate H-Gly-Phe-AFC (amino-fluoro-coumarin). This cell-based fluorescence assay can be performed in a 96-well plate format and is ideally suited for determining the cell potency of potential DPP1 enzyme inhibitors.

  19. Transgelin-2 in B-Cells Controls T-Cell Activation by Stabilizing T Cell - B Cell Conjugates

    Science.gov (United States)

    Chae, Myoung-Won; Kim, Hye-Ran; Kim, Chang-Hyun; Jun, Chang-Duk; Park, Zee-Yong

    2016-01-01

    The immunological synapse (IS), a dynamic and organized junction between T-cells and antigen presenting cells (APCs), is critical for initiating adaptive immunity. The actin cytoskeleton plays a major role in T-cell reorganization during IS formation, and we previously reported that transgelin-2, an actin-binding protein expressed in T-cells, stabilizes cortical F-actin, promoting T-cell activation in response to antigen stimulation. Transgelin-2 is also highly expressed in B-cells, although no specific function has been reported. In this study, we found that deficiency in transgelin-2 (TAGLN2-/-) in B-cells had little effect on B-cell development and activation, as measured by the expression of CD69, MHC class II molecules, and CD80/86. Nevertheless, in B-cells, transgelin-2 accumulated in the IS during the interaction with T-cells. These results led us to hypothesize that transgelin-2 may also be involved in IS stability in B-cells, thereby influencing T-cell function. Notably, we found that transgelin-2 deficiency in B-cells reduced T-cell activation, as determined by the release of IL-2 and interferon-γ and the expression of CD69. Furthermore, the reduced T-cell activation was correlated with reduced B-cell–T-cell conjugate formation. Collectively, these results suggest that actin stability in B-cells during IS formation is critical for the initiation of adaptive T-cell immunity. PMID:27232882

  20. Characterization of normal and cancer stem cells: One experimental paradigm for two kinds of stem cells

    OpenAIRE

    Mayol, Jean-François; Loeuillet, Corinne; Hérodin, Francis; Wion, Didier

    2009-01-01

    The characterization of normal stem cells and cancer stem cells uses the same paradigm. These cells are isolated by a Fluorescent-Activated Cell Sorting step and their stemness is assayed following implantation into animals. However, differences exist between these two kinds of stem cells. Therefore, the translation of the experimental procedures used for normal stem cell isolation into the cancer stem cell research field is a potential source of artefacts. In addition, normal stem cell thera...

  1. Development of a Prototype Automated Sorting System for Plastic Recycling

    Directory of Open Access Journals (Sweden)

    D. A. Wahab

    2006-01-01

    Full Text Available Automated sorting for plastic recyclables has been seen as the way forward in the plastic recycling industry. Automated sorting provides significant improvements in terms of efficiency and consistency in the sorting process. In the case of macro sorting, which is the most common type of automated sorting, efficiency is determined by the mechanical details of the material handling system as well as the detection system. This paper provides a review on the state of-the-art technologies that have been deployed by some of the recycling facilities abroad. The design and development of a cost effective prototype automated system for sorting plastic recyclables is proposed and discussed.

  2. A differential role for CXCR4 in the regulation of normal versus malignant breast stem cell activity.

    Science.gov (United States)

    Ablett, Matthew P; O'Brien, Ciara S; Sims, Andrew H; Farnie, Gillian; Clarke, Robert B

    2014-02-15

    C-X-C chemokine receptor type 4 (CXCR4) is known to regulate lung, pancreatic and prostate cancer stem cells. In breast cancer, CXCR4 signalling has been reported to be a mediator of metastasis, and is linked to poor prognosis. However its role in normal and malignant breast stem cell function has not been investigated. Anoikis resistant (AR) cells were collected from immortalised (MCF10A, 226L) and malignant (MCF7, T47D, SKBR3) breast cell lines and assessed for stem cell enrichment versus unsorted cells. AR cells had significantly higher mammosphere forming efficiency (MFE) than unsorted cells. The AR normal cells demonstrated increased formation of 3D structures in Matrigel compared to unsorted cells. In vivo, SKBR3 and T47D AR cells had 7- and 130-fold enrichments for tumour formationrespectively, compared with unsorted cells. AR cells contained significantly elevated CXCR4 transcript and protein levels compared to unsorted cells. Importantly, CXCR4 mRNA was higher in stem cell-enriched CD44+/CD24- patient-derived breast cancer cells compared to non-enriched cells. CXCR4 stimulation by its ligand SDF-1 reduced MFE of the normal breast cells lines but increased the MFE in T47D and patient-derived breast cancer cells. CXCR4 inhibition by AMD3100 increased stem cell activity but reduced the self-renewal capacity of the malignant breast cell line T47D. CXCR4+ FACS sorted MCF7 cells demonstrated a significantly increased MFE compared with CXCR4- cells. This significant increase in MFE was further demonstrated in CXCR4 over-expressing MCF7 cells which also had an increase in self-renewal compared to parental cells. A greater reduction in self-renewal following CXCR4 inhibition in the CXCR4 over-expressing cells compared with parental cells was also observed. Our data establish for the first time that CXCR4 signalling has contrasting effects on normal and malignant breast stem cell activity. Here, we demonstrate that CXCR4 signalling specifically regulates breast

  3. Regulatory activity of azabisphosphonate-capped dendrimers on human CD4+ T cell proliferation enhances ex-vivo expansion of NK cells from PBMCs for immunotherapy

    Directory of Open Access Journals (Sweden)

    Caminade Anne-Marie

    2009-09-01

    Full Text Available Abstract Background Adoptive cell therapy with allogenic NK cells constitutes a promising approach for the treatment of certain malignancies. Such strategies are currently limited by the requirement of an efficient protocol for NK cell expansion. We have developed a method using synthetic nanosized phosphonate-capped dendrimers allowing such expansion. We are showing here that this is due to a specific inhibitory activity towards CD4+ T cell which could lead to further medical applications of this dendrimer. Methods Mononuclear cells from human peripheral blood were used to investigate the immunomodulatory effects of nanosized phosphonate-capped dendrimers on interleukin-2 driven CD4+T cell expansion. Proliferation status was investigated using flow cytometry analysis of CFSE dilution and PI incorporation experiments. Magnetic bead cell sorting was used to address activity towards individual or mixed cell sub-populations. We performed equilibrium binding assay to assess the interaction of fluorescent dendrimers with pure CD4+ T cells. Results Phosphonate-capped dendrimers are inhibiting the activation, and therefore the proliferation; of CD4+ T cells in IL-2 stimulated PBMCs, without affecting their viability. This allows a rapid enrichment of NK cells and further expansion. We found that dendrimer acts directly on T cells, as their regulatory property is maintained when stimulating purified CD4+ T cells with anti-CD3/CD28 microbeads. Performing equilibrium binding assays using a fluorescent analogue, we show that the phosphonate capped-dendrimers are specifically interacting with purified CD4+ T cells. Ultimately, we found that our protocol prevents the IL-2 related expansion of regulatory T cells that would be deleterious for the activity of infused NK cells. Conclusion High yield expansion of NK cells from human PBMCs by phosphonate-capped dendrimers and IL-2 occurs through the specific inhibition of the CD4+ lymphocyte compartment. Given the

  4. Activation of cell divisions in legume nodulation

    DEFF Research Database (Denmark)

    Nadzieja, Marcin

    2016-01-01

    Leguminous plants engage into symbiotic relationships with soil bacteria, rhizobia, and develop root nodules. This process initiates with recognition of bacteria derived signalling molecules called nod factors. The subsequent events lead to symbiotic infection and, occurring in parallel, de novo...... was shown to require auxin signalling. Cytokinin, in contrast, exert a negative regulation of bacterial entry into the root. During organogenesis, auxin and cytokinin maxima are known to accompany nodule primordia development and together regulate progression through the cell cycle. Moreover, application...... observations of the DR5 reporter showed activity maxima situated in pericycle and endodermis cells specifically below infection sites. Using gravitropic bending auxin responses in the pericycle could be induced, specifically on the outer side of the gravitropic bend in uninoculated plants. When conducted...

  5. Uncoupling the functions of CALM in VAMP sorting and clathrin-coated pit formation.

    Directory of Open Access Journals (Sweden)

    Daniela A Sahlender

    Full Text Available CALM (clathrin assembly lymphoid myeloid leukemia protein is a cargo-selective adaptor for the post-Golgi R-SNAREs VAMPs 2, 3, and 8, and it also regulates the size of clathrin-coated pits and vesicles at the plasma membrane. The present study has two objectives: to determine whether CALM can sort additional VAMPs, and to investigate whether VAMP sorting contributes to CALM-dependent vesicle size regulation. Using a flow cytometry-based endocytosis efficiency assay, we demonstrate that CALM is also able to sort VAMPs 4 and 7, even though they have sorting signals for other clathrin adaptors. CALM homologues are present in nearly every eukaryote, suggesting that the CALM family may have evolved as adaptors for retrieving all post-Golgi VAMPs from the plasma membrane. Using a knockdown/rescue system, we show that wild-type CALM restores normal VAMP sorting in CALM-depleted cells, but that two non-VAMP-binding mutants do not. However, when we assayed the effect of CALM depletion on coated pit morphology, using a fluorescence microscopy-based assay, we found that the two mutants were as effective as wild-type CALM. Thus, we can uncouple the sorting function of CALM from its structural role.

  6. Uncoupling the functions of CALM in VAMP sorting and clathrin-coated pit formation.

    Science.gov (United States)

    Sahlender, Daniela A; Kozik, Patrycja; Miller, Sharon E; Peden, Andrew A; Robinson, Margaret S

    2013-01-01

    CALM (clathrin assembly lymphoid myeloid leukemia protein) is a cargo-selective adaptor for the post-Golgi R-SNAREs VAMPs 2, 3, and 8, and it also regulates the size of clathrin-coated pits and vesicles at the plasma membrane. The present study has two objectives: to determine whether CALM can sort additional VAMPs, and to investigate whether VAMP sorting contributes to CALM-dependent vesicle size regulation. Using a flow cytometry-based endocytosis efficiency assay, we demonstrate that CALM is also able to sort VAMPs 4 and 7, even though they have sorting signals for other clathrin adaptors. CALM homologues are present in nearly every eukaryote, suggesting that the CALM family may have evolved as adaptors for retrieving all post-Golgi VAMPs from the plasma membrane. Using a knockdown/rescue system, we show that wild-type CALM restores normal VAMP sorting in CALM-depleted cells, but that two non-VAMP-binding mutants do not. However, when we assayed the effect of CALM depletion on coated pit morphology, using a fluorescence microscopy-based assay, we found that the two mutants were as effective as wild-type CALM. Thus, we can uncouple the sorting function of CALM from its structural role.

  7. Antibody Reactivity of B Cells in Lupus Patients with Increased Disease Activity and ARID3a Expression

    Directory of Open Access Journals (Sweden)

    Julie M. Ward

    2015-11-01

    Full Text Available Earlier studies showed that the DNA-binding protein, Bright/ARID3a bound to a subset of human and mouse immunoglobulin heavy chain promoters where it enhanced expression. Indeed, mice with transgenic expression of ARID3a in all B lymphocytes have expanded MZ B cells and produce anti-nuclear antibodies (ANAs. Consistent with our findings in mice, we observed that human systemic lupus erythematosus (SLE patients had expanded numbers of peripheral blood ARID3a+ B cells that were associated with increased disease activity (p = 0.0038. We hypothesized that ARID3a+ naïve B cells would eventually produce autoantibodies, explaining associations between ARID3a expression and disease activity in lupus. Unlike healthy controls, ARID3a was expressed in the naïve B cell population in SLE patients, and we hypothesized that these might represent expansions of autoreactive cells. Therefore, monoclonal antibodies were generated from single-sorted naïve B cells derived from patients with normal (ARID3aN and high (ARID3aH numbers of ARID3a+ B cells. We found that ARID3a expression did not correlate with autoantibody expression. Furthermore, measures of antigen specificities of autoreactive antibodies did not reveal skewing toward particular proteins. These data suggest that the association of increased disease activity in SLE with numbers of ARID3a+ B lymphocytes may be mediated by an antibody-independent mechanism.

  8. Dielectrophoresis microsystem with integrated flow cytometers for on-line monitoring of sorting efficiency.

    Science.gov (United States)

    Wang, Zhenyu; Hansen, Ole; Petersen, Peter K; Rogeberg, Anders; Kutter, Jörg P; Bang, Dang D; Wolff, Anders

    2006-12-01

    Dielectrophoresis (DEP) and flow cytometry are powerful technologies and widely applied in microfluidic systems for handling and measuring cells and particles. Here, we present a novel microchip with a DEP selective filter integrated with two microchip flow cytometers (FCs) for on-line monitoring of cell sorting processes. On the microchip, the DEP filter is integrated in a microfluidic channel network to sort yeast cells by positive DEP. The two FCs detection windows are set upstream and downstream of the DEP filter. When a cell passes through the detection windows, the light scattered by the cell is measured by integrated polymer optical elements (waveguide, lens, and fiber coupler). By comparing the cell counting rates measured by the two FCs, the collection efficiency of the DEP filter can be determined. The chips were used for quantitative determination of the effect of flow rate, applied voltage, conductivity of the sample, and frequency of the electric field on the sorting efficiency. A theoretical model for the capture efficiency was developed and a reasonable agreement with the experimental results observed. Viable and non-viable yeast cells showed different frequency dependencies and were sorted with high efficiency. At 2 MHz, more than 90% of the viable and less than 10% of the non-viable cells were captured on the DEP filter. The presented approach provides quantitative real-time data for sorting a large number of cells and will allow optimization of the conditions for, e.g., collecting cancer cells on a DEP filter while normal cells pass through the system. Furthermore, the microstructure is simple to fabricate and can easily be integrated with other microstructures for lab-on-a-chip applications. PMID:17161009

  9. Sertoli cells maintain Leydig cell number and peritubular myoid cell activity in the adult mouse testis.

    Directory of Open Access Journals (Sweden)

    Diane Rebourcet

    Full Text Available The Sertoli cells are critical regulators of testis differentiation and development. In the adult, however, their known function is restricted largely to maintenance of spermatogenesis. To determine whether the Sertoli cells regulate other aspects of adult testis biology we have used a novel transgenic mouse model in which Amh-Cre induces expression of the receptor for Diphtheria toxin (iDTR specifically within Sertoli cells. This causes controlled, cell-specific and acute ablation of the Sertoli cell population in the adult animal following Diphtheria toxin injection. Results show that Sertoli cell ablation leads to rapid loss of all germ cell populations. In addition, adult Leydig cell numbers decline by 75% with the remaining cells concentrated around the rete and in the sub-capsular region. In the absence of Sertoli cells, peritubular myoid cell activity is reduced but the cells retain an ability to exclude immune cells from the seminiferous tubules. These data demonstrate that, in addition to support of spermatogenesis, Sertoli cells are required in the adult testis both for retention of the normal adult Leydig cell population and for support of normal peritubular myoid cell function. This has implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health.

  10. A mower detector to judge soil sorting

    Energy Technology Data Exchange (ETDEWEB)

    Bramlitt, E.T.; Johnson, N.R. [Thermo Nuclear Services, Inc., Albuquerque, NM (United States)

    1995-12-31

    Thermo Nuclear Services (TNS) has developed a mower detector as an inexpensive and fast means for deciding potential value of soil sorting for cleanup. It is a shielded detector box on wheels pushed over the ground (as a person mows grass) at 30 ft/min with gamma-ray counts recorded every 0.25 sec. It mirror images detection by the TNS transportable sorter system which conveys soil at 30 ft/min and toggles a gate to send soil on separate paths based on counts. The mower detector shows if contamination is variable and suitable for sorting, and by unique calibration sources, it indicates detection sensitivity. The mower detector has been used to characterize some soil at Department of Energy sites in New Jersey and South Carolina.

  11. Sorting Network for Reversible Logic Synthesis

    CERN Document Server

    Islam, Md Saiful; Mahmud, Abdullah Al; karim, Muhammad Rezaul

    2010-01-01

    In this paper, we have introduced an algorithm to implement a sorting network for reversible logic synthesis based on swapping bit strings. The algorithm first constructs a network in terms of n*n Toffoli gates read from left to right. The number of gates in the circuit produced by our algorithm is then reduced by template matching and removing useless gates from the network. We have also compared the efficiency of the proposed method with the existing ones.

  12. A VIN1 GUS::GFP fusion reveals activated sucrose metabolism programming occurring in interspersed cells during tomato fruit ripening.

    Science.gov (United States)

    Estornell, Leandro Hueso; Pons, Clara; Martínez, Alicia; O'Connor, José Enrique; Orzaez, Diego; Granell, Antonio

    2013-08-15

    The tomato is a model for fleshy fruit development and ripening. Here we report on the identification of a novel unique cell autonomous/cellular pattern of expression that was detected in fruits of transgenic tomato lines carrying a GFP GUS driven by the fruit specific vacuolar invertase promoter VIN1. The VIN1 promoter sequence faithfully reproduced the global endogenous VIN expression by conferring a biphasic pattern of expression with a second phase clearly associated to fruit ripening. A closer view revealed a salt and pepper pattern of expression characterized by individual cells exhibiting a range of expression levels (from high to low) surrounded by cells with no expression. This type of pattern was detected across different fruit tissues and cell types with some preferences for vascular, sub-epidermal layer and the inner part of the fruit. Cell ability to show promoter activity was neither directly associated with overall ripening - as we find VIN+ and - VIN- cells at all stages of ripening, nor with cell size. Nevertheless the number of cells with active VIN-driven expression increased with ripening and the activity of the VIN promoter seems to be inversely correlated with cell size in VIN+ cells. Gene expression analysis of FACS-sorted VIN+ cells revealed a transcriptionally distinct subpopulation of cells defined by increased expression of genes related to sucrose metabolism, and decreased activity in protein synthesis and chromatin remodeling. This finding suggests that local micro heterogeneity may underlie some aspects (i.e. the futile cycles involving sucrose metabolism) of an otherwise more uniform looking ripening program.

  13. Kinase Activity Studied in Living Cells Using an Immunoassay

    Science.gov (United States)

    Bavec, Aljos?a

    2014-01-01

    This laboratory exercise demonstrates the use of an immunoassay for studying kinase enzyme activity in living cells. The advantage over the classical method, in which students have to isolate the enzyme from cell material and measure its activity in vitro, is that enzyme activity is modulated and measured in living cells, providing a more…

  14. Differential expression of axon-sorting molecules in mouse olfactory sensory neurons.

    Science.gov (United States)

    Ihara, Naoki; Nakashima, Ai; Hoshina, Naosuke; Ikegaya, Yuji; Takeuchi, Haruki

    2016-08-01

    In the mouse olfactory system, the axons of olfactory sensory neurons that express the same type of odorant receptor (OR) converge to a specific set of glomeruli in the olfactory bulb (OB). It is widely accepted that expressed OR molecules instruct glomerular segregation by regulating the expression of axon-sorting molecules. Although the relationship between the expression of axon-sorting molecules and OR types has been analyzed in detail, those between the expressions of axon-sorting molecules remain to be elucidated. Here we collected the expression profiles of four axon-sorting molecules from a large number of glomeruli in the OB. These molecules demonstrated position-independent mosaic expressions, but their patterns were not identical in the OB. Comparing their expressions identified positive and negative correlations between several pairs of genes even though they showed various expressions. Furthermore, the principal component analysis revealed that the factor loadings in the principal component 1, which explain the largest amount of variation, were most likely to reflect the degree of the cyclic nucleotide-gated (CNG) channel dependence on the expression of axon-sorting molecules. Thus, neural activity generated through the CNG channel is a major component in the generation of a wide variety of expressions of axon-sorting molecules in glomerular segregation. PMID:27207328

  15. Random mitotic activities across human embryonic stem cell colonies.

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Q.; Duggan, R.; Dasa, S.; Li, F.; Chen, L. (Biosciences Division)

    2010-08-01

    A systemic and quantitative study was performed to examine whether different levels of mitotic activities, assessed by the percentage of S-phase cells at any given time point, existed at different physical regions of human embryonic stem (hES) cell colonies at 2, 4, 6 days after cell passaging. Mitotically active cells were identified by the positive incorporation of 5-bromo-2-deoxyuridine (BrdU) within their newly synthesized DNA. Our data indicated that mitotically active cells were often distributed as clusters randomly across the colonies within the examined growth period, presumably resulting from local deposition of newly divided cells. This latter notion was further demonstrated by the confined growth of enhanced green florescence protein (EGFP) expressing cells amongst non-GFP expressing cells. Furthermore, the overall percentage of mitotically active cells remained constantly at about 50% throughout the 6-day culture period, indicating mitotic activities of hES cell cultures were time-independent under current growth conditions.

  16. Human retinal pigment epithelial cells inhibit proliferation and IL2R expression of activated T cells

    DEFF Research Database (Denmark)

    Kaestel, Charlotte G; Jørgensen, Annette; Nielsen, Mette;

    2002-01-01

    The purpose of this study was to characterize the effects of human retinal pigment epithelial (RPE) cells on activated T cells. Activated T cells were cocultured with adult and foetal human RPE cells whereafter apoptosis and proliferation were determined by flow cytometry and (3)H......-Thymidine incorporation assay, respectively. T cells and RPE cells were cultured directly together or in a transwell system for determination of the effect of cell contact. The importance of cell surface molecules was examined by application of a panel of blocking antibodies (CD2, CD18, CD40, CD40L, CD54, CD58......) in addition to use of TCR negative T cell lines. The expression of IL2R-alpha -beta and -gamma chains of activated T cells was analysed by flow cytometry after incubation of T cells alone or with RPE cells. Human RPE cells were found to inhibit the proliferation of activated T cells by a cell contact...

  17. Categorizing Variations of Student-Implemented Sorting Algorithms

    Science.gov (United States)

    Taherkhani, Ahmad; Korhonen, Ari; Malmi, Lauri

    2012-01-01

    In this study, we examined freshmen students' sorting algorithm implementations in data structures and algorithms' course in two phases: at the beginning of the course before the students received any instruction on sorting algorithms, and after taking a lecture on sorting algorithms. The analysis revealed that many students have insufficient…

  18. Order-sorted Algebraic Specifications with Higher-order Functions

    DEFF Research Database (Denmark)

    Haxthausen, Anne Elisabeth

    1995-01-01

    This paper gives a proposal for how order-sorted algebraic specification languages can be extended with higher-order functions. The approach taken is a generalisation to the order-sorted case of an approach given by Mller, Tarlecki and Wirsing for the many-sorted case. The main idea in the proposal...

  19. My eSorts and Digital Extensions of Word Study

    Science.gov (United States)

    Zucker, Tricia A.; Invernizzi, Marcia

    2008-01-01

    "My eSorts" is a strategy for helping children learn to read and spell in a socially motivated context. It is based on developmental spelling research and the word study approach to teaching phonics and spelling. "eSorting" employs digital desktop publishing tools that allow children to author their own electronic word sorts and then share these…

  20. A Comparison of Card-sorting Analysis Methods

    DEFF Research Database (Denmark)

    Nawaz, Ather

    2012-01-01

    This study investigates how the choice of analysis method for card sorting studies affects the suggested information structure for websites. In the card sorting technique, a variety of methods are used to analyse the resulting data. The analysis of card sorting data helps user experience (UX...

  1. Age-related neurogenesis decline in the subventricular zone is associated with specific cell cycle regulation changes in activated neural stem cells.

