Sample records for rapid cell shrinkage

  1. Longitudinal shrinkage of compression wood in dependence on water content and cell wall structure

    Directory of Open Access Journals (Sweden)

    Wiesław Włoch


    Full Text Available Compression wood from branches of Pinus silvestris L. was examined. Wide differences were noted in longitudinal shrinkage of the wood when dried from water saturated state. A relation was found between shrinkage and cell wall thickness, particularly of layer S2, and the degree of callose accumulation in the wall. No dependence could be revealed between the shrinkage and the fibril angle in S2.

  2. Mechanisms of activation of NHE by cell shrinkage and by calyculin A in Ehrlich ascites tumor cells

    DEFF Research Database (Denmark)

    Pedersen, Stine Helene Falsig; Varming, Camilla; Hoffmann, E K


    The Na+/H+ exchanger isoforms NHE1, NHE2, and NHE3 were all found to be expressed in Ehrlich ascites tumor cells, as evaluated by Western blotting and confocal microscopy. Under unstimulated conditions, NHE1 was found predominantly in the plasma membrane, NHE3 intracellularly, and NHE2 in both...... compartments. Osmotic cell shrinkage elicited a rapid intracellular alkalinization, the sensitivity of which to EIPA (IC50 0.19 microM) and HOE 642 (IC50 0.85 microM) indicated that it predominantly reflected activation of NHE1. NHE activation by osmotic shrinkage was inhibited by the protein kinase C...... inhibitors chelerythrine (IC50 12.5 microM), Gö 6850 (5 microM), and Gö 6976 (1 microM), and by the p38 MAPK inhibitor SB 203580 (10 microM). Furthermore, hypertonic cell shrinkage elicited a biphasic increase in p38 MAPK phosphorylation, with the first significant increase detectable 2 minutes after...

  3. Study of SEM preparation artefacts with correlative microscopy: Cell shrinkage of adherent cells by HMDS-drying. (United States)

    Katsen-Globa, Alisa; Puetz, Norbert; Gepp, Michael M; Neubauer, Julia C; Zimmermann, Heiko


    One of the often reported artefacts during cell preparation to scanning electron microscopy (SEM) is the shrinkage of cellular objects, that mostly occurs at a certain time-dependent stage of cell drying. Various methods of drying for SEM, such as critical point drying, freeze-drying, as well as hexamethyldisilazane (HMDS)-drying, were usually used. The latter becomes popular since it is a low cost and fast method. However, the correlation of drying duration and real shrinkage of objects was not investigated yet. In this paper, cell shrinkage at each stage of preparation for SEM was studied. We introduce a shrinkage coefficient using correlative light microscopy (LM) and SEM of the same human mesenchymal stem cells (hMSCs). The influence of HMDS-drying duration on the cell shrinkage is shown: the longer drying duration, the more shrinkage is observed. Furthermore, it was demonstrated that cell shrinkage is inversely proportional to cultivation time: the longer cultivation time, the more cell spreading area and the less cell shrinkage. Our results can be applicable for an exact SEM quantification of cell size and determination of cell spreading area in engineering of artificial cellular environments using biomaterials. SCANNING 38:625-633, 2016. © 2016 Wiley Periodicals, Inc. © Wiley Periodicals, Inc.

  4. Shrinkage insensitivity of NKCC1 in myosin II-depleted cytoplasts from Ehrlich ascites tumor cells

    DEFF Research Database (Denmark)

    Hoffmann, Else K; Pedersen, Stine F


    -actin organization was disrupted, and myosin II, which in shrunken EATC translocates to the cortical region, was absent. Moreover, NKCC1 activity was essentially insensitive to the myosin light chain kinase (MLCK) inhibitor ML-7, a potent blocker of shrinkage-induced NKCC1 activity in intact EATC. Cytoplast NKCC1...... to the substantial activation in shrunken intact cells, p38 MAPK could not be further activated by shrinkage of the cytoplasts. Together these findings indicate that shrinkage activation of NKCC1 in EATC is dependent on the cortical F-actin network, myosin II, and MLCK....

  5. Cell shrinkage as a signal to apoptosis in NIH 3T3 fibroblasts

    DEFF Research Database (Denmark)

    Friis, Martin B; Friborg, Christel R; Schneider, Linda


    Cell shrinkage is a hallmark of the apoptotic mode of programmed cell death, but it is as yet unclear whether a reduction in cell volume is a primary activation signal of apoptosis. Here we studied the effect of an acute elevation of osmolarity (NaCl or sucrose additions, final osmolarity 687...... mosmol l(-1)) on NIH 3T3 fibroblasts to identify components involved in the signal transduction from shrinkage to apoptosis. After 1.5 h the activity of caspase-3 started to increase followed after 3 h by the appearance of many apoptotic-like bodies. The caspase-3 activity increase was greatly enhanced...

  6. Shrinkage of freeze-dried cryosections of cells: Investigations by EFTEM and cryo-CLEM. (United States)

    Casanova, G; Nolin, F; Wortham, L; Ploton, D; Banchet, V; Michel, J


    Freeze-drying of cryosections of cells or tissues is considered to be the most efficient preparation for microanalysis purpose related to transmission electron microscopy. It allows the measurements of ions and water contents at the ultrastructural level. However an important drawback is associated to freeze-drying: the shrinkage of the cryosections. The aim of this paper is the investigation of this phenomenon by means of three different methods applied to both hydrated and dehydrated cryosections: direct distance measurements on fiducial points, thickness measurements by energy filtered transmission microscopy (EFTEM) and cryo-correlative light electron microscopy (cryo-CLEM). Measurements in our experimental conditions reveal a lateral shrinkage around 10% but the most important result concerns the lack of differential shrinkage between most of the cellular compartments. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Minimal volume regulation after shrinkage of red blood cells from five species of reptiles

    DEFF Research Database (Denmark)

    Kristensen, Karina; Berenbrink, Michael; Koldkjær, Pia


    cell function. The present study demonstrates that oxygenated RBCs in all major groups of reptiles exhibit no or a very reduced RVI upon ~ 25% calculated hyperosmotic shrinkage. Thus, RBCs from the snakes Crotalus durissus and Python regius, the turtle Trachemys scripta and the alligator Alligator...

  8. Hygroscopic swelling and shrinkage of latewood cell wall micropillars reveal ultrastructural anisotropy. (United States)

    Rafsanjani, Ahmad; Stiefel, Michael; Jefimovs, Konstantins; Mokso, Rajmund; Derome, Dominique; Carmeliet, Jan


    We document the hygroscopic swelling and shrinkage of the central and the thickest secondary cell wall layer of wood (named S2) in response to changes in environmental humidity using synchrotron radiation-based phase contrast X-ray tomographic nanoscopy. The S2 layer is a natural fibre-reinforced nano-composite polymer and is strongly reactive to water. Using focused ion beam, micropillars with a cross section of few micrometres are fabricated from the S2 layer of the latewood cell walls of Norway spruce softwood. The thin neighbouring cell wall layers are removed to prevent hindering or restraining of moisture-induced deformation during swelling or shrinkage. The proposed experiment intended to get further insights into the microscopic origin of the anisotropic hygro-expansion of wood. It is found that the swelling/shrinkage strains are highly anisotropic in the transverse plane of the cell wall, larger in the normal than in the direction parallel to the cell wall's thickness. This ultrastructural anisotropy may be due to the concentric lamellation of the cellulose microfibrils as the role of the cellulose microfibril angle in the transverse swelling anisotropy is negligible. The volumetric swelling of the cell wall material is found to be substantially larger than the one of wood tissues within the growth ring and wood samples made of several growth rings. The hierarchical configuration in wood optimally increases its dimensional stability in response to a humid environment with higher scales of complexity.

  9. Porcine intestinal mast cells. Evaluation of different fixatives for histochemical staining techniques considering tissue shrinkage

    Directory of Open Access Journals (Sweden)

    J. Rieger


    Full Text Available Staining of mast cells (MCs, including porcine ones, is critically dependent upon the fixation and staining technique. In the pig, mucosal and submucosal MCs do not stain or stain only faintly after formalin fixation. Some fixation methods are particularly recommended for MC staining, for example the fixation with Carnoy or lead salts. Zinc salt fixation (ZSF has been reported to work excellently for the preservation of fixation-sensitive antigens. The aim of this study was to establish a reliable histological method for counting of MCs in the porcine intestinum. For this purpose, different tissue fixation and staining methods that also allow potential subsequent immunohistochemical investigations were evaluated in the porcine mucosa, as well as submucosa of small and large intestine. Tissues were fixed in Carnoy, lead acetate, lead nitrate, Zamboni and ZSF and stained subsequently with either polychromatic methylene blue, alcian blue or toluidine blue. For the first time our study reveals that ZSF, a heavy metal fixative, preserves metachromatic staining of porcine MCs. Zamboni fixation was not suitable for histochemical visualization of MCs in the pig intestine. All other tested fixatives were suitable. Alcian blue and toluidine blue co-stained intestinal goblet cells which made a prima facie identification of MCs difficult. The polychromatic methylene blue proved to be the optimal staining. In order to compare MC counting results of the different fixation methods, tissue shrinkage was taken into account. As even the same fixation caused shrinkage-differences between tissue from small and large intestine, different factors for each single fixation and intestinal localization had to be calculated. Tissue shrinkage varied between 19% and 57%, the highest tissue shrinkage was found after fixation with ZSF in the large intestine, the lowest one in the small intestine after lead acetate fixation. Our study emphasizes that MC counting results from

  10. High-throughput deterministic single-cell encapsulation and droplet pairing, fusion, and shrinkage in a single microfluidic device

    NARCIS (Netherlands)

    Schoeman, R.M.; Kemna, Evelien; Wolbers, F.; van den Berg, Albert

    In this article, we present a microfluidic device capable of successive high-yield single-cell encapsulation in droplets, with additional droplet pairing, fusion, and shrinkage. Deterministic single-cell encapsulation is realized using Dean-coupled inertial ordering of cells in a Yin-Yang-shaped

  11. Rapid shrinkage of a pancreatic serous cystadenoma with cystic degeneration: report of a case. (United States)

    Sumiyoshi, Tatsuaki; Shima, Yasuo; Okabayashi, Takehiro; Kozuki, Akihito; Nakamura, Toshio; Iwata, Jun; Noda, Yoshihiro; Hata, Yasuhiro; Murata, Yoriko; Uka, Kiminori; Morita, Sojiro


    We report a rare case of pancreatic serous cystadenoma, which shrank remarkably from 6 to 1.5 cm in diameter, with cystic degeneration, over a period of only 3 weeks. A 29-year-old woman who presented with epigastric pain and jaundice underwent computed tomography, which showed a 6-cm monolocular cystic tumor in the pancreatic head. Endoscopic retrograde cholangiopancreatography showed stenosis and deviation of the inferior part of the extrahepatic bile duct. We performed surgery 3 weeks later for suspected mucinous cystadenoma or macroscopic serous cystadenoma of the pancreas. At laparotomy, the tumor in the pancreatic head was found to have shrunk remarkably. We excised the tumor completely by performing the Whipple procedure. Macroscopically, the mass was a 1.5-cm monolocular cyst. Microscopically, the cystic tumor was composed of a thick fibrous wall with granulation tissue and hemorrhage. Although epithelial cells were not found inside the cystic wall, numerous grossly invisible microcysts with glycogen-containing epithelial cells were seen at its periphery. Based on these findings, the tumor was diagnosed as a serous cystadenoma with cystic degeneration.

  12. Slow and steady cell shrinkage reduces osmotic stress in bovine and murine oocyte and zygote vitrification. (United States)

    Lai, D; Ding, J; Smith, G W; Smith, G D; Takayama, S


    Does the use of a new cryoprotectant agent (CPA) exchange protocol designed to minimize osmotic stress improve oocyte or zygote vitrification by reducing sublethal cryodamage? The use of a new CPA exchange protocol made possible by automated microfluidics improved oocyte and zygote vitrification with superior morphology as indicated by a smoother cell surface, higher sphericity, higher cytoplasmic lipid retention, less cytoplasmic leakage and higher developmental competence compared with conventional methods. The use of more 'steps' of CPA exposure during the vitrification protocol increases cryosurvival and development in the bovine model. However, such an attempt to eliminate osmotic stress is limited by the practicality of performing numerous precise pipetting steps in a short amount of time. Murine meiotically competent germinal vesicle intact oocytes and zygotes were harvested from the antral follicles in ovaries and ampulla, respectively. Bovine ovaries were obtained from a local abattoir at random stages of the estrous cycle. A total of 110 murine oocytes, 802 murine zygotes and 52 bovine oocytes were used in this study. Microfluidic devices were fabricated using conventional photo- and soft-lithography. CPAs used were 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for equilibration solution and 15% EG, 15% DMSO and 0.5 M sucrose for vitrification solution. End-point analyses include mathematical modeling using Kedem-Katchalsky equations, morphometrics assessed by conventional and confocal microscopy, cytoplasmic lipid quantification by nile red staining, cytoplasmic leakage quantification by fluorescent dextran intercalation and developmental competence analysis by 96 h embryo culture and blastomere quantification. The automated microfluidics protocol decreased the shrinkage rate of the oocyte and zygote by 13.8 times over its manual pipetting alternative. Oocytes and zygotes with a lower shrinkage rate during CPA exposure experienced less

  13. A cell shrinkage-induced non-selective cation conductance with a novel pharmacology in Ehrlich-Lettre-ascites tumour cells

    DEFF Research Database (Denmark)

    Lawonn, Peter; Hoffmann, Else K; Hougaard, Charlotte


    In whole-cell recordings on Ehrlich-Lettre-ascites tumour (ELA) cells, the shrinkage-induced activation of a cation conductance with a selectivity ratio P(Na):P(Li):P(K):P(choline):P(NMDG) of 1.00:0.97:0.88:0.03:0.01 was observed. In order of potency, this conductance was blocked by Gd(3+)=benzamil......>amiloride>ethyl-isopropyl-amiloride (EIPA). In patch-clamp studies using the cell-attached configuration, a 14 pS channel became detectable that was reversibly activated upon hypertonic cell shrinkage. It is concluded that ELA cells express a shrinkage-induced cation channel that may reflect a molecular link between amiloride...

