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Sample records for nyh cells selected

  1. In vitro cross-resistance and collateral sensitivity in seven resistant small-cell lung cancer cell lines: preclinical identification of suitable drug partners to taxotere, taxol, topotecan and gemcitabin.

    PubMed Central

    Jensen, P. B.; Holm, B.; Sorensen, M.; Christensen, I. J.; Sehested, M.

    1997-01-01

    The acquisition of drug-resistant tumour cells is the main problem in the medical treatment of a range of malignant diseases. In recent years, three new classes of anti-cancer agents, each with a novel mechanism of action, have been brought forward to clinical trials. These are the topoisomerase I (topo I) poisons topotecan and irinotecan, which are both camptothecin derivatives, the taxane tubulin stabilizers taxol and taxotere and, finally, the antimetabolite gemcitabin, which is active in solid tumours. The process of optimizing their use in a combination with established agents is very complex, with numerous possible drug and schedule regimens. We describe here how a broad panel of drug-resistant small-cell lung cancer (SCLC) cell lines can be used as a model of tumour heterogeneity to aid in the selection of non-cross-resistant regimens. We have selected low-fold (3-10x) drug-resistant sublines from a classic (NCI-H69) and a variant (OC-NYH) SCLC cell line. The resistant cell lines include two sublines with different phenotypes towards alkylating agents (H69/BCNU and NYH/CIS), two sublines with different phenotypes against topo I poisons (NYH/CAM and NYH/TPT) and three multidrug resistant (MDR) sublines (H69/DAU, NYH/VM, and H69/VP) with combinations of mdr1 and MRP overexpression as well as topoisomerase II (topo II) down-regulation or mutation. Sensitivity to 20 established and new agents was measured in a standardized clonogenic assay. Resistance was highly drug specific. Thus, none of the cell lines was resistant to all drugs. In fact, all resistant cell lines exhibited patterns of collateral sensitivity to various different classes of drugs. The most intriguing pattern was collateral sensitivity to gemcitabin in two cell lines and to ara-C in five drug-resistant cell lines, i.e. in all lines except the lines resistant to topo I poisons. Next, all sensitivity patterns in the nine cell lines were compared by correlation analysis. A high correlation

  2. Sickle Cell: A Selected Resource Bibliography.

    ERIC Educational Resources Information Center

    National Center for Education in Maternal and Child Health, Washington, DC.

    This annotated, selective bibliography lists the following types of educational and informational material on both sickle cell disease and trait: (1) professional education materials; (2) fact sheets, pamphlets, and brochures; and (3) audiovisual material. A selected list of references is provided for the following topic areas: (1) genetic…

  3. Bead-Selected Antitumor Genetic Cell Vaccines

    PubMed Central

    Herrero, MJ; R, Botella; R, Algás; Marco, FM; Aliño, SF

    2008-01-01

    Cancer vaccines have always been in the scope of gene therapy research. One of the most successful approaches has been working with genetically modified tumor cells. However, to become a clinical reality, tumor cells must suffer a long and risky process from the extraction from the patient to the reimplantation as a vaccine. In this work, we explain our group’s approach to reduce the cell number required to achieve an immune response against a melanoma murine model, employing bead-selected B16 tumor cells expressing GM-CSF and B7.2. PMID:21892287

  4. Broadening cell selection criteria with micropallet arrays of adherent cells.

    PubMed

    Wang, Yuli; Young, Grace; Aoto, Phillip C; Pai, Jeng-Hao; Bachman, Mark; Li, G P; Sims, Christopher E; Allbritton, Nancy L

    2007-10-01

    A host of technologies exists for the separation of living, nonadherent cells, with separation decisions typically based on fluorescence or immunolabeling of cells. Methods to separate adherent cells as well as to broaden the range of possible sorting criteria would be of high value and complementary to existing strategies. Cells were cultured on arrays of releasable pallets. The arrays were screened and individual cell(s)/pallets were released and collected. Conventional fluorescence and immunolabeling of cells were compatible with the pallet arrays, as were separations based on gene expression. By varying the size of the pallet and the number of cells cultured on the array, single cells or clonal colonies of cells were isolated from a heterogeneous population. Since cells remained adherent throughout the isolation process, separations based on morphologic characteristics, for example cell shape, were feasible. Repeated measurements of each cell in an array were performed permitting the selection of cells based on their temporal behavior, e.g. growth rate. The pallet array system provides the flexibility to select and collect adherent cells based on phenotypic and temporal criteria and other characteristics not accessible by alternative methods. PMID:17559133

  5. Engineering novel cell surface chemistry for selective tumor cell targeting

    SciTech Connect

    Bertozzi, C.R. |

    1997-12-31

    A common feature of many different cancers is the high expression level of the two monosaccharides sialic acid and fucose within the context of cell-surface associated glycoconjugates. A correlation has been made between hypersialylation and/or hyperfucosylation and the highly metastatic phenotype. Thus, a targeting strategy based on sialic acid or fucose expression would be a powerful tool for the development of new cancer cell-selective therapies and diagnostic agents. We have discovered that ketone groups can be incorporated metabolically into cell-surface associated sialic acids. The ketone is can be covalently ligated with hydrazide functionalized proteins or small molecules under physiological conditions. Thus, we have discovered a mechanism to selectively target hydrazide conjugates to highly sialylated cells such as cancer cells. Applications of this technology to the generation of novel cancer cell-selective toxins and MRI contrast reagents will be discussed, in addition to progress towards the use of cell surface fucose residues as vehicles for ketone expression.

  6. Microgravity-Enhanced Stem Cell Selection

    NASA Technical Reports Server (NTRS)

    Claudio, Pier Paolo; Valluri, Jagan

    2011-01-01

    Stem cells, both embryonic and adult, promise to revolutionize the practice of medicine in the future. In order to realize this potential, a number of hurdles must be overcome. Most importantly, the signaling mechanisms necessary to control the differentiation of stem cells into tissues of interest remain to be elucidated, and much of the present research on stem cells is focused on this goal. Nevertheless, it will also be essential to achieve large-scale expansion and, in many cases, assemble cells in 3D as transplantable tissues. To this end, microgravity analog bioreactors can play a significant role. Microgravity bioreactors were originally conceived as a tool to study the cellular responses to microgravity. However, the technology can address some of the shortcomings of conventional cell culture systems; namely, the deficiency of mass transport in static culture and high mechanical shear forces in stirred systems. Unexpectedly, the conditions created in the vessel were ideal for 3D cell culture. Recently, investigators have demonstrated the capability of the microgravity bioreactors to expand hematopoietic stem cells compared to static culture, and facilitate the differentiation of umbilical cord stem cells into 3D liver aggregates. Stem cells are capable of differentiating into functional cells. However, there are no reliable methods to induce the stem cells to form specific cells or to gain enough cells for transplantation, which limits their application in clinical therapy. The aim of this study is to select the best experimental setup to reach high proliferation levels by culturing these cells in a microgravity-based bioreactor. In typical cell culture, the cells sediment to the bottom surface of their container and propagate as a one-cell-layer sheet. Prevention of such sedimentation affords the freedom for self-assembly and the propagation of 3D tissue arrays. Suspension of cells is easily achievable using stirred technologies. Unfortunately, in

  7. Sodium selectivity of Reissner's membrane epithelial cells

    PubMed Central

    2011-01-01

    Background Sodium absorption by Reissner's membrane is thought to contribute to the homeostasis of the volume of cochlear endolymph. It was previously shown that the absorptive transepithelial current was blocked by amiloride and benzamil. The most commonly-observed target of these drugs is the epithelial sodium channel (ENaC), which is composed of the three subunits α-,β- and γ-ENaC. However, other less-selective cation channels have also been observed to be sensitive to benzamil and amiloride. The aim of this study was to determine whether Reissner's membrane epithelial cells could support parasensory K+ absorption via amiloride- and benzamil-sensitive electrogenic pathways. Results We determined the molecular and functional expression of candidate cation channels with gene array (GEO GSE6196), RT-PCR, and whole-cell patch clamp. Transcript expression analysis of Reissner's membrane detected no amiloride-sensitive acid-sensing ion channels (ASIC1a, ASIC2a, ASIC2b) nor amiloride-sensitive cyclic-nucleotide gated channels (CNGA1, CNGA2, CNGA4, CNGB3). By contrast, α-,β- and γ-ENaC were all previously reported as present in Reissner's membrane. The selectivity of the benzamil-sensitive cation currents was observed in whole-cell patch clamp recordings under Cl--free conditions where cations were the only permeant species. The currents were carried by Na+ but not K+, and the permeability of Li+ was greater than that of Na+ in Reissner's membrane. Complete replacement of bath Na+ with the inpermeable cation NMDG+ led to the same inward current as with benzamil in a Na+ bath. Conclusions These results are consistent with the amiloride/benzamil-sensitive absorptive flux of Reissner's membrane mediated by a highly Na+-selective channel that has several key characteristics in common with αβγ-ENaC. The amiloride-sensitive pathway therefore absorbs only Na+ in this epithelium and does not provide a parasensory K+ efflux route from scala media. PMID:21284860

  8. Particle compositions with a pre-selected cell internalization mode

    NASA Technical Reports Server (NTRS)

    Decuzzi, Paolo (Inventor); Ferrari, Mauro (Inventor)

    2012-01-01

    A method of formulating a particle composition having a pre-selected cell internalization mode involves selecting a target cell having surface receptors and obtaining particles that have i) surface moieties, that have an affinity for or are capable of binding to the surface receptors of the cell and ii) a preselected shape, where a surface distribution of the surface moieties on the particles and the shape of the particles are effective for the pre-selected cell internalization mode.

  9. Selectivity of Direct Methanol Fuel Cell Membranes

    PubMed Central

    Aricò, Antonino S.; Sebastian, David; Schuster, Michael; Bauer, Bernd; D’Urso, Claudia; Lufrano, Francesco; Baglio, Vincenzo

    2015-01-01

    Sulfonic acid-functionalized polymer electrolyte membranes alternative to Nafion® were developed. These were hydrocarbon systems, such as blend sulfonated polyetheretherketone (s-PEEK), new generation perfluorosulfonic acid (PFSA) systems, and composite zirconium phosphate–PFSA polymers. The membranes varied in terms of composition, equivalent weight, thickness, and filler and were investigated with regard to their methanol permeation characteristics and proton conductivity for application in direct methanol fuel cells. The behavior of the membrane electrode assemblies (MEA) was investigated in fuel cell with the aim to individuate a correlation between membrane characteristics and their performance in a direct methanol fuel cell (DMFC). The power density of the DMFC at 60 °C increased according to a square root-like function of the membrane selectivity. This was defined as the reciprocal of the product between area specific resistance and crossover. The power density achieved at 60 °C for the most promising s-PEEK-based membrane-electrode assembly (MEA) was higher than the benchmark Nafion® 115-based MEA (77 mW·cm−2 vs. 64 mW·cm−2). This result was due to a lower methanol crossover (47 mA·cm−2 equivalent current density for s-PEEK vs. 120 mA·cm−2 for Nafion® 115 at 60 °C as recorded at OCV with 2 M methanol) and a suitable area specific resistance (0.15 Ohm cm2 for s-PEEK vs. 0.22 Ohm cm2 for Nafion® 115). PMID:26610582

  10. Nylon-3 Polymers that Enable Selective Culture of Endothelial Cells

    PubMed Central

    Liu, Runhui; Chen, Xinyu; Gellman, Samuel H.; Masters, Kristyn S.

    2014-01-01

    Substrates that selectively encourage the growth of specific cell types are valuable for the engineering of complex tissues. Some cell-selective peptides have been identified from extracellular matrix proteins; these peptides have proven useful for biomaterials-based approaches to tissue repair or regeneration. However, there are very few examples of synthetic materials that display selectivity in supporting cell growth. We describe nylon-3 polymers that support in vitro culture of endothelial cells, but do not support the culture of smooth muscle cells or fibroblasts. These materials may be promising for vascular biomaterials applications. PMID:24156536

  11. Modeling Selective Elimination of Quiescent Cancer Cells from Bone Marrow

    PubMed Central

    Cavnar, Stephen P.; Rickelmann, Andrew D.; Meguiar, Kaille F.; Xiao, Annie; Dosch, Joseph; Leung, Brendan M.; Cai Lesher-Perez, Sasha; Chitta, Shashank; Luker, Kathryn E.; Takayama, Shuichi; Luker, Gary D.

    2015-01-01

    Patients with many types of malignancy commonly harbor quiescent disseminated tumor cells in bone marrow. These cells frequently resist chemotherapy and may persist for years before proliferating as recurrent metastases. To test for compounds that eliminate quiescent cancer cells, we established a new 384-well 3D spheroid model in which small numbers of cancer cells reversibly arrest in G1/G0 phase of the cell cycle when cultured with bone marrow stromal cells. Using dual-color bioluminescence imaging to selectively quantify viability of cancer and stromal cells in the same spheroid, we identified single compounds and combination treatments that preferentially eliminated quiescent breast cancer cells but not stromal cells. A treatment combination effective against malignant cells in spheroids also eliminated breast cancer cells from bone marrow in a mouse xenograft model. This research establishes a novel screening platform for therapies that selectively target quiescent tumor cells, facilitating identification of new drugs to prevent recurrent cancer. PMID:26408255

  12. Personalized chemotherapy profiling using cancer cell lines from selectable mice

    PubMed Central

    Kamiyama, Hirohiko; Rauenzahn, Sherri; Shim, Joong Sup; Karikari, Collins A.; Feldmann, Georg; Hua, Li; Kamiyama, Mihoko; Schuler, F. William; Lin, Ming-Tseh; Beaty, Robert M.; Karanam, Balasubramanyam; Liang, Hong; Mullendore, Michael E.; Mo, Guanglan; Hidalgo, Manuel; Jaffee, Elizabeth; Hruban, Ralph H.; Jinnah, H. A.; Roden, Richard B. S.; Jimeno, Antonio; Liu, Jun O.; Maitra, Anirban; Eshleman, James R.

    2013-01-01

    Purpose High-throughput chemosensitivity testing of low-passage cancer cell lines can be used to prioritize agents for personalized chemotherapy. However, generating cell lines from primary cancers is difficult, because contaminating stromal cells overgrow the malignant cells. Experimental Design We produced a series of hypoxanthine phosphoribosyl transferase (hprt)-null immunodeficient mice. During growth of human cancers in these mice, hprt-null murine stromal cells replace their human counterparts. Results Pancreatic and ovarian cancers explanted from these mice were grown in selection media to produce pure human cancer cell lines. We screened one cell line with a 3,131-drug panel and identified seventy-seven FDA approved drugs with activity, including two novel drugs to which the cell line was uniquely sensitive. Xenografts of this carcinoma were selectively responsive to both drugs. Conclusion Chemotherapy can be personalized using patient-specific cell lines derived in biochemically selectable mice. PMID:23340293

  13. How do I perform hematopoietic progenitor cell selection?

    PubMed

    Avecilla, Scott T; Goss, Cheryl; Bleau, Sharon; Tonon, Jo-Ann; Meagher, Richard C

    2016-05-01

    Graft-versus-host disease remains the most important source of morbidity and mortality associated with allogeneic stem cell transplantation. The implementation of hematopoietic progenitor cell (HPC) selection is employed by some stem cell processing facilities to mitigate this complication. Current cell selection methods include reducing the number of unwanted T cells (negative selection) and/or enriching CD34+ hematopoietic stem/progenitors (positive selection) using immunomagnetic beads subjected to magnetic fields within columns to separate out targeted cells. Unwanted side effects of cell selection as a result of T-cell reduction are primary graft failure, increased infection rates, delayed immune reconstitution, possible disease relapse, and posttransplant lymphoproliferative disease. The Miltenyi CliniMACS cell isolation system is the only device currently approved for clinical use by the Food and Drug Administration. It uses magnetic microbeads conjugated with a high-affinity anti-CD34 monoclonal antibody capable of binding to HPCs in marrow, peripheral blood, or umbilical cord blood products. The system results in significantly improved CD34+ cell recoveries (50%-100%) and consistent 3-log CD3+ T-cell reductions compared to previous generations of CD34+ cell selection procedures. In this article, the CliniMACS procedure is described in greater detail and the authors provide useful insight into modifications of the system. Successful implementation of cell selection procedures can have a significant positive clinical effect by greatly increasing the pool of donors for recipients requiring transplants. However, before a program implements cell selection techniques, it is important to consider the time and financial resources required to properly and safely perform these procedures. PMID:26919388

  14. Targeting Mitochondria with Avocatin B Induces Selective Leukemia Cell Death.

    PubMed

    Lee, Eric A; Angka, Leonard; Rota, Sarah-Grace; Hanlon, Thomas; Mitchell, Andrew; Hurren, Rose; Wang, Xiao Ming; Gronda, Marcela; Boyaci, Ezel; Bojko, Barbara; Minden, Mark; Sriskanthadevan, Shrivani; Datti, Alessandro; Wrana, Jeffery L; Edginton, Andrea; Pawliszyn, Janusz; Joseph, Jamie W; Quadrilatero, Joe; Schimmer, Aaron D; Spagnuolo, Paul A

    2015-06-15

    Treatment regimens for acute myeloid leukemia (AML) continue to offer weak clinical outcomes. Through a high-throughput cell-based screen, we identified avocatin B, a lipid derived from avocado fruit, as a novel compound with cytotoxic activity in AML. Avocatin B reduced human primary AML cell viability without effect on normal peripheral blood stem cells. Functional stem cell assays demonstrated selectivity toward AML progenitor and stem cells without effects on normal hematopoietic stem cells. Mechanistic investigations indicated that cytotoxicity relied on mitochondrial localization, as cells lacking functional mitochondria or CPT1, the enzyme that facilitates mitochondria lipid transport, were insensitive to avocatin B. Furthermore, avocatin B inhibited fatty acid oxidation and decreased NADPH levels, resulting in ROS-dependent leukemia cell death characterized by the release of mitochondrial proteins, apoptosis-inducing factor, and cytochrome c. This study reveals a novel strategy for selective leukemia cell eradication based on a specific difference in mitochondrial function. PMID:26077472

  15. Ion-Selective Detection with Glass Nanopipette for Living Cells

    NASA Astrophysics Data System (ADS)

    Takami, T.; Son, J. W.; Kang, E. J.; Deng, X. L.; Kawai, T.; Lee, S.-W.; Park, B. H.

    2013-05-01

    We developed a method to probe local ion concentration with glass nanopipette in which poly(vinyl chloride) membrane containing ionophore for separate ion detection is prepared. Here we demonstrate how ion-selective detections are available for living cells such as HeLa cell, rat vascular myocyte, and neuron cell.

  16. Comparative analysis of selected fuel cell vehicles

    SciTech Connect

    1993-05-07

    Vehicles powered by fuel cells operate more efficiently, more quietly, and more cleanly than internal combustion engines (ICEs). Furthermore, methanol-fueled fuel cell vehicles (FCVs) can utilize major elements of the existing fueling infrastructure of present-day liquid-fueled ICE vehicles (ICEVs). DOE has maintained an active program to stimulate the development and demonstration o fuel cell technologies in conjunction with rechargeable batteries in road vehicles. The purpose of this study is to identify and assess the availability of data on FCVs, and to develop a vehicle subsystem structure that can be used to compare both FCVs and ICEV, from a number of perspectives--environmental impacts, energy utilization, materials usage, and life cycle costs. This report focuses on methanol-fueled FCVs fueled by gasoline, methanol, and diesel fuel that are likely to be demonstratable by the year 2000. The comparative analysis presented covers four vehicles--two passenger vehicles and two urban transit buses. The passenger vehicles include an ICEV using either gasoline or methanol and an FCV using methanol. The FCV uses a Proton Exchange Membrane (PEM) fuel cell, an on-board methanol reformer, mid-term batteries, and an AC motor. The transit bus ICEV was evaluated for both diesel and methanol fuels. The transit bus FCV runs on methanol and uses a Phosphoric Acid Fuel Cell (PAFC) fuel cell, near-term batteries, a DC motor, and an on-board methanol reformer. 75 refs.

  17. T Cell Adolescence: Maturation Events Beyond Positive Selection.

    PubMed

    Hogquist, Kristin A; Xing, Yan; Hsu, Fan-Chi; Shapiro, Virginia Smith

    2015-08-15

    Single-positive thymocytes that successfully complete positive and negative selection must still undergo one final step, generally termed T cell maturation, before they gain functional competency and enter the long-lived T cell pool. Maturation initiates after positive selection in single-positive thymocytes and continues in the periphery in recent thymic emigrants, before these newly produced T cells gain functional competency and are ready to participate in the immune response as peripheral naive T cells. Recent work using genetically altered mice demonstrates that T cell maturation is not a single process, but a series of steps that occur independently and sequentially after positive selection. This review focuses on the changes that occur during T cell maturation, as well as the molecules and pathways that are critical at each step. PMID:26254267

  18. Cell selectivity to laser-induced photoacoustic injury of skin.

    PubMed

    Yashima, Y; McAuliffe, D J; Flotte, T J

    1990-01-01

    Cell selectivity to photoacoustic injury induced by argon-fluoride excimer laser (193 nm) was studied. Rats were irradiated through air or water and a 2.5 mm aperture. The laser was adjusted to deliver 150 mJ/cm2 at the skin surface with 12 and 24 pulses. Immediate damage was assessed by transmission electron microscopy. Cell selectivity was observed in dermis and epidermis. Fibroblasts showed alteration of nuclear chromatin and cytoplasmic organelles, while some of the migratory cells adjacent to fibroblasts did not. Similar difference of damage was observed between keratinocytes and Langerhans cells in epidermis. Considering the relationship between cells and their microenvironment in tissue, this selectivity may be due to the difference of acoustical coupling of propagation of acoustic waves rather than to differential sensitivity of the cells to damage. PMID:2345477

  19. Advancing tandem solar cells by spectrally selective multilayer intermediate reflectors.

    PubMed

    Hoffmann, Andre; Paetzold, Ulrich W; Zhang, Chao; Merdzhanova, Tsvetelina; Lambertz, Andreas; Ulbrich, Carolin; Bittkau, Karsten; Rau, Uwe

    2014-08-25

    Thin-film silicon tandem solar cells are composed of an amorphous silicon top cell and a microcrystalline silicon bottom cell, stacked and connected in series. In order to match the photocurrents of the top cell and the bottom cell, a proper photon management is required. Up to date, single-layer intermediate reflectors of limited spectral selectivity are applied to match the photocurrents of the top and the bottom cell. In this paper, we design and prototype multilayer intermediate reflectors based on aluminum doped zinc oxide and doped microcrystalline silicon oxide with a spectrally selective reflectance allowing for improved current matching and an overall increase of the charge carrier generation. The intermediate reflectors are successfully integrated into state-of-the-art tandem solar cells resulting in an increase of overall short-circuit current density by 0.7 mA/cm(2) in comparison to a tandem solar cell with the standard single-layer intermediate reflector. PMID:25322181

  20. Target selection: invasion, mapping and cell choice.

    PubMed

    Holt, C E; Harris, W A

    1998-02-01

    Recent research has shown that changes in the concentration of particular molecules lead axons to invade their target, and that concentration changes in other molecules at the borders of the target prevent axons from leaving the target area. After invasion, topographic and lamina-specific cues guide axons to the correct location within the target field. At the level of a single cell or part of a cell, the evidence raises the possibility that axon targeting might be a combinatorial affair whereby specific axons compare the relative concentrations of several molecules on the surface of postsynaptic cells in order to choose a particular target. Both proteins and carbohydrates of various classes play major roles in these processes. PMID:9568397

  1. Selectable-Tip Corrosion-Testing Electrochemical Cell

    NASA Technical Reports Server (NTRS)

    Lomness, Janice; Hintze, Paul

    2008-01-01

    The figure depicts aspects of an electrochemical cell for pitting- corrosion tests of material specimens. The cell is designed to generate a region of corrosion having a pit diameter determined by the diameter of a selectable tip. The average depth of corrosion is controlled by controlling the total electric charge passing through the cell in a test. The cell is also designed to produce minimal artifacts associated with crevice corrosion. There are three selectable tips, having diameters of 0.1 in. (0.254 cm), 0.3 in. (0.762 cm), and 0.6 in. (1.524 cm), respectively.

  2. Distinct interactions select and maintain a specific cell fate

    PubMed Central

    Dončić, Andreas; Falleur-Fettig, Melody; Skotheim, Jan M.

    2011-01-01

    The ability to specify and maintain discrete cell fates is essential for development. However, the dynamics underlying selection and stability of distinct cell types remains poorly understood. Here, we provide a quantitative single-cell analysis of commitment dynamics during the mating-mitosis switch in budding yeast. Commitment to division corresponds precisely to activating the G1 cyclin positive feedback loop in competition with the cyclin inhibitor Far1. Cyclin-dependent phosphorylation and inhibition of the mating pathway scaffold Ste5 is required to ensure exclusive expression of the mitotic transcriptional program after cell cycle commitment. Failure to commit exclusively results in coexpression of both cell cycle and pheromone-induced genes, and a morphologically-mixed inviable cell fate. Thus, specification and maintenance of a cellular state are performed by distinct interactions, which is likely a consequence of disparate reaction rates and may be a general feature of the interlinked regulatory networks responsible for selecting cell fates. PMID:21855793

  3. T cell adolescence: maturation events beyond positive selection1

    PubMed Central

    Hogquist, Kristin A.; Xing, Yan; Hsu, Fan-Chi; Shapiro, Virginia Smith

    2015-01-01

    Single positive (SP) thymocytes that successfully complete positive and negative selection must still undergo one final step, generally termed T cell maturation, before they gain functional competency and enter the long-lived T cell pool. Maturation initiates after positive selection in SP thymocytes, and continues in the periphery in recent thymic emigrants (RTEs), before these newly produced T cells gain functional competency and are ready to participate in the immune response as peripheral naïve T cells. Recent work using genetically altered mice demonstrates that T cell maturation is not a single process, but a series of steps that occur independently and sequentially after positive selection. This review will focus on the changes that occur during T maturation, and the molecules and pathways that are critical at each step. PMID:26254267

  4. Selective cell proliferation can be controlled with CPC particle coatings

    PubMed Central

    Szivek, J.A.; Margolis, D.S.; Schnepp, A.B.; Grana, W.A.; Williams, S.K.

    2008-01-01

    To develop implantable, engineered, cartilage constructs supported by a scaffold, techniques to encourage rapid tissue growth into, and on the scaffold are essential. Preliminary studies indicated that human endothelial cells proliferated at different rates on different calcium phosphate ceramic (CPC) particles. Judicious selection of particles may encourage specific cell proliferation, leading to an ordered growth of tissues for angiogenesis, osteogenesis, and chondrogenesis. The goal of this study was to identify CPC surfaces that encourage bone and vascular cell growth, and other surfaces that support chondrocyte growth while inhibiting proliferation of vascular cells. Differences in bone and vascular cell proliferation were observed when using epoxy without embedded CPCs to encourage bone cells, and when three CPCs were tested, which encouraged vascular cell proliferation. One of these (CPC 7) also substantially depressed cartilage cell proliferation. Only one small-diameter crystalline CPC (CPC 2) supported rapid chondrocyte proliferation, and maintained the cartilage cell phenotype. PMID:17252549

  5. Sickle cell disease: selected aspects of pathophysiology.

    PubMed

    Alexy, T; Sangkatumvong, S; Connes, P; Pais, E; Tripette, J; Barthelemy, J C; Fisher, T C; Meiselman, H J; Khoo, M C; Coates, T D

    2010-01-01

    Sickle cell disease (SCD), a genetically-determined pathology due to an amino acid substitution (i.e., valine for glutamic acid) on the beta-chain of hemoglobin, is characterized by abnormal blood rheology and periods of painful vascular occlusive crises. Sickle cell trait (SCT) is a typically benign variant in which only one beta chain is affected by the mutation. Although both SCD and SCT have been the subject of numerous studies, information related to neurological function and transfusion therapy is still incomplete: an overview of these areas is presented. An initial section provides pertinent background information on the pathology and clinical significance of these diseases. The roles of three factors in the clinical manifestations of the diseases are then discussed: hypoxia, autonomic nervous system regulation and blood rheology. The possibility of a causal relationship between these three factors and sudden death is also examined. It is concluded that further studies in these specific areas are warranted. It is anticipated that the outcome of such research is likely to provide valuable insights into the pathophysiology of SCD and SCT and will lead to improved clinical management and enhanced quality of life. PMID:20364061

  6. Peptide fibrils with altered stability, activity, and cell selectivity

    PubMed Central

    Chen, Long; Liang, Jun F.

    2014-01-01

    Peptides have some unique and superior features compared to proteins. However, the use of peptides as therapeutics is hampered by their low stability and cell selectivity. In this study, a new lytic peptide (CL-1, FLGALFRALSRLL) was constructed. Under the physiological condition, peptide CL-1 self-assembled into dynamically stable aggregates with fibrils-like structures. Aggregated CL-1 demonstrated dramatically altered activity and stability in comparison with single molecule CL-1 and other lytic peptides: when incubated with co-cultured bacteria and tissue cells, CL-1 aggregates killed bacteria selectively but spared co-cultured human cells; CL-1 aggregates kept intact in human serum for more than five hours. Peptide-cell interaction studies performed on lipid monolayers and live human tissue cells revealed that in comparison with monomeric CL-1, aggregated CL-1 had decreased cell affinity and membrane insertion capability on tissue cells. A dynamic process involving aggregate dissociation and rearrangement seemed to be an essential step for membrane bound CL-1 aggregates to realize its cytotoxicity to tissue cells. Our study suggests that peptide aggregation could be as important as the charge and secondary structure of a peptide in affecting peptide-cell interactions. Controlling peptide self-assembly represents a new way to increase the stability and cell selectivity of bioactive peptides for wide biomedical applications. PMID:23713839

  7. Oncotripsy: Targeting cancer cells selectively via resonant harmonic excitation

    NASA Astrophysics Data System (ADS)

    Heyden, S.; Ortiz, M.

    2016-07-01

    We investigate a method of selectively targeting cancer cells by means of ultrasound harmonic excitation at their resonance frequency, which we refer to as oncotripsy. The geometric model of the cells takes into account the cytoplasm, nucleus and nucleolus, as well as the plasma membrane and nuclear envelope. Material properties are varied within a pathophysiologically-relevant range. A first modal analysis reveals the existence of a spectral gap between the natural frequencies and, most importantly, resonant growth rates of healthy and cancerous cells. The results of the modal analysis are verified by simulating the fully-nonlinear transient response of healthy and cancerous cells at resonance. The fully nonlinear analysis confirms that cancerous cells can be selectively taken to lysis by the application of carefully tuned ultrasound harmonic excitation while simultaneously leaving healthy cells intact.

  8. Soft fibrin gels promote selection and growth of tumorigenic cells

    NASA Astrophysics Data System (ADS)

    Liu, Jing; Tan, Youhua; Zhang, Huafeng; Zhang, Yi; Xu, Pingwei; Chen, Junwei; Poh, Yeh-Chuin; Tang, Ke; Wang, Ning; Huang, Bo

    2012-08-01

    The identification of stem-cell-like cancer cells through conventional methods that depend on stem cell markers is often unreliable. We developed a mechanical method for selecting tumorigenic cells by culturing single cancer cells in fibrin matrices of ~100 Pa in stiffness. When cultured within these gels, primary human cancer cells or single cancer cells from mouse or human cancer cell lines grew within a few days into individual round colonies that resembled embryonic stem cell colonies. Subcutaneous or intravenous injection of 10 or 100 fibrin-cultured cells in syngeneic or severe combined immunodeficiency mice led to the formation of solid tumours at the site of injection or at the distant lung organ much more efficiently than control cancer cells selected using conventional surface marker methods or cultured on conventional rigid dishes or on soft gels. Remarkably, as few as ten such cells were able to survive and form tumours in the lungs of wild-type non-syngeneic mice.

  9. Select forms of tumor cell apoptosis induce dendritic cell maturation.

    PubMed

    Demaria, Sandra; Santori, Fabio R; Ng, Bruce; Liebes, Leonard; Formenti, Silvia C; Vukmanovic, Stanislav

    2005-03-01

    Dendritic cells (DC) play a crucial role in initiating immune responses to tumors. DC can efficiently present antigens from apoptotic tumor cells, but apoptotic cells are thought to lack the inflammatory signals required to induce DC maturation. Here, we show that apoptosis of 67NR mouse carcinoma cells via the Fas (CD95) pathway or induced by the anticancer drug bortezomib (PS-341) but not by ultraviolet irradiation is associated with the production of maturation signals for DC. These data have important implications for the effects of chemotherapy on antitumor immunity in solid and hematologic malignancies. PMID:15569694

  10. CD6 modulates thymocyte selection and peripheral T cell homeostasis.

    PubMed

    Orta-Mascaró, Marc; Consuegra-Fernández, Marta; Carreras, Esther; Roncagalli, Romain; Carreras-Sureda, Amado; Alvarez, Pilar; Girard, Laura; Simões, Inês; Martínez-Florensa, Mario; Aranda, Fernando; Merino, Ramón; Martínez, Vanesa-Gabriela; Vicente, Rubén; Merino, Jesús; Sarukhan, Adelaida; Malissen, Marie; Malissen, Bernard; Lozano, Francisco

    2016-07-25

    The CD6 glycoprotein is a lymphocyte surface receptor putatively involved in T cell development and activation. CD6 facilitates adhesion between T cells and antigen-presenting cells through its interaction with CD166/ALCAM (activated leukocyte cell adhesion molecule), and physically associates with the T cell receptor (TCR) at the center of the immunological synapse. However, its precise role during thymocyte development and peripheral T cell immune responses remains to be defined. Here, we analyze the in vivo consequences of CD6 deficiency. CD6(-/-) thymi showed a reduction in both CD4(+) and CD8(+) single-positive subsets, and double-positive thymocytes exhibited increased Ca(2+) mobilization to TCR cross-linking in vitro. Bone marrow chimera experiments revealed a T cell-autonomous selective disadvantage of CD6(-/-) T cells during development. The analysis of TCR-transgenic mice (OT-I and Marilyn) confirmed that abnormal T cell selection events occur in the absence of CD6. CD6(-/-) mice displayed increased frequencies of antigen-experienced peripheral T cells generated under certain levels of TCR signal strength or co-stimulation, such as effector/memory (CD4(+)TEM and CD8(+)TCM) and regulatory (T reg) T cells. The suppressive activity of CD6(-/-) T reg cells was diminished, and CD6(-/-) mice presented an exacerbated autoimmune response to collagen. Collectively, these data indicate that CD6 modulates the threshold for thymocyte selection and the generation and/or function of several peripheral T cell subpopulations, including T reg cells. PMID:27377588

  11. A Pathway Toward Tumor Cell-Selective CPPs?

    PubMed

    Alves, Isabel D; Carré, Manon; Lavielle, Solange

    2015-01-01

    Despite the great potential of CPPs in therapeutics and diagnosis, their application still suffers from a non-negligible drawback: a complete lack of cell-type specificity. In the innumerous routes proposed for CPP cell entry there is common agreement that electrostatic interactions between cationic CPPs and anionic components in membranes, including lipids and glycosaminoglycans, play a crucial role. Tumor cells have been shown to overexpress certain glycosaminoglycans at the cell membrane surface and to possess a higher amount of anionic lipids in their outer leaflet when compared with healthy cells. Such molecules confer tumor cell membranes an enhanced anionic character, a property that could be exploited by CPPs to preferentially target these cells. Herein, these aspects are discussed in an attempt to confer CPPs certain selectivity toward cancer cells. PMID:26202276

  12. Positive selection of self-antigen-specific CD8+ T cells by hematopoietic cells.

    PubMed

    Yamada, Hisakata; Shibata, Kensuke; Sakuraba, Koji; Fujimura, Kenjiro; Yoshikai, Yasunobu

    2013-08-01

    In contrast to thymic epithelial cells, which induce the positive selection of conventional CD8(+) T cells, hematopoietic cells (HCs) select innate CD8(+) T cells whose Ag specificity is not fully understood. Here we show that CD8(+) T cells expressing an H-Y Ag-specific Tg TCR were able to develop in mice in which only HCs expressed MHC class I, when HCs also expressed the H-Y Ag. These HC-selected self-specific CD8(+) T cells resemble innate CD8(+) T cells in WT mice in terms of the expression of memory markers and effector functions, but are phenotypically distinct from the thymus-independent CD8(+) T-cell population. The peripheral maintenance of H-Y-specific CD8(+) T cells required presentation of the self-Ag and IL-15 on HCs. HC-selected CD8(+) T cells in mice lacking the Tg TCR also showed these features. Furthermore, by using MHC class I tetramers with a male Ag peptide, we found that self-Ag-specific CD8(+) T cells in TCR non-Tg mice could develop via HC-induced positive selection, supporting results obtained from H-Y TCR Tg mice. These findings indicate the presence of self-specific CD8(+) T cells that are positively selected by HCs in the peripheral T-cell repertoire. PMID:23636825

  13. An analysis of B cell selection mechanisms in germinal centers.

    PubMed

    Meyer-Hermann, Michael E; Maini, Philip K; Iber, Dagmar

    2006-09-01

    Affinity maturation of antibodies during immune responses is achieved by multiple rounds of somatic hypermutation and subsequent preferential selection of those B cells that express B cell receptors with improved binding characteristics for the antigen. The mechanism underlying B cell selection has not yet been defined. By employing an agent-based model, we show that for physiologically reasonable parameter values affinity maturation can be driven by competition for neither binding sites nor antigen--even in the presence of competing secreted antibodies. Within the tested mechanisms, only clonal competition for T cell help or a refractory time for the interaction of centrocytes with follicular dendritic cells is found to enable affinity maturation while generating the experimentally observed germinal centre characteristics and tolerating large variations in the initial antigen density. PMID:16707510

  14. Building Cell Selectivity into CPP-Mediated Strategies

    PubMed Central

    Martín, Irene; Teixidó, Meritxell; Giralt, Ernest

    2010-01-01

    There is a pressing need for more effective and selective therapies for cancer and other diseases. Consequently, much effort is being devoted to the development of alternative experimental approaches based on selective systems, which are designed to be specifically directed against target cells. In addition, a large number of highly potent therapeutic molecules are being discovered. However, they do not reach clinical trials because of their low delivery, poor specificity or their incapacity to bypass the plasma membrane. Cell-penetrating peptides (CPPs) are an open door for cell-impermeable compounds to reach intracellular targets. Putting all these together, research is sailing in the direction of the design of systems with the capacity to transport new drugs into a target cell. Some CPPs show cell type specificity while others require modifications or form part of more sophisticated drug delivery systems. In this review article we summarize several strategies for directed drug delivery involving CPPs that have been reported in the literature.

  15. Ozone selectively inhibits growth of human cancer cells

    SciTech Connect

    Sweet, F.; Kao, M.S.; Lee, S.C.; Hagar, W.L.; Sweet, W.E.

    1980-08-01

    The growth of human cancer cells from lung, breast, and uterine tumors was selectively inhibited in a dose-dependent manner by ozone at 0.3 to 0.8 part per million of ozone in ambient air during 8 days of culture. Human lung diploid fibroblasts served as noncancerous control cells. The presence of ozone at 0.3 to 0.5 part per million inhibited cancer cell growth 40 and 60 percent, respectively. The noncancerous lung cells were unaffected at these levels. Exposure to ozone at 0.8 part per million inhibited cancer cell growth more than 90 percent and control cell growth less than 50 percent. Evidently, the mechanisms for defense against ozone damage are impaired in human cancer cells.

  16. Identification of compounds selectively killing multidrug resistant cancer cells

    PubMed Central

    Türk, Dóra; Hall, Matthew D.; Chu, Benjamin F.; Ludwig, Joseph A.; Fales, Henry M.; Gottesman, Michael M.; Szakács, Gergely

    2009-01-01

    There is a great need for the development of novel chemotherapeutic agents that overcome the emergence of multidrug resistance in cancer. We catalogued the National Cancer Institute’s Developmental Therapeutics Program (DTP) drug repository in search of compounds showing increased toxicity in multidrug resistant (MDR) cells. By comparing the sensitivity of parental cell lines with multidrug resistant derivatives, we identified 22 compounds possessing MDR-selective activity. Analysis of structural congeners led to the identification of 15 additional drugs showing increased toxicity in Pgp-expressing cells. Analysis of MDR-selective compounds led to the formulation of structure activity relationships (SAR) and pharmacophore models. This data mining coupled with experimental data points to a possible mechanism of action linked to metal chelation. Taken together, the discovery of the MDR-selective compound set demonstrates the robustness of the developing field of MDR-targeting therapy as a new strategy for resolving Pgp-mediated multidrug resistance. PMID:19843850

  17. Selective Cell Growth on Fibronectin-Carbon Nanotube Hybrid Nanostructures

    NASA Astrophysics Data System (ADS)

    Namgung, Seon; Park, Sung Young; Lee, Byung Yang; Lee, Minbaek; Nam, Jwa-Min; Hong, Seunghun

    2008-03-01

    Carbon nanotubes (CNT) have been considered a promising material for biological applications including biosensors, therapeutic application, and nano-structured scaffolds. However, there are still controversies associated with toxicity and biocompatibility of CNTs on live cells. Here, we report general strategy to functionalize CNTs with cell adhesion molecules (fibronectins) for selective and stable adhesion of cells on CNTs. Interestingly, more fibronectins were adsorbed and activated on CNTs rather than on hydrophobic self assembled monolayers (SAMs) or bare substrates (SiO2). We demonstrate the functionality of fibronectins on CNTs with immunofluorescence and molecule-level force measurement study using atomic force microscopy (AFM). These fibronectin-CNT hybrid nanostructures were successfully applied to attract cells selectively onto predefined regions on the substrate. Our strategy was generally available on various cell types including mesenchymal stem cells, KB cells, and NIH3T3 fibroblast cells (Advanced Materials 19, 2530-2534 (2007)). We will also discuss about its impacts on cell biology combined with CNTs.