    Science.gov (United States)

    Daynac, Mathieu; Morizur, Lise; Chicheportiche, Alexandra; Mouthon, Marc-André; Boussin, François D

    2016-01-01

    Although neural stem cells (NSCs) sustain continuous neurogenesis throughout the adult lifespan of mammals, they progressively exhibit proliferation defects that contribute to a sharp reduction in subventricular neurogenesis during aging. However, little is known regarding the early age-related events in neurogenic niches. Using a fluorescence-activated cell sorting technique that allows for the prospective purification of the main neurogenic populations from the subventricular zone (SVZ), we demonstrated an early decline in adult neurogenesis with a dramatic loss of progenitor cells in 4 month-old young adult mice. Whereas the activated and quiescent NSC pools remained stable up to 12 months, the proliferative status of activated NSCs was already altered by 6 months, with an overall extension of the cell cycle resulting from a specific lengthening of G1. Whole genome analysis of activated NSCs from 2- and 6-month-old mice further revealed distinct transcriptomic and molecular signatures, as well as a modulation of the TGFβ signalling pathway. Our microarray study constitutes a cogent identification of new molecular players and signalling pathways regulating adult neurogenesis and its early modifications. PMID:26893147

  2. Activation of the sonic hedgehog signaling pathway occurs in the CD133 positive cells of mouse liver cancer Hepa 1–6 cells

    Directory of Open Access Journals (Sweden)

    Jeng KS

    2013-08-01

    Full Text Available Kuo-Shyang Jeng,1 I-Shyan Sheen,2 Wen-Juei Jeng,2 Ming-Che Yu,3 Hsin-I Hsiau,3 Fang-Yu Chang,3 Hsin-Hua Tsai31Department of Surgery, Far Eastern Memorial Hospital, Taipei, 2Department of Hepato-Gastroenterology, Chang Gung Memorial Hospital, Linkou Medical Center, Chang Gung University, 3Department of Medical Research, Far Eastern Memorial Hospital, Taipei, Taiwan, Republic of ChinaBackground: The important role of cancer stem cells in carcinogenesis has been emphasized in research. CD133+ cells have been mentioned as liver cancer stem cells in hepatocellular carcinoma (HCC. Some researchers have proposed that the sonic hedgehog (Shh pathway contributes to hepatocarcinogenesis and that the pathway activation occurs mainly in cancer stem cells. We investigated whether the activation of the Shh pathway occurs in CD133+ cells from liver cancer.Materials and methods: We used magnetic sorting to isolate CD133+ cells from mouse cancer Hepa 1–6 cells. To examine the clonogenicity, cell culture and soft agar colony formation assay were performed between CD133+ and CD133- cells. To study the activation of the Shh pathway, we examined the mRNA expressions of Shh, patched homolog 1 (Ptch-1, glioma-associated oncogene homolog 1 (Gli-1, and smoothened homolog (Smoh by real-time polymerase chain reaction of both CD133+ and CD133- cells.Results: The number (mean ± standard deviation of colonies of CD133+ cells and CD133- cells was 1,031.0 ± 104.7 and 119.7 ± 17.6 respectively. This difference was statistically significant (P < 0.001. Their clonogenicity was 13.7% ± 1.4% and 1.6% ± 0.2% respectively with a statistically significant difference found (P < 0.001. CD133+ cells and CD133– cells were found to have statistically significant differences in Shh mRNA and Smoh mRNA (P = 0.005 and P = 0.043 respectively.Conclusion: CD133+ Hepa 1–6 cells have a significantly higher colony proliferation and clonogenicity. The Shh pathway is activated in these

  3. Sorting and targeting of melanosomal membrane proteins: signals, pathways, and mechanisms.

    Science.gov (United States)

    Setaluri, V

    2000-06-01

    Newly synthesized melanosomal proteins, like many other cellular proteins, traverse through a series of intracellular compartments en route to melanosomes. Entry and exit of proteins through these compartments is orchestrated by cellular sorting machinery that recognize specific sorting signals. Melanosomal membrane proteins begin their intracellular journey upon co-translational importation into the endoplasmic reticulum (ER). The biosynthetic output of tyrosinase, the key melanogenic enzyme, appears to be regulated by quality-control events at the ER, the 'port of entry' to the secretory pathway. Following maturation in the ER and through the Golgi, the sorting of these proteins in the trans-Golgi network for intracellular retention and transport along endosome/lysosome pathway requires cytoplasmically exposed signals. A di-leucine motif, present in the cytoplasmic tails of most melanosomal proteins, and its interaction with adaptor protein (AP) complexes, specifically AP-3, are critical for these events. Defects in sorting signals and the cytosolic components that interact with these signals result in a number of murine coat color phenotypes and cause human pigmentary disorders. Thus, missense or frame-shift mutations that produce truncated tyrosinase lacking the melanosomal sorting signal(s) appear to be responsible for murine platinum coat color phenotypes and a proportion of human oculocutaneous albinism-1; mutations in AP-3 appear to be responsible for the mocha phenotype in mice and Hermansky-Pudlak-like syndrome in man. Additional signals and sorting steps downstream of AP-3 appear to be required for endosomal sorting and targeting proteins to melanosomes. Signals and mechanisms that sequester melanosomal proteins from endosomes/lysosomes are not understood. Potential candidates that mediate such processes include proteins encoded by lyst and pallid genes. The common occurrence of abnormalities in melanosomes in many storage-pool disorders suggests that

  4. Discovery of a dual-targeting organometallic ruthenium complex with high activity inducing early stage apoptosis of cancer cells.

    Science.gov (United States)

    Du, Jun; Zhang, Erlong; Zhao, Yao; Zheng, Wei; Zhang, Yang; Lin, Yu; Wang, Zhaoying; Luo, Qun; Wu, Kui; Wang, Fuyi

    2015-12-01

    Ruthenium based complexes are promising antitumour candidates due to their lower toxicity and better water-solubility compared to the platinum antitumour complexes. An epidermal growth factor receptor (EGFR) has been found to be overexpressed in a large set of tumour cells. In this work, a series of organoruthenium complexes containing EGFR-inhibiting 4-anilinoquinazoline pharmacophores were synthesised and characterised. These complexes exhibited excellent inhibitory activity against EGFR and high affinity to interact with DNA via minor groove binding, featuring dual-targeting properties. In vitro screening demonstrated that the as-prepared ruthenium complexes are anti-proliferating towards a series of cancer cell lines, in particular the non-small-cell lung cancer cell line A549. Fluorescence-activated cell sorting analysis and fluorescence microscopy revealed that the most active complex 3 induced much more early-stage cell apoptosis than its cytotoxic arene ruthenium analogue and the EGFR-inhibiting 4-anilinoquinazolines, verifying the synergetic effect of the two mono-functional pharmacophores. PMID:26446567

  5. Ly-1 B cells and disease activity in (New Zealand black x New Zealand white)F1 mice. Effect of total lymphoid irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Farinas, M.C.; Stall, A.M.; Solovera, J.J.; Tarlinton, D.M.; Herzenberg, L.A.; Strober, S. (Stanford Univ. School of Medicine, CA (USA))

    1990-04-01

    The treatment of female (New Zealand black x New Zealand white)F1 mice with total lymphoid irradiation resulted in a prolonged remission of autoimmune disease activity. Total lymphoid irradiation-treated mice also showed a marked reduction of Ly-1 B cells, which lasted up to 3 months. The subsequent return of Ly-1 B cells to preirradiation levels was not associated with a simultaneous return of disease when measured by parameters such as IgG anti-DNA antibodies and spontaneous secretion of IgG by splenic cells. In cell sorting experiments, most of the cells spontaneously secreting IgG were found within the Ly-1- (CD5-) splenic B cell population.

  6. Microvortex for focusing, guiding and sorting of particles.

    Science.gov (United States)

    Hsu, Chia-Hsien; Di Carlo, Dino; Chen, Chihchen; Irimia, Daniel; Toner, Mehmet

    2008-12-01

    We report a microvortex manipulator (MVM) that is a passive, scalable system with great potential for the manipulation and separation of particulate samples in microfluidic environments. The movement of particles is determined by a unique combination of helical flow, buoyant, and gravitational forces. Helical flows are induced by topographically patterned microchannel surfaces, which have previously been used for molecular mixing in microfluidic devices. We illustrate the mechanism of MVM and its applications in passive focusing of beads and cells into parallel streams and guiding of particles and cells. We also explore the application of the unique density-selectivity of microvortex focusing and successfully sort a mixture of two bead populations whose density difference is as small as 0.1 g cm(-3). PMID:19023476

  7. Enhanced casein kinase II activity in human tumour cell cultures

    DEFF Research Database (Denmark)

    Prowald, K; Fischer, H; Issinger, O G

    1984-01-01

    Casein kinase II (CKII) activity is enhanced as much as 2-3 fold in established and 4-5-fold in transformed human cell lines when compared to that of fibroblasts and primary human tumour cell cultures where CKII activity never exceeded a basic level. The high activity of CKII in transformed cells...... and in established cell lines was reduced to about the same basic level after treatment with heparin, a highly specific inhibitor of CKII activity. The activity of the cAMP-dependent protein kinase was virtually the same in fibroblasts and various human tumour cell lines investigated....

  8. Sorting Nexin 11 Regulates Lysosomal Degradation of Plasma Membrane TRPV3.

    Science.gov (United States)

    Li, Caiyue; Ma, Wenbo; Yin, Shikui; Liang, Xin; Shu, Xiaodong; Pei, Duanqing; Egan, Terrance M; Huang, Jufang; Pan, Aihua; Li, Zhiyuan

    2016-05-01

    The trafficking of ion channels to/from the plasma membrane is considered an important mechanism for cellular activity and an interesting approach for disease therapies. The transient receptor potential vanilloid 3 (TRPV3) ion channel is widely expressed in skin keratinocytes, and its trafficking mechanism to/from the plasma membrane is unknown. Here, we report that the vesicular trafficking protein sorting nexin 11 (SNX11) downregulates the level of the TRPV3 plasma membrane protein. Overexpression of SNX11 causes a decrease in the level of TRPV3 current and TRPV3 plasma membrane protein in TRPV3-transfected HEK293T cells. Subcellular localizations and western blots indicate that SNX11 interacts with TRPV3 and targets it to lysosomes for degradation, which is blocked by the lysosomal inhibitors chloroquine and leupeptin. Both TRPV3 and SNX11 are highly expressed in HaCaT cells. We show that TRPV3 agonists-activated Ca(2+) influxes and the level of native TRPV3 total protein in HaCaT cells are decreased by overexpression of SNX11 and increased by knockdown of SNX11. Our findings reveal that SNX11 promotes the trafficking of TRPV3 from the plasma membrane to lysosomes for degradation via protein-protein interactions, which demonstrates a previously unknown function of SNX11 as a regulator of TRPV3 trafficking from the plasma membrane to lysosomes. PMID:26818531

  9. Natural killer cell activity during premedication, anaesthesia and surgery

    DEFF Research Database (Denmark)

    Tønnesen, E; Mickley, H; Grunnet, N

    1983-01-01

    Natural killer (NK) cell activity of peripheral blood mononuclear cells was measured against K-562 target cells in a 51Cr release assay in eight patients undergoing total hip replacement surgery. Eight consecutive blood samples were taken from each patient. A significant increase of NK cell...... days. The findings of this study indicate that premedication, anaesthesia and surgery cause a rapid and transient increase in NK cell activity, followed by a decline in activity postoperatively. The transient increase in activity may be explained by mobilization of natural killer cells from extravasal...... activity was observed after premedication with diazepam per os. The activity increased further during a combined anaesthesia (thiopentone + N2O + O2 + buprenorphene + pancuronium) and remained increased during surgery. Postoperatively, NK cell activity fell and remained depressed for a period of at least 5...

  10. Cache-Aware and Cache-Oblivious Adaptive Sorting

    DEFF Research Database (Denmark)

    Brodal, Gerth Stølting; Fagerberg, Rolf; Moruz, Gabriel

    2005-01-01

    Two new adaptive sorting algorithms are introduced which perform an optimal number of comparisons with respect to the number of inversions in the input. The first algorithm is based on a new linear time reduction to (non-adaptive) sorting. The second algorithm is based on a new division protocol ...... for the GenericSort algorithm by Estivill-Castro and Wood. From both algorithms we derive I/O-optimal cache-aware and cache-oblivious adaptive sorting algorithms. These are the first I/O-optimal adaptive sorting algorithms....

  11. Pancreatic and pulmonary mast cells activation during experimental acute pancreatitis

    Institute of Scientific and Technical Information of China (English)

    Inmaculada; Lopez-Font; Sabrina; Gea-Sorlí; Enrique; de-Madaria; Luis; M; Gutiérrez; Miguel; Pérez-Mateo; Daniel; Closa

    2010-01-01

    AIM:To study the activation of pancreatic and pulmonary mast cells and the effect of mast cell inhibition on the activation of peritoneal and alveolar macrophages during acute pancreatitis.METHODS:Pancreatitis was induced by intraductal infusion of 5% sodium taurodeoxycholate in rats.The mast cell inhibitor cromolyn was administered intraperitoneally(i.p.) 30 min before pancreatitis induction.The pancreatic and pulmonary tissue damage was evaluated histologically and mast cells and their state of activation...

  12. Structural and functional robustness of the adaptive-sorting signaling network

    Science.gov (United States)

    Pang, Ning-Ning

    2016-06-01

    A major task of study on ligand discrimination by T cells is the construction of a mechanistic model to account for threshold setting in response to variant ligands interacting with the same T-cell receptors. Recently, Lalanne and Francois in a seminal paper (2013 Phys. Rev. Lett. 110 218102) have addressed this question by constructing minimal core circuits such that the biological outputs can satisfy the essential properties of early T-cell activation. To make this core set of network topology a valuable tool for synthetic biologists to robustly engineer biological circuits, we are motivated to ask a general question: is adaptive response encoded by the proposed circuit topology structurally stable, regardless of the values of the kinetic parameters? This has particularly relevant effects for the network reliability, since failures in ligand discrimination result in either infection or autoimmune diseases. To the best of our knowledge, a rigorous and complete mathematical proof of this issue is still lacking in the literature. In this paper, by giving a rigorous mathematical proof, we have shown that this regulatory circuitry is appropriately designed and the existence, uniqueness, and globally asymptotic attractiveness of the steady state are preserved. Moreover, we further generalize the adaptive sorting module and undertake an extensive analysis on the trade-off between antagonism and sensitivity of T-cell ligand discrimination in various cellular conditions. Notably, the optimal phosphorylation step in which to place the regulatory motif is analytically obtained and numerically confirmed. Finally, relevant experimental facts and biological implications are discussed.

  13. Flow cytometric and near-infrared Raman spectroscopic investigation of quality in stained, sorted, and frozen-thawed buffalo sperm.

    Science.gov (United States)

    Li, Xiao-Xia; Wang, Meng; Chen, Huan-Hua; Li, Qing-Yang; Yang, Huan; Xu, Hui-Yan; Lu, Yang-Qing; Zhang, Ming; Yang, Xiao-Gan; Lu, Sheng-Sheng; Lu, Ke-Huan

    2016-07-01

    Flow cytometry and Laser Tweezers Raman spectroscopy have been used to investigate Nili-Ravi buffalo (Bubalus bubalis) sperm from different samples (fresh, stained, sorted and frozen-thawed) of the flow-sorting process to optimize sperm sex sorting procedures. During the sorting and freezing-thawing processes, the two detection methods both indicated there were differences in mitochondrial activity and membrane integrity. Moreover, a dispersive-type NIR (Near Infrared Reflection) use of the Raman system resulted in the ability to detect a variety of sperm components, including relative DNA, lipid, carbohydrates and protein contents. The use of the Raman system allowed for PCA (principal components analysis) and DFA (discriminant function analysis) of fresh, stained, sorted and frozen-thawed sperm. The methodology, therefore, allows for distinguishing sperm from different samples (fresh, stained, sorted and frozen-thawed), and demonstrated the great discriminative power of ANN (artificial neural network) classification models for the differentiating sperm from different phases of the flow-sorting process. In conclusion, the damage induced by sperm sorting and freezing-thawing procedures can be quantified, and in the present research it is demonstrated that Raman spectroscopy is a valuable technology for assessing sperm quality. PMID:27095613

  14. Online sorting of recovered wood waste by automated XRF-technology: part II. Sorting efficiencies.

    Science.gov (United States)

    Hasan, A Rasem; Solo-Gabriele, Helena; Townsend, Timothy

    2011-04-01

    Sorting of waste wood is an important process practiced at recycling facilities in order to detect and divert contaminants from recycled wood products. Contaminants of concern include arsenic, chromium and copper found in chemically preserved wood. The objective of this research was to evaluate the sorting efficiencies of both treated and untreated parts of the wood waste stream, and metal (As, Cr and Cu) mass recoveries by the use of automated X-ray fluorescence (XRF) systems. A full-scale system was used for experimentation. This unit consisted of an XRF-detection chamber mounted on the top of a conveyor and a pneumatic slide-way diverter which sorted wood into presumed treated and presumed untreated piles. A randomized block design was used to evaluate the operational conveyance parameters of the system, including wood feed rate and conveyor belt speed. Results indicated that online sorting efficiencies of waste wood by XRF technology were high based on number and weight of pieces (70-87% and 75-92% for treated wood and 66-97% and 68-96% for untreated wood, respectively). These sorting efficiencies achieved mass recovery for metals of 81-99% for As, 75-95% for Cu and 82-99% of Cr. The incorrect sorting of wood was attributed almost equally to deficiencies in the detection and conveyance/diversion systems. Even with its deficiencies, the system was capable of producing a recyclable portion that met residential soil quality levels established for Florida, for an infeed that contained 5% of treated wood. PMID:21194917

  15. Sorting N-Elements Using Natural Order: A New Adaptive Sorting Approach

    Directory of Open Access Journals (Sweden)

    Shamim Akhter

    2010-01-01

    Full Text Available Problem statement: Researchers focused their attention on optimally adaptive sorting algorithm and illustrated a need to develop tools for constructing adaptive algorithms for large classes of measures. In adaptive sorting algorithm the run time for n input data smoothly varies from O(n to O(nlogn, with respect to several measures of disorder. Questions were raised whether any approach or technique would reduce the run time of adaptive sorting algorithm and provide an easier way of implementation for practical applications. Approach: The objective of this study is to present a new method on natural sorting algorithm with a run time for n input data O(n to O(nlogm, where m defines a positive value and surrounded by 50% of n. In our method, a single pass over the inputted data creates some blocks of data or buffers according to their natural sequential order and the order can be in ascending or descending. Afterward, a bottom up approach is applied to merge the naturally sorted subsequences or buffers. Additionally, a parallel merging technique is successfully aggregated in our proposed algorithm. Results: Experiments are provided to establish the best, worst and average case runtime behavior of the proposed method. The simulation statistics provide same harmony with the theoretical calculation and proof the method efficiency. Conclusion: The results indicated that our method uses less time as well as acceptable memory to sort a data sequence considering the natural order behavior and applicable to the realistic researches. The parallel implementation can make the algorithm for efficient in time domain and will be the future research issue.

  16. Hyperoxia Inhibits T Cell Activation in Mice

    Science.gov (United States)

    Hughes-Fulford, M.; Meissler, J.; Aguayo, E. T.; Globus, R.; Aguado, J.; Candelario, T.

    2013-02-01

    , spleens were removed and the splenocytes were isolated and kept as individual biological samples. We have also examined transcription factors (JASPAR) and pathways of the immune system to help us understand the mechanism of regulation. Results: Our recent mouse immunology experiment aboard STS-131 suggests that the early T cell immune response was inhibited in animals that have been exposed to spaceflight, even 24 hours after return to earth. Moreover, recent experiments in hyperoxic mice show that many of the same genes involved in early T cell activation were altered. Specifically, expression of IL-2Rα, Cxcl2, TNFα, FGF2, LTA and BCL2 genes are dysregulated in mice exposed to hyperoxia. Conclusions: If these hyperoxia-induced changes of gene expression in early T cell activation are additive to the changes seen in the microgravity of spaceflight, there could be an increased infection risk to EVA astronauts, which should be addressed prior to conducting a Mars or other long-term mission.

  17. Lateral chirality-sorting optical forces

    Science.gov (United States)

    Hayat, Amaury; Mueller, J. P. Balthasar; Capasso, Federico

    2015-01-01

    The transverse component of the spin angular momentum of evanescent waves gives rise to lateral optical forces on chiral particles, which have the unusual property of acting in a direction in which there is neither a field gradient nor wave propagation. Because their direction and strength depends on the chiral polarizability of the particle, they act as chirality-sorting and may offer a mechanism for passive chirality spectroscopy. The absolute strength of the forces also substantially exceeds that of other recently predicted sideways optical forces. PMID:26453555

  18. Multidimensional Skills, Sorting, and Human Capital Accumulation

    OpenAIRE

    Fabien Postel-Vinay; Jeremy Lise

    2015-01-01

    We construct a structural model of on-the-job search in which workers differ in skills along several dimensions (cognitive, manual, interpersonal) and sort themselves into jobs with heterogeneous skill requirements along those same dimensions. We further allow for skills to be accumulated when used, and eroded away when not used. We estimate the model using occupation-level measures of skill requirements based on O*NET data, combined with a worker-level panel from the NLSY79. We use the estim...

  19. Stochastic Models of Vesicular Sorting in Cellular Organelles

    CERN Document Server

    Vagne, Quentin

    2016-01-01

    The proper sorting of membrane components by regulated exchange between cellular organelles is crucial to intra-cellular organization. This process relies on the budding and fusion of transport vesicles, and should be strongly influenced by stochastic fluctuations considering the relatively small size of many organelles. We identify the perfect sorting of two membrane components initially mixed in a single compartment as a first passage process, and we show that the mean sorting time exhibits two distinct regimes as a function of the ratio of vesicle fusion to budding rates. Low ratio values leads to fast sorting, but results in a broad size distribution of sorted compartments dominated by small entities. High ratio values result in two well defined sorted compartments but is exponentially slow. Our results suggests an optimal balance between vesicle budding and fusion for the rapid and efficient sorting of membrane components, and highlight the importance of stochastic effects for the steady-state organizati...

  20. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M;

    1998-01-01

    induced apoptosis in several activated T-cell populations and T-cell lines, including T-cell antigen receptor (TCR)-CD3-negative T-cell lines. In contrast, RPE cells induced little or no apoptosis in resting peripheral T cells. Major histocompatibility complex (MHC) class II monoclonal antibodies, which...

  1. >Effect of progesterone hormon on cell viability and stem cell activation in dental pulp cells

    Directory of Open Access Journals (Sweden)

    Segah Altuntaş

    2016-03-01

    Full Text Available Objective: The dental pulp is the part in the center of a tooth made up of living connective tissue and cells called odontoblasts. The vitality of the dentin structure, both during health and after injury, depends on pulp cell activity and the signaling processes that regulate the cell’s behavior. Dental pulp tissue has condensed stem cell activity. Dental pulp stem cells are multipotent stem cells that have the potential to differentiate into a variety of cell types. Several publications have stressed the importance of the expression of pluripotentiality associated markers: the transcription factors Nanog, Sox2, Oct3/4, SSEA4, CD13, Stro1 are indispensable for the stem cells to divide indefinitely without affecting their differentiation potential (self renewal capacity. Progesterone is a steroid hormone leading to menstrual cycle and gestation. There is a widespread rumor among people that pregnancy causes toothy loss. Method: So, progesterone was applied in different concentrations on human dental pulp cells in cell culture. Cell viability assay was applied 24th hour later with trypan blue. RNA isolation, cDNA synthesis and Real Time PCR analysis were applied on selected transcription factors (Nanog and Oct4 (POU5F1 genes which have role on steamness of stem cells. Gene expression analyses results were correlated with the cell viability assay results. Results: Cell viability assay results were 80% viable in control, 82% viable in 7 ml progesterone application, 81% viable in 14 ml progesterone application, 83% viable in 21 ml progesterone application. Due to our findings, progesterone in different concentrations did not chance the cell viability in dental pulpa cells. On gene expression analyses, preliminary results supported that high concentrations of progesterone enhance the gene expressions of steamness genes (Nanog, and Oct4 in dental pulp cells. Conclusions: So, progesterone did not change cell viability in high concentrations. We

  2. A new multicolor bioluminescence imaging platform to investigate NF-κB activity and apoptosis in human breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Laura Mezzanotte

    Full Text Available BACKGROUND: Evaluation of novel drugs for clinical development depends on screening technologies and informative preclinical models. Here we developed a multicolor bioluminescent imaging platform to simultaneously investigate transcription factor NF-κB signaling and apoptosis. METHODS: The human breast cancer cell line (MDA-MB-231 was genetically modified to express green, red and blue light emitting luciferases to monitor cell number and viability, NF-κB promoter activity and to perform specific cell sorting and detection, respectively. The pro-luciferin substrate Z-DEVD-animoluciferin was employed to determine apoptotic caspase 3/7 activity. We used the cell line for the in vitro evaluation of natural compounds and in vivo optical imaging of tumor necrosis factor TNFα-induced NF-κB activation. RESULTS: Celastrol, resveratrol, sulphoraphane and curcumin inhibited the NF-κB promoter activity significantly and in a dose dependent manner. All compounds except resveratrol induced caspase 3/7 dependent apoptosis. Multicolor bioluminescence in vivo imaging allowed the investigation of tumor growth and NF-κB induction in a mouse model of breast cancer. CONCLUSION: Our new method provides an imaging platform for the identification, validation, screening and optimization of compounds acting on NF-κB signaling and apoptosis both in vitro and in vivo.