  14. Heat shock protein 70 inhibits shrinkage-induced programmed cell death via mechanisms independent of effects on cell volume-regulatory membrane transport proteins

    DEFF Research Database (Denmark)

    Nylandsted, J; Jäättelä, M; Hoffmann, E K


    Cell shrinkage is a ubiquitous feature of programmed cell death (PCD), but whether it is an obligatory signalling event in PCD is unclear. Heat shock protein 70 (Hsp70) potently counteracts PCD in many cells, by mechanisms that are incompletely understood. In the present investigation, we found...

  15. High-throughput deterministic single-cell encapsulation and droplet pairing, fusion, and shrinkage in a single microfluidic device. (United States)

    Schoeman, Rogier M; Kemna, Evelien W M; Wolbers, Floor; van den Berg, Albert


    In this article, we present a microfluidic device capable of successive high-yield single-cell encapsulation in droplets, with additional droplet pairing, fusion, and shrinkage. Deterministic single-cell encapsulation is realized using Dean-coupled inertial ordering of cells in a Yin-Yang-shaped curved microchannel using a double T-junction, with a frequency over 2000 Hz, followed by controlled droplet pairing with a 100% success rate. Subsequently, droplet fusion is realized using electrical actuation resulting in electro-coalescence of two droplets, each containing a single HL60 cell, with 95% efficiency. Finally, volume reduction of the fused droplet up to 75% is achieved by a triple pitchfork structure. This droplet volume reduction is necessary to obtain close cell-cell membrane contact necessary for final cell electrofusion, leading to hybridoma formation, which is the ultimate aim of this research. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Xeno-free and shrinkage-free preparation of scaffold-free cartilage-like disc-shaped cell sheet using human bone marrow mesenchymal stem cells. (United States)

    Sato, Yasushi; Wakitani, Shigeyuki; Takagi, Mutsumi


    Aiming for the clinical application of cartilage regeneration, the xeno-free cultivation method to obtain a scaffold-free cartilage-like disc-shaped cell sheet using mesenchymal stem cells (MSCs) derived from human bone marrow without the shrinkage of the sheet was investigated. MSCs were inoculated into Cell Culture Insert (0.3 cm(2), pore size; 0.4 μm, pore density; 1.0 × 10(8)/cm(2)) using serum-free chondrogenic differentiation medium containing TGF-β3, IGF-1 and dexamethasone or other modified media, and cultured at 37 °C in 5% CO2 for 3 weeks. Sheet thickness, cartilage specific genes expression, ECM accumulation were determined, and the sections of sheets were stained with alcian blue. A novel mixed medium consisting of a growth medium (10% FCS) with a serum-free chondrogenic differentiation medium could prevent the shrinkage of the sheet and produced a disc-shaped cell sheet. The depth of the sheet was approximately 0.7 mm and the gene expression levels were higher than those in cells in normal human cartilage. The use of human serum instead of FCS did not cause shrinkage and did not decrease the accumulation levels of sGAG and type 2 collagen in the sheet. The cultivation of MSCs grown with completely xeno-free materials using the mixed medium containing human serum in a cell culture insert showed a sheet depth of 1.0 mm and gene expression levels higher than those in normal cartilage. The scaffold-free and xeno-free cartilage-like cell sheet was successfully formed without shrinkage using human bone marrow MSCs and the chondrogenic differentiation medium containing human serum. Copyright © 2013. Published by Elsevier B.V.

  17. The triple angiokinase inhibitor nintedanib directly inhibits tumor cell growth and induces tumor shrinkage via blocking oncogenic receptor tyrosine kinases. (United States)

    Hilberg, Frank; Tontsch-Grunt, Ulrike; Baum, Anke; Le, Anh T; Doebele, Robert C; Lieb, Simone; Dianni, Davide; Voss, Tilman; Garin-Chesa, Pilar; Haslinger, Christian; Kraut, Norbert


    The triple angiokinase inhibitor nintedanib is an orally available, potent and selective inhibitor of tumor angiogenesis by blocking the tyrosine kinase activities of VEGFR 1-3, PDGFR α and β and FGFR 1-3. Nintedanib has received regulatory approval in second line adenocarcinoma NSCLC in combination with docetaxel. In addition, nintedanib has been approved for the treatment of idiopathic lung fibrosis. Here we report the results from a broad kinase screen that identified additional kinases as targets for nintedanib in the low nanomolar range. Several of these kinases are known to be mutated or overexpressed and are involved in tumor development (DDR1 and 2, TRKA and C, Ret) as well as in fibrotic diseases (eg. DDRs). In tumor cell lines displaying molecular alterations in potential nintedanib targets, the inhibitor demonstrates direct anti-proliferative effects: in the NSCLC cell line NCI-H1703 carrying a PDGFRα amplification; the gastric cancer cell line KatoIII and the breast cancer cell line MFM223 both driven by a FGFR2 amplification; AN3CA (endometrial carcinoma) bearing a mutated FGFR2; the AML cell lines MOLM-13 and MV-4-11-B with FLT3 mutations; the NSCLC adenocarcinoma LC-2/ad harboring a CCDC6-RET fusion. However, potent kinase inhibition does not strictly translate into anti-proliferative activity as demonstrated in the TRKA dependent cell lines CUTO-3 and KM-12. Importantly, nintedanib treatment of NCI-H1703 tumor xenografts triggered effective tumor shrinkage, indicating the direct effect on the tumor cells on top of the antiangiogenic effect on the tumor stroma. These findings will be instructive to guide future genome-based clinical trials with nintedanib. The American Society for Pharmacology and Experimental Therapeutics.

  18. Dual response of human leukemia U937 cells to hypertonic shrinkage: initial regulatory volume increase (RVI) and delayed apoptotic volume decrease (AVD). (United States)

    Yurinskaya, Valentina E; Moshkov, Alexey V; Wibberley, Anna V; Lang, Florian; Model, Michael A; Vereninov, Alexey A


    Osmotic cell shrinkage is a powerful trigger of suicidal cell death or apoptosis, which is paralleled and enforced by apoptotic volume decrease (AVD). Cells counteract cell shrinkage by volume regulatory increase (RVI). The present study explored the response of human U937 cells to hypertonic solution thus elucidating the relationship between RVI and AVD. Cell water, concentration of monovalent ions and the appearance of apoptotic markers were followed for 0.5-4 h after the cells were transferred to a hypertonic medium. Intracellular water, K+, Na+, and Cl- content, ouabain-sensitive and -resistant Rb+ influxes were determined by measurement of the cell buoyant density in Percoll density gradient, flame emission analysis and 36Cl- assay, respectively. Fluorescent microscopy of live cells stained by acridine orange and ethidium bromide was used to verify apoptosis. After 2-4 h incubation in hypertonic media the cell population was split into light (L) and heavy (H) fractions. According to microscopy and analysis of monovalent ions the majority of cells in the L population were healthy, while the H fractions were enriched with apoptotic cells. The density of L cells was decreasing with time, while the density of H cells was increasing, thus reflecting the opposite effects of RVI and AVD. At the same time, some of the cells were shifting from L to H fractions, indicating that apoptosis was gradually extending to cells that were previously displaying normal RVI. The findings suggest that apoptosis can develop in cells capable of RVI. Copyright © 2012 S. Karger AG, Basel.

  19. An Understanding of the Band Gap Shrinkage in Sn-Doped ZnO for Dye-Sensitized Solar Cells (United States)

    Yildiz, Abdullah; Ozturk, Elif; Atilgan, Abdullah; Sbeta, Mohamed; Atli, Aycan; Serin, Tulay


    Sn-doped ZnO (TZO) films were deposited onto glass substrates by a spray pyrolysis technique. The temperature-dependent conductivity measurements and room-temperature Hall effect measurements were carried out, which indicated that the films exhibited a degenerate semiconductor behavior. Band gap energy of the films was studied by transmission measurements. With increasing Sn content, band gap shrinkage was observed. It was determined that this shrinkage was associated with the competition between many body interactions and the Burstein-Moss effect. We concluded that there is a good agreement between experimental results and theoretical calculations in terms of the shift in band gap. Furthermore, the effective mass value based on parabolic band considerations needed to be replaced by one based on nonparabolic band structure of ZnO for higher content of Sn (>3% at.) to correlate the calculations with the results.

  20. Successful Application of Edoxaban in the Treatment of Venous Thromboembolism Recurrence in a Patient with Non-small Cell Lung Cancer after Tumor Shrinkage. (United States)

    Shoji, Tetsuaki; Mizugaki, Hidenori; Ikezawa, Yasuyuki; Furuta, Megumi; Takashima, Yuta; Kikuchi, Hajime; Goudarzi, Houman; Asahina, Hajime; Kikuchi, Junko; Kikuchi, Eiki; Sakakibara-Konishi, Jun; Shinagawa, Naofumi; Tsujino, Ichizo; Nishimura, Masaharu


    This report describes the case of a 66-year-old man with non-small cell lung cancer and venous thromboembolism (VTE). Unfractionated heparin (UFH) was initially used to control VTE before chemotherapy. However, switching UFH to warfarin or edoxaban, a novel oral anticoagulant (NOAC), failed. Chemotherapy was then administered to control the tumor which was thought to have been the main cause of VTE, which had been treated by UFH. After tumor shrinkage was achieved by chemotherapy, we were able to successfully switch from UFH to edoxaban. Controlling the tumor size and activity enabled the use of edoxaban as maintenance therapy for VTE.

  1. Radial shrinkage and ultrasound acoustic emissions of fresh versus pre-dried Norway spruce sapwood. (United States)

    Rosner, Sabine; Konnerth, Johannes; Plank, Bernhard; Salaberger, Dietmar; Hansmann, Christian


    Acoustic emission (AE) and radial shrinkage were compared between fully saturated fresh and pre-dried Norway spruce sapwood during dehydration at ambient temperature. Hydraulic conductivity measurements, anatomical investigations on bordered pits and X-ray computed tomography (CT) scans were done to search for possible AE sources other than the breakage of the water columns inside the tracheids. Both fresh and pre-dried specimens showed radial shrinkage due to drying surface layers right from the beginning of dehydration, which induced almost no AE. Whereas no dimensional changes occurred in pre-dried wood thereafter, fresh wood showed a rapid shrinkage increase starting at 25% relative water loss. This dimensional change ceased when further moisture got lost and was even partially reversed. AE of fresh wood showed much higher activity and energy, which is a waveform feature that describes the strength of the acoustic signal. Extremely high single AE energy events were detected at this critical stage of dehydration. After partial recovery from shrinkage, neither dimensional changes nor AE activity showed differences between fresh and pre-dried wood after more than 80% relative moisture loss. Our results suggested that fresh sapwood is more prone to dehydration stresses than pre-dried sapwood. Differences in AE and shrinkage behavior might be due to the weakening or distortion of the pit membranes (cavitation fatigue), pit aspiration, structural changes of the cell walls and micro-checks, which occurred during the first dehydration cycle.

  2. Cure shrinkage in casting resins

    Energy Technology Data Exchange (ETDEWEB)

    Spencer, J. Brock [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)


    A method is described whereby the shrinkage of a casting resin can be determined. Values for the shrinkage of several resin systems in frequent use by Sandia have been measured. A discussion of possible methods for determining the stresses generated by cure shrinkage and thermal contraction is also included.

  3. Total shrinkage versus partial shrinkage in multiple linear regression ...

    African Journals Online (AJOL)

    The paper discusses the merits of partial shrinkage of the ordinary least square estimator of the coefficients of the multiple regression model of full rank. Theoretical comparisons of scalar and matrix-valued risks of the partially shrunken and totally shrunken estimators are given. The strategy of partial shrinkage is applied to ...

  4. Development of concrete shrinkage performance specifications. (United States)


    During its service life, concrete undergoes volume changes. One of the types of deformation is shrinkage. The four main types of shrinkage associated with concrete are plastic, autogenous, carbonation, and drying shrinkage. The volume changes in conc...

  5. Method of Preventing Shrinkage of Aluminum Foam Using Carbonates

    Directory of Open Access Journals (Sweden)

    Takashi Nakamura


    Full Text Available Metallic foams are commonly produced using titanium hydride as a foaming agent. Carbonates produce aluminum foam with a fine and homogenous cell structure. However, foams produced using carbonates show marked shrinkage, which is clearly different from those produced using titanium hydride. It is essential for practical applications to clarify foam shrinkage and establish a method of preventing it. In this research, cell structures were observed to study the shrinkage of aluminum foam produced using carbonates. The cells of foam produced using dolomite as a foaming agent connected to each other with maximum expansion. It was estimated that foaming gas was released through connected cells to the outside. It was assumed that cell formation at different sites is effective in preventing shrinkage induced by cell connection. The multiple additions of dolomite and magnesium carbonate, which have different decomposition temperatures, were applied. The foam in the case with multiple additions maintained a density of 0.66 up to 973 K, at which the foam produced using dolomite shrank. It was verified that the multiple additions of carbonates are effective in preventing shrinkage.

  6. Plastic shrinkage of mortars with shrinkage reducing admixture and lightweight aggregates studied by neutron tomography

    Energy Technology Data Exchange (ETDEWEB)

    Wyrzykowski, Mateusz, E-mail: [Empa, Swiss Federal Laboratories for Materials Science and Technology, Concrete and Construction Chemistry Laboratory, Dübendorf (Switzerland); Lodz University of Technology, Department of Building Physics and Building Materials, Lodz (Poland); Trtik, Pavel [Paul Scherrer Institute, Laboratory for Neutron Scattering and Imaging, Villigen (Switzerland); Empa, Swiss Federal Laboratories for Materials Science and Technology, Concrete and Construction Chemistry Laboratory, Dübendorf (Switzerland); Münch, Beat [Empa, Swiss Federal Laboratories for Materials Science and Technology, Concrete and Construction Chemistry Laboratory, Dübendorf (Switzerland); Weiss, Jason [Purdue University, School of Civil Engineering, West Lafayette (United States); Vontobel, Peter [Paul Scherrer Institute, Laboratory for Neutron Scattering and Imaging, Villigen (Switzerland); Lura, Pietro [Empa, Swiss Federal Laboratories for Materials Science and Technology, Concrete and Construction Chemistry Laboratory, Dübendorf (Switzerland); ETH Zurich, Institute for Building Materials (IfB), Zurich (Switzerland)


    Water transport in fresh, highly permeable concrete and rapid water evaporation from the concrete surface during the first few hours after placement are the key parameters influencing plastic shrinkage cracking. In this work, neutron tomography was used to determine both the water loss from the concrete surface due to evaporation and the redistribution of fluid that occurs in fresh mortars exposed to external drying. In addition to the reference mortar with a water to cement ratio (w/c) of 0.30, a mortar with the addition of pre-wetted lightweight aggregates (LWA) and a mortar with a shrinkage reducing admixture (SRA) were tested. The addition of SRA reduced the evaporation rate from the mortar at the initial stages of drying and reduced the total water loss. The pre-wetted LWA released a large part of the absorbed water as a consequence of capillary pressure developing in the fresh mortar due to evaporation.