  18. Selective Label-free Electrokinetic Cell Tracker (SELECT): a novel liquid platform for cell characterization

    NASA Astrophysics Data System (ADS)

    Taruvai Kalyana Kumar, Rajeshwari; de Mello Gindri, Izabelle; Kinnamon, David; Kanchustambham, Pradyotha; Rodrigues, Danieli; Prasad, Shalini; BiomaterialsOsseointegration; Novel Engineering Lab Collaboration

    2015-03-01

    Characterization and analysis of rare cells provide critical cues for early diagnosis of diseases. Electrokinetic cell separation has been previously established to have greater efficiency when compared to traditional flow cytometry methods. It has been shown by many researchers that buffer solutions in which cells are suspended in, have enormous effects on producing required dielectrophoretic (DEP) forces to characterize cells. Most commonly used suspension buffers used are deionized water and cell media. However, these solutions exhibit high level of intrinsic noise, which greatly masks the electrokinetic signals from cells under study. Ionic liquids (ILs) show promise towards the creation of conductive fluids with required electrical properties. The goal of this project is to design and test ILs for enhancing DEP forces on cells while creating an environment for preserving their integrity. We analyzed two methylimidazolium based ILs as suspension medium for cell separation. These dicationic ILs possess slight electrical and structural differences with high thermal stability. The two ILs were tested for cytotoxicity using HeLa and bone cells. The effects of electrical neutrality, free charge screening due to ILs towards enhanced electrokinetic signals from cells were studied with improved system resolution and no harmful effects.

  19. The selection and function of cell type-specific enhancers.

    PubMed

    Heinz, Sven; Romanoski, Casey E; Benner, Christopher; Glass, Christopher K

    2015-03-01

    The human body contains several hundred cell types, all of which share the same genome. In metazoans, much of the regulatory code that drives cell type-specific gene expression is located in distal elements called enhancers. Although mammalian genomes contain millions of potential enhancers, only a small subset of them is active in a given cell type. Cell type-specific enhancer selection involves the binding of lineage-determining transcription factors that prime enhancers. Signal-dependent transcription factors bind to primed enhancers, which enables these broadly expressed factors to regulate gene expression in a cell type-specific manner. The expression of genes that specify cell type identity and function is associated with densely spaced clusters of active enhancers known as super-enhancers. The functions of enhancers and super-enhancers are influenced by, and affect, higher-order genomic organization. PMID:25650801

  20. Directional summation in non-direction selective retinal ganglion cells.

    PubMed

    Abbas, Syed Y; Hamade, Khaldoun C; Yang, Ellen J; Nawy, Scott; Smith, Robert G; Pettit, Diana L

    2013-01-01

    Retinal ganglion cells receive inputs from multiple bipolar cells which must be integrated before a decision to fire is made. Theoretical studies have provided clues about how this integration is accomplished but have not directly determined the rules regulating summation of closely timed inputs along single or multiple dendrites. Here we have examined dendritic summation of multiple inputs along On ganglion cell dendrites in whole mount rat retina. We activated inputs at targeted locations by uncaging glutamate sequentially to generate apparent motion along On ganglion cell dendrites in whole mount retina. Summation was directional and dependent13 on input sequence. Input moving away from the soma (centrifugal) resulted in supralinear summation, while activation sequences moving toward the soma (centripetal) were linear. Enhanced summation for centrifugal activation was robust as it was also observed in cultured retinal ganglion cells. This directional summation was dependent on hyperpolarization activated cyclic nucleotide-gated (HCN) channels as blockade with ZD7288 eliminated directionality. A computational model confirms that activation of HCN channels can override a preference for centripetal summation expected from cell anatomy. This type of direction selectivity could play a role in coding movement similar to the axial selectivity seen in locust ganglion cells which detect looming stimuli. More generally, these results suggest that non-directional retinal ganglion cells can discriminate between input sequences independent of the retina network. PMID:23516351

  1. Directional Summation in Non-direction Selective Retinal Ganglion Cells

    PubMed Central

    Abbas, Syed Y.; Hamade, Khaldoun C.; Yang, Ellen J.; Nawy, Scott; Smith, Robert G.; Pettit, Diana L.

    2013-01-01

    Retinal ganglion cells receive inputs from multiple bipolar cells which must be integrated before a decision to fire is made. Theoretical studies have provided clues about how this integration is accomplished but have not directly determined the rules regulating summation of closely timed inputs along single or multiple dendrites. Here we have examined dendritic summation of multiple inputs along On ganglion cell dendrites in whole mount rat retina. We activated inputs at targeted locations by uncaging glutamate sequentially to generate apparent motion along On ganglion cell dendrites in whole mount retina. Summation was directional and dependent13 on input sequence. Input moving away from the soma (centrifugal) resulted in supralinear summation, while activation sequences moving toward the soma (centripetal) were linear. Enhanced summation for centrifugal activation was robust as it was also observed in cultured retinal ganglion cells. This directional summation was dependent on hyperpolarization activated cyclic nucleotide-gated (HCN) channels as blockade with ZD7288 eliminated directionality. A computational model confirms that activation of HCN channels can override a preference for centripetal summation expected from cell anatomy. This type of direction selectivity could play a role in coding movement similar to the axial selectivity seen in locust ganglion cells which detect looming stimuli. More generally, these results suggest that non-directional retinal ganglion cells can discriminate between input sequences independent of the retina network. PMID:23516351

  2. Cold Atmospheric Plasma for Selectively Ablating Metastatic Breast Cancer Cells

    PubMed Central

    Wang, Mian; Holmes, Benjamin; Cheng, Xiaoqian; Zhu, Wei; Keidar, Michael; Zhang, Lijie Grace

    2013-01-01

    Traditional breast cancer treatments such as surgery and radiotherapy contain many inherent limitations with regards to incomplete and nonselective tumor ablation. Cold atomospheric plasma (CAP) is an ionized gas where the ion temperature is close to room temperature. It contains electrons, charged particles, radicals, various excited molecules, UV photons and transient electric fields. These various compositional elements have the potential to either enhance and promote cellular activity, or disrupt and destroy them. In particular, based on this unique composition, CAP could offer a minimally-invasive surgical approach allowing for specific cancer cell or tumor tissue removal without influencing healthy cells. Thus, the objective of this research is to investigate a novel CAP-based therapy for selectively bone metastatic breast cancer treatment. For this purpose, human metastatic breast cancer (BrCa) cells and bone marrow derived human mesenchymal stem cells (MSCs) were separately treated with CAP, and behavioral changes were evaluated after 1, 3, and 5 days of culture. With different treatment times, different BrCa and MSC cell responses were observed. Our results showed that BrCa cells were more sensitive to these CAP treatments than MSCs under plasma dose conditions tested. It demonstrated that CAP can selectively ablate metastatic BrCa cells in vitro without damaging healthy MSCs at the metastatic bone site. In addition, our study showed that CAP treatment can significantly inhibit the migration and invasion of BrCa cells. The results suggest the great potential of CAP for breast cancer therapy. PMID:24040051

  3. Non PN junction solar cells using carrier selective contacts

    NASA Astrophysics Data System (ADS)

    Bowden, Stuart; Ghosh, Kunal; Honsberg, Christiana

    2013-03-01

    A novel device concept utilizing the approach of selectively extracting carriers at the respective contacts is outlined in the work. The dominant silicon solar cell technology is based on a diffused, top-contacted p-n junction on a relatively thick silicon wafer for both commercial and laboratory solar cells. The VOC and hence the efficiency of a diffused p-n junction solar cell is limited by the emitter recombination current and a value of 720 mV is considered to be the upper limit. The value is more than 100 mV smaller than the thermodynamic limit of VOC as applicable for silicon based solar cells. Also, in diffused junction the use of thin wafers (< 50 um) are problematic because of the requirement of high temperature processing steps. But a number of roadmaps have identified solar cells manufactured on thinner silicon wafers to achieve lower cost and higher efficiency. The carrier selective contact device provides a novel alternative to diffused p-n junction solar cells by eliminating the need for complementary doping to form the emitter and hence it allows the solar cells to achieve a VOC of greater than 720 mV. Also, the complete device structure can be fabricated with low temperature thin film deposition or organic coating on silicon substrates and thus epitaxially grown silicon or kerfless silicon, in addition to standard silicon wafers can be utilized.

  4. Metabolic selection of glycosylation defects in human cells

    SciTech Connect

    Yarema, Kevin J.; Goon, Scarlett; Bertozzi, Carolyn R.

    2000-08-01

    Changes in glycosylation are often associated with disease progression, but the genetic and metabolic basis of these events is rarely understood in detail at a molecular level. This report describes a novel metabolism-based approach to the selection of mutants in glycoconjugate biosynthesis that has provided insight into regulatory mechanisms for oligosaccharide expression and metabolic flux. Unnatural intermediates are used to challenge a specific pathway and cell-surface expression of their metabolic products provides a readout of flux in that pathway and a basis for selecting genetic mutants. The approach was applied to the sialic acid metabolic pathway in human cells, yielding novel mutants with phenotypes related to the inborn metabolic defect sialuria and metastatic tumor cells.

  5. Selecting agonists from single cells infected with combinatorial antibody libraries.

    PubMed

    Zhang, Hongkai; Yea, Kyungmoo; Xie, Jia; Ruiz, Diana; Wilson, Ian A; Lerner, Richard A

    2013-05-23

    We describe a system for direct selection of antibodies that are receptor agonists. Combinatorial antibody libraries in lentiviruses are used to infect eukaryotic cells that contain a fluorescent reporter system coupled to the receptor for which receptor agonist antibodies are sought. In this embodiment of the method, very large numbers of candidate antibodies expressing lentivirus and eukaryotic reporter cells are packaged together in a format where each is capable of replication, thereby forging a direct link between genotype and phenotype. Following infection, cells that fluoresce are sorted and the integrated genes encoding the agonist antibodies recovered. We validated the system by illustrating its ability to generate rapidly potent antibody agonists that are complete thrombopoietin phenocopies. The system should be generalizable to any pathway where its activation can be linked to production of a selectable phenotype. PMID:23706638

  6. Selectivity for multiple stimulus features in retinal ganglion cells.

    PubMed

    Fairhall, Adrienne L; Burlingame, C Andrew; Narasimhan, Ramesh; Harris, Robert A; Puchalla, Jason L; Berry, Michael J

    2006-11-01

    Under normal viewing conditions, retinal ganglion cells transmit to the brain an encoded version of the visual world. The retina parcels the visual scene into an array of spatiotemporal features, and each ganglion cell conveys information about a small set of these features. We study the temporal features represented by salamander retinal ganglion cells by stimulating with dynamic spatially uniform flicker and recording responses using a multi-electrode array. While standard reverse correlation methods determine a single stimulus feature--the spike-triggered average--multiple features can be relevant to spike generation. We apply covariance analysis to determine the set of features to which each ganglion cell is sensitive. Using this approach, we found that salamander ganglion cells represent a rich vocabulary of different features of a temporally modulated visual stimulus. Individual ganglion cells were sensitive to at least two and sometimes as many as six features in the stimulus. While a fraction of the cells can be described by a filter-and-fire cascade model, many cells have feature selectivity that has not previously been reported. These reverse models were able to account for 80-100% of the information encoded by ganglion cells. PMID:16914609

  7. Phototoxic aptamers selectively enter and kill epithelial cancer cells

    PubMed Central

    Ferreira, Cátia S. M.; Cheung, Melissa C.; Missailidis, Sotiris; Bisland, Stuart; Gariépy, Jean

    2009-01-01

    The majority of cancers arise from malignant epithelial cells. We report the design of synthetic oligonucleotides (aptamers) that are only internalized by epithelial cancer cells and can be precisely activated by light to kill such cells. Specifically, phototoxic DNA aptamers were selected to bind to unique short O-glycan-peptide signatures on the surface of breast, colon, lung, ovarian and pancreatic cancer cells. These surface antigens are not present on normal epithelial cells but are internalized and routed through endosomal and Golgi compartments by cancer cells, thus providing a focused mechanism for their intracellular delivery. When modified at their 5′ end with the photodynamic therapy agent chlorin e6 and delivered to epithelial cancer cells, these aptamers exhibited a remarkable enhancement (>500-fold increase) in toxicity upon light activation, compared to the drug alone and were not cytotoxic towards cell types lacking such O-glycan-peptide markers. Our findings suggest that these synthetic oligonucleotide aptamers can serve as delivery vehicles in precisely routing cytotoxic cargoes to and into epithelial cancer cells. PMID:19103663

  8. Carrier-selective contacts for Si solar cells

    NASA Astrophysics Data System (ADS)

    Feldmann, F.; Simon, M.; Bivour, M.; Reichel, C.; Hermle, M.; Glunz, S. W.

    2014-05-01

    Carrier-selective contacts (i.e., minority carrier mirrors) are one of the last remaining obstacles to approaching the theoretical efficiency limit of silicon solar cells. In the 1980s, it was already demonstrated that n-type polysilicon and semi-insulating polycrystalline silicon emitters form carrier-selective emitters which enabled open-circuit voltages (Voc) of up to 720 mV. Albeit promising, to date a polysilicon emitter solar cell having a high fill factor (FF) has not been demonstrated yet. In this work, we report a polysilicon emitter related solar cell achieving both a high Voc = 694 mV and FF = 81%. The passivation mechanism of these so-called tunnel oxide passivated contacts will be outlined and the impact of TCO (transparent conductive oxide) deposition on the injection-dependent lifetime characteristic of the emitter as well as its implications on FF will be discussed. Finally, possible transport paths across the tunnel oxide barrier will be discussed and it will be shown that the passivating oxide layer does not lead to a relevant resistive loss and thus does not limit the solar cell's carrier transport. Contrary to amorphous silicon-based heterojunction solar cells, this structure also shows a good thermal stability and, thus, could be a very appealing option for next generation high-efficiency silicon solar cells.

  9. Subtractive Cell-SELEX Selection of DNA Aptamers Binding Specifically and Selectively to Hepatocellular Carcinoma Cells with High Metastatic Potential

    PubMed Central

    Chen, Hao; Yuan, Chun-Hui; Yang, Yi-Fei; Yin, Chang-Qing; Guan, Qing; Wang, Fu-Bing; Tu, Jian-Cheng

    2016-01-01

    Relapse and metastasis are two key risk factors of hepatocellular carcinoma (HCC) prognosis; thus, it is emergent to develop an early and accurate detection method for prognostic evaluation of HCC after surgery. In this study, we sought to acquire oligonucleotide DNA aptamers that specifically bind to HCC cells with high metastatic potential. Two HCC cell lines derived from the same genetic background but with different metastatic potential were employed: MHCC97L (low metastatic properties) as subtractive targets and HCCLM9 (high metastatic properties) as screening targets. To mimic a fluid combining environment, initial DNA aptamers library was firstly labelled with magnetic nanoparticles using biotin-streptavidin system and then applied for aptamers selection. Through 10-round selection with subtractive Cell-SELEX, six aptamers, LY-1, LY-13, LY-46, LY-32, LY-27/45, and LY-7/43, display high affinity to HCCLM9 cells and do not bind to MHCC97L cells, as well as other tumor cell lines, including breast cancer, lung cancer, colon adenocarcinoma, gastric cancer, and cervical cancer, suggesting high specificity for HCCLM9 cells. Thus, the aptamers generated here will provide solid basis for identifying new diagnostic targets to detect HCC metastasis and also may provide valuable clues for developing new targeted therapeutics. PMID:27119081

  10. Selective Cell Targeting with Light-Absorbing Microparticles and Nanoparticles

    PubMed Central

    Pitsillides, Costas M.; Joe, Edwin K.; Wei, Xunbin; Anderson, R. Rox; Lin, Charles P.

    2003-01-01

    We describe a new method for selective cell targeting based on the use of light-absorbing microparticles and nanoparticles that are heated by short laser pulses to create highly localized cell damage. The method is closely related to chromophore-assisted laser inactivation and photodynamic therapy, but is driven solely by light absorption, without the need for photochemical intermediates (particularly singlet oxygen). The mechanism of light-particle interaction was investigated by nanosecond time-resolved microscopy and by thermal modeling. The extent of light-induced damage was investigated by cell lethality, by cell membrane permeability, and by protein inactivation. Strong particle size dependence was found for these interactions. A technique based on light to target endogenous particles is already being exploited to treat pigmented cells in dermatology and ophthalmology. With exogenous particles, phamacokinetics and biodistribution studies are needed before the method can be evaluated against photodynamic therapy for cancer treatment. However, particles are unique, unlike photosensitizers, in that they can remain stable and inert in cells for extended periods. Thus they may be particularly useful for prelabeling cells in engineered tissue before implantation. Subsequent irradiation with laser pulses will allow control of the implanted cells (inactivation or modulation) in a noninvasive manner. PMID:12770906

  11. Regulated selection of germinal-center cells into the memory B cell compartment.

    PubMed

    Shinnakasu, Ryo; Inoue, Takeshi; Kometani, Kohei; Moriyama, Saya; Adachi, Yu; Nakayama, Manabu; Takahashi, Yoshimasa; Fukuyama, Hidehiro; Okada, Takaharu; Kurosaki, Tomohiro

    2016-07-01

    Despite the importance of memory B cells in protection from reinfection, how such memory cells are selected and generated during germinal-center (GC) reactions remains unclear. We found here that light-zone (LZ) GC B cells with B cell antigen receptors (BCRs) of lower affinity were prone to enter the memory B cell pool. Mechanistically, cells in this memory-prone fraction had higher expression of the transcriptional repressor Bach2 than that of their counterparts with BCRs of higher affinity. Haploinsufficiency of Bach2 resulted in reduced generation of memory B cells, independently of suppression of the gene encoding the transcription factor Blimp-1. Bach2 expression in GC cells was inversely correlated with the strength of help provided by T cells. Thus, we propose an instructive model in which weak help from T cells maintains relatively high expression of Bach2, which predisposes GC cells to enter the memory pool. PMID:27158841

  12. Adipose-derived stem cells: selecting for translational success

    PubMed Central

    Johal, Kavan S; Lees, Vivien C; Reid, Adam J

    2016-01-01

    We have witnessed a rapid expansion of in vitro characterization and differentiation of adipose-derived stem cells, with increasing translation to both in vivo models and a breadth of clinical specialties. However, an appreciation of the truly heterogeneous nature of this unique stem cell group has identified a need to more accurately delineate subpopulations by any of a host of methods, to include functional properties or surface marker expression. Cells selected for improved proliferative, differentiative, angiogenic or ischemia-resistant properties are but a few attributes that could prove beneficial for targeted treatments or therapies. Optimizing cell culture conditions to permit re-introduction to patients is critical for clinical translation. PMID:25562354

  13. Selection of Phage Display Peptides Targeting Human Pluripotent Stem Cell-Derived Progenitor Cell Lines.

    PubMed

    Bignone, Paola A; Krupa, Rachel A; West, Michael D; Larocca, David

    2016-01-01

    The ability of human pluripotent stem cells (hPS) to both self-renew and differentiate into virtually any cell type makes them a promising source of cells for cell-based regenerative therapies. However, stem cell identity, purity, and scalability remain formidable challenges that need to be overcome for translation of pluripotent stem cell research into clinical applications. Directed differentiation from hPS cells is inefficient and residual contamination with pluripotent cells that have the potential to form tumors remains problematic. The derivation of scalable (self-renewing) embryonic progenitor stem cell lines offers a solution because they are well defined and clonally pure. Clonally pure progenitor stem cell lines also provide a means for identifying cell surface targeting reagents that are useful for identification, tracking, and repeated derivation of the corresponding progenitor stem cell types from additional hPS cell sources. Such stem cell targeting reagents can then be applied to the manufacture of genetically diverse banks of human embryonic progenitor cell lines for drug screening, disease modeling, and cell therapy. Here we present methods to identify human embryonic progenitor stem cell targeting peptides by selection of phage display libraries on clonal embryonic progenitor cell lines and demonstrate their use for targeting quantum dots (Qdots) for stem cell labeling. PMID:25410289

  14. Cell biology, molecular embryology, Lamarckian and Darwinian selection as evolvability.

    PubMed

    Hoenigsberg, H

    2003-01-01

    The evolvability of vertebrate systems involves various mechanisms that eventually generate cooperative and nonlethal functional variation on which Darwinian selection can operate. It is a truism that to get vertebrate animals to develop a coherent machine they first had to inherit the right multicellular ontogeny. The ontogeny of a metazoan involves cell lineages that progressively deny their own capacity for increase and for totipotency in benefit of the collective interest of the individual. To achieve such cell altruism Darwinian dynamics rescinded its original unicellular mandate to reproduce. The distinction between heritability at the level of the cell lineage and at the level of the individual is crucial. However, its implications have seldom been explored in depth. While all out reproduction is the Darwinian measure of success among unicellular organisms, a high replication rate of cell lineages within the organism may be deleterious to the individual as a functional unit. If a harmoniously functioning unit is to evolve, mechanisms must have evolved whereby variants that increase their own replication rate by failing to accept their own somatic duties are controlled. For questions involving organelle origins, see Godelle and Reboud, 1995 and Hoekstra, 1990. In other words, modifiers of conflict that control cell lineages with conflicting genes and new mutant replication rates that deviate from their somatic duties had to evolve. Our thesis is that selection at the level of the (multicellular) individual must have opposed selection at the level of the cell lineage. The metazoan embryo is not immune to this conflict especially with the evolution of set-aside cells and other modes of self-policing modifiers (Blackstone and Ellison, 1998; Ransick et al., 1996. In fact, the conflict between the two selection processes permitted a Lamarckian soma-to-germline feedback loop. This new element in metazoan ontogeny became the evolvability of the vertebrate adaptive

  15. CRK proteins selectively regulate T cell migration into inflamed tissues

    PubMed Central

    Huang, Yanping; Clarke, Fiona; Karimi, Mobin; Roy, Nathan H.; Williamson, Edward K.; Okumura, Mariko; Mochizuki, Kazuhiro; Chen, Emily J.H.; Park, Tae-Ju; Debes, Gudrun F.; Zhang, Yi; Curran, Tom; Kambayashi, Taku; Burkhardt, Janis K.

    2015-01-01

    Effector T cell migration into inflamed sites greatly exacerbates tissue destruction and disease severity in inflammatory diseases, including graft-versus-host disease (GVHD). T cell migration into such sites depends heavily on regulated adhesion and migration, but the signaling pathways that coordinate these functions downstream of chemokine receptors are largely unknown. Using conditional knockout mice, we found that T cells lacking the adaptor proteins CRK and CRK-like (CRKL) exhibit reduced integrin-dependent adhesion, chemotaxis, and diapedesis. Moreover, these two closely related proteins exhibited substantial functional redundancy, as ectopic expression of either protein rescued defects in T cells lacking both CRK and CRKL. We determined that CRK proteins coordinate with the RAP guanine nucleotide exchange factor C3G and the adhesion docking molecule CASL to activate the integrin regulatory GTPase RAP1. CRK proteins were required for effector T cell trafficking into sites of inflammation, but not for migration to lymphoid organs. In a murine bone marrow transplantation model, the differential migration of CRK/CRKL-deficient T cells resulted in efficient graft-versus-leukemia responses with minimal GVHD. Together, the results from our studies show that CRK family proteins selectively regulate T cell adhesion and migration at effector sites and suggest that these proteins have potential as therapeutic targets for preventing GVHD. PMID:25621495

  16. Thymic Selection of T Cells as Diffusion with Intermittent Traps

    NASA Astrophysics Data System (ADS)

    Košmrlj, Andrej

    2011-04-01

    T cells orchestrate adaptive immune responses by recognizing short peptides derived from pathogens, and by distinguishing them from self-peptides. To ensure the latter, immature T cells (thymocytes) diffuse within the thymus gland, where they encounter an ensemble of self-peptides presented on (immobile) antigen presenting cells. Potentially autoimmune T cells are eliminated if the thymocyte binds sufficiently strongly with any such antigen presenting cell. We model thymic selection of T cells as a random walker diffusing in a field of immobile traps that intermittently turn "on" and "off". The escape probability of potentially autoimmune T cells is equivalent to the survival probability of such a random walker. In this paper we describe the survival probability of a random walker on a d-dimensional cubic lattice with randomly placed immobile intermittent traps, and relate it to the result of a well-studied problem where traps are always "on". Additionally, when switching between the trap states is slow, we find a peculiar caging effect for the survival probability.

  17. Formulation of selected renal cells for implantation into a kidney.

    PubMed

    Halberstadt, Craig; Robbins, Neil; McCoy, Darell W; Guthrie, Kelly I; Bruce, Andrew T; Knight, Toyin A; Payne, Richard G

    2013-01-01

    Delivery of cells to organs has primarily relied on formulating the cells in a nonviscous liquid carrier. We have developed a methodology to isolate selected renal cells (SRC) that have provided functional stability to damaged kidneys in preclinical models (Kelley et al. Poster presentation at 71st scientific sessions of American diabetes association , 2011; Kelley et al. Oral presentation given at Tissue Engineering and Regenerative Medicine International Society (TERMIS)-North America annual conference, 2010; Presnell et al. Tissue Eng Part C Methods 17:261-273, 2011; Kelley et al. Am J Physiol Renal Physiol 299:F1026-F1039, 2010). In order to facilitate SRC injection into the kidney of patients who have chronic kidney disease, we have developed a strategy to immobilize the cells in a hydrogel matrix. This hydrogel (gelatin) supports cells by maintaining them in a three-dimensional state during storage and shipment (both at cold temperatures) while facilitating the delivery of cells by liquefying when engrafting into the kidney. This chapter will define a method for the formulation of the kidney epithelial cells within a hydrogel. PMID:23494437

  18. Class I MHC molecules on hematopoietic cells can support intrathymic positive selection of T cell receptor transgenic T cells

    PubMed Central

    Zerrahn, Jens; Volkmann, Ariane; Coles, Mark C.; Held, Werner; Lemonnier, Francois A.; Raulet, David H.

    1999-01-01

    The identity of cells that mediate positive selection of CD8+ T cells was investigated in two T cell receptor (TCR) transgenic systems. Irradiated β2-microglobulin mutant mice or mice with mutations in both the Kb and Db genes were repopulated with fetal liver cells from class I+ TCR transgenic mice. In the case of the 2C TCR, mature transgene-expressing CD8+ T cells appeared in the thymuses of the chimeras and in larger numbers in the peripheral lymphoid organs. These CD8+ T cells were functional, exhibited a naive, resting phenotype, and were mostly thymus-dependent. Their development depended on donor cell class I expression. These results establish that thymic hematopoietic cells can direct positive selection of CD8+ T cells expressing a conventional TCR. In contrast, no significant development of HY (male antigen)–TCR+ CD8+ T cells was observed in class I+ into class I-deficient chimeras. These data suggest that successful positive selection directed by hematopoietic cells depends on specific properties of the TCR or its thymic ligands. The possibility that hematopoietic cell-induced, positive selection occurs only with TCRs that exhibit relatively high avidity interactions with selecting ligands in the thymus is discussed. PMID:10500200

  19. Autophagy variation within a cell population determines cell fate through selective degradation of Fap-1.

    PubMed

    Gump, Jacob M; Staskiewicz, Leah; Morgan, Michael J; Bamberg, Alison; Riches, David W H; Thorburn, Andrew

    2014-01-01

    Autophagy regulates cell death both positively and negatively, but the molecular basis for this paradox remains inadequately characterized. We demonstrate here that transient cell-to-cell variations in autophagy can promote either cell death or survival depending on the stimulus and cell type. By separating cells with high and low basal autophagy using flow cytometry, we demonstrate that autophagy determines which cells live or die in response to death receptor activation. We have determined that selective autophagic degradation of the phosphatase Fap-1 promotes Fas apoptosis in Type I cells, which do not require mitochondrial permeabilization for efficient apoptosis. Conversely, autophagy inhibits apoptosis in Type II cells (which require mitochondrial involvement) or on treatment with TRAIL in either Type I or II cells. These data illustrate that differences in autophagy in a cell population determine cell fate in a stimulus- and cell-type-specific manner. This example of selective autophagy of an apoptosis regulator may represent a general mechanism for context-specific regulation of cell fate by autophagy. PMID:24316673

  20. T cell receptor interactions with class I heavy-chain influence T cell selection

    PubMed Central

    Kuhns, Scott T.; Tallquist, Michelle D.; Johnson, Aaron J.; Mendez-Fernandez, Yanice; Pease, Larry R.

    2000-01-01

    The interaction of the T cell receptor (TCR) with peptide in the binding site of the major histocompatibility complex molecule provides the basis for T cell recognition during immune surveillance, repertoire development, and tolerance. Little is known about the extent to which repertoire selection is influenced directly by variation of the structure of the class I heavy chain. We find that the 2C TCR, normally positively selected in the context of the Kb molecule, is minimally selected into the CD8 lineage in the absence of antigen-processing genes. This finding underscores the importance of peptides in determining the positive-selecting class I ligands in the thymus. In contrast, Kbm3, a variant class I molecule that normally exerts a negative selection pressure on 2C-bearing T cells, positively selects 2C transgenic T cells into the CD8 lineage in an antigen-processing gene-deficient environment. These findings indicate that structural changes in the heavy chain can have direct influence in T cell recognition, from which we conclude that the nature of TCR interaction with class I heavy chain influences the array of TCRs selected during development of the functional adult repertoire. PMID:10639152

  1. Prolonged Drug Selection of Breast Cancer Cells and Enrichment of Cancer Stem Cell Characteristics

    PubMed Central

    Calcagno, Anna Maria; Salcido, Crystal D.; Gillet, Jean-Pierre; Wu, Chung-Pu; Fostel, Jennifer M.; Mumau, Melanie D.; Gottesman, Michael M.; Varticovski, Lyuba

    2010-01-01

    Background Cancer stem cells are presumed to have virtually unlimited proliferative and self-renewal abilities and to be highly resistant to chemotherapy, a feature that is associated with overexpression of ATP-binding cassette transporters. We investigated whether prolonged continuous selection of cells for drug resistance enriches cultures for cancer stem–like cells. Methods Cancer stem cells were defined as CD44+/CD24− cells that could self-renew (ie, generate cells with the tumorigenic CD44+/CD24− phenotype), differentiate, invade, and form tumors in vivo. We used doxorubicin-selected MCF-7/ADR cells, weakly tumorigenic parental MCF-7 cells, and MCF-7/MDR, an MCF-7 subline with forced expression of ABCB1 protein. Cells were examined for cell surface markers and side-population fractions by microarray and flow cytometry, with in vitro invasion assays, and for ability to form mammospheres. Xenograft tumors were generated in mice to examine tumorigenicity (n = 52). The mRNA expression of multidrug resistance genes was examined in putative cancer stem cells and pathway analysis of statistically significantly differentially expressed genes was performed. All statistical tests were two-sided. Results Pathway analysis showed that MCF-7/ADR cells express mRNAs from ABCB1 and other genes also found in breast cancer stem cells (eg, CD44, TGFB1, and SNAI1). MCF-7/ADR cells were highly invasive, formed mammospheres, and were tumorigenic in mice. In contrast to parental MCF-7 cells, more than 30% of MCF-7/ADR cells had a CD44+/CD24− phenotype, could self-renew, and differentiate (ie, produce CD44+/CD24− and CD44+/CD24+ cells) and overexpressed various multidrug resistance–linked genes (including ABCB1, CCNE1, and MMP9). MCF-7/ADR cells were statistically significantly more invasive in Matrigel than parental MCF-7 cells (MCF-7 cells = 0.82 cell per field and MCF-7/ADR = 7.51 cells per field, difference = 6.69 cells per field, 95% confidence interval = 4.82 to 8

  2. A highly selective telomerase inhibitor limiting human cancer cell proliferation

    PubMed Central

    Damm, Klaus; Hemmann, Ulrike; Garin-Chesa, Pilar; Hauel, Norbert; Kauffmann, Iris; Priepke, Henning; Niestroj, Claudia; Daiber, Christine; Enenkel, Barbara; Guilliard, Bernd; Lauritsch, Ines; Müller, Elfriede; Pascolo, Emanuelle; Sauter, Gabriele; Pantic, Milena; Martens, Uwe M.; Wenz, Christian; Lingner, Joachim; Kraut, Norbert; Rettig, Wolfgang J.; Schnapp, Andreas

    2001-01-01

    Telomerase, the ribonucleoprotein enzyme maintaining the telomeres of eukaryotic chromosomes, is active in most human cancers and in germline cells but, with few exceptions, not in normal human somatic tissues. Telomere maintenance is essential to the replicative potential of malignant cells and the inhibition of telomerase can lead to telomere shortening and cessation of unrestrained proliferation. We describe novel chemical compounds which selectively inhibit telomerase in vitro and in vivo. Treatment of cancer cells with these inhibitors leads to progressive telomere shortening, with no acute cytotoxicity, but a proliferation arrest after a characteristic lag period with hallmarks of senescence, including morphological, mitotic and chromosomal aberrations and altered patterns of gene expression. Telomerase inhibition and telomere shortening also result in a marked reduction of the tumorigenic potential of drug-treated tumour cells in a mouse xenograft model. This model was also used to demonstrate in vivo efficacy with no adverse side effects and uncomplicated oral administration of the inhibitor. These findings indicate that potent and selective, non-nucleosidic telomerase inhibitors can be designed as novel cancer treatment modalities. PMID:11742973

  3. Selective-emitter crystalline silicon solar cells using phosphorus paste

    NASA Astrophysics Data System (ADS)

    Jeong, Kyung Taek; Kang, Min Gu; Song, Hee-eun

    2014-11-01

    Selective-emitter structures have been studied to improve the conversion efficiency of crystalline silicon solar cells. However, such structures require additional complicated processes and incur extra cost. In this work, we used phosphorus paste (P-paste) to form a heavily-doped region beneath the grid and POCl3 to create a shallow emitter area. This method should be convenient to use in the solar-cell industry because it requires only additional P paste printing, compared to the case of homogeneous solar cells. Diffusion parameters including the temperature, diffusion time, and ambient gases were optimized. We observed that the spreading of the P paste was affected by the pyramidal size of the textured wafer due to the low viscosity of the P paste. The pyramidal height of the textured silicon surface was optimized at 3 μm to counterbalance the surface reflectance and the spreading of the P paste. The short-circuit current density of the completed selective emitter solar cell was increased, and an improvement of blue response in the internal quantum efficiency was seen while contact properties such as the fill factor deteriorated due to the spreading of the P paste and the thin emitter on top of the pyramid of the textured silicon surface. Double printing of the P paste was applied to solve this contact problem; a fill factor improvement of 2.4% was obtained.

  4. Selective Interlayers and Contacts in Organic Photovoltaic Cells.

    PubMed

    Ratcliff, Erin L; Zacher, Brian; Armstrong, Neal R

    2011-06-01

    Organic photovoltaic cells (OPVs) are promising solar electric energy conversion systems with impressive recent optimization of active layers. OPV optimization must now be accompanied by the development of new charge-selective contacts and interlayers. This Perspective considers the role of interface science in energy harvesting using OPVs, looking back at early photoelectrochemical (photogalvanic) energy conversion platforms, which suffered from a lack of charge carrier selectivity. We then examine recent platforms and the fundamental aspects of selective harvesting of holes and electrons at opposite contacts. For blended heterojunction OPVs, contact/interlayer design is especially critical because charge harvesting competes with recombination at these same contacts. New interlayer materials can modify contacts to both control work function and introduce selectivity and chemical compatibility with nonpolar active layers and add thermodynamic and kinetic selectivity to charge harvesting. We briefly discuss the surface and interface science required for the development of new interlayer materials and take a look ahead at the challenges yet to be faced in their optimization. PMID:26295432

  5. Selective modulation of cell response on engineered fractal silicon substrates

    PubMed Central

    Gentile, Francesco; Medda, Rebecca; Cheng, Ling; Battista, Edmondo; Scopelliti, Pasquale E.; Milani, Paolo; Cavalcanti-Adam, Elisabetta A.; Decuzzi, Paolo

    2013-01-01

    A plethora of work has been dedicated to the analysis of cell behavior on substrates with ordered topographical features. However, the natural cell microenvironment is characterized by biomechanical cues organized over multiple scales. Here, randomly rough, self-affinefractal surfaces are generated out of silicon,where roughness Ra and fractal dimension Df are independently controlled. The proliferation rates, the formation of adhesion structures, and the morphology of 3T3 murine fibroblasts are monitored over six different substrates. The proliferation rate is maximized on surfaces with moderate roughness (Ra ~ 40 nm) and large fractal dimension (Df ~ 2.4); whereas adhesion structures are wider and more stable on substrates with higher roughness (Ra ~ 50 nm) and lower fractal dimension (Df ~ 2.2). Higher proliferation occurson substrates exhibiting densely packed and sharp peaks, whereas more regular ridges favor adhesion. These results suggest that randomly roughtopographies can selectively modulate cell behavior. PMID:23492898

  6. Cell-selective labelling of proteomes in Drosophila melanogaster

    PubMed Central

    Erdmann, Ines; Marter, Kathrin; Kobler, Oliver; Niehues, Sven; Abele, Julia; Müller, Anke; Bussmann, Julia; Storkebaum, Erik; Ziv, Tamar; Thomas, Ulrich; Dieterich, Daniela C.

    2015-01-01

    The specification and adaptability of cells rely on changes in protein composition. Nonetheless, uncovering proteome dynamics with cell-type-specific resolution remains challenging. Here we introduce a strategy for cell-specific analysis of newly synthesized proteomes by combining targeted expression of a mutated methionyl-tRNA synthetase (MetRS) with bioorthogonal or fluorescent non-canonical amino-acid-tagging techniques (BONCAT or FUNCAT). Substituting leucine by glycine within the MetRS-binding pocket (MetRSLtoG) enables incorporation of the non-canonical amino acid azidonorleucine (ANL) instead of methionine during translation. Newly synthesized proteins can thus be labelled by coupling the azide group of ANL to alkyne-bearing tags through ‘click chemistry'. To test these methods for applicability in vivo, we expressed MetRSLtoG cell specifically in Drosophila. FUNCAT and BONCAT reveal ANL incorporation into proteins selectively in cells expressing the mutated enzyme. Cell-type-specific FUNCAT and BONCAT, thus, constitute eligible techniques to study protein synthesis-dependent processes in complex and behaving organisms. PMID:26138272

  7. Selective Inhibition of Collagen Prolyl 4-Hydroxylase in Human Cells

    PubMed Central

    Vasta, James D.; Andersen, Kristen A.; Deck, Kathryn M.; Nizzi, Christopher P.; Eisenstein, Richard S.; Raines, Ronald T.

    2016-01-01

    Collagen is the most abundant protein in animals. Its overproduction is associated with fibrosis and cancer metastasis. The stability of collagen relies on post-translational modifications, the most prevalent being the hydroxylation of collagen strands by collagen prolyl 4-hydroxylases (CP4Hs). Catalysis by CP4Hs enlists an iron cofactor to convert proline residues to 4 hydroxyproline residues, which are essential for the conformational stability of mature collagen. Ethyl 3,4-dihydroxybenzoate (EDHB) is commonly used as a “P4H” inhibitor in cells, but suffers from low potency, poor selectivity, and off-target effects that cause iron deficiency. Dicarboxylates of 2,2′-bipyridine are among the most potent known CP4H inhibitors but suffer from a high affinity for free iron. A screen of biheteroaryl compounds revealed that replacing one pyridyl group with a thiazole moiety retains potency and enhances selectivity. A diester of 2 (5-carboxythiazol-2-yl)pyridine-5-carboxylic acid is bioavailable to human cells and inhibits collagen biosynthesis at concentrations that neither cause general toxicity nor disrupt iron homeostasis. These data anoint a potent and selective probe for CP4H and a potential lead for the development of a new class of antifibrotic and antimetastatic agents. PMID:26535807

  8. Subcortical orientation biases explain orientation selectivity of visual cortical cells

    PubMed Central

    Vidyasagar, Trichur R; Jayakumar, Jaikishan; Lloyd, Errol; Levichkina, Ekaterina V

    2015-01-01

    The primary visual cortex of carnivores and primates shows an orderly progression of domains of neurons that are selective to a particular orientation of visual stimuli such as bars and gratings. We recorded from single-thalamic afferent fibers that terminate in these domains to address the issue whether the orientation sensitivity of these fibers could form the basis of the remarkable orientation selectivity exhibited by most cortical cells. We first performed optical imaging of intrinsic signals to obtain a map of orientation domains on the dorsal aspect of the anaesthetized cat's area 17. After confirming using electrophysiological recordings the orientation preferences of single neurons within one or two domains in each animal, we pharmacologically silenced the cortex to leave only the afferent terminals active. The inactivation of cortical neurons was achieved by the superfusion of either kainic acid or muscimol. Responses of single geniculate afferents were then recorded by the use of high impedance electrodes. We found that the orientation preferences of the afferents matched closely with those of the cells in the orientation domains that they terminated in (Pearson's r = 0.633, n = 22, P = 0.002). This suggests a possible subcortical origin for cortical orientation selectivity. PMID:25855249

  9. SHAPE SELECTIVE NANOCATALYSTS FOR DIRECT METHANOL FUEL CELL APPLICATIONS

    SciTech Connect

    Murph, S.