  3. A New Multicolor Bioluminescence Imaging Platform to Investigate NF-κB Activity and Apoptosis in Human Breast Cancer Cells

    Science.gov (United States)

    Mezzanotte, Laura; An, Na; Mol, Isabel M.; Löwik, Clemens W. G. M.; Kaijzel, Eric L.

    2014-01-01

    Background Evaluation of novel drugs for clinical development depends on screening technologies and informative preclinical models. Here we developed a multicolor bioluminescent imaging platform to simultaneously investigate transcription factor NF-κB signaling and apoptosis. Methods The human breast cancer cell line (MDA-MB-231) was genetically modified to express green, red and blue light emitting luciferases to monitor cell number and viability, NF-κB promoter activity and to perform specific cell sorting and detection, respectively. The pro-luciferin substrate Z-DEVD-animoluciferin was employed to determine apoptotic caspase 3/7 activity. We used the cell line for the in vitro evaluation of natural compounds and in vivo optical imaging of tumor necrosis factor TNFα-induced NF-κB activation. Results Celastrol, resveratrol, sulphoraphane and curcumin inhibited the NF-κB promoter activity significantly and in a dose dependent manner. All compounds except resveratrol induced caspase 3/7 dependent apoptosis. Multicolor bioluminescence in vivo imaging allowed the investigation of tumor growth and NF-κB induction in a mouse model of breast cancer. Conclusion Our new method provides an imaging platform for the identification, validation, screening and optimization of compounds acting on NF-κB signaling and apoptosis both in vitro and in vivo. PMID:24465597

  4. Depressed natural killer cell activity in acute myocardial infarction

    DEFF Research Database (Denmark)

    Klarlund, K; Pedersen, B K; Theander, T G;

    1987-01-01

    Natural killer (NK) cell activity against K562 target cells was measured in patients within 24 h of acute myocardial infarction (AMI) and regularly thereafter for 6 weeks. NK cell activity was suppressed on days 1, 3, and 7 (P less than 0.01), day 14 (P less than 0.05) and at 6 weeks (P = 0...

  5. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M;

    1998-01-01

    human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...... induced apoptosis in several activated T-cell populations and T-cell lines, including T-cell antigen receptor (TCR)-CD3-negative T-cell lines. In contrast, RPE cells induced little or no apoptosis in resting peripheral T cells. Major histocompatibility complex (MHC) class II monoclonal antibodies, which...

  6. Sorting waste - A question of good will

    CERN Multimedia

    TS Department - FM Group

    2006-01-01

    In order to minimise waste-sorting costs, CERN provides two types of container at the entrance of buildings: a green plastic container for paper/cardboard and a metal container for household-type waste. We regret that recently there has been a significant decrease in the extent to which these types of waste are sorted, for example green containers have been found to hold assorted waste such as cardboard boxes filled with polystyrene, bubble-wrap or even plastic bottles, yoghurt pots, etc. Checks have shown that this 'non-compliant' waste does not come from the rubbish bins emptied by the cleaners but is deposited there directly by inconsiderate users. During the months of October and November alone, for example, only 15% of the waste from the paper/cardboard containers was recycled and the remaining 85% had to be incinerated, which entails a high cost for CERN. You should note that once an item of non-compliant waste is found in a green container its contents are immediately sent as waste to be incinerated ...

  7. Glycolipid-dependent sorting of melanosomal from lysosomal membrane proteins by lumenal determinants

    NARCIS (Netherlands)

    Groux-Degroote, S.; Dijk, S.M. van; Wolthoorn, J.; Neumann, S.; Theos, A.C.; Mazière, A.M. de; Klumperman, J.; Meer, G. van; Sprong, H.

    2008-01-01

    Melanosomes are lysosome-related organelles that coexist with lysosomes in mammalian pigment cells. Melanosomal and lysosomal membrane proteins share similar sorting signals in their cytoplasmic tail, raising the question how they are segregated. We show that in control melanocytes, the melanosomal

  8. Aquaporin-3 and aquaporin-4 are sorted differently and separately in the trans-Golgi network

    DEFF Research Database (Denmark)

    Christensen, Eva Arnspang; Sundbye, Sabrina Maria Gade; Nelson, W. James;

    2013-01-01

    , it is unknown if they are sorted together in the Golgi, or arrive in the same or different vesicles at the plasma membrane. We addressed these questions using high resolution deconvolution imaging, spinning disk and laser scanning confocal microscopy of cells expressing AQP3 and AQP4. AQP3 and AQP4 were...

  9. Single cell multiplexed assay for proteolytic activity using droplet microfluidics.

    Science.gov (United States)

    Ng, Ee Xien; Miller, Miles A; Jing, Tengyang; Chen, Chia-Hung

    2016-07-15

    Cellular enzymes interact in a post-translationally regulated fashion to govern individual cell behaviors, yet current platform technologies are limited in their ability to measure multiple enzyme activities simultaneously in single cells. Here, we developed multi-color Förster resonance energy transfer (FRET)-based enzymatic substrates and use them in a microfluidics platform to simultaneously measure multiple specific protease activities from water-in-oil droplets that contain single cells. By integrating the microfluidic platform with a computational analytical method, Proteolytic Activity Matrix Analysis (PrAMA), we are able to infer six different protease activity signals from individual cells in a high throughput manner (~100 cells/experimental run). We characterized protease activity profiles at single cell resolution for several cancer cell lines including breast cancer cell line MDA-MB-231, lung cancer cell line PC-9, and leukemia cell line K-562 using both live-cell and in-situ cell lysis assay formats, with special focus on metalloproteinases important in metastasis. The ability to measure multiple proteases secreted from or expressed in individual cells allows us to characterize cell heterogeneity and has potential applications including systems biology, pharmacology, cancer diagnosis and stem cell biology. PMID:26995287

  10. Hydrogen peroxide activates activator protein-1 and mitogen-activated protein kinases in pancreatic stellate cells.

    Science.gov (United States)

    Kikuta, Kazuhiro; Masamune, Atsushi; Satoh, Masahiro; Suzuki, Noriaki; Satoh, Kennichi; Shimosegawa, Tooru

    2006-10-01

    Activated pancreatic stellate cells (PSCs) are implicated in the pathogenesis of pancreatic inflammation and fibrosis, where oxidative stress is thought to play a key role. Reactive oxygen species such as hydrogen peroxide (H(2)O(2)) may act as a second messenger to mediate the actions of growth factors and cytokines. But the role of reactive oxygen species in the activation and regulation of cell functions in PSCs remains largely unknown. We here examined the effects of H(2)O(2) on the activation of signal transduction pathways and cell functions in PSCs. PSCs were isolated from the pancreas of male Wistar rats, and used in their culture-activated, myofibroblast-like phenotype unless otherwise stated. Activation of transcription factors was examined by electrophoretic mobility shift assay and luciferase assay. Activation of mitogen-activated protein (MAP) kinases was assessed by Western blotting using anti-phosphospecific antibodies. The effects of H(2)O(2) on proliferation, alpha(1)(I)procollagen gene expression, and monocyte chemoattractant protein-1 production were evaluated. The effect of H(2)O(2) on the transformation of freshly isolated PSCs in culture was also assessed. H(2)O(2) at non-cytotoxic concentrations (up to 100 microM) induced oxidative stress in PSCs. H(2)O(2) activated activator protein-1, but not nuclear factor kappaB. In addition, H(2)O(2) activated three classes of MAP kinases: extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAP kinase. H(2)O(2) induced alpha(1)(I)procollagen gene expression but did not induce proliferation or monocyte chemoattractant protein-1 production. H(2)O(2) did not initiate the transformation of freshly isolated PSCs to myofibroblast-like phenotype. Specific activation of these signal transduction pathways and collagen gene expression by H(2)O(2) may play a role in the pathogenesis of pancreatic fibrosis.

  11. Myeloid-derived Suppressor Cells Inhibit T Cell Activation by Depleting Cystine and Cysteine

    OpenAIRE

    Minu K Srivastava; Sinha, Pratima; Clements, Virginia K.; Rodriguez, Paulo; Ostrand-Rosenberg, Suzanne

    2009-01-01

    Myeloid-derived suppressor cells (MDSC) are present in most cancer patients and are potent inhibitors of T-cell-mediated anti-tumor immunity. Their inhibitory activity is attributed to production of arginase, reactive oxygen species, inducible nitric oxide synthase, and IL-10. We now report that MDSC also block T cell activation by sequestering cystine and limiting the availability of cysteine. Cysteine is an essential amino acid for T cell activation because T cells lack cystathionase, which...

  12. The adhesion receptor CD44 promotes atherosclerosis by mediating inflammatory cell recruitment and vascular cell activation

    OpenAIRE

    Cuff, Carolyn A.; Kothapalli, Devashish; Azonobi, Ijeoma; Chun, Sam; Zhang, Yuanming; Belkin, Richard; Yeh, Christine; Secreto, Anthony; Richard K Assoian; Rader, Daniel J; Puré, Ellen

    2001-01-01

    Atherosclerosis causes most acute coronary syndromes and strokes. The pathogenesis of atherosclerosis includes recruitment of inflammatory cells to the vessel wall and activation of vascular cells. CD44 is an adhesion protein expressed on inflammatory and vascular cells. CD44 supports the adhesion of activated lymphocytes to endothelium and smooth muscle cells. Furthermore, ligation of CD44 induces activation of both inflammatory and vascular cells. To assess the potential contribution of CD4...

  13. Recruitment of activation receptors at inhibitory NK cell immune synapses.

    Directory of Open Access Journals (Sweden)

    Nicolas Schleinitz

    Full Text Available Natural killer (NK cell activation receptors accumulate by an actin-dependent process at cytotoxic immune synapses where they provide synergistic signals that trigger NK cell effector functions. In contrast, NK cell inhibitory receptors, including members of the MHC class I-specific killer cell Ig-like receptor (KIR family, accumulate at inhibitory immune synapses, block actin dynamics, and prevent actin-dependent phosphorylation of activation receptors. Therefore, one would predict inhibition of actin-dependent accumulation of activation receptors when inhibitory receptors are engaged. By confocal imaging of primary human NK cells in contact with target cells expressing physiological ligands of NK cell receptors, we show here that this prediction is incorrect. Target cells included a human cell line and transfected Drosophila insect cells that expressed ligands of NK cell activation receptors in combination with an MHC class I ligand of inhibitory KIR. The two NK cell activation receptors CD2 and 2B4 accumulated and co-localized with KIR at inhibitory immune synapses. In fact, KIR promoted CD2 and 2B4 clustering, as CD2 and 2B4 accumulated more efficiently at inhibitory synapses. In contrast, accumulation of KIR and of activation receptors at inhibitory synapses correlated with reduced density of the integrin LFA-1. These results imply that inhibitory KIR does not prevent CD2 and 2B4 signaling by blocking their accumulation at NK cell immune synapses, but by blocking their ability to signal within inhibitory synapses.

  14. Corner Sort for Pareto-Based Many-Objective Optimization.

    Science.gov (United States)

    Wang, Handing; Yao, Xin

    2014-01-01

    Nondominated sorting plays an important role in Pareto-based multiobjective evolutionary algorithms (MOEAs). When faced with many-objective optimization problems multiobjective optimization problems (MOPs) with more than three objectives, the number of comparisons needed in nondominated sorting becomes very large. In view of this, a new corner sort is proposed in this paper. Corner sort first adopts a fast and simple method to obtain a nondominated solution from the corner solutions, and then uses the nondominated solution to ignore the solutions dominated by it to save comparisons. Obtaining the nondominated solutions requires much fewer objective comparisons in corner sort. In order to evaluate its performance, several state-of-the-art nondominated sorts are compared with our corner sort on three kinds of artificial solution sets of MOPs and the solution sets generated from MOEAs on benchmark problems. On one hand, the experiments on artificial solution sets show the performance on the solution sets with different distributions. On the other hand, the experiments on the solution sets generated from MOEAs show the influence that different sorts bring to MOEAs. The results show that corner sort performs well, especially on many-objective optimization problems. Corner sort uses fewer comparisons than others.

  15. Activation of intracellular angiotensin AT2 receptors induces rapid cell death in human uterine leiomyosarcoma cells

    DEFF Research Database (Denmark)

    Zhao, Yi; Lützen, Ulf; Fritsch, Jürgen;

    2015-01-01

    densities in mitochondria. Activation of the cell membrane AT2 receptors by a concomitant treatment with angiotensin II and the AT1 receptor antagonist, losartan, induces apoptosis but does not affect the rate of cell death. We demonstrate for the first time that the high-affinity, non-peptide AT2 receptor...... of apoptosis and cell death in cultured human uterine leiomyosarcoma (SK-UT-1) cells and control human uterine smooth muscle cells (HutSMC). The intracellular levels of the AT2 receptor are low in proliferating SK-UT-1 cells but the receptor is substantially up-regulated in quiescent SK-UT-1 cells with high...... agonist, Compound 21 (C21) penetrates the cell membrane of quiescent SK-UT-1 cells, activates intracellular AT2 receptors and induces rapid cell death; approximately 70% of cells died within 24 h. The cells, which escaped from the cell death, displayed activation of the mitochondrial apoptotic pathway, i...

  16. Human Liver Stem Cells Suppress T-Cell Proliferation, NK Activity, and Dendritic Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Stefania Bruno

    2016-01-01

    Full Text Available Human liver stem cells (HLSCs are a mesenchymal stromal cell-like population resident in the adult liver. Preclinical studies indicate that HLSCs could be a good candidate for cell therapy. The aim of the present study was to evaluate the immunogenicity and the immunomodulatory properties of HLSCs on T-lymphocytes, natural killer cells (NKs, and dendritic cells (DCs in allogeneic experimental settings. We found that HLSCs inhibited T-cell proliferation by a mechanism independent of cell contact and dependent on the release of prostaglandin E2 (PGE2 and on indoleamine 2,3-dioxygenase activity. When compared with mesenchymal stromal cells (MSCs, HLSCs were more efficient in inhibiting T-cell proliferation. At variance with MSCs, HLSCs did not elicit NK degranulation. Moreover, HLSCs inhibited NK degranulation against K562, a NK-sensitive target, by a mechanism dependent on HLA-G release. When tested on DC generation from monocytes, HLSCs were found to impair DC differentiation and DCs ability to induce T-cell proliferation through PGE2. This study shows that HLSCs have immunomodulatory properties similar to MSCs, but, at variance with MSCs, they do not elicit a NK response.

  17. Interleukin 4 (B cell stimulatory factor 1) can mediate the induction of lymphokine-activated killer cell activity directed against fresh tumor cells

    OpenAIRE

    1987-01-01

    Interleukin 4 (IL-4) expresses multiple biologic activities, including B cell, mast cell, and T cell stimulation. We showed that the incubation of resting splenocytes from C57BL/6 mice solely in purified native or recombinant mouse IL-4 results in the generation of lymphokine-activated killer (LAK) activity directed against fresh, syngeneic sarcoma cells. The precursor activated by IL-4 expresses surface asialo-GM1. In addition, IL-4 is capable of amplifying the splenic LAK activity induced b...

  18. Mycoplasma pneumoniae induces cytotoxic activity in guinea pig bronchoalveolar cells

    International Nuclear Information System (INIS)

    Precultured guinea pig alveolar macrophages (AM) and freshly harvested alveolar cells (FHAC) activated by interaction with Mycoplasma pneumoniae were cytotoxic for xenogeneic 75selenomethionine-labeled tumor target cells. Phagocytosis of whole opsonized or nonopsonized M. pneumoniae cells was more effective in eliciting cytotoxicity than uptake of sonicated microorganisms. The addition of living mycoplasma cells to the assay system enhanced the cytotoxic effect considerably. Target cells were significantly more susceptible to the cytotoxic action of phagocytes if they were coated with mycoplasma antigen or cocultured together with M. pneumoniae. The activation of the phagocytes could be inhibited by 2-deoxy-D-glucose but not by antimicrobial substances suppressing mycoplasma protein synthesis. It was accompanied by 51Cr release without detectable signs of cell damage. The supernatants of activated cells were cytotoxic for approximately 24 h. Inhibition, release, and cytotoxic activity indicate the necessity of an intact metabolism of the effector cells and suggest a secretion of cytotoxic substances

  19. Tolerogenicity of resting and activated B cells

    OpenAIRE

    1994-01-01

    Antigen presentation by resting splenic B cells has been shown previously to induce T helper 1 cell (Th1) anergy. In contrast to expectations, it was found here that B cells treated with F(ab')2 goat anti-mouse immunoglobulin (IgM) for 24 or 48 h also presented antigen (Ag) to Th1 cells in a manner that induced dramatic Ag-specific proliferative inactivation. The tolerogenicity of the anti-Ig-treated B cells was consistent with the observation that these B cells were only slightly more effici...

  20. Cholangiocarcinoma-derived exosomes inhibit the antitumor activity of cytokine-induced killer cells by down-regulating the secretion of tumor necrosis factor-α and perforin*

    Science.gov (United States)

    Chen, Jiong-huang; Xiang, Jian-yang; Ding, Guo-ping; Cao, Li-ping

    2016-01-01

    Objective: The aim of our study is to observe the impact of cholangiocarcinoma-derived exosomes on the antitumor activities of cytokine-induced killer (CIK) cells and then demonstrate the appropriate mechanism. Methods: Tumor-derived exosomes (TEXs), which are derived from RBE cells (human cholangiocarcinoma line), were collected by ultracentrifugation. CIK cells induced from peripheral blood were stimulated by TEXs. Fluorescence-activated cell sorting (FACS) was performed to determine the phenotypes of TEX-CIK and N-CIK (normal CIK) cells. The concentrations of tumor necrosis factor-α (TNF-α) and perforin in the culture medium supernatant were examined by using an enzyme-linked immunosorbent assay (ELISA) kit. A CCK-8 kit was used to evaluate the cytotoxic activity of the CIK cells to the RBE cell line. Results: The concentrations of TNF-α and perforin of the group TEX-CIK were 138.61 pg/ml and 2.41 ng/ml, respectively, lower than those of the group N-CIK 194.08 pg/ml (Pexosomes inhibit the antitumor activity of CIK cells by down-regulating the population of CD3+, CD8+, NK (CD56+), and CD3+CD56+ cells and the secretion of TNF-α and perforin. TEX may play an important role in cholangiocarcinoma immune escape. PMID:27381730

  1. Dengue Virus Directly Stimulates Polyclonal B Cell Activation

    Science.gov (United States)

    Papa, Michelle Premazzi; de Morais, Ana Theresa Silveira; Peçanha, Ligia Maria Torres; de Arruda, Luciana Barros

    2015-01-01

    Dengue infection is associated to vigorous inflammatory response, to a high frequency of activated B cells, and to increased levels of circulating cross-reactive antibodies. We investigated whether direct infection of B cells would promote activation by culturing primary human B lymphocytes from healthy donors with DENV in vitro. B cells were susceptible, but poorly permissive to infection. Even though, primary B cells cultured with DENV induced substantial IgM secretion, which is a hallmark of polyclonal B cell activation. Notably, DENV induced the activation of B cells obtained from either DENV immune or DENV naïve donors, suggesting that it was not dependent on DENV-specific secondary/memory response. B cell stimulation was dependent on activation of MAPK and CD81. B cells cultured with DENV also secreted IL-6 and presented increased expression of CD86 and HLA-DR, which might contribute to B lymphocyte co-stimulatory function. Indeed, PBMCs, but not isolated B cells, secreted high amounts of IgG upon DENV culture, suggesting that interaction with other cell types in vivo might promote Ig isotype switching and IgG secretion from different B cell clones. These findings suggest that activation signaling pathways triggered by DENV interaction with non-specific receptors on B cells might contribute to the exacerbated response observed in dengue patients. PMID:26656738

  2. The DNA methylation profile of activated human natural killer cells.

    Science.gov (United States)

    Wiencke, John K; Butler, Rondi; Hsuang, George; Eliot, Melissa; Kim, Stephanie; Sepulveda, Manuel A; Siegel, Derick; Houseman, E Andres; Kelsey, Karl T

    2016-05-01

    Natural killer (NK) cells are now recognized to exhibit characteristics akin to cells of the adaptive immune system. The generation of adaptive memory is linked to epigenetic reprogramming including alterations in DNA methylation. The study herein found reproducible genome wide DNA methylation changes associated with human NK cell activation. Activation led predominately to CpG hypomethylation (81% of significant loci). Bioinformatics analysis confirmed that non-coding and gene-associated differentially methylated sites (DMS) are enriched for immune related functions (i.e., immune cell activation). Known DNA methylation-regulated immune loci were also identified in activated NK cells (e.g., TNFA, LTA, IL13, CSF2). Twenty-one loci were designated high priority and further investigated as potential markers of NK activation. BHLHE40 was identified as a viable candidate for which a droplet digital PCR assay for demethylation was developed. The assay revealed high demethylation in activated NK cells and low demethylation in naïve NK, T- and B-cells. We conclude the NK cell methylome is plastic with potential for remodeling. The differentially methylated region signature of activated NKs revealed similarities with T cell activation, but also provided unique biomarker candidates of NK activation, which could be useful in epigenome-wide association studies to interrogate the role of NK subtypes in global methylation changes associated with exposures and/or disease states. PMID:26967308

  3. Remote Control of T Cell Activation Using Magnetic Janus Particles.

    Science.gov (United States)

    Lee, Kwahun; Yi, Yi; Yu, Yan

    2016-06-20

    We report a strategy for using magnetic Janus microparticles to control the stimulation of T cell signaling with single-cell precision. To achieve this, we designed Janus particles that are magnetically responsive on one hemisphere and stimulatory to T cells on the other side. By manipulating the rotation and locomotion of Janus particles under an external magnetic field, we could control the orientation of the particle-cell recognition and thereby the initiation of T cell activation. This study demonstrates a step towards employing anisotropic material properties of Janus particles to control single-cell activities without the need of complex magnetic manipulation devices.

  4. Passive chip-based droplet sorting

    Energy Technology Data Exchange (ETDEWEB)

    Beer, Neil Reginald; Lee, Abraham P; Hatch, Andrew C; Fisher, Jeffrey S

    2015-11-05

    An apparatus for passive sorting of microdroplets including a main flow channel, a flow stream of microdroplets in the main flow channel wherein the microdroplets have substantially the same diameter and wherein the flow stream of microdroplets includes first microdroplets having a first degree of stiffness and second microdroplets having a second degree of stiffness wherein the second degree of stiffness is different than the first degree of stiffness. A second flow channel is connected to the main flow channel for the second microdroplets having a second degree of stiffness. A separator separates the second microdroplets having a second degree of stiffness from the first microdroplets and directs the second microdroplets having a second degree of stiffness into the second flow channel.

  5. Radiometric sorting of Rio Algom uranium ore

    International Nuclear Information System (INIS)

    An ore sample of about 0.2 percent uranium from Quirke Mine was subjected to radiometric sorting by Ore Sorters Limited. Approximately 60 percent of the sample weight fell within the sortable size range: -150 + 25 mm. Rejects of low uranium content (2 (2 counts/in2) but only 7.6 percent of the ore, by weight, was discarded. At 0.8-0.9 counts/cm2 (5-6 counts/in2) a significant amount of rejects was removed (> 25 percent) but the uranium loss was unacceptably high (7.7 percent). Continuation of the testwork to improve the results is proposed by trying to extend the sortable size range and to reduce the amount of fines during crushing

  6. Utjecaj sorte jabuke na kvalitetu suhog proizvoda

    OpenAIRE

    Dobričević, Nadica; Voća, Sandra; Pliestić, Stjepan; Magdić, D.