  7. Magnitude, modeling and significance of swelling and shrinkage processes in clay soils

    NARCIS (Netherlands)

    Bronswijk, J.J.B.


    The dynamic process of swelling and shrinkage in clay soils has significant practical consequences, such as the rapid transport of water and solutes via shrinkage cracks to the subsoil, and the destruction of buildings and roads on clay soils. In order to develop measuring methods and

  8. The potential of computer vision, optical backscattering parameters and artificial neural network modelling in monitoring the shrinkage of sweet potato (Ipomoea batatas L.) during drying. (United States)

    Onwude, Daniel I; Hashim, Norhashila; Abdan, Khalina; Janius, Rimfiel; Chen, Guangnan


    Drying is a method used to preserve agricultural crops. During the drying of products with high moisture content, structural changes in shape, volume, area, density and porosity occur. These changes could affect the final quality of dried product and also the effective design of drying equipment. Therefore, this study investigated a novel approach in monitoring and predicting the shrinkage of sweet potato during drying. Drying experiments were conducted at temperatures of 50-70 °C and samples thicknesses of 2-6 mm. The volume and surface area obtained from camera vision, and the perimeter and illuminated area from backscattered optical images were analysed and used to evaluate the shrinkage of sweet potato during drying. The relationship between dimensionless moisture content and shrinkage of sweet potato in terms of volume, surface area, perimeter and illuminated area was found to be linearly correlated. The results also demonstrated that the shrinkage of sweet potato based on computer vision and backscattered optical parameters is affected by the product thickness, drying temperature and drying time. A multilayer perceptron (MLP) artificial neural network with input layer containing three cells, two hidden layers (18 neurons), and five cells for output layer, was used to develop a model that can monitor, control and predict the shrinkage parameters and moisture content of sweet potato slices under different drying conditions. The developed ANN model satisfactorily predicted the shrinkage and dimensionless moisture content of sweet potato with correlation coefficient greater than 0.95. Combined computer vision, laser light backscattering imaging and artificial neural network can be used as a non-destructive, rapid and easily adaptable technique for in-line monitoring, predicting and controlling the shrinkage and moisture changes of food and agricultural crops during drying. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  9. Preliminary test-shrinkage estimators

    Directory of Open Access Journals (Sweden)

    H. H. Lemmer


    Full Text Available The advantages of using the very simple shrinkage estimator TL proposed by Lemmer rather than that proposed by Mehta and Srivivasan in the case of preliminary test estimators for parameters of the normal, binomial and Poisson distributions are examined.

  10. Smooth Adaptation by Sigmoid Shrinkage

    Directory of Open Access Journals (Sweden)

    Atto AbdourrahmaneM


    Full Text Available This paper addresses the properties of a subclass of sigmoid-based shrinkage functions: the non zeroforcing smooth sigmoid-based shrinkage functions or SigShrink functions. It provides a SURE optimization for the parameters of the SigShrink functions. The optimization is performed on an unbiased estimation risk obtained by using the functions of this subclass. The SURE SigShrink performance measurements are compared to those of the SURELET (SURE linear expansion of thresholds parameterization. It is shown that the SURE SigShrink performs well in comparison to the SURELET parameterization. The relevance of SigShrink is the physical meaning and the flexibility of its parameters. The SigShrink functions performweak attenuation of data with large amplitudes and stronger attenuation of data with small amplitudes, the shrinkage process introducing little variability among data with close amplitudes. In the wavelet domain, SigShrink is particularly suitable for reducing noise without impacting significantly the signal to recover. A remarkable property for this class of sigmoid-based functions is the invertibility of its elements. This propertymakes it possible to smoothly tune contrast (enhancement, reduction.

  11. Rapid magnetic cell delivery for large tubular bioengineered constructs. (United States)

    Gonzalez-Molina, J; Riegler, J; Southern, P; Ortega, D; Frangos, C C; Angelopoulos, Y; Husain, S; Lythgoe, M F; Pankhurst, Q A; Day, R M


    Delivery of cells into tubular tissue constructs with large diameters poses significant spatial and temporal challenges. This study describes preliminary findings for a novel process for rapid and uniform seeding of cells onto the luminal surface of large tubular constructs. Fibroblasts, tagged with superparamagnetic iron oxide nanoparticles (SPION), were directed onto the luminal surface of tubular constructs by a magnetic field generated by a k4-type Halbach cylinder device. The spatial distribution of attached cells, as measured by the mean number of cells, was compared with a conventional, dynamic, rotational cell-delivery technique. Cell loading onto the constructs was measured by microscopy and magnetic resonance imaging. The different seeding techniques employed had a significant effect on the spatial distribution of the cells (p same construct was significantly different for the dynamic rotation technique (p delivery techniques and is amenable to a variety of tubular organs where rapid loading and uniform distribution of cells for therapeutic applications are required.

  12. The influence of shrinkage reducing admixtures on plastic shrinkage

    Directory of Open Access Journals (Sweden)

    Mora, J.


    Full Text Available Shrinkage reducing admixtures (SRAs are viable alternatives for reducing plastic shrinkage cracking in concrete. The objective of the present paper is to study early age plastic shrinkage in restrained concrete elements, where three different SRAs have been used. The influence of the admixture is analyzed through the following measurements: capillary pressure, evaporation, temperature evolution, crack evolution and settlement. The tests for studying the cracking and deformation were made on two different configurations (i.e., restrained prisms with reduced cross-section and restrained panel, in a wind tunnel, with controlled wind temperature and velocity. The conclusions obtained indicate the viability of the use of this type of admixture and the usefulness of the test methods.

    Los aditivos reductores de retracción (SRAs se plantean, hoy en día, como una alternativa viable para reducir la fisuración por retracción plástica. El objetivo del presente artículo es conocer mejor y predecir el comportamiento a primeras edades de la retracción plástica en elementos estructurales coaccionados, a los que se les ha añadido diversos aditivos reductores de retracción (tres tipos diferentes. Esta influencia se analiza a través de las siguientes propiedades: presión capilar, evaporación, evolución de temperaturas, evolución de fisuración, y deformaciones verticales de asentamiento. Los ensayos para estudiar la fisuración y las deformaciones se han realizado sobre diferentes configuraciones (prisma restringido con estrangulamiento y panel restringido, en un túnel de viento, con temperaturas y velocidades de viento controladas. Las conclusiones obtenidas señalan la viabilidad del empleo de este tipo de aditivos y la bondad de los métodos experimentales utilizados.

  13. Modelling desiccation shrinkage of large structures


    Torrenti J.-M.; Benboudjema F.


    Drying of cement-based materials induces drying shrinkage, which may cause prestress loss or/and cracking if strains are (self or externally) restrained. Drying shrinkage is difficult to predict, since it depends on the material mix, mechanical and hygral boundary conditions, geometry ... This paper focuses on the study of size effect on final drying shrinkage, which is not well documented in the literature. In the Eurocode 2 (European code model), a reduction factor is applied for large stru...

  14. Accounting for PDMS shrinkage when replicating structures

    DEFF Research Database (Denmark)

    Madsen, Morten Hannibal; Feidenhans'l, Nikolaj Agentoft; Hansen, Poul-Erik


    are seldom applied to counteract the shrinkage of PDMS. Also, to perform metrological measurements using replica techniques one has to take the shrinkage into account. Thus we report a study of the shrinkage of PDMS with several different mixing ratios and curing temperatures. The shrinkage factor, with its...... associated uncertainty, for PDMS in the range 40 to 120 °C is provided. By applying this correction factor, it is possible to replicate structures with a standard uncertainty of less than 0.2% in lateral dimensions using typical curing temperatures and PDMS mixing ratios in the range 1:6 to 1:20 (agent:base)....

  15. Rapid Mobilization Reveals a Highly Engraftable Hematopoietic Stem Cell. (United States)

    Hoggatt, Jonathan; Singh, Pratibha; Tate, Tiffany A; Chou, Bin-Kuan; Datari, Shruti R; Fukuda, Seiji; Liu, Liqiong; Kharchenko, Peter V; Schajnovitz, Amir; Baryawno, Ninib; Mercier, Francois E; Boyer, Joseph; Gardner, Jason; Morrow, Dwight M; Scadden, David T; Pelus, Louis M


    Hematopoietic stem cell transplantation is a potential curative therapy for malignant and nonmalignant diseases. Improving the efficiency of stem cell collection and the quality of the cells acquired can broaden the donor pool and improve patient outcomes. We developed a rapid stem cell mobilization regimen utilizing a unique CXCR2 agonist, GROβ, and the CXCR4 antagonist AMD3100. A single injection of both agents resulted in stem cell mobilization peaking within 15 min that was equivalent in magnitude to a standard multi-day regimen of granulocyte colony-stimulating factor (G-CSF). Mechanistic studies determined that rapid mobilization results from synergistic signaling on neutrophils, resulting in enhanced MMP-9 release, and unexpectedly revealed genetic polymorphisms in MMP-9 that alter activity. This mobilization regimen results in preferential trafficking of stem cells that demonstrate a higher engraftment efficiency than those mobilized by G-CSF. Our studies suggest a potential new strategy for the rapid collection of an improved hematopoietic graft. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. A rapid mitochondrial toxicity assay utilizing rapidly changing cell energy metabolism. (United States)

    Sanuki, Yosuke; Araki, Tetsuro; Nakazono, Osamu; Tsurui, Kazuyuki


    Drug-induced liver injury is a major cause of safety-related drug-marketing withdrawals. Several drugs have been reported to disrupt mitochondrial function, resulting in hepatotoxicity. The development of a simple and effective in vitro assay to identify the potential for mitochondrial toxicity is thus desired to minimize the risk of causing hepatotoxicity and subsequent drug withdrawal. An in vitro test method called the "glucose-galactose" assay is often used in drug development but requires prior-culture of cells over several passages for mitochondrial adaptation, thereby restricting use of the assay. Here, we report a rapid version of this method with the same predictability as the original method. We found that replacing the glucose in the medium with galactose resulted in HepG2 cells immediately shifting their energy metabolism from glycolysis to oxidative phosphorylation due to drastic energy starvation; in addition, the intracellular concentration of ATP was reduced by mitotoxicants when glucose in the medium was replaced with galactose. Using our proposed rapid method, mitochondrial dysfunction in HepG2 cells can be evaluated by drug exposure for one hour without a pre-culture step. This rapid assay for mitochondrial toxicity may be more suitable for high-throughput screening than the original method at an early stage of drug development.

  17. Polymerization shrinkage strain of interocclusal recording materials. (United States)

    Chun, Jung-hyun; Pae, Ahran; Kim, Sung-hun


    The aim of this in vitro study was to investigate the polymerization shrinkage behavior and to measure the polymerization shrinkage-strain of interocclusal recording materials. The materials investigated in this study were five polyvinylsiloxane (Imprint Bite, Silagum Automix Bite, O-Bite, Blu-Mousse Classic and Exabite II), one polyether (Ramitec) and one dimethacrylatebased (Luxabite) materials. The polymerization shrinkage values of ten specimens for each material were measured by the Bonded-disk method at 1, 3, 5, 7 and 10 min after mixing at 37 degrees C. The amount of shrinkage-strain (%) was derived and all data were statistically analyzed by one-way ANOVA and the multiple comparison Scheffé test (alpha=0.05). The representative shrinkage-strain kinetic graphs showed that all specimens shrank immediately, except Luxabite which expanded for the initial few seconds. After that, the shrinkage-strain values increased in the magnitude up to 10 min, but its rate decreased gradually with time. The shrinkage-strain values (0.18+/-0.03-0.16+/-0.03%) of O-Bite at 5, 7 and 10 min were significantly lower than the other materials, but Luxabite exhibited the highest values (3.10+/-0.17-3.30+/-0.16%). The interocclusal recording materials investigated presented significantly different polymerization shrinkage-strain kinetics and showed dimensional changes even after the setting time indicated by respective manufacturers.

  18. Modelling desiccation shrinkage of large structures

    Directory of Open Access Journals (Sweden)

    Torrenti J.-M.


    Full Text Available Drying of cement-based materials induces drying shrinkage, which may cause prestress loss or/and cracking if strains are (self or externally restrained. Drying shrinkage is difficult to predict, since it depends on the material mix, mechanical and hygral boundary conditions, geometry ... This paper focuses on the study of size effect on final drying shrinkage, which is not well documented in the literature. In the Eurocode 2 (European code model, a reduction factor is applied for large structure, which is in agreement with experimental data of one campaign (found in the literature. Using numerical simulations, it is shown that a large panel of models, including phenomenological models as physical ones (which takes into account of (aging creep under capillary pressure (assumed to be the physical mechanism for drying shrinkage, do not predict size effect on final value of drying shrinkage.

  19. Mitigation strategies for autogenous shrinkage cracking

    DEFF Research Database (Denmark)

    Bentz, Dale P.; Jensen, Ole Mejlhede


    As the use of high-performance concrete has increased, problems with early-age cracking have become prominent. The reduction in water-to-cement ratio, the incorporation of silica fume, and the increase in binder content of high-performance concretes all contribute to this problem. In this paper......, the fundamental parameters contributing to the autogenous shrinkage and resultant early-age cracking of concrete are presented. Basic characteristics of the cement paste that contribute to or control the autogenous shrinkage response include the surface tension of the pore solution, the geometry of the pore...... of early-age cracking due to autogenous shrinkage. Mitigation strategies discussed in this paper include: the addition of shrinkage-reducing admixtures more commonly used to control drying shrinkage, control of the cement particle size distribution, modification of the mineralogical composition...