    2012-09-12

    While gold and platinum have long been recognized for their beauty and value, researchers at the Savannah River National Laboratory (SRNL) are working on the nano-level to use these elements for creative solutions to our nation's energy and security needs. Multiinterdisciplinary teams consisting of chemists, materials scientists, physicists, computational scientists, and engineers are exploring unchartered territories with shape-selective nanocatalysts for the development of novel, cost effective and environmentally friendly energy solutions to meet global energy needs. This nanotechnology is vital, particularly as it relates to fuel cells.SRNL researchers have taken process, chemical, and materials discoveries and translated them for technological solution and deployment. The group has developed state-of-the art shape-selective core-shell-alloy-type gold-platinum nanostructures with outstanding catalytic capabilities that address many of the shortcomings of the Direct Methanol Fuel Cell (DMFC). The newly developed nanostructures not only busted the performance of the platinum catalyst, but also reduced the material cost and overall weight of the fuel cell.

  10. Kinetics of drug selection systems in mouse embryonic stem cells

    PubMed Central

    2013-01-01

    Background Stable expression of transgenes is an important technique to analyze gene function. Various drug resistance genes, such as neo, pac, hph, zeo, bsd, and hisD, have been equally used as selection markers to isolate a transfectant without considering their dose-dependent characters. Results We quantitatively measured the variation of transgene expression levels in mouse embryonic stem (mES) cells, using a series of bi-cistronic expression vectors that contain Egfp expression cassette linked to each drug resistant gene via IRES with titration of the selective drugs, and found that the transgene expression levels achieved in each system with this vector design are in order, in which pac and zeo show sharp selection of transfectants with homogenously high expression levels. We also showed the importance of the choice of the drug selection system in gene-trap or gene targeting according to this order. Conclusions The results of the present study clearly demonstrated that an appropriate choice of the drug resistance gene(s) is critical for a proper design of the experimental strategy. PMID:23919313

  11. Polymer selection and cell design for electric-vehicle supercapacitors

    SciTech Connect

    Mastragostino, M.; Arbizzani, C.; Paraventi, R.; Zanelli, A.

    2000-02-01

    Supercapacitors are devices for applications requiring high operating power levels, such as secondary power sources in electric vehicles (EVs) to provide peak power for acceleration and hill climbing. While electronically conducting polymers yield different redox supercapacitor configurations, devices with the n-doped polymer as the negative electrode and the p-doped polymer as the positive one are the most promising for EV applications. Indeed, this type of supercapacitor has a high operating potential, is able to deliver all the doping charge and, when charged, has both electrodes in the conducting (p- and n-doped) states. This study reports selection criteria for polymer materials and cell design for high performance EV supercapacitors and experimental results of selected polymer materials.

  12. Selection of mammalian cells based on their cell-cycle phase using dielectrophoresis

    PubMed Central

    Kim, Unyoung; Shu, Chih-Wen; Dane, Karen Y.; Daugherty, Patrick S.; Wang, Jean Y. J.; Soh, H. T.

    2007-01-01

    An effective, noninvasive means of selecting cells based on their phase within the cell cycle is an important capability for biological research. Current methods of producing synchronous cell populations, however, tend to disrupt the natural physiology of the cell or suffer from low synchronization yields. In this work, we report a microfluidic device that utilizes the dielectrophoresis phenomenon to synchronize cells by exploiting the relationship between the cell's volume and its phase in the cell cycle. The dielectrophoresis activated cell synchronizer (DACSync) device accepts an asynchronous mixture of cells at the inlet, fractionates the cell populations according to the cell-cycle phase (G1/S and G2/M), and elutes them through different outlets. The device is gentle and efficient; it utilizes electric fields that are 1–2 orders of magnitude below those used in electroporation and enriches asynchronous tumor cells in the G1 phase to 96% in one round of sorting, in a continuous flow manner at a throughput of 2 × 105 cells per hour per microchannel. This work illustrates the feasibility of using laminar flow and electrokinetic forces for the efficient, noninvasive separation of living cells. PMID:18093921

  13. Selective apoptotic cell death effects of oral cancer cells treated with destruxin B

    PubMed Central

    2014-01-01

    Background Recent studies have revealed that destruxins (Dtx) have potent cytotoxic activities on individual cancer cells, however, data on oral cancer cells especial human are absent. Methods Destruxin B (DB) was isolated and used to evaluate the selective cytotoxicity with human oral cancer cell lines, GNM (Neck metastasis of gingival carcinoma) and TSCCa (Tongue squamous cell carcinoma) cells, and normal gingival fibroblasts (GF) were also included as controls. Cells were tested with different concentrations of DB for 24, 48, and 72 h by MTT assay. Moreover, the mechanism of cytotoxicity was investigated using caspase-3 Immunofluorescence, annexin V/PI staining, and the expression of caspase-3, Bax, and Bcl-2 by western blotting after treated with different concentrations of DB for 72 h as parameters for apoptosis analyses. Results The results show that DB exhibited significant (p < 0.01) and selective time- and dose-dependent inhibitory effects on GNM and TSCCa cells viability but not on GF cells. The data suggested that DB is capable to induce tumor specific growth inhibition in oral GNM and TSCCa cancer cells via Bax/Bcl-2-mediated intrinsic mitochondrial apoptotic pathway in time- and dose-dependent manners. Conclusions This is the first report on the anti-proliferation effect of DB in oral cancer cells. The results reported here may offer further evidences to the development of DB as a potential complementary chemotherapeutic target for oral cancer complications. PMID:24972848

  14. Preparation of functional human factor V111 in mammalian cells using methotrexate based selection

    SciTech Connect

    Capon, D.J.; Lawn, R.M.; Levinson, A.D.; Vehar, G.A.; Wood, W.I.

    1990-10-23

    This patent describes a process for producing factor VII. It comprises: cotransfecting a mammalian host cell with a DNA sequence encoding factor VIII, a second DNA sequence encoding an amplifiable marker, and a third DNA sequence encoding a selectable marker; growing the transfected cell in a non-selection medium and selecting for such selectable marker resistant cells; and amplifying the amplifiable marker DNA sequence by culturing the selected cells in media containing increasing amounts of selection agent, wherein the host cell is not deficient in the amplifiable marker.

  15. Selective Destruction Of Cells Infected With The Human Immunodeficiency Virus

    DOEpatents

    Keener, William K.; Ward, Thomas E.

    2006-03-28

    Compositions and methods for selectively killing a cell containing a viral protease are disclosed. The composition is a varient of a protein synthesis inactivating toxin wherein a viral protease cleavage site is interposed between the A and B chains. The variant of the type II ribosome-inactivating protein is activated by digestion of the viral protease cleavage site by the specific viral protease. The activated ribosome-inactivating protein then kills the cell by inactivating cellular ribosomes. A preferred embodiment of the invention is specific for human immunodeficiency virus (HIV) and uses ricin as the ribosome-inactivating protein. In another preferred embodiment of the invention, the variant of the ribosome-inactivating protein is modified by attachment of one or more hydrophobic agents. The hydrophobic agent facilitates entry of the variant of the ribosome-inactivating protein into cells and can lead to incorporation of the ribosome-inactivating protein into viral particles. Still another preferred embodiment of the invention includes a targeting moiety attached to the variants of the ribosome-inactivating protein to target the agent to HIV infectable cells.

  16. Selective destruction of cells infected with human immunodeficiency virus

    DOEpatents

    Keener, William K.; Ward, Thomas E.

    2003-09-30

    Compositions and methods for selectively killing a cell containing a viral protease are disclosed. The composition is a variant of a protein synthesis inactivating toxin wherein a viral protease cleavage site is interposed between the A and B chains. The variant of the type II ribosome-inactivating protein is activated by digestion of the viral protease cleavage site by the specific viral protease. The activated ribosome-inactivating protein then kills the cell by inactivating cellular ribosomes. A preferred embodiment of the invention is specific for human immunodeficiency virus (HIV) and uses ricin as the ribosome-inactivating protein. In another preferred embodiment of the invention, the variant of the ribosome-inactivating protein is modified by attachment of one or more hydrophobic agents. The hydrophobic agent facilitates entry of the variant of the ribosome-inactivating protein into cells and can lead to incorporation of the ribosome-inactivating protein into viral particles. Still another preferred embodiment of the invention includes a targeting moiety attached to the variants of the ribosome-inactivating protein to target the agent to HIV infectable cells.

  17. Selective Calcium Sensitivity in Immature Glioma Cancer Stem Cells

    PubMed Central

    Marinescu, Voichita Dana; Segerman, Anna; Schmidt, Linnéa; Hermansson, Annika; Dirks, Peter; Forsberg-Nilsson, Karin; Westermark, Bengt; Uhrbom, Lene; Linnarsson, Sten; Nelander, Sven; Andäng, Michael

    2014-01-01

    Tumor-initiating cells are a subpopulation in aggressive cancers that exhibit traits shared with stem cells, including the ability to self-renew and differentiate, commonly referred to as stemness. In addition, such cells are resistant to chemo- and radiation therapy posing a therapeutic challenge. To uncover stemness-associated functions in glioma-initiating cells (GICs), transcriptome profiles were compared to neural stem cells (NSCs) and gene ontology analysis identified an enrichment of Ca2+ signaling genes in NSCs and the more stem-like (NSC-proximal) GICs. Functional analysis in a set of different GIC lines regarding sensitivity to disturbed homeostasis using A23187 and Thapsigargin, revealed that NSC-proximal GICs were more sensitive, corroborating the transcriptome data. Furthermore, Ca2+ drug sensitivity was reduced in GICs after differentiation, with most potent effect in the NSC-proximal GIC, supporting a stemness-associated Ca2+ sensitivity. NSCs and the NSC-proximal GIC line expressed a larger number of ion channels permeable to potassium, sodium and Ca2+. Conversely, a higher number of and higher expression levels of Ca2+ binding genes that may buffer Ca2+, were expressed in NSC-distal GICs. In particular, expression of the AMPA glutamate receptor subunit GRIA1, was found to associate with Ca2+ sensitive NSC-proximal GICs, and decreased as GICs differentiated along with reduced Ca2+ drug sensitivity. The correlation between high expression of Ca2+ channels (such as GRIA1) and sensitivity to Ca2+ drugs was confirmed in an additional nine novel GIC lines. Calcium drug sensitivity also correlated with expression of the NSC markers nestin (NES) and FABP7 (BLBP, brain lipid-binding protein) in this extended analysis. In summary, NSC-associated NES+/FABP7+/GRIA1+ GICs were selectively sensitive to disturbances in Ca2+ homeostasis, providing a potential target mechanism for eradication of an immature population of malignant cells. PMID:25531110

  18. Galactosylated poly(ethyleneglycol)-lithocholic Acid selectively kills hepatoma cells, while sparing normal liver cells.

    PubMed

    Gankhuyag, Nomundelger; Singh, Bijay; Maharjan, Sushila; Choi, Yun-Jaie; Cho, Chong-Su; Cho, Myung-Haing

    2015-06-01

    Delivering drugs selectively to cancer cells but not to nearby normal cells is a major obstacle in drug therapy. In this study, lithocholic acid (LCA), a potent anti-cancer drug, is converted to two forms of poly(ethyleneglycol) (PEG) conjugates, viz., PEG-LCA (PL) and lactobionic acid (LBA) conjugated PEG-LCA (LPL). The latter form contains a galactose ligand in LBA to target the hepatocytes. Both forms are self-assembled to form nanoparticle formulation, and they have high potency than LCA to kill HepG2 cancer cells, sparing normal LO2 cells. Besides, LPL has high specificity to mouse liver cells in vivo. Western blot results confirm that the cell death is occurred through apoptosis induced by LPL nanoparticles. In conclusion, the induction of apoptosis and cell death is much more efficient with LPL nanoparticles than LCA molecules. PMID:25657071

  19. Wood-fired fuel cells in selected buildings

    NASA Astrophysics Data System (ADS)

    McIlveen-Wright, D. R.; McMullan, J. T.; Guiney, D. J.

    of selected buildings in rural areas, with regard to the high cost of importing other fuel, and/or lack of grid electricity, could still make these systems attractive options. Any economic analysis of these systems is beset with severe difficulties. Capital costs of the major system components are not known with any great precision. However, a guideline assessment of the payback period for such CHP systems was made. When the best available capital costs for system components were used, most of these systems were found to have unacceptably long payback periods, particularly where the fuel cell lifetimes are short, but the larger systems show the potential for a reasonable economic return.

  20. 21 CFR 866.6020 - Immunomagnetic circulating cancer cell selection and enumeration system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Immunomagnetic circulating cancer cell selection... Associated Antigen immunological Test Systems § 866.6020 Immunomagnetic circulating cancer cell selection and enumeration system. (a) Identification. An immunomagnetic circulating cancer cell selection and...

  1. 21 CFR 866.6020 - Immunomagnetic circulating cancer cell selection and enumeration system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Immunomagnetic circulating cancer cell selection... Associated Antigen immunological Test Systems § 866.6020 Immunomagnetic circulating cancer cell selection and enumeration system. (a) Identification. An immunomagnetic circulating cancer cell selection and...

  2. 21 CFR 866.6020 - Immunomagnetic circulating cancer cell selection and enumeration system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Immunomagnetic circulating cancer cell selection... Associated Antigen immunological Test Systems § 866.6020 Immunomagnetic circulating cancer cell selection and enumeration system. (a) Identification. An immunomagnetic circulating cancer cell selection and...

  3. 21 CFR 866.6020 - Immunomagnetic circulating cancer cell selection and enumeration system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Immunomagnetic circulating cancer cell selection... Associated Antigen immunological Test Systems § 866.6020 Immunomagnetic circulating cancer cell selection and enumeration system. (a) Identification. An immunomagnetic circulating cancer cell selection and...

  4. 21 CFR 866.6020 - Immunomagnetic circulating cancer cell selection and enumeration system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Immunomagnetic circulating cancer cell selection... Associated Antigen immunological Test Systems § 866.6020 Immunomagnetic circulating cancer cell selection and enumeration system. (a) Identification. An immunomagnetic circulating cancer cell selection and...

  5. A rapid and selective assay for measuring cell surface hydrophobicity of brewer's yeast cells.

    PubMed

    Straver, M H; Kijne, J W

    1996-03-15

    A rapid and selective assay was developed to measure cell surface hydrophobicity of brewer's yeast cells. During this so-called magnobead assay, bottom-fermenting yeast cells adhere to paramagnetic, polystyrene-coated latex beads which can easily be removed from the cell suspension by using a (samarium-cobalt) magnet. At pH 4 center dot 5, electrostatic repulsion between yeast cells and latex beads was found to be minimal and yeast cell adhesion was predominantly based on hydrophobic interactions. The percentage of cells adhering to the beads could be calculated and provided a measure for cell surface hydrophobicity. Cell surface hydrophobicity measured by the magnobead assay was found to yield similar results, as did determination of contact angles of water droplets on a layer of yeast cells, a standard method for measuring surface hydrophobicity. However, the magnobead assay has the following advantages: (i) it is a quick and simple method, and, more significantly, (ii) hydrophobicity can be measured under physiological conditions. Use of the magnobead assay confirmed that a higher level of cell surface hydrophobicity is correlated with stronger flocculence of brewer's lager yeast cells. PMID:8904332

  6. A Novel Selectable Islet 1 Positive Progenitor Cell Reprogrammed to Expandable and Functional Smooth Muscle Cells.

    PubMed

    Turner, Elizabeth C; Huang, Chien-Ling; Sawhney, Neha; Govindarajan, Kalaimathi; Clover, Anthony J P; Martin, Kenneth; Browne, Tara C; Whelan, Derek; Kumar, Arun H S; Mackrill, John J; Wang, Shaohua; Schmeckpeper, Jeffrey; Stocca, Alessia; Pierce, William G; Leblond, Anne-Laure; Cai, Liquan; O'Sullivan, Donnchadh M; Buneker, Chirlei K; Choi, Janet; MacSharry, John; Ikeda, Yasuhiro; Russell, Stephen J; Caplice, Noel M

    2016-05-01

    Disorders affecting smooth muscle structure/function may require technologies that can generate large scale, differentiated and contractile smooth muscle cells (SMC) suitable for cell therapy. To date no clonal precursor population that provides large numbers of differentiated SMC in culture has been identified in a rodent. Identification of such cells may also enhance insight into progenitor cell fate decisions and the relationship between smooth muscle precursors and disease states that implicate differentiated SMC.  In this study, we used classic clonal expansion techniques to identify novel self-renewing Islet 1 (Isl-1) positive primitive progenitor cells (PPC) within rat bone marrow that exhibited canonical stem cell markers and preferential differentiation towards a smooth muscle-like fate. We subsequently used molecular tagging to select Isl-1 positive clonal populations from expanded and de novo marrow cell populations. We refer to these previously undescribed cells as the PPC given its stem cell marker profile, and robust self-renewal capacity. PPC could be directly converted into induced smooth muscle cells (iSMC) using single transcription factor (Kruppel-like factor 4) knockdown or transactivator (myocardin) overexpression in contrast to three control cells (HEK 293, endothelial cells and mesenchymal stem cells) where such induction was not possible. iSMC exhibited immuno- and cytoskeletal-phenotype, calcium signaling profile and contractile responses similar to bona fide SMC. Passaged iSMC could be expanded to a scale sufficient for large scale tissue replacement.  PPC and reprogramed iSMC so derived may offer future opportunities to investigate molecular, structure/function and cell-based replacement therapy approaches to diverse cardiovascular, respiratory, gastrointestinal, and genitourinary diseases that have as their basis smooth muscle cell functional aberrancy or numerical loss. Stem Cells 2016;34:1354-1368. PMID:26840832

  7. Neisseria lactamica selectively induces mitogenic proliferation of the naive B cell pool via cell surface Ig.

    PubMed

    Vaughan, Andrew T; Brackenbury, Louise S; Massari, Paola; Davenport, Victoria; Gorringe, Andrew; Heyderman, Robert S; Williams, Neil A

    2010-09-15

    Neisseria lactamica is a commensal bacteria that colonizes the human upper respiratory tract mucosa during early childhood. In contrast to the closely related opportunistic pathogen Neisseria meningitidis, there is an absence of adaptive cell-mediated immunity to N. lactamica during the peak age of carriage. Instead, outer membrane vesicles derived from N. lactamica mediate a B cell-dependent proliferative response in mucosal mononuclear cells that is associated with the production of polyclonal IgM. We demonstrate in this study that this is a mitogenic human B cell response that occurs independently of T cell help and any other accessory cell population. The ability to drive B cell proliferation is a highly conserved property and is present in N. lactamica strains derived from diverse clonal complexes. CFSE staining of purified human tonsillar B cells demonstrated that naive IgD(+) and CD27(-) B cells are selectively induced to proliferate by outer membrane vesicles, including the innate CD5(+) subset. Neither purified lipooligosaccharide nor PorB from N. lactamica is likely to be responsible for this activity. Prior treatment of B cells with pronase to remove cell-surface Ig or treatment with BCR-specific Abs abrogated the proliferative response to N. lactamica outer membrane vesicles, suggesting that this mitogenic response is dependent upon the BCR. PMID:20709949

  8. Targeted erythropoietin selectively stimulates red blood cell expansion in vivo.

    PubMed

    Burrill, Devin R; Vernet, Andyna; Collins, James J; Silver, Pamela A; Way, Jeffrey C

    2016-05-10

    The design of cell-targeted protein therapeutics can be informed by natural protein-protein interactions that use cooperative physical contacts to achieve cell type specificity. Here we applied this approach in vivo to the anemia drug erythropoietin (EPO), to direct its activity to EPO receptors (EPO-Rs) on red blood cell (RBC) precursors and prevent interaction with EPO-Rs on nonerythroid cells, such as platelets. Our engineered EPO molecule was mutated to weaken its affinity for EPO-R, but its avidity for RBC precursors was rescued via tethering to an antibody fragment that specifically binds the human RBC marker glycophorin A (huGYPA). We systematically tested the impact of these engineering steps on in vivo markers of efficacy, side effects, and pharmacokinetics. huGYPA transgenic mice dosed with targeted EPO exhibited elevated RBC levels, with only minimal platelet effects. This in vivo selectivity depended on the weakening EPO mutation, fusion to the RBC-specific antibody, and expression of huGYPA. The terminal plasma half-life of targeted EPO was ∼28.3 h in transgenic mice vs. ∼15.5 h in nontransgenic mice, indicating that huGYPA on mature RBCs acted as a significant drug sink but did not inhibit efficacy. In a therapeutic context, our targeting approach may allow higher restorative doses of EPO without platelet-mediated side effects, and also may improve drug pharmacokinetics. These results demonstrate how rational drug design can improve in vivo specificity, with potential application to diverse protein therapeutics. PMID:27114509

  9. Single-cell Migration Chip for Chemotaxis-based Microfluidic Selection of Heterogeneous Cell Populations

    NASA Astrophysics Data System (ADS)

    Chen, Yu-Chih; Allen, Steven G.; Ingram, Patrick N.; Buckanovich, Ronald; Merajver, Sofia D.; Yoon, Euisik

    2015-05-01

    Tumor cell migration toward and intravasation into capillaries is an early and key event in cancer metastasis, yet not all cancer cells are imbued with the same capability to do so. This heterogeneity within a tumor is a fundamental property of cancer. Tools to help us understand what molecular characteristics allow a certain subpopulation of cells to spread from the primary tumor are thus critical for overcoming metastasis. Conventional in vitro migration platforms treat populations in aggregate, which leads to a masking of intrinsic differences among cells. Some migration assays reported recently have single-cell resolution, but these platforms do not provide for selective retrieval of the distinct migrating and non-migrating cell populations for further analysis. Thus, to study the intrinsic differences in cells responsible for chemotactic heterogeneity, we developed a single-cell migration platform so that individual cells’ migration behavior can be studied and the heterogeneous population sorted based upon chemotactic phenotype. Furthermore, after migration, the highly chemotactic and non-chemotactic cells were retrieved and proved viable for later molecular analysis of their differences. Moreover, we modified the migration channel to resemble lymphatic capillaries to better understand how certain cancer cells are able to move through geometrically confining spaces.

  10. An Electrochemical Cell for Selective Lithium Capture from Seawater.

    PubMed

    Kim, Joo-Seong; Lee, Yong-Hee; Choi, Seungyeon; Shin, Jaeho; Dinh, Hung-Cuong; Choi, Jang Wook

    2015-08-18

    Lithium (Li) is a core element of Li-ion batteries (LIBs). Recent developments in mobile electronics such as smartphones and tablet PCs as well as advent of large-scale LIB applications including electrical vehicles and grid-level energy storage systems have led to an increase in demand for LIBs, giving rise to a concern on the availability and market price of Li resources. However, the current Lime-Soda process that is responsible for greater than 80% of worldwide Li resource supply is applicable only in certain regions on earth where the Li concentrations are sufficiently high (salt lakes or salt pans). Moreover, not only is the process time-consuming (12-18 months), but post-treatments are also required for the purification of Li. Here, we have devised a location-independent electrochemical system for Li capture, which can operate within a short time period (a few hours to days). By engaging olivine LiFePO4 active electrode that improves interfacial properties via polydopamine coating, the electrochemical cell achieves 4330 times amplification in Li/Na ion selectivity (Li/Na molar ratio of initial solution = 0.01 and Li/Na molar ratio of final electrode = 43.3). In addition, the electrochemical system engages an I(-)/I3(-) redox couple in the other electrode for balancing of the redox states on both electrode sides and sustainable operations of the entire cell. Based on the electrochemical results, key material and interfacial properties that affect the selectivity in Li capture are identified. PMID:25920476

  11. Hedgehog inhibitors selectively target cell migration and adhesion of mantle cell lymphoma in bone marrow microenvironment

    PubMed Central

    Zhang, Han; Chen, Zheng; Neelapu, Sattva S.; Romaguera, Jorge; McCarty, Nami

    2016-01-01

    The clinical benefits of a Hedgehog (Hh) inhibitor, LDE225 (NPV-LDE-225, Erismodegib), have been unclear in hematological cancers. Here, we report that LDE225 selectively inhibited migration and adhesion of mantle cell lymphoma (MCL) to bone marrows via very late antigen-4 (VLA-4) mediated inactivation of focal adhesion kinase (FAK) signaling. LDE225 treatment not only affected MCL cells, but also modulated stromal cells within the bone marrow microenvironment by decreasing their production of SDF-1, IL-6 and VCAM-1, the ligand for VLA-4. Surprisingly, LDE225 treatment alone did not suppress cell proliferation due to increased CXCR4 expression mediated by reactive oxygen species (ROS). The increased ROS/CXCR4 further stimulated autophagy formation. The combination of LDE225 with the autophagy inhibitors further enhanced MCL cell death. Our data, for the first time, revealed LDE225 selectively targets MCL cells migration and adhesion to bone marrows. The ineffectiveness of LDE225 in MCL is due to autophagy formation, which in turn increases cell viability. Inhibiting autophagy will be an effective adjuvant therapy for LDE225 in MCL, especially for advanced MCL patients with bone marrow involvement. PMID:26885608

  12. Hedgehog inhibitors selectively target cell migration and adhesion of mantle cell lymphoma in bone marrow microenvironment.

    PubMed

    Zhang, Han; Chen, Zheng; Neelapu, Sattva S; Romaguera, Jorge; McCarty, Nami

    2016-03-22

    The clinical benefits of a Hedgehog (Hh) inhibitor, LDE225 (NPV-LDE-225, Erismodegib), have been unclear in hematological cancers. Here, we report that LDE225 selectively inhibited migration and adhesion of mantle cell lymphoma (MCL) to bone marrows via very late antigen-4 (VLA-4) mediated inactivation of focal adhesion kinase (FAK) signaling. LDE225 treatment not only affected MCL cells, but also modulated stromal cells within the bone marrow microenvironment by decreasing their production of SDF-1, IL-6 and VCAM-1, the ligand for VLA-4. Surprisingly, LDE225 treatment alone did not suppress cell proliferation due to increased CXCR4 expression mediated by reactive oxygen species (ROS). The increased ROS/CXCR4 further stimulated autophagy formation. The combination of LDE225 with the autophagy inhibitors further enhanced MCL cell death. Our data, for the first time, revealed LDE225 selectively targets MCL cells migration and adhesion to bone marrows. The ineffectiveness of LDE225 in MCL is due to autophagy formation, which in turn increases cell viability. Inhibiting autophagy will be an effective adjuvant therapy for LDE225 in MCL, especially for advanced MCL patients with bone marrow involvement. PMID:26885608

  13. Repair of Ischemic Injury by Pluripotent Stem Cell Based Cell Therapy without Teratoma through Selective Photosensitivity.

    PubMed

    Cho, Seung-Ju; Kim, So-Yeon; Jeong, Ho-Chang; Cheong, Hyeonsik; Kim, Doseok; Park, Soon-Jung; Choi, Jong-Jin; Kim, Hyongbum; Chung, Hyung-Min; Moon, Sung-Hwan; Cha, Hyuk-Jin

    2015-12-01

    Stem-toxic small molecules have been developed to induce selective cell death of pluripotent stem cells (PSCs) to lower the risk of teratoma formation. However, despite their high efficacies, chemical-based approaches may carry unexpected toxicities on specific differentiated cell types. Herein, we took advantage of KillerRed (KR) as a suicide gene, to selectively induce phototoxicity using visible light via the production of reactive oxygen species. PSCs in an undifferentiated state that exclusively expressed KR (KR-PSCs) were eliminated by a single exposure to visible light. This highly selective cell death in KR-PSCs was exploited to successfully inhibit teratoma formation. In particular, endothelial cells from KR-mPSCs remained fully functional in vitro and sufficient to repair ischemic injury in vivo regardless of light exposure, suggesting that a genetic approach in which KR is expressed in a tightly controlled manner would be a viable strategy to inhibit teratoma formation for future safe PSC-based therapies. PMID:26584542

  14. Repair of Ischemic Injury by Pluripotent Stem Cell Based Cell Therapy without Teratoma through Selective Photosensitivity

    PubMed Central

    Cho, Seung-Ju; Kim, So-Yeon; Jeong, Ho-Chang; Cheong, Hyeonsik; Kim, Doseok; Park, Soon-Jung; Choi, Jong-Jin; Kim, Hyongbum; Chung, Hyung-Min; Moon, Sung-Hwan; Cha, Hyuk-Jin

    2015-01-01

    Summary Stem-toxic small molecules have been developed to induce selective cell death of pluripotent stem cells (PSCs) to lower the risk of teratoma formation. However, despite their high efficacies, chemical-based approaches may carry unexpected toxicities on specific differentiated cell types. Herein, we took advantage of KillerRed (KR) as a suicide gene, to selectively induce phototoxicity using visible light via the production of reactive oxygen species. PSCs in an undifferentiated state that exclusively expressed KR (KR-PSCs) were eliminated by a single exposure to visible light. This highly selective cell death in KR-PSCs was exploited to successfully inhibit teratoma formation. In particular, endothelial cells from KR-mPSCs remained fully functional in vitro and sufficient to repair ischemic injury in vivo regardless of light exposure, suggesting that a genetic approach in which KR is expressed in a tightly controlled manner would be a viable strategy to inhibit teratoma formation for future safe PSC-based therapies. PMID:26584542

  15. Curcumin and cancer cells: how many ways can curry kill tumor cells selectively?

    PubMed

    Ravindran, Jayaraj; Prasad, Sahdeo; Aggarwal, Bharat B

    2009-09-01

    Cancer is a hyperproliferative disorder that is usually treated by chemotherapeutic agents that are toxic not only to tumor cells but also to normal cells, so these agents produce major side effects. In addition, these agents are highly expensive and thus not affordable for most. Moreover, such agents cannot be used for cancer prevention. Traditional medicines are generally free of the deleterious side effects and usually inexpensive. Curcumin, a component of turmeric (Curcuma longa), is one such agent that is safe, affordable, and efficacious. How curcumin kills tumor cells is the focus of this review. We show that curcumin modulates growth of tumor cells through regulation of multiple cell signaling pathways including cell proliferation pathway (cyclin D1, c-myc), cell survival pathway (Bcl-2, Bcl-xL, cFLIP, XIAP, c-IAP1), caspase activation pathway (caspase-8, 3, 9), tumor suppressor pathway (p53, p21) death receptor pathway (DR4, DR5), mitochondrial pathways, and protein kinase pathway (JNK, Akt, and AMPK). How curcumin selectively kills tumor cells, and not normal cells, is also described in detail. PMID:19590964

  16. A PCNA-Derived Cell Permeable Peptide Selectively Inhibits Neuroblastoma Cell Growth

    PubMed Central

    Gu, Long; Smith, Shanna; Li, Caroline; Hickey, Robert J.; Stark, Jeremy M.; Fields, Gregg B.; Lang, Walter H.; Sandoval, John A.; Malkas, Linda H.

    2014-01-01

    Proliferating cell nuclear antigen (PCNA), through its interaction with various proteins involved in DNA synthesis, cell cycle regulation, and DNA repair, plays a central role in maintaining genome stability. We previously reported a novel cancer associated PCNA isoform (dubbed caPCNA), which was significantly expressed in a broad range of cancer cells and tumor tissues, but not in non-malignant cells. We found that the caPCNA-specific antigenic site lies between L126 and Y133, a region within the interconnector domain of PCNA that is known to be a major binding site for many of PCNA's interacting proteins. We hypothesized that therapeutic agents targeting protein-protein interactions mediated through this region may confer differential toxicity to normal and malignant cells. To test this hypothesis, we designed a cell permeable peptide containing the PCNA L126-Y133 sequence. Here, we report that this peptide selectively kills human neuroblastoma cells, especially those with MYCN gene amplification, with much less toxicity to non-malignant human cells. Mechanistically, the peptide is able to block PCNA interactions in cancer cells. It interferes with DNA synthesis and homologous recombination-mediated double-stranded DNA break repair, resulting in S-phase arrest, accumulation of DNA damage, and enhanced sensitivity to cisplatin. These results demonstrate conceptually the utility of this peptide for treating neuroblastomas, particularly, the unfavorable MYCN-amplified tumors. PMID:24728180

  17. Microselection – affinity selecting antibodies against a single rare cell in a heterogeneous population

    PubMed Central

    Sørensen, Morten Dræby; Agerholm, Inge Errebo; Christensen, Britta; Kølvraa, Steen; Kristensen, Peter

    2010-01-01

    Abstract Rare cells not normally present in the peripheral bloodstream, such as circulating tumour cells, have potential applications for development of non-invasive methods for diagnostics or follow up. Obtaining these cells however require some means of discrimination, achievable by cell type specific antibodies. Here we have generated a microselection method allowing antibody selection, by phage display, targeting a single cell in a heterogeneous population. One K562 cell (female origin) was positioned on glass slide among millions of lymphocytes from male donor, identifying the K562 cell by FISH (XX). Several single cell selections were performed on such individual slides. The phage particles bound to the target cell is protected by a minute disc, while inactivating all remaining phage by UV-irradiation; leaving only the phage bound to the target cell viable. We hereby retrieved up to eight antibodies per single cell selection, including three highly K562 cell type specific. PMID:20726925

  18. Intraclonal Cell Expansion and Selection Driven by B Cell Receptor in Chronic Lymphocytic Leukemia

    PubMed Central

    Colombo, Monica; Cutrona, Giovanna; Reverberi, Daniele; Fabris, Sonia; Neri, Antonino; Fabbi, Marina; Quintana, Giovanni; Quarta, Giovanni; Ghiotto, Fabio; Fais, Franco; Ferrarini, Manlio

    2011-01-01

    The mutational status of the immunoglobulin heavy-chain variable region (IGHV) genes utilized by chronic lymphocytic leukemia (CLL) clones defines two disease subgroups. Patients with unmutated IGHV have a more aggressive disease and a worse outcome than patients with cells having somatic IGHV gene mutations. Moreover, up to 30% of the unmutated CLL clones exhibit very similar or identical B cell receptors (BcR), often encoded by the same IG genes. These “stereotyped” BcRs have been classified into defined subsets. The presence of an IGHV gene somatic mutation and the utilization of a skewed gene repertoire compared with normal B cells together with the expression of stereotyped receptors by unmutated CLL clones may indicate stimulation/selection by antigenic epitopes. This antigenic stimulation may occur prior to or during neoplastic transformation, but it is unknown whether this stimulation/selection continues after leukemogenesis has ceased. In this study, we focused on seven CLL cases with stereotyped BcR Subset #8 found among a cohort of 700 patients; in six, the cells expressed IgG and utilized IGHV4-39 and IGKV1-39/IGKV1D-39 genes, as reported for Subset #8 BcR. One case exhibited special features, including expression of IgM or IgG by different subclones consequent to an isotype switch, allelic inclusion at the IGH locus in the IgM-expressing cells and a particular pattern of cytogenetic lesions. Collectively, the data indicate a process of antigenic stimulation/selection of the fully transformed CLL cells leading to the expansion of the Subset #8 IgG-bearing subclone. PMID:21541442

  19. Cytomegalovirus Infection after CD34(+)-Selected Hematopoietic Cell Transplantation.

    PubMed

    Huang, Yao-Ting; Neofytos, Dionysios; Foldi, Julia; Kim, Seong Jin; Maloy, Molly; Chung, Dick; Castro-Malaspina, Hugo; Giralt, Sergio A; Papadopoulos, Esperanza; Perales, Miguel-Angel; Jakubowski, Ann A; Papanicolaou, Genovefa A

    2016-08-01

    The effectiveness of preemptive treatment (PET) for cytomegalovirus (CMV) in recipients of ex vivo T cell-depleted (TCD) hematopoietic cell transplantation (HCT) by CD34(+) selection is not well defined. We analyzed 213 adults who received TCD-HCT at our institution from June 2010 through May 2014. Patients were monitored by a CMV quantitative PCR assay if recipient (R) or donor (D) were CMV seropositive. CMV viremia occurred early (median, 27 days after HCT) in 91 of 213 (42.7%) patients for a 180-day cumulative incidence of 84.5%, 61.8%, and 0 for R+/D+, R+/D-, and R-/D+ patients, respectively. CMV disease occurred in 5% of patients. In Cox regression analysis, R+/D+ status was associated with increased risk for CMV viremia compared with R+/D- (hazard ratio [HR], 1.79, 95% confidence interval [CI], 1.16 to 2.76, P = .01), whereas matched unrelated donor allograft was associated with decreased risk (HR, .62; 95% CI, .39 to .97, P = .04). Of 91 patients with CMV viremia, 52 (57%) had persistent viremia (>28 days duration). Time lag from detection of CMV viremia to PET was associated with incremental risk for persistent viremia (HR, 1.09; 95% CI, 1.01 to 1.18; P = .03). Overall, 166 of 213 (77.9%) patients were alive 1 year after HCT, with no difference between patients with and without CMV viremia or among the different CMV serostatus pairs (P = not significant). CMV viremia occurred in 70% of R + TCD-HCT. Delay in PET initiation was associated with persistent viremia. With PET, CMV R/D serostatus did not adversely impact survival in TCD-HCT on 1-year survival in the present cohort. PMID:27178374

  20. Genomic instability, driver genes and cell selection: Projections from cancer to stem cells.

    PubMed

    Ben-David, Uri

    2015-04-01

    Cancer cells and stem cells share many traits, including a tendency towards genomic instability. Human cancers exhibit tumor-specific genomic aberrations, which often affect their malignancy and drug response. During their culture propagation, human pluripotent stem cells (hPSCs) also acquire characteristic genomic aberrations, which may have significant impact on their molecular and cellular phenotypes. These aberrations vary in size from single nucleotide alterations to copy number alterations to whole chromosome gains. A prominent challenge in both cancer and stem cell research is to identify "driver aberrations" that confer a selection advantage, and "driver genes" that underlie the recurrence of these aberrations. Following principles that are already well-established in cancer research, candidate driver genes have also been suggested in hPSCs. Experimental validation of the functional role of such candidates can uncover whether these are bona fide driver genes. The identification of driver genes may bring us closer to a mechanistic understanding of the genomic instability of stem cells. Guided by terminologies and methodologies commonly applied in cancer research, such understanding may have important ramifications for both stem cell and cancer biology. This article is part of a Special Issue entitled: Stress as a fundamental theme in cell plasticity. PMID:25132386

  1. Selection of insulin-producing rat insulinoma (RINm) cells with improved resistance to oxidative stress.

    PubMed

    Bloch, Konstantin O; Vorobeychik, Marina; Yavrians, Karina; Vardi, Pnina

    2003-06-01

    The defense system against reactive oxygen species is believed to be crucial for the survival of insulin-producing cells after various injuries. The aim of our study was to select a subpopulation of insulin-producing RINm cells with higher resistance to oxidative stress. The cells resistant to hydrogen peroxide (RINmHP) were obtained by repeated exposure of parental RINm cells to 100 and 200 microM hydrogen peroxide (HP). The increased resistance of RINmHP cells to HP was confirmed by three different cytotoxicity assays. In addition, the selected cells also were resistant to the cytotoxic effect of activated rat splenocytes compared to parental cells. The half-life of HP in the RINmHP cell culture medium was about 2.5 times lower than that of the parental cells, corresponding to the increased level of catalase expression and activity in selected cells. The increased defense property of the selected cells was not associated with any significant changes in insulin content and insulin response to a mixture of glucose with isobutyl methyl xanthine or potassium chloride. In conclusion, repeated exposure to HP induces selection of RINm cells with improved resistance to oxidative stress. This improved defense characteristic probably is due to an increased level of catalase expression and activity in the selected cells. PMID:12781331

  2. Depolarization Controls TRAIL-Sensitization and Tumor-Selective Killing of Cancer Cells: Crosstalk with ROS

    PubMed Central

    Suzuki-Karasaki, Yoshihiro; Suzuki-Karasaki, Miki; Uchida, Mayumi; Ochiai, Toyoko

    2014-01-01

    Conventional genotoxic anti-cancer drugs target the proliferative advantage of tumor cells over normal cells. This kind of approach lacks the selectivity of treatment to cancer cells, because most of the targeted pathways are essential for the survival of normal cells. As a result, traditional cancer treatments are often limited by undesirable damage to normal cells (side-effects). Ideal anti-cancer drugs are expected to be highly effective against malignant tumor cells with minimal cytotoxicity toward normal cells. Such selective killing can be achieved by targeting pathways essential for the survival of cancer cells, but not normal cells. As cancer cells are characterized by their resistance to apoptosis, selective apoptosis induction is a promising approach for selective killing of cancer cells. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising tumor-selective anti-cancer drug. However, the congenital and acquired resistance of some cancer cell types, including malignant melanoma cells, currently impedes effective TRAIL therapy, and an innovative approach that can override TRAIL resistance is urgently required. Apoptosis is characterized by cell shrinkage caused by disruption of the maintenance of the normal physiological concentrations of K+ and Na+ and intracellular ion homeostasis. The disrupted ion homeostasis leads to depolarization and apoptosis. Recent evidence suggests that depolarization is an early and prerequisite event during TRAIL-induced apoptosis. Moreover, diverse natural products and synthetic chemicals capable of depolarizing the cell membrane exhibit tumor-selective killing and TRAIL-sensitizing effects. Here, we discuss the role of depolarization in selective killing of cancer cells in connection with the emerging concept that oxidative stress is a critical mediator of mitochondrial and endoplasmic reticulum dysfunctions and serves as a tumor-selective target in cancer treatment. PMID:24910845

  3. Mechanosensory calcium-selective cation channels in epidermal cells

    NASA Technical Reports Server (NTRS)

    Ding, J. P.; Pickard, B. G.