    2008-01-01

    Sorte jabuka Gloster, Jonagold, Idared, Melrose i Mutsu s pro-izvodnog područja sjeverozapadne Hrvatske ubrane su u tehnološkoj zriobi i skladištene u rashladnom prostoru na prosječnoj temperaturi od 2,0 oC i relativnoj vlazi zraka od 98% do užitne dospjelosti. Plodovi jabuka namijenjeni za sušenje očišćeni su i rezani na kockice veličine 1x1x1 cm. Količina mesa u analiziranim sortama je 63,37-75,46 %, količina kožica je 9,11-12,85 %, količina sjemene lože je 10,82-20,34 % i količina neupo...

  7. Passive chip-based droplet sorting

    Energy Technology Data Exchange (ETDEWEB)

    Beer, Neil Reginald; Lee, Abraham P; Hatch, Andrew C; Fisher, Jeffrey S

    2015-03-03

    An apparatus for passive sorting of microdroplets including a main flow channel, a flow stream of microdroplets in the main flow channel wherein the microdroplets have substantially the same diameter and wherein the flow stream of microdroplets includes first microdroplets having a first degree of stiffness and second microdroplets having a second degree of stiffness wherein the second degree of stiffness is different than the first degree of stiffness. A second flow channel is connected to the main flow channel for the second microdroplets having a second degree of stiffness. A separator separates the second microdroplets having a second degree of stiffness from the first microdroplets and directs the second microdroplets having a second degree of stiffness into the second flow channel.

  8. Carbon Nanotube–Purification and Sorting Protocols

    Directory of Open Access Journals (Sweden)

    Poornendu Chaturvedi

    2008-09-01

    Full Text Available Carbon nanotubes (CNTs have shown extraordinary mechanical, thermal, electrical, and electronic properties. Electronic properties of CNT are very sensitive to its diameter and chirality, making it metallicor semiconducting, depending upon its chiral vector. The extraordinary properties of CNTs have led to demonstration of several applications but commercial realisation of these devices require consistent qualityof CNTs, and these should be  free of any impurity. For development of electronic devices, CNTs should notjust be pure but also of similar length, diameter, and electronic behaviour. Such demanding requirements need development of elaborate purification and sorting protocols. In this paper,  a brief review of the existing technologies and the research done is presented.Defence Science Journal, 2008, 58(5, pp.591-599, DOI:http://dx.doi.org/10.14429/dsj.58.1694

  9. Solution structure of human sorting nexin 22.

    Science.gov (United States)

    Song, Jikui; Zhao, Kate Qin; Newman, Carrie L Loushin; Vinarov, Dmitriy A; Markley, John L

    2007-05-01

    The sorting nexins (SNXs) constitute a large group of PX domain-containing proteins that play critical roles in protein trafficking. We report here the solution structure of human sorting nexin 22 (SNX22). Although SNX22 has <30% sequence identity with any PX domain protein of known structure, it was found to contain the alpha/beta fold and compact structural core characteristic of PX domains. Analysis of the backbone dynamics of SNX22 by NMR relaxation measurements revealed that the two walls of the ligand binding cleft undergo internal motions: on the picosecond timescale for the beta1/beta2 loop and on the micro- to millisecond timescale for the loop between the polyproline motif and helix alpha2. Regions of the SNX22 structure that differ from those of other PX domains include the loop connecting strands beta1 and beta2 and the loop connecting helices alpha1 and alpha2, which appear to be more mobile than corresponding loops in other known structures. The interaction of dibutanoyl-phosphatidylinositol-3-phosphate (dibutanoyl-PtdIns(3)P) with SNX22 was investigated by an NMR titration experiment, which identified the binding site in a basic cleft and indicated that ligand binding leads only to a local structural rearrangement as has been found with other PX domains. Because motions in the loops are damped out when dibutanoyl-PtdIns(3)P binds, entropic effects could contribute to the lower affinity of SNX22 for this ligand compared to other PX domains. PMID:17400918

  10. Preferences and Choice Constraints in Marital Sorting: Evidence From Korea

    OpenAIRE

    Soohyung Lee

    2008-01-01

    Marital sorting along education, income and other salient dimensions is well-documented for many countries. The degree of marital sorting may influence income inequality, intergenerational mobility, and household labor supply, and other economic outcomes. Marital sorting is thought to arise from some combination of people’s preferences and constraints on their choice sets. However, separating these two causes is difficult because typical data sets provide information on either a person’s spou...

  11. Sort sourceof pea in Ukraine and in the world

    OpenAIRE

    ШЕВЧЕНКО А.М.

    2006-01-01

    The sorts different by economic-biologic character of unshed seed, breeded in Ukraine and abroad obtained wide spreading in production. Expediency using lugansky or samarsky type of determinant stability to healthy growth. Breeding sorts with tendril type of leaf give the positive results in increase stability of the plants to damping-off. The sorts combining named recessive character with another aconomic-value traits of grain pea made and suggested to production.

  12. Retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Zhang Yingang; Guo Xiong; Liu Zheng; Wang Shijie

    2007-01-01

    Objective To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells. Methods Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-BMP2 vector by the optimized retroviral transduction protocol. Fluorescent microscopy's examination was to evaluate the results of the transduction, flow cytometer's analysis was to evaluate the transduction efficiency and the Fluorescence-activated cell sorting method was to sort the transduced cells. Bioactivity test from C2C12K4 cells was to show the expression and bio-activity of the fusion gene. Results Fluorescent microscopy showed the success of the transduction. By flow cytometer's analysis, the mean efficiency of the transduction with EGFP was (42.8±6.1)% SD. Transduced cells were sorted efficiently by the fluorescence-activated cell sorting method and after sorting, almost of those showed the expression of BMP2. Fluorescently and strongly bioactivity test for C2C12K4 cells demonstrated that fluorescent materials were located the surface of cells and the activity of luciferase increased compared with the control. Analysis of long-term expression showed there was no difference between 2 week-time point and 3 month-time point of culture post-sorting. Conclusion Mesenchymal stem cells can be transduced efficiently by retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2, the highly pure transduced cells are obtained by the fluorescence-activated cell sorting technique, the expressed chimeric protein embraced the double bioactivity of EGFP and BMP2, and moreover, the expression had not attenuated over time.

  13. Cisplatin-induced Casepase-3 activation in different tumor cells

    Science.gov (United States)

    Shi, Hua; Li, Xiao; Su, Ting; Zhang, Yu-Hai

    2008-12-01

    Apoptosis plays an essential role in normal organism development which is one of the main types of programmed cell death to help tissues maintain homeostasis. Defective apoptosis can result in cell accumulation and therefore effects on tumor pathogenesis, progression and therapy resistance. A family of proteins, known as caspases, is typically activated in the early stages of apoptosis. Therefore, studying the kinetics of activation of caspases induced by antitumor drugs can contribute to antitumor drug discovery and explanation of the molecular mechanisms. This paper detected the Caspase-3 activity induced by cisplatin in human adenoid cystic carcinoma cell line (ACC-M), human hepatocellular liver carcinoma cell line (HepG2) and human epithelial carcinoma cell line (Hela) with stably expressing ECFP-DEVDDsRed (CD3) probe, a fluorescent probe consisting of Enhanced Cyan Fluorescent Protein (ECFP), red fluorescent protein (DsRed) and a linker with a recognition site of Caspase-3, by using the capillary electrophoresis (CE) and fluorescence resonance energy transfer (FRET) imaging system. Under the same concentration of cisplatin, ACC-M cells responded the most rapidly, and then HepG2 cells and Hela cells, respectively, in the early 30 hours. Later, HepG2 cells represented acceleration in the Caspase-3 activation speed and reached full activation the earliest comparing to other two cell types. The results demonstrated that ACC-M cell is more sensitive than the other two cell types under the treatment of cisplatin.

  14. Cortisol patterns are associated with T cell activation in HIV.

    Directory of Open Access Journals (Sweden)

    Sarah Patterson

    Full Text Available The level of T cell activation in untreated HIV disease is strongly and independently associated with risk of immunologic and clinical progression. The factors that influence the level of activation, however, are not fully defined. Since endogenous glucocorticoids are important in regulating inflammation, we sought to determine whether less optimal diurnal cortisol patterns are associated with greater T cell activation.We studied 128 HIV-infected adults who were not on treatment and had a CD4(+ T cell count above 250 cells/µl. We assessed T cell activation by CD38 expression using flow cytometry, and diurnal cortisol was assessed with salivary measurements.Lower waking cortisol levels correlated with greater T cell immune activation, measured by CD38 mean fluorescent intensity, on CD4(+ T cells (r = -0.26, p = 0.006. Participants with lower waking cortisol also showed a trend toward greater activation on CD8(+ T cells (r = -0.17, p = 0.08. A greater diurnal decline in cortisol, usually considered a healthy pattern, correlated with less CD4(+ (r = 0.24, p = 0.018 and CD8(+ (r = 0.24, p = 0.017 activation.These data suggest that the hypothalamic-pituitary-adrenal (HPA axis contributes to the regulation of T cell activation in HIV. This may represent an important pathway through which psychological states and the HPA axis influence progression of HIV.

  15. Interfering with interferon receptor sorting and trafficking: impact on signaling.

    Science.gov (United States)

    Claudinon, Julie; Monier, Marie-Noëlle; Lamaze, Christophe

    2007-01-01

    Interferons (IFNs) and their receptors (IFN-Rs) play fundamental roles in a multitude of biological functions. Many articles and reviews emphasize that the JAK/STAT machinery is obligatory for relay of the information transmitted by IFNs after binding to their cognate receptors at the plasma membrane. In contrast, very few studies have addressed the endocytosis and the intracellular trafficking of IFN-Rs, the immediate step following IFN binding. However, recent findings have shed light on the importance of IFN-R sorting and trafficking in the control of IFN signaling. Thus, IFN-Rs can be included in the growing family of signaling receptors for which regulation of biological activity critically involves endocytosis and trafficking. PMID:17493737

  16. A many-sorted calculus based on resolution and paramodulation

    CERN Document Server

    Walther, Christoph

    1987-01-01

    A Many-Sorted Calculus Based on Resolution and Paramodulation emphasizes the utilization of advantages and concepts of many-sorted logic for resolution and paramodulation based automated theorem proving.This book considers some first-order calculus that defines how theorems from given hypotheses by pure syntactic reasoning are obtained, shifting all the semantic and implicit argumentation to the syntactic and explicit level of formal first-order reasoning. This text discusses the efficiency of many-sorted reasoning, formal preliminaries for the RP- and ?RP-calculus, and many-sorted term rewrit

  17. 宫颈不典型鳞状细胞232例临床分流方法探讨%Clinical Sorting Method of 232 Cases with Atypical Squamous Cells

    Institute of Scientific and Technical Information of China (English)

    杨冬; 诸雪峻

    2011-01-01

    Objective:To explore the correct diagnosis and proper sorting method in women with ASC. Methods:232 Women diagnosed as ASC were included in this study, and underwent colposcopy and hr-HPV test (Hybrid Capture 2). Results:(I)ln the 232 cases of ASC, 214 cases of ASCUS ( 92.2%)and 18 cases of ASCH (7. 8%) were found. ?The positive result of cervical biopsy and CIN11 or higher grade lesion in ASCUS or ASCH groups were 98 cases(45. 8%) ,18 caes(8.4%) ,and 15 cases(83.3%), 6 cases (33. 3%) ,the differences between the two groups were significant (P=0. 003;P=0. 005). In cases with ASCUS, 97 cases of hr-HPV Test were positive, in which 77.3% cases were with positive biopsy, 16. 5% cases were CIN U or higher grade disease. While, 117 cases of HPV test were negative, in which 19.7% cases were positive biopsy, 1.7% cases were CIN n , no higher grade disease. (3)The sensitivity and specificity of hr-HPV test for CIN and cervical cancer were 76. 5% and 81.0%, while those of colposcopy were 83.7% and 77.6% respectively, no differences were found between the two groups( P=0.283; P=0.627). Conclusions: ASCH strongly predicts the presence of HSIL and cervical cancer, colposcopic examination is the proper choice. HPV test and colposcopic examination are effective sorting methods for patients with ASCUS. For HPV negative patients, repeat pap smear in 6 month is proper, while for HPV positive patients,colposcopic examination is recommended.%目的:探讨宫颈不典型鳞状细胞(ASC)正确诊断、合理临床分流方法.方法:232例ASC患者接受阴道镜下宫颈活检,并采用第二代杂交捕获法检测高危型人乳头瘤病毒(HPV).结果:①232例ASC中未明确诊断意义的不典型鳞状上皮细胞(ASCUS)Z14例(92.2%),不排除高度病变的不典型鳞状上皮细胞(ASCH)18例(7.8%).②ASCUS组和ASCH组宫颈活检阳性患者、CINⅡ-Ⅲ及宫颈浸润癌患者分别为98例(45.8%)、18例(8.4%)和15例(83.3%)、6例(33.3%),两组间比较差

  18. Heteronomous rhythmic activity of neurosecretory cells in the silkmoth.

    Science.gov (United States)

    Ichikawa, Toshio; Kamimoto, Satoshi

    2003-08-21

    Electrical action potentials of neurosecretory cells producing pheromone biosynthesis-activating neuropeptide (PBAN) and electrocardiograms were recorded from female pupae of Bombyx mori and the correlation between firing activity of the cells and cardiac activity was analyzed. PBAN producing cells localized in the suboesophageal ganglion (SOG) generated clusters of action potentials at an interval of 30-60 min. The firing activity rhythm at a middle pupal period was closely related to heartbeat reversal rhythm: an active phase of the cells was usually apparent during anterograde pulse phases. Electrocardiograms at a late pupal period often revealed brief oscillatory potentials (15-25 Hz in frequency) of unknown origin. The firing activity rhythm of PBAN cells closely correlated with the rhythmic appearance of clustered oscillatory potentials. Transection of connectives between the brain and SOG abolished rhythmic activity of the cells. These results suggest that a rhythmic firing activity of the PBAN cell system is heteronomously generated by a cerebral neuronal mechanism and the cerebral mechanism relates the cell system to other neuronal mechanisms controlling cardiac activity and oscillatory potential rhythms. PMID:12873731

  19. Activated Notch Causes Deafness by Promoting a Supporting Cell Phenotype in Developing Auditory Hair Cells

    OpenAIRE

    Grace Savoy-Burke; Felicia A Gilels; Wei Pan; Diana Pratt; Jianwen Que; Lin Gan; White, Patricia M.; Kiernan, Amy E.

    2014-01-01

    Purpose To determine whether activated Notch can promote a supporting cell fate during sensory cell differentiation in the inner ear. Methods An activated form of the Notch1 receptor (NICD) was expressed in early differentiating hair cells using a Gfi1-Cre mouse allele. To determine the effects of activated Notch on developing hair cells, Gfi1-NICD animals and their littermate controls were assessed at 5 weeks for hearing by measuring auditory brainstem responses (ABRs) and distortion product...

  20. A minimal model for spontaneous cell polarization and edge activity in oscillating, rotating and migrating cells

    CERN Document Server

    Raynaud, Franck; Gabella, Chiara; Bornert, Alicia; Sbalzarini, Ivo F; Meister, Jean-Jacques; Verkhovsky, Alexander B

    2016-01-01

    How the cells break symmetry and organize their edge activity to move directionally is a fun- damental question in cell biology. Physical models of cell motility commonly rely on gradients of regulatory factors and/or feedback from the motion itself to describe polarization of edge activity. Theses approaches, however, fail to explain cell behavior prior to the onset of polarization. Our analysis using the model system of polarizing and moving fish epidermal keratocytes suggests a novel and simple principle of self-organization of cell activity in which local cell-edge dynamics depends on the distance from the cell center, but not on the orientation with respect to the front-back axis. We validate this principle with a stochastic model that faithfully reproduces a range of cell-migration behaviors. Our findings indicate that spontaneous polarization, persistent motion, and cell shape are emergent properties of the local cell-edge dynamics controlled by the distance from the cell center.

  1. Exosomes released from Mycoplasma infected tumor cells activate inhibitory B cells.

    Directory of Open Access Journals (Sweden)

    Chenjie Yang

    Full Text Available Mycoplasmas cause numerous human diseases and are common opportunistic pathogens in cancer patients and immunocompromised individuals. Mycoplasma infection elicits various host immune responses. Here we demonstrate that mycoplasma-infected tumor cells release exosomes (myco+ exosomes that specifically activate splenic B cells and induce splenocytes cytokine production. Induction of cytokines, including the proinflammatory IFN-γ and the anti-inflammatory IL-10, was largely dependent on the presence of B cells. B cells were the major IL-10 producers. In splenocytes from B cell deficient μMT mice, induction of IFN-γ+ T cells by myco+ exosomes was greatly increased compared with wild type splenocytes. In addition, anti-CD3-stimulated T cell proliferation was greatly inhibited in the presence of myco+ exosome-treated B cells. Also, anti-CD3-stimulated T cell signaling was impaired by myco+ exosome treatment. Proteomic analysis identified mycoplasma proteins in exosomes that potentially contribute to the effects. Our results demonstrate that mycoplasma-infected tumor cells release exosomes carrying mycoplasma components that preferentially activate B cells, which in turn, are able to inhibit T cell activity. These results suggest that mycoplasmas infecting tumor cells can exploit the exosome pathway to disseminate their own components and modulate the activity of immune cells, in particular, activate B cells with inhibitory activity.

  2. SORT:Ed - An Interactive Educational Game for HealthCare Students

    OpenAIRE

    Currell, Karen

    2011-01-01

    This short paper outlines an academic’s entrepreneurial journey from idea conception to the market place. Sort Ed is an interactive board game designed for paediatric student nurses and it set in a child’s ward. There is a huge market demand for this type of educational games in the UK especially by institutions that run healthcare management courses. This learning tool is a major contribution to the limited number of interactive educational games currently available to healthcare tutors and ...

  3. In vitro separation and expansion of CD4 lymphocytes from HIV-infected individuals without activation of HIV infection

    DEFF Research Database (Denmark)

    Nielsen, S D; Nielsen, Jens Ole; Hansen, J E

    1997-01-01

    In order to offer a gene therapy-based treatment against AIDS, it is likely to be necessary to harvest and culture CD4 cells from HIV-positive patients without activating the HIV infection. We have used a magnetic cell sorting (MACS) system to enrich CD4 cells. Using positive selection, CD4 cells...

  4. Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high-resolution flow cytometry.

    Science.gov (United States)

    Groot Kormelink, Tom; Arkesteijn, Ger J A; Nauwelaers, Frans A; van den Engh, Ger; Nolte-'t Hoen, Esther N M; Wauben, Marca H M

    2016-02-01

    Submicron-sized vesicles released by cells are increasingly recognized for their role in intercellular communication and as biomarkers of disease. Methods for high-throughput, multi-parameter analysis of such extracellular vesicles (EVs) are crucial to further investigate their diversity and function. We recently developed a high-resolution flow cytometry-based method (using a modified BD Influx) for quantitative and qualitative analysis of EVs. The fact that the majority of EVs is particle concentrations affect high-resolution flow cytometry-based particle quantification and characterization. Increasing concentrations of submicron-sized particles (beads, liposomes, and EVs) were measured to identify coincidence and swarm effects, caused by the concurrent presence of multiple particles in the measuring spot. As a result, we demonstrate that analysis of highly concentrated samples resulted in an underestimation of the number of particles and an interdependent overestimation of light scattering and fluorescence signals. On the basis of this knowledge, and by varying nozzle size and sheath pressure, we developed a strategy for high-resolution flow cytometric sorting of submicron-sized particles. Using the adapted sort settings, subsets of EVs differentially labeled with two fluorescent antibodies could be sorted to high purity. Moreover, sufficient numbers of EVs could be sorted for subsequent analysis by western blotting. In conclusion, swarm effects that occur when measuring high particle concentrations severely hamper EV quantification and characterization. These effects can be easily overlooked without including proper controls (e.g., sample dilution series) or tools (e.g., oscilloscope). Providing that the event rate is well controlled, the sorting strategy we propose here indicates that high-resolution flow cytometric sorting of different EV subsets is feasible. PMID:25688721

  5. Looking for a Symphony: A Sort of Essay with a Perspective on Activity Theories and the Ontology of Psychology: Learning from Danish and Russian Experiences by Jens Mammen & Irina Mironenko.

    Science.gov (United States)

    Neumann, Asger

    2016-06-01

    As a perspective on Mammen and Miroenkos the article is reflecting on the possibility of Activity Theory being a foundation on which Psychology could be integrated. Mammen and Miroenkos point that directed activity not only is towards objects "defined as a sum of qualities, but by individual reference" is a starting point. As a specific example the phenomenon Love, as "significant object relations", is related to the concept "choice categories". It is stated that relations of affection and love can't be understood independent of history of common activity, and that this makes the concept "choice categories" central in a psychological understanding of what love is. PMID:26476531

  6. Borrelia burgdorferi Spirochetes Induce Mast Cell Activation and Cytokine Release

    Science.gov (United States)

    Talkington, Jeffrey; Nickell, Steven P.

    1999-01-01

    The Lyme disease spirochete, Borrelia burgdorferi, is introduced into human hosts via tick bites. Among the cell types present in the skin which may initially contact spirochetes are mast cells. Since spirochetes are known to activate a variety of cell types in vitro, we tested whether B. burgdorferi spirochetes could activate mast cells. We report here that freshly isolated rat peritoneal mast cells or mouse MC/9 mast cells cultured in vitro with live or freeze-thawed B. burgdorferi spirochetes undergo low but detectable degranulation, as measured by [5-3H] hydroxytryptamine release, and they synthesize and secrete the proinflammatory cytokine tumor necrosis factor alpha (TNF-α). In contrast to findings in previous studies, where B. burgdorferi-associated activity was shown to be dependent upon protein lipidation, mast cell TNF-α release was not induced by either lipidated or unlipidated recombinant OspA. This activity was additionally shown to be protease sensitive and surface expressed. Finally, comparisons of TNF-α-inducing activity in known low-, intermediate-, and high-passage B. burgdorferi B31 isolates demonstrated passage-dependent loss of activity, indicating that the activity is probably plasmid encoded. These findings document the presence in low-passage B. burgdorferi spirochetes of a novel lipidation-independent activity capable of inducing cytokine release from host cells. PMID:10024550

  7. Activation of Natural Killer cells during microbial infections

    Directory of Open Access Journals (Sweden)

    Amir eHorowitz

    2012-01-01

    Full Text Available Natural killer (NK cells are large granular lymphocytes that express a diverse array of germline encoded inhibitory and activating receptors for MHC Class I and Class I-like molecules, classical co-stimulatory ligands and cytokines. The ability of NK cells to be very rapidly activated by inflammatory cytokines, to secrete effector cytokines and to kill infected or stressed host cells, suggests that they may be among the very early responders during infection. Recent studies have also identified a small number of pathogen-derived ligands that can bind to NK cell surface receptors and directly induce their activation. Here we review recent studies that have begun to elucidate the various pathways by which viral, bacterial and parasite pathogens activate NK cells. We also consider two emerging themes of NK cell-pathogen interactions, namely their contribution to adaptive immune responses and their potential to take on regulatory and immunomodulatory functions.