  20. Rapid induction of senescence in human cervical carcinoma cells (United States)

    Goodwin, Edward C.; Yang, Eva; Lee, Chan-Jae; Lee, Han-Woong; Dimaio, Daniel; Hwang, Eun-Seong


    Expression of the bovine papillomavirus E2 regulatory protein in human cervical carcinoma cell lines repressed expression of the resident human papillomavirus E6 and E7 oncogenes and within a few days caused essentially all of the cells to synchronously display numerous phenotypic markers characteristic of cells undergoing replicative senescence. This process was accompanied by marked but in some cases transient alterations in the expression of cell cycle regulatory proteins and by decreased telomerase activity. We propose that the human papillomavirus E6 and E7 proteins actively prevent senescence from occurring in cervical carcinoma cells, and that once viral oncogene expression is extinguished, the senescence program is rapidly executed. Activation of endogenous senescence pathways in cancer cells may represent an alternative approach to treat human cancers.

  1. Rapid neurogenesis through transcriptional activation in human stem cells. (United States)

    Busskamp, Volker; Lewis, Nathan E; Guye, Patrick; Ng, Alex H M; Shipman, Seth L; Byrne, Susan M; Sanjana, Neville E; Murn, Jernej; Li, Yinqing; Li, Shangzhong; Stadler, Michael; Weiss, Ron; Church, George M


    Advances in cellular reprogramming and stem cell differentiation now enable ex vivo studies of human neuronal differentiation. However, it remains challenging to elucidate the underlying regulatory programs because differentiation protocols are laborious and often result in low neuron yields. Here, we overexpressed two Neurogenin transcription factors in human-induced pluripotent stem cells and obtained neurons with bipolar morphology in 4 days, at greater than 90% purity. The high purity enabled mRNA and microRNA expression profiling during neurogenesis, thus revealing the genetic programs involved in the rapid transition from stem cell to neuron. The resulting cells exhibited transcriptional, morphological and functional signatures of differentiated neurons, with greatest transcriptional similarity to prenatal human brain samples. Our analysis revealed a network of key transcription factors and microRNAs that promoted loss of pluripotency and rapid neurogenesis via progenitor states. Perturbations of key transcription factors affected homogeneity and phenotypic properties of the resulting neurons, suggesting that a systems-level view of the molecular biology of differentiation may guide subsequent manipulation of human stem cells to rapidly obtain diverse neuronal types. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

  2. Exploiting tumor shrinkage through temporal optimization of radiotherapy

    CERN Document Server

    Unkelbach, Jan; Hong, Theodore; Papp, David; Ramakrishnan, Jagdish; Salari, Ehsan; Wolfgang, John; Bortfeld, Thomas


    In multi-stage radiotherapy, a patient is treated in several stages separated by weeks or months. This regimen has been motivated mostly by radiobiological considerations, but also provides an approach to reduce normal tissue dose by exploiting tumor shrinkage. The paper considers the optimal design of multi-stage treatments, motivated by the clinical management of large liver tumors for which normal liver dose constraints prohibit the administration of an ablative radiation dose in a single treatment. We introduce a dynamic tumor model that incorporates three factors: radiation induced cell kill, tumor shrinkage, and tumor cell repopulation. The design of multi-stage radiotherapy is formulated as a mathematical optimization problem in which the total dose to the liver is minimized, subject to delivering the prescribed dose to the tumor. Based on the model, we gain insight into the optimal administration of radiation over time, i.e. the optimal treatment gaps and dose levels. We analyze treatments consisting ...

  3. Potato shrinkage during hot air drying. (United States)

    Frías, A; Clemente, G; Mulet, A


    Shrinkage is one of the most important physical changes that occur during the dehydration of foods. In this work, the effect of the temperature (35, 40, 50, 60 and 70 °C) and air velocity (7, 8, 9 and 10 m/s) on bulk volumetric shrinkage was investigated. Volume changes were evaluated by image analysis. It was found that neither temperature nor air velocity had any significant effects on bulk shrinkage in this system. The bulk shrinkage of the potato cubes was well correlated with the moisture content of the sample during drying (R(2) = 97.28). Volume varied linearly with the moisture content changes under the studied conditions. The volume of lost water and the decrease in volume of the samples during dehydration were similar.

  4. Breast specimen shrinkage following formalin fixation

    Directory of Open Access Journals (Sweden)

    Horn CL


    Full Text Available Christopher L Horn, Christopher Naugler Department of Pathology and Laboratory Medicine, University of Calgary, and Calgary Laboratory Services, Calgary, AB, Canada Abstract: Accurate measurement of primary breast tumors and subsequent surgical margin assessment is critical for pathology reporting and resulting patient therapy. Anecdotal observations from pathology laboratory staff indicate possible shrinkage of breast cancer specimens due to the formalin fixation process. As a result, we conducted a prospective study to investigate the possible shrinkage effects of formalin fixation on breast cancer specimens. The results revealed no significant changes in tumor size, but there were significant changes in the distance to all surgical resection margins from the unfixed to fixed state. This shrinkage effect could interfere with the accuracy of determining distance to margin assessment and tumor-free margin assessment. Thus, changes in these measurements due to the formalin fixation process have the potential to alter treatment options for the patient. Keywords: breast margins, formalin, shrinkage, cancer

  5. Volumetric polymerization shrinkage of contemporary composite resins

    Directory of Open Access Journals (Sweden)

    Halim Nagem Filho


    Full Text Available The polymerization shrinkage of composite resins may affect negatively the clinical outcome of the restoration. Extensive research has been carried out to develop new formulations of composite resins in order to provide good handling characteristics and some dimensional stability during polymerization. The purpose of this study was to analyze, in vitro, the magnitude of the volumetric polymerization shrinkage of 7 contemporary composite resins (Definite, Suprafill, SureFil, Filtek Z250, Fill Magic, Alert, and Solitaire to determine whether there are differences among these materials. The tests were conducted with precision of 0.1 mg. The volumetric shrinkage was measured by hydrostatic weighing before and after polymerization and calculated by known mathematical equations. One-way ANOVA (a or = 0.05 was used to determine statistically significant differences in volumetric shrinkage among the tested composite resins. Suprafill (1.87±0.01 and Definite (1.89±0.01 shrank significantly less than the other composite resins. SureFil (2.01±0.06, Filtek Z250 (1.99±0.03, and Fill Magic (2.02±0.02 presented intermediate levels of polymerization shrinkage. Alert and Solitaire presented the highest degree of polymerization shrinkage. Knowing the polymerization shrinkage rates of the commercially available composite resins, the dentist would be able to choose between using composite resins with lower polymerization shrinkage rates or adopting technical or operational procedures to minimize the adverse effects deriving from resin contraction during light-activation.

  6. The Na+/H+ exchanger, NHE1, differentially regulates mitogen-activated protein kinase subfamilies after osmotic shrinkage in Ehrlich Lettre Ascites cells

    DEFF Research Database (Denmark)

    Petersen, Stine Helene Falsig; Rasmussen, Maria; Darborg, Barbara Vasek


    Osmotic stress modulates mitogen activated protein kinase (MAPK) activities, leading to altered gene transcription and cell death/survival balance, however, the mechanisms involved are incompletely elucidated. Here, we show, using a combination of biochemical and molecular biology approaches...

  7. Activation of intracellular angiotensin AT2 receptors induces rapid cell death in human uterine leiomyosarcoma cells

    DEFF Research Database (Denmark)

    Zhao, Yi; Lützen, Ulf; Fritsch, Jürgen


    of apoptosis and cell death in cultured human uterine leiomyosarcoma (SK-UT-1) cells and control human uterine smooth muscle cells (HutSMC). The intracellular levels of the AT2 receptor are low in proliferating SK-UT-1 cells but the receptor is substantially up-regulated in quiescent SK-UT-1 cells with high...... densities in mitochondria. Activation of the cell membrane AT2 receptors by a concomitant treatment with angiotensin II and the AT1 receptor antagonist, losartan, induces apoptosis but does not affect the rate of cell death. We demonstrate for the first time that the high-affinity, non-peptide AT2 receptor...... agonist, Compound 21 (C21) penetrates the cell membrane of quiescent SK-UT-1 cells, activates intracellular AT2 receptors and induces rapid cell death; approximately 70% of cells died within 24 h. The cells, which escaped from the cell death, displayed activation of the mitochondrial apoptotic pathway, i...

  8. Rapid flow-induced responses in endothelial cells (United States)

    Stamatas, G. N.; McIntire, L. V.


    Endothelial cells alter their morphology, growth rate, and metabolism in response to fluid shear stress. To study rapid flow-induced responses in the 3D endothelial cell morphology and calcium distribution, coupled fluorescence microscopy with optical sectioning, digital imaging, and numerical deconvolution techniques have been utilized. Results demonstrate that within the first minutes of flow application nuclear calcium is increasing. In the same time frame whole cell height and nuclear height are reduced by about 1 microm. Whole cell height changes may facilitate reduction of shear stress gradients on the luminal surface, whereas nuclear structural changes may be important for modulating endothelial growth rate and metabolism. To study the role of the cytoskeleton in these responses, endothelial cells have been treated with specific disrupters (acrylamide, cytochalasin D, and colchicine) of each of the cytoskeleton elements (intermediate filaments, microfilaments, and microtubules, respectively). None of these compounds had any effect on the shear-induced calcium response. Cytochalasin D and acrylamide did not affect the shear-induced nuclear morphology changes. Colchicine, however, completely abrogated the response, indicating that microtubules may be implicated in force transmission from the plasma membrane to the nucleus. A pedagogical model based on tensegrity theory principles is presented that is consistent with the results on the 3D endothelial morphology.

  9. Rapid thermal sintering of the metallizations of silicon solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Laugier, A.; El Omari, H.; Boyeaux, J.P. [Institut National des Sciences Appliquees de Lyon, Villeurbanne (France). Lab. de Physique de la Matiere; Hartiti, B.; Muller, J.C. [CNRS, Strasbourg (France). Lab. de Physique et Applications des Semiconducteurs; Nam, L.Q.; Sarti, D. [Photowatt International S.A., Bourgoin Jallieu (France)


    Rapid Thermal Processing (RTP) using radiation from tungsten halogen lamps as a heat source is a very promising candidate to replace conventional furnace annealing as it offers many advantages such as a reduced overall thermal budget and a lower gas consumption. In this paper the authors show that with moderate temperature, RTP can be used to obtain screen printed contacts with low contacts resistivity and without degrading the transport properties of the solar cell base region. They investigate on Polix multicrystalline solar cells the possibility to replace the conventional sintering by a RTP annealing of the Ag front grid and of the back Al/Ag contact in a single step performed after the antireflection coating deposition.

  10. Heat shrinkage of electron beam modified EVA

    Energy Technology Data Exchange (ETDEWEB)

    Datta, S.K.; Chaki, T.K.; Bhowmick, A.K. [Indian Institute of Technology, Kharagpur (India). Rubber Technology Center; Tikku, V.K.; Pradhan, N.K. [NICCO Corporation Ltd., (Cable Div.), Calcutta (India)


    Heat shrinkage of electron beam modified ethylene vinyl acetate copolymer (EVA) has been investigated over a range of times, temperatures, stretching, irradiation doses and trimethylolpropane trimethacrylate (TMPTMA) levels. The irradiated (radiation dose 50 kGy and TMPTMA level 1%) and stretched (100% elongation) sample shrinks to a maximum level when kept at 453K temperature for 60 s. The heat shrinkage of samples irradiated with radiation doses of 20, 50, 100 and 150 kGy increases sharply with increasing stretching in the initial stage. Amnesia rating decreases with increasing radiation dose and TMPTMA level as well as gel content. The high radiation dose and TMPTMA level lower the heat shrinkage due to the chain scission. The effect of temperature at which extension is carried out on heat shrinkage is marginal. The irradiated (radiation dose 50 kGy and TMPTMA level 1%) EVA tubes of different dimensions expanded in a laboratory grade tube expander show similar behaviour at 453K and 60 s. The X-ray and DSC studies reveal that the crystallinity increases on stretching due to orientation of chains and it decreases to a considerable extent on heat shrinking. The theoretical and experimental values of heat shrinkage for tubes and rectangular strips are in good accord, when the radiation dose is 50 kGy and TMPTMA level 1%. (author).

  11. Designing Multicomponent Nanosystems for Rapid Detection of Circulating Tumor Cells. (United States)

    Banerjee, Shashwat S; Khobragade, Vrushali; Khandare, Jayant


    Detection of circulating tumor cells (CTCs) in the blood circulation holds immense promise as it predicts the overall probability of patient survival. Therefore, CTC-based technologies are gaining prominence as a "liquid biopsy" for cancer diagnostics and prognostics. Here, we describe the design and synthesis of two distinct multicomponent magnetic nanosystems for rapid capture and detection of CTCs. The multifunctional Magneto-Dendrimeric Nano System (MDNS) composed of an anchoring dendrimer that is conjugated to multiple agents such as near infrared (NIR) fluorescent cyanine 5 NHS (Cy5), glutathione (GSH), transferrin (Tf), and iron oxide (Fe3O4) magnetic nanoparticle (MNP) for simultaneous tumor cell-specific affinity, multimodal high resolution confocal imaging, and cell isolation. The second nanosystem is a self-propelled microrocket that is composed of carbon nanotube (CNT), chemically conjugated with targeting ligand such as transferrin on the outer surface and Fe3O4 nanoparticles in the inner surface. The multicomponent nanosystems described here are highly efficient in targeting and isolating cancer cells thus benefiting early diagnosis and therapy of cancer.