    1993-01-01

    This paper explores the properties and likely functions of an epidermal Ca(2+)-selective cation channel complex activated by tension. As many as eight or nine linked or linkable equivalent conductance units or co-channels can open together. Open time for co-channel quadruplets and quintuplets tends to be relatively long with millimolar Mg2+ (but not millimolar Ca2+) at the cytosolic face of excised plasma membrane. Sensitivity to tension is regulated by transmembrane voltage and temperature. Under some circumstances channel activity is sychronized in rhythmic pulses. Certain lanthanides and a cytoskeleton-disturbing herbicide that inhibit gravitropic reception act on the channel system at low concentrations. Specifically, ethyl-N-phenylcarbamate promotes tension-dependent activity at micromolar levels. With moderate suction, Gd3+ provided at about 0.5 micromole at the extracellular face of the membrane promotes for several seconds but may then become inhibitory. Provision at 1-2 micromoles promotes and subsequently inhibits more vigorously (often abruptly and totally), and at high levels inhibits immediately. La3+, a poor gravitropic inhibitor, acts similarly but much more gradually and only at much higher concentrations. These properties, particularly these susceptibilities to modulation, indicate that in vivo the mechanosensitive channel must be mechanosensory and mechanoregulatory. It could serve to transduce the shear forces generated in the integrated wall-membrane-cytoskeleton system during turgor changes and cell expansion as well as transducing the stresses induced by gravity, touch and flexure. In so far as such transduction is modulated by voltage and temperature, the channels would also be sensors for these modalities as long as the wall-membrane-cytoskeleton system experiences mechanical stress.

  4. Photostick: a method for selective isolation of target cells from culture

    PubMed Central

    Chien, Miao-Ping; Werley, Christopher A.; Farhi, Samouil L.

    2015-01-01

    Sorting of target cells from a heterogeneous pool is technically difficult when the selection criterion is complex, e.g. a dynamic response, a morphological feature, or a combination of multiple parameters. At present, mammalian cell selections are typically performed either via static fluorescence (e.g. fluorescence activated cell sorter), via survival (e.g. antibiotic resistance), or via serial operations (flow cytometry, laser capture microdissection). Here we present a simple protocol for selecting cells based on any static or dynamic property that can be identified by video microscopy and image processing. The “photostick” technique uses a cell-impermeant photochemical crosslinker and digital micromirror array-based patterned illumination to immobilize selected cells on the culture dish. Other cells are washed away with mild protease treatment. The crosslinker also labels the selected cells with a fluorescent dye and a biotin for later identification. The photostick protocol preserves cell viability, permits genetic profiling of selected cells, and can be performed with complex functional selection criteria such as neuronal firing patterns. PMID:25705368

  5. Selective activation of functional suppressor cells by human seminal fluid.

    PubMed Central

    Witkin, S S

    1986-01-01

    The ability of seminal fluid (SF) to induce suppressor cell activity from peripheral blood mononuclear cells (PBMN) was examined. PBMN were incubated with SF for 48 h, washed to remove SF components, treated with mitomycin C (mit C) and co-cultured with Raji cells, a lymphoblastoid cell line. Raji cell proliferation was inhibited by SF-treated PBMN proportionally to SF concentration. SF (50-200 micrograms), mit C-treated Raji cells or mit C-treated PBMN pre-incubated with phytohaemagglutinin were without effect on Raji cell growth. Suppressor T lymphocytes generated by incubation of PBMN with concanavalin A inhibited Raji cells to the same extent as did SF-treated PBMN. All activity was lost following heating at 56 degrees C for 30 min; freezing and thawing reduced the ability of SF to induce suppression by 50%. Dialysis of SF or treatment with antibody to prostaglandin E2 led to a 50% reduction in suppression. PMID:2943541

  6. Evolution and phenotypic selection of cancer stem cells.

    PubMed

    Poleszczuk, Jan; Hahnfeldt, Philip; Enderling, Heiko

    2015-03-01

    Cells of different organs at different ages have an intrinsic set of kinetics that dictates their behavior. Transformation into cancer cells will inherit these kinetics that determine initial cell and tumor population progression dynamics. Subject to genetic mutation and epigenetic alterations, cancer cell kinetics can change, and favorable alterations that increase cellular fitness will manifest themselves and accelerate tumor progression. We set out to investigate the emerging intratumoral heterogeneity and to determine the evolutionary trajectories of the combination of cell-intrinsic kinetics that yield aggressive tumor growth. We develop a cellular automaton model that tracks the temporal evolution of the malignant subpopulation of so-called cancer stem cells(CSC), as these cells are exclusively able to initiate and sustain tumors. We explore orthogonal cell traits, including cell migration to facilitate invasion, spontaneous cell death due to genetic drift after accumulation of irreversible deleterious mutations, symmetric cancer stem cell division that increases the cancer stem cell pool, and telomere length and erosion as a mitotic counter for inherited non-stem cancer cell proliferation potential. Our study suggests that cell proliferation potential is the strongest modulator of tumor growth. Early increase in proliferation potential yields larger populations of non-stem cancer cells(CC) that compete with CSC and thus inhibit CSC division while a reduction in proliferation potential loosens such inhibition and facilitates frequent CSC division. The sub-population of cancer stem cells in itself becomes highly heterogeneous dictating population level dynamics that vary from long-term dormancy to aggressive progression. Our study suggests that the clonal diversity that is captured in single tumor biopsy samples represents only a small proportion of the total number of phenotypes. PMID:25742563

  7. Deformability-based cell selection with downstream immunofluorescence analysis.

    PubMed

    Shaw Bagnall, Josephine; Byun, Sangwon; Miyamoto, David T; Kang, Joon Ho; Maheswaran, Shyamala; Stott, Shannon L; Toner, Mehmet; Manalis, Scott R

    2016-05-16

    Mechanical properties of single cells have been shown to relate to cell phenotype and malignancy. However, until recently, it has been difficult to directly correlate each cell's biophysical characteristics to its molecular traits. Here, we present a cell sorting technique for use with a suspended microchannel resonator (SMR), which can measure biophysical characteristics of a single cell based on the sensor's record of its buoyant mass as well as its precise position while it traverses through a constricted microfluidic channel. The measurement provides information regarding the amount of time a cell takes to pass through a constriction (passage time), as related to the cell's deformability and surface friction, as well as the particular manner in which it passes through. In the method presented here, cells of interest are determined based on passage time, and are collected off-chip for downstream immunofluorescence imaging. The biophysical single-cell SMR measurement can then be correlated to the molecular expression of the collected cell. This proof-of-principle is demonstrated by sorting and collecting tumor cells from cell line-spiked blood samples as well as a metastatic prostate cancer patient blood sample, identifying them by their surface protein expression and relating them to distinct SMR signal trajectories. PMID:26999591

  8. Rhodamine-123 Selectively Reduces Clonal Growth of Carcinoma Cells in vitro

    NASA Astrophysics Data System (ADS)

    1982-12-01

    Rhodamine-123, a cationic laser dye, markedly reduced the clonal growth of carcinoma cells but had little effect on nontumorigenic epithelial cells in vitro. This selective inhibitory effect of Rhodamine-123 on some carcinomas is unusual since known anticancer drugs, such as arabinosyl cytosine and methotrexate, have not been shown to exhibit such selectivity in vitro.

  9. Isolation of melanoma cell subpopulations using negative selection

    PubMed Central

    Slipicevic, Ana; Somasundaram, Rajasekharan; Sproesser, Katrin; Herlyn, Meenhard

    2014-01-01

    Melanomas are phenotypically and functiwonally heterogeneous tumors comprising of distinct subpopulations that drive disease progression and are responsible for resistance to therapy. Identification and characterization of such subpopulations are highly important to develop novel targeted therapies. However, this can be a challenging task as there is a lack of clearly defined markers to distinguish the melanoma subpopulations from a general tumor cell population. Also, there is a lack of optimal isolation methods and functional assays that can fully recapitulate their phenotype. Here we describe a method for isolating tumor cells from fresh human tumor tissue specimens using an antibody coupled magnetic bead sorting technique that is well established in our laboratory. Thus, melanoma cells are enriched by negative cell sorting and elimination of non-tumor cell population such as erythrocytes, leukocytes, and endothelial cells. Enriched unmodified tumor cells can be further used for phenotypic and functional characterization of melanoma subpopulations. PMID:24258995

  10. New Strategies for Designing Inexpensive but Selective Bioadsorbants for Environmental Pollutants: Selection of specific Ligands and Their Cell Surface Expression

    SciTech Connect

    Brent L. Iverson; George Georgiou; Mohammad M. Ataai; Richard R. Koepsel

    2001-02-22

    The Broad, long term objective of the research plan is to develop exquisitely selective polypeptide metal chelators for the remediation of aqueous systems. A variety of polypeptide chelators will be developed and optimized ranging from antibodies to small peptides. Then, through unique molecular engineering approaches developed in our laboratories, the polypeptide chelators will be anchored directly on the surface of the cells that produce them. Thus, instead of using isolated biomolecules we will employ inexpensive genetically engineered whole cell adsorbents. Following a simple, easily scaleable treatment, the engineered cells can be used to manufacture an inexpensive, particulate adsorbent for metal removal.

  11. Selectivity of pyramidal cells and interneurons in the human medial temporal lobe

    PubMed Central

    Mormann, Florian; Cerf, Moran; Koch, Christof; Fried, Itzhak; Quiroga, Rodrigo Quian

    2011-01-01

    Neurons in the medial temporal lobe (MTL) respond selectively to pictures of specific individuals, objects, and places. However, the underlying mechanisms leading to such degree of stimulus selectivity are largely unknown. A necessary step to move forward in this direction involves the identification and characterization of the different neuron types present in MTL circuitry. We show that putative principal cells recorded in vivo from the human MTL are more selective than putative interneurons. Furthermore, we report that putative hippocampal pyramidal cells exhibit the highest degree of selectivity within the MTL, reflecting the hierarchical processing of visual information. We interpret these differences in selectivity as a plausible mechanism for generating sparse responses. PMID:21715671

  12. Apoptotic cells selectively uptake minor glycoforms of vitronectin from serum.

    PubMed

    Malagolini, Nadia; Catera, Mariangela; Osorio, Hugo; Reis, Celso A; Chiricolo, Mariella; Dall'Olio, Fabio

    2013-04-01

    Apoptosis profoundly alters the carbohydrate layer coating the membrane of eukaryotic cells. Previously we showed that apoptotic cells became reactive with the α2,6-sialyl-specific lectin from Sambucus nigra agglutinin (SNA), regardless of their histological origin and the nature of the apoptotic stimulus. Here we reveal the basis of the phenomenon by showing that in apoptotic cancer cell lines SNA reactivity was mainly associated with a 67 kDa glycoprotein which we identified by MALDI-TOF/TOF and immunoblot analysis as bovine vitronectin (bVN). bVN was neither present in non-apoptotic cells, nor in cells induced to apoptosis in serum-free medium, indicating that its uptake from the cell culture serum occurred only during apoptosis. The bVN molecules associated with apoptotic cancer cell lines represented minor isoforms, lacking the carboxyterminal sequence and paradoxically containing a few α2,6-linked sialic acid residues. Despite their poor α2,6-sialylation, these bVN molecules were sufficient to turn apoptotic cells to SNA reactivity, which is a late apoptotic event occurring in cells positive to both annexin-V and propidium iodide. Unlike in cancer cell lines, the major bVN form taken up by apoptotic neutrophils and mononuclear cells was a 80 kDa form. In apoptotic SW948 cells we also detected the α2,6-sialylated forms of the stress-70 mitochondrial precursor (mortalin) and of tubulin-β2C. These data indicate that the acquisition of vitronectin isoforms from the environment is a general, although cell specific phenomenon, potentially playing an important role in post-apoptotic events and that the α2,6-sialylation of intracellular proteins is a new kind of posttranslational modification associated with apoptosis. PMID:23381642

  13. Selective cytotoxicity of benzyl isothiocyanate in the proliferating fibroblastoid cells.

    PubMed

    Miyoshi, Noriyuki; Uchida, Koji; Osawa, Toshihiko; Nakamura, Yoshimasa

    2007-02-01

    In the present study, experiments using presynchronization culture cells demonstrated that benzyl ITC (BITC), previously isolated from a tropical papaya fruit extract, induced the cytotoxic effect preferentially in the proliferating human colon CCD-18Co cells to the quiescent ones. Quiescent CCD-18Co cells were virtually unaffected by BITC and marginal cytotoxicity was observed at 15 microM. We observed that BITC dramatically induced the p53 phosphorylation and stabilization only in the quiescent (G(0)/G(1) phase-arrested) cells, but not significantly in the proliferating human colon CCD-18Co cells when compared with quiescent ones. We also observed ataxia telangiectasia-mutated (ATM) phosphorylation in the quiescent cells. The BITC-induced p53 phosphorylation was counteracted by caffeine treatment, implying the involvement of an ATM/ataxia telangiectasia and Rad3-related kinase signaling pathway. Moreover, downregulation of p53 by a siRNA resulted in the enhancement of susceptibility to undergo apoptosis by BITC. We also showed here that depletion of p53 abrogated G(0)/G(1) arrest accompanied by the declined expression of p21(waf1/cip1) and p27(kip1) in CCD-18Co cells. In conclusion, we identified p53 as a potential negative regulator of the apoptosis induction by BITC in the normal colon CCD-18Co cells through the inhibition of cell-cycle progression at the G(0)/G(1) phase. PMID:17096346

  14. Bacterial IMPDH gene used for the selection of mammalian cell transfectants.

    SciTech Connect

    Baccam, M.; Huberman, E.; Energy Systems

    2003-06-01

    Stable cell transfection is used for the expression of exogenous genes or cDNAs in eukaryotic cells. Selection of these transfectants requires a dominant selectable marker. A variety of such markers has been identified and is currently in use. However, many of these are not suitable for all cell types or require unique conditions. Here we describe a simple and versatile dominant selectable marker that involves bacterial IMP dehydrogenase (IMPDH), an enzyme essential for the replication of mammalian and bacterial cells. Although IMPDH is evolutionarily conserved, the bacterial enzyme is orders of magnitude more resistant to the toxic effect of the drug mycophenolic acid, which is an IMPDH inhibitor. We have demonstrated that transfection of human, monkey or Chinese hamster cell lines with an expression vector containing bacterial IMPDH and mycophenolic acid treatment resulted in the selection of colonies with a strikingly increased resistance to mycophenolic acid toxicity. Analysis of cells derived from these colonies indicated that the acquisition of this resistance was associated with bacterial IMPDH protein expression. As a proof of principle, we showed that mammalian cell transfection with a hicistronic IMPDH/GFP expression vector and mycophenolic acid treatment can he used to successfully select transfectants that express the fluorescent protein. These results indicate that bacterial IMPDH is a practical dominant selectable marker that can be used for the selection of transfectants that express exogenous genes or cDNAs in mammalian cells.

  15. Selection for intrabody solubility in mammalian cells using GFP fusions.

    PubMed

    Guglielmi, Laurence; Denis, Vincent; Vezzio-Vié, Nadia; Bec, Nicole; Dariavach, Piona; Larroque, Christian; Martineau, Pierre

    2011-12-01

    Single-chain antibody fragments (scFv) expressed in the cytoplasm of mammalian cells, also called intrabodies, have many applications in functional proteomics. These applications are, however, limited by the aggregation-prone behaviour of many intrabodies. We show here that two scFv with highly homologous sequences and comparable soluble expression levels in Escherichia coli cytoplasm have different behaviours in mammalian cells. When over-expressed, one of the scFv aggregates in the cytoplasm whereas the second one is soluble and active. When expressed at low levels, using a retroviral vector, as a fusion with the green fluorescent protein (GFP) the former does not form aggregates and is degraded, resulting in weakly fluorescent cells, whereas the latter is expressed as a soluble protein, resulting in strongly fluorescent cells. These data suggest that the GFP signal can be used to evaluate the soluble expression of intrabodies in mammalian cells. When applied to a subset of an E.coli-optimised intrabody library, we showed that the population of GFP+ cells contains indeed soluble mammalian intrabodies. Altogether, our data demonstrate that the requirements for soluble intrabody expression are different in E.coli and mammalian cells, and that intrabody libraries can be directly optimised in human cells using a simple GFP-based assay. PMID:21997307

  16. Cell-selective encapsulation in hydrogel sheaths via biospecific identification and biochemical cross-linking.

    PubMed

    Sakai, Shinji; Liu, Yang; Sengoku, Mikako; Taya, Masahito

    2015-01-01

    Selective encapsulation of a particular cell population from heterogeneous cell populations has potential applications such as studies in cell-to-cell communication, regenerative medicine, and cell therapies. However, there are no versatile methods for realizing this. Here we report a method based on biospecific identification of the target cells through antigen-antibody reaction and subsequent enzymatic hydrogel sheath formation on the cell surfaces by horseradish peroxidase (HRP). Human hepatoma cell line HepG2 cells were selectively encapsulated in alginate-based hydrogel sheath from the mixture with mouse embryo fibroblast-like cell line 10T1/2 fibroblasts using anti-human CD326 antibody conjugated with HRP. The viability of the encapsulated cells was 93%. The cells released at 6 days of the encapsulation by degrading the sheath using alginate lyase grew almost the same as those free from encapsulation. The versatility of the method was confirmed using another antibody, cells, and hydrogel sheath material: Only human vein endothelial cells were encapsulated in gelatin-based hydrogel sheath from the mixture with 10T1/2 fibroblasts using anti-human CD31 antibody conjugated with HRP. The cell-selective encapsulation was also achieved by a system using a primary antibody with a secondary antibody conjugated with HRP. PMID:25890746

  17. Effect of selected flavones on cancer and endothelial cells.

    PubMed

    Pilátová, Martina; Stupáková, Viktória; Varinská, Lenka; Sarisský, Marek; Mirossay, Ladislav; Mirossay, Andrej; Gál, Peter; Kraus, Vladimír; Dianisková, Katarína; Mojzis, Ján

    2010-06-01

    In our study we used quercetin (3,3 ,4 ,5,7-pentahydroxyflavone) as the reference standard to compare antiproliferative and antiangiogenic effects of chrysin (5,7-dihydroxyflavone) and 3-hydroxyflavone. Our data indicates that chrysin and 3-hydroxyflavone showed significantly higher cytotoxic effect than reference standard quercetin. These tested agents significantly decreased cell survival with the efficacy of 65-85% at the concentration 100 micromol/l for HUVEC, lung carcinoma and leukemic cells being the most sensitive. Cell cycle analysis indicates that quercetin and 3-hydroxyflavone might affect the cell cycle of Jurkat cells by a similar or the same mechanism of action which lead to G2/M arrest as well as to an increase in sub-G0/G1 fraction. Treatment of Jurkat cells with chrysin resulted only increase in the fraction of cells with sub-G0/G1 DNA content, which is considered to be a marker of apoptotic cell death. Apoptosis was confirmed by DNA fragmentation and by staining with annexin V. All three tested flavones inhibited endothelial cell migration after 24 h of incubation at a concentration 100 micromol/l. At a lower concentration (10 micromol/l) only quercetin significantly inhibited migration of endothelial cells. Furthermore, in our experiments decreased secretion of matrix metalloproteinases (MMP-2 and MMP-9) was observed after a 72 h treatment with quercetin. No decrease in secretion of MMP-2 and MMP-9 was seen after chrysin and 3-hydroxyflavone treatment. On the other hand, our results showed that none of three flavonoids blocked microcapillary tube formation. Further studies are necessary to investigate the mechanism of action and to find out the relationship between the structure, character and position of substituents of natural substances and their biological activities. PMID:20577025

  18. Bisphosphonamidate Clodronate Prodrug Exhibits Selective Cytotoxic Activity Against Melanoma Cell Lines

    PubMed Central

    Webster, Marie R.; Kamat, Chandrashekhar; Connis, Nick; Zhao, Ming; Weeraratna, Ashani T.; Rudek, Michelle A.; Hann, Christine L.; Freel Meyers, Caren L.

    2014-01-01

    Bisphosphonates are used clinically to treat disorders of calcium metabolism and malignant bone disease and are known to inhibit cancer cell growth, adhesion, and invasion. However, clinical use of these agents for the treatment of extraskeletal disease is limited due to low cell permeability. We recently described a bisphosphonamidate prodrug strategy for efficient intracellular release of bisphosphonates, including clodronate (CLO), in NSCLC cells. To evaluate anticancer activity of this prodrug class across many cancer cell types, the bisphosphonamidate clodronate prodrug (CLO prodrug) was screened against the NCI-60 cell line panel, and was found to exhibit selectivity toward melanoma cell lines. Here, we confirm efficient cellular uptake and intracellular activation of this prodrug class in melanoma cells. We further demonstrate inhibition of melanoma cell proliferation, induction of apoptosis, and an anti-tumor effect of CLO prodrug in a xenograft model. These data suggest a novel therapeutic application for the CLO prodrug and potential to selectively target melanoma cells. PMID:24310621

  19. Eat this, not that! How selective autophagy helps cancer cells survive

    PubMed Central

    Mathew, Robin; White, Eileen

    2015-01-01

    Autophagy degrades the cellular proteome to promote survival, but the underlying mechanism and substrates of consequence are poorly understood. We found that autophagy selectively remodels the proteome in cancer cells by eliminating proinflammatory signaling proteins. Autophagy ablation causes aberrant accumulation of these proteins that primes cancer cells for interferon-dependent cell death, explaining how autophagy suppresses inflammation and promotes tumor maintenance. PMID:27308434

  20. Ionene polymers for selectively inhibiting the vitro growth of malignant cells

    NASA Technical Reports Server (NTRS)

    Rembaum, Alan (Inventor)

    1977-01-01

    Ionene polymers of the structure ##STR1## WHERE X AND Y ARE INTEGERS FROM 3 TO 16, Z.sup.- is an anion such as a halogen and n is an integer from 50 to 150 are found to bind negatively charged mammalian cells such as malignant cells and can be utilized to selectively inhibit the growth of malignant cells in vitro.

  1. Positive genetic selection for gene disruption in mammalian cells by homologous recombination.

    PubMed Central

    Sedivy, J M; Sharp, P A

    1989-01-01

    Efficient modification of genes in mammalian cells by homologous recombination has not been possible because of the high frequency of nonhomologous recombination. An efficient method for targeted gene disruption has been developed. Cells with substitution of exogenous sequences into a chromosomal locus were enriched, by a factor of 100, using a positive genetic selection that specifically selects for homologous recombination at the targeted site. The selection is based on the conditional expression of a dominant selectable marker by virtue of in-frame gene fusion with the target gene. The dominant selectable marker was derived by modification of the Escherichia coli neo gene so that it retains significant activity in mammalian cells after in-frame fusion with heterologous coding sequences. In the example presented here, homologous recombinants were efficiently recovered from a pool in which the targeted gene was disrupted in 1 per 10,000 cells incorporating exogenous DNA. Images PMID:2536156

  2. Piperlongumine selectively kills cancer cells and increases cisplatin antitumor activity in head and neck cancer

    PubMed Central

    Roh, Jong-Lyel; Kim, Eun Hye; Park, Jin Young; Kim, Ji Won; Kwon, Minsu; Lee, Byung-Heon

    2014-01-01

    Adaptation to cellular stress is not a vital function of normal cells but is required of cancer cells, and as such might be a sensible target in cancer therapy. Piperlongumine is a naturally occurring small molecule selectively toxic to cancer cells. This study assesses the cytotoxicity of piperlongumine and its combination with cisplatin in head-and-neck cancer (HNC) cells in vitro and in vivo. The effect of piperlongumine, alone and in combination with cisplatin, was assessed in human HNC cells and normal cells by measuring growth, death, cell cycle progression, reactive oxygen species (ROS) production, and protein expression, and in tumor xenograft mouse models. Piperlongumine killed HNC cells regardless of p53 mutational status but spared normal cells. It increased ROS accumulation in HNC cells, an effect that can be blocked by the antioxidant N-acetyl-L-cysteine. Piperlongumine induced selective cell death in HNC cells by targeting the stress response to ROS, leading to the induction of death pathways involving JNK and PARP. Piperlongumine increased cisplatin-induced cytotoxicity in HNC cells in a synergistic manner in vitro and in vivo. Piperlongumine might be a promising small molecule with which to selectively kill HNC cells and increase cisplatin antitumor activity by targeting the oxidative stress response. PMID:25193861

  3. Piperlongumine selectively kills cancer cells and increases cisplatin antitumor activity in head and neck cancer.

    PubMed

    Roh, Jong-Lyel; Kim, Eun Hye; Park, Jin Young; Kim, Ji Won; Kwon, Minsu; Lee, Byung-Heon

    2014-10-15

    Adaptation to cellular stress is not a vital function of normal cells but is required of cancer cells, and as such might be a sensible target in cancer therapy. Piperlongumine is a naturally occurring small molecule selectively toxic to cancer cells. This study assesses the cytotoxicity of piperlongumine and its combination with cisplatin in head-and-neck cancer (HNC) cells in vitro and in vivo. The effect of piperlongumine, alone and in combination with cisplatin, was assessed in human HNC cells and normal cells by measuring growth, death, cell cycle progression, reactive oxygen species (ROS) production, and protein expression, and in tumor xenograft mouse models. Piperlongumine killed HNC cells regardless of p53 mutational status but spared normal cells. It increased ROS accumulation in HNC cells, an effect that can be blocked by the antioxidant N-acetyl-L-cysteine. Piperlongumine induced selective cell death in HNC cells by targeting the stress response to ROS, leading to the induction of death pathways involving JNK and PARP. Piperlongumine increased cisplatin-induced cytotoxicity in HNC cells in a synergistic manner in vitro and in vivo. Piperlongumine might be a promising small molecule with which to selectively kill HNC cells and increase cisplatin antitumor activity by targeting the oxidative stress response. PMID:25193861

  4. Properties of pattern and component direction-selective cells in area MT of the macaque.

    PubMed

    Wang, Helena X; Movshon, J Anthony

    2016-06-01

    Neurons in area MT/V5 of the macaque visual cortex encode visual motion. Some cells are selective for the motion of oriented features (component direction-selective, CDS); others respond to the true direction of complex patterns (pattern-direction selective, PDS). There is a continuum of selectivity in MT, with CDS cells at one extreme and PDS cells at the other; we compute a pattern index that captures this variation. It is unknown how a neuron's pattern index is related to its other tuning characteristics. We therefore analyzed the responses of 792 MT cells recorded in the course of other experiments from opiate-anesthetized macaque monkeys, as a function of the direction, spatial frequency, drift rate, size, and contrast of sinusoidal gratings and of the direction and speed of random-dot textures. We also compared MT responses to those of 718 V1 cells. As expected, MT cells with higher pattern index tended to have stronger direction selectivity and broader direction tuning to gratings, and they responded better to plaids than to gratings. Strongly PDS cells also tended to have smaller receptive fields and stronger surround suppression. Interestingly, they also responded preferentially to higher drift rates and higher speeds of moving dots. The spatial frequency preferences of PDS cells depended strongly on their preferred temporal frequencies, whereas these preferences were independent in component-selective cells. Pattern direction selectivity is statistically associated with many response properties of MT cells but not strongly associated with any particular property. Pattern-selective signals are thus available in association with most other signals exported by MT. PMID:26561603

  5. Selection of Metastatic Breast Cancer Cells Based on Adaptability of Their Metabolic State

    PubMed Central

    Singh, Balraj; Tai, Karen; Madan, Simran; Raythatha, Milan R.; Cady, Amanda M.; Braunlin, Megan; Irving, LaTashia R.; Bajaj, Ankur; Lucci, Anthony

    2012-01-01

    A small subpopulation of highly adaptable breast cancer cells within a vastly heterogeneous population drives cancer metastasis. Here we describe a function-based strategy for selecting rare cancer cells that are highly adaptable and drive malignancy. Although cancer cells are dependent on certain nutrients, e.g., glucose and glutamine, we hypothesized that the adaptable cancer cells that drive malignancy must possess an adaptable metabolic state and that such cells could be identified using a robust selection strategy. As expected, more than 99.99% of cells died upon glutamine withdrawal from the aggressive breast cancer cell line SUM149. The rare cells that survived and proliferated without glutamine were highly adaptable, as judged by additional robust adaptability assays involving prolonged cell culture without glucose or serum. We were successful in isolating rare metabolically plastic glutamine-independent (Gln-ind) variants from several aggressive breast cancer cell lines that we tested. The Gln-ind cells overexpressed cyclooxygenase-2, an indicator of tumor aggressiveness, and they were able to adjust their glutaminase level to suit glutamine availability. The Gln-ind cells were anchorage-independent, resistant to chemotherapeutic drugs doxorubicin and paclitaxel, and resistant to a high concentration of a COX-2 inhibitor celecoxib. The number of cells being able to adapt to non-availability of glutamine increased upon prior selection of cells for resistance to chemotherapy drugs or resistance to celecoxib, further supporting a linkage between cellular adaptability and therapeutic resistance. Gln-ind cells showed indications of oxidative stress, and they produced cadherin11 and vimentin, indicators of mesenchymal phenotype. Gln-ind cells were more tumorigenic and more metastatic in nude mice than the parental cell line as judged by incidence and time of occurrence. As we decreased the number of cancer cells in xenografts, lung metastasis and then primary

  6. Hematopoietic stem cell transplantation in sickle cell disease: patient selection and special considerations

    PubMed Central

    Bhatia, Monica; Sheth, Sujit

    2015-01-01

    Hematopoietic stem cell transplantation remains the only curative treatment currently in use for patients with sickle cell disease (SCD). The first successful hematopoietic stem cell transplantation was performed in 1984. To date, approximately 1,200 transplants have been reported. Given the high prevalence of this disorder in Africa, and its emergence in the developed world through immigration, this number is relatively small. There are many reasons for this; primary among them are the availability of a donor, the risks associated with this complex procedure, and the cost and availability of resources in the developing world. Of these, it is fair to say that the risks associated with the procedure have steadily decreased to the point where, if currently performed in a center with experience using a matched sibling donor, overall survival is close to 100% and event-free survival is over 90%. While there is little controversy around offering hematopoietic stem cell transplantation to symptomatic SCD patients with a matched sibling donor, there is much debate surrounding the use of this modality in “less severe” patients. An overview of the current state of our understanding of the pathology and treatment of SCD is important to show that our current strategy is not having the desired impact on survival of homozygous SCD patients, and should be changed to significantly impact the small proportion of these patients who have matched siblings and could be cured, especially those without overt clinical manifestations. Both patient families and providers must be made to understand the progressive nature of SCD, and should be encouraged to screen full siblings of patients with homozygous SCD for their potential to be donors. Matched siblings should be referred to an experienced transplant center for evaluation and counseling. In this review, we will discuss the rationale for these opinions and make recommendations for patient selection. PMID:26203293

  7. In Vitro Selection of Cancer Cell-Specific Molecular Recognition Elements from Amino Acid Libraries

    PubMed Central

    Williams, Ryan M.; Sooter, Letha J.

    2015-01-01

    Differential cell systematic evolution of ligands by exponential enrichment (SELEX) is an in vitro selection method for obtaining molecular recognition elements (MREs) that specifically bind to individual cell types with high affinity. MREs are selected from initial large libraries of different nucleic or amino acids. This review outlines the construction of peptide and antibody fragment libraries as well as their different host types. Common methods of selection are also reviewed. Additionally, examples of cancer cell MREs are discussed, as well as their potential applications. PMID:26436100

  8. Circulating cancer stem cells: the importance to select

    PubMed Central

    Yang, Ming-Hsin; Imrali, Ahmet

    2015-01-01

    It has been demonstrated that even localized tumors without clinically apparent metastasis give rise to circulating tumor cells (CTCs). A growing number of technically diverse platforms are being developed for detecting/isolating CTCs in the circulating blood. Despite the technical challenges of isolating rare CTCs from blood, recent studies have already shown the predictive value of CTCs enumeration. Thus, it is becoming increasingly accepted that CTC numbers are linked to patients’ outcome and may also be used to monitor treatment response and disease relapse, respectively. Further CTCs provide a non-invasive source for tumor material, ‘liquid biopsy’, which is particularly important for patients, where no biopsy material can be obtained or where serial biopsies of the tumor, e.g., following treatment, are practically impossible. On the other hand the molecular and biological characterization of CTCs has still remained at a rather experimental stage. Future studies are necessary to define CTC heterogeneity to establish the crucial role of circulating cancer stem cells for driving metastasis, which represent a distinct subpopulation of CTCs that bear metastasis-initiating capabilities based on their stemness properties and invasiveness and thus are critical for the patients’ clinical outcome. As compared to non-tumorigenic/metastatic bulk CTCs, circulating cancer stem cells may not only be capable of evading from the primary tumor, but also escape from immune surveillance, survive in the circulating blood and subsequently form metastases in distant organs. Thus, circulating cancer stem cells represent a subset of exclusively tumorigenic cancer stem cells characterized by their invasive characteristics and are potential therapeutic targets for preventing disease progression. To date, only a few original reports and reviews have been published focusing on circulating cancer stem cells. This review discusses the potential importance of isolating and

  9. Selection of Mesenchymal-Like Metastatic Cells in Primary Tumors – An in silico Investigation

    PubMed Central

    Narang, Vipin; Wong, Shek Yoon; Leong, Shiang Rong; Harish, Bindu; Abastado, Jean-Pierre; Gouaillard, Alexandre

    2012-01-01

    In order to metastasize, cancer cells must undergo phenotypic transition from an anchorage-dependent form to a motile form via a process referred to as epithelial to mesenchymal transition. It is currently unclear whether metastatic cells emerge late during tumor progression by successive accumulation of mutations, or whether they derive from distinct cell populations already present during the early stages of tumorigenesis. Similarly, the selective pressures that drive metastasis are poorly understood. Selection of cancer cells with increased proliferative capacity and enhanced survival characteristics may explain how some transformations promote a metastatic phenotype. However, it is difficult to explain how cancer cells that disseminate can emerge due to such selective pressure, since these cells usually remain dormant for prolonged periods of time. In the current study, we have used in silico modeling and simulation to investigate the hypothesis that mesenchymal-like cancer cells evolve during the early stages of primary tumor development, and that these cells exhibit survival and proliferative advantages within the tumor microenvironment. In an agent-based tumor microenvironment model, cancer cell agents with distinct sets of attributes governing nutrient consumption, proliferation, apoptosis, random motility, and cell adhesion were allowed to compete for space and nutrients. These simulation data indicated that mesenchymal-like cancer cells displaying high motility and low adhesion proliferate more rapidly and display a survival advantage over epithelial-like cancer cells. Furthermore, the presence of mesenchymal-like cells within the primary tumor influences the macroscopic properties, emergent morphology, and growth rate of tumors. PMID:22566967

  10. Prediction of cell-penetrating peptides with feature selection techniques.

    PubMed

    Tang, Hua; Su, Zhen-Dong; Wei, Huan-Huan; Chen, Wei; Lin, Hao

    2016-08-12

    Cell-penetrating peptides are a group of peptides which can transport different types of cargo molecules such as drugs across plasma membrane and have been applied in the treatment of various diseases. Thus, the accurate prediction of cell-penetrating peptides with bioinformatics methods will accelerate the development of drug delivery systems. The study aims to develop a powerful model to accurately identify cell-penetrating peptides. At first, the peptides were translated into a set of vectors with the same dimension by using dipeptide compositions. Secondly, the Analysis of Variance-based technique was used to reduce the dimension of the vector and explore the optimized features. Finally, the support vector machine was utilized to discriminate cell-penetrating peptides from non-cell-penetrating peptides. The five-fold cross-validated results showed that our proposed method could achieve an overall prediction accuracy of 83.6%. Based on the proposed model, we constructed a free webserver called C2Pred (http://lin.uestc.edu.cn/server/C2Pred). PMID:27291150

  11. Selectivity of biopolymer membranes using HepG2 cells

    PubMed Central

    Lü, Dongyuan; Gao, Yuxin; Luo, Chunhua; Lü, Shouqian; Wang, Qian; Xu, Xianghong; Sun, Shujin; Wang, Chengzhi; Long, Mian

    2015-01-01

    Bioartificial liver (BAL) system has emerged as an alternative treatment to bridge acute liver failure to either liver transplantation or liver regeneration. One of the main reasons that the efficacy of the current BAL systems was not convincing in clinical trials is attributed to the lack of friendly interface between the membrane and the hepatocytes in liver bioreactor, the core unit of BAL system. Here, we systematically compared the biological responses of hepatosarcoma HepG2 cells seeded on eight, commercially available biocompatible membranes made of acetyl cellulose-nitrocellulose mixed cellulose (CA-NC), acetyl cellulose (CA), nylon (JN), polypropylene (PP), nitrocellulose (NC), polyvinylidene fluoride (PVDF), polycarbonate (PC) and polytetrafluoroethylene (PTFE). Physicochemical analysis and mechanical tests indicated that CA, JN and PP membranes yield high adhesivity and reasonable compressive and/or tensile features with friendly surface topography for cell seeding. Cells prefer to adhere on CA, JN, PP or PTFE membranes with high proliferation rate in spheriod-like shape. Actin, albumin and cytokeratin 18 expressions are favorable for cells on CA or PP membrane, whereas protein filtration is consistent among all the eight membranes. These results further the understandings of cell growth, morphology and spreading, as well as protein filtration on distinct membranes in designing a liver bioreactor. PMID:26816630

  12. Selective photodepletion of malignant T cells in extracorporeal photopheresis with selenorhodamine photosensitizers.

    PubMed

    McIver, Zachariah A; Kryman, Mark W; Choi, Young; Coe, Benjamin N; Schamerhorn, Gregory A; Linder, Michelle K; Davies, Kellie S; Hill, Jacqueline E; Sawada, Geri A; Grayson, Jason M; Detty, Michael R

    2016-09-01

    Extracorporeal photopheresis (ECP) has been used successfully in the treatment of erythrodermic cutaneous T cell lymphoma (CTCL), and other T cell-mediated disorders. Not all patients obtain a significant or durable response from ECP. The design of a selective photosensitizer that spares desirable lymphocytes while targeting malignant T cells may promote cytotoxic T cell responses and improve outcomes after ECP. A series of selenorhodamines built with variations of the Texas red core targeted the mitochondria of malignant T cells, were phototoxic to malignant T cells presumably via their ability to generate singlet oxygen, and were transported by P-glycoprotein (P-gp). To determine the selectivity of the photosensitizers in the ECP milieu, staphylococcal enterotoxin B (SEB)-stimulated and non-stimulated human lymphocytes were combined with HUT-78 cells (a CTCL) to simulate ECP. The amide-containing analogues of the selenorhodamines were transported more rapidly than the thioamide analogues in monolayers of MDCKII-MDR1 cells and, consequently, were extruded more rapidly from P-gp-expressing T cells than the corresponding thioamide analogues. Selenorhodamine 6 with the Texas red core and a piperidylamide functionality was phototoxic to >90% of malignant T cells while sparing >60% of both stimulated and non-stimulated T cells. In the resting T cells, (63±7)% of the CD4+ T cell compartment, and (78±2.5)% of the CD8+ cytotoxic T cell population were preserved, resulting in an enrichment of healthy and cytotoxic T cells after photodepletion. PMID:27301678

  13. Glimpse of natural selection of long-lived T-cell clones in healthy life.

    PubMed

    Zhang, Baojun; Jia, Qingzhu; Bock, Cheryl; Chen, Gang; Yu, Haili; Ni, Qingshan; Wan, Ying; Li, Qijing; Zhuang, Yuan

    2016-08-30

    Homeostatic maintenance of T cells with broad clonal diversity is influenced by both continuing output of young T cells from the thymus and ongoing turnover of preexisting clones in the periphery. In the absence of infection, self and commensal antigens are thought to play important roles in selection and homeostatic maintenance of the T-cell pool. Most naïve T cells are short-lived due to lack of antigen encounter, whereas antigen-experienced T cells may survive and persist as long-lived clones. Thus far, little is known about the homeostasis, antigenic specificity, and clonal diversity of long-lived T-cell clones in peripheral lymphoid organs under healthy living conditions. To identify long-lived T-cell clones in mice, we designed a lineage-tracing method to label a wave of T cells produced in the thymus of young mice. After aging the mice for 1.5 y, we found that lineage-tracked T cells consisted of primarily memory-like T cells and T regulatory cells. T-cell receptor repertoire analysis revealed that the lineage-tracked CD4 memory-like T cells and T regulatory cells exhibited age-dependent enrichment of shared clonotypes. Furthermore, these shared clonotypes were found across different mice maintained in the same housing condition. These findings suggest that nonrandom and shared antigens are involved in controlling selection, retention, and immune tolerance of long-lived T-cell clones under healthy living conditions. PMID:27535935

  14. Selective killing of methotrexate-resistant cells carrying amplified dihydrofolate reductase genes

    SciTech Connect

    Urlaub, G.; Landzberg, M.; Chasin, L.A.