  8. Receptor-mediated sorting of soluble vacuolar proteins ends at the trans-Golgi network/early endosome.

    Science.gov (United States)

    Künzl, Fabian; Früholz, Simone; Fäßler, Florian; Li, Beibei; Pimpl, Peter

    2016-01-01

    The sorting of soluble proteins for degradation in the vacuole is of vital importance in plant cells, and relies on the activity of vacuolar sorting receptors (VSRs). In the plant endomembrane system, VSRs bind vacuole-targeted proteins and facilitate their transport to the vacuole. Where exactly these interactions take place has remained controversial, however. Here, we examine the potential for VSR-ligand interactions in all compartments of the vacuolar transport system in tobacco mesophyll protoplasts. To do this, we developed compartment-specific VSR sensors that assemble as a result of a nanobody-epitope interaction, and monitored the degree of ligand binding by analysing Förster resonance energy transfer using fluorescence lifetime imaging microscopy (FRET-FLIM). We show that VSRs bind ligands in the endoplasmic reticulum (ER) and in the Golgi, but not in the trans-Golgi network/early endosome (TGN/EE) or multivesicular late endosomes, suggesting that the post-TGN/EE trafficking of ligands towards the vacuole is VSR independent. We verify this by showing that non-VSR-ligands are also delivered to the vacuole from the TGN/EE after endocytic uptake. We conclude that VSRs are required for the transport of ligands from the ER and the Golgi to the TGN/EE, and suggest that the onward transport to the vacuole occurs by default. PMID:27249560

  9. Cystatin F regulates proteinase activity in IL-2-activated natural killer cells.

    Science.gov (United States)

    Maher, Katarina; Konjar, Spela; Watts, Colin; Turk, Boris; Kopitar-Jerala, Natasa

    2014-01-01

    Cystatin F is a unique member of the cystatin family of cysteine protease inhibitors, which is synthesized as an inactive dimer and it is activated by N-terminal cleavage in the endolysosomes. It is expressed in the cells of the immune system: myeloid cells and the cells involved in target cell killing: natural killer (NK) cells and cytotoxic T cells (CTLs). Upon activation of the NK cells with interleukin 2 (IL-2), cystatin F was found upregulated and co-localized in cytotoxic granules with cathepsin C (CatC) and CatV. However, cystatin F inhibits the CatC in cells only when its N-terminal part is processed. Although cystatin F could inhibit both CatV and CatC, the IL-2 stimulation of the YT cells resulted in an increased CatV activity, while the CatC activity was unchanged. The incubation of IL-2 activated NK cells with a cysteine proteinase inhibitor E-64d increased the cystatin F dimer formation. Our results suggest that cystatin F not only inhibits CatV, but it is processed by the CatV in order to inhibit the CatC activity in cytotoxic granules. The regulation of the CatC activity in the cytotoxic granules of the NK cells by the cystatin F could be important for the processing and activation of granule-associated serine proteases - granzymes.

  10. Preparation of cell lines for single-cell analysis of transcriptional activation dynamics.

    Science.gov (United States)

    Rafalska-Metcalf, Ilona U; Janicki, Susan M

    2013-01-01

    Imaging molecularly defined regions of chromatin in single living cells during transcriptional activation has the potential to provide new insight into gene regulatory mechanisms. Here, we describe a method for isolating cell lines with multi-copy arrays of reporter transgenes, which can be used for real-time high-resolution imaging of transcriptional activation dynamics in single cells.

  11. Neural progenitor cells regulate microglia functions and activity.

    Science.gov (United States)

    Mosher, Kira I; Andres, Robert H; Fukuhara, Takeshi; Bieri, Gregor; Hasegawa-Moriyama, Maiko; He, Yingbo; Guzman, Raphael; Wyss-Coray, Tony

    2012-11-01

    We found mouse neural progenitor cells (NPCs) to have a secretory protein profile distinct from other brain cells and to modulate microglial activation, proliferation and phagocytosis. NPC-derived vascular endothelial growth factor was necessary and sufficient to exert at least some of these effects in mice. Thus, neural precursor cells may not only be shaped by microglia, but also regulate microglia functions and activity.

  12. Effects of Neuroendocrine CB1 Activity on Adult Leydig Cells.

    Science.gov (United States)

    Cobellis, Gilda; Meccariello, Rosaria; Chianese, Rosanna; Chioccarelli, Teresa; Fasano, Silvia; Pierantoni, Riccardo

    2016-01-01

    Endocannabinoids control male reproduction acting at central and local level via cannabinoid receptors. The cannabinoid receptor CB1 has been characterized in the testis, in somatic and germ cells of mammalian and non-mammalian animal models, and its activity related to Leydig cell differentiation, steroidogenesis, spermiogenesis, sperm quality, and maturation. In this short review, we provide a summary of the insights concerning neuroendocrine CB1 activity in male reproduction focusing on adult Leydig cell ontogenesis and steroid biosynthesis. PMID:27375550

  13. Effects of Neuroendocrine CB1 Activity on Adult Leydig Cells

    Science.gov (United States)

    Cobellis, Gilda; Meccariello, Rosaria; Chianese, Rosanna; Chioccarelli, Teresa; Fasano, Silvia; Pierantoni, Riccardo

    2016-01-01

    Endocannabinoids control male reproduction acting at central and local level via cannabinoid receptors. The cannabinoid receptor CB1 has been characterized in the testis, in somatic and germ cells of mammalian and non-mammalian animal models, and its activity related to Leydig cell differentiation, steroidogenesis, spermiogenesis, sperm quality, and maturation. In this short review, we provide a summary of the insights concerning neuroendocrine CB1 activity in male reproduction focusing on adult Leydig cell ontogenesis and steroid biosynthesis. PMID:27375550

  14. Telomere elongation in immortal human cells without detectable telomerase activity.

    OpenAIRE

    Bryan, T M; Englezou, A; J Gupta; Bacchetti, S; Reddel, R. R.

    1995-01-01

    Immortalization of human cells is often associated with reactivation of telomerase, a ribonucleoprotein enzyme that adds TTAGGG repeats onto telomeres and compensates for their shortening. We examined whether telomerase activation is necessary for immortalization. All normal human fibroblasts tested were negative for telomerase activity. Thirteen out of 13 DNA tumor virus-transformed cell cultures were also negative in the pre-crisis (i.e. non-immortalized) stage. Of 35 immortalized cell line...

  15. Microwave-induced thermogenetic activation of single cells

    Energy Technology Data Exchange (ETDEWEB)

    Safronov, N. A. [Physics Department, International Laser Center, M.V. Lomonosov Moscow State University, Moscow 119992 (Russian Federation); Fedotov, I. V. [Physics Department, International Laser Center, M.V. Lomonosov Moscow State University, Moscow 119992 (Russian Federation); Department of Physics and Astronomy, Texas A and M University, College Station, Texas 77843 (United States); Russian Quantum Center, ul. Novaya 100, Skolkovo, Moscow Region 143025 (Russian Federation); Ermakova, Yu. G.; Matlashov, M. E.; Belousov, V. V. [M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997 (Russian Federation); Sidorov-Biryukov, D. A.; Fedotov, A. B. [Physics Department, International Laser Center, M.V. Lomonosov Moscow State University, Moscow 119992 (Russian Federation); Russian Quantum Center, ul. Novaya 100, Skolkovo, Moscow Region 143025 (Russian Federation); Zheltikov, A. M. [Physics Department, International Laser Center, M.V. Lomonosov Moscow State University, Moscow 119992 (Russian Federation); Department of Physics and Astronomy, Texas A and M University, College Station, Texas 77843 (United States); Russian Quantum Center, ul. Novaya 100, Skolkovo, Moscow Region 143025 (Russian Federation); Kurchatov Institute National Research Center, Moscow 123182 (Russian Federation)

    2015-04-20

    Exposure to a microwave field is shown to enable thermogenetic activation of individual cells in a culture of cell expressing thermosensitive ion channels. Integration of a microwave transmission line with an optical fiber and a diamond quantum thermometer has been shown to allow thermogenetic single-cell activation to be combined with accurate local online temperature measurements based on an optical detection of electron spin resonance in nitrogen–vacancy centers in diamond.

  16. The sorting protein PACS-2 promotes ErbB signalling by regulating recycling of the metalloproteinase ADAM17

    Science.gov (United States)

    Dombernowsky, Sarah Louise; Samsøe-Petersen, Jacob; Petersen, Camilla Hansson; Instrell, Rachael; Hedegaard, Anne-Mette Bornhardt; Thomas, Laurel; Atkins, Katelyn Mae; Auclair, Sylvain; Albrechtsen, Reidar; Mygind, Kasper Johansen; Fröhlich, Camilla; Howell, Michael; Parker, Peter; Thomas, Gary; Kveiborg, Marie

    2015-01-01

    The metalloproteinase ADAM17 activates ErbB signalling by releasing ligands from the cell surface, a key step underlying epithelial development, growth, and tumour progression. However, mechanisms acutely controlling ADAM17 cell-surface availability to modulate the extent of ErbB ligand release are poorly understood. Here, through a functional genome-wide siRNA screen, we identify the sorting protein PACS-2 as a regulator of ADAM17 trafficking and ErbB signalling. PACS-2 loss reduces ADAM17 cell-surface levels and ADAM17-dependent ErbB ligand shedding, without apparent effects on related proteases. PACS-2 co-localizes with ADAM17 on early endosomes and PACS-2 knockdown decreases the recycling and stability of internalized ADAM17. Hence, PACS-2 sustains ADAM17 cell-surface activity by diverting ADAM17 away from degradative pathways. Interestingly, Pacs2-deficient mice display significantly reduced levels of phosphorylated EGFR and intestinal proliferation. We suggest that this mechanism controlling ADAM17 cell-surface availability and EGFR signalling may play a role in intestinal homeostasis, with potential implications for cancer biology. PMID:26108729

  17. Phorbol ester and vasopressin activate phospholipase D in Leydig cells

    DEFF Research Database (Denmark)

    Vinggaard, Anne Marie; Hansen, Harald S.

    1991-01-01

    In the present study evidence is provided for the existence of phospholipase D (PLD) activity in rat Leydig cells. Leydig cells were cultured and labelled with [H]myristic acid. In the presence of ethanol, phorbol 12-myristate 13-acetate (PMA) stimulated the formation of [H]phosphatidylethanol ([...... support the notion that activation of PLD by PMA is dependent on PKC. Arginine vasopressin (AVP) caused a rapid stimulation of PLD activity in the cells. This activation was inhibited after downregulation of PKC, indicating that the agonist acts by a mechanism similar to that of PMA....

  18. Effects of dexamethasone on palate mesenchymal cell phospholipase activity

    International Nuclear Information System (INIS)

    Corticosteroids will induce cleft palate in mice. One suggested mechanism for this effect is through inhibition of phospholipase activity. This hypothesis was tested by measuring the effects of dexamethasone, a synthetic corticosteroid, on phospholipase activity in cultures of palate mesenchymal cells. Palate mesenchymal cells were prelabeled with [3H]arachidonic acid. The cells were subsequently treated with various concentrations of dexamethasone. Concurrently, cultures of M-MSV-transformed 3T3 cells were prepared identically. After treatment, phospholipase activity was stimulated by the addition of serum or epidermal growth factor (EGF), and radioactivity released into the medium was taken as a measure of phospholipase activity. Dexamethasone (1 X 10(-5) or 1 X 10(-4) M) could inhibit serum-stimulated phospholipase activity in transformed 3T3 cells after 1 to 24 hr of treatment. However, no inhibition of activity was measured in palate mesenchymal cells following this period of treatment. Not until 120 hr of treatment with dexamethasone (1 X 10(-4) M) was any significant inhibition of serum-stimulated phospholipase activity observed in palate mesenchymal cells. When EGF was used to stimulate phospholipase activity, dexamethasone (1 X 10(-5) M) caused an increase in phospholipase activity in palate mesenchymal cells. These observations suggested that phospholipase in transformed 3T3 cells was sensitive to inhibition by dexamethasone. However, palate mesenchymal cell phospholipase is only minimally sensitive to dexamethasone, and in certain instances can be enhanced. These results cannot support the hypothesis that corticosteroids mediate their teratogenic effect via inhibition of phospholipase activity

  19. Mitogen-activated Tasmanian devil blood mononuclear cells kill devil facial tumour disease cells.

    Science.gov (United States)

    Brown, Gabriella K; Tovar, Cesar; Cooray, Anne A; Kreiss, Alexandre; Darby, Jocelyn; Murphy, James M; Corcoran, Lynn M; Bettiol, Silvana S; Lyons, A Bruce; Woods, Gregory M

    2016-08-01

    Devil facial tumour disease (DFTD) is a transmissible cancer that has brought the host species, the Tasmanian devil, to the brink of extinction. The cancer cells avoid allogeneic immune recognition by downregulating cell surface major histocompatibility complex (MHC) I expression. This should prevent CD8(+) T cell, but not natural killer (NK) cell, cytotoxicity. The reason why NK cells, normally reactive to MHC-negative cells, are not activated to kill DFTD cells has not been determined. The immune response of wild devils to DFTD, if it occurs, is uncharacterised. To investigate this, we tested 12 wild devils with DFTD, and found suggestive evidence of low levels of antibodies against DFTD cells in one devil. Eight of these devils were also analysed for cytotoxicity, however, none showed evidence for cytotoxicity against cultured DFTD cells. To establish whether mimicking activation of antitumour responses could induce cytotoxic activity against DFTD, Tasmanian devil peripheral blood mononuclear cells (PBMCs) were treated with either the mitogen Concanavalin A, the Toll-like receptor agonist polyinosinic:polycytidylic acid or recombinant Tasmanian devil IL-2. All induced the PBMC cells to kill cultured DFTD cells, suggesting that activation does not occur after encounter with DFTD cells in vivo, but can be induced. The identification of agents that activate cytotoxicity against DFTD target cells is critical for developing strategies to protect against DFTD. Such agents could function as adjuvants to induce functional immune responses capable of targeting DFTD cells and tumours in vivo. PMID:27089941

  20. The Protease-associated Domain and C-terminal Extension Are Required for Zymogen Processing, Sorting within the Secretory Pathway, and Activity of Tomato Subtilase 3 (SlSBT3)*S⃞

    OpenAIRE

    Cedzich, Anna; Huttenlocher, Franziska; Kuhn, Benjamin M.; Pfannstiel, Jens; Gabler, Leszek; Stintzi, Annick; Schaller, Andreas

    2009-01-01

    A transgenic plant cell suspension culture was established as a versatile and efficient expression system for the subtilase SlSBT3 from tomato. The recombinant protease was purified to homogeneity from culture supernatants by fractionated ammonium sulfate precipitation, batch adsorption to cation exchange material, and anion exchange chromatography. Purified SlSBT3 was identified as a 79-kDa glycoprotein with both complex and paucimannosidic type glycan chains at Asn17...

  1. BMP2 Transfer to Neighboring Cells and Activation of Signaling.

    Science.gov (United States)

    Alborzinia, Hamed; Shaikhkarami, Marjan; Hortschansky, Peter; Wölfl, Stefan

    2016-09-01

    Morphogen gradients and concentration are critical features during early embryonic development and cellular differentiation. Previously we reported the preparation of biologically active, fluorescently labeled BMP2 and quantitatively analyzed their binding to the cell surface and followed BMP2 endocytosis over time on the level of single endosomes. Here we show that this internalized BMP2 can be transferred to neighboring cells and, moreover, also activates downstream BMP signaling in adjacent cells, indicated by Smad1/5/8 phosphorylation and activation of the downstream target gene id1. Using a 3D matrix to modulate cell-cell contacts in culture we could show that direct cell-cell contact significantly increased BMP2 transfer. Using inhibitors of vesicular transport, transfer was strongly inhibited. Interestingly, cotreatment with the physiological BMP inhibitor Noggin increased BMP2 uptake and transfer, albeit activation of Smad signaling in neighboring cells was completely suppressed. Our findings present a novel and interesting mechanism by which morphogens such as BMP2 can be transferred between cells and how this is modulated by BMP antagonists such as Noggin, and how this influences activation of Smad signaling by BMP2 in neighboring cells. PMID:27306974

  2. Enhancement of intracellular concentration and biological activity of PNA after conjugation with a cell-penetrating synthetic model peptide.

    Science.gov (United States)

    Oehlke, Johannes; Wallukat, Gerd; Wolf, Yvonne; Ehrlich, Angelika; Wiesner, Burkhard; Berger, Hartmut; Bienert, Michael

    2004-07-01

    In order to evaluate the ability of the cell-penetrating alpha-helical amphipathic model peptide KLALKLALKALKAALKLA-NH(2) (MAP) to deliver peptide nucleic acids (PNAs) into mammalian cells, MAP was covalently linked to the 12-mer PNA 5'-GGAGCAGGAAAG-3' directed against the mRNA of the nociceptin/orphanin FQ receptor. The cellular uptake of both the naked PNA and its MAP-conjugate was studied by means of capillary electrophoresis combined with laser-induced fluorescence detection, confocal laser scanning microscopy and fluorescence-activated cell sorting. Incubation with the fluorescein-labelled PNA-peptide conjugate led to three- and eightfold higher intracellular concentrations in neonatal rat cardiomyocytes and CHO cells, respectively, than found after exposure of the cells to the naked PNA. Correspondingly, pretreatment of spontaneously-beating neonatal rat cardiomyocytes with the PNA-peptide conjugate and the naked PNA slowed down the positive chronotropic effect elicited by the neuropeptide nociceptin by 10- and twofold, respectively. The main reasons for the higher bioavailability of the PNA-peptide conjugate were found to be a more rapid cellular uptake in combination with a lowered re-export and resistance against influences of serum.

  3. Tubular solid oxide fuel cell demonstration activities

    Energy Technology Data Exchange (ETDEWEB)

    Veyo, S.E.

    1995-08-01

    The development of a viable fuel cell driven electrical power generation system involves not only the development of cell and stack technology, but also the development of the overall system concept, the strategy for control, and the ancillary subsystems. The design requirements used to guide system development must reflect a customer focus in order to evolve a commercial product. In order to obtain useful customer feedback, Westinghouse has practiced the deployment with customers of fully integrated, automatically controlled, packaged solid oxide fuel cell power generation systems. These field units have served to demonstrate to customers first hand the beneficial attributes of the SOFC, to expose deficiencies through experience in order to guide continued development, and to garner real world feedback and data concerning not only cell and stack parameters, but also transportation, installation, permitting and licensing, start-up and shutdown, system alarming, fault detection, fault response, and operator interaction.

  4. Multiple pathways for vacuolar sorting of yeast proteinase A

    DEFF Research Database (Denmark)

    Westphal, V; Marcusson, E G; Winther, Jakob R.;

    1996-01-01

    The sorting of the yeast proteases proteinase A and carboxypeptidase Y to the vacuole is a saturable, receptor-mediated process. Information sufficient for vacuolar sorting of the normally secreted protein invertase has in fusion constructs previously been found to reside in the propeptide...

  5. Supramolecular fibres: Self-sorting shows its true colours

    Science.gov (United States)

    Draper, Emily R.; Adams, Dave J.

    2016-08-01

    Self-sorting events in supramolecular assembly lead to complex systems that are attractive for the design of functional materials, but have remained difficult to understand and control. Now, the growth of self-sorted supramolecular nanofibres has been elucidated by direct imaging through real-time in situ confocal microscopy.

  6. NEW TOBACCO SORTS POTENTIAL FOR TOBACCO INDUSTRY DEVELOPMENT

    Directory of Open Access Journals (Sweden)

    Homutova S. A.

    2014-10-01

    Full Text Available The review of the latest tobacco selection researches is given in the article. The basic aim of these researches is creation of new sort material, which is ecologically stable, and corresponding to energy conservation demands. New tobacco sorts are presented

  7. The Use of Binary Search Trees in External Distribution Sorting.

    Science.gov (United States)

    Cooper, David; Lynch, Michael F.

    1984-01-01

    Suggests new method of external distribution called tree partitioning that involves use of binary tree to split incoming file into successively smaller partitions for internal sorting. Number of disc accesses during a tree-partitioning sort were calculated in simulation using files extracted from British National Bibliography catalog files. (19…

  8. Magnetic fluid equipment for sorting of secondary polyolefins from waste

    NARCIS (Netherlands)

    Rem, P.C.; Di Maio, F.; Hu, B.; Houzeaux, G...; Baltes, L.; Tierean, M.

    2012-01-01

    The paper presents the researches made on the FP7 project „Magnetic Sorting and Ultrasound Sensor Technologies for Production of High Purity Secondary Polyolefins from Waste” in order to develop a magnetic fluid equipment for sorting of polypropylene (PP) and polyethylene (PE) from polymers mixed wa

  9. Finding all sorting tandem duplication random loss operations

    DEFF Research Database (Denmark)

    Bernt, Matthias; Chen, Kuan Yu; Chen, Ming Chiang;

    2011-01-01

    a theoretical point of view. Of particular interest are sorting TDRLs which are TDRLs that, when applied to a permutation representing a genome, reduce the distance towards another given permutation. The identification of sorting genome rearrangement operations in general is a key ingredient of many algorithms...

  10. A FORTRAN Computer Program for Q Sort Calculations

    Science.gov (United States)

    Dunlap, William R.

    1978-01-01

    The Q Sort method is a rank order procedure. A FORTRAN program is described which calculates a total value for any group of cases for the items in the Q Sort, and rank orders the items according to this composite value. (Author/JKS)

  11. Cloning of aminopeptidase Npromoter and its activity in hematopoietic cell and different tumor cell lines

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Aminopeptidase N (APN) promoter region was cloned and sequenced from peripheral blood mononuclear cells. The recombinant reporter construct containing the promoter and luciferase gene, designated pXP1-APNLuc, was introduced into myeloblastic cell line, T lymphocyte cell line and various tumor cell lines. Luciferase assay showed that APN upstream promoter is myeloid-specific for high expression in myeloblastic cell line and much lower expres sion in T lymphocyte cell line. The promoter activity was relatively high in lung adenoma cell line compared with other tumor cell lines including hepatoma cell line, tong cancer cell line and esophageal cancer cell line in which the promoter activity significantly diminished or was almost undetectable. The characteristics of APN promoter may pro vide a new strategy for specific myeloprotection while tumor patients are being treated with chemotherapy and/or radio therapy.

  12. Help the planet by sorting your waste!

    CERN Multimedia

    2012-01-01

    Paper and cardboard waste comes in various forms, from newspapers to the toughest cardboard. Every year CERN dispatches about 200 tonnes of paper and cardboard to a recycling plant, but this is still too little when you take into consideration the tonnes of paper and cardboard that are still thrown out as part of ordinary rubbish or are incorrectly sorted into other rubbish skips.   Each office is equipped with a wastepaper bin, and a paper and cardboard container is available near every building. Cardboard boxes should be folded before they are placed in the containers in order to save space. Please note: Here are some sobering statistics: - 2 to 3 tonnes of wood pulp are required to manufacture 1 tonne of paper. - Each tonne of recycled paper means that we can save approximately 15 trees and substantial amounts of the water that is needed to extract cellulose (60 litres of water per kilo of paper). - A production of 100% recycled paper represents a 90% saving in water. - 5000 kWh of e...

  13. Substrate stiffness regulates filopodial activities in lung cancer cells.

    Directory of Open Access Journals (Sweden)

    Yu-Ren Liou

    Full Text Available Microenvironment stiffening plays a crucial role in tumorigenesis. While filopodia are generally thought to be one of the cellular mechanosensors for probing environmental stiffness, the effects of environmental stiffness on filopodial activities of cancer cells remain unclear. In this work, we investigated the filopodial activities of human lung adenocarcinoma cells CL1-5 cultured on substrates of tunable stiffness using a novel platform. The platform consists of an optical system called structured illumination nano-profilometry, which allows time-lapsed visualization of filopodial activities without fluorescence labeling. The culturing substrates were composed of polyvinyl chloride mixed with an environmentally friendly plasticizer to yield Young's modulus ranging from 20 to 60 kPa. Cell viability studies showed that the viability of cells cultured on the substrates was similar to those cultured on commonly used elastomers such as polydimethylsiloxane. Time-lapsed live cell images were acquired and the filopodial activities in response to substrates with varying degrees of stiffness were analyzed. Statistical analyses revealed that lung cancer cells cultured on softer substrates appeared to have longer filopodia, higher filopodial densities with respect to the cellular perimeter, and slower filopodial retraction rates. Nonetheless, the temporal analysis of filopodial activities revealed that whether a filopodium decides to extend or retract is purely a stochastic process without dependency on substrate stiffness. The discrepancy of the filopodial activities between lung cancer cells cultured on substrates with different degrees of stiffness vanished when the myosin II activities were inhibited by treating the cells with blebbistatin, which suggests that the filopodial activities are closely modulated by the adhesion strength of the cells. Our data quantitatively relate filopodial activities of lung cancer cells with environmental stiffness and

  14. Expansion and long-term culture of human spermatogonial stem cells via the activation of SMAD3 and AKT pathways

    Science.gov (United States)

    Guo, Ying; Liu, Linhong; Sun, Min; Hai, Yanan; Li, Zheng

    2015-01-01

    Spermatogonial stem cells (SSCs) can differentiate into spermatids, reflecting that they could be used in reproductive medicine for treating male infertility. SSCs are able to become embryonic stem-like cells with the potentials of differentiating into numerous cell types of the three germ layers and they can transdifferentiate to mature and functional cells of other lineages, highlighting significant applications of human SSCs for treating human diseases. However, human SSCs are very rare and a long-term culture system of human SSCs has not yet established. This aim of study was to isolate, identify and culture human SSCs for a long period. We isolated GPR125-positive spermatogonia with high purity and viability from adult human testicular tissues utilizing the two-step enzymatic digestion and magnetic-activated cell sorting with antibody against GPR125. These freshly isolated cells expressed a number of markers for SSCs, including GPR125, PLZF, GFRA1, RET, THY1, UCHL1 and MAGEA4, but not the hallmarks for spermatocytes and spermatozoa, e.g. SYCP1, SYCP3, PRM1, and TNP1. The isolated human SSCs could be cultured for two months with a significant increase of cell number with the defined medium containing growth factors and hydrogel. Notably, the expression of numerous SSC markers was maintained during the cultivation of human SSCs. Furthermore, SMAD3 and AKT phosphorylation was enhanced during the culture of human SSCs. Collectively, these results suggest that human SSCs can be cultivated for a long period and expanded whilst retaining an undifferentiated status via the activation of SMAD3 and AKT pathways. This study could provide sufficient cells of SSCs for their basic research and clinic applications in reproductive and regenerative medicine. PMID:26088866

  15. Telomerase activity in germline and embryonic cells of Xenopus.

    OpenAIRE

    Mantell, L L; Greider, C W

    1994-01-01

    Telomerase is a ribonucleoprotein which synthesizes telomere repeats onto chromosome ends. Telomerase activity is involved in telomere length maintenance. We used Xenopus laevis as a model system to study the expression of telomerase activity in germline cells and during early development. We identified a non-processive telomerase activity in manually dissected nuclei of Xenopus stage VI oocytes. Telomerase activity was detected throughout oogenesis and embryogenesis. Telomerase was active in...