  12. Shrinkage Properties of Cement Stabilized Gravel

    DEFF Research Database (Denmark)

    Lund, Mia Schou Møller; Hansen, Kurt Kielsgaard


    Cement stabilized gravel is an attractive material in road construction because its strength prop-erties are accommodating the increasingly higher requirements to the bearing capacity of a base course. However, reflection cracking of cement stabilized gravel is a major concern. In this pa......-per the shrinkage properties of cement stabilized gravel have been documented under various temperature and relative humidity conditions. Two cement contents corresponding to a 28-days compressive strength of 6.2 MPa and 12.3 MPa have been tested and compared. It is found that the coefficient of linear expansion...... for the two cement contents is 9.9 × 10-6 ⁰C-1 and 11.3 × 10-6 ⁰C-1, respectively. Furthermore, it is found that reflecting cracking can mainly be explained by temperature dependent shrinkage rather than moisture dependent shrinkage....

  13. Dry shrinkage characteristics of buffer materials

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, H. [ITC, Tokai, Ibaraki (Japan); Fujita, A.


    Generation of cracks due to drying of compressed bentonite was observed by changing the initial water content to obtain shrinkage constants such as shrinkage limit and shrinking rate. As a result, generation of practically no cracks was observed when the initial water content of samples was below 13%. The volume change due to drying increased with the water content in the sample, and the shrinkage constants were found to depend on the initial water content. Further, the one-dimensional compression strength after drying was compared with that before drying in order to clarify the effect of cracks generated by drying on the mechanical strength. As a result, the dry sample with cracks proved to have large one-dimensional compression strength or E{sub 50} compared to wet samples, so that the mechanical strength was kept even after drying. (H. Baba)

  14. Plasmonic cell nanocoating: a new concept for rapid microbial screening. (United States)

    Xu, Ke; Bui, Minh-Phuong N; Fang, Aiqin; Abbas, Abdennour


    Nanocoating of single microbial cells with gold nanostructures can confer optical, electrical, thermal, and mechanical properties to microorganisms, thus enabling new avenues for their control, study, application, and detection. Cell nanocoating is often performed using layer-by-layer (LbL) deposition. LbL is time-consuming and relies on nonspecific electrostatic interactions, which limit potential applications for microbial diagnostics. Here, we show that, by taking advantage of surface molecules densely present in the microbial outer layers, cell nanocoating with gold nanoparticles can be achieved within seconds using surface molecules, including disulfide- bond-containing (Dsbc) proteins and chitin. A simple activation of these markers and their subsequent interaction with gold nanoparticles allow specific microbial screening and quantification of bacteria and fungi within 5 and 30 min, respectively. The use of plasmonics and fluorescence as transduction methods offers a limit of detection below 35 cfu mL-1 for E. coli bacteria and 1500 cfu mL-1 for M. circinelloides fungi using a hand-held fluorescent reader. Graphical abstract A new concept for rapid microbial screening by targeting disulfide - bond-containing (Dsbc) proteins and chitin with reducing agents and gold nanoparticles.

  15. Optimized Tetrazine Derivatives for Rapid Bioorthogonal Decaging in Living Cells. (United States)

    Fan, Xinyuan; Ge, Yun; Lin, Feng; Yang, Yi; Zhang, Gong; Ngai, William Shu Ching; Lin, Zhi; Zheng, Siqi; Wang, Jie; Zhao, Jingyi; Li, Jie; Chen, Peng R


    The inverse-electron-demand Diels-Alder (iDA) reaction has recently been repurposed as a bioorthogonal decaging reaction by accelerating the elimination process after an initial cycloaddition between trans-cyclooctene (TCO) and tetrazine (TZ). Herein, we systematically surveyed 3,6-substituted TZ derivatives by using a fluorogenic TCO-coumarin reporter followed by LC-MS analysis, which revealed that the initial iDA cycloaddition step was greatly accelerated by electron-withdrawing groups (EWGs) while the subsequent elimination step was strongly suppressed by EWGs. In addition, smaller substituents facilitated the decaging process. These findings promoted us to design and test unsymmetric TZs bearing an EWG group and a small non-EWG group at the 3- and 6-position, respectively. These TZs showed remarkably enhanced decaging rates, enabling rapid iDA-mediated protein activation in living cells. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Quantitative analyses of shrinkage characteristics of neem ...

    African Journals Online (AJOL)

    Quantitative analyses of shrinkage characteristics of neem (Azadirachta indica A. Juss.) wood were carried out. Forty five wood specimens were prepared from the three ecological zones of north eastern Nigeria, viz: sahel savanna, sudan savanna and guinea savanna for the research. The results indicated that the wood ...

  17. A rapid method of fruit cell isolation for cell size and shape measurements

    Directory of Open Access Journals (Sweden)

    Johnston Jason W


    Full Text Available Abstract Background Cell size is a structural component of fleshy fruit, contributing to important traits such as fruit size and texture. There are currently a number of methods for measuring cell size; most rely either on tissue sectioning or digestion of the tissue with cell wall degrading enzymes or chemicals to release single cells. Neither of these approaches is ideal for assaying large fruit numbers as both require a considerable time to prepare the tissue, with current methods of cell wall digestions taking 24 to 48 hours. Additionally, sectioning can lead to a measurement of a plane that does not represent the widest point of the cell. Results To develop a more rapid way of measuring fruit cell size we have developed a protocol that solubilises pectin in the middle lamella of the plant cell wall releasing single cells into a buffered solution. Gently boiling small fruit samples in a 0.05 M Na2CO3 solution, osmotically balanced with 0.3 M mannitol, produced good cell separation with little cellular damage in less than 30 minutes. The advantage of combining a chemical treatment with boiling is that the cells are rapidly killed. This stopped cell shape changes that could potentially occur during separation. With this method both the rounded and angular cells of the apple cultivars SciRos 'Pacific Rose' and SciFresh 'Jazz'™ were observed in the separated cells. Using this technique, an in-depth analysis was performed measuring cell size from 5 different apple cultivars. Cell size was measured using the public domain ImageJ software. For each cultivar a minimum of 1000 cells were measured and it was found that each cultivar displayed a different distribution of cell size. Cell size within cultivars was similar and there was no correlation between flesh firmness and cell size. This protocol was tested on tissue from other fleshy fruit including tomato, rock melon and kiwifruit. It was found that good cell separation was achieved with flesh

  18. Drying shrinkage problems in high PI subgrade soils. (United States)


    The main objective of this study was to investigate the longitudinal cracking in pavements due to drying : shrinkage of high PI subgrade soils. The study involved laboartory soil testing and modeling. The : shrinkage cracks usually occur within the v...

  19. Leaf shrinkage with dehydration: coordination with hydraulic vulnerability and drought tolerance. (United States)

    Scoffoni, Christine; Vuong, Christine; Diep, Steven; Cochard, Hervé; Sack, Lawren


    Leaf shrinkage with dehydration has attracted attention for over 100 years, especially as it becomes visibly extreme during drought. However, little has been known of its correlation with physiology. Computer simulations of the leaf hydraulic system showed that a reduction of hydraulic conductance of the mesophyll pathways outside the xylem would cause a strong decline of leaf hydraulic conductance (K(leaf)). For 14 diverse species, we tested the hypothesis that shrinkage during dehydration (i.e. in whole leaf, cell and airspace thickness, and leaf area) is associated with reduction in K(leaf) at declining leaf water potential (Ψ(leaf)). We tested hypotheses for the linkage of leaf shrinkage with structural and physiological water relations parameters, including modulus of elasticity, osmotic pressure at full turgor, turgor loss point (TLP), and cuticular conductance. Species originating from moist habitats showed substantial shrinkage during dehydration before reaching TLP, in contrast with species originating from dry habitats. Across species, the decline of K(leaf) with mild dehydration (i.e. the initial slope of the K(leaf) versus Ψ(leaf) curve) correlated with the decline of leaf thickness (the slope of the leaf thickness versus Ψ(leaf) curve), as expected based on predictions from computer simulations. Leaf thickness shrinkage before TLP correlated across species with lower modulus of elasticity and with less negative osmotic pressure at full turgor, as did leaf area shrinkage between full turgor and oven desiccation. These findings point to a role for leaf shrinkage in hydraulic decline during mild dehydration, with potential impacts on drought adaptation for cells and leaves, influencing plant ecological distributions.


    NARCIS (Netherlands)



    In this article a method is presented for dentine shrinkage measurements. The relative shrinkage of sound dentine slabs is assessed using a Perthometer with respect to a steel reference. The relative shrinkage of lesions in dentine slabs can be estimated using combined Perthometer/microradiography

  1. Identification of microcracks caused by autogenous shrinkage

    DEFF Research Database (Denmark)

    Lura, Pietro; Jensen, Ole Mejlhede; Guang, Ye


    Detection and quantification of microcracks caused by restrained autogenous shrinkage in high-performance concrete is difficult. Available techniques either lack the required resolution or may cause further cracks indistinguishable from the original ones. The new technique presented in this paper...... allows detection of microcracks while avoiding artifacts induced by unwanted restraint, drying or temperature variations. The technique consists of casting small circular cylindrical samples of cement pastes in silicone moulds. Steel rods of different diameters are cast into the cement paste to restrain...... the autogenous shrinkage and cause crack formation during hardening. Subsequently, liquid gallium is intruded into the cracks under pressure. After solidification of the gallium, the crack pattern is frozen and can be analyzed after plane polishing of the samples. The microcracks are identified by optical...

  2. Compensating For Shrinkage In A Cryogenic Seal (United States)

    Hill, Arnold E.


    Proposed design for seals in liquid-hydrogen plumbing eliminates leaks caused by contraction of seals at low operating temperature. Each seal consists of rubber, polytetrafluorethylene, or lead O-ring including hollow core filled with water. At temperature of liquid hydrogen, anomalous expansion of water keeps seal gland filled and leaktight despite shrinkage of surrounding O-ring material. Design also used in systems using cryogenic fluids other than liquid hydrogen.

  3. Shrinkage measurement for holographic recording materials (United States)

    Fernández, R.; Gallego, S.; Márquez, A.; Francés, J.; Navarro Fuster, V.; Neipp, C.; Ortuño, M.; Beléndez, A.; Pascual, I.


    There is an increasing demand for new holographic recording materials. One of them are photopolymers, which are becoming a classic media in this field. Their versatility is well known and new possibilities are being created by including new components, such as nanoparticles or dispersed liquid crystal molecules in classical formulations, making them interesting for additional applications in which the thin film preparation and the structural modification have a fundamental importance. Prior to obtaining a wide commercialization of displays based on photopolymers, one of the key aspects is to achieve a complete characterization of them. In this sense, one of the main parameters to estimate and control is the shrinkage of these materials. The volume variations change the angular response of the hologram in two aspects, the angular selectivity and the maximum diffraction efficiency. One criteria for the recording material to be used in a holographic data storage application is the shrinkage, maximum of 0.5%. Along this work, we compare two different methods to measure the holographic recording material shrinkage. The first one is measuring the angle of propagation for both diffracted orders +/-1 when slanted gratings are recorded, so that an accurate value of the grating vector can be calculated. The second one is based on interference measurements at zero spatial frequency limit. We calculate the shrinkage for three different photopolymers: a polyvinyl alcohol acrylamide (PVA/AA) based photopolymer, one of the greenest photopolymers whose patent belongs to the Alicante University called Biophotopol and on the last place a holographic-dispersed liquid crystal photopolymer (H-PDLC).

  4. Nearest shrunken centroids via alternative genewise shrinkages.

    Directory of Open Access Journals (Sweden)

    Byeong Yeob Choi

    Full Text Available Nearest shrunken centroids (NSC is a popular classification method for microarray data. NSC calculates centroids for each class and "shrinks" the centroids toward 0 using soft thresholding. Future observations are then assigned to the class with the minimum distance between the observation and the (shrunken centroid. Under certain conditions the soft shrinkage used by NSC is equivalent to a LASSO penalty. However, this penalty can produce biased estimates when the true coefficients are large. In addition, NSC ignores the fact that multiple measures of the same gene are likely to be related to one another. We consider several alternative genewise shrinkage methods to address the aforementioned shortcomings of NSC. Three alternative penalties were considered: the smoothly clipped absolute deviation (SCAD, the adaptive LASSO (ADA, and the minimax concave penalty (MCP. We also showed that NSC can be performed in a genewise manner. Classification methods were derived for each alternative shrinkage method or alternative genewise penalty, and the performance of each new classification method was compared with that of conventional NSC on several simulated and real microarray data sets. Moreover, we applied the geometric mean approach for the alternative penalty functions. In general the alternative (genewise penalties required fewer genes than NSC. The geometric mean of the class-specific prediction accuracies was improved, as well as the overall predictive accuracy in some cases. These results indicate that these alternative penalties should be considered when using NSC.

  5. Polymerization shrinkage assessment of dental resin composites: a literature review. (United States)

    Kaisarly, Dalia; Gezawi, Moataz El


    Composite restorations are widely used worldwide, but the polymerization shrinkage is their main disadvantage that may lead to clinical failures and adverse consequences. This review reports, currently available in vitro techniques and methods used for assessing the polymerization shrinkage. The focus lies on recent methods employing three-dimensional micro-CT data for the evaluation of polymerization shrinkage: volumetric measurement and the shrinkage vector evaluation through tracing particles before and after polymerization. Original research articles reporting in vitro shrinkage measurements and shrinkage stresses were included in electronic and hand-search. Earlier methods are easier, faster and less expensive. The procedures of scanning the samples in the micro-CT and performing the shrinkage vector evaluation are time consuming and complicated. Moreover, the respective software is not commercially available and the various methods for shrinkage vector evaluation are based on different mathematical principles. Nevertheless, these methods provide clinically relevant information and give insight into the internal shrinkage behavior of composite applied in cavities and how boundary conditions affect the shrinkage vectors. The traditional methods give comparative information on polymerization shrinkage of resin composites, whereas using three-dimensional micro-CT data for volumetric shrinkage measurement and the shrinkage vector evaluation is a highly accurate method. The methods employing micro-CT data give the researchers knowledge related to the application method and the boundary conditions of restorations for visualizing the shrinkage effects that could not be seen otherwise. Consequently, this knowledge can be transferred to the clinical situation to optimize the material manipulation and application techniques for improved outcomes.

  6. Comparative Study of Shrinkage and Non-Shrinkage Model of Food Drying (United States)

    Shahari, N.; Jamil, N.; Rasmani, KA.