    1981-05-01

    A method for the selective killing of methotrexate (MTX)-resistant cells has been developed. The selection is based on the incorporation of tritiated deoxyuridine into the DNA of MTX-resistant cells but not normal MTX-sensitive cells in the presence of the drug. A Chinese hamster ovary cell mutant that overproduces dihydrofolate reductase was used as an example of a MTX-resistant cell line. In this system, a 10,000-fold enrichment for wild-type MTX-sensitive cells could be achieved after 24 hr of exposure to the drug combination. This selection technique was applied to the isolation of MTX-sensitive segregants from hybrid cells formed between the MTX-resistant mutant and wild-type cells. The loss of MTX resistance and dihydrofolate reductase overproduction was always accompanied by the loss of a homogeneously staining region on chromosome 2 of the resistant parent that contains the amplified genes specifying this enzyme. While this region is always lost, other parts of chromosome 2 are almost always retained, suggesting that deletion rather than chromosome loss underlies marker segregation in this case. When the selection was applied to the resistant mutant itself, no MTX-sensitive revertants were obtained among 10(5) cells screened, attesting to the stability of gene amplification in this clone. It is suggested that this combination of drugs may be useful for the elimination of MTX-resistant tumor cells that develop after MTX chemotherapy.

  15. Multifunctionalized iron oxide nanoparticles for selective drug delivery to CD44-positive cancer cells.

    PubMed

    Aires, Antonio; Ocampo, Sandra M; Simões, Bruno M; Josefa Rodríguez, María; Cadenas, Jael F; Couleaud, Pierre; Spence, Katherine; Latorre, Alfonso; Miranda, Rodolfo; Somoza, Álvaro; Clarke, Robert B; Carrascosa, José L; Cortajarena, Aitziber L

    2016-02-12

    Nanomedicine nowadays offers novel solutions in cancer therapy and diagnosis by introducing multimodal treatments and imaging tools in one single formulation. Nanoparticles acting as nanocarriers change the solubility, biodistribution and efficiency of therapeutic molecules, reducing their side effects. In order to successfully  apply these novel therapeutic approaches, efforts are focused on the biological functionalization of the nanoparticles to improve the selectivity towards cancer cells. In this work, we present the synthesis and characterization of novel multifunctionalized iron oxide magnetic nanoparticles (MNPs) with antiCD44 antibody and gemcitabine derivatives, and their application for the selective treatment of CD44-positive cancer cells. The lymphocyte homing receptor CD44 is overexpressed in a large variety of cancer cells, but also in cancer stem cells (CSCs) and circulating tumor cells (CTCs). Therefore, targeting CD44-overexpressing cells is a challenging and promising anticancer strategy. Firstly, we demonstrate the targeting of antiCD44 functionalized MNPs to different CD44-positive cancer cell lines using a CD44-negative non-tumorigenic cell line as a control, and verify the specificity by ultrastructural characterization and downregulation of CD44 expression. Finally, we show the selective drug delivery potential of the MNPs by the killing of CD44-positive cancer cells using a CD44-negative non-tumorigenic cell line as a control. In conclusion, the proposed multifunctionalized MNPs represent an excellent biocompatible nanoplatform for selective CD44-positive cancer therapy in vitro. PMID:26754042

  16. Multifunctionalized iron oxide nanoparticles for selective drug delivery to CD44-positive cancer cells

    NASA Astrophysics Data System (ADS)

    Aires, Antonio; Ocampo, Sandra M.; Simões, Bruno M.; Josefa Rodríguez, María; Cadenas, Jael F.; Couleaud, Pierre; Spence, Katherine; Latorre, Alfonso; Miranda, Rodolfo; Somoza, Álvaro; Clarke, Robert B.; Carrascosa, José L.; Cortajarena, Aitziber L.

    2016-02-01

    Nanomedicine nowadays offers novel solutions in cancer therapy and diagnosis by introducing multimodal treatments and imaging tools in one single formulation. Nanoparticles acting as nanocarriers change the solubility, biodistribution and efficiency of therapeutic molecules, reducing their side effects. In order to successfully apply these novel therapeutic approaches, efforts are focused on the biological functionalization of the nanoparticles to improve the selectivity towards cancer cells. In this work, we present the synthesis and characterization of novel multifunctionalized iron oxide magnetic nanoparticles (MNPs) with antiCD44 antibody and gemcitabine derivatives, and their application for the selective treatment of CD44-positive cancer cells. The lymphocyte homing receptor CD44 is overexpressed in a large variety of cancer cells, but also in cancer stem cells (CSCs) and circulating tumor cells (CTCs). Therefore, targeting CD44-overexpressing cells is a challenging and promising anticancer strategy. Firstly, we demonstrate the targeting of antiCD44 functionalized MNPs to different CD44-positive cancer cell lines using a CD44-negative non-tumorigenic cell line as a control, and verify the specificity by ultrastructural characterization and downregulation of CD44 expression. Finally, we show the selective drug delivery potential of the MNPs by the killing of CD44-positive cancer cells using a CD44-negative non-tumorigenic cell line as a control. In conclusion, the proposed multifunctionalized MNPs represent an excellent biocompatible nanoplatform for selective CD44-positive cancer therapy in vitro.

  17. A Dimeric Mutant of Human Pancreatic Ribonuclease with Selective Cytotoxicity toward Malignant Cells

    NASA Astrophysics Data System (ADS)

    Piccoli, Renata; di Gaetano, Sonia; de Lorenzo, Claudia; Grauso, Michela; Monaco, Carmen; Spalletti-Cernia, Daniela; Laccetti, Paolo; Cinatl, Jaroslav; Matousek, Josef; D'Alessio, Giuseppe

    1999-07-01

    Monomeric human pancreatic RNase, devoid of any biological activity other than its RNA degrading ability, was engineered into a dimeric protein with a cytotoxic action on mouse and human tumor cells, but lacking any appreciable toxicity on mouse and human normal cells. This dimeric variant of human pancreas RNase selectively sensitizes to apoptotic death cells derived from a human thyroid tumor. Because of its selectivity for tumor cells, and because of its human origin, this protein represents a potentially very attractive, novel tool for anticancer therapy.

  18. Selective binding of human cumulus cell-secreted glycoproteins to human spermatozoa during capacitation in vitro

    SciTech Connect

    Tesarik, J.; Kopecny, V.; Dvorak, M.

    1984-06-01

    The results of this study demonstrate that glycoproteins manufactured by human cumulus cells can be detected bound to human spermatozoa incubated in capacitational medium containing the labeled cumulus-cell secretions. Cumulus-cell-secreted glycoproteins were labeled with a mixture of /sup 3/H-methionine and /sup 3/H-tryptophan or with 3H-fucose, and the binding of the labeled compounds to spermatozoa was evaluated by autoradiography. The binding was highly selective, involving only approximately 1% of the samples of spermatozoa used. The results suggest that the binding of cumulus-cell-secreted glycoproteins to spermatozoa may represent a final and highly selective step in human sperm capacitation.

  19. Solid tumor therapy by selectively targeting stromal endothelial cells.

    PubMed

    Liu, Shihui; Liu, Jie; Ma, Qian; Cao, Liu; Fattah, Rasem J; Yu, Zuxi; Bugge, Thomas H; Finkel, Toren; Leppla, Stephen H

    2016-07-12

    Engineered tumor-targeted anthrax lethal toxin proteins have been shown to strongly suppress growth of solid tumors in mice. These toxins work through the native toxin receptors tumor endothelium marker-8 and capillary morphogenesis protein-2 (CMG2), which, in other contexts, have been described as markers of tumor endothelium. We found that neither receptor is required for tumor growth. We further demonstrate that tumor cells, which are resistant to the toxin when grown in vitro, become highly sensitive when implanted in mice. Using a range of tissue-specific loss-of-function and gain-of-function genetic models, we determined that this in vivo toxin sensitivity requires CMG2 expression on host-derived tumor endothelial cells. Notably, engineered toxins were shown to suppress the proliferation of isolated tumor endothelial cells. Finally, we demonstrate that administering an immunosuppressive regimen allows animals to receive multiple toxin dosages and thereby produces a strong and durable antitumor effect. The ability to give repeated doses of toxins, coupled with the specific targeting of tumor endothelial cells, suggests that our strategy should be efficacious for a wide range of solid tumors. PMID:27357689

  20. Selective glucocorticoid control of Rho kinase isoforms regulate cell-cell interactions

    PubMed Central

    Rubenstein, Nicola M.; Callahan, Joseph A.; Lo, Daniel H.; Firestone, Gary L.

    2007-01-01

    The two Rho kinase isoforms ROCK1 and ROCK2 are downstream effectors of the small GTPase RhoA, although relatively little is known about potential isoform specific functions or the selective control of their cellular activities. Using Con8 rat mammary epithelial cells, we show that the synthetic glucocorticoid dexamethasone strongly stimulates the level of ROCK2 protein, which accounts for the increase in total cellular ROCK2 activity, whereas, steroid treatment down-regulated ROCK1 specific kinase activity without altering ROCK1 protein levels. In Con8 cells, the glucocorticoid induced formation of tight junctions requires the steroid-mediated down-regulation RhoA and function of the RhoA antagonist Rnd3. Treatment with the ROCK inhibitor Y-27632 ablated both the glucocorticoid-induced and Rnd3-mediated stimulation in tight junction sealing. Taken together, our results demonstrate that the expression and activity of ROCK1 and ROCK2 can be uncoupled in a signal-dependent manner, and further implicate a new function for ROCK2 in the steroid control of tight junction dynamics. PMID:17240358

  1. T-cell selection in the thymus: a spatial and temporal perspective.

    PubMed

    Kurd, Nadia; Robey, Ellen A

    2016-05-01

    The ability of T cells to respond to a wide array of foreign antigens while avoiding reactivity to self is largely determined by cellular selection of developing T cells in the thymus. While a great deal is known about the cell types and molecules involved in T-cell selection in the thymus, our understanding of the spatial and temporal aspects of this process remain relatively poorly understood. Thymocytes are highly motile within the thymus and travel between specialized microenvironments at different phases of their development while interacting with distinct sets of self-peptides and peptide presenting cells. A knowledge of when, where, and how thymocytes encounter self-peptide MHC ligands at different stages of thymic development is key to understanding T-cell selection. In the past several years, our laboratory has investigated this topic using two-photon time-lapse microscopy to directly visualize thymocyte migration and signaling events, together with a living thymic slice preparation to provide a synchronized experimental model of T-cell selection in situ. Here, we discuss recent advances in our understanding of the temporal and spatial aspects of T-cell selection, highlighting our own work, and placing them in the context of work from other groups. PMID:27088910

  2. Energy deposition in selected-mammalian cell for several-MeV single-proton beam

    NASA Astrophysics Data System (ADS)

    Ding, K.; Yu, Z.

    2007-05-01

    The phenomena resulting from interaction between ion beam and mammalian cell pose important problems for biological applications. Classic Bethe-Bloch theory utilizing attached V79 mammalian cell has been conducted in order to establish the stopping powers of the mammalian cell for several-MeV single-proton microbeam. Based on the biological structure of the mammalian cell, a physical model is proposed which presumes that the attached cell is simple MWM model. According to this model and Monte Carlo simulation, we studied the energy deposition and its ratio on the selected attached mammalian cell for MeV proton implantation.

  3. Advances in clinical NK cell studies: Donor selection, manufacturing and quality control

    PubMed Central

    Koehl, U.; Kalberer, C.; Spanholtz, J.; Lee, D. A.; Miller, J. S.; Cooley, S.; Lowdell, M.; Uharek, L.; Klingemann, H.; Curti, A.; Leung, W.; Alici, E.

    2016-01-01

    ABSTRACT Natural killer (NK) cells are increasingly used in clinical studies in order to treat patients with various malignancies. The following review summarizes platform lectures and 2013–2015 consortium meetings on manufacturing and clinical use of NK cells in Europe and United States. A broad overview of recent pre-clinical and clinical results in NK cell therapies is provided based on unstimulated, cytokine-activated, as well as genetically engineered NK cells using chimeric antigen receptors (CAR). Differences in donor selection, manufacturing and quality control of NK cells for cancer immunotherapies are described and basic recommendations are outlined for harmonization in future NK cell studies. PMID:27141397

  4. A highly selective fluorescent probe for direct detection and isolation of mouse embryonic stem cells.

    PubMed

    Chandran, Yogeswari; Kang, Nam-Young; Park, Sung-Jin; Alamudi, Samira Husen; Kim, Jun-Young; Sahu, Srikanta; Su, Dongdong; Lee, Jungyeol; Vendrell, Marc; Chang, Young-Tae

    2015-11-01

    Stem cell research has gathered immense attention in the past decade due to the remarkable ability of stem cells for self-renewal and tissue-specific differentiation. Despite having numerous advancements in stem cell isolation and manipulation techniques, there is a need for highly reliable probes for the specific detection of live stem cells. Herein we developed a new fluorescence probe (CDy9) with high selectivity for mouse embryonic stem cells. CDy9 allows the detection and isolation of intact stem cells with marginal impact on their function and capabilities. PMID:26115574

  5. Synaptic Basis for Differential Orientation Selectivity between Complex and Simple Cells in Mouse Visual Cortex

    PubMed Central

    Li, Ya-tang; Liu, Bao-hua; Chou, Xiao-lin; Zhang, Li I.

    2015-01-01

    In the primary visual cortex (V1), orientation-selective neurons can be categorized into simple and complex cells primarily based on their receptive field (RF) structures. In mouse V1, although previous studies have examined the excitatory/inhibitory interplay underlying orientation selectivity (OS) of simple cells, the synaptic bases for that of complex cells have remained obscure. Here, by combining in vivo loose-patch and whole-cell recordings, we found that complex cells, identified by their overlapping on/off subfields, had significantly weaker OS than simple cells at both spiking and subthreshold membrane potential response levels. Voltage-clamp recordings further revealed that although excitatory inputs to complex and simple cells exhibited a similar degree of OS, inhibition in complex cells was more narrowly tuned than excitation, whereas in simple cells inhibition was more broadly tuned than excitation. The differential inhibitory tuning can primarily account for the difference in OS between complex and simple cells. Interestingly, the differential synaptic tuning correlated well with the spatial organization of synaptic input: the inhibitory visual RF in complex cells was more elongated in shape than its excitatory counterpart and also was more elongated than that in simple cells. Together, our results demonstrate that OS of complex and simple cells is differentially shaped by cortical inhibition based on its orientation tuning profile relative to excitation, which is contributed at least partially by the spatial organization of RFs of presynaptic inhibitory neurons. SIGNIFICANCE STATEMENT Simple and complex cells, two classes of principal neurons in the primary visual cortex (V1), are generally thought to be equally selective for orientation. In mouse V1, we report that complex cells, identified by their overlapping on/off subfields, has significantly weaker orientation selectivity (OS) than simple cells. This can be primarily attributed to the

  6. Methotrexate supports in vivo selection of human embryonic stem cell derived-hematopoietic cells expressing dihydrofolate reductase

    PubMed Central

    Gori, Jennifer L; McIvor, R Scott

    2010-01-01

    Human embryonic stem cells (hES Cs) are an attractive alternative cell source for hematopoietic gene therapy applications as the cells are easily modified with lentiviral or other vectors and can be subsequently induced to differentiate into hematopoietic progenitor cells. However, demonstration of the full hematopoietic potential of hESC-derived progeny is challenging due to low marrow engraftment and the difficulty of detecting cells in the peripheral blood of human/mouse xenografts. Methotrexate (MTX) chemotherapy coupled with expression of a drug resistant dihydrofolate reductase such as Tyr22 (Tyr22DHFR) has the potential to selectively increase engraftment of gene-modified human hematopoietic cells in mice, which would allow for better phenotypic characterization of hESC-derived cells in vivo. We showed that hES Cs transduced with Tyr22DHFR-GFP encoding lentivirus vectors differentiate into MTX resistant (MTXr) hemato-endothelial cells. MTX treatment of immunodeficient mice infused with Tyr22DHFR hESC-derived hemato-endothelial cells increased the long-term engraftment of human cells in the bone marrow of MTX-treated mice. In contrast to previous studies, these results indicate that MTX administration has the potential to support in vivo selection that is maintained after cessation of treatment. The MTX/Tyr22DHFR system may therefore be useful for enrichment of gene-modified cell populations in human stem cell and gene therapy applications. PMID:21468213

  7. Selective Migration of Subpopulations of Bone Marrow Cells along an SDF-1α and ATP Gradient.

    PubMed

    Laupheimer, Michael; Skorska, Anna; Große, Jana; Tiedemann, Gudrun; Steinhoff, Gustav; David, Robert; Lux, Cornelia A

    2014-01-01

    Both stem cell chemokine stromal cell-derived factor-1α (SDF-1α) and extracellular nucleotides such as adenosine triphosphate (ATP) are increased in ischemic myocardium. Since ATP has been reported to influence cell migration, we analysed the migratory response of bone marrow cells towards a combination of SDF-1 and ATP. Total nucleated cells (BM-TNCs) were isolated from bone marrow of cardiac surgery patients. Migration assays were performed in vitro. Subsequently, migrated cells were subjected to multicolor flow cytometric analysis of CD133, CD34, CD117, CD184, CD309, and CD14 expression. BM-TNCs migrated significantly towards a combination of SDF-1 and ATP. The proportions of CD34+ cells as well as subpopulations coexpressing multiple stem cell markers were selectively enhanced after migration towards SDF-1 or SDF-1 + ATP. After spontaneous migration, significantly fewer stem cells and CD184+ cells were detected. Direct incubation with SDF-1 led to a reduction of CD184+ but not stem cell marker-positive cells, while incubation with ATP significantly increased CD14+ percentage. In summary, we found that while a combination of SDF-1 and ATP elicited strong migration of BM-TNCs in vitro, only SDF-1 was responsible for selective attraction of hematopoietic stem cells. Meanwhile, spontaneous migration of stem cells was lower compared to BM-TNCs or monocytes. PMID:25610653

  8. Selective Migration of Subpopulations of Bone Marrow Cells along an SDF-1α and ATP Gradient

    PubMed Central

    Laupheimer, Michael; Skorska, Anna; Große, Jana; Tiedemann, Gudrun; Steinhoff, Gustav; David, Robert; Lux, Cornelia A.

    2014-01-01

    Both stem cell chemokine stromal cell-derived factor-1α (SDF-1α) and extracellular nucleotides such as adenosine triphosphate (ATP) are increased in ischemic myocardium. Since ATP has been reported to influence cell migration, we analysed the migratory response of bone marrow cells towards a combination of SDF-1 and ATP. Total nucleated cells (BM-TNCs) were isolated from bone marrow of cardiac surgery patients. Migration assays were performed in vitro. Subsequently, migrated cells were subjected to multicolor flow cytometric analysis of CD133, CD34, CD117, CD184, CD309, and CD14 expression. BM-TNCs migrated significantly towards a combination of SDF-1 and ATP. The proportions of CD34+ cells as well as subpopulations coexpressing multiple stem cell markers were selectively enhanced after migration towards SDF-1 or SDF-1 + ATP. After spontaneous migration, significantly fewer stem cells and CD184+ cells were detected. Direct incubation with SDF-1 led to a reduction of CD184+ but not stem cell marker-positive cells, while incubation with ATP significantly increased CD14+ percentage. In summary, we found that while a combination of SDF-1 and ATP elicited strong migration of BM-TNCs in vitro, only SDF-1 was responsible for selective attraction of hematopoietic stem cells. Meanwhile, spontaneous migration of stem cells was lower compared to BM-TNCs or monocytes. PMID:25610653

  9. Pyrvinium selectively induces apoptosis of lymphoma cells through impairing mitochondrial functions and JAK2/STAT5.

    PubMed

    Xiao, Meifang; Zhang, Liming; Zhou, Yizheng; Rajoria, Pasupati; Wang, Changfu

    2016-01-15

    Targeting mitochondrial respiration has emerged as an attractive therapeutic strategy in blood cancer due to their unique metabolic dependencies. In this study, we show that pyrvinium, a FDA-approved anthelmintic drug, selectively targets lymphoma T-cells though inhibition of mitochondrial functions and JAK2/STAT5. Pyrvinium induces apoptosis of malignant T-cell line Jurkat and primary T-cells from lymphoma patients while sparing T-cells from healthy donors. Increased level of active caspase-3 and decreased levels of Bcl-2 and Mcl-1 were also observed in Jurkat and lymphoma T-cells but not normal T-cells treated with pyrvinium. In addition, pyrvinium impairs mitochondrial functions by inhibit mitochondrial respiration, suppressing mitochondrial respiratory complex I activity, increasing ROS and decreasing ATP levels. However, the effects of pyrvinium were abolished in mitochondrial respiration-deficient Jurkat ρ(0) cells, confirming that pyrvinium acts on lymphoma T-cells via targeting mitochondrial respiration. We further show that lymphoma T-cells derived from patients depend more on mitochondrial respiration than normal T-cells, and this explains the selective toxicity of pyrvinium in lymphoma versus normal T-cells. Finally, we demonstrate that pyrvinium also suppresses JAK2/STAT5 signaling pathway in Jurkat cells. Our study suggests that pyrvinium is a useful addition to T-cell lymphoma treatment, and emphasizes the potential therapeutic value of the differences in the mitochondrial characteristics between malignant and normal T-cells in blood cancer. PMID:26707639

  10. Purging peripheral blood progenitor cell grafts from lymphoma cells: quantitative comparison of immunomagnetic CD34+ selection systems.

    PubMed

    Paulus, U; Dreger, P; Viehmann, K; von Neuhoff, N; Schmitz, N

    1997-01-01

    Autologous peripheral blood progenitor cell (PBPC) transplantation is increasingly being used for treatment of indolent lymphomas. Since involvement of bone marrow and peripheral blood is frequent and methods to reduce the lymphoma cell load of PBPC grafts are thus highly desirable, we have studied purging of PBPC comparing two immunomagnetic CD34+ selection systems (VarioMACS, Miltenyi Biotech; Bergisch Gladbach, Germany, and Isolex50 System, Baxter; Irvine, CA). Samples of freshly collected mobilized PBPCs were contaminated with BALM-3 or KARPAS422 lymphoma cells that had been labeled with the fluorescent DNA stain Hoechst 33342. The mixture was subjected to separation with the two devices and the resulting "CD34+" fractions were screened for lymphoma cells by limiting dilution using fluorescence microscopy and by polymerase chain reaction amplification of t(14;18) or CDRIII-rearrangements. Both devices yielded comparable purities (MACS 97% [87%-99%]; Isolex 97% [84%-99%]) and recoveries of CD34+ cells (MACS 56% [30%-81%]; Isolex 45% [24%-63%]). The overall depletion of lymphoma cells was 3.9 log (2.6-5.9), however, residual contaminating cells were seen in every single experiment. The purging efficacy was dependent on the type of contaminating lymphoma cell (BALM-3: 4.4 log [3.7-4.8]; KARPAS422: 3.2 log [2.6-4.2]; p = 0.018), whereas the type of selection system used or the percentage of CD34+ cells in the starting material had no influence. We conclude that excellent purification of CD34+ cells leading to a vigorous depletion of lymphoma cells can be achieved with both CD34+ selection systems investigated. However, the efficacy of purging may greatly differ between individual lymphomas, and complete eradication of contaminating cells from PBPC grafts may rarely be achieved with CD34+ selection alone. PMID:9253114

  11. Antibody repertoire deep sequencing reveals antigen-independent selection in maturing B cells

    PubMed Central

    Kaplinsky, Joseph; Li, Anthony; Sun, Amy; Coffre, Maryaline; Koralov, Sergei B.; Arnaout, Ramy

    2014-01-01

    Antibody repertoires are known to be shaped by selection for antigen binding. Unexpectedly, we now show that selection also acts on a non–antigen-binding antibody region: the heavy-chain variable (VH)–encoded “elbow” between variable and constant domains. By sequencing 2.8 million recombined heavy-chain genes from immature and mature B-cell subsets in mice, we demonstrate a striking gradient in VH gene use as pre-B cells mature into follicular and then into marginal zone B cells. Cells whose antibodies use VH genes that encode a more flexible elbow are more likely to mature. This effect is distinct from, and exceeds in magnitude, previously described maturation-associated changes in heavy-chain complementarity determining region 3, a key antigen-binding region, which arise from junctional diversity rather than differential VH gene use. Thus, deep sequencing reveals a previously unidentified mode of B-cell selection. PMID:24927543

  12. Impact of selection for decreased somatic cell score on productive life and culling for mastitis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Impact of continued selection for decreased somatic cell score (SCS) was examined to determine if such selection resulted in greater mastitis susceptibility and shorter productive life (PL). Holstein artificial-insemination bulls with a predicted transmitting ability (PTA) for SCS based on >=35 daug...

  13. Beyond Helper Phage: Using "Helper Cells" to Select Peptide Affinity Ligands.

    PubMed

    Phipps, M Lisa; Lillo, Antoinetta M; Shou, Yulin; Schmidt, Emily N; Paavola, Chad D; Naranjo, Leslie; Bemdich, Sara; Swanson, Basil I; Bradbury, Andrew R M; Martinez, Jennifer S

    2016-01-01

    Peptides are important affinity ligands for microscopy, biosensing, and targeted delivery. However, because they can have low affinity for their targets, their selection from large naïve libraries can be challenging. When selecting peptidic ligands from display libraries, it is important to: 1) ensure efficient display; 2) maximize the ability to select high affinity ligands; and 3) minimize the effect of the display context on binding. The "helper cell" packaging system has been described as a tool to produce filamentous phage particles based on phagemid constructs with varying display levels, while remaining free of helper phage contamination. Here we report on the first use of this system for peptide display, including the systematic characterization and optimization of helper cells, their inefficient use in antibody display and their use in creating and selecting from a set of phage display peptide libraries. Our libraries were analyzed with unprecedented precision by standard or deep sequencing, and shown to be superior in quality than commercial gold standards. Using our helper cell libraries, we have obtained ligands recognizing Yersinia pestis surface antigen F1V and L-glutamine-binding periplasmic protein QBP. In the latter case, unlike any of the peptide library selections described so far, we used a combination of phage and yeast display to select intriguing peptide ligands. Based on the success of our selections we believe that peptide libraries obtained with helper cells are not only suitable, but preferable to traditional phage display libraries for selection of peptidic ligands. PMID:27626637

  14. Benzothiophene inhibitors of MK2. Part 2: Improvements in kinase selectivity and cell potency

    SciTech Connect

    Anderson, David R.; Meyers, Marvin J.; Kurumbail, Ravi G.; Caspers, Nicole; Poda, Gennadiy I.; Long, Scott A.; Pierce, Betsy S.; Mahoney, Matthew W.; Mourey, Robert J.; Parikh, Mihir D.; Pfizer

    2010-10-01

    Optimization of kinase selectivity for a set of benzothiophene MK2 inhibitors provided analogs with potencies of less than 500 nM in a cell based assay. The selectivity of the inhibitors can be rationalized by examination of X-ray crystal structures of inhibitors bound to MK2.

  15. The Effect of BAFF Inhibition on Autoreactive B-Cell Selection in Murine Systemic Lupus Erythematosus

    PubMed Central

    Boneparth, Alexis; Woods, Megan; Huang, Weiqing; Akerman, Meredith; Lesser, Martin; Davidson, Anne

    2016-01-01

    The goal of this study was to determine how B-cell–activating factor of the TNF family (BAFF) availability influences selection of the autoreactive B-cell repertoire in NZB/W and NZW/BXSB lupus-prone mice bearing the site-directed heavy-chain transgene 3H9 that encodes for anti-dsDNA and anti-cardiolipin (CL) autoantibodies. We used a bone marrow chimera system in which autoreactive 3H9 transgenic B cells were allowed to mature in competition with wild-type cells and could be identified by green fluorescent protein. The light-chain repertoire associated with the 3H9 heavy chain in naive and antigen-activated B-cell subsets was assessed using single-cell polymerase chain reaction. We found that deletion of autoreactive transgenic B cells occurred in the bone marrow of both strains regardless of BAFF availability, and there were only modest and physiologically non-relevant effects on the naive B-cell repertoire. BAFF inhibition had different effects on selection of the germinal center repertoire in the two strains. In the NZW/BXSB strain, BAFF inhibition phenocopied the loss of one TLR7 allele in that it influenced the selection of 3H9-encoded autoreactive B cells in the germinal center but did not prevent somatic mutation. In the NZB/W strain, BAFF inhibition did not alter the selection of 3H9-encoded B cells in the germinal center, but it influenced selection of a subset of germinal center cells into the plasma cell compartment. Our data underscore the complexity of regulation of the autoreactive B-cell repertoire by BAFF and may help to explain the heterogeneity of responses observed after BAFF inhibition in humans. PMID:26882090

  16. Signalling thresholds and negative B-cell selection in acute lymphoblastic leukaemia.

    PubMed

    Chen, Zhengshan; Shojaee, Seyedmehdi; Buchner, Maike; Geng, Huimin; Lee, Jae Woong; Klemm, Lars; Titz, Björn; Graeber, Thomas G; Park, Eugene; Tan, Ying Xim; Satterthwaite, Anne; Paietta, Elisabeth; Hunger, Stephen P; Willman, Cheryl L; Melnick, Ari; Loh, Mignon L; Jung, Jae U; Coligan, John E; Bolland, Silvia; Mak, Tak W; Limnander, Andre; Jumaa, Hassan; Reth, Michael; Weiss, Arthur; Lowell, Clifford A; Müschen, Markus

    2015-05-21

    B cells are selected for an intermediate level of B-cell antigen receptor (BCR) signalling strength: attenuation below minimum (for example, non-functional BCR) or hyperactivation above maximum (for example, self-reactive BCR) thresholds of signalling strength causes negative selection. In ∼25% of cases, acute lymphoblastic leukaemia (ALL) cells carry the oncogenic BCR-ABL1 tyrosine kinase (Philadelphia chromosome positive), which mimics constitutively active pre-BCR signalling. Current therapeutic approaches are largely focused on the development of more potent tyrosine kinase inhibitors to suppress oncogenic signalling below a minimum threshold for survival. We tested the hypothesis that targeted hyperactivation--above a maximum threshold--will engage a deletional checkpoint for removal of self-reactive B cells and selectively kill ALL cells. Here we find, by testing various components of proximal pre-BCR signalling in mouse BCR-ABL1 cells, that an incremental increase of Syk tyrosine kinase activity was required and sufficient to induce cell death. Hyperactive Syk was functionally equivalent to acute activation of a self-reactive BCR on ALL cells. Despite oncogenic transformation, this basic mechanism of negative selection was still functional in ALL cells. Unlike normal pre-B cells, patient-derived ALL cells express the inhibitory receptors PECAM1, CD300A and LAIR1 at high levels. Genetic studies revealed that Pecam1, Cd300a and Lair1 are critical to calibrate oncogenic signalling strength through recruitment of the inhibitory phosphatases Ptpn6 (ref. 7) and Inpp5d (ref. 8). Using a novel small-molecule inhibitor of INPP5D (also known as SHIP1), we demonstrated that pharmacological hyperactivation of SYK and engagement of negative B-cell selection represents a promising new strategy to overcome drug resistance in human ALL. PMID:25799995

  17. Induction of tissue-specific stem cells by reprogramming factors, and tissue-specific selection.

    PubMed

    Noguchi, H; Saitoh, I; Tsugata, T; Kataoka, H; Watanabe, M; Noguchi, Y

    2015-01-01

    Although induced pluripotent stem (iPS) cells have significant implications for overcoming most of the ethical issues associated with embryonic stem (ES) cells, there are still several unresolved issues related to the use of iPS cells for clinical applications, such as teratoma formation. In this study, we were able to generate tissue-specific stem (induced tissue-specific stem; iTS) cells from the pancreas (iTS-P) or liver (iTS-L) by transient overexpression of reprogramming factors, combined with tissue-specific selection. The generation of iTS cells was easier than that of iPS cells. The iTS-P/iTS-L cells express genetic markers of endoderm and pancreatic/hepatic progenitors and were able to differentiate into insulin-producing cells/hepatocytes more efficiently than ES cells. Subcutaneous transplantation of both types of iTS cells into immunodeficient mice resulted in no teratoma formation. The technology used for the transient overexpression of reprogramming factors and tissue-specific selection may be useful for the generation of other tissue-specific stem cells, and the generation of iTS cells could have important implications for the clinical application of stem cells. PMID:25190146

  18. Selective culling of high avidity antigen-specific CD4+ T cells after virulent Salmonella infection

    PubMed Central

    Ertelt, James M; Johanns, Tanner M; Mysz, Margaret A; Nanton, Minelva R; Rowe, Jared H; Aguilera, Marijo N; Way, Sing Sing

    2011-01-01

    Typhoid fever is a persistent infection caused by host-adapted Salmonella strains adept at circumventing immune-mediated host defences. Given the importance of T cells in protection, the culling of activated CD4+ T cells after primary infection has been proposed as a potential immune evasion strategy used by this pathogen. We demonstrate that the purging of activated antigen-specific CD4+ T cells after virulent Salmonella infection requires SPI-2 encoded virulence determinants, and is not restricted only to cells with specificity to Salmonella-expressed antigens, but extends to CD4+ T cells primed to expand by co-infection with recombinant Listeria monocytogenes. Unexpectedly, however, the loss of activated CD4+ T cells during Salmonella infection demonstrated using a monoclonal population of adoptively transferred CD4+ T cells was not reproduced among the endogenous repertoire of antigen-specific CD4+ T cells identified with MHC class II tetramer. Analysis of T-cell receptor variable segment usage revealed the selective loss and reciprocal enrichment of defined CD4+ T-cell subsets after Salmonella co-infection that is associated with the purging of antigen-specific cells with the highest intensity of tetramer staining. Hence, virulent Salmonella triggers the selective culling of high avidity activated CD4+ T-cell subsets, which re-shapes the repertoire of antigen-specific T cells that persist later after infection. PMID:22044420

  19. Induction of tissue-specific stem cells by reprogramming factors, and tissue-specific selection

    PubMed Central

    Noguchi, H; Saitoh, I; Tsugata, T; Kataoka, H; Watanabe, M; Noguchi, Y

    2015-01-01

    Although induced pluripotent stem (iPS) cells have significant implications for overcoming most of the ethical issues associated with embryonic stem (ES) cells, there are still several unresolved issues related to the use of iPS cells for clinical applications, such as teratoma formation. In this study, we were able to generate tissue-specific stem (induced tissue-specific stem; iTS) cells from the pancreas (iTS-P) or liver (iTS-L) by transient overexpression of reprogramming factors, combined with tissue-specific selection. The generation of iTS cells was easier than that of iPS cells. The iTS-P/iTS-L cells express genetic markers of endoderm and pancreatic/hepatic progenitors and were able to differentiate into insulin-producing cells/hepatocytes more efficiently than ES cells. Subcutaneous transplantation of both types of iTS cells into immunodeficient mice resulted in no teratoma formation. The technology used for the transient overexpression of reprogramming factors and tissue-specific selection may be useful for the generation of other tissue-specific stem cells, and the generation of iTS cells could have important implications for the clinical application of stem cells. PMID:25190146

  20. Cell penetration and cell-selective drug delivery using α-helix peptides conjugated with gold nanoparticles.

    PubMed

    Park, Hyejin; Tsutsumi, Hiroshi; Mihara, Hisakazu

    2013-07-01

    Cell penetrating peptides (CPPs) have been developed as vectors for molecular delivery into various cells for use in drug delivery, gene therapy and cancer treatment by their property transporting various molecules into cytoplasm. CPPs with high internalization, cell specificity, and low cytotoxicity have been considered to increase the applicability for cell engineering. Gold nanospheres (GNSs) are a useful tool for molecular imaging, because they are non-cytotoxic and have high solubility, ease of synthesis and excellent light scattering property. Here, we investigated the cell penetrability using α-helix peptides of 17-amino acids conjugated to gold nanospheres (P-GNS). Depending on the peptide sequence had the different cell penetrating (CP) activity for three kinds of cell lines. P-GNS showed low cytotoxicity and high selectivity against three cell types, despite just one amino acid difference between the peptide. We studied the cytotoxic activity of an anti-cancer drug doxorubicin (DOX) conjugated to the P-GNS. They showed different cytotoxicity against the three cell lines, depending on the peptide sequence, with a higher efficiency than free DOX at the same concentration. The cytotoxicity by DOX was correlated with the CP activity of the peptides against the three cell lines. These results demonstrated that P-GNS would be a useful tool for the development of a new cell-selective drug delivery system. PMID:23545289

  1. Galectin-4 Controls Intestinal Inflammation by Selective Regulation of Peripheral and Mucosal T Cell Apoptosis and Cell Cycle

    PubMed Central

    Paclik, Daniela; Danese, Silvio; Berndt, Uta; Wiedenmann, Bertram; Dignass, Axel; Sturm, Andreas

    2008-01-01

    Galectin-4 is a carbohydrate-binding protein belonging to the galectin family. Here we provide novel evidence that galectin-4 is selectively expressed and secreted by intestinal epithelial cells and binds potently to activated peripheral and mucosal lamina propria T-cells at the CD3 epitope. The carbohydrate-dependent binding of galectin-4 at the CD3 epitope is fully functional and inhibited T cell activation, cycling and expansion. Galectin-4 induced apoptosis of activated peripheral and mucosal lamina propria T cells via calpain-, but not caspase-dependent, pathways. Providing further evidence for its important role in regulating T cell function, galectin-4 blockade by antisense oligonucleotides reduced TNF-alpha inhibitor induced T cell death. Furthermore, in T cells, galectin-4 reduced pro-inflammatory cytokine secretion including IL-17. In a model of experimental colitis, galectin-4 ameliorated mucosal inflammation, induced apoptosis of mucosal T-cells and decreased the secretion of pro-inflammatory cytokines. Our results show that galectin-4 plays a unique role in the intestine and assign a novel role of this protein in controlling intestinal inflammation by a selective induction of T cell apoptosis and cell cycle restriction. Conclusively, after defining its biological role, we propose Galectin-4 is a novel anti-inflammatory agent that could be therapeutically effective in diseases with a disturbed T cell expansion and apoptosis such as inflammatory bowel disease. PMID:18612433

  2. IL-15 inhibits pre-B cell proliferation by selectively expanding Mac-1{sup +}B220{sup +} NK cells

    SciTech Connect

    Nakajima, Shinsuke; Hida, Shigeaki; Taki, Shinsuke

    2008-05-16

    Natural killer (NK) cells are the cells critical for inhibition of repopulation of allogenic bone marrow cells. However, it is not well known if NK cells affect autologous lymphopoiesis. Here, we observed that NK cells could inhibit pre-B cell proliferation in vitro driven by interleukin (IL)-7 in a manner dependent on IL-15. Interestingly, the great majority of expanding NK cells were Mac-1{sup +}B220{sup +}, a recently identified potent interferon (IFN)-{gamma} producer. Indeed, IFN-{gamma} was produced in those cultures, and pre-B cells lacking IFN-{gamma} receptors, but not those lacking type I IFN receptors, were resistant to such an inhibition. Furthermore, even NK cells from mice lacking {beta}2-microglobulin, which were known to be functionally dampened, inhibited pre-B cell proliferation as well. Thus, activated NK cells, which were expanded selectively by IL-15, could potentially regulate B lymphopoiesis through IFN-{gamma} beyond the selection imposed upon self-recognition.

  3. A signal integration model of thymic selection and natural regulatory T cell commitment.

    PubMed

    Khailaie, Sahamoddin; Robert, Philippe A; Toker, Aras; Huehn, Jochen; Meyer-Hermann, Michael

    2014-12-15

    The extent of TCR self-reactivity is the basis for selection of a functional and self-tolerant T cell repertoire and is quantified by repeated engagement of TCRs with a diverse pool of self-peptides complexed with self-MHC molecules. The strength of a TCR signal depends on the binding properties of a TCR to the peptide and the MHC, but it is not clear how the specificity to both components drives fate decisions. In this study, we propose a TCR signal-integration model of thymic selection that describes how thymocytes decide among distinct fates, not only based on a single TCR-ligand interaction, but taking into account the TCR stimulation history. These fates are separated based on sustained accumulated signals for positive selection and transient peak signals for negative selection. This spans up the cells into a two-dimensional space where they are either neglected, positively selected, negatively selected, or selected as natural regulatory T cells (nTregs). We show that the dynamics of the integrated signal can serve as a successful basis for extracting specificity of thymocytes to MHC and detecting the existence of cognate self-peptide-MHC. It allows to select a self-MHC-biased and self-peptide-tolerant T cell repertoire. Furthermore, nTregs in the model are enriched with MHC-specific TCRs. This allows nTregs to be more sensitive to activation and more cross-reactive than conventional T cells. This study provides a mechanistic model showing that time integration of TCR-mediated signals, as opposed to single-cell interaction events, is needed to gain a full view on the properties emerging from thymic selection. PMID:25392533

  4. Strategies for selecting recombinant CHO cell lines for cGMP manufacturing: improving the efficiency of cell line generation.