  16. Sorting Pairs of Points Based on Their Distances

    Directory of Open Access Journals (Sweden)

    Mohammad Farshi

    2016-02-01

    Full Text Available Sorting data is one of the main problems in computer science which studied vastly and used in several places. In several geometric problems, like problems on point sets or lines in the plane or Euclidean space with higher dimensions, the problem of sorting pairs of points based on the distance (between them is used. Using general sorting algorithms, sorting n 2 distances between n points can be done in O(n2 log n time. Ofcourse, sorting (n2 independent numbers does not have a faster solution, but since we have dependency between numbers in this case, finding a faster algorithm or showing that the problem in this case has O(n2 log n time complexity is interesting. In this paper, we try to answer this question.

  17. Efficient External Sorting on Flash Memory Embedded Devices

    Directory of Open Access Journals (Sweden)

    Tyler Cossentine

    2013-03-01

    Full Text Available Many embedded system applications involve storing and querying large datasets. Existing research in thisarea has focused on adapting and applying conventional database algorithms to embedded devices.Algorithms designed for processing queries on embedded devices must be able to execute given the smallamount of available memory and energy constraints. Most embedded devices use flash memory to storelarge amounts of data. Flash memory has unique performance characteristics that can be exploited toimprove algorithm performance. In this paper, we describe the Flash MinSort external sorting algorithmthat uses an index, generated at runtime, to take advantage of fast random reads in flash memory. Thisalgorithm adapts to the amount of memory available and performs best in applications where sort keys areclustered. Experimental results show that Flash MinSort is two to ten times faster than previousapproaches for small memory sizes where external merge sort is not executable.

  18. Multiphase ferrofluid flows for micro-particle sorting

    Science.gov (United States)

    Zhou, Ran; Wang, Cheng

    2015-11-01

    Utilizing negative magnetophoresis, ferrofluids have demonstrated great potential for sorting nonmagnetic micro-particles by size. Most of the existing techniques use single phase ferrofluids by pushing micro-particles to channel walls; the sorting speed is thus hindered. We demonstrate a novel sorting strategy by co-flowing a ferrofluid and a non-magnetic fluid in microchannels. Due to the magnetic force, the particles migrate across the ferrofluid stream at size-dependent velocities as they travel downstream. The laminar interface between the two fluids functions as a virtual boundary to accumulate particles, resulting in effective separation of particles. A stable and sharp interface is important to the success of this sorting technique. We investigate several factors that affect sorting efficiency, including magnetic field, susceptibility difference of the fluids, flow velocity, and channel geometry.

  19. Electrode activation and passivation of solid oxide fuel cell electrodes

    DEFF Research Database (Denmark)

    Koch, Søren; Mogensen, Mogens Bjerg; Hendriksen, P.V.;

    2006-01-01

    resistance increased significantly over the next four days at open circuit conditions. Apparently, at OCV conditions cell passivation occurs. The cell gradually reactivates, once the current is switched on again. Part of this activation/passivation process is fast enough to influence the resistance...... of the cell during i-V measurements (over less than 1 hour) and a considerable hysteresis is observed in the cell voltage during these measurements. Impedance spectroscopy was used to investigate the activation/passivation process. It was found that the series resistance and the part of the polarisation...... impedance above approximately 100 Hz were not influenced by the activation/passivation process. The part of the polarisation impedance between I and 100 Hz was highly influenced by the activation/passivation process and during cell polarisation this part of the polarisation impedance was up to 40% lower...

  20. Automatic online spike sorting with singular value decomposition and fuzzy C-mean clustering

    Directory of Open Access Journals (Sweden)

    Oliynyk Andriy

    2012-08-01

    Full Text Available Abstract Background Understanding how neurons contribute to perception, motor functions and cognition requires the reliable detection of spiking activity of individual neurons during a number of different experimental conditions. An important problem in computational neuroscience is thus to develop algorithms to automatically detect and sort the spiking activity of individual neurons from extracellular recordings. While many algorithms for spike sorting exist, the problem of accurate and fast online sorting still remains a challenging issue. Results Here we present a novel software tool, called FSPS (Fuzzy SPike Sorting, which is designed to optimize: (i fast and accurate detection, (ii offline sorting and (iii online classification of neuronal spikes with very limited or null human intervention. The method is based on a combination of Singular Value Decomposition for fast and highly accurate pre-processing of spike shapes, unsupervised Fuzzy C-mean, high-resolution alignment of extracted spike waveforms, optimal selection of the number of features to retain, automatic identification the number of clusters, and quantitative quality assessment of resulting clusters independent on their size. After being trained on a short testing data stream, the method can reliably perform supervised online classification and monitoring of single neuron activity. The generalized procedure has been implemented in our FSPS spike sorting software (available free for non-commercial academic applications at the address: http://www.spikesorting.com using LabVIEW (National Instruments, USA. We evaluated the performance of our algorithm both on benchmark simulated datasets with different levels of background noise and on real extracellular recordings from premotor cortex of Macaque monkeys. The results of these tests showed an excellent accuracy in discriminating low-amplitude and overlapping spikes under strong background noise. The performance of our method is

  1. Human immunodeficiency syndromes affecting human natural killer cell cytolytic activity

    Directory of Open Access Journals (Sweden)

    Daniel Denis Billadeau

    2014-01-01

    Full Text Available NK cells are lymphocytes of the innate immune system that secrete cytokines upon activation and mediate the killing of tumor cells and virus-infected cells, especially those that escape the adaptive T-cell response caused by the down regulation of MHC-I. The induction of cytotoxicity requires that NK cells contact target cells through adhesion receptors, and initiate activation signaling leading to increased adhesion and accumulation of F-actin at the NK cell cytotoxic synapse. Concurrently, lytic granules undergo minus-end directed movement and accumulate at the microtubule-organizing center (MTOC through the interaction with microtubule motor proteins, followed by polarization of the lethal cargo toward the target cell. Ultimately, myosin-dependent movement of the lytic granules toward the NK cell plasma membrane through F-actin channels, along with SNARE-dependent fusion promotes lytic granule release into the cleft between the NK cell and target cell resulting in target cell killing. Herein, we will discuss several disease-causing mutations in primary immunodeficiency syndromes and how they impact NK cell-mediated killing by disrupting distinct steps of this tightly regulated process.

  2. Monocytic cells become less compressible but more deformable upon activation.

    Directory of Open Access Journals (Sweden)

    Agnese Ravetto

    Full Text Available Monocytes play a significant role in the development of atherosclerosis. During the process of inflammation, circulating monocytes become activated in the blood stream. The consequent interactions of the activated monocytes with the blood flow and endothelial cells result in reorganization of cytoskeletal proteins, in particular of the microfilament structure, and concomitant changes in cell shape and mechanical behavior. Here we investigate the full elastic behavior of activated monocytes in relation to their cytoskeletal structure to obtain a better understanding of cell behavior during the progression of inflammatory diseases such as atherosclerosis.The recently developed Capillary Micromechanics technique, based on exposing a cell to a pressure difference in a tapered glass microcapillary, was used to measure the deformation of activated and non-activated monocytic cells. Monitoring the elastic response of individual cells up to large deformations allowed us to obtain both the compressive and the shear modulus of a cell from a single experiment. Activation by inflammatory chemokines affected the cytoskeletal organization and increased the elastic compressive modulus of monocytes with 73-340%, while their resistance to shape deformation decreased, as indicated by a 25-88% drop in the cell's shear modulus. This decrease in deformability is particularly pronounced at high strains, such as those that occur during diapedesis through the vascular wall.Overall, monocytic cells become less compressible but more deformable upon activation. This change in mechanical response under different modes of deformation could be important in understanding the interplay between the mechanics and function of these cells. In addition, our data are of direct relevance for computational modeling and analysis of the distinct monocytic behavior in the circulation and the extravascular space. Lastly, an understanding of the changes of monocyte mechanical properties

  3. Inhibition of Leukemic Cell Telomerase Activity by Antisense Phosphorothioate Oligodeoxynucleotides

    Institute of Scientific and Technical Information of China (English)

    HEDongmei; ZHANGYuan

    2002-01-01

    Objective To evaluate the effect of human telomerase reverse transcriptase(hTERT) gene antisense oligodeoxynucleotide (ASON) on telomerase activity in K562 cells.Methods Telomerase activity was detemined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA) in K562 cells treated with ASODN and hTERTmRNA expression was detected by reverse transcriptase polymerase chain reaction (RT-PCR). Results The hTERTmRNA level was decreased,and telomerase activity was significantly inhibited when the K562 cells were treated with ASODN for 48 h. Conclusion It is suggested that hTETR ASODN might specifically inhibit telomrase activity of K562 cells at translation level,and it is further proved that hTERT gene has significant correlation with telopmerase activity.

  4. Anticancer Activities of Trichostatin A on Maligant Lymphoid Cells

    Institute of Scientific and Technical Information of China (English)

    SUN Chunyan; LIU Xinyue; CHEN Yan; LIU Fang

    2006-01-01

    The anticancer activity of trichostain A (TSA) on human B cell non-Hodgkin's lymphoma and its mechanism were explored. The effect of TSA on the growth of Raji cells and normal peripheral blood mononuclear cells (NPBMNC) was studied by MTT assay. The effect of TSA on the apoptosis of Raji cells and NPBMNC was studied by flow cytometry and TDT-mediated dUTP nick end labeling (TUNEL). The effect of TSA on the cell cycle of Raji cells was studied by propidium iodide method. The results showed that TSA potently inhibited proliferation of Raji cells at microgram concentrations and induced apoptosis of Raji cells in a time- and concentration-dependent manner.Treatment with TSA induced accumulation of cells in G0/G1 or G2/M and a concomitant decrease of cell population in S phase. However, NPBMNC was less sensitive to the cytotoxic effect of TSA than Raji cells. It was concluded that TSA may inhibit the proliferation of Raji cells by regulating the cell cycle and inducing the cell apoptosis. Moreover, TSA demonstrates low toxicity in NPBMNC but selectively induces apoptosis of Raji cells.

  5. p53 regulation and activity in mouse embryonic stem cells

    OpenAIRE

    Solozobova, Valeriya

    2010-01-01

    P53 is a tumour development p53. The aim of this work was to study the regulation of p53 in embryonic stem cells and its activation in response to DNA damage. p53 was found that p53 becomes transcriptionally active in ES cells after DNA damage. Embryonic stem cells contain a relatively high amount of p53 protein and p53 RNA. After differentiation p53 level is rapidly downregulated. The high abundance of p53 in undifferentiated ES cells is a result of enhanced translation.

  6. Cytotoxic activity of allogeneic natural killer cells on U251 glioma cells in vitro.

    Science.gov (United States)

    Guo, Meng; Wu, Tingting; Wan, Lixin

    2016-07-01

    The present study aimed to observe the cytotoxic activity of allogeneic natural killer (NK) cells on U251 glioma cells and to investigate their mechanism of action to establish an effective treatment strategy for neuroglioma. Cell survival curves, colony formation assays and karyotype analysis were performed to investigate the characteristics of U251 glioma cells. The present study demonstrated that natural killer group 2, member D (NKG2D)‑major histocompatibility complex class I‑related chain A/B (MICA/B) interactions contributed to the cytotoxic effect of NK cells on K562 and U251 cells. In antibody‑blocking assays to inhibit NKG2D ligands, the cytotoxic activity was not completely attenuated, which suggested that other signaling pathways contribute to the cytotoxic activity of NK cells on tumor cells in addition to the NKG2D‑mediated activity. The present study identified that the expression levels of NKG2D ligands on the surface of target cells influenced the strength of the NK cell immune response. Furthermore, allogeneic NK cells were observed to kill glioma cells in vitro, and this anticancer activity is associated with the rate of NKG2D expression on the surface of glioma cells.

  7. Cytotoxic activity of allogeneic natural killer cells on U251 glioma cells in vitro.

    Science.gov (United States)

    Guo, Meng; Wu, Tingting; Wan, Lixin

    2016-07-01

    The present study aimed to observe the cytotoxic activity of allogeneic natural killer (NK) cells on U251 glioma cells and to investigate their mechanism of action to establish an effective treatment strategy for neuroglioma. Cell survival curves, colony formation assays and karyotype analysis were performed to investigate the characteristics of U251 glioma cells. The present study demonstrated that natural killer group 2, member D (NKG2D)‑major histocompatibility complex class I‑related chain A/B (MICA/B) interactions contributed to the cytotoxic effect of NK cells on K562 and U251 cells. In antibody‑blocking assays to inhibit NKG2D ligands, the cytotoxic activity was not completely attenuated, which suggested that other signaling pathways contribute to the cytotoxic activity of NK cells on tumor cells in addition to the NKG2D‑mediated activity. The present study identified that the expression levels of NKG2D ligands on the surface of target cells influenced the strength of the NK cell immune response. Furthermore, allogeneic NK cells were observed to kill glioma cells in vitro, and this anticancer activity is associated with the rate of NKG2D expression on the surface of glioma cells. PMID:27175912

  8. In situ real-time imaging of self-sorted supramolecular nanofibres

    Science.gov (United States)

    Onogi, Shoji; Shigemitsu, Hajime; Yoshii, Tatsuyuki; Tanida, Tatsuya; Ikeda, Masato; Kubota, Ryou; Hamachi, Itaru

    2016-08-01

    Self-sorted supramolecular nanofibres—a multicomponent system that consists of several types of fibre, each composed of distinct building units—play a crucial role in complex, well-organized systems with sophisticated functions, such as living cells. Designing and controlling self-sorting events in synthetic materials and understanding their structures and dynamics in detail are important elements in developing functional artificial systems. Here, we describe the in situ real-time imaging of self-sorted supramolecular nanofibre hydrogels consisting of a peptide gelator and an amphiphilic phosphate. The use of appropriate fluorescent probes enabled the visualization of self-sorted fibres entangled in two and three dimensions through confocal laser scanning microscopy and super-resolution imaging, with 80 nm resolution. In situ time-lapse imaging showed that the two types of fibre have different formation rates and that their respective physicochemical properties remain intact in the gel. Moreover, we directly visualized stochastic non-synchronous fibre formation and observed a cooperative mechanism.

  9. Microfluidic EmbryoSort technology: towards in flow analysis, sorting and dispensing of individual vertebrate embryos

    Science.gov (United States)

    Fuad, Nurul M.; Wlodkowic, Donald

    2013-12-01

    The demand to reduce the numbers of laboratory animals has facilitated the emergence of surrogate models such as tests performed on zebrafish (Danio rerio) or African clawed frog's (Xenopus levis) eggs, embryos and larvae. Those two model organisms are becoming increasingly popular replacements to current adult animal testing in toxicology, ecotoxicology and also in drug discovery. Zebrafish eggs and embryos are particularly attractive for toxicological analysis due their size (diameter 1.6 mm), optical transparency, large numbers generated per fish and very straightforward husbandry. The current bottleneck in using zebrafish embryos for screening purposes is, however, a tedious manual evaluation to confirm the fertilization status and subsequent dispensing of single developing embryos to multitier plates to perform toxicity analysis. Manual procedures associated with sorting hundreds of embryos are very monotonous and as such prone to significant analytical errors due to operator's fatigue. In this work, we present a proofof- concept design of a continuous flow embryo sorter capable of analyzing, sorting and dispensing objects ranging in size from 1.5 - 2.5 mm. The prototypes were fabricated in polymethyl methacrylate (PMMA) transparent thermoplastic using infrared laser micromachining. The application of additive manufacturing processes to prototype Lab-on-a-Chip sorters using both fused deposition manufacturing (FDM) and stereolithography (SLA) were also explored. The operation of the device was based on a revolving receptacle capable of receiving, holding and positioning single fish embryos for both interrogation and subsequent sorting. The actuation of the revolving receptacle was performed using a DC motor and/or microservo motor. The system was designed to separate between fertilized (LIVE) and non-fertilized (DEAD) eggs, based on optical transparency using infrared (IR) emitters and receivers.

  10. Tim-3: An activation marker and activation limiter of innate immune cells

    Directory of Open Access Journals (Sweden)

    Gencheng eHan

    2013-12-01

    Full Text Available Tim-3 was initially identified on activated Th1, Th17, and Tc1 cells and induces T cell death or exhaustion after binding to its ligand, Gal-9. The observed relationship between dysregulated Tim-3 expression on T cells and the progression of many clinical diseases has identified this molecule as an important target for intervention in adaptive immunity. Recent data have shown that it also plays critical roles in regulating the activities of macrophages, monocytes, dendritic cells, mast cells, natural killer cells, and endothelial cells. Although the underlying mechanisms remain unclear, dysregulation of Tim-3 expression on these innate immune cells leads to an excessive or inhibited inflammatory response and subsequent autoimmune damage or viral or tumor evasion. In this review, we focus on the expression and function of Tim-3 on innate immune cells and discuss 1 how Tim-3 is expressed and regulated on different innate immune cells; 2 how it affects the activity of different innate immune cells; and 3 how dysregulated Tim-3 expression on innate immune cells affects adaptive immunity and disease progression. Tim-3 is involved in the optimal activation of innate immune cells through its varied expression. A better understanding of the physiopathological role of the Tim-3 pathway in innate immunity will shed new light on the pathogenesis of clinical diseases, such as autoimmune diseases, chronic viral infections, and cancer, and suggest new approaches to intervention.

  11. Role of SKD1 Regulators LIP5 and IST1-LIKE1 in Endosomal Sorting and Plant Development1[OPEN

    Science.gov (United States)

    Paez-Valencia, Julio; Miller, Nathan D.; Goodman, Kaija

    2016-01-01

    SKD1 is a core component of the mechanism that degrades plasma membrane proteins via the Endosomal Sorting Complex Required for Transport (ESCRT) pathway. Its ATPase activity and endosomal recruitment are regulated by the ESCRT components LIP5 and IST1. How LIP5 and IST1 affect ESCRT-mediated endosomal trafficking and development in plants is not known. Here we use Arabidopsis mutants to demonstrate that LIP5 controls the constitutive degradation of plasma membrane proteins and the formation of endosomal intraluminal vesicles. Although lip5 mutants were able to polarize the auxin efflux facilitators PIN2 and PIN3, both proteins were mis-sorted to the tonoplast in lip5 root cells. In addition, lip5 root cells over-accumulated PIN2 at the plasma membrane. Consistently with the trafficking defects of PIN proteins, the lip5 roots showed abnormal gravitropism with an enhanced response within the first 4 h after gravistimulation. LIP5 physically interacts with IST1-LIKE1 (ISTL1), a protein predicted to be the Arabidopsis homolog of yeast IST1. However, we found that Arabidopsis contains 12 genes coding for predicted IST1-domain containing proteins (ISTL1–12). Within the ISTL1–6 group, ISTL1 showed the strongest interaction with LIP5, SKD1, and the ESCRT-III-related proteins CHMP1A in yeast two hybrid assays. Through the analysis of single and double mutants, we found that the synthetic interaction of LIP5 with ISTL1, but not with ISTL2, 3, or 6, is essential for normal plant growth, repression of spontaneous cell death, and post-embryonic lethality. PMID:26983994

  12. Activated microglia cause reversible apoptosis of pheochromocytoma cells, inducing their cell death by phagocytosis.

    Science.gov (United States)

    Hornik, Tamara C; Vilalta, Anna; Brown, Guy C

    2016-01-01

    Some apoptotic processes, such as phosphatidylserine exposure, are potentially reversible and do not necessarily lead to cell death. However, phosphatidylserine exposure can induce phagocytosis of a cell, resulting in cell death by phagocytosis: phagoptosis. Phagoptosis of neurons by microglia might contribute to neuropathology, whereas phagoptosis of tumour cells by macrophages might limit cancer. Here, we examined the mechanisms by which BV-2 microglia killed co-cultured pheochromocytoma (PC12) cells that were either undifferentiated or differentiated into neuronal cells. We found that microglia activated by lipopolysaccharide rapidly phagocytosed PC12 cells. Activated microglia caused reversible phosphatidylserine exposure on and reversible caspase activation in PC12 cells, and caspase inhibition prevented phosphatidylserine exposur and decreased subsequent phagocytosis. Nitric oxide was necessary and sufficient to induce the reversible phosphatidylserine exposure and phagocytosis. The PC12 cells were not dead at the time they were phagocytised, and inhibition of their phagocytosis left viable cells. Cell loss was inhibited by blocking phagocytosis mediated by phosphatidylserine, MFG-E8, vitronectin receptors or P2Y6 receptors. Thus, activated microglia can induce reversible apoptosis of target cells, which is insufficient to cause apoptotic cell death, but sufficient to induce their phagocytosis and therefore cell death by phagoptosis.

  13. Hsp60 is actively secreted by human tumor cells.

    Directory of Open Access Journals (Sweden)

    Anna M Merendino

    Full Text Available BACKGROUND: Hsp60, a Group I mitochondrial chaperonin, is classically considered an intracellular chaperone with residence in the mitochondria; nonetheless, in the last few years it has been found extracellularly as well as in the cell membrane. Important questions remain pertaining to extracellular Hsp60 such as how generalized is its occurrence outside cells, what are its extracellular functions and the translocation mechanisms that transport the chaperone outside of the cell. These questions are particularly relevant for cancer biology since it is believed that extracellular chaperones, like Hsp70, may play an active role in tumor growth and dissemination. METHODOLOGY/PRINCIPAL FINDINGS: Since cancer cells may undergo necrosis and apoptosis, it could be possible that extracellular Hsps are chiefly the result of cell destruction but not the product of an active, physiological process. In this work, we studied three tumor cells lines and found that they all release Hsp60 into the culture media by an active mechanism independently of cell death. Biochemical analyses of one of the cell lines revealed that Hsp60 secretion was significantly reduced, by inhibitors of exosomes and lipid rafts. CONCLUSIONS/SIGNIFICANCE: Our data suggest that Hsp60 release is the result of an active secretion mechanism and, since extracellular release of the chaperone was demonstrated in all tumor cell lines investigated, our observations most likely reflect a general physiological phenomenon, occurring in many tumors.