    A single phase heat and mass model has always been used to represent the moisture and temperature distribution during the drying of food. Several effects of the drying process, such as physical and structural changes, have been considered in order to increase understanding of the movement of water and temperature. However, the comparison between the heat and mass equation with and without structural change (in terms of shrinkage), which can affect the accuracy of the prediction model, has been little investigated. In this paper, two mathematical models to describe the heat and mass transfer in food, with and without the assumption of structural change, were analysed. The equations were solved using the finite difference method. The converted coordinate system was introduced within the numerical computations for the shrinkage model. The result shows that the temperature with shrinkage predicts a higher temperature at a specific time compared to that of the non-shrinkage model. Furthermore, the predicted moisture content decreased faster at a specific time when the shrinkage effect was included in the model.

  7. Polymerization Behavior and Mechanical Properties of High-Viscosity Bulk Fill and Low Shrinkage Resin Composites. (United States)

    Shibasaki, S; Takamizawa, T; Nojiri, K; Imai, A; Tsujimoto, A; Endo, H; Suzuki, S; Suda, S; Barkmeier, W W; Latta, M A; Miyazaki, M

    The present study determined the mechanical properties and volumetric polymerization shrinkage of different categories of resin composite. Three high viscosity bulk fill resin composites were tested: Tetric EvoCeram Bulk Fill (TB, Ivoclar Vivadent), Filtek Bulk Fill posterior restorative (FB, 3M ESPE), and Sonic Fill (SF, Kerr Corp). Two low-shrinkage resin composites, Kalore (KL, GC Corp) and Filtek LS Posterior (LS, 3M ESPE), were used. Three conventional resin composites, Herculite Ultra (HU, Kerr Corp), Estelite ∑ Quick (EQ, Tokuyama Dental), and Filtek Supreme Ultra (SU, 3M ESPE), were used as comparison materials. Following ISO Specification 4049, six specimens for each resin composite were used to determine flexural strength, elastic modulus, and resilience. Volumetric polymerization shrinkage was determined using a water-filled dilatometer. Data were evaluated using analysis of variance followed by Tukey's honestly significant difference test (α=0.05). The flexural strength of the resin composites ranged from 115.4 to 148.1 MPa, the elastic modulus ranged from 5.6 to 13.4 GPa, and the resilience ranged from 0.70 to 1.0 MJ/m 3 . There were significant differences in flexural properties between the materials but no clear outliers. Volumetric changes as a function of time over a duration of 180 seconds depended on the type of resin composite. However, for all the resin composites, apart from LS, volumetric shrinkage began soon after the start of light irradiation, and a rapid decrease in volume during light irradiation followed by a slower decrease was observed. The low shrinkage resin composites KL and LS showed significantly lower volumetric shrinkage than the other tested materials at the measuring point of 180 seconds. In contrast, the three bulk fill resin composites showed higher volumetric change than the other resin composites. The findings from this study provide clinicians with valuable information regarding the mechanical properties and

  8. Polymerization and polymerization shrinkage stress: fast cure versus conventional cure. (United States)

    Strydom, C


    Dentists nowadays have a choice of conventional halogen lights, halogen lights with more sophisticated curing cycles (step-cure, rapid-cure, ramp-cure & pulse-cure), fast halogen lights, laser lights, plasma arc lights (PAC) and, lately, LED lights. While the manufacturers of some of the curing units try to improve on the operational reliability of their lights with a slower initial rate of cure, other manufacturers simply wish to offer as fast a curing time as possible. The conventional approach to cure accepts that sufficient light intensity of at least 400 mW/cm2 at a wavelength of 400-500 nm, and an exposure time of at least 40 seconds is needed to cure a 2-mm layer of composite. When a halogen light with higher or very high intensity is used, alternative curing strategies provide for an initial slower cure to allow flow, and after that a higher-intensity cure to improve the degree of cure. In contrast, in the fast-cure or rapid-cure approach it is suggested that a layer of composite can be cured for only 5- 10 seconds at >2000 mW/cm2. Some go so far as to say that an exposure time of 3 seconds per layer may be enough. This contradictory approach is compounded by the fact that this support for fast cure does not seem to consider the negative consequences. Therefore, to address these concerns, this review discusses the possible effects of a fast cure approach compared to a more conventional approach in polymerization and polymerization shrinkage, and the consequences there-off. Other factors that play an influencing role in polymerization shrinkage stress are also included in the discussion.

  9. Distinct spontaneous shrinkage of a sporadic vestibular schwannoma

    DEFF Research Database (Denmark)

    Huang, Xiaowen; Cayé-Thomasen, Per; Stangerup, Sven-Eric


    We present a case with outspoken spontaneous vestibular schwannoma shrinkage and review the related literature. The patient was initially diagnosed with a left-sided, intrameatal vestibular schwannoma, which subsequently grew into the cerebello-pontine angle (CPA), followed by total shrinkage...... of the CPA component without any intervention over a 12-year observation period. The literature on spontaneous tumor shrinkage was retrieved by searching the subject terms "vestibular schwannoma, conservative management" in PubMed/MEDLINE database, without a time limit. Of the published data, the articles...... on "shrinkage" or "negative growth" or "regression" or "involution" of the tumor were selected, and the contents on the rate, extent and mechanism of spontaneous tumor shrinkage were extracted and reviewed. The reported rate of spontaneous shrinkage of vestibular schwannoma is 5-10% of patients managed...

  10. Influence of Shrinkage-Reducing Admixtures on the Development of Plastic Shrinkage Cracks

    DEFF Research Database (Denmark)

    Lura, Pietro; Pease, Bradley Justin; Mazzotta, Guy


    -reducing admixture (SRA). Mortars containing SRA show fewer and narrower plastic shrinkage cracks than plain mortars when exposed to the same environmental conditions. It is proposed that the lower surface tension of the pore fluid in the mortars containing SRA results in less evaporation, reduced settlement...

  11. Static and dynamic photoresist shrinkage effects in EUV photoresists (United States)

    Bunday, Benjamin; Montgomery, Cecilia; Montgomery, Warren; Cordes, Aaron


    Photoresist shrinkage (a.k.a. line slimming) is an important systematic uncertainty source in critical dimension-scanning electron microscope (CD-SEM) metrology of lithographic features [1][2][3][4][5]. In terms of metrology gauge metrics, it influences both the precision and the accuracy of CD-SEM measurements, while locally damaging the sample. Minimization or elimination of shrinkage is desirable, yet elusive. This error source will furthermore be a factor in CDSEM metrology on such polymer materials into the era of EUV lithography, such that learning to work around this issue will continue to be necessary. Recent work has demonstrated improved understanding of the trends in the shrinkage response depending on electron beam and target parameters in the static measurement case [2][3][4][5][6]. Another recent work has highlighted a second mode of shrinkage that is apparent over time and progresses as a function of time between consecutive measurements, a form of "dynamic shrinkage" that appears to be activated by electron beam, in which the activated feature perpetually and logarithmically shrinks [7][8]. In this work, we will explore both the static and dynamic shrinkage behaviors of various EUV photoresists. The static shrinkage behaviors will be tested for compliance with the SEMATECH shrinkage model [5][6], and further studies will confirm whether or not the dynamic effects are observable. Knowledge of secondary trends in dynamic shrinkage will also be further explored, including how these vary with electron beam energy, activation dose, feature size, and other parameters.

  12. Rapid white blood cell detection for peritonitis diagnosis (United States)

    Wu, Tsung-Feng; Mei, Zhe; Chiu, Yu-Jui; Cho, Sung Hwan; Lo, Yu-Hwa


    A point-of-care and home-care lab-on-a-chip (LoC) system that integrates a microfluidic spiral device as a concentrator with an optical-coding device as a cell enumerator is demonstrated. The LoC system enumerates white blood cells from dialysis effluent of patients receiving peritoneal dialysis. The preliminary results show that the white blood cell counts from our system agree well with the results from commercial flow cytometers. The LoC system can potentially bring significant benefits to end stage renal disease (ESRD) patients that are on peritoneal dialysis (PD).

  13. T cells suppress memory-dependent rapid mucous cell metaplasia in mouse airways. (United States)

    Chand, Hitendra S; Mebratu, Yohannes A; Montera, Marena; Tesfaigzi, Yohannes


    Airway epithelial cells (AECs) are crucial for mucosal and adaptive immunity but whether these cells respond in a memory-dependent manner is poorly studied. Previously, we have reported that LPS intratracheal instillation in rodents causes extensive neutrophilic inflammation and airway epithelial cell hyperplasia accompanied by mucous cell metaplasia (MCM). And the resolution process required a period of 40 d for the inflammation to subside and the lung epithelia to resemble the non-exposed condition. Therefore, the present study investigated the memory-dependent response of airway epithelial cells to a secondary LPS challenge after the initial inflammation was resolved. Airway epithelial and mucous cells were assessed in response to a secondary LPS challenge in F344/N rats, and in C57BL/6 wild-type (Foxn1(WT)) and T cell-deficient athymic (Foxn1(nu)) mice that were instilled with LPS or saline 40 d earlier. Epithelial expression of TLR4, EGFR, and phosphorylated-ERK1/2 (pERK) were also analyzed. LPS-pretreated F344/N rats responded with elevated numbers of AECs after saline challenge and with 3-4-fold increased MCM following the LPS challenge in LPS- compared with saline-pretreated rats. LPS-pretreated rats showed 5-fold higher number of AECs expressing TLR4 apically than saline-pretreated rats. Also, the expression of EGFR was increased in LPS-pretreated rats along with the number of AECs with active or nuclear pERK, and the levels were further increased upon LPS challenge. LPS-pretreated Foxn1(nu) compared with Foxn1(WT) mice showed increased MCM and elevated levels of TLR4, EGFR, and nuclear pERK at 40 d after LPS instillation. LPS challenge further augmented MCM rapidly in Foxn1(nu) compared with Foxn1(WT) mice. Together, these data suggest that AECs preserve an 'innate memory' that drives a rapid mucous phenotype via spatiotemporal regulation of TLR4 and EGFR. Further, T cells may suppress the sustained elevated expression of TLR4 and EGFR and thereby the

  14. CD8 memory T cells have a bioenergetic advantage that underlies their rapid recall ability

    NARCIS (Netherlands)

    van der Windt, Gerritje J. W.; O'Sullivan, David; Everts, Bart; Huang, Stanley Ching-Cheng; Buck, Michael D.; Curtis, Jonathan D.; Chang, Chih-Hao; Smith, Amber M.; Ai, Teresa; Faubert, Brandon; Jones, Russell G.; Pearce, Edward J.; Pearce, Erika L.


    A characteristic of memory T (T-M) cells is their ability to mount faster and stronger responses to reinfection than naive T (T-N) cells do in response to an initial infection. However, the mechanisms that allow this rapid recall are not completely understood. We found that CD8 T-M cells have more

  15. Prediction of transverse shrinkages of young-growth Sitka spruce (Picea sitchensis) and western hemlock (Tsuga heterophylla) with ultrasonic measurements (United States)

    Turker Dundar; Xiping Wang; Robert J. Ross


    The objective of this study was to examine the potential of acoustic measurement as a rapid and nondestructive method to predict the dimensional stability of young-growth Sitka spruce and western hemlock. Ultrasonic velocity, peak energy, specific gravity, and radial and tangential shrinkages were measured on twenty-four 25- x

  16. Haemolysis following rapid experimental red blood cell transfusion--an evaluation of two infusion pumps

    DEFF Research Database (Denmark)

    Hansen, Tom Giedsing; Sprogøe-Jakobsen, U; Pedersen, C M


    The vast majority of infusion pumps used for rapid transfusion of large amounts of blood have never been properly examined regarding their influence on the quality of the red blood cells (RBCs) infused. In this study, we evaluated the effect of two different infusion pumps on the degree of RBC...... destruction following rapid experimental blood transfusion....

  17. Clinical scale rapid expansion of lymphocytes for adoptive cell transfer therapy in the WAVE® bioreactor (United States)


    Background To simplify clinical scale lymphocyte expansions, we investigated the use of the WAVE®, a closed system bioreactor that utilizes active perfusion to generate high cell numbers in minimal volumes. Methods We have developed an optimized rapid expansion protocol for the WAVE bioreactor that produces clinically relevant numbers of cells for our adoptive cell transfer clinical protocols. Results TIL and genetically modified PBL were rapidly expanded to clinically relevant scales in both static bags and the WAVE bioreactor. Both bioreactors produced comparable numbers of cells; however the cultures generated in the WAVE bioreactor had a higher percentage of CD4+ cells and had a less activated phenotype. Conclusions The WAVE bioreactor simplifies the process of rapidly expanding tumor reactive lymphocytes under GMP conditions, and provides an alternate approach to cell generation for ACT protocols. PMID:22475724

  18. IL-6 trans-Signaling-Dependent Rapid Development of Cytotoxic CD8+ T Cell Function

    Directory of Open Access Journals (Sweden)

    Jan P. Böttcher


    Full Text Available Immune control of infections with viruses or intracellular bacteria relies on cytotoxic CD8+ T cells that use granzyme B (GzmB for elimination of infected cells. During inflammation, mature antigen-presenting dendritic cells instruct naive T cells within lymphoid organs to develop into effector T cells. Here, we report a mechanistically distinct and more rapid process of effector T cell development occurring within 18 hr. Such rapid acquisition of effector T cell function occurred through cross-presenting liver sinusoidal endothelial cells (LSECs in the absence of innate immune stimulation and known costimulatory signaling. Rather, interleukin-6 (IL-6 trans-signaling was required and sufficient for rapid induction of GzmB expression in CD8+ T cells. Such LSEC-stimulated GzmB-expressing CD8+ T cells further responded to inflammatory cytokines, eliciting increased and protracted effector functions. Our findings identify a role for IL-6 trans-signaling in rapid generation of effector function in CD8+ T cells that may be beneficial for vaccination strategies.

  19. Drying Shrinkage Microcracking in Cement-based Materials

    NARCIS (Netherlands)

    Bisschop, J.; Van Mier, J.G.M.