    PubMed

    Porter, Alison J; Racher, Andrew J; Preziosi, Richard; Dickson, Alan J

    2010-01-01

    Transfectants with a wide range of cellular phenotypes are obtained during the process of cell line generation. For the successful manufacture of a therapeutic protein, a means is required to identify a cell line with desirable growth and productivity characteristics from this phenotypically wide-ranging transfectant population. This identification process is on the critical path for first-in-human studies. We have stringently examined a typical selection strategy used to isolate cell lines suitable for cGMP manufacturing. One-hundred and seventy-five transfectants were evaluated as they progressed through the different assessment stages of the selection strategy. High producing cell lines, suitable for cGMP manufacturing, were identified. However, our analyses showed that the frequency of isolation of the highest producing cell lines was low and that ranking positions were not consistent between each assessment stage, suggesting that there is potential to improve upon the strategy. Attempts to increase the frequency of isolation of the 10 highest producing cell lines, by in silico analysis of alternative selection strategies, were unsuccessful. We identified alternative strategies with similar predictive capabilities to the typical selection strategy. One alternate strategy required fewer cell lines to be progressed at the assessment stages but the stochastic nature of the models means that cell line numbers are likely to change between programs. In summary, our studies illuminate the potential for improvement to this and future selection strategies, based around use of assessments that are more informative or that reduce variance, paving the way to improved efficiency of generation of manufacturing cell lines. PMID:20623584

  5. Sickle cell disease: Selected aspects of pathophysiology1

    PubMed Central

    Alexy, T.; Sangkatumvong, S.; Connes, P.; Pais, E.; Tripette, J.; Barthelemy, J.C.; Fisher, T.C.; Meiselman, H.J.; Khoo, M.C.; Coates, T.D.

    2010-01-01

    Sickle cell disease (SCD), a genetically-determined pathology due to an amino acid substitution (i.e., valine for glutamic acid) on the beta-chain of hemoglobin, is characterized by abnormal blood rheology and periods of painful vascular occlusive crises. Sickle cell trait (SCT) is a typically benign variant in which only one beta chain is affected by the mutation. Although both SCD and SCT have been the subject of numerous studies, information related to neurological function and transfusion therapy is still incomplete: an overview of these areas is presented. An initial section provides pertinent background information on the pathology and clinical significance of these diseases. The roles of three factors in the clinical manifestations of the diseases are then discussed: hypoxia, autonomic nervous system regulation and blood rheology. The possibility of a causal relationship between these three factors and sudden death is also examined. It is concluded that further studies in these specific areas are warranted. It is anticipated that the outcome of such research is likely to provide valuable insights into the pathophysiology of SCD and SCT and will lead to improved clinical management and enhanced quality of life. PMID:20364061

  6. Selective advantage of trisomic human cells cultured in non-standard conditions.

    PubMed

    Rutledge, Samuel D; Douglas, Temple A; Nicholson, Joshua M; Vila-Casadesús, Maria; Kantzler, Courtney L; Wangsa, Darawalee; Barroso-Vilares, Monika; Kale, Shiv D; Logarinho, Elsa; Cimini, Daniela

    2016-01-01

    An abnormal chromosome number, a condition known as aneuploidy, is a ubiquitous feature of cancer cells. A number of studies have shown that aneuploidy impairs cellular fitness. However, there is also evidence that aneuploidy can arise in response to specific challenges and can confer a selective advantage under certain environmental stresses. Cancer cells are likely exposed to a number of challenging conditions arising within the tumor microenvironment. To investigate whether aneuploidy may confer a selective advantage to cancer cells, we employed a controlled experimental system. We used the diploid, colorectal cancer cell line DLD1 and two DLD1-derived cell lines carrying single-chromosome aneuploidies to assess a number of cancer cell properties. Such properties, which included rates of proliferation and apoptosis, anchorage-independent growth, and invasiveness, were assessed both under standard culture conditions and under conditions of stress (i.e., serum starvation, drug treatment, hypoxia). Similar experiments were performed in diploid vs. aneuploid non-transformed human primary cells. Overall, our data show that aneuploidy can confer selective advantage to human cells cultured under non-standard conditions. These findings indicate that aneuploidy can increase the adaptability of cells, even those, such as cancer cells, that are already characterized by increased proliferative capacity and aggressive tumorigenic phenotypes. PMID:26956415

  7. Selective advantage of trisomic human cells cultured in non-standard conditions

    PubMed Central

    Rutledge, Samuel D.; Douglas, Temple A.; Nicholson, Joshua M.; Vila-Casadesús, Maria; Kantzler, Courtney L.; Wangsa, Darawalee; Barroso-Vilares, Monika; Kale, Shiv D.; Logarinho, Elsa; Cimini, Daniela

    2016-01-01

    An abnormal chromosome number, a condition known as aneuploidy, is a ubiquitous feature of cancer cells. A number of studies have shown that aneuploidy impairs cellular fitness. However, there is also evidence that aneuploidy can arise in response to specific challenges and can confer a selective advantage under certain environmental stresses. Cancer cells are likely exposed to a number of challenging conditions arising within the tumor microenvironment. To investigate whether aneuploidy may confer a selective advantage to cancer cells, we employed a controlled experimental system. We used the diploid, colorectal cancer cell line DLD1 and two DLD1-derived cell lines carrying single-chromosome aneuploidies to assess a number of cancer cell properties. Such properties, which included rates of proliferation and apoptosis, anchorage-independent growth, and invasiveness, were assessed both under standard culture conditions and under conditions of stress (i.e., serum starvation, drug treatment, hypoxia). Similar experiments were performed in diploid vs. aneuploid non-transformed human primary cells. Overall, our data show that aneuploidy can confer selective advantage to human cells cultured under non-standard conditions. These findings indicate that aneuploidy can increase the adaptability of cells, even those, such as cancer cells, that are already characterized by increased proliferative capacity and aggressive tumorigenic phenotypes. PMID:26956415

  8. Select plant tannins induce IL-2Ralpha up-regulation and augment cell division in gammadelta T cells.

    PubMed

    Holderness, Jeff; Jackiw, Larissa; Kimmel, Emily; Kerns, Hannah; Radke, Miranda; Hedges, Jodi F; Petrie, Charles; McCurley, Patrick; Glee, Pati M; Palecanda, Aiyappa; Jutila, Mark A

    2007-11-15

    Gammadelta T cells are innate immune cells that participate in host responses against many pathogens and cancers. Recently, phosphoantigen-based drugs, capable of expanding gammadelta T cells in vivo, entered clinical trials with the goal of enhancing innate immune system functions. Potential shortcomings of these drugs include the induction of nonresponsiveness upon repeated use and the expansion of only the Vdelta2 subset of human gammadelta T cells. Vdelta1 T cells, the major tissue subset, are unaffected by phosphoantigen agonists. Using FACS-based assays, we screened primary bovine cells for novel gammadelta T cell agonists with activities not encompassed by the current treatments in an effort to realize the full therapeutic potential of gammadelta T cells. We identified gammadelta T cell agonists derived from the condensed tannin fractions of Uncaria tomentosa (Cat's Claw) and Malus domestica (apple). Based on superior potency, the apple extract was selected for detailed analyses on human cells. The apple extract was a potent agonist for both human Vdelta1 and Vdelta2 T cells and NK cells. Additionally, the extract greatly enhanced phosphoantigen-induced gammadelta T cell expansion. Our analyses suggest that a tannin-based drug may complement the phosphoantigen-based drugs, thereby enhancing the therapeutic potential of gammadelta T cells. PMID:17982035

  9. Selection of representative volume elements for pore-scale analysis of transport in fuel cell materials

    NASA Astrophysics Data System (ADS)

    Wargo, E. A.; Hanna, A. C.; Çeçen, A.; Kalidindi, S. R.; Kumbur, E. C.

    2012-01-01

    Pore-scale modeling has become a quite popular tool for evaluating the impact of material structure on fuel cell performance. However, the computational complexity of these models often limits simulations to analyze only a small volume of material, which is typically selected randomly from a much larger microstructure dataset. When considering the heterogeneous internal structure of fuel cell materials, it is highly unlikely that such a randomly selected volume (i.e., model domain) would adequately reflect the salient features of the material structure. The objective of this work is to utilize the recent advances in microstructure quantification to select small representative volume elements (RVEs) that accurately reflect the overall microstructure and transport properties of fuel cell materials. The micro-porous layer (MPL) in polymer electrolyte fuel cells is chosen for initial demonstration of the approach. Dual-beam focused ion beam scanning electron microscopy is utilized to obtain a 3-D structural dataset of the selected MPL sample. The RVEs are selected using the new approach of weighted sets of optimally selected statistical volume elements, and the key structure and transport metrics are evaluated using advanced microstructure algorithms developed in-house. Metric comparisons between the RVEs and the full dataset indicate that the RVEs selected by this approach offer a very good representation of the full dataset, albeit in a volume that is significantly smaller in spatial extent, therefore providing a computationally efficient and reliable model domain for pore-scale analyses.

  10. Selective Gene Transfection of Individual Cells In Vitro with Plasmonic Nanobubbles

    PubMed Central

    Lukianova-Hleb, Ekaterina; Samaniego, Adam P.; Wen, Jianguo; Metelitsa, Leonid; Chang, Chung-Che; Lapotko, Dmitri

    2011-01-01

    Gene delivery and transfection of eukaryotic cells is widely used for research and for developing gene cell therapy. However, the existing methods lack selectivity, efficacy and safety when heterogeneous cell systems must be treated. We report a new method that employs plasmonic nanobubbles (PNBs) for delivery and transfection. A PNB is a novel, tunable cellular agent with a dual mechanical and optical action due to the formation of the vapor nanobubble around a transiently heated gold nanoparticle upon its exposure to a laser pulse. PNBs enabled the mechanical injection of the extracellular cDNA plasmid into the cytoplasm of individual target living cells, cultured leukemia cells and human CD34+CD117+ stem cells and expression of a green fluorescent protein (GFP) in those cells. PNB generation and lifetime correlated with the expression of green fluorescent protein in PNB-treated cells. Optical scattering by PNBs additionally provided the detection of the target cells and the guidance of cDNA injection at single cell level. In both cell models PNBs demonstrated a gene transfection effect in a single pulse treatment with high selectivity, efficacy and safety. Thus, PNBs provided targeted gene delivery at the single cell level in a single pulse procedure that can be used for safe and effective gene therapy. PMID:21315120

  11. Selective effect of cell membrane on synaptic neurotransmission.

    PubMed

    Postila, Pekka A; Vattulainen, Ilpo; Róg, Tomasz

    2016-01-01

    Atomistic molecular dynamics simulations were performed with 13 non-peptidic neurotransmitters (NTs) in three different membrane environments. The results provide compelling evidence that NTs are divided into membrane-binding and membrane-nonbinding molecules. NTs adhere to the postsynaptic membrane surface whenever the ligand-binding sites of their synaptic receptors are buried in the lipid bilayer. In contrast, NTs that have extracellular ligand-binding sites do not have a similar tendency to adhere to the membrane surface. This finding is a seemingly simple yet important addition to the paradigm of neurotransmission, essentially dividing it into membrane-independent and membrane-dependent mechanisms. Moreover, the simulations also indicate that the lipid composition especially in terms of charged lipids can affect the membrane partitioning of NTs. The revised paradigm, highlighting the importance of cell membrane and specific lipids for neurotransmission, should to be of interest to neuroscientists, drug industry and the general public alike. PMID:26782980

  12. Selective effect of cell membrane on synaptic neurotransmission

    NASA Astrophysics Data System (ADS)

    Postila, Pekka A.; Vattulainen, Ilpo; Róg, Tomasz

    2016-01-01

    Atomistic molecular dynamics simulations were performed with 13 non-peptidic neurotransmitters (NTs) in three different membrane environments. The results provide compelling evidence that NTs are divided into membrane-binding and membrane-nonbinding molecules. NTs adhere to the postsynaptic membrane surface whenever the ligand-binding sites of their synaptic receptors are buried in the lipid bilayer. In contrast, NTs that have extracellular ligand-binding sites do not have a similar tendency to adhere to the membrane surface. This finding is a seemingly simple yet important addition to the paradigm of neurotransmission, essentially dividing it into membrane-independent and membrane-dependent mechanisms. Moreover, the simulations also indicate that the lipid composition especially in terms of charged lipids can affect the membrane partitioning of NTs. The revised paradigm, highlighting the importance of cell membrane and specific lipids for neurotransmission, should to be of interest to neuroscientists, drug industry and the general public alike.

  13. Fine tuning of the threshold of T cell selection by the Nck adapters.

    PubMed

    Roy, Edwige; Togbe, Dieudonnée; Holdorf, Amy; Trubetskoy, Dmitry; Nabti, Sabrina; Küblbeck, Günter; Schmitt, Sabine; Kopp-Schneider, Annette; Leithäuser, Frank; Möller, Peter; Bladt, Friedhelm; Hämmerling, Günter J; Arnold, Bernd; Pawson, Tony; Tafuri, Anna

    2010-12-15

    Thymic selection shapes the T cell repertoire to ensure maximal antigenic coverage against pathogens while preventing autoimmunity. Recognition of self-peptides in the context of peptide-MHC complexes by the TCR is central to this process, which remains partially understood at the molecular level. In this study we provide genetic evidence that the Nck adapter proteins are essential for thymic selection. In vivo Nck deletion resulted in a reduction of the thymic cellularity, defective positive selection of low-avidity T cells, and impaired deletion of thymocytes engaged by low-potency stimuli. Nck-deficient thymocytes were characterized by reduced ERK activation, particularly pronounced in mature single positive thymocytes. Taken together, our findings identify a crucial role for the Nck adapters in enhancing TCR signal strength, thereby fine-tuning the threshold of thymocyte selection and shaping the preimmune T cell repertoire. PMID:21078909

  14. Selective, rapid and optically switchable regulation of protein function in live mammalian cells

    NASA Astrophysics Data System (ADS)

    Tsai, Yu-Hsuan; Essig, Sebastian; James, John R.; Lang, Kathrin; Chin, Jason W.

    2015-07-01

    The rapid and selective regulation of a target protein within living cells that contain closely related family members is an outstanding challenge. Here we introduce genetically directed bioorthogonal ligand tethering (BOLT) and demonstrate selective inhibition (iBOLT) of protein function. In iBOLT, inhibitor-conjugate/target protein pairs are created where the target protein contains a genetically encoded unnatural amino acid with bioorthogonal reactivity and the inhibitor conjugate contains a complementary bioorthogonal group. iBOLT enables the first rapid and specific inhibition of MEK isozymes, and introducing photoisomerizable linkers in the inhibitor conjugate enables reversible, optical regulation of protein activity (photo-BOLT) in live mammalian cells. We demonstrate that a pan kinase inhibitor conjugate allows selective and rapid inhibition of the lymphocyte specific kinase, indicating the modularity and scalability of BOLT. We anticipate that BOLT will enable the rapid and selective regulation of diverse proteins for which no selective small-molecule ligands exist.

  15. Active and Selective Transcytosis of Cell-Free Human Immunodeficiency Virus through a Tight Polarized Monolayer of Human Endometrial Cells

    PubMed Central

    Hocini, Hakim; Becquart, Pierre; Bouhlal, Hicham; Chomont, Nicolas; Ancuta, Petronela; Kazatchkine, Michel D.; Bélec, Laurent

    2001-01-01

    We report that both primary and laboratory-adapted infectious human immunodeficiency virus type 1 (HIV-1) isolates in a cell-free form are capable of transcytosis through a tight and polarized monolayer of human endometrial cells. Trancytosis of cell-free HIV occurs in a strain-selective fashion and appears to be dependent on interactions between HIV envelope glycoproteins and lectins on the apical membrane of the epithelial cells. These findings provide new insights into the initial events occurring during heterosexual transmission of the virus. PMID:11333919

  16. Cell surface thiol isomerases may explain the platelet-selective action of S-nitrosoglutathione.

    PubMed

    Xiao, Fang; Gordge, Michael P

    2011-10-30

    S-nitrosoglutathione (GSNO) at low concentration inhibits platelet aggregation without causing vasodilation, suggesting platelet-selective nitric oxide delivery. The mechanism of this selectivity is unknown, but may involve cell surface thiol isomerases, in particular protein disulphide isomerase (csPDI) (EC 5.3.4.1). We have now compared csPDI expression and activity on platelets, endothelial cells and vascular smooth muscle cells, and the dependence on thiol reductase activity of these cell types for NO uptake from GSNO. csPDI expression was measured by flow cytometry and its reductase activity using the pseudosubstrate dieosin glutathione disulphide. This activity assay was adapted and validated for 96-well plate format. Flow cytometry revealed csPDI on all three cell types, but percentage positivity of expression was higher on platelets than on vascular cells. Consistent with this, thiol isomerase-related reductase activity was higher on platelets (P<0.01), and cellular activation (with either phorbol myristate acetate or ionomycin) increased csPDI activity on both platelets and smooth muscle cells, but not on endothelium. Intracellular NO delivery from GSNO was greater in platelets than in vascular cells (P<0.002), and was more sensitive to thiol isomerase inhibition using phenylarsine oxide (P<0.05). Increased surface thiol isomerase activity on platelets, compared with cells of the vascular wall, may explain the platelet-selective actions of GSNO and help define its antithrombotic potential. PMID:21642008

  17. Evodiamine selectively targets cancer stem-like cells through the p53-p21-Rb pathway.

    PubMed

    Han, Seula; Woo, Jong Kyu; Jung, Yuchae; Jeong, Dawoon; Kang, Minsook; Yoo, Young-Ji; Lee, Hani; Oh, Seung Hyun; Ryu, Jae-Ha; Kim, Woo-Young

    2016-01-22

    In spite of the recent improvements, the resistance to chemotherapy/radiotherapy followed by relapse is the main hurdle for the successful treatment of breast cancer, a leading cause of death in women. A small population of breast cancer cells that have stem-like characteristics (cancer stem-like cells; CSLC) may contribute to this resistance and relapse. Here, we report on a component of a traditional Chinese medicine, evodiamine, which selectively targets CSLC of breast cancer cell lines MCF7 and MDAMB 231 at a concentration that does show a little or no cytotoxic effect on bulk cancer cells. While evodiamine caused the accumulation of bulk cancer cells at the G2/M phase, it did not hold CSLC in a specific cell cycle phase but instead, selectively killed CSLC. This was not due to the culture of CSLC in suspension or without FBS. A proteomic analysis and western blotting revealed that evodiamine changed the expression of cell cycle regulating molecules more efficiently in CSLC cells than in bulk cancer cells. Surprisingly, evodiamine selectively activated p53 and p21 and decreased inactive Rb, the master molecules in G1/S checkpoint. These data collectively suggest a novel mechanism involving CSLC-specific targeting by evodiamine and its possible use to the therapy of breast cancer. PMID:26713361

  18. Inhibins Tune the Thymocyte Selection Process by Regulating Thymic Stromal Cell Differentiation

    PubMed Central

    Carbajal-Franco, Ebzadrel; de la Fuente-Granada, Marisol; Alemán-Muench, Germán R.; García-Zepeda, Eduardo A.; Soldevila, Gloria

    2015-01-01

    Inhibins and Activins are members of the TGF-β superfamily that regulate the differentiation of several cell types. These ligands were initially identified as hormones that regulate the hypothalamus-pituitary-gonadal axis; however, increasing evidence has demonstrated that they are key regulators in the immune system. We have previously demonstrated that Inhibins are the main Activin ligands expressed in the murine thymus and that they regulate thymocyte differentiation, promoting the DN3-DN4 transition and the selection of SP thymocytes. As Inhibins are mainly produced by thymic stromal cells, which also express Activin receptors and Smad proteins, we hypothesized that Inhibins might play a role in stromal cell differentiation and function. Here, we demonstrate that, in the absence of Inhibins, thymic conventional dendritic cells display reduced levels of MHC Class II (MHCII) and CD86. In addition, the ratio between cTECs and mTECs was affected, indicating that mTEC differentiation was favoured and cTEC diminished in the absence of Inhibins. These changes appeared to impact thymocyte selection leading to a decreased selection of CD4SP thymocytes and increased generation of natural regulatory T cells. These findings demonstrate that Inhibins tune the T cell selection process by regulating both thymocyte and stromal cell differentiation. PMID:25973437

  19. Selection and expansion of natural killer cells for NK cell-based immunotherapy.

    PubMed

    Becker, Petra S A; Suck, Garnet; Nowakowska, Paulina; Ullrich, Evelyn; Seifried, Erhard; Bader, Peter; Tonn, Torsten; Seidl, Christian

    2016-04-01

    Natural killer (NK) cells have been used in several clinical trials as adaptive immunotherapy. The low numbers of these cells in peripheral blood mononuclear cells (PBMC) have resulted in various approaches to preferentially expand primary NK cells from PBMC. While some clinical trials have used the addition of interleukin 2 (IL-2) to co-stimulate the expansion of purified NK cells from allogeneic donors, recent studies have shown promising results in achieving in vitro expansion of NK cells to large numbers for adoptive immunotherapy. NK cell expansion requires multiple cell signals for survival, proliferation and activation. Thus, expansion strategies have been focused either to substitute these factors using autologous feeder cells or to use genetically modified allogeneic feeder cells. Recent developments in the clinical use of genetically modified NK cell lines with chimeric antigen receptors, the development of expansion protocols for the clinical use of NK cell from human embryonic stem cells and induced pluripotent stem cells are challenging improvements for NK cell-based immunotherapy. Transfer of several of these protocols to clinical-grade production of NK cells necessitates adaptation of good manufacturing practice conditions, and the development of freezing conditions to establish NK cell stocks will require some effort and, however, should enhance the therapeutic options of NK cells in clinical medicine. PMID:26810567

  20. Optofluidic Cell Selection from Complex Microbial Communities for Single-Genome Analysis

    PubMed Central

    Landry, Zachary C.; Giovanonni, Stephen J.; Quake, Stephen R.; Blainey, Paul C.

    2013-01-01

    Genetic analysis of single cells is emerging as a powerful approach for studies of heterogeneous cell populations. Indeed, the notion of homogeneous cell populations is receding as approaches to resolve genetic and phenotypic variation between single cells are applied throughout the life sciences. A key step in single-cell genomic analysis today is the physical isolation of individual cells from heterogeneous populations, particularly microbial populations, which often exhibit high diversity. Here, we detail the construction and use of instrumentation for optical trapping inside microfluidic devices to select individual cells for analysis by methods including nucleic acid sequencing. This approach has unique advantages for analyses of rare community members, cells with irregular morphologies, small quantity samples, and studies that employ advanced optical microscopy. PMID:24060116

  1. Novel Pyrrolidine Diketopiperazines Selectively Inhibit Melanoma Cells via Induction of Late-Onset Apoptosis

    PubMed Central

    2015-01-01

    A common liability of cancer drugs is toxicity to noncancerous cells. Thus, molecules are needed that are potent toward cancer cells while sparing healthy cells. The cost of traditional cell-based HTS is dictated by the library size, which is typically in the hundreds of thousands of individual compounds. Mixture-based combinatorial libraries offer a cost-effective alternative to single-compound libraries while eliminating the need for molecular target validation. Presently, lung cancer and melanoma cells were screened in parallel with healthy cells using a mixture-based library. A novel class of compounds was discovered that selectively inhibited melanoma cell growth via apoptosis with submicromolar potency while sparing healthy cells. Additionally, the cost of screening and biological follow-up experiments was significantly lower than in typical HTS. Our findings suggest that mixture-based phenotypic HTS can significantly reduce cost and hit-to-lead time while yielding novel compounds with promising pharmacology. PMID:24471466

  2. Positive selection of B10 cells is determined by BCR specificity and signaling strength.

    PubMed

    Zhang, Jigang; Wan, Ming; Ren, Jing; Gao, Jixin; Fu, Meng; Wang, Gang; Liu, Yufeng; Li, Wei

    2016-01-01

    B10 cells, a regulatory B cell subset, negatively regulate immune responses in an IL-10-dependent manner. However, the mechanism of B10 cell development is unclear. We found that B10 cells mainly identified self-antigens. TgVH3B4 transgenic mice, whose VH was derived from an actin-reactive natural antibody, exhibit elevated numbers of actin-binding B10 cells. Immunization of TgVH3B4 mice with actin induced elevated B10 cell numbers in an antigen-specific manner, indicating positive selection of B10 cells by self-antigens. Furthermore, higher BCR signaling strength facilitated B10 cell development. We also observed that actin-reactive IgG levels were unchanged in TgVH3B4 mice after immunization with actin in contrast to the elevated OVA-reactive IgG level after immunization with OVA, indicating that B10 cells acted in an antigen-specific manner to inhibit the immune response. Our data demonstrate for the first time that B10 cells are positively selected by self-reactivity and that higher BCR signaling strength promotes B10 cell development. PMID:27132875

  3. Cdc42 explores the cell periphery for mate selection in fission yeast.

    PubMed

    Bendezú, Felipe O; Martin, Sophie G

    2013-01-01

    How cells polarize in response to external cues is a fundamental biological problem. For mating, yeast cells orient growth toward the source of a pheromone gradient produced by cells of the opposite mating type. Polarized growth depends on the small GTPase Cdc42, a central eukaryotic polarity regulator that controls signaling, cytoskeleton polarization, and vesicle trafficking. However, the mechanisms of polarity establishment and mate selection in complex cellular environments are poorly understood. Here we show that, in fission yeast, low-level pheromone signaling promotes a novel polarization state, where active Cdc42, its GEF Scd1, and scaffold Scd2 form colocalizing dynamic zones that sample the periphery of the cell. Two direct Cdc42 effectors--actin cables marked by myosin V Myo52 and the exocyst complex labeled by Sec6 and Sec8--also dynamically colocalize with active Cdc42. However, these cells do not grow due to a block in the exocytosis of cell wall synthases Bgs1 and Bgs4. High-level pheromone stabilizes active Cdc42 zones and promotes cell wall synthase exocytosis and polarized growth. However, in the absence of prior low-level pheromone signaling, exploration fails, and cells polarize growth at cell poles by default. Consequently, these cells show altered partner choice, mating preferentially with sister rather than nonsister cells. Thus, Cdc42 exploration serves to orient growth for partner selection. This process may also promote genetic diversification. PMID:23200991

  4. Suppression of cancer-initiating cells and selection of adipose-derived stem cells cultured on biomaterials having specific nanosegments.

    PubMed

    Kao, Ta-Chun; Lee, Henry Hsin-Chung; Higuchi, Akon; Ling, Qing-Dong; Yu, Wan-Chun; Chou, Yu-Hsuan; Wang, Pin-Yu; Suresh Kumar, S; Chang, Yu; Hung Chen, Yung; Chang, Yung; Chen, Da-Chung; Hsu, Shih-Tien

    2014-04-01

    Cancer-initiating cells [cancer stem cells (CSCs)] in colon cancer cells can be selectively suppressed when they are cultured on Pluronic (nanosegment)-grafted dishes, whereas CSCs are maintained on conventional tissue culture dishes and extracellular matrix-coated dishes. CSCs persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumorigenic clones. The purification or depletion (suppression) of CSCs should be useful for analyzing CSC characteristics and for clinical application. CSCs can be selectively suppressed from colon cancer cells containing adipose-derived stem cells (ADSCs) on Pluronic-grafted dishes, while ADSCs remain on the dishes. ADSCs on Pluronic-grafted dishes after the suppression of the CSCs can differentiate into osteoblasts, chondrocytes, adipocytes, cardiomyocytes, and neuronal cells. The CSCs and ADSCs exhibited different characteristics. The selection of ADSCs was possible on Pluronic-grafted dishes that suppressed the CSCs from the fat tissues of cancer patients (i.e., cell-sorting dishes), which was explained by specific biomedical characteristics of Pluronic. PMID:24039170

  5. In vivo selection of human embryonic stem cell-derived cells expressing methotrexate-resistant dihydrofolate reductase

    PubMed Central

    Gori, Jennifer L.; Tian, Xinghui; Swanson, Debra; Gunther, Roland; Shultz, Leonard D.; McIvor, R. Scott; Kaufman, Dan S.

    2009-01-01

    SUMMARY Human embryonic stem cells (hESCs) provide a novel source of hematopoietic and other cell populations suitable for gene therapy applications. Preclinical studies to evaluate engraftment of hESC-derived hematopoietic cells transplanted into immunodeficient mice demonstrate only limited repopulation. Expression of a drug resistance gene, such as Tyr22-dihydrofolate reductase (Tyr22-DHFR), coupled to methotrexate (MTX) chemotherapy has the potential to selectively increase engraftment of gene-modified hESC-derived cells in mouse xenografts. Here, we describe the generation of Tyr22-DHFR – GFP expressing hESCs that maintain pluripotency, produce teratomas and can differentiate into MTXr-hemato-endothelial cells. We demonstrate that MTX administered to nonobese diabetic/severe combined immunodeficient/IL-2Rγcnull (NSG) mice after injection of Tyr22-DHFR-derived cells significantly increases human CD34+ and CD45+ cell engraftment in the bone marrow (BM) and peripheral blood of transplanted MTX-treated mice. These results demonstrate that MTX treatment supports selective, long-term engraftment of Tyr22-DHFR-cells in vivo, and provides a novel approach for combined human cell and gene therapy. PMID:19829316

  6. Infection Profiles of Selected Aquabirnavirus Isolates in CHSE Cells

    PubMed Central

    Gamil, Amr A. A.; Evensen, Øystein; Mutoloki, Stephen

    2015-01-01

    The wide host range and antigenic diversity of aquabirnaviruses are reflected by the presence of a collection of isolates with different sero- and genotypic properties that have previously been classified as such. Differences in cytopathogenic mechanisms and host responses induced by these isolates have not been previously examined. In the present study, we investigated infection profiles induced by genetically and serologically closely related as well as distant isolates in-vitro. CHSE-214 cells were infected with either E1S (serotype A3, genogroup 3), VR-299 (serotype A1, genogroup 1), highly virulent Sp (TA) or avirulent Sp (PT) (serotype A2, genogroup 5). The experiments were performed at temperatures most optimum for each of the isolates namely 15°C for VR-299, TA and PT strains and 20°C for E1S. Differences in virus loads and ability to induce cytopathic effect, inhibition of protein synthesis, apoptosis, and induction of IFNa, Mx1, PKR or TNFα gene expression at different times post infection were examined. The results showed on one hand, E1S with the highest ability to replicate, induce apoptosis and IFNa gene expression while VR-299 inhibited protein synthesis and induced Mx1 and PKR gene expression the most. The two Sp isolates induced the highest TNFα gene expression but differed in their ability to replicate, inhibit protein synthesis, and induce gene expression, with TA being more superior. Collectively, these findings point towards the adaptation by different virus isolates to suit environments and hosts that they patronize. Furthermore, the results also suggest that genetic identity is not prerequisite to functional similarities thus results of one aquabirnavirus isolate cannot necessarily be extrapolated to another. PMID:26263557

  7. Stimulus design for model selection and validation in cell signaling.

    PubMed

    Apgar, Joshua F; Toettcher, Jared E; Endy, Drew; White, Forest M; Tidor, Bruce

    2008-02-01

    Mechanism-based chemical kinetic models are increasingly being used to describe biological signaling. Such models serve to encapsulate current understanding of pathways and to enable insight into complex biological processes. One challenge in model development is that, with limited experimental data, multiple models can be consistent with known mechanisms and existing data. Here, we address the problem of model ambiguity by providing a method for designing dynamic stimuli that, in stimulus-response experiments, distinguish among parameterized models with different topologies, i.e., reaction mechanisms, in which only some of the species can be measured. We develop the approach by presenting two formulations of a model-based controller that is used to design the dynamic stimulus. In both formulations, an input signal is designed for each candidate model and parameterization so as to drive the model outputs through a target trajectory. The quality of a model is then assessed by the ability of the corresponding controller, informed by that model, to drive the experimental system. We evaluated our method on models of antibody-ligand binding, mitogen-activated protein kinase (MAPK) phosphorylation and de-phosphorylation, and larger models of the epidermal growth factor receptor (EGFR) pathway. For each of these systems, the controller informed by the correct model is the most successful at designing a stimulus to produce the desired behavior. Using these stimuli we were able to distinguish between models with subtle mechanistic differences or where input and outputs were multiple reactions removed from the model differences. An advantage of this method of model discrimination is that it does not require novel reagents, or altered measurement techniques; the only change to the experiment is the time course of stimulation. Taken together, these results provide a strong basis for using designed input stimuli as a tool for the development of cell signaling models. PMID

  8. Tocotrienol-rich fraction of palm oil induces cell cycle arrest and apoptosis selectively in human prostate cancer cells

    SciTech Connect

    Srivastava, Janmejai K.; Gupta, Sanjay . E-mail: sanjay.gupta@case.edu

    2006-07-28

    One of the requisite of cancer chemopreventive agent is elimination of damaged or malignant cells through cell cycle inhibition or induction of apoptosis without affecting normal cells. In this study, employing normal human prostate epithelial cells (PrEC), virally transformed normal human prostate epithelial cells (PZ-HPV-7), and human prostate cancer cells (LNCaP, DU145, and PC-3), we evaluated the growth-inhibitory and apoptotic effects of tocotrienol-rich fraction (TRF) extracted from palm oil. TRF treatment to PrEC and PZ-HPV-7 resulted in almost identical growth-inhibitory responses of low magnitude. In sharp contrast, TRF treatment resulted in significant decreases in cell viability and colony formation in all three prostate cancer cell lines. The IC{sub 5} values after 24 h TRF treatment in LNCaP, PC-3, and DU145 cells were in the order 16.5, 17.5, and 22.0 {mu}g/ml. TRF treatment resulted in significant apoptosis in all the cell lines as evident from (i) DNA fragmentation (ii) fluorescence microscopy, and (iii) cell death detection ELISA, whereas the PrEC and PZ-HPV-7 cells did not undergo apoptosis, but showed modestly decreased cell viability only at a high dose of 80 {mu}g/ml. In cell cycle analysis, TRF (10-40 {mu}g/ml) resulted in a dose-dependent G0/G1 phase arrest and sub G1 accumulation in all three cancer cell lines but not in PZ-HPV-7 cells. These results suggest that the palm oil derivative TRF is capable of selectively inhibiting cellular proliferation and accelerating apoptotic events in prostate cancer cells. TRF offers significant promise as a chemopreventive and/or therapeutic agent against prostate cancer.

  9. T cell-specific inhibition of multiple apoptotic pathways blocks negative selection and causes autoimmunity

    PubMed Central

    Burger, Megan L; Leung, Kenneth K; Bennett, Margaux J; Winoto, Astar

    2014-01-01

    T cell self-tolerance is thought to involve peripheral tolerance and negative selection, involving apoptosis of autoreactive thymocytes. However, evidence supporting an essential role for negative selection is limited. Loss of Bim, a Bcl-2 BH3-only protein essential for thymocyte apoptosis, rarely results in autoimmunity on the C57BL/6 background. Mice with T cell-specific over-expression of Bcl-2, that blocks multiple BH3-only proteins, are also largely normal. The nuclear receptor Nur77, also implicated in negative selection, might function redundantly to promote apoptosis by associating with Bcl-2 and exposing its potentially pro-apoptotic BH3 domain. Here, we report that T cell-specific expression of a Bcl2 BH3 mutant transgene results in enhanced rescue of thymocytes from negative selection. Concomitantly, Treg development is increased. However, aged BH3 mutant mice progressively accumulate activated, autoreactive T cells, culminating in development of multi-organ autoimmunity and lethality. These data provide strong evidence that negative selection is crucial for establishing T cell tolerance. DOI: http://dx.doi.org/10.7554/eLife.03468.001 PMID:25182415

  10. Designing the nanobiointerface of fluorescent nanodiamonds: highly selective targeting of glioma cancer cells

    NASA Astrophysics Data System (ADS)

    Slegerova, Jitka; Hajek, Miroslav; Rehor, Ivan; Sedlak, Frantisek; Stursa, Jan; Hruby, Martin; Cigler, Petr

    2014-12-01

    Core-shell nanoparticles based on fluorescent nanodiamonds coated with a biocompatible N-(2-hydroxypropyl)methacrylamide copolymer shell were developed for background-free near-infrared imaging of cancer cells. The particles showed excellent colloidal stability in buffers and culture media. After conjugation with a cyclic RGD peptide they selectively targeted integrin αvβ3 receptors on glioblastoma cells with high internalization efficacy.Core-shell nanoparticles based on fluorescent nanodiamonds coated with a biocompatible N-(2-hydroxypropyl)methacrylamide copolymer shell were developed for background-free near-infrared imaging of cancer cells. The particles showed excellent colloidal stability in buffers and culture media. After conjugation with a cyclic RGD peptide they selectively targeted integrin αvβ3 receptors on glioblastoma cells with high internalization efficacy. Electronic supplementary information (ESI) available: Materials and methods, colloidal stability studies and cell viability studies. See DOI: 10.1039/c4nr02776k

  11. Selective advantage for multicellular replicative strategies: A two-cell example

    NASA Astrophysics Data System (ADS)

    Tannenbaum, Emmanuel

    2006-01-01

    This paper develops a quasispecies model where cells can adopt a two-cell survival strategy. Within this strategy, pairs of cells join together, at which point one of the cells sacrifices its own replicative ability for the sake of the other cell. We develop a simplified model for the evolutionary dynamics of this process, allowing us to solve for the steady state using standard approaches from quasispecies theory. We find that our model exhibits two distinct regimes of behavior: At low concentrations of limiting resource, the two-cell strategy outcompetes the single-cell survival strategy, while at high concentrations of limiting resource, the single-cell survival strategy dominates. The single-cell survival strategy becomes disadvantageous at low concentrations of limiting resource because the energetic costs of maintaining reproductive and metabolic pathways approach, and may even exceed, the rate of energy production, leaving little excess energy for the purposes of replicating a new cell. However, if the rate of energy production exceeds the energetic costs of maintaining metabolic pathways, then the excess energy, if shared among several cells, can pay for the reproductive costs of a single cell, leaving energy to replicate a new cell. Associated with the two solution regimes of our model is a localization to delocalization transition over the portion of the genome coding for the multicell strategy, analogous to the error catastrophe in standard quasispecies models. The existence of such a transition indicates that multicellularity can emerge because natural selection does not act on specific cells, but rather on replicative strategies. Within this framework, individual cells become the means by which replicative strategies are propagated. Such a framework is therefore consistent with the concept that natural selection does not act on individuals, but rather on populations.

  12. Engineering Multi-Walled Carbon Nanotube Therapeutic Bionanofluids to Selectively Target Papillary Thyroid Cancer Cells

    PubMed Central

    Paliouras, Miltiadis; Mitmaker, Elliot J.; Trifiro, Mark A.

    2016-01-01

    Background The incidence of papillary thyroid carcinoma (PTC) has risen steadily over the past few decades as well as the recurrence rates. It has been proposed that targeted ablative physical therapy could be a therapeutic modality in thyroid cancer. Targeted bio-affinity functionalized multi-walled carbon nanotubes (BioNanofluid) act locally, to efficiently convert external light energy to heat thereby specifically killing cancer cells. This may represent a promising new cancer therapeutic modality, advancing beyond conventional laser ablation and other nanoparticle approaches. Methods Thyroid Stimulating Hormone Receptor (TSHR) was selected as a target for PTC cells, due to its wide expression. Either TSHR antibodies or Thyrogen or purified TSH (Thyrotropin) were chemically conjugated to our functionalized Bionanofluid. A diode laser system (532 nm) was used to illuminate a PTC cell line for set exposure times. Cell death was assessed using Trypan Blue staining. Results TSHR-targeted BioNanofluids were capable of selectively ablating BCPAP, a TSHR-positive PTC cell line, while not TSHR-null NSC-34 cells. We determined that a 2:1 BCPAP cell:α-TSHR-BioNanofluid conjugate ratio and a 30 second laser exposure killed approximately 60% of the BCPAP cells, while 65% and >70% of cells were ablated using Thyrotropin- and Thyrogen-BioNanofluid conjugates, respectively. Furthermore, minimal non-targeted killing was observed using selective controls. Conclusion A BioNanofluid platform offering a potential therapeutic path for papillary thyroid cancer has been investigated, with our in vitro results suggesting the development of a potent and rapid method of selective cancer cell killing. Therefore, BioNanofluid treatment emphasizes the need for new technology to treat patients with local recurrence and metastatic disease who are currently undergoing either re-operative neck explorations, repeated administration of radioactive iodine and as a last resort external beam

  13. Selective photothermal efficiency of citrate capped gold nanoparticles for destruction of cancer cells

    SciTech Connect

    Raji, V.; Kumar, Jatish; Rejiya, C.S.; Vibin, M.; Shenoi, Vinesh N.; Abraham, Annie

    2011-08-15

    Gold nanoparticles are recently having much attention because of their increased applications in biomedical fields. In this paper, we demonstrated the photothermal efficacy of citrate capped gold nanoparticles (AuNPs) for the destruction of A431 cancer cells. Citrate capped AuNPs were synthesized successfully and characterized by UV-visible-NIR spectrophotometry and High Resolution Transmission Electron Microscopy (HR-TEM). Further, AuNPs were conjugated with epidermal growth factor receptor antibody (anti-EGFR) and applied for the selective photothermal therapy (PTT) of human epithelial cancer cells, A431. PTT experiments were conducted in four groups, Group I-control cells, Group II-cells treated with laser light alone, Group III-cells treated with unconjugated AuNP and further laser irradiation and Group IV-anti-EGFR conjugated AuNP treated cells irradiated by laser light. After laser irradiation, cell morphology changes that were examined using phase contrast microscopy along with the relevant biochemical parameters like lactate dehydrogenase activity, reactive oxygen species generation and caspase-3 activity were studied for all the groups to determine whether cell death occurs due to necrosis or apoptosis. From these results we concluded that, these immunotargeted nanoparticles could selectively induce cell death via ROS mediated apoptosis when cells were exposed to a low power laser light.