  14. Real-time transposable element activity in individual live cells

    Science.gov (United States)

    Lee, Gloria; Martini, K. Michael

    2016-01-01

    The excision and reintegration of transposable elements (TEs) restructure their host genomes, generating cellular diversity involved in evolution, development, and the etiology of human diseases. Our current knowledge of TE behavior primarily results from bulk techniques that generate time and cell ensemble averages, but cannot capture cell-to-cell variation or local environmental and temporal variability. We have developed an experimental system based on the bacterial TE IS608 that uses fluorescent reporters to directly observe single TE excision events in individual cells in real time. We find that TE activity depends upon the TE’s orientation in the genome and the amount of transposase protein in the cell. We also find that TE activity is highly variable throughout the lifetime of the cell. Upon entering stationary phase, TE activity increases in cells hereditarily predisposed to TE activity. These direct observations demonstrate that real-time live-cell imaging of evolution at the molecular and individual event level is a powerful tool for the exploration of genome plasticity in stressed cells. PMID:27298350

  15. Real-time transposable element activity in individual live cells.

    Science.gov (United States)

    Kim, Neil H; Lee, Gloria; Sherer, Nicholas A; Martini, K Michael; Goldenfeld, Nigel; Kuhlman, Thomas E

    2016-06-28

    The excision and reintegration of transposable elements (TEs) restructure their host genomes, generating cellular diversity involved in evolution, development, and the etiology of human diseases. Our current knowledge of TE behavior primarily results from bulk techniques that generate time and cell ensemble averages, but cannot capture cell-to-cell variation or local environmental and temporal variability. We have developed an experimental system based on the bacterial TE IS608 that uses fluorescent reporters to directly observe single TE excision events in individual cells in real time. We find that TE activity depends upon the TE's orientation in the genome and the amount of transposase protein in the cell. We also find that TE activity is highly variable throughout the lifetime of the cell. Upon entering stationary phase, TE activity increases in cells hereditarily predisposed to TE activity. These direct observations demonstrate that real-time live-cell imaging of evolution at the molecular and individual event level is a powerful tool for the exploration of genome plasticity in stressed cells. PMID:27298350

  16. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M;

    1998-01-01

    PURPOSE: The immune privilege of the eye has been thought to be dependent on physical barriers and absence of lymphatic vessels. However, the immune privilege may also involve active immunologic processes, as recent studies have indicated. The purpose of the present study was to investigate whether...... human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...

  17. Salinomycin activates AMP-activated protein kinase-dependent autophagy in cultured osteoblastoma cells: a negative regulator against cell apoptosis.

    Directory of Open Access Journals (Sweden)

    Lun-qing Zhu

    Full Text Available BACKGROUND: The malignant osteoblastoma has poor prognosis, thus the search for novel and more efficient chemo-agents against this disease is urgent. Salinomycin induces broad anti-cancer effects both in vivo and in vitro, however, its role in osteoblastoma is still not clear. KEY FINDINGS: Salinomycin induced both apoptosis and autophagy in cultured U2OS and MG-63 osteoblastoma cells. Inhibition of autophagy by 3-methyladenine (3-MA, or by RNA interference (RNAi of light chain 3B (LC3B, enhanced salinomycin-induced cytotoxicity and apoptosis. Salinomycin induced a profound AMP-activated protein kinase (AMPK activation, which was required for autophagy induction. AMPK inhibition by compound C, or by AMPKα RNAi prevented salinomycin-induced autophagy activation, while facilitating cancer cell death and apoptosis. On the other hand, the AMPK agonist AICAR promoted autophagy activation in U2OS cells. Salinomycin-induced AMPK activation was dependent on reactive oxygen species (ROS production in osteoblastoma cells. Antioxidant n-acetyl cysteine (NAC significantly inhibited salinomycin-induced AMPK activation and autophagy induction. CONCLUSIONS: Salinomycin activates AMPK-dependent autophagy in osteoblastoma cells, which serves as a negative regulator against cell apoptosis. AMPK-autophagy inhibition might be a novel strategy to sensitize salinomycin's effect in cancer cells.

  18. Advanced research on separating prostate cancer stem cells

    International Nuclear Information System (INIS)

    Prostate cancer is a common malignant tumor in male urinary system,and may easily develop into the hormone refractory prostate cancer which can hardly be cured. Recent studies had found that the prostate cancer stem cells may be the source of the prostate cancer's occurrence,development, metastasis and recurrence. The therapy targeting the prostate cancer stem cells may be the effective way to cure prostate cancer. But these cells is too low to be detected. The difficulty lies in the low separation efficiency of prostate cancer stem cell, so the effectively separating prostate cancer stem cells occupied the main position for the more in-depth research of prostate cancer stem cells. This paper reviews the research progress and existing problems on the several main separating methods of prostate cancer stem cells, includes the fluorescence activated cells sorting and magnetic activated cells sorting based on prostate cancer stem cell surface markers, the side-population sorting and serum-free medium sphere forming sorting based on prostate cancer stem cell's biology. (authors)

  19. Acetaminophen induces human neuroblastoma cell death through NFKB activation.

    Directory of Open Access Journals (Sweden)

    Inmaculada Posadas

    Full Text Available Neuroblastoma resistance to apoptosis may contribute to the aggressive behavior of this tumor. Therefore, it would be relevant to activate endogenous cellular death mechanisms as a way to improve neuroblastoma therapy. We used the neuroblastoma SH-SY5Y cell line as a model to study the mechanisms involved in acetaminophen (AAP-mediated toxicity by measuring CYP2E1 enzymatic activity, NFkB p65 subunit activation and translocation to the nucleus, Bax accumulation into the mitochondria, cytochrome c release and caspase activation. AAP activates the intrinsic death pathway in the SH-SY5Y human neuroblastoma cell line. AAP metabolism is partially responsible for this activation, because blockade of the cytochrome CYP2E1 significantly reduced but did not totally prevent, AAP-induced SH-SY5Y cell death. AAP also induced NFkB p65 activation by phosphorylation and its translocation to the nucleus, where NFkB p65 increased IL-1β production. This increase contributed to neuroblastoma cell death through a mechanism involving Bax accumulation into the mitochondria, cytochrome c release and caspase3 activation. Blockade of NFkB translocation to the nucleus by the peptide SN50 prevented AAP-mediated cell death and IL-1β production. Moreover, overexpression of the antiapoptotic protein Bcl-x(L did not decrease AAP-mediated IL-1β production, but prevented both AAP and IL-1β-mediated cell death. We also confirmed the AAP toxic actions on SK-N-MC neuroepithelioma and U87MG glioblastoma cell lines. The results presented here suggest that AAP activates the intrinsic death pathway in neuroblastoma cells through a mechanism involving NFkB and IL-1β.

  20. Lactic Acid Bacteria Differentially Activate Natural Killer Cells

    DEFF Research Database (Denmark)

    Fink, Lisbeth Nielsen; Christensen, Hanne Risager; Frøkiær, Hanne

    proliferation of the NK cells and induced IFN-gamma production, both to levels comparable to PHA stimulation. The proliferative response was further enhanced when autologous monocytes were present, probably because cytokines secreted by monocytes having engulfed bacteria stimulated the growth of the NK cells...... antigen presenting cells and T-cells. Bacteria translocating across the gastrointestinal mucosa are presumed to gain access to NK cell compartments, as consumption of certain strains of lactic acid bacteria has been shown to increase in vivo NK cytotoxic activity. On-going research in our lab aims...... at describing strain-dependent effects of lactic acid bacteria on regulatory functions of NK-cells. Here, we have investigated how human gut flora-derived non-pathogenic lactic acid bacteria affect NK cells in vitro, by measuring proliferation and IFN-gamma production of human peripheral blood NK cells upon...

  1. Strategies for Profiling Single Mouse Intestinal Epithelial Cells by Targeted Gene Expression

    OpenAIRE

    McDowell, W.; Box, A. (Antonio); Staehling, K.; Wang, F.; Li, L.; Zueckert-Gaudenz, K.

    2014-01-01

    Targeted gene expression profiling of single cells permits the study of heterogeneity in cell populations. Here, a pool of mouse intestinal crypt-base CD44+/GRP78- cells was collected by fluorescence activated cell sorting. Aliquots were either loaded onto Fluidigm's C1 System for microfluidic cell capture and cDNA synthesis in nanoliter volumes, or flow-sorted directly into individual PCR plate wells for cDNA synthesis in microliter volumes. The pre-amplified cDNAs were transferred to the Bi...

  2. Specific Sorting and Post-Golgi trafficking of Dendritic Potassium Channels in Living Neurons

    DEFF Research Database (Denmark)

    Jensen, Camilla Stampe; Watanabe, Shoji; Rasmussen, Hanne Borger;

    2014-01-01

    localization in distinct dendritic sub-compartments are largely unknown. Here, we developed a quantitative live-cell imaging method to analyze protein sorting and post-Golgi vesicular trafficking. We focused on two dendritic voltage-gated potassium channels which exhibit distinct localizations; Kv2.1 in......, cytoskeletal elements, and motor proteins. By live-cell and super-resolution imaging, we identified a novel trafficking machinery important for the localization of Kv2.1 channels. Particularly, we identified non-muscle myosin II as an important factor in Kv2.1 trafficking. These findings reveal that the...... sorting of ion channels at the Golgi apparatus and their subsequent trafficking by unique molecular mechanisms, are crucial for their specific localizations within dendrites....

  3. Cell volume and membrane stretch independently control K+ channel activity

    DEFF Research Database (Denmark)

    Bomholtz, Sofia Hammami; Willumsen, Niels J; Olsen, Hervør L;

    2009-01-01

    A number of potassium channels including members of the KCNQ family and the Ca(2+) activated IK and SK, but not BK, are strongly and reversibly regulated by small changes in cell volume. It has been argued that this general regulation is mediated through sensitivity to changes in membrane stretch....... To test this hypothesis we have studied the regulation of KCNQ1 and BK channels after expression in Xenopus oocytes. Results from cell-attached patch clamp studies (approximately 50 microm(2) macropatches) in oocytes expressing BK channels demonstrate that the macroscopic volume-insensitive BK current...... was not affected by membrane stretch. The results indicate that (1) activation of BK channels by local membrane stretch is not mimicked by membrane stress induced by cell swelling, and (2) activation of KCNQ1 channels by cell volume increase is not mediated by local tension in the cell membrane. We conclude...

  4. Mast Cell-activated Bone Marrow Mesenchymal Stromal Cells Regulate Proliferation and Lineage Commitment of CD34+ Progenitor cells

    Directory of Open Access Journals (Sweden)

    Zoulfia eAllakhverdi

    2013-12-01

    Full Text Available Background: Shortly after allergen exposure, the number of bone marrow and circulating CD34+ progenitors increases. We aim to analyze the possible mechanism whereby the allergic reaction stimulates bone marrow to release these effector cells in increased numbers. We hypothesize that mast cells may play a predominant role in this process. Objective: To examine the effect of IgE-activated mast cells on bone marrow mesenchymal stromal cells which regulate proliferation and differentiation of CD34+ progenitors. Methods: Primary mast cells were derived from CD34+ precursors and activated with IgE/anti-IgE. Bone marrow mesenchymal stromal cells were co-cultured with CD34+ progenitor cells and stimulated with IL1/TNF or IgE/anti-IgE activated mast cells in Transwell system. Results: Bone marrow mesenchymal stromal cells produce low level of TSLP under steady state conditions, which is markedly increased by stimulation with proinflammatory cytokines IL-1 and TNF or IgE-activated mast cells. The latter also triggers BM-MSCs production of G-CSF, and GM-CSF while inhibiting SDF-1. Mast cell-activated mesenchymal stromal cells stimulate CD34+ cells to proliferate and to regulate their expression of early allergy-associated genes. Conclusion and Clinical Relevance: This in vitro study indicates that IgE-activated mast cells trigger bone marrow mesenchymal stromal cells to release TSLP and hematopoietic growth factors and to regulate the proliferation and lineage commitment of CD34+ precursor cells. The data predict that the effective inhibition of mast cells should impair mobilization and accumulation of allergic effector cells and thereby reduce the severity of allergic diseases.

  5. γδ T Cells Support Pancreatic Oncogenesis by Restraining αβ T Cell Activation.

    Science.gov (United States)

    Daley, Donnele; Zambirinis, Constantinos Pantelis; Seifert, Lena; Akkad, Neha; Mohan, Navyatha; Werba, Gregor; Barilla, Rocky; Torres-Hernandez, Alejandro; Hundeyin, Mautin; Mani, Vishnu Raj Kumar; Avanzi, Antonina; Tippens, Daniel; Narayanan, Rajkishen; Jang, Jung-Eun; Newman, Elliot; Pillarisetty, Venu Gopal; Dustin, Michael Loran; Bar-Sagi, Dafna; Hajdu, Cristina; Miller, George

    2016-09-01

    Inflammation is paramount in pancreatic oncogenesis. We identified a uniquely activated γδT cell population, which constituted ∼40% of tumor-infiltrating T cells in human pancreatic ductal adenocarcinoma (PDA). Recruitment and activation of γδT cells was contingent on diverse chemokine signals. Deletion, depletion, or blockade of γδT cell recruitment was protective against PDA and resulted in increased infiltration, activation, and Th1 polarization of αβT cells. Although αβT cells were dispensable to outcome in PDA, they became indispensable mediators of tumor protection upon γδT cell ablation. PDA-infiltrating γδT cells expressed high levels of exhaustion ligands and thereby negated adaptive anti-tumor immunity. Blockade of PD-L1 in γδT cells enhanced CD4(+) and CD8(+) T cell infiltration and immunogenicity and induced tumor protection suggesting that γδT cells are critical sources of immune-suppressive checkpoint ligands in PDA. We describe γδT cells as central regulators of effector T cell activation in cancer via novel cross-talk.

  6. Inhibition of Cell Growth and Telomerase Activity in Osteosarcoma Cells by DN-hTERT

    Institute of Scientific and Technical Information of China (English)

    XU Tao; RAO Yaojian; ZHU Wentao; GUO Fengjin

    2006-01-01

    In order to study the effects of dominant negative human telomerase reverse transcriptase (DN-hTERT) on cell growth and telomerase activity in osteosarcoma cell line MG63, MG63 cells were transfected with DN-hTERT-IRES2-EGFP9 (DN) or IRES2-EGF (I, blank vector) with lipofectamine 2000. The stably transfected cells were selected with G-418. Cell growth properties were examined under a fluorescence microscope. The hTERT mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). Telomerase activities were measured by TRAP-ELISE. The tumorigenicity was studied with tumor xenografts by subcutaneous injection of cancer cells into nude mice. The results showed that cell growth was suppressed in MG63 cells transfected with DN-hTERT. The hTERT mRNA was increased in N-hTERT transfected-MG63 cells (MG63/DN). The telomerase activity was 2.45±0.11 in MG63/DN cells, while 3.40±0.12 in the cells transfected with blank vector (MG63/I), (P<0.05); DN-hTERT-expressing clones did not form tumors in 2 weeks, but the ratio of tumorigenesis was 30 % in nude mice bearing MG63/I (P<0.01). It was concluded that DN-hTERT could specifically inhibit the cell growth and telomerase activity in MG63 cells.

  7. Microbial solar cells: applying photosynthetic and electrochemically active organisms

    NARCIS (Netherlands)

    Strik, D.P.B.T.B.; Timmers, R.A.; Helder, M.; Steinbusch, K.J.J.; Hamelers, H.V.M.; Buisman, C.J.N.

    2011-01-01

    Microbial solar cells (MSCs) are recently developed technologies that utilize solar energy to produce electricity or chemicals. MSCs use photoautotrophic microorganisms or higher plants to harvest solar energy, and use electrochemically active microorganisms in the bioelectrochemical system to gener

  8. Activation of human tonsil and skin mast cells by agonists of proteinase activated receptor-2

    Institute of Scientific and Technical Information of China (English)

    Shao-heng HE; Hua XIE; Yi-ling FU

    2005-01-01

    Aim: To investigate the effects of the agonists of proteinase activated receptor (PAR)-2,and histamine on degranulation of human mast cells. Methods: Human mast cells were enzymatically dispersed from tonsil and skin tissues. The dis persed cells were then cultured with various stimuli, and tryptase and histamine levels in cell supernatants collected from challenge tubes were measured. Results:PAR-2 agonist peptide SLIGKV provoked a dose-dependent release of histamine from skin mast cells. It also induced tryptase release from tonsil mast cells, tcLIGRLO appeared less potent than SLIGKV in induction of release of histamine and tryptase. Trypsin was able to induce a "bell" shape increase in tryptase release from tonsil mast cells. It was also able to induce a dose-dependent release of histamine from both tonsil and skin mast cells. The actions of trypsin on mast cells were inhibited by soy bean trypsin inhibitor (SBTI) or α1-antitrypsin (α1-AT).Time course study revealed that both stimulated tryptase or histamine release initiated within 10 s and reached their peak release between 4 and 6 min. Pretreatment of cells with metabolic inhibitors or pertussis toxin reduced the ability of mast cells to release tryptase or histamine. Conclusion: It was demonstrated that the in vitro tryptase release properties of human tonsil and skin mast cells suggested a novel type of mast cell heterogeneity. The activation of mast cells by PAR-2 agonists indicated a self-amplification mechanism of mast cell degranulation.

  9. Microspherical photonics: Sorting resonant photonic atoms by using light

    Energy Technology Data Exchange (ETDEWEB)

    Maslov, Alexey V., E-mail: avmaslov@yandex.ru [University of Nizhny Novgorod, Nizhny Novgorod (Russian Federation); Astratov, Vasily N., E-mail: astratov@uncc.edu [Department of Physics and Optical Science, Center for Optoelectronics and Optical Communications, University of North Carolina at Charlotte, Charlotte, North Carolina 28223-0001 (United States)

    2014-09-22

    A method of sorting microspheres by resonant light forces in vacuum, air, or liquid is proposed. Based on a two-dimensional model, it is shown that the sorting can be realized by allowing spherical particles to traverse a focused beam. Under resonance with the whispering gallery modes, the particles acquire significant velocity along the beam direction. This opens a unique way of large-volume sorting of nearly identical photonic atoms with 1/Q accuracy, where Q is the resonance quality factor. This is an enabling technology for developing super-low-loss coupled-cavity structures and devices.

  10. Efficient Sorting of Free Electron Orbital Angular Momentum

    CERN Document Server

    McMorran, Benjamin J; Lavery, Martin P J

    2016-01-01

    We propose a method for sorting electrons by orbital angular momentum (OAM). Several methods now exist to prepare electron wavefunctions in OAM states, but no technique has been developed for efficient, parallel measurement of pure and mixed electron OAM states. The proposed technique draws inspiration from the recent demonstration of the sorting of OAM through modal transformation. We show that the same transformation can be performed with electrostatic electron optical elements. Specifically, we show that a charged needle and an array of electrodes perform the transformation and phase correction necessary to sort orbital angular momentum states. This device may enable the analysis of the spatial mode distribution of inelastically scattered electrons.

  11. Let’s limit our waste production and let’s’ sort it!

    CERN Multimedia

    HSE Unit

    2013-01-01

    Let’s limit our waste production! – Why ? Preventing the production of waste is the best solution to avoid environmental issues, economic impacts and technical constraints. So, whenever you are involved in the design, manufacturing, distribution, use or dismantling of a product or an activity in general, always remember that the best waste is that which is not produced. The limitation of waste production being an HSE objective declared in 2013 by the CERN Director-General, we encourage everyone to help limit the amount of waste produced through CERN activities. Let’s sort it! – Why ? Since the 90s, CERN has implemented a policy to promote recovery of the waste* generated by its activities. Nowadays, CERN is committed to continuously improving its sorting and recovery and therefore various initiatives have been started by GS-IS to improve the recovery of waste (e.g. recovery of organic waste from restaurants; implementation of solar trash compactors - see Bulletin 27-...

  12. Surface kinematics of periglacial sorted circles using Structure-from-Motion technology

    Directory of Open Access Journals (Sweden)

    A. Kääb

    2013-12-01

    Full Text Available Sorted soil circles are a conspicuous form of periglacial patterned ground. Numerical modelling suggests that these features develop from a convection-like circulation of material in the active layer of permafrost. The related iterative burying and resurfacing of material is believed to play an important role in the soil carbon cycle of high latitudes. The connection of sorted circles to permafrost conditions and its changes over time make these ground forms to a potential paleoclimatic indicator. In this study we apply the photogrammetric Structure-from-Motion technology (SfM to large sets of overlapping terrestrial photos taken in Augusts 2007 and 2010 over three sorted circles at Kvadehuksletta, Western Spitsbergen. We retrieve repeat digital elevation models (DEMs and orthoimages with millimetre-resolution and accuracy. Changes in microrelief over the three years are obtained from DEM-differencing and horizontal displacement fields from tracking features between the orthoimages. In the inner domains of the circles, consisting of fines, material moves radially outside with horizontal surface speeds of up to 2 cm yr−1. The outer circle ridges consist of coarse stones that displace towards the inner circle domain at similar rates. A number of substantial deviations from this overall radial symmetry, both in horizontal displacements and in microrelief, shed new light on the potential spatio-temporal evolution of sorted soil circles, and periglacial patterned ground in general.

  13. Heavy mineral sorting in downwards injected Palaeocene sandstone, Siri Canyon, Danish North Sea

    Science.gov (United States)

    Kazerouni, Afsoon Moatari; Friis, Henrik; Svendsen, Johan Byskov; Weibel, Rikke

    2011-05-01

    Post-depositional remobilization and injection of sand are often seen in deep-water clastic systems and have been recently recognised as a significant modifier of deep-water sandstone geometry. Large scale injectite complexes have been interpreted from borehole data in the Palaeocene Siri Canyon near the Danish Central Graben of the North Sea hydrocarbon province. The emplacement of large scale injectite complexes has been commonly attributed to seismic activity and consequent sand liquefaction. However, due to very small differences in textural and compositional properties, and the lack of depositional structures in deep-water sandstones, the distinction between "in situ" and injected or remobilized sandstones is often ambiguous. Large scale heavy mineral sorting (in 10 m thick units) is observed in several reservoir units in the Siri Canyon and has been interpreted to represent the depositional sorting. In this study we describe an example of effective shear-zone sorting of heavy minerals in a thin downwards injected sandstone dyke which was encountered in one of the cores in the Cecilie Field, Siri Canyon. Differences in sorting pattern of heavy minerals are suggested as a tool for petrographic/geochemical distinction between "in situ" sandstones and their related injectites, especially where primary sedimentary structures are removed by fluidization or minor remobilization.

  14. Resident macrophages influence stem cell activity in the mammary gland

    OpenAIRE

    Gyorki, D.E.; Asselin-Labat, M.L.; Rooijen, van, J.; Lindeman, G J; Visvader, J E

    2009-01-01

    Introduction Macrophages in the mammary gland are essential for morphogenesis of the ductal epithelial tree and have been implicated in promoting breast tumor metastasis. Although it is well established that macrophages influence normal mammopoiesis, the mammary cell types that these accessory cells influence have not been determined. Here we have explored a role for macrophages in regulating mammary stem cell (MaSC) activity, by assessing the ability of MaSCs to reconstitute a mammary gland ...

  15. Mediated Electrochemical Measurements of Intracellular Catabolic Activities of Yeast Cells

    Institute of Scientific and Technical Information of China (English)

    Jin Sheng ZHAO; Zhen Yu YANG; Yao LU; Zheng Yu YANG

    2005-01-01

    Coupling with the dual mediator system menadione/ferricyanide, microelectrode voltammetric measurements were undertaken to detect the ferrocyanide accumulations arising from the mediated reduction of ferricyanide by yeast cells. The results indicate that the dual mediator system menadione/ferricyanide could be used as a probe to detect cellular catabolic activities in yeast cells and the electrochemical response has a positive relationship with the specific growth rate of yeast cells.

  16. MicroRNA expression profiling identifies activated B cell status in chronic lymphocytic leukemia cells.

    Directory of Open Access Journals (Sweden)

    Shuqiang Li

    Full Text Available Chronic lymphocytic leukemia (CLL is thought to be a disease of resting lymphocytes. However, recent data suggest that CLL cells may more closely resemble activated B cells. Using microRNA (miRNA expression profiling of highly-enriched CLL cells from 38 patients and 9 untransformed B cells from normal donors before acute CpG activation and 5 matched B cells after acute CpG activation, we demonstrate an activated B cell status for CLL. Gene set enrichment analysis (GSEA identified statistically-significant similarities in miRNA expression between activated B cells and CLL cells including upregulation of miR-34a, miR-155, and miR-342-3p and downregulation of miR-103, miR-181a and miR-181b. Additionally, decreased levels of two CLL signature miRNAs miR-29c and miR-223 are associated with ZAP70(+ and IgV(H unmutated status and with shorter time to first therapy. These data indicate an activated B cell status for CLL cells and suggest that the direction of change of individual miRNAs may predict clinical course in CLL.