    In this paper the nature of drying shrinkage microcracking in a variety of model cementbased materials, as well as in more practical types of concrete is described. The model mixtures were studied to elucidate the mechanisms of drying shrinkage microcracking and the factors that influence these

  20. Finite element analysis of moisture migration, creep, shrinkage and cracking

    NARCIS (Netherlands)

    Zijl, G.P.A.G. van; Borst, R. de; Rots, J.G.


    A finite element formulation is presented for the analysis of moisture migra-tion, creep, shrinkage and cracking in cementitious materials. A one-way coupled approach is followed, where the pore humidity, the driving force for shrinkage, is solved for from a diffusion equation. The evolution of the

  1. Coping with shrinkage in Europe's cities and towns

    NARCIS (Netherlands)

    Hospers, Gerrit J.


    An increasing number of cities and towns in Europe are facing population decline. In this article we focus on the challenges of this urban shrinkage process from a policy perspective. After a short review of the main causes and consequences of urban shrinkage in Europe, two common public policy

  2. Rapid cell-surface prion protein conversion revealed using a novel cell system (United States)

    Goold, R.; Rabbanian, S.; Sutton, L.; Andre, R.; Arora, P.; Moonga, J.; Clarke, A.R.; Schiavo, G.; Jat, P.; Collinge, J.; Tabrizi, S.J.


    Prion diseases are fatal neurodegenerative disorders with unique transmissible properties. The infectious and pathological agent is thought to be a misfolded conformer of the prion protein. Little is known about the initial events in prion infection because the infecting prion source has been immunologically indistinguishable from normal cellular prion protein (PrPC). Here we develop a unique cell system in which epitope-tagged PrPC is expressed in a PrP knockdown (KD) neuroblastoma cell line. The tagged PrPC, when expressed in our PrP-KD cells, supports prion replication with the production of bona fide epitope-tagged infectious misfolded PrP (PrPSc). Using this epitope-tagged PrPSc, we study the earliest events in cellular prion infection and PrP misfolding. We show that prion infection of cells is extremely rapid occurring within 1 min of prion exposure, and we demonstrate that the plasma membrane is the primary site of prion conversion. PMID:21505437

  3. Subsets of CD34+ cells and rapid hematopoietic recovery after peripheral-blood stem-cell transplantation

    NARCIS (Netherlands)

    Dercksen, M. W.; Rodenhuis, S.; Dirkson, M. K.; Schaasberg, W. P.; Baars, J. W.; van der Wall, E.; Slaper-Cortenbach, I. C.; Pinedo, H. M.; von dem Borne, A. E.; van der Schoot, C. E.


    To study whether there is a relationship between transplanted cell dose and rate of hematopoietic recovery after peripheral-blood stem-cell (PBSC) transplantation, and to obtain an indication whether specific subsets of CD34+ cell populations contribute to rapid recovery of neutrophils or platelets.

  4. Rapid method for culturing embryonic neuron-glial cell cocultures

    DEFF Research Database (Denmark)

    Svenningsen, Åsa Fex; Shan, Wei-Song; Colman, David R


    to cultures first treated with antimitotic agents. It also ensures that all the cells present in vivo will be present in the culture. Myelination commences after approximately 2 weeks in culture for dissociated DRG and 3-4 weeks in cerebellar cultures. In enteric cultures, glial wrapping of the enteric...... neurons is seen after 3 weeks (2 weeks in ascorbic acid), suggesting that basal lamina production is important even for glial ensheathment in the enteric nervous system. No overgrowth of fibroblasts or other nonneuronal cells was noted in any cultures, and myelination of the peripheral nervous system...

  5. Rapid prototyping methods for the manufacture of fuel cells

    Directory of Open Access Journals (Sweden)

    Dudek Piotr


    The potential for the application of this method for the manufacture of metallic bipolar plates (BPP for use in proton exchange membrane fuel cells (PEMFCs is presented and discussed. Special attention is paid to the fabrication of light elements for the construction of PEMFC stacks designed for mobile applications such as aviation technology and unmanned aerial vehicles (UAVs.

  6. Reduction of the Early Autogenous Shrinkage of High Strength Concrete

    Directory of Open Access Journals (Sweden)

    Drago Saje


    Full Text Available The results of a laboratory investigation on the early autogenous shrinkage of high strength concrete, and the possibilities of its reduction, are presented. Such concrete demonstrates significant autogenous shrinkage, which should, however, be limited in the early stages of its development in order to prevent the occurrence of cracks and/or drop in the load-carrying capacity of concrete structures. The following possibilities for reducing autogenous shrinkage were investigated: the use of low-heat cement, a shrinkage-reducing admixture, steel fibres, premoistened polypropylene fibres, and presoaked lightweight aggregate. In the case of the use of presoaked natural lightweight aggregate, with a fraction from 2 to 4 mm, the early autogenous shrinkage of one-day-old high strength concrete decreased by about 90%, with no change to the concrete's compressive strength in comparison with that of the reference concrete.

  7. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow (United States)

    Colter, David C.; Class, Reiner; DiGirolamo, Carla M.; Prockop, Darwin J.


    Cultures of plastic-adherent cells from bone marrow have attracted interest because of their ability to support growth of hematopoietic stem cells, their multipotentiality for differentiation, and their possible use for cell and gene therapy. Here we found that the cells grew most rapidly when they were initially plated at low densities (1.5 or 3.0 cells/cm2) to generate single-cell derived colonies. The cultures displayed a lag phase of about 5 days, a log phase of rapid growth of about 5 days, and then a stationary phase. FACS analysis demonstrated that stationary cultures contained a major population of large and moderately granular cells and a minor population of small and agranular cells here referred to as recycling stem cells or RS-1 cells. During the lag phase, the RS-1 cells gave rise to a new population of small and densely granular cells (RS-2 cells). During the late log phase, the RS-2 cells decreased in number and regenerated the pool of RS-1 cells found in stationary cultures. In repeated passages in which the cells were plated at low density, they were amplified about 109-fold in 6 wk. The cells retained their ability to generate single-cell derived colonies and therefore apparently retained their multipotentiality for differentiation. PMID:10725391

  8. Evaluation of rapid volume changes of substrate-adherent cells by conventional microscopy 3D imaging. (United States)

    Boudreault, F; Grygorczyk, R


    Precise measurement of rapid volume changes of substrate-adherent cells is essential to understand many aspects of cell physiology, yet techniques to evaluate volume changes with sufficient precision and high temporal resolution are limited. Here, we describe a novel imaging method that surveys the rapid morphology modifications of living, substrate-adherent cells based on phase-contrast, digital video microscopy. Cells grown on a glass substrate are mounted in a custom-designed, side-viewing chamber and subjected to hypotonic swelling. Side-view images of the rapidly swelling cell, and at the end of the assay, an image of the same cell viewed from a perpendicular direction through the substrate, are acquired. Based on these images, off-line reconstruction of 3D cell morphology is performed, which precisely measures cell volume, height and surface at different points during cell volume changes. Volume evaluations are comparable to those obtained by confocal laser scanning microscopy (DeltaVolume microscopy without the need for cell staining or intense illumination to monitor cell volume make this system a promising new tool to investigate the fundamentals of cell volume physiology.

  9. Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity. (United States)

    Davenport, A J; Cross, R S; Watson, K A; Liao, Y; Shi, W; Prince, H M; Beavis, P A; Trapani, J A; Kershaw, M H; Ritchie, D S; Darcy, P K; Neeson, P J; Jenkins, M R


    Chimeric antigen receptor T (CAR-T) cells are effective serial killers with a faster off-rate from dying tumor cells than CAR-T cells binding target cells through their T cell receptor (TCR). Here we explored the functional consequences of CAR-mediated signaling using a dual-specific CAR-T cell, where the same cell was triggered via TCR (tcrCTL) or CAR (carCTL). The carCTL immune synapse lacked distinct LFA-1 adhesion rings and was less reliant on LFA to form stable conjugates with target cells. carCTL receptors associated with the synapse were found to be disrupted and formed a convoluted multifocal pattern of Lck microclusters. Both proximal and distal receptor signaling pathways were induced more rapidly and subsequently decreased more rapidly in carCTL than in tcrCTL. The functional consequence of this rapid signaling in carCTL cells included faster lytic granule recruitment to the immune synapse, correlating with faster detachment of the CTL from the target cell. This study provides a mechanism for how CAR-T cells can debulk large tumor burden quickly and may contribute to further refinement of CAR design for enhancing the quality of signaling and programming of the T cell. Copyright © 2018 the Author(s). Published by PNAS.

  10. Durability and Shrinkage Characteristics of Self-Compacting Concretes Containing Recycled Coarse and/or Fine Aggregates

    Directory of Open Access Journals (Sweden)

    Mehmet Gesoglu


    Full Text Available This paper addresses durability and shrinkage performance of the self-compacting concretes (SCCs in which natural coarse aggregate (NCA and/or natural fine aggregate (NFA were replaced by recycled coarse aggregate (RCA and/or recycled fine aggregate (RFA, respectively. A total of 16 SCCs were produced and classified into four series, each of which included four mixes designed with two water to binder (w/b ratios of 0.3 and 0.43 and two silica fume replacement levels of 0 and 10%. Durability properties of SCCs were tested for rapid chloride penetration, water sorptivity, gas permeability, and water permeability at 56 days. Also, drying shrinkage accompanied by the water loss and restrained shrinkage of SCCs were monitored over 56 days of drying period. Test results revealed that incorporating recycled coarse and/or fine aggregates aggravated the durability properties of SCCs tested in this study. The drying shrinkage and restrained shrinkage cracking of recycled aggregate (RA concretes had significantly poorer performance than natural aggregate (NA concretes. The time of cracking greatly prolonged as the RAs were used along with the increase in water/binder ratio.

  11. Effective Expansion: Balance between Shrinkage and Hygroscopic Expansion. (United States)

    Suiter, E A; Watson, L E; Tantbirojn, D; Lou, J S B; Versluis, A


    The purpose of this study was to investigate the relationship between hygroscopic expansion and polymerization shrinkage for compensation of polymerization shrinkage stresses in a restored tooth. One resin-modified glass-ionomer (RMGI) (Ketac Nano, 3M ESPE), 2 compomers (Dyract, Dentsply; Compoglass, Ivoclar), and a universal resin-based composite (Esthet•X HD, Dentsply) were tested. Volumetric change after polymerization ("total shrinkage") and during 4 wk of water storage at 37°C was measured using an optical method (n= 10). Post-gel shrinkage was measured during polymerization using a strain gauge method (n= 10). Extracted human molars with large mesio-occluso-distal slot preparations were restored with the tested restorative materials. Tooth surfaces at baseline (preparation), after restoration, and during 4 wk of 37°C water storage were scanned with an optical scanner to determine cuspal flexure (n= 8). Occlusal interface integrity was measured using dye penetration. Data were analyzed using analysis of variance and post hoc tests (significance level 0.05). All tested materials shrunk after polymerization. RMGI had the highest total shrinkage (4.65%) but lowest post-gel shrinkage (0.35%). Shrinkage values dropped significantly during storage in water but had not completely compensated polymerization shrinkage after 4 wk. All restored teeth initially exhibited inward (negative) cuspal flexure due to polymerization shrinkage. Cuspal flexure with the RMGI restoration was significantly less (-6.4 µm) than with the other materials (-12.1 to -14.1 µm). After 1 d, cuspal flexure reversed to +5.0 µm cuspal expansion with the RMGI and increased to +9.3 µm at 4 wk. After 4 wk, hygroscopic expansion compensated cuspal flexure in a compomer (Compoglass) and reduced flexure with Dyract and resin-based composite. Marginal integrity (93.7% intact restoration wall) was best for the Compoglass restorations and lowest (73.1%) for the RMGI restorations. Hygroscopic

  12. Rapid and Efficient Generation of Regulatory T Cells to Commensal Antigens in the Periphery

    Directory of Open Access Journals (Sweden)

    Katherine Nutsch


    Full Text Available Commensal bacteria shape the colonic regulatory T (Treg cell population required for intestinal tolerance. However, little is known about this process. Here, we use the transfer of naive commensal-reactive transgenic T cells expressing colonic Treg T cell receptors (TCRs to study peripheral Treg (pTreg cell development in normal hosts. We found that T cells were activated primarily in the distal mesenteric lymph node. Treg cell induction was rapid, generating >40% Foxp3+ cells 1 week after transfer. Contrary to prior reports, Foxp3+ cells underwent the most cell divisions, demonstrating that pTreg cell generation can be the dominant outcome from naive T cell activation. Moreover, Notch2-dependent, but not Batf3-dependent, dendritic cells were involved in Treg cell selection. Finally, neither deletion of the conserved nucleotide sequence 1 (CNS1 region in Foxp3 nor blockade of TGF-β (transforming growth factor-β-receptor signaling completely abrogated Foxp3 induction. Thus, these data show that pTreg cell selection to commensal bacteria is rapid, is robust, and may be specified by TGF-β-independent signals.

  13. Fundamentals of rapid injection molding for microfluidic cell-based assays. (United States)

    Lee, Ulri N; Su, Xiaojing; Guckenberger, David J; Dostie, Ashley M; Zhang, Tianzi; Berthier, Erwin; Theberge, Ashleigh B


    Microscale cell-based assays have demonstrated unique capabilities in reproducing important cellular behaviors for diagnostics and basic biological research. As these assays move beyond the prototyping stage and into biological and clinical research environments, there is a need to produce microscale culture platforms more rapidly, cost-effectively, and reproducibly. 'Rapid' injection molding is poised to meet this need as it enables some of the benefits of traditional high volume injection molding at a fraction of the cost. However, rapid injection molding has limitations due to the material and methods used for mold fabrication. Here, we characterize advantages and limitations of rapid injection molding for microfluidic device fabrication through measurement of key features for cell culture applications including channel geometry, feature consistency, floor thickness, and surface polishing. We demonstrate phase contrast and fluorescence imaging of cells grown in rapid injection molded devices and provide design recommendations to successfully utilize rapid injection molding methods for microscale cell-based assay development in academic laboratory settings.