  14. A Small Molecule Inhibitor Selectively Induces Apoptosis in Cells Transformed by High Risk Human Papilloma Viruses

    PubMed Central

    Lee, Min S.; Qi, Huilin; Chaniewski, Susan; Zheng, Xiaofan; Farr, Glen A.; Esposito, Kim; Harden, David; Lei, Ming; Schweizer, Liang; Friborg, Jacques; Agler, Michele; McPhee, Fiona; Gentles, Robert; Beno, Brett R.; Chupak, Lou; Mason, Stephen

    2016-01-01

    A phenotypic high-throughput cell culture screen was performed to identify compounds that prevented proliferation of the human Papilloma virus type 16 (HPV-16) transformed cell line Ca Ski. A series of quinoxaline compounds exemplified by Compound 1 was identified. Testing against a panel of cell lines demonstrated that Compound 1 selectively inhibited replication of all HPV-16, HPV-18, and HPV-31 transformed cell lines tested with 50% Inhibitory Concentration (IC50) values of 2 to 8 μM relative to IC50 values of 28 to 73 μM in HPV-negative cell lines. Treatment with Compound 1 resulted in a cascade of multiple apoptotic events, including selective activation of effector caspases 3 and 7, fragmentation of cellular DNA, and PARP (poly(ADP-ribose) polymerase) cleavage in HPV-positive cells relative to HPV-negative cells. Unregulated proliferation of HPV transformed cells is dependent on the viral oncogenes, E6 and E7. Treatment with Compound 1 resulted in a decrease in HPV E7 protein in Ca Ski cells. However, the timing of this reduction relative to other effects of compound treatment suggests that this was a consequence, rather than a cause, of the apoptotic cascade. Likewise, compound treatment resulted in no obvious effects on the E6- and E7- mediated down regulation of p53 and Rb, or their downstream effectors, p21 or PCNA. Further investigation of apoptotic signals induced by Compound 1 revealed cleavage of Caspase-8 in HPV-positive cells as early as 2 hours post-treatment, suggesting the compound initiates apoptosis through the extrinsic, death receptor-mediated, pathway of cell death. These studies provide proof of concept that cells transformed by oncogenic Papillomaviruses can be selectively induced to undergo apoptosis by compound treatment. PMID:27280728

  15. A Small Molecule Inhibitor Selectively Induces Apoptosis in Cells Transformed by High Risk Human Papilloma Viruses.

    PubMed

    Sheaffer, Amy K; Lee, Min S; Qi, Huilin; Chaniewski, Susan; Zheng, Xiaofan; Farr, Glen A; Esposito, Kim; Harden, David; Lei, Ming; Schweizer, Liang; Friborg, Jacques; Agler, Michele; McPhee, Fiona; Gentles, Robert; Beno, Brett R; Chupak, Lou; Mason, Stephen

    2016-01-01

    A phenotypic high-throughput cell culture screen was performed to identify compounds that prevented proliferation of the human Papilloma virus type 16 (HPV-16) transformed cell line Ca Ski. A series of quinoxaline compounds exemplified by Compound 1 was identified. Testing against a panel of cell lines demonstrated that Compound 1 selectively inhibited replication of all HPV-16, HPV-18, and HPV-31 transformed cell lines tested with 50% Inhibitory Concentration (IC50) values of 2 to 8 μM relative to IC50 values of 28 to 73 μM in HPV-negative cell lines. Treatment with Compound 1 resulted in a cascade of multiple apoptotic events, including selective activation of effector caspases 3 and 7, fragmentation of cellular DNA, and PARP (poly(ADP-ribose) polymerase) cleavage in HPV-positive cells relative to HPV-negative cells. Unregulated proliferation of HPV transformed cells is dependent on the viral oncogenes, E6 and E7. Treatment with Compound 1 resulted in a decrease in HPV E7 protein in Ca Ski cells. However, the timing of this reduction relative to other effects of compound treatment suggests that this was a consequence, rather than a cause, of the apoptotic cascade. Likewise, compound treatment resulted in no obvious effects on the E6- and E7- mediated down regulation of p53 and Rb, or their downstream effectors, p21 or PCNA. Further investigation of apoptotic signals induced by Compound 1 revealed cleavage of Caspase-8 in HPV-positive cells as early as 2 hours post-treatment, suggesting the compound initiates apoptosis through the extrinsic, death receptor-mediated, pathway of cell death. These studies provide proof of concept that cells transformed by oncogenic Papillomaviruses can be selectively induced to undergo apoptosis by compound treatment. PMID:27280728

  16. A novel steroidal saponin glycoside from Fagonia indica induces cell-selective apoptosis or necrosis in cancer cells.

    PubMed

    Waheed, Abdul; Barker, James; Barton, Stephen J; Owen, Caroline P; Ahmed, Sabbir; Carew, Mark A

    2012-09-29

    Fagonia indica is a small spiny shrub of great ethnopharmacological importance in folk medicine. The aqueous decoction of aerial parts is a popular remedy against various skin lesions, including cancer. We used a biological activity-guided fractionation approach to isolate the most potent fraction of the crude extract on three cancer cell lines: MCF-7 oestrogen-dependent breast cancer, MDA-MB-468 oestrogen-independent breast cancer, and Caco-2 colon cancer cells. A series of chromatographic and spectroscopic procedures were utilised on the EtOAc fraction, which resulted in the isolation of a new steroidal saponin glycoside. The cytotoxic activity of the saponin glycoside was determined in cancer cells using the MTT and neutral red uptake assays. After 24h treatment, the observed IC(50) values of the saponin glycoside were 12.5 μM on MDA-MB-468 and Caco-2 cells, but 100 μM on MCF-7 cells. Several lines of evidence: PARP cleavage, caspase-3 cleavage, DNA ladder assays, and reversal of growth inhibition with the pan-caspase inhibitor Z-VAD-fmk, suggested stimulation of apoptosis in MDA-MB-468 and Caco-2 cells, but not in MCF-7 cells, which do not express caspase-3. The haemolytic activity of the saponin glycoside was confirmed in sheep red blood cells, with cell lysis observed at >100 μM, suggesting that, at this concentration, the saponin glycoside caused necrosis through cell lysis in MCF-7 cells. Using the DNA ladder assay, the saponin glycoside (12.5 μM) was not toxic to HUVEC (human umbilical vein endothelial cells) or U937 cells, indicating some selectivity between malignant and normal cells. We conclude that the steroidal saponin glycoside isolated from F. indica is able to induce apoptosis or necrosis in cancer cells depending on the cell type. PMID:22800968

  17. Development of JNK2-Selective Peptide Inhibitors that Inhibit Breast Cancer Cell Migration

    PubMed Central

    Kaoud, Tamer S.; Mitra, Shreya; Lee, Sunbae; Taliaferro, Juliana; Cantrell, Michael; Linse, Klaus D.; Van Den Berg, Carla L.; Dalby, Kevin N.

    2012-01-01

    Despite their lack of selectivity towards c-Jun N-terminal kinase (JNK) isoforms, peptides derived from the JIP (JNK Interacting Protein) scaffolds linked to the cell-penetrating peptide TAT are widely used to investigate JNK-mediated signaling events. To engineer an isoform-selective peptide inhibitor, several JIP-based peptide sequences were designed and tested. A JIP sequence connected through a flexible linker to either the N-terminus of an inverted TAT sequence (JIP10-Δ-TATi), or to a poly-arginine sequence (JIP10-Δ-R9) enabled the potent inhibition of JNK2 (IC50~90 nM) and exhibited 10-fold selectivity for JNK2 over JNK1 and JNK3. Examination of both peptides in HEK293 cells revealed a potent ability to inhibit the induction of both JNK activation and c-Jun phosphorylation in cells treated with anisomycin. Notably, Western blot analysis indicates that only a fraction of total JNK must be activated to elicit robust c-Jun phosphorylation. To examine the potential of each peptide to selectively modulate JNK2 signaling in vivo, their ability to inhibit the migration of Polyoma Middle-T Antigen Mammary Tumor (PyVMT) cells was assessed. PyVMTjnk2-/- cells exhibit a lower migration potential compared to PyVMTjnk2+/+ cells, and this migration potential is restored through the over-expression of GFP-JNK2α. Both JIP10-Δ-TATi and JIP10-Δ-R9 inhibit the migration of PyVMTjnk2+/+ cells and PyVMTjnk2-/- cells expressing GFP-JNK2α. However, neither peptide inhibits the migration of PyVMTjnk2-/- cells. A control form of JIP10-Δ-TATi containing a single leucine to arginine mutation lacks ability to inhibit JNK2 in vitro cell-free and cell-based assays and does not inhibit the migration of PyVMTjnk2+/+ cells. Together, these data suggest that JIP10-Δ-TATi and JIP10-Δ-R9 inhibit the migration of PyVMT cells through the selective inhibition of JNK2. Finally, the mechanism of inhibition of a D-retro-inverso JIP peptide, previously reported to inhibit JNK, was examined

  18. Lectin of Abelmoschus esculentus (okra) promotes selective antitumor effects in human breast cancer cells.

    PubMed

    Monte, Leonardo G; Santi-Gadelha, Tatiane; Reis, Larissa B; Braganhol, Elizandra; Prietsch, Rafael F; Dellagostin, Odir A; E Lacerda, Rodrigo Rodrigues; Gadelha, Carlos A A; Conceição, Fabricio R; Pinto, Luciano S

    2014-03-01

    The anti-tumor effects of a newly-discovered lectin, isolated from okra, Abelmoschus esculentus (AEL), were investigated in human breast cancer (MCF7) and skin fibroblast (CCD-1059 sk) cells. AEL induced significant cell growth inhibition (63 %) in MCF7 cells. The expression of pro-apoptotic caspase-3, caspase-9, and p21 genes was increased in MCF7 cells treated with AEL, compared to those treated with controls. In addition, AEL treatment increased the Bax/Bcl-2 ratio in MCF7 cells. Flow cytometry also indicated that cell death (72 %) predominantly occurred through apoptosis. Thus, AEL in its native form promotes selective antitumor effects in human breast cancer cells and may represent a potential therapeutic to combat human breast cancer. PMID:24129958

  19. Selective Conditions Are Required for the Induction of Invariant NKT Cell Hyporesponsiveness by Antigenic Stimulation.

    PubMed

    Wingender, Gerhard; Birkholz, Alysia M; Sag, Duygu; Farber, Elisa; Chitale, Sampada; Howell, Amy R; Kronenberg, Mitchell

    2015-10-15

    Activation of invariant (i)NKT cells with the model Ag α-galactosylceramide induces rapid production of multiple cytokines, impacting a wide variety of different immune reactions. In contrast, following secondary activation with α-galactosylceramide, the behavior of iNKT cells is altered for months, with the production of most cytokines being strongly reduced. The requirements for the induction of this hyporesponsive state, however, remain poorly defined. In this study, we show that Th1-biasing iNKT cell Ags could induce iNKT cell hyporesponsiveness, as long as a minimum antigenic affinity was reached. In contrast, the Th2-biasing Ag OCH did not induce a hyporesponsive state, nor did cytokine-driven iNKT cell activation by LPS or infections. Furthermore, although dendritic cells and B cells have been reported to be essential for iNKT cell stimulation, neither dendritic cells nor B cells were required to induce iNKT cell hyporesponsiveness. Therefore, our data indicate that whereas some bone marrow-derived cells could induce iNKT cell hyporesponsiveness, selective conditions, dependent on the structure and potency of the Ag, were required to induce hyporesponsiveness. PMID:26355152

  20. Antithymidylate resistance enables transgene selection and cell survival for T cells in the presence of 5-fluorouracil and antifolates.

    PubMed

    Rushworth, D; Alpert, A; Santana-Carrero, R; Olivares, S; Spencer, D; Cooper, L J N

    2016-02-01

    Antithymidylates (AThy) constitute a class of drugs used in the treatment of cancers such as lung, colon, breast and pancreas. These drugs inhibit DNA synthesis by targeting the enzymes dihydrofolate reductase (DHFR) and/or thymidylate synthase (TYMS). AThys effectively inhibit cancer cells, and also inhibit T cells, preventing anticancer immunity, which might otherwise develop from AThy-induced cancer destruction. We establish that T cells expressing mutant DHFR--DHFR L22F, F31S (DHFR(FS))--and/or mutant TYMS--TYMS T51S, G52S (TYMS(SS))-effectively survive in toxic concentrations of AThys methotrexate, pemetrexed and 5-fluorouracil. Furthermore, we show that DHFR(FS) permitted rapid selection of an inducible suicide transgene in T cells. These findings demonstrate that AThy resistances prevent AThy cytotoxicity to T cells while permitting selection of important transgenes. This technological development could enhance in vitro and in vivo survival and selection of T-cell therapeutics being designed for a broad range of cancers. PMID:26273805

  1. Toward chelerythrine optimization: Analogues designed by molecular simplification exhibit selective growth inhibition in non-small-cell lung cancer cells.

    PubMed

    Yang, Rosania; Tavares, Maurício T; Teixeira, Sarah F; Azevedo, Ricardo A; C Pietro, Diego; Fernandes, Thais B; Ferreira, Adilson K; Trossini, Gustavo H G; Barbuto, José A M; Parise-Filho, Roberto

    2016-10-01

    A series of novel chelerythrine analogues was designed and synthesized. Antitumor activity was evaluated against A549, NCI-H1299, NCI-H292, and NCI-H460 non-small-cell lung cancer (NSCLC) cell lines in vitro. The selectivity of the most active analogues and chelerythrine was also evaluated, and we compared their cytotoxicity in NSCLC cells and non-tumorigenic cell lines, including human umbilical vein endothelial cells (HUVECs) and LL24 human lung fibroblasts. In silico studies were performed to establish structure-activity relationships between chelerythrine and the analogues. The results showed that analogue compound 3f induced significant dose-dependent G0/G1 cell cycle arrest in A549 and NCI-H1299 cells. Theoretical studies indicated that the molecular arrangement and electron characteristics of compound 3f were closely related to the profile of chelerythrine, supporting its activity. The present study presents a new and simplified chelerythrinoid scaffold with enhanced selectivity against NSCLC tumor cells for further optimization. PMID:27561984

  2. An excitatory amacrine cell detects object motion and provides feature-selective input to ganglion cells in the mouse retina

    PubMed Central

    Kim, Tahnbee; Soto, Florentina; Kerschensteiner, Daniel

    2015-01-01

    Retinal circuits detect salient features of the visual world and report them to the brain through spike trains of retinal ganglion cells. The most abundant ganglion cell type in mice, the so-called W3 ganglion cell, selectively responds to movements of small objects. Where and how object motion sensitivity arises in the retina is incompletely understood. In this study, we use 2-photon-guided patch-clamp recordings to characterize responses of vesicular glutamate transporter 3 (VGluT3)-expressing amacrine cells (ACs) to a broad set of visual stimuli. We find that these ACs are object motion sensitive and analyze the synaptic mechanisms underlying this computation. Anatomical circuit reconstructions suggest that VGluT3-expressing ACs form glutamatergic synapses with W3 ganglion cells, and targeted recordings show that the tuning of W3 ganglion cells' excitatory input matches that of VGluT3-expressing ACs' responses. Synaptic excitation of W3 ganglion cells is diminished, and responses to object motion are suppressed in mice lacking VGluT3. Object motion, thus, is first detected by VGluT3-expressing ACs, which provide feature-selective excitatory input to W3 ganglion cells. DOI: http://dx.doi.org/10.7554/eLife.08025.001 PMID:25988808

  3. Selective suppression of Notch1 inhibits proliferation of renal cell carcinoma cells through JNK/p38 pathway.

    PubMed

    Wu, Kerong; Hu, Linkun; Hou, Jianquan

    2016-05-01

    The present study was performed to explore the effects of Notch1 inhibition selectively by siRNA on the proliferation and cell cycle of renal carcinoma cells. Human renal carcinoma cell lines, 786-0 and Caki-1, were treated with Si-Notch1 or negative control (NC). RT-PCR and western blotting were used to confirm the efficiency of siRNA on Notch1 expression. MTT, cell cycle analysis, colony formation as well as migration and invasion assays were performed. The expression levels of p38 and SAPK/JNK were measured by western blotting. For both cell lines, as compared with the NC group, the cell growth was markedly reduced, and colony formation was restricted in the Si-Notch1‑treated group. After incubated with Si-Notch1 or NC for 48 h, Si-Notch1-treated cells arrested the cell cycle at G1/S phase. The Si-Notch1 group also had a reduced rate of migration as well as invasion. Moreover, we observed a reduction in p-SAPK/JNK and p-p38 in Si-Notch1 transfected cells. The present study indicated that Notch signaling is important in the tumorigenesis of renal cell carcinoma. Notch1 may be a potential therapeutic regimen towards renal cell carcinoma, and JNK/p38 may serve as an important molecular mechanism for Notch1-mediated carcinogenesis. PMID:26986634

  4. Analysis of selective reflection spectrum in cholesteric liquid crystal cells for solar-ray controller

    NASA Astrophysics Data System (ADS)

    Ogiwara, Akifumi; Kakiuchida, Hiroshi

    2015-09-01

    The cholesteric liquid crystal (CLC) cells are fabricated by varying the concentration of various chiral dopants and liquid crystal (LC) diacrylate monomers. The wavelength and bandwidth of selective reflection spectrum in CLC cells are measured by a spectroscopic technique. The variation of the selective reflection spectrum in CLC cells is investigated by doping the different kinds of liquid crystal (LC) diacrylate monomers which stabilize a helical twisting structure by photopolymerization. The effects of the selective reflection spectrum on the visible and infrared lights in spectral solar irradiance are explained by the performance for a solar-ray controller based on the spectral solar irradiance for air mass 1.5 and the standard luminous efficiency function for photopic vision.

  5. Cidofovir selectivity is based on the different response of normal and cancer cells to DNA damage

    PubMed Central

    2013-01-01

    Background Cidofovir (CDV) proved efficacious in treatment of human papillomaviruses (HPVs) hyperplasias. Antiproliferative effects of CDV have been associated with apoptosis induction, S-phase accumulation, and increased levels of tumor suppressor proteins. However, the molecular mechanisms for the selectivity and antitumor activity of CDV against HPV-transformed cells remain unexplained. Methods We evaluated CDV drug metabolism and incorporation into cellular DNA, in addition to whole genome gene expression profiling by means of microarrays in two HPV+ cervical carcinoma cells, HPV- immortalized keratinocytes, and normal keratinocytes. Results Determination of the metabolism and drug incorporation of CDV into genomic DNA demonstrated a higher rate of drug incorporation in HPV+ tumor cells and immortalized keratinocytes compared to normal keratinocytes. Gene expression profiling clearly showed distinct and specific drug effects in the cell types investigated. Although an effect on inflammatory response was seen in all cell types, different pathways were identified in normal keratinocytes compared to immortalized keratinocytes and HPV+ tumor cells. Notably, Rho GTPase pathways, LXR/RXR pathways, and acute phase response signaling were exclusively activated in immortalized cells. CDV exposed normal keratinocytes displayed activated cell cycle regulation upon DNA damage signaling to allow DNA repair via homologous recombination, resulting in genomic stability and survival. Although CDV induced cell cycle arrest in HPV- immortalized cells, DNA repair was not activated in these cells. In contrast, HPV+ cells lacked cell cycle regulation, leading to genomic instability and eventually apoptosis. Conclusions Taken together, our data provide novel insights into the mechanism of action of CDV and its selectivity for HPV-transformed cells. The proposed mechanism suggests that this selectivity is based on the inability of HPV+ cells to respond to DNA damage, rather than on a

  6. Conditional Cytotoxic Anti-HIV Gene Therapy for Selectable Cell Modification.

    PubMed

    Garg, Himanshu; Joshi, Anjali

    2016-05-01

    Gene therapy remains one of the potential strategies to achieve a cure for HIV infection. One of the major limitations of anti-HIV gene therapy concerns recovering an adequate number of modified cells to generate an HIV-proof immune system. Our study addresses this issue by developing a methodology that can mark conditional vector-transformed cells for selection and subsequently target HIV-infected cells for elimination by treatment with ganciclovir (GCV). We used the herpes simplex virus thymidine kinase (TK) mutant SR39, which is highly potent at killing cells at low GCV concentrations. This gene was cloned into a conditional HIV vector, pNL-GFPRRESA, which expresses the gene of interest as well as green fluorescent protein (GFP) in the presence of HIV Tat protein. We show here that TK-SR39 was more potent that wild-type TK (TK-WT) at eliminating infected cells at lower concentrations of GCV. As the vector expresses GFP in the presence of Tat, transient expression of Tat either by Tat RNA transfection or transduction by a nonintegrating lentiviral (NIL) vector marked the cells with GFP for selection. In cells selected by this strategy, TK-SR39 was more potent at limiting virus replication than TK-WT. Finally, in Jurkat cells modified and selected by this approach, infection with CXCR4-tropic Lai virus could be suppressed by treatment with GCV. GCV treatment limited the number of HIV-infected cells, virus production, as well as virus-induced cytopathic effects in this model. We provide proof of principle that TK-SR39 in a conditional HIV vector can provide a safe and effective anti-HIV strategy. PMID:26800572

  7. Targeted toxin-based selectable drug-free enrichment of Mammalian cells with high transgene expression.

    PubMed

    Sato, Masahiro; Akasaka, Eri; Saitoh, Issei; Ohtsuka, Masato; Nakamura, Shingo; Sakurai, Takayuki; Watanabe, Satoshi

    2013-01-01

    Almost all transfection protocols for mammalian cells use a drug resistance gene for the selection of transfected cells. However, it always requires the characterization of each isolated clone regarding transgene expression, which is time-consuming and labor-intensive. In the current study, we developed a novel method to selectively isolate clones with high transgene expression without drug selection. Porcine embryonic fibroblasts were transfected with pCEIEnd, an expression vector that simultaneously expresses enhanced green fluorescent protein (EGFP) and endo-b-galactosidase C(EndoGalC; an enzyme capable of digesting cell surface a-Gal epitope) upon transfection. After transfection, the surviving cells were briefly treated with IB4SAP (a-Gal epitope-specific BS-I-B4 lectin conjugated with a toxin saporin). The treated cells were then allowed to grow in normal medium, during which only cells strongly expressing EndoGalC and EGFP would survive because of the absence of a-Gal epitopes on their cell surface. Almost all the surviving colonies after IB4SAP treatment were in fact negative for BS-I-B4 staining, and also strongly expressed EGFP. This system would be particularly valuable for researchers who wish to perform large-scale production of therapeutically important recombinant proteins. PMID:24832665

  8. Bezielle Selectively Targets Mitochondria of Cancer Cells to Inhibit Glycolysis and OXPHOS

    PubMed Central

    Chen, Vivian; Staub, Richard E.; Fong, Sylvia; Tagliaferri, Mary; Cohen, Isaac; Shtivelman, Emma

    2012-01-01

    Bezielle (BZL101) is a candidate oral drug that has shown promising efficacy and excellent safety in the early phase clinical trials for advanced breast cancer. Bezielle is an aqueous extract from the herb Scutellaria barbata. We have reported previously that Bezielle was selectively cytotoxic to cancer cells while sparing non-transformed cells. In tumor, but not in non-transformed cells, Bezielle induced generation of ROS and severe DNA damage followed by hyperactivation of PARP, depletion of the cellular ATP and NAD, and inhibition of glycolysis. We show here that tumor cells' mitochondria are the primary source of reactive oxygen species induced by Bezielle. Treatment with Bezielle induces progressively higher levels of mitochondrial superoxide as well as peroxide-type ROS. Inhibition of mitochondrial respiration prevents generation of both types of ROS and protects cells from Bezielle-induced death. In addition to glycolysis, Bezielle inhibits oxidative phosphorylation in tumor cells and depletes mitochondrial reserve capacity depriving cells of the ability to produce ATP. Tumor cells lacking functional mitochondria maintain glycolytic activity in presence of Bezielle thus supporting the hypothesis that mitochondria are the primary target of Bezielle. The metabolic effects of Bezielle towards normal cells are not significant, in agreement with the low levels of oxidative damage that Bezielle inflicts on them. Bezielle is therefore a drug that selectively targets cancer cell mitochondria, and is distinguished from other such drugs by its ability to induce not only inhibition of OXPHOS but also of glycolysis. This study provides a better understanding of the mechanism of Bezielle's cytotoxicity, and the basis of its selectivity towards cancer cells. PMID:22319564

  9. Engineering a BCR-ABL–activated caspase for the selective elimination of leukemic cells

    PubMed Central

    Kurokawa, Manabu; Ito, Takahiro; Yang, Chih-Sheng; Zhao, Chen; Macintyre, Andrew N.; Rizzieri, David A.; Rathmell, Jeffrey C.; Deininger, Michael W.; Reya, Tannishtha; Kornbluth, Sally

    2013-01-01

    Increased understanding of the precise molecular mechanisms involved in cell survival and cell death signaling pathways offers the promise of harnessing these molecules to eliminate cancer cells without damaging normal cells. Tyrosine kinase oncoproteins promote the genesis of leukemias through both increased cell proliferation and inhibition of apoptotic cell death. Although tyrosine kinase inhibitors, such as the BCR-ABL inhibitor imatinib, have demonstrated remarkable efficacy in the clinic, drug-resistant leukemias emerge in some patients because of either the acquisition of point mutations or amplification of the tyrosine kinase, resulting in a poor long-term prognosis. Here, we exploit the molecular mechanisms of caspase activation and tyrosine kinase/adaptor protein signaling to forge a unique approach for selectively killing leukemic cells through the forcible induction of apoptosis. We have engineered caspase variants that can directly be activated in response to BCR-ABL. Because we harness, rather than inhibit, the activity of leukemogenic kinases to kill transformed cells, this approach selectively eliminates leukemic cells regardless of drug-resistant mutations. PMID:23324740

  10. Simple avarone mimetics as selective agents against multidrug resistant cancer cells.

    PubMed

    Jeremić, Marko; Pešić, Milica; Dinić, Jelena; Banković, Jasna; Novaković, Irena; Šegan, Dejan; Sladić, Dušan

    2016-08-01

    In this work, synthesis of alkylamino and aralkylamino derivatives of sesquiterpene quinone avarone and its model compound tert-butylquinone was described. For all obtained derivatives biological activity was studied. Cytotoxic activity of the synthesized derivatives towards multidrug resistant MDR human non-small cell lung carcinoma NCI-H460/R cells, their sensitive counterpart NCI-H460 and human normal keratinocytes (HaCaT) as well as detection of cell death superoxide anion generation were investigated. Antimicrobial activity towards Gram positive and Gram negative bacteria and fungal cultures was determined. The results showed that strong cytotoxic activity toward cancer cells was improved with simple avarone mimetics. Some derivatives were selective towards MDR cancer cells. The most active derivatives induced apoptosis in both cancer cell lines, but not in normal cells. Superoxide production was induced by 2,6-disubstituted compounds in MDR cancer cells and not by less active 2,5-disubstituted compounds and was accompanied by the collapse of the mitochondrial transmembrane potential. Two tert-butylquinone derivatives were particularly selective towards MDR cancer cells. Some tert-butylquinone derivatives exhibited a strong antimicrobial activity. PMID:27128177

  11. Isolation of homozygous mutant mouse embryonic stem cells using a dual selection system

    PubMed Central

    Huang, Yue; Pettitt, Stephen J.; Guo, Ge; Liu, Guang; Li, Meng Amy; Yang, Fengtang; Bradley, Allan

    2012-01-01

    Obtaining random homozygous mutants in mammalian cells for forward genetic studies has always been problematic due to the diploid genome. With one mutation per cell, only one allele of an autosomal gene can be disrupted, and the resulting heterozygous mutant is unlikely to display a phenotype. In cells with a genetic background deficient for the Bloom's syndrome helicase, such heterozygous mutants segregate homozygous daughter cells at a low frequency due to an elevated rate of crossover following mitotic recombination between homologous chromosomes. We constructed DNA vectors that are selectable based on their copy number and used these to isolate these rare homozygous mutant cells independent of their phenotype. We use the piggyBac transposon to limit the initial mutagenesis to one copy per cell, and select for cells that have increased the transposon copy number to two or more. This yields homozygous mutants with two allelic mutations, but also cells that have duplicated the mutant chromosome and become aneuploid during culture. On average, 26% of the copy number gain events occur by the mitotic recombination pathway. We obtained homozygous cells from 40% of the heterozygous mutants tested. This method can provide homozygous mammalian loss-of-function mutants for forward genetic applications. PMID:22127858

  12. Spatially selective sampling of single cells using optically trapped fusogenic emulsion droplets: a new single-cell proteomic tool

    PubMed Central

    Lanigan, Peter M.P.; Chan, Karen; Ninkovic, Tanya; Templer, Richard H.; French, P.M.W.; de Mello, A.J.; Willison, K.R.; Parker, P.J.; Neil, M.A.A.; Ces, Oscar; Klug, D.R.

    2008-01-01

    We present a platform for the spatially selective sampling of the plasma membrane of single cells. Optically trapped lipid-coated oil droplets (smart droplet microtools, SDMs), typically 0.5–5 μm in size, composed of a hexadecane hydrocarbon core and fusogenic lipid outer coating (mixture of 1,2-dioleoyl-phosphatidylethanolamine and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) were brought into controlled contact with target colon cancer cells leading to the formation of connecting membrane tethers. Material transfer from the cell to the SDM across the membrane tether was monitored by tracking membrane-localized enhanced green fluorescent protein. PMID:18664432

  13. Selective killing of G2 decatenation checkpoint defective colon cancer cells by catalytic topoisomerase II inhibitor.

    PubMed

    Jain, Chetan Kumar; Roychoudhury, Susanta; Majumder, Hemanta Kumar

    2015-05-01

    Cancer cells with defective DNA decatenation checkpoint can be selectively targeted by the catalytic inhibitors of DNA topoisomerase IIα (topo IIα) enzyme. Upon treatment with catalytic topo IIα inhibitors, cells with defective decatenation checkpoint fail to arrest their cell cycle in G2 phase and enter into M phase with catenated and under-condensed chromosomes resulting into impaired mitosis and eventually cell death. In the present work we analyzed decatenation checkpoint in five different colon cancer cell lines (HCT116, HT-29, Caco2, COLO 205 and SW480) and in one non-cancerous cell line (HEK293T). Four out of the five colon cancer cell lines i.e. HCT116, HT-29, Caco2, and COLO 205 were found to be compromised for the decatenation checkpoint function at different extents, whereas SW480 and HEK293T cell lines were found to be proficient for the checkpoint function. Upon treatment with ICRF193, decatenation checkpoint defective cell lines failed to arrest the cell cycle in G2 phase and entered into M phase without proper chromosomal decatenation, resulting into the formation of tangled mass of catenated and under-condensed chromosomes. Such cells underwent mitotic catastrophe and rapid apoptosis like cell death and showed higher sensitivity for ICRF193. Our study suggests that catalytic inhibitors of topoisomerase IIα are promising therapeutic agents for the treatment of colon cancers with defective DNA decatenation checkpoint. PMID:25746763

  14. Sulindac sulfide selectively increases sensitivity of ABCC1 expressing tumor cells to doxorubicin and glutathione depletion

    PubMed Central

    Whitt, Jason D.; Keeton, Adam B.; Gary, Bernard D.; Sklar, Larry A.; Sodani, Kamlesh; Chen, Zhe-Sheng; Piazza, Gary A.

    2016-01-01

    Abstract ATP-binding cassette (ABC) transpo rters ABCC1 (MRP1), ABCB1 (P-gp), and ABCG2 (BCRP) contribute to chemotherapy failure. The primary goals of this study were to characterize the efficacy and mechanism of the non­steroidal anti-inflammatory drug (NSAID), sulindac sulfide, to reverse ABCC1 mediated resistance to chemother­apeutic drugs and to determine if sulindac sulfide can influence sensitivity to chemotherapeutic drugs independently of drug efflux. Cytotoxicity assays were performed to measure resistance of ABC-expressing cell lines to doxoru­bicin and other chemotherapeutic drugs. NSAIDs were tested for the ability to restore sensitivity to resistance selected tumor cell lines, as well as a large panel of standard tumor cell lines. Other experiments characterized the mechanism by which sulindac sulfide inhibits ABCC1 substrate and co-substrate (GSH) transport in isolated membrane vesicles and intact cells. Selective reversal of multi-drug resistance (MDR), decreased efflux of doxor­ubicin, and fluorescent substrates were demonstrated by sulindac sulfide and a related NSAID, indomethacin, in resistance selected and engineered cell lines expressing ABCC1, but not ABCB1 or ABCG2. Sulindac sulfide also inhibited transport of leukotriene C4 into membrane vesicles. Sulindac sulfide enhanced the sensitivity to doxoru­bicin in 24 of 47 tumor cell lines, including all melanoma lines tested (7-7). Sulindac sulfide also decreased intra­cellular GSH in ABCC1 expressing cells, while the glutathione synthesis inhibitor, BSO, selectively increased sensitivity to sulindac sulfide induced cytotoxicity. Sulindac sulfide potently and selectively reverses ABCC1-mediated MDR at clinically achievable concentrations. ABCC1 expressing tumors may be highly sensitive to the direct cytotoxicity of sulindac sulfide, and in combination with chemotherapeutic drugs that induce oxidative stress.

  15. Selective cell recruitment and spatially controlled cell attachment on instructive chitosan surfaces functionalized with antibodies.

    PubMed

    Custódio, C A; Frias, A M; del Campo, A; Reis, R L; Mano, J F

    2012-12-01

    Bioactive constructs to guide cellular mobilization and function have been proposed as an approach for a new generation of biomaterials in functional tissue engineering. Adult mesenchymal stem cells have been widely used as a source for cell based therapeutic strategies, namely tissue engineering. This is a heterogeneous cell population containing many subpopulations with distinct regenerative capacity. Thus, one of the issues for the effective clinical use of stem cells in tissue engineering is the isolation of a highly purified, expandable specific subpopulation of stem cells. Antibody functionalized biomaterials could be promising candidates to isolate and recruit specific cell types. Here we propose a new concept of instructive biomaterials that are able to recruit and purify specific cell types from a mixed cell population. This biomimetic concept uses a target-specific chitosan substrate to capture specific adipose derived stem cells. Specific antibodies were covalently immobilized onto chitosan membranes using bis[sulfosuccinimidyl] suberate (BS3). Quartz crystal microbalance (QCM) was used to monitor antibody immobilization/adsorption onto the chitosan films. Specific antibodies covalently immobilized, kept their bioactivity and captured specific cell types from a mixed cell population. Microcontact printing allowed to covalently immobilize antibodies in patterns and simultaneously a spatial control in cell attachment. PMID:23109106

  16. Photothermolysis by laser-induced microbubbles generated around gold nanorod clusters selectively formed in leukemia cells

    NASA Astrophysics Data System (ADS)

    Lapotko, Dmitri; Lukianova-Hleb, Ekaterina; Zhdanok, Sergei; Rostro, Betty; Simonette, Rebecca; Hafner, Jason; Konopleva, Marina; Andreeff, Michael; Conjusteau, Andre; Oraevsky, Alexander

    2008-02-01

    In an effort of developing clinical LANTCET (laser-activated nano-thermolysis as cell elimination technology) we achieved selective destruction of individual tumor cells through laser generation of vapor microbubbles around clusters of light absorbing gold nanorods (GNR) selectively formed in target tumor cells. Among all gold nanoparticles, nanorods offer the highest optical absorption in the near-infrared. We applied covalent conjugates of gold nanorods with targeting vectors such as monoclonal antibodies CD33 (specific for Acute Myeloid Leukemia), while GNR conjugates with polyethylene-glycol (PEG) were used as nonspecific targeting control. GNR clusters were formed inside the tumor cells at 37 °C due to endocytosis of large concentration of nanorods accumulated on the surface of tumor cells targeted at 4 °C. Formation of GNR clusters significantly reduces the threshold of tumor cell damage making LANTCET safe for normal cells. Appearance of GNR clusters was verified directly with optical resonance scattering microscopy. LANTCET was performed in vitro with living cells of (1) model myeloid K562 cells (CD33 positive), (2) primary human bone marrow CD33-positive blast cells from patients diagnosed with acute myeloid leukemia. Laser-induced microbubbles were generated and detected with a photothermal microscope equipped with a tunable Ti-Sa pulsed laser. GNT cluster formation caused a 100-fold decrease in the threshold optical fluence for laser microbubble generation in tumor cells compared with that in normal cells under the same targeting and irradiation conditions. Combining imaging based on resonance optical scattering with photothermal imaging of microbubbles, we developed a method for detection, image-guided treatment and monitoring of LANTCET. Pilot experiments were performed in flow mode bringing LANTCET closer to reality of clinical procedure of purging tumor cells from bone marrow grafts.

  17. Selective and potent furin inhibitors protect cells from anthrax without significant toxicity.

    PubMed

    Remacle, Albert G; Gawlik, Katarzyna; Golubkov, Vladislav S; Cadwell, Gregory W; Liddington, Robert C; Cieplak, Piotr; Millis, Sherri Z; Desjardins, Roxane; Routhier, Sophie; Yuan, Xue Wen; Neugebauer, Witold A; Day, Robert; Strongin, Alex Y

    2010-06-01

    Furin and related proprotein convertases cleave the multibasic motifs R-X-R/K/X-R in the precursor proteins and, as a result, transform the latent proproteins into biologically active proteins and peptides. Furin is present both in the intracellular secretory pathway and at the cell surface. Intracellular furin processes its multiple normal cellular targets in the Golgi and secretory vesicle compartments while cell-surface furin appears to be essential only for the processing of certain pathogenic proteins and, importantly, anthrax. To design potent, safe and selective inhibitors of furin, we evaluated the potency and selectivity of the derivatized peptidic inhibitors modeled from the extended furin cleavage sequence of avian influenza A H5N1. We determined that the N- and C-terminal modifications of the original RARRRKKRT inhibitory scaffold produced selective and potent, nanomolar range, inhibitors of furin. These inhibitors did not interfere with the normal cellular function of furin because of the likely functional redundancy existing between furin and other proprotein convertases. These furin inhibitors, however, were highly potent in blocking the furin-dependent cell-surface processing of anthrax protective antigen-83 both in vitro and cell-based assays and in vivo. We conclude that the inhibitors we have designed have a promising potential as selective anthrax inhibitors, without affecting major cell functions. PMID:20197107

  18. Selective and potent furin inhibitors protect cells from anthrax without significant toxicity

    PubMed Central

    Remacle, Albert G.; Gawlik, Katarzyna; Golubkov, Vladislav S.; Cadwell, Gregory W.; Liddington, Robert C.; Cieplak, Piotr; Millis, Sherri Z.; Desjardins, Roxane; Routhier, Sophie; Yuan, Xue Wen; Neugebauer, Witold A.; Day, Robert; Strongin, Alex Y.

    2010-01-01

    Furin and related proprotein convertases cleave the multibasic motifs R-X-R/K/X-R in the precursor proteins and, as a result, transform the latent proproteins into biologically active proteins and peptides. Furin is present both in the intracellular secretory pathway and at the cell surface. Intracellular furin processes its multiple normal cellular targets in the Golgi and secretory vesicle compartments while cell-surface furin appears to be essential only for the processing of certain pathogenic proteins and, importantly, anthrax. To design potent, safe and selective inhibitors of furin, we evaluated the potency and selectivity of the derivatized peptidic inhibitors modeled from the extended furin cleavage sequence of avian influenza A H5N1. We determined that the N- and C-terminal modifications of the original RARRRKKRT inhibitory scaffold produced selective and potent, nanomolar range, inhibitors of furin. These inhibitors did not interfere with the normal cellular function of furin because of the likely functional redundancy existing between furin and other proprotein convertases. These furin inhibitors, however, were highly potent in blocking the furin-dependent cell-surface processing of anthrax protective antigen-83 both in vitro and cell-based assays and in vivo. We conclude that the inhibitors we have designed have a promising potential as selective anthrax inhibitors, without affecting major cell functions. PMID:20197107

  19. Selective CDK7 inhibition with BS-181 suppresses cell proliferation and induces cell cycle arrest and apoptosis in gastric cancer

    PubMed Central

    Wang, Bo-Yong; Liu, Quan-Yan; Cao, Jun; Chen, Ji-Wei; Liu, Zhi-Su

    2016-01-01

    Cyclin-dependent kinase (CDK) family members have been considered as attractive therapeutic targets for cancer. In this study, we aim to investigate the anticancer effects of a selective CDK7 inhibitor, BS-181, in gastric cancer (GC) cell line. Human GC cells (BGC823) were cultured with or without BS-181 at different concentrations for 24–72 hours. BS-181 significantly reduced the activity of CDK7 with downregulation of cyclin D1 and XIAP in GC cells. Treatment with BS-181 induced cell cycle arrest and apoptosis. The expression of Bax and caspase-3 was significantly increased, while Bcl-2 expression was decreased in cells treated with BS-181. In addition, the inhibition of CDK7 with BS-181 resulted in reduced rates of proliferation, migration, and invasion of gastric cells. Those results demonstrated the anticancer activities of selective CDK7 inhibitor BS-181 in BGC823 cells, suggesting that CDK7 may serve as a novel therapeutic target or the treatment of GC. PMID:27042010

  20. From basic network principles to neural architecture: emergence of orientation-selective cells.

    PubMed Central

    Linsker, R

    1986-01-01

    This is the second paper in a series of three that explores the emergence of several prominent features of the functional architecture of visual cortex, in a "modular self-adaptive network" containing several layers of cells with parallel feedforward connections whose strengths develop according to a Hebb-type correlation-rewarding rule. In the present paper I show that orientation-selective cells, similar to the "simple" cortical cells of Hubel and Wiesel [Hubel, D. H. & Wiesel, T. N. (1962) J. Physiol. 160, 106-154], emerge in such a network. No orientation preference is specified to the system at any stage, the orientation-selective cell layer emerges even in the absence of environmental input to the system, and none of the basic developmental rules is specific to visual processing. PMID:3464958

  1. Electrospun Nanofibrous Sheets for Selective Cell Capturing in Continuous Flow in Microchannels.

    PubMed

    Son, Young Ju; Kang, Jihyun; Kim, Hye Sung; Yoo, Hyuk Sang

    2016-03-14

    Electrospun nanofibrous meshes were surface-modified for selective capturing of specific cells from a continuous flow in PDMS microchannels. We electrospun nanofibrous mats composed of poly(ε-carprolactone) (PCL) and amine-functionalized block copolymers composed of PCL and poly(ethylenimine) (PEI). A mixture of biotinylated PEG and blunt PEG was chemically tethered to the nanofibrous mats via the surface-exposed amines on the mat. The degree of biotinylation was fluorescently and quantitatively assayed for confirming the surface-biotinylation levels for avidin-specific binding. The incorporation level of avidin gradually increased when the blend ratio of biotinylated PEG on the mat increased, confirming the manipulated surfaces with various degree of biotinylation. Biotinylated cells were incubated with avidin-coated biotinylated mats and the specific binding of biotinylated cells was monitored in a microfluidic channel with a continuous flow of culture medium, which suggests efficient and selective capturing of the biotinylated cells on the nanofibrous mat. PMID:26812501

  2. Aptamers selected by cell-SELEX for application in cancer studies

    PubMed Central

    Zhang, Yunfei; Chen, Yan; Han, Da; Ocsoy, Ismail; Tan, Weihong

    2010-01-01

    Rapid development of anticancer therapies has occurred, but many challenges remain, including difficulties in early detection and the side effects from chemotherapy. To address these problems, aptamers, which are single-stranded DNA or RNA oligonucleotides with high selectivity, affinity and stability, have attracted considerable attention for biomedical applications. These oligonucleotides, which are selected by an in vitro process known as cell systematic evolution of ligands by exponential enrichment (cell-SELEX), have demonstrated the merits required to recognize disease cells. As such, they show great potential for applications in both clinical and laboratory settings. This review focuses on recently developed techniques utilizing aptamers in cancer research, including cancer cell detection, sorting and enrichment, as well as targeted drug delivery for cancer therapy. PMID:20657791

  3. Label-free selection and enrichment of liver cancer stem cells by surface niches build up with polyelectrolyte multilayer films.