  17. DMPD: Activation of lymphokine genes in T cells: role of cis-acting DNA elements thatrespond to T cell activation signals. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 1492121 Activation of lymphokine genes in T cells: role of cis-acting DNA elements ...html) (.csml) Show Activation of lymphokine genes in T cells: role of cis-acting ...DNA elements thatrespond to T cell activation signals. PubmedID 1492121 Title Activation of lymphokine genes in T cells: role

  18. Cellular, Molecular Consequences of Peroxisome Proliferator- Activated Receptor-δ Activation in Ovarian Cancer Cells

    Directory of Open Access Journals (Sweden)

    Sara Vignati

    2006-10-01

    Full Text Available Peroxisome proliferator-activated receptor-δ (PPAR-δ is a ligand-activated transcription factor. In addition to its canonical role in lipid, glucose metabolism, PPAR-δ controls cell proliferation, death, differentiation in several tissues. Here we have examined the expression of PPAR-δ in ovarian tumors, the cellular, molecular consequences of its activation in ovarian cancer cells. PPAR-δ was expressed in a large number of epithelial ovarian tumors, cell lines. The PPAR-δ lig, ciglitazone inhibited the growth, clonogenic survival of ovarian cancer cells, inducing cell cycle arrest, cell death. Growth inhibition by ciglitazone was reversed by the PPAR-δ antagonist GW9662, indicating the involvement of PPAR-δ- dependent mechanisms. Microarray-based gene profiling revealed complex changes in the transcriptional program of ovarian cancer cells on treatment with ciglitazone, identified multiple pathways that may contribute to PPAR-δ ligands' antitumor activity. Genes upregulated by ciglitazone were predominantly associated with metabolic, differentiation, tumorsuppressor pathways, whereas downregulated genes were involved in cell proliferation, cell cycle, cell organization, steroid biosynthesis. Collectively, our data indicate that PPAR-δ activation by selective agonists is a valid strategy for ovarian cancer therapy, prevention, should be tested alone, in combination with other anticancer drugs.

  19. H pylori stimulates proliferation of gastric cancer cells through activating mitogen-activated protein kinase cascade

    Institute of Scientific and Technical Information of China (English)

    Yong-Chang Chen; Ying Wang; Jing-Yan Li; Wen-Rong Xu; You-Li Zhang

    2006-01-01

    AIM: To explore the mechanism by which H pylori causes activation of gastric epithelial cells.METHODS: A VacA (+) and CagA (+) standard Hpyloriline NCTC 11637 and a human gastric adenocarcinoma derived gastric epithelial cell line BGC-823 were applied in the study. MTT assay and 3H-TdR incorporation test were used to detect the proliferation of BGC-823 cells and Western blotting was used to detect the activity and existence of related proteins.RESULTS: Incubation with Hpylori extract increased the proliferation of gastric epithelial cells, reflected by both live cell number and DNA synthesis rate. The activity of extracellular signal-regulated protein kinase (ERK) signal transduction cascade increased within 20 min after incubation with Hpylori extract and appeared to be a sustained event. MAPK/ERK kinase (MEK) inhibitor PD98059abolished the action of H pylori extract on both ERK activity and cell proliferation. Incubation with H pyloriextract increased c-Fos expression and SRE-dependentgene expression. H pylori extract caused phosphorylation of several proteins including a protein with molecular size of 97.4 kDa and tyrosine kinase inhibitor genistein inhibited the activation of ERK and the proliferation of cells caused by H pylori extract.CONCLUSION: Biologically active elements in H pylori extract cause proliferation of gastric epithelial cells through activating tyrosine kinase and ERK signal transduction cascade.

  20. Activated T cells sustain myeloid-derived suppressor cell-mediated immune suppression.

    Science.gov (United States)

    Pinton, Laura; Solito, Samantha; Damuzzo, Vera; Francescato, Samuela; Pozzuoli, Assunta; Berizzi, Antonio; Mocellin, Simone; Rossi, Carlo Riccardo; Bronte, Vincenzo; Mandruzzato, Susanna

    2016-01-12

    The expansion of myeloid derived suppressor cells (MDSCs), a suppressive population able to hamper the immune response against cancer, correlates with tumor progression and overall survival in several cancer types. We have previously shown that MDSCs can be induced in vitro from precursors present in the bone marrow and observed that these cells are able to actively proliferate in the presence of activated T cells, whose activation level is critical to drive the suppressive activity of MDSCs. Here we investigated at molecular level the mechanisms involved in the interplay between MDSCs and activated T cells. We found that activated T cells secrete IL-10 following interaction with MDSCs which, in turn, activates STAT3 phosphorylation on MDSCs then leading to B7-H1 expression. We also demonstrated that B7-H1+ MDSCs are responsible for immune suppression through a mechanism involving ARG-1 and IDO expression. Finally, we show that the expression of ligands B7-H1 and MHC class II both on in vitro-induced MDSCs and on MDSCs in the tumor microenvironment of cancer patients is paralleled by an increased expression of their respective receptors PD-1 and LAG-3 on T cells, two inhibitory molecules associated with T cell dysfunction. These findings highlight key molecules and interactions responsible for the extensive cross-talk between MDSCs and activated T cells that are at the basis of immune suppression.

  1. Programmed Cell-to-Cell Variability in Ras Activity Triggers Emergent Behaviors during Mammary Epithelial Morphogenesis

    Directory of Open Access Journals (Sweden)

    Jennifer S. Liu

    2012-11-01

    Full Text Available Variability in signaling pathway activation between neighboring epithelial cells can arise from local differences in the microenvironment, noisy gene expression, or acquired genetic changes. To investigate the consequences of this cell-to-cell variability in signaling pathway activation on coordinated multicellular processes such as morphogenesis, we use DNA-programmed assembly to construct three-dimensional MCF10A microtissues that are mosaic for low-level expression of activated H-Ras. We find two emergent behaviors in mosaic microtissues: cells with activated H-Ras are basally extruded or lead motile multicellular protrusions that direct the collective motility of their wild-type neighbors. Remarkably, these behaviors are not observed in homogeneous microtissues in which all cells express the activated Ras protein, indicating that heterogeneity in Ras activity, rather than the total amount of Ras activity, is critical for these processes. Our results directly demonstrate that cell-to-cell variability in pathway activation within local populations of epithelial cells can drive emergent behaviors during epithelial morphogenesis.

  2. Autofluorescence as a Signal to Sort Developing Glandular Trichomes by Flow Cytometry.

    Science.gov (United States)

    Bergau, Nick; Navarette Santos, Alexander; Henning, Anja; Balcke, Gerd U; Tissier, Alain

    2016-01-01

    The industrial relevance of a number of metabolites produced in plant glandular trichomes (GTs) has spurred research on these specialized organs for a number of years. Most of the research, however, has focused on the elucidation of secondary metabolite pathways and comparatively little has been undertaken on the development and differentiation of GTs. One way to gain insight into these developmental processes is to generate stage-specific transcriptome and metabolome data. The difficulty for this resides in the isolation of early stages of development of the GTs. Here we describe a method for the separation and isolation of intact young and mature type VI trichomes from the wild tomato species Solanum habrochaites. The final and key step of the method uses cell sorting based on distinct autofluorescence signals of the young and mature trichomes. We demonstrate that sorting by flow cytometry allows recovering pure fractions of young and mature trichomes. Furthermore, we show that the sorted trichomes can be used for transcript and metabolite analyses. Because many plant tissues or cells have distinct autofluorescence components, the principles of this method can be generally applicable for the isolation of specific cell types without prior labeling. PMID:27446176

  3. Autofluorescence as a Signal to Sort Developing Glandular Trichomes by Flow Cytometry

    Science.gov (United States)

    Bergau, Nick; Navarette Santos, Alexander; Henning, Anja; Balcke, Gerd U.; Tissier, Alain

    2016-01-01

    The industrial relevance of a number of metabolites produced in plant glandular trichomes (GTs) has spurred research on these specialized organs for a number of years. Most of the research, however, has focused on the elucidation of secondary metabolite pathways and comparatively little has been undertaken on the development and differentiation of GTs. One way to gain insight into these developmental processes is to generate stage-specific transcriptome and metabolome data. The difficulty for this resides in the isolation of early stages of development of the GTs. Here we describe a method for the separation and isolation of intact young and mature type VI trichomes from the wild tomato species Solanum habrochaites. The final and key step of the method uses cell sorting based on distinct autofluorescence signals of the young and mature trichomes. We demonstrate that sorting by flow cytometry allows recovering pure fractions of young and mature trichomes. Furthermore, we show that the sorted trichomes can be used for transcript and metabolite analyses. Because many plant tissues or cells have distinct autofluorescence components, the principles of this method can be generally applicable for the isolation of specific cell types without prior labeling. PMID:27446176

  4. Transmembrane protein sorting driven by membrane curvature

    NARCIS (Netherlands)

    H. Strahl; S. Ronneau; B. Solana González; D. Klutsch; C. Schaffner-Barbero; L.W. Hamoen

    2015-01-01

    The intricate structure of prokaryotic and eukaryotic cells depends on the ability to target proteins to specific cellular locations. In most cases, we have a poor understanding of the underlying mechanisms. A typical example is the assembly of bacterial chemoreceptors at cell poles. Here we show th

  5. Enhancement of endothelial cell migration by constitutively active LPA{sub 1}-expressing tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Kitayoshi, Misaho; Kato, Kohei; Tanabe, Eriko; Yoshikawa, Kyohei; Fukui, Rie [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Fukushima, Nobuyuki [Division of Molecular Neurobiology, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Tsujiuchi, Toshifumi, E-mail: ttujiuch@life.kindai.ac.jp [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan)

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer Mutated LPA{sub 1} stimulates cell migration of endothelial cells. Black-Right-Pointing-Pointer VEGF expressions are increased by mutated LPA{sub 1}. Black-Right-Pointing-Pointer LPA signaling via mutated LPA{sub 1} is involved in angiogenesis. Black-Right-Pointing-Pointer Mutated LPA{sub 1} promotes cancer cell progression. -- Abstract: Lysophosphatidic acid (LPA) receptors belong to G protein-coupled transmembrane receptors (LPA receptors; LPA{sub 1} to LPA{sub 6}). They indicate a variety of cellular response by the interaction with LPA, including cell proliferation, migration and differentiation. Recently, we have reported that constitutive active mutated LPA{sub 1} induced the strong biological effects of rat neuroblastoma B103 cells. In the present study, we examined the effects of mutated LPA{sub 1} on the interaction between B103 cells and endothelial F-2 cells. Each LPA receptor expressing B103 cells were maintained in serum-free DMEM and cell motility assay was performed with a Cell Culture Insert. When F-2 cells were cultured with conditioned medium from Lpar1 and Lpar3-expressing cells, the cell motility of F-2 cells was significantly higher than control cells. Interestingly, the motile activity of F-2 cells was strongly induced by mutated LPA{sub 1} than other cells, correlating with the expression levels of vascular endothelial growth factor (Vegf)-A and Vegf-C. Pretreatment of LPA signaling inhibitors inhibited F-2 cell motility stimulated by mutated LPA{sub 1}. These results suggest that activation of LPA signaling via mutated LPA{sub 1} may play an important role in the promotion of angiogenesis in rat neuroblastoma cells.

  6. Cbl negatively regulates JNK activation and cell death

    Institute of Scientific and Technical Information of China (English)

    Andrew A Sproul; Zhiheng Xu; Michael Wilhelm; Stephen Gire; Lloyd A Greene

    2009-01-01

    Here, we explore the role of Cbl proteins in regulation of neuronal apoptosis. In two paradigms of neuron apopto-sis--nerve growth factor (NGF) deprivation and DNA damage--cellular levels of c-Cbl and Cbl-b fell well before the onset of cell death. NGF deprivation also induced rapid loss of tyrosine phosphorylation (and most likely, activa-tion) of c-Cbl. Targeting e-Cbl and Cbl-b with siRNAs to mimic their loss/inactivation sensitized neuronal cells to death promoted by NGF deprivation or DNA damage. One potential mechanism by which Cbl proteins might affect neuronal death is by regulation of apoptotic c-Jun N-terminal kinase (JNK) signaling. We demonstrate that Cbl pro-teins interact with the JNK pathway components mixed lineage kinase (MLK) 3 and POSH and that knockdown of Cbl proteins is sufficient to increase JNK pathway activity. Furthermore, expression of c-Cbl blocks the ability of MLKs to signal to downstream components of the kinase cascade leading to JNK activation and protects neuronal cells from death induced by MLKs, but not from downstream JNK activators. On the basis of these findings, we propose that Cbls suppress cell death in healthy neurons at least in part by inhibiting the ability of MLKs to activate JNK signaling. Apoptotic stimuli lead to loss of Cbl protein/activity, thereby removing a critical brake on JNK acti-vation and on cell death.

  7. Sleep-active cells in the cerebral cortex and their role in slow-wave activity

    OpenAIRE

    Gerashchenko, Dmitry; Wisor, Jonathan P.; Kilduff, Thomas S.

    2011-01-01

    We recently identified neurons in the cerebral cortex that become activated during sleep episodes with high slow-wave activity (SWA). The distinctive properties of these neurons are the ability to produce nitric oxide and their long-range projections within the cortex. In this review, we discuss how these characteristics of sleep-active cells could be relevant to SWA production in the cortex. We also discuss possible models of the role of nNOS cells in SWA production.

  8. Chip-based device for parallel sorting, amplification, detection, and identification of nucleic acid subsequences

    Energy Technology Data Exchange (ETDEWEB)

    Beer, Neil Reginald; Colston, Jr, Billy W.

    2016-08-09

    An apparatus for chip-based sorting, amplification, detection, and identification of a sample having a planar substrate. The planar substrate is divided into cells. The cells are arranged on the planar substrate in rows and columns. Electrodes are located in the cells. A micro-reactor maker produces micro-reactors containing the sample. The micro-reactor maker is positioned to deliver the micro-reactors to the planar substrate. A microprocessor is connected to the electrodes for manipulating the micro-reactors on the planar substrate. A detector is positioned to interrogate the sample contained in the micro-reactors.

  9. Minimal model for spontaneous cell polarization and edge activity in oscillating, rotating and migrating cells

    Science.gov (United States)

    Raynaud, Franck; Ambühl, Mark E.; Gabella, Chiara; Bornert, Alicia; Sbalzarini, Ivo F.; Meister, Jean-Jacques; Verkhovsky, Alexander B.

    2016-04-01

    How cells break symmetry and organize activity at their edges to move directionally is a fundamental question in cell biology. Physical models of cell motility commonly incorporate gradients of regulatory proteins and/or feedback from the motion itself to describe the polarization of this edge activity. These approaches, however, fail to explain cell behaviour before the onset of polarization. We use polarizing and moving fish epidermal cells as a model system to bridge the gap between cell behaviours before and after polarization. Our analysis suggests a novel and simple principle of self-organizing cell activity, in which local cell-edge dynamics depends on the distance from the cell centre, but not on the orientation with respect to the front-back axis. We validate this principle with a stochastic model that faithfully reproduces a range of cell-migration behaviours. Our findings indicate that spontaneous polarization, persistent motion and cell shape are emergent properties of the local cell-edge dynamics controlled by the distance from the cell centre.

  10. The calcium-activated potassium channels of turtle hair cells

    OpenAIRE

    1995-01-01

    A major factor determining the electrical resonant frequency of turtle cochlear hair cells is the time course of the Ca-activated K current (Art, J. J., and R. Fettiplace. 1987. Journal of Physiology. 385:207- 242). We have examined the notion that this time course is dictated by the K channel kinetics by recording single Ca-activated K channels in inside-out patches from isolated cells. A hair cell's resonant frequency was estimated from its known correlation with the dimensions of the hair ...

  11. Volume changes during active shape fluctuations in cells

    CERN Document Server

    Taloni, Alessandro; Salman, Oguz Umut; Truskinovsky, Lev; Zapperi, Stefano; La Porta, Caterina A M

    2015-01-01

    Cells modify their volume in response to changes in osmotic pressure but it is usually assumed that other active shape variations do not involve significant volume fluctuations. Here we report experiments demonstrating that water transport in and out of the cell is needed for the formation of blebs, commonly observed protrusions in the plasma membrane driven by cortex contraction. We develop and simulate a model of fluid mediated membrane-cortex deformations and show that a permeable membrane is necessary for bleb formation which is otherwise impaired. Taken together our experimental and theoretical results emphasize the subtle balance between hydrodynamics and elasticity in actively driven cell morphological changes.

  12. Lymphokine-activated killer cell phenomenon. III. Evidence that IL-2 is sufficient for direct activation of peripheral blood lymphocytes into lymphokine-activated killer cells

    OpenAIRE

    1983-01-01

    Purified interleukin 2 (IL-2) was found to be sufficient for direct activation of peripheral blood lymphocytes into lymphokine-activated killer (LAK) cells. The LAK activation factor was directly and consistently associated with IL-2 activity using classic protein purification techniques, adsorption to IL-2-dependent cell lines, and inhibition with anti-Tac antibody. As yet, no other cytokines have been found that perform the same role.

  13. Running worms: C. elegans self-sorting by electrotaxis.

    Directory of Open Access Journals (Sweden)

    Xavier Manière

    Full Text Available The nematode C. elegans displays complex dynamical behaviors that are commonly used to identify relevant phenotypes. Although its maintenance is straightforward, sorting large populations of worms when looking for a behavioral phenotype is difficult, time consuming and hardly quantitative when done manually. Interestingly, when submitted to a moderate electric field, worms move steadily along straight trajectories. Here, we report an inexpensive method to measure worms crawling velocities and sort them within a few minutes by taking advantage of their electrotactic skills. This method allows to quantitatively measure the effect of mutations and aging on worm's crawling velocity. We also show that worms with different locomotory phenotypes can be spatially sorted, fast worms traveling away from slow ones. Group of nematodes with comparable locomotory fitness could then be isolated for further analysis. C. elegans is a growing model for neurodegenerative diseases and using electrotaxis for self-sorting can improve the high-throughput search of therapeutic bio-molecules.

  14. Brazil Nuts on Eros: Size-Sorting of Asteroid Regolith

    Science.gov (United States)

    Asphaug, E.; King, P. J.; Swift, M. R.; Merrifield, M. R.

    2001-01-01

    We consider the hypothesis that frequent cratering produces size- or compositionally-sorted asteroid regolith, affecting the structure, texture, and in extreme cases the shape of asteroids. Additional information is contained in the original extended abstract.

  15. Unsupervised Spike Sorting Based on Discriminative Subspace Learning

    CERN Document Server

    Keshtkaran, Mohammad Reza

    2014-01-01

    Spike sorting is a fundamental preprocessing step for many neuroscience studies which rely on the analysis of spike trains. In this paper, we present two unsupervised spike sorting algorithms based on discriminative subspace learning. The first algorithm simultaneously learns the discriminative feature subspace and performs clustering. It uses histogram of features in the most discriminative projection to detect the number of neurons. The second algorithm performs hierarchical divisive clustering that learns a discriminative 1-dimensional subspace for clustering in each level of the hierarchy until achieving almost unimodal distribution in the subspace. The algorithms are tested on synthetic and in-vivo data, and are compared against two widely used spike sorting methods. The comparative results demonstrate that our spike sorting methods can achieve substantially higher accuracy in lower dimensional feature space, and they are highly robust to noise. Moreover, they provide significantly better cluster separab...

  16. Using Sorting Networks for Skill Building and Reasoning

    Science.gov (United States)

    Andre, Robert; Wiest, Lynda R.

    2007-01-01

    Sorting networks, used in graph theory, have instructional value as a skill- building tool as well as an interesting exploration in discrete mathematics. Students can practice mathematics facts and develop reasoning and logic skills with this topic. (Contains 4 figures.)

  17. The EARP Complex and Its Interactor EIPR-1 Are Required for Cargo Sorting to Dense-Core Vesicles.

    Science.gov (United States)

    Topalidou, Irini; Cattin-Ortolá, Jérôme; Pappas, Andrea L; Cooper, Kirsten; Merrihew, Gennifer E; MacCoss, Michael J; Ailion, Michael

    2016-05-01

    The dense-core vesicle is a secretory organelle that mediates the regulated release of peptide hormones, growth factors, and biogenic amines. Dense-core vesicles originate from the trans-Golgi of neurons and neuroendocrine cells, but it is unclear how this specialized organelle is formed and acquires its specific cargos. To identify proteins that act in dense-core vesicle biogenesis, we performed a forward genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We previously reported the identification of two conserved proteins that interact with the small GTPase RAB-2 to control normal dense-core vesicle cargo-sorting. Here we identify several additional conserved factors important for dense-core vesicle cargo sorting: the WD40 domain protein EIPR-1 and the endosome-associated recycling protein (EARP) complex. By assaying behavior and the trafficking of dense-core vesicle cargos, we show that mutants that lack EIPR-1 or EARP have defects in dense-core vesicle cargo-sorting similar to those of mutants in the RAB-2 pathway. Genetic epistasis data indicate that RAB-2, EIPR-1 and EARP function in a common pathway. In addition, using a proteomic approach in rat insulinoma cells, we show that EIPR-1 physically interacts with the EARP complex. Our data suggest that EIPR-1 is a new interactor of the EARP complex and that dense-core vesicle cargo sorting depends on the EARP-dependent trafficking of cargo through an endosomal sorting compartment. PMID:27191843

  18. NK-cell activity in immunotoxicity drug evaluation

    International Nuclear Information System (INIS)

    NK-cell activity as a tool for detection of immunotoxic effects of new human drugs has gained further attention when the recent European note for guidance CPMP/SWP/1042/99 was adopted. The inclusion of NK-cell activity plus distribution of lymphocyte subsets were suggested as an alternative to the primary antibody response to a T-cell dependent antigen. Either of the two test alternatives should be included as a routine parameter in at least one repeated dose-toxicity study, rats or mice being the species of choice. The standard procedure for measuring NK-cell activity is the 51Cr-release assay. However, a new flow-cytometric assay, adapted for rat peripheral blood, does not require dedicated groups of animals, offers the possibility of repeated testing, and shows at least as sensitive as the conventional 51Cr-release assay

  19. Sorting and hardware assisted rendering for volume visualization

    Energy Technology Data Exchange (ETDEWEB)

    Stein, C.; Becker, B.; Max, N.

    1994-03-01

    We present some techniques for volume rendering unstructured data. Interpolation between vertex colors and opacities is performed using hardware assisted texture mapping, and color is integrated for use with a volume rendering system. We also present an O(n{sup 2}) method for sorting n arbitrarily shaped convex polyhedra prior to visualization. It generalizes the Newell, Newell and Sancha sort for polygons to 3-D volume elements.

  20. Selective Antitumor Activity of Ibrutinib in EGFR-Mutant Non–Small Cell Lung Cancer Cells

    OpenAIRE

    Gao, Wen; Wang, Michael; Wang, Li; Lu, Haibo; Wu, Shuhong; Dai, Bingbing; Ou, Zhishuo; Zhang, Liang; Heymach, John V.; Gold, Kathryn A.; Minna, John ,; Roth, Jack A.; Hofstetter, Wayne L.; Swisher, Stephen G.; Fang, Bingliang

    2014-01-01

    Ibrutinib, which irreversibly inhibits Bruton tyrosine kinase, was evaluated for antitumor activity in a panel of non–small cell lung cancer (NSCLC) cell lines and found to selectively inhibit growth of NSCLC cells carrying mutations in the epidermal growth factor receptor (EGFR) gene, including T790M mutant and erlotinib-resistant H1975 cells. Ibrutinib induced dose-dependent inhibition of phosphor-EGFR at both Y1068 and Y1173 sites, suggesting ibrutinib functions as an EGFR inhibitor. Survi...