    PubMed

    Lee, I-Chi; Chang, Jen-Fu

    2015-01-01

    Recent studies indicate that a small population of cancer cells exhibits stem cell properties and are referred to as cancer-initiating or cancer stem cells (CSCs). The selection and identification of cancer stem cells through methods require well-defined biomarkers and immunolabeling procedures are complicated and often unreliable. Herein, we fabricated a series of microenviroment by using polyelectrolyte multilayers (PEM) nanofilms to program and mimic hepatocellular carcinoma CSCs niches for CSCs selection with a label-free method. When cultured on PEM substrates, human cancer cell lines-Huh7 cells grew into individual round colonies and these cells displayed high marker expression of CSCs. Especially, these selected cells demonstrated significant chemo-resistant property in comparison with normal population. Therefore, we believed that niches selection and colony formation method may provide a new strategy on CSCs selection and drug evaluation for cancer therapy. PMID:25461919

  4. Altered and dynamic ion selectivity of K+ channels in cell development and excitability

    PubMed Central

    Chen, Haijun; Chatelain, Franck C.; Lesage, Florian

    2015-01-01

    K+ channels play a key role in regulating cellular excitability. It was thought that the strong K+-selectivity of these channels was static, only altered by mutations in their selectivity filter, which can cause severe genetic disorders. Recent studies demonstrate that selectivity of K+ channels can also exhibit dynamic changes. Under acidic conditions or in low extracellular K+ concentrations, the two-pore domain K+ channel (K2P) TWIK1 becomes permeable to Na+, shifting from an inhibitory role to an excitatory role. This phenomenon is responsible for the paradoxical depolarization of human cardiomyocytes in pathological hypokalemia, and therefore may contribute to cardiac arrhythmias. In other cell types, TWIK1 produces depolarizing leak currents under physiological conditions. Dynamic ion selectivity also occurs in other K2P channels. Here we review evidence that dynamic selectivity of K2P channels constitutes a new regulatory mechanism of cellular excitability, whose significance is only now becoming appreciated. PMID:25023607

  5. Low Selection Pressure Aids the Evolution of Cooperative Ribozyme Mutations in Cells*

    PubMed Central

    Amini, Zhaleh N.; Müller, Ulrich F.

    2013-01-01

    Understanding the evolution of functional RNA molecules is important for our molecular understanding of biology. Here we tested experimentally how two evolutionary parameters, selection pressure and recombination, influenced the evolution of an evolving RNA population. This was done using four parallel evolution experiments that employed low or gradually increasing selection pressure, and recombination events either at the end or dispersed throughout the evolution. As model system, a trans-splicing group I intron ribozyme was evolved in Escherichia coli cells over 12 rounds of selection and amplification, including mutagenesis and recombination. The low selection pressure resulted in higher efficiency of the evolved ribozyme populations, whereas differences in recombination did not have a strong effect. Five mutations were responsible for the highest efficiency. The first mutation swept quickly through all four evolving populations, whereas the remaining four mutations accumulated later and more efficiently under low selection pressure. To determine why low selection pressure aided this evolution, all evolutionary intermediates between the wild type and the 5-mutation variant were constructed, and their activities at three different selection pressures were determined. The resulting fitness profiles showed a high cooperativity among the four late mutations, which can explain why high selection pressure led to inefficient evolution. These results show experimentally how low selection pressure can benefit the evolution of cooperative mutations in functional RNAs. PMID:24089519

  6. Cardinal difference between the orientation-selective retinal ganglion cells projecting to the fish tectum and the orientation-selective complex cells of the mammalian striate cortex.

    PubMed

    Damjanović, Ilija; Maximova, Elena; Maximov, Paul; Maximov, Vadim

    2012-06-01

    Responses from two types of orientation-selective units of retinal origin were recorded extracellularly from their axon terminals in the medial sublaminae of tectal retinorecipient layer of immobilized cyprinid fish Carassius gibelio. Excitatory and inhibitory interactions in the receptive field were analyzed with two narrow stripes of optimal orientation flashing synchronously, one in the center and the other in different parts of the periphery. The general pattern of results was that the influence of the remote peripheral stripe was inhibitory, irrespective of the polarity of each stripe (light or dark). In this regard, the orientation-selective ganglion cells of the fish retina differ from the classical orientation-selective complex cells of the mammalian cortex, where the remote paired stripes of the opposite polarity (one light and one dark) interact in a facilitatory fashion. The consequence of these differences may be a weaker lateral inhibition in the latter case in response to stimulation by periodic gratings, which may contribute to a better spatial frequency tuning in the visual cortex. PMID:22744823

  7. Patterning pallet arrays for cell selection based on high-resolution measurements of fluorescent biosensors.

    PubMed

    Shadpour, Hamed; Zawistowski, Jon S; Herman, Annadele; Hahn, Klaus; Allbritton, Nancy L

    2011-06-24

    Pallet arrays enable cells to be separated while they remain adherent to a surface and provide a much greater range of cell selection criteria relative to that of current technologies. However there remains a need to further broaden cell selection criteria to include dynamic intracellular signaling events. To demonstrate the feasibility of measuring cellular protein behavior on the arrays using high resolution microscopy, the surfaces of individual pallets were modified to minimize the impact of scattered light at the pallet edges. The surfaces of the three-dimensional pallets on an array were patterned with a coating such as fibronectin using a customized stamping tool. Micropatterns of varying shape and size were printed in designated regions on the pallets in single or multiple steps to demonstrate the reliability and precision of patterning molecules on the pallet surface. Use of a fibronectin matrix stamped at the center of each pallet permitted the localization of H1299 and mouse embryonic fibroblast (MEF) cells to the pallet centers and away from the edges. Compared to pallet arrays with fibronectin coating the entire top surface, arrays with a central fibronectin pattern increased the percentage of cells localized to the pallet center by 3-4-fold. Localization of cells to the pallet center also enabled the physical separation of cells from optical artifacts created by the rough pallet side walls. To demonstrate the measurement of dynamic intracellular signaling on the arrays, fluorescence measurements of high spatial resolution were performed using a RhoA GTPase biosensor. This biosensor utilized fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) to measure localized RhoA activity in cellular ruffles at the cell periphery. These results demonstrated the ability to perform spatially resolved measurements of fluorescence-based sensors on the pallet arrays. Thus, the patterned pallet arrays

  8. Butyrate-rich colonic microenvironment is a relevant selection factor for metabolically adapted tumor cells.

    PubMed

    Serpa, Jacinta; Caiado, Francisco; Carvalho, Tânia; Torre, Cheila; Gonçalves, Luís G; Casalou, Cristina; Lamosa, Pedro; Rodrigues, Margarida; Zhu, Zhenping; Lam, Eric W F; Dias, Sérgio

    2010-12-10

    The short chain fatty acid (SCFA) butyrate is a product of colonic fermentation of dietary fibers. It is the main source of energy for normal colonocytes, but cannot be metabolized by most tumor cells. Butyrate also functions as a histone deacetylase (HDAC) inhibitor to control cell proliferation and apoptosis. In consequence, butyrate and its derived drugs are used in cancer therapy. Here we show that aggressive tumor cells that retain the capacity of metabolizing butyrate are positively selected in their microenvironment. In the mouse xenograft model, butyrate-preselected human colon cancer cells gave rise to subcutaneous tumors that grew faster and were more angiogenic than those derived from untreated cells. Similarly, butyrate-preselected cells demonstrated a significant increase in rates of homing to the lung after intravenous injection. Our data showed that butyrate regulates the expression of VEGF and its receptor KDR at the transcriptional level potentially through FoxM1, resulting in the generation of a functional VEGF:KDR autocrine growth loop. Cells selected by chronic exposure to butyrate express higher levels of MMP2, MMP9, α2 and α3 integrins, and lower levels of E-cadherin, a marker for epithelial to mesenchymal transition. The orthotopic model of colon cancer showed that cells preselected by butyrate are able to colonize the animals locally and at distant organs, whereas control cells can only generate a local tumor in the cecum. Together our data shows that a butyrate-rich microenvironment may select for tumor cells that are able to metabolize butyrate, which are also phenotypically more aggressive. PMID:20926374

  9. A Traceless Selection: Counter-selection System That Allows Efficient Generation of Transposon and CRISPR-modified T-cell Products.

    PubMed

    Mezzadra, Riccardo; Hollenstein, Andreas; Gomez-Eerland, Raquel; Schumacher, Ton N

    2016-01-01

    Recent years have seen major breakthroughs in genome-engineering systems, such as transposon-mediated gene delivery systems and CRISPR-Cas9-mediated genome-editing tools. In these systems, transient expression of auxiliary genes is responsible for permanent genomic modification. For both systems, it would be valuable to select for cells that are likely to undergo stable genome modification. Importantly, in particular for clinical applications of genome-engineered cell products, it will also be of importance to remove those cells that, due to random vector integration, display an unwanted stable expression of the auxiliary gene. Here, we develop a traceless selection system that on the one hand allows efficient enrichment of modified cells, and on the other hand can be used to select against cells that retain expression of the auxiliary gene. The value of this system to produce highly enriched-auxiliary gene-free cell products is demonstrated. PMID:27003756

  10. Can Selective MHC Downregulation Explain the Specificity and Genetic Diversity of NK Cell Receptors?

    PubMed Central

    Carrillo-Bustamante, Paola; Kesmir, Can; de Boer, Rob J.

    2015-01-01

    Natural killer (NK) cells express inhibiting receptors (iNKRs), which specifically bind MHC-I molecules on the surface of healthy cells. When the expression of MHC-I on the cell surface decreases, which might occur during certain viral infections and cancer, iNKRs lose inhibiting signals and the infected cells become target for NK cell activation (missing-self detection). Although the detection of MHC-I deficient cells can be achieved by conserved receptor-ligand interactions, several iNKRs are encoded by gene families with a remarkable genetic diversity, containing many haplotypes varying in gene content and allelic polymorphism. So far, the biological function of this expansion within the NKR cluster has remained poorly understood. Here, we investigate whether the evolution of diverse iNKRs genes can be driven by a specific viral immunoevasive mechanism: selective MHC downregulation. Several viruses, including EBV, CMV, and HIV, decrease the expression of MHC-I to escape from T cell responses. This downregulation does not always affect all MHC loci in the same way, as viruses target particular MHC molecules. To study the selection pressure of selective MHC downregulation on iNKRs, we have developed an agent-based model simulating an evolutionary scenario of hosts infected with herpes-like viruses, which are able to selectively downregulate the expression of MHC-I molecules on the cell surface. We show that iNKRs evolve specificity and, depending on the similarity of MHC alleles within each locus and the differences between the loci, they can specialize to a particular MHC-I locus. The easier it is to classify an MHC allele to its locus, the lower the required diversity of the NKRs. Thus, the diversification of the iNKR cluster depends on the locus specific MHC structure. PMID:26136746

  11. Frequency-selective REDOR and spin-diffusion relays in uniformly labeled whole cells.

    PubMed

    Rice, David M; Romaniuk, Joseph A H; Cegelski, Lynette

    2015-11-01

    Solid-state NMR is a powerful and non-perturbative method to measure and define chemical composition and architecture in bacterial cell walls, even in the context of whole cells. Most NMR studies on whole cells have used selectively labeled samples. Here, we introduce an NMR sequence relay using frequency-selective REDOR (fsREDOR) and spin diffusion elements to probe a unique amine contribution in uniformly (13)C- and (15)N-labeled Staphylococcus aureus whole cells that we attribute to the d-alanine of teichoic acid. In addition to the primary peptidoglycan structural scaffold, cell walls can contain significant amounts of teichoic acid that contribute to cell-wall function. When incorporated into teichoic acid, d-alanine is present as an ester, connected via its carbonyl to a ribitol carbon, and thus has a free amine. Teichoic acid d-Ala is removed during cell-wall isolations and can only be detected in the context of whole cells. The sequence presented here begins with fsREDOR and a chemical shift evolution period for 2D data acquisition, followed by DARR spin diffusion and then an additional fsREDOR period. fsREDOR elements were used for (13)C observation to avoid complications from (13)C-(13)C couplings due to uniform labeling and for (15)N dephasing to achieve selectivity in the nitrogens serving as dephasers. The results show that the selected amine nitrogen of interest is near to teichoic acid ribitol carbons and also the methyl group carbon associated with alanine. In addition, its carbonyl is not significantly dephased by amide nitrogens, consistent with the expected microenvironment around teichoic acid. PMID:26493462

  12. The Autoimmunity-Associated Gene CLEC16A Modulates Thymic Epithelial Cell Autophagy and Alters T Cell Selection.

    PubMed

    Schuster, Cornelia; Gerold, Kay D; Schober, Kilian; Probst, Lilli; Boerner, Kevin; Kim, Mi-Jeong; Ruckdeschel, Anna; Serwold, Thomas; Kissler, Stephan

    2015-05-19

    CLEC16A variation has been associated with multiple immune-mediated diseases, including type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, celiac disease, Crohn's disease, Addison's disease, primary biliary cirrhosis, rheumatoid arthritis, juvenile idiopathic arthritis, and alopecia areata. Despite strong genetic evidence implicating CLEC16A in autoimmunity, this gene's broad association with disease remains unexplained. We generated Clec16a knock-down (KD) mice in the nonobese diabetic (NOD) model for type 1 diabetes and found that Clec16a silencing protected against autoimmunity. Disease protection was attributable to T cell hyporeactivity, which was secondary to changes in thymic epithelial cell (TEC) stimuli that drive thymocyte selection. Our data indicate that T cell selection and reactivity were impacted by Clec16a variation in thymic epithelium owing to Clec16a's role in TEC autophagy. These findings provide a functional link between human CLEC16A variation and the immune dysregulation that underlies the risk of autoimmunity. PMID:25979422

  13. Aneuploidy impairs hematopoietic stem cell fitness and is selected against in regenerating tissues in vivo.

    PubMed

    Pfau, Sarah J; Silberman, Rebecca E; Knouse, Kristin A; Amon, Angelika

    2016-06-15

    Aneuploidy, an imbalanced karyotype, is a widely observed feature of cancer cells that has long been hypothesized to promote tumorigenesis. Here we evaluate the fitness of cells with constitutional trisomy or chromosomal instability (CIN) in vivo using hematopoietic reconstitution experiments. We did not observe cancer but instead found that aneuploid hematopoietic stem cells (HSCs) exhibit decreased fitness. This reduced fitness is due at least in part to the decreased proliferative potential of aneuploid hematopoietic cells. Analyses of mice with CIN caused by a hypomorphic mutation in the gene Bub1b further support the finding that aneuploidy impairs cell proliferation in vivo. Whereas nonregenerating adult tissues are highly aneuploid in these mice, HSCs and other regenerative adult tissues are largely euploid. These findings indicate that, in vivo, mechanisms exist to select against aneuploid cells. PMID:27313317

  14. Repurposing a Prokaryotic Toxin-Antitoxin System for the Selective Killing of Oncogenically Stressed Human Cells.

    PubMed

    Preston, Mark A; Pimentel, Belén; Bermejo-Rodríguez, Camino; Dionne, Isabelle; Turnbull, Alice; de la Cueva-Méndez, Guillermo

    2016-07-15

    Prokaryotes express intracellular toxins that pass unnoticed to carrying cells until coexpressed antitoxin partners are degraded in response to stress. Although not evolved to function in eukaryotes, one of these toxins, Kid, induces apoptosis in mammalian cells, an effect that is neutralized by its cognate antitoxin, Kis. Here we engineered this toxin-antitoxin pair to create a synthetic system that becomes active in human cells suffering a specific oncogenic stress. Inspired by the way Kid becomes active in bacterial cells, we produced a Kis variant that is selectively degraded in human cells expressing oncoprotein E6. The resulting toxin-antitoxin system functions autonomously in human cells, distinguishing those that suffer the oncogenic insult, which are killed by Kid, from those that do not, which remain protected by Kis. Our results provide a framework for developing personalized anticancer strategies avoiding off-target effects, a challenge that has been hardly tractable by other means thus far. PMID:26230535

  15. Modulation of Igβ is essential for the B cell selection in germinal center

    PubMed Central

    Todo, Kagefumi; Koga, Orie; Nishikawa, Miwako; Hikida, Masaki

    2015-01-01

    The positive and negative selection of antigen-reactive B cells take place in the germinal center (GC) during an immune responses. However, the precise molecular mechanisms underlying these selection machineries, including the involvement of antigen receptor signaling molecules, remain to be elucidated. We found that expression levels of Igα and Igβ, which are the essential components of B cell antigen-receptor complex, were differentially regulated in GC B cells and that the expression of Igβ was more prominently down-regulated in a portion of GC B cells. The suppression of Igβ down-regulation reduced the number of GL7+GC B cells and the affinity maturation in T-dependent responses was markedly impaired. In addition, the disease phenotypes in autoimmune-prone mice were ameliorated by blocking of Igβ down-regulation. These results suggest that Igβ down-regulation is involved in the normal positive selection in GC and the accumulation of autoreactive B cells in autoimmune-prone mice. PMID:25980548

  16. Silicon cells made by self-aligned selective-emitter plasma-etchback process

    DOEpatents

    Ruby, Douglas S.; Schubert, William K.; Gee, James M.; Zaidi, Saleem H.

    2000-01-01

    Photovoltaic cells and methods for making them are disclosed wherein the metallized grids of the cells are used to mask portions of cell emitter regions to allow selective etching of phosphorus-doped emitter regions. The preferred etchant is SF.sub.6 or a combination of SF.sub.6 and O.sub.2. This self-aligned selective etching allows for enhanced blue response (versus cells with uniform heavy doping of the emitter) while preserving heavier doping in the region beneath the gridlines needed for low contact resistance. Embodiments are disclosed for making cells with or without textured surfaces. Optional steps include plasma hydrogenation and PECVD nitride deposition, each of which are suited to customized applications for requirements of given cells to be manufactured. The techniques disclosed could replace expensive and difficult alignment methodologies used to obtain selectively etched emitters, and they may be easily integrated with existing plasma processing methods and techniques of the invention may be accomplished in a single plasma-processing chamber.

  17. MicroRNA-203 represses selection and expansion of oncogenic Hras transformed tumor initiating cells

    PubMed Central

    Riemondy, Kent; Wang, Xiao-jing; Torchia, Enrique C; Roop, Dennis R; Yi, Rui

    2015-01-01

    In many mouse models of skin cancer, only a few tumors typically form even though many cells competent for tumorigenesis receive the same oncogenic stimuli. These observations suggest an active selection process for tumor-initiating cells. Here, we use quantitative mRNA- and miR-Seq to determine the impact of HrasG12V on the transcriptome of keratinocytes. We discover that microRNA-203 is downregulated by HrasG12V. Using a knockout mouse model, we demonstrate that loss of microRNA-203 promotes selection and expansion of tumor-initiating cells. Conversely, restoration of microRNA-203 using an inducible model potently inhibits proliferation of these cells. We comprehensively identify microRNA-203 targets required for Hras-initiated tumorigenesis. These targets include critical regulators of the Ras pathway and essential genes required for cell division. This study establishes a role for the loss of microRNA-203 in promoting selection and expansion of Hras mutated cells and identifies a mechanism through which microRNA-203 antagonizes Hras-mediated tumorigenesis. DOI: http://dx.doi.org/10.7554/eLife.07004.001 PMID:26203562

  18. Hard tissue formation of STRO-1-selected rat dental pulp stem cells in vivo.

    PubMed

    Yang, Xuechao; Walboomers, X Frank; van den Beucken, Jeroen J J P; Bian, Zhuan; Fan, Mingwen; Jansen, John A

    2009-02-01

    The objective of this study was to examine hard tissue formation of STRO-1-selected rat dental pulp-derived stem cells, seeded into a calcium phosphate ceramic scaffold, and implanted subcutaneously in mice. Previously, STRO-1 selection was used to obtain a mesenchymal stem cell progenitor subpopulation from primary dental pulp-derived stem cells. In the current study, these cells were cultured with three different media: "BMP-plus" medium containing dexamethasone and 100 ng/mL of rhBMP-2, "odontogenic" medium containing dexamethasone, and "control" medium without supplements. The cell-scaffold complexes were cultured in these media for 1, 4, or 8 days before implantation. Histological analysis demonstrated that the cultures with BMP-plus and 4 days of culture gave the highest percentage of hard tissue formation per implant (36 +/- 9% of pore area). Real-time PCR confirmed these results. In conclusion, STRO-1-selected dental pulp stem cells show effective hard tissue formation in vivo, and a short in vitro culture period and addition of BMP-2 can enhance this effect. PMID:18652538

  19. Selective inhibitory effect of HPMA copolymer-cyclopamine conjugate on prostate cancer stem cells

    PubMed Central

    Zhou, Yan; Yang, Jiyuan; Kopeček, Jindřich

    2011-01-01

    Improved treatments for prostate cancer are in great need to overcome lethal recurrence and metastasis. Targeting the tumorigenic cancer stem cells (CSCs) with self-renewal and differentiation capacity appears to be a promising strategy. Blockade of the hedgehog (Hh) signaling pathway, an important pathway involved in stem cell self-renewal, by cyclopamine leads to long-term prostate cancer regression without recurrence, strongly suggesting the connection between Hh pathway and prostate CSCs. Here we designed a HPMA (N-(2-hydroxypropyl)methacrylamide)-based cyclopamine delivery system as a CSC-selective macromolecular therapeutics with improved drug solubility and decreased systemic toxicity. To this end, HPMA and N-methacryloylglycylphenylalanylleucylglycyl thiazolidine-2-thione were copolymerized using the RAFT (reversible addition-fragmentation chain transfer) process, followed by polymer-analogous attachment of cyclopamine. The selectivity of the conjugate toward CSCs was evaluated on RC-92a/hTERT cells, the human prostate cancer epithelial cells with human telomerase reverse transcriptase transduction. The use of RC-92a/hTERT cells as an in vitro CSC model was validated by stem cell marker expression and prostasphere culture. The bioactivity of cyclopamine was retained after conjugation to the polymer. Furthermore, HPMA polymer-conjugated cyclopamine showed anti-CSC efficacy on RC-92a/hTERT cells as evaluated by decreased stem cell marker expression and CSC viability. PMID:22138033

  20. Aging-associated inflammation promotes selection for adaptive oncogenic events in B cell progenitors

    PubMed Central

    Henry, Curtis J.; Casás-Selves, Matias; Kim, Jihye; Zaberezhnyy, Vadym; Aghili, Leila; Daniel, Ashley E.; Jimenez, Linda; Azam, Tania; McNamee, Eoin N.; Clambey, Eric T.; Klawitter, Jelena; Serkova, Natalie J.; Tan, Aik Choon; Dinarello, Charles A.; DeGregori, James

    2015-01-01

    The incidence of cancer is higher in the elderly; however, many of the underlying mechanisms for this association remain unexplored. Here, we have shown that B cell progenitors in old mice exhibit marked signaling, gene expression, and metabolic defects. Moreover, B cell progenitors that developed from hematopoietic stem cells (HSCs) transferred from young mice into aged animals exhibited similar fitness defects. We further demonstrated that ectopic expression of the oncogenes BCR-ABL, NRASV12, or Myc restored B cell progenitor fitness, leading to selection for oncogenically initiated cells and leukemogenesis specifically in the context of an aged hematopoietic system. Aging was associated with increased inflammation in the BM microenvironment, and induction of inflammation in young mice phenocopied aging-associated B lymphopoiesis. Conversely, a reduction of inflammation in aged mice via transgenic expression of α-1-antitrypsin or IL-37 preserved the function of B cell progenitors and prevented NRASV12-mediated oncogenesis. We conclude that chronic inflammatory microenvironments in old age lead to reductions in the fitness of B cell progenitor populations. This reduced progenitor pool fitness engenders selection for cells harboring oncogenic mutations, in part due to their ability to correct aging-associated functional defects. Thus, modulation of inflammation — a common feature of aging — has the potential to limit aging-associated oncogenesis. PMID:26551682

  1. 5-Fluorouracil targets thymidylate synthase in the selective suppression of TH17 cell differentiation

    PubMed Central

    Wang, Juan; Peng, Liang; Zhang, Ruihua; Zheng, Zihan; Chen, Chun; Cheung, Ka Lung; Cui, Miao; Bian, Guanglin; Xu, Feihong; Chiang, David; Hu, Yuan; Chen, Ye; Lu, Geming; Yang, Jianjun; Zhang, Hui; Yang, Jianfei; Zhu, Hongfa; Chen, Shu-hsia; Liu, Kebin; Zhou, Ming-Ming; Sikora, Andrew G.; Li, Liwu; Jiang, Bo; Xiong, Huabao

    2016-01-01

    While it is well established that treatment of cancer patients with 5-Fluorouracil (5-FU) can result in immune suppression, the exact function of 5-FU in the modulation of immune cells has not been fully established. We found that low dose 5-FU selectively suppresses TH17 and TH1 cell differentiation without apparent effect on Treg, TH2, and significantly suppresses thymidylate synthase (TS) expression in TH17 and TH1 cells but has a lesser effect in tumor cells and macrophages. Interestingly, the basal expression of TS varies significantly between T helper phenotypes and knockdown of TS significantly impairs TH17 and TH1 cell differentiation without affecting the differentiation of either Treg or TH2 cells. Finally, low dose 5-FU is effective in ameliorating colitis development by suppressing TH17 and TH1 cell development in a T cell transfer colitis model. Taken together, the results highlight the importance of the anti-inflammatory functions of low dose 5-FU by selectively suppressing TH17 and TH1 immune responses. PMID:27027355

  2. Murine hematopoietic reconstitution after tagging and selection of retrovirally transduced bone marrow cells

    PubMed Central

    García-Hernández, B.; Castellanos, A.; López, A.; Orfao, A.; Sánchez-García, I.

    1997-01-01

    A major problem facing the effective treatment of patients with cancer is how to get the specific antitumor agent into every tumor cell. In this report we describe the use of a strategy that, by using retroviral vectors encoding a truncated human CD5 cDNA, allows the selection of only the infected cells, and we show the ability to obtain, before bone marrow transplantation, a population of 5-fluouraci-treated murine bone marrow cells that are 100% marked. This marked population of bone marrow cells is able to reconstitute the hematopoietic system in lethally irradiated mice, indicating that the surface marker lacks deleterious effects on the functionality of bone marrow cells. No gross abnormalities in hematopoiesis were detected in mice repopulated with CD5-expressing cells. Nevertheless, a significant proportion of the hematopoietic cells no longer expresses the surface marker CD5 in the 9-month-old recipient mice. This transcriptional inactivity of the proviral long terminal repeat (LTR) was accompanied by de novo methylation of the proviral sequences. Our results show that the use of the CD5 as a retrovirally encoded marker enables the rapid, efficient, and nontoxic selection in vitro of infected primary cells, which can entirely reconstitute the hematopoietic system in mice. These results should now greatly enhance the power of studies aimed at addressing questions such as generation of cancer-negative hematopoiesis. PMID:9371830

  3. Molecular mechanisms of apoptosis and cell selectivity of zinc dithiocarbamates functionalized with hydroxyethyl substituents.

    PubMed

    Tan, Yee Seng; Ooi, Kah Kooi; Ang, Kok Pian; Akim, Abdah Md; Cheah, Yoke-Kqueen; Halim, Siti Nadiah Abdul; Seng, Hoi-Ling; Tiekink, Edward R T

    2015-09-01

    In the solid state each of three binuclear zinc dithiocarbamates bearing hydroxyethyl groups, {Zn[S2CN(R)CH2CH2OH]2}2 for R = iPr (1), CH2CH2OH (2), and Me (3), and an all alkyl species, [Zn(S2CNEt2)2]2 (4), features a centrosymmetric {ZnSCS}2 core with a step topology; both 1 and 3 were isolated as monohydrates. All compounds were broadly cytotoxic, specifically against human cancer cell lines compared with normal cells, with greater potency than cisplatin. Notably, some selectivity were indicated with 2 being the most potent against human ovarian carcinoma cells (cisA2780), and 4 being more cytotoxic toward multidrug resistant human breast carcinoma cells (MCF-7R), human colon adenocarcinoma cells (HT-29), and human lung adenocarcinoma epithelial cells (A549). Based on human apoptosis PCR-array analysis, caspase activities, DNA fragmentation, cell apoptotic assays, intracellular reactive oxygen species (ROS) measurements and human topoisomerase I inhibition, induction of apoptosis in HT-29 cells is demonstrated via both extrinsic and intrinsic pathways. Compounds 2-4 activate the p53 gene while 1 activates both p53 and p73. Cell cycle arrest at the S and G2/M phases correlates with inhibition of HT-29 cell growth. Cell invasion is also inhibited by 1-4 which is correlated with down-regulation of NF-κB. PMID:26086852

  4. Selective local lysis and sampling of live cells for nucleic acid analysis using a microfluidic probe

    PubMed Central

    Kashyap, Aditya; Autebert, Julien; Delamarche, Emmanuel; Kaigala, Govind V.

    2016-01-01

    Heterogeneity is inherent to biology, thus it is imperative to realize methods capable of obtaining spatially-resolved genomic and transcriptomic profiles of heterogeneous biological samples. Here, we present a new method for local lysis of live adherent cells for nucleic acid analyses. This method addresses bottlenecks in current approaches, such as dilution of analytes, one-sample-one-test, and incompatibility to adherent cells. We make use of a scanning probe technology - a microfluidic probe - and implement hierarchical hydrodynamic flow confinement (hHFC) to localize multiple biochemicals on a biological substrate in a non-contact, non-destructive manner. hHFC enables rapid recovery of nucleic acids by coupling cell lysis and lysate collection. We locally lysed ~300 cells with chemical systems adapted for DNA or RNA and obtained lysates of ~70 cells/μL for DNA analysis and ~15 cells/μL for mRNA analysis. The lysates were introduced into PCR-based workflows for genomic and transcriptomic analysis. This strategy further enabled selective local lysis of subpopulations in a co-culture of MCF7 and MDA-MB-231 cells, validated by characteristic E-cadherin gene expression in individually extracted cell types. The developed strategy can be applied to study cell-cell, cell-matrix interactions locally, with implications in understanding growth, progression and drug response of a tumor. PMID:27411740

  5. Cold atmospheric plasma jet-generated RONS and their selective effects on normal and carcinoma cells

    PubMed Central

    Kim, Sun Ja; Chung, T. H.

    2016-01-01

    Cold atmospheric helium plasma jets were fabricated and utilized for plasma–cell interactions. The effect of operating parameters and jet design on the generation of specific reactive oxygen and nitrogen species (RONS) within cells and cellular response were investigated. It was found that plasma treatment induced the overproduction of RONS in various cancer cell lines selectively. The plasma under a relatively low applied voltage induced the detachment of cells, a reduction in cell viability, and apoptosis, while the plasma under higher applied voltage led to cellular necrosis in our case. To determine whether plasma-induced reactive oxygen species (ROS) generation occurs through interfering with mitochondria-related cellular response, we examined the plasma effects on ROS generation in both parental A549 cells and A549 ρ0 cells. It was observed that cancer cells were more susceptible to plasma-induced RONS (especially nitric oxide (NO) and nitrogen dioxide (NO2−) radicals) than normal cells, and consequently, plasma induced apoptotic cell responses mainly in cancer cells. PMID:26838306

  6. Selective local lysis and sampling of live cells for nucleic acid analysis using a microfluidic probe.

    PubMed

    Kashyap, Aditya; Autebert, Julien; Delamarche, Emmanuel; Kaigala, Govind V

    2016-01-01

    Heterogeneity is inherent to biology, thus it is imperative to realize methods capable of obtaining spatially-resolved genomic and transcriptomic profiles of heterogeneous biological samples. Here, we present a new method for local lysis of live adherent cells for nucleic acid analyses. This method addresses bottlenecks in current approaches, such as dilution of analytes, one-sample-one-test, and incompatibility to adherent cells. We make use of a scanning probe technology - a microfluidic probe - and implement hierarchical hydrodynamic flow confinement (hHFC) to localize multiple biochemicals on a biological substrate in a non-contact, non-destructive manner. hHFC enables rapid recovery of nucleic acids by coupling cell lysis and lysate collection. We locally lysed ~300 cells with chemical systems adapted for DNA or RNA and obtained lysates of ~70 cells/μL for DNA analysis and ~15 cells/μL for mRNA analysis. The lysates were introduced into PCR-based workflows for genomic and transcriptomic analysis. This strategy further enabled selective local lysis of subpopulations in a co-culture of MCF7 and MDA-MB-231 cells, validated by characteristic E-cadherin gene expression in individually extracted cell types. The developed strategy can be applied to study cell-cell, cell-matrix interactions locally, with implications in understanding growth, progression and drug response of a tumor. PMID:27411740

  7. Intercellular redistribution of cAMP underlies selective suppression of cancer cell growth by connexin26.

    PubMed

    Chandrasekhar, Anjana; Kalmykov, Edward A; Polusani, Srikanth R; Mathis, Sandra A; Zucker, Shoshanna N; Nicholson, Bruce J

    2013-01-01

    Connexins (Cx), which constitute gap junction intercellular channels in vertebrates, have been shown to suppress transformed cell growth and tumorigenesis, but the mechanism(s) still remain largely speculative. Here, we define the molecular basis by which Cx26, but less frequently Cx43 or Cx32, selectively confer growth suppression on cancer cells. Functional intercellular coupling is shown to be required, producing partial blocks of the cell cycle due to prolonged activation of several mitogenic kinases. PKA is both necessary and sufficient for the Cx26 induced growth inhibition in low serum and the absence of anchorage. Activation of PKA was not associated with elevated cAMP levels, but appeared to result from a redistribution of cAMP throughout the cell population, eliminating the cell cycle oscillations in cAMP required for efficient cell cycle progression. Cx43 and Cx32 fail to mediate this redistribution as, unlike Cx26, these channels are closed during the G2/M phase of the cell cycle when cAMP levels peak. Comparisons of tumor cell lines indicate that this is a general pattern, with growth suppression by connexins occurring whenever cAMP oscillates with the cell cycle, and the gap junction remain open throughout the cell cycle. Thus, gap junctional coupling, in the absence of any external signals, provides a general means to limit the mitotic rate of cell populations. PMID:24312655

  8. A Skin-selective Homing Mechanism for Human Immune Surveillance T Cells

    PubMed Central

    Schaerli, Patrick; Ebert, Lisa; Willimann, Katharina; Blaser, Andrea; Roos, Regula Stuber; Loetscher, Pius; Moser, Bernhard

    2004-01-01

    Effective immune surveillance is essential for maintaining protection and homeostasis of peripheral tissues. However, mechanisms controlling memory T cell migration to peripheral tissues such as the skin are poorly understood. Here, we show that the majority of human T cells in healthy skin express the chemokine receptor CCR8 and respond to its selective ligand I-309/CCL1. These CCR8+ T cells are absent in small intestine and colon tissue, and are extremely rare in peripheral blood, suggesting healthy skin as their physiological target site. Cutaneous CCR8+ T cells are preactivated and secrete proinflammatory cytokines such as tumor necrosis factor–α and interferon-γ, but lack markers of cytolytic T cells. Secretion of interleukin (IL)-4, IL-10, and transforming growth factor–β was low to undetectable, arguing against a strict association of CCR8 expression with either T helper cell 2 or regulatory T cell subsets. Potential precursors of skin surveillance T cells in peripheral blood may correspond to the minor subset of CCR8+CD25− T cells. Importantly, CCL1 is constitutively expressed at strategic cutaneous locations, including dermal microvessels and epidermal antigen-presenting cells. For the first time, these findings define a chemokine system for homeostatic T cell traffic in normal human skin. PMID:15123746

  9. Intercellular Redistribution of cAMP Underlies Selective Suppression of Cancer Cell Growth by Connexin26

    PubMed Central

    Polusani, Srikanth R.; Mathis, Sandra A.; Zucker, Shoshanna N.; Nicholson, Bruce J.

    2013-01-01

    Connexins (Cx), which constitute gap junction intercellular channels in vertebrates, have been shown to suppress transformed cell growth and tumorigenesis, but the mechanism(s) still remain largely speculative. Here, we define the molecular basis by which Cx26, but less frequently Cx43 or Cx32, selectively confer growth suppression on cancer cells. Functional intercellular coupling is shown to be required, producing partial blocks of the cell cycle due to prolonged activation of several mitogenic kinases. PKA is both necessary and sufficient for the Cx26 induced growth inhibition in low serum and the absence of anchorage. Activation of PKA was not associated with elevated cAMP levels, but appeared to result from a redistribution of cAMP throughout the cell population, eliminating the cell cycle oscillations in cAMP required for efficient cell cycle progression. Cx43 and Cx32 fail to mediate this redistribution as, unlike Cx26, these channels are closed during the G2/M phase of the cell cycle when cAMP levels peak. Comparisons of tumor cell lines indicate that this is a general pattern, with growth suppression by connexins occurring whenever cAMP oscillates with the cell cycle, and the gap junction remain open throughout the cell cycle. Thus, gap junctional coupling, in the absence of any external signals, provides a general means to limit the mitotic rate of cell populations. PMID:24312655

  10. A Selective and Purification-Free Strategy for Labeling Adherent Cells with Inorganic Nanoparticles.

    PubMed

    Gao, Yu; Lim, Jing; Yeo, David Chen Loong; Liao, Shanshan; Lans, Malin; Wang, Yaqi; Teoh, Swee-Hin; Goh, Bee Tin; Xu, Chenjie

    2016-03-01

    Cellular labeling with inorganic nanoparticles such as magnetic iron oxide nanoparticles, quantum dots, and fluorescent silica nanoparticles is an important method for the noninvasive visualization of cells using various imaging modalities. Currently, this is mainly achieved through the incubation of cultured cells with the nanoparticles that eventually reach the intracellular compartment through specific or nonspecific internalization. This classic method is advantageous in terms of simplicity and convenience, but it suffers from issues such as difficulties in fully removing free nanoparticles (suspended in solution) and the lack of selectivity on cell types. This article reports an innovative strategy for the specific labeling of adherent cells without the concern of freely suspended nanoparticles. This method relies on a nanocomposite film that is prepared by homogeneously dispersing nanoparticles within a biodegradable polymeric film. When adherent cells are seeded on the film, they adhere, spread, and filtrate into the film through the micropores formed during the film fabrication. The pre-embedded nanoparticles are thus internalized by the cells during this infiltration process. As an example, fluorescent silica nanoparticles were homogeneously distributed within a polycaprolactone film by utilizing cryomilling and heat pressing. Upon incubation within physiological buffer, no silica nanoparticles were released from the nanocomposite film even after 20 d of incubation. However, when adherent cells (e.g., human mesenchymal stem cells) were grown on the film, they became fluorescent after 3 d, which suggests internalization of silica nanoparticles by cells. In comparison, the suspension cells (e.g., monocytes) in the medium remained nonfluorescent no matter whether there was the presence of adherent cells or not. This strategy eventually allowed the selective and concomitant labeling of mesenchymal stem cells during their harvest from bone marrow aspiration