  14. Shrinkage singularities of amplitudes and weak interaction cross- section asymptotic

    CERN Document Server

    Dolgov, A D; Okun, Lev Borisovich


    The so called shrinkage singularities of amplitudes caused by shrinkage of diffraction peak at asymptotically high energies are discussed given the condition that the amplitude singularities are not stronger than t/sup 2/ ln t (as is case for neutrino pair exchange diagrams) then total cross-section sigma /sub tot/ cannot increase faster at s to infinity than s/sup 1/3/. If shrinkage singularities are absent then sigma /sub tot/ cannot increase as any power of s. All the conclusions are valid, if the dispersion relations with finite number of subtractions exist at t

  15. Near infrared photoimmunotherapy rapidly elicits specific host immunity against cancer cells (Conference Presentation) (United States)

    Kobayashi, Hisataka


    Near infrared photoimmunotherapy (NIR-PIT) is a new molecularly-targeted cancer photo-therapy based on conjugating a near infrared silica-phthalocyanine dye, IR700, to a monoclonal antibody (mAb) targeting cell-surface molecules. When exposed to NIR light, the conjugate induces a highly-selective necrotic/immunogenic cell death (ICD) only in target-positive, mAb-IR700-bound cancer cells. This cell death occurs as early as 1 minute after exposure to NIR light. Meanwhile, immediately adjacent target-negative cells are unharmed. Dynamic 3D-microscopy of live tumor cells undergoing NIR-PIT showed rapid swelling in treated cells immediately after light exposure, followed by irreversible morphologic changes such as bleb formation, and rupture of vesicles within several minutes. Furthermore, biological markers of ICD including relocation of HSP70/90 and calreticulin, and release of ATP and High Mobility Group Box 1 (HMGB1), were clearly detected immediately after NIR-PIT. When NIR-PIT was performed in a mixture of cancer cells and immature dendritic cells, maturation of immature dendritic cells was strongly induced rapidly after NIR-PIT. Alternatively, NIR-PIT can also target negative regulatory immune cells such as Treg only in the tumor bed. Treg targeting NIR-PIT against CD25 can deplete >80% of Treg in tumor bed within 20 min that induces activation of tumor cell-specific CD8+-T and NK cells within 1.5 hour, and then these activated cells killed cancer cells in local tumor within 1 day and also in distant tumors of the same cell origin within 2 days. In summary, cancer cell-targeting and immuno-suppressor cell-targeting NIR-PITs effectively induce innate and acquired immunity specifically against cancer cells growing in patients, respectively.

  16. Rapid transcriptional pulsing dynamics of high expressing retroviral transgenes in embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Mandy Y M Lo

    Full Text Available Single cell imaging studies suggest that transcription is not continuous and occurs as discrete pulses of gene activity. To study mechanisms by which retroviral transgenes can transcribe to high levels, we used the MS2 system to visualize transcriptional dynamics of high expressing proviral integration sites in embryonic stem (ES cells. We established two ES cell lines each bearing a single copy, self-inactivating retroviral vector with a strong ubiquitous human EF1α gene promoter directing expression of mRFP fused to an MS2-stem-loop array. Transfection of MS2-EGFP generated EGFP focal dots bound to the mRFP-MS2 stem loop mRNA. These transcription foci colocalized with the transgene integration site detected by immunoFISH. Live tracking of single cells for 20 minutes detected EGFP focal dots that displayed frequent and rapid fluctuations in transcription over periods as short as 25 seconds. Similarly rapid fluctuations were detected from focal doublet signals that colocalized with replicated proviral integration sites by immunoFISH, consistent with transcriptional pulses from sister chromatids. We concluded that retroviral transgenes experience rapid transcriptional pulses in clonal ES cell lines that exhibit high level expression. These events are directed by a constitutive housekeeping gene promoter and may provide precedence for rapid transcriptional pulsing at endogenous genes in mammalian stem cells.

  17. Hyperkalemia caused by rapid red cell transfusion and the potassium absorption filter

    Directory of Open Access Journals (Sweden)

    Yasuhiko Imashuku


    Full Text Available We report a case of transient hyperkalemia during hysterectomy after cesarean section, due to preoperatively undiagnosed placenta accreta that caused unforeseen massive hemorrhage and required rapid red cell transfusion. Hyperkalemia-induced by rapid red cell transfusion is a well-known severe complication of transfusion; however, in patients with sudden massive hemorrhage, rapid red cell transfusion is necessary to save their life. In such cases, it is extremely important to monitor serum potassium levels. For an emergency situation, a system should be developed to ensure sufficient preparation for immediate transfusion and laboratory tests. Furthermore, sufficient stock of preparations to treat hyperkalemia, such as calcium preparations, diuretics, glucose, and insulin is required. Moreover, a transfusion filter that absorbs potassium has been developed and is now available for clinical use in Japan. The filter is easy to use and beneficial, and should be prepared when it is available.

  18. Multiple-Shrinkage Multinomial Probit Models with Applications to Simulating Geographies in Public Use Data. (United States)

    Burgette, Lane F; Reiter, Jerome P


    Multinomial outcomes with many levels can be challenging to model. Information typically accrues slowly with increasing sample size, yet the parameter space expands rapidly with additional covariates. Shrinking all regression parameters towards zero, as often done in models of continuous or binary response variables, is unsatisfactory, since setting parameters equal to zero in multinomial models does not necessarily imply "no effect." We propose an approach to modeling multinomial outcomes with many levels based on a Bayesian multinomial probit (MNP) model and a multiple shrinkage prior distribution for the regression parameters. The prior distribution encourages the MNP regression parameters to shrink toward a number of learned locations, thereby substantially reducing the dimension of the parameter space. Using simulated data, we compare the predictive performance of this model against two other recently-proposed methods for big multinomial models. The results suggest that the fully Bayesian, multiple shrinkage approach can outperform these other methods. We apply the multiple shrinkage MNP to simulating replacement values for areal identifiers, e.g., census tract indicators, in order to protect data confidentiality in public use datasets.

  19. Multiple-Shrinkage Multinomial Probit Models with Applications to Simulating Geographies in Public Use Data (United States)

    Burgette, Lane F.; Reiter, Jerome P.


    Multinomial outcomes with many levels can be challenging to model. Information typically accrues slowly with increasing sample size, yet the parameter space expands rapidly with additional covariates. Shrinking all regression parameters towards zero, as often done in models of continuous or binary response variables, is unsatisfactory, since setting parameters equal to zero in multinomial models does not necessarily imply “no effect.” We propose an approach to modeling multinomial outcomes with many levels based on a Bayesian multinomial probit (MNP) model and a multiple shrinkage prior distribution for the regression parameters. The prior distribution encourages the MNP regression parameters to shrink toward a number of learned locations, thereby substantially reducing the dimension of the parameter space. Using simulated data, we compare the predictive performance of this model against two other recently-proposed methods for big multinomial models. The results suggest that the fully Bayesian, multiple shrinkage approach can outperform these other methods. We apply the multiple shrinkage MNP to simulating replacement values for areal identifiers, e.g., census tract indicators, in order to protect data confidentiality in public use datasets. PMID:24358073

  20. Mesoscopic analysis of drying shrinkage damage in a cementitious material

    DEFF Research Database (Denmark)

    Moonen, P.; Pedersen, R.R.; Simone, A.


    Concrete and cement-based materials exhibit shrinkage when exposed to drying. Structural effects and inhomogeneity of material properties adverse free shrinkage, hereby inducing stress concentrations and possibly damage. In this contribution, the magnitude of shrinkage- induced damage during...... a typical sample preparation procedure is assessed. To this extent, a coupled hygro-thermo-mechanical model, incorporating rate-effects, is developed. The constitutive model is applied at a mesoscopic level where the aggregates and the interfacial transition zone (ITZ) are explicitly modelled. Two drying...... temperatures are considered: 35 °C and 50 °C. Significantly more micro-damage and higher internal stresses are found for the latter, revealing the importance of drying shrinkage damage, even at laboratory scale....

  1. Development of spraying agent for reducing drying shrinkage of mortar (United States)

    Fujiwara, Hiromi; Maruoka, Masanori; Liu, Lingling


    Mortar used to repair is sometimes exposed to drying state in early ages after construction and a few days later water is sprayed frequently on the surface of the mortar in order to prevent cracks. This research studied on shrinkage characteristic of mortar subjected to drying conditions like this. The result showed that the water spraying on the mortar after initial drying did not have any effect to prevent shrinkage, but increased. And it also showed when various chemical agents are mixed and used in watersprayingit had the prevention effect on shrinkage. This report was to understand this kind of phenomenon and clarify the mechanism. In addition, based on the results, the new spraying agent was developed to reduce drying shrinkage.

  2. Creep and Shrinkage of High Strength Concretes: an Experimental Analysis

    Directory of Open Access Journals (Sweden)

    Berenice Martins Toralles carbonari


    Full Text Available The creep and shrinkage behaviour of high strength silica fume concretes is significantly different from that of conventional concretes. In order to represent the proper time-dependent response of the material in structural analysis and design, these aspects should be adequately quantified. This paper discusses an experimental setup that is able to determine the creep and shrinkage of concrete from the time of placing. It also compares different gages that can be used for measuring the strains. The method is applied to five different concretes in the laboratory under controlled environmental conditions. The phenomena that are quantified can be classified as basic shrinkage, drying shrinkage, basic creep and drying creep. The relative importance of these mechanisms in high strength concrete will also be presented.

  3. The oncogene c-Myc coordinates regulation of metabolic networks to enable rapid cell cycle entry. (United States)

    Morrish, Fionnuala; Neretti, Nicola; Sedivy, John M; Hockenbery, David M


    The c-myc proto-oncogene is rapidly activated by serum and regulates genes involved in metabolism and cell cycle progression. This gene is thereby uniquely poised to coordinate both the metabolic and cell cycle regulatory events required for cell cycle entry. However, this function of Myc has not been evaluated. Using a rat fibroblast model of isogenic cell lines, myc(-/-), myc(+/-), myc(+/+) and myc(-/-) cells with an inducible c-myc transgene (mycER), we show that the Myc protein programs cells to utilize both oxidative phosphorylation and glycolysis to drive cell cycle progression. We demonstrate this coordinate regulation of metabolic networks is essential, as specific inhibitors of these pathways block Myc-induced proliferation. Metabolic events temporally correlated with cell cycle entry include increased oxygen consumption, mitochondrial function, pyruvate and lactate production, and ATP generation. Treatment of normal cells with inhibitors of oxidative phosphorylation recapitulates the myc(-/-) phenotype, resulting in impaired cell cycle entry and reduced metabolism. Combined with a kinetic expression profiling analysis of genes linked to mitochondrial function, our study indicates that Myc's ability to coordinately regulate the mitochondrial metabolic network transcriptome is required for rapid cell cycle entry. This function of Myc may underlie the pervasive presence of Myc in many human cancers.

  4. Rapid and non-enzymatic in vitro retrieval of tumour cells from surgical specimens.

    Directory of Open Access Journals (Sweden)

    Brigitte Mack

    Full Text Available The study of tumourigenesis commonly involves the use of established cell lines or single cell suspensions of primary tumours. Standard methods for the generation of short-term tumour cell cultures include the disintegration of tissue based on enzymatic and mechanical stress. Here, we describe a simple and rapid method for the preparation of single cells from primary carcinomas, which is independent of enzymatic treatment and feeder cells. Tumour biopsies are processed to 1 mm(3 cubes termed explants, which are cultured 1-3 days on agarose-coated well plates in specified medium. Through incisions generated in the explants, single cells are retrieved and collected from the culture supernatant and can be used for further analysis including in vitro and in vivo studies. Collected cells retain tumour-forming capacity in xenotransplantation assays, mimic the phenotype of the primary tumour, and facilitate the generation of cell lines.

  5. Sudden collapse of vacuoles in Saintpaulia sp. palisade cells induced by a rapid temperature decrease. (United States)

    Kadohama, Noriaki; Goh, Tatsuaki; Ohnishi, Miwa; Fukaki, Hidehiro; Mimura, Tetsuro; Suzuki, Yoshihiro


    It is well known that saintpaulia leaf is damaged by the rapid temperature decrease when cold water is irrigated onto the leaf surface. We investigated this temperature sensitivity and the mechanisms of leaf damage in saintpaulia (Saintpaulia sp. cv. 'Iceberg') and other Gesneriaceae plants. Saintpaulia leaves were damaged and discolored when subjected to a rapid decrease in temperature, but not when the temperature was decreased gradually. Sensitivity to rapid temperature decrease increased within 10 to 20 min during pre-incubation at higher temperature. Injury was restricted to the palisade mesophyll cells, where there was an obvious change in the color of the chloroplasts. During a rapid temperature decrease, chlorophyll fluorescence monitored by a pulse amplitude modulated fluorometer diminished and did not recover even after rewarming to the initial temperature. Isolated chloroplasts were not directly affected by the rapid temperature decrease. Intracellular pH was monitored with a pH-dependent fluorescent dye. In palisade mesophyll cells damaged by rapid temperature decrease, the cytosolic pH decreased and the vacuolar membrane collapsed soon after a temperature decrease. In isolated chloroplasts, chlorophyll fluorescence declined when the pH of the medium was lowered. These results suggest that a rapid temperature decrease directly or indirectly affects the vacuolar membrane, resulting in a pH change in the cytosol that subsequently affects the chloroplasts in palisade mesophyll cells. We further confirmed that the same physiological damage occurs in other Gesneriaceae plants. These results strongly suggested that the vacuoles of palisade mesophyll cells collapsed during the initial phase of leaf injury.

  6. Sudden collapse of vacuoles in Saintpaulia sp. palisade cells induced by a rapid temperature decrease.

    Directory of Open Access Journals (Sweden)

    Noriaki Kadohama

    Full Text Available It is well known that saintpaulia leaf is damaged by the rapid temperature decrease when cold water is irrigated onto the leaf surface. We investigated this temperature sensitivity and the mechanisms of leaf damage in saintpaulia (Saintpaulia sp. cv. 'Iceberg' and other Gesneriaceae plants. Saintpaulia leaves were damaged and discolored when subjected to a rapid decrease in temperature, but not when the temperature was decreased gradually. Sensitivity to rapid temperature decrease increased within 10 to 20 min during pre-incubation at higher temperature. Injury was restricted to the palisade mesophyll cells, where there was an obvious change in the color of the chloroplasts. During a rapid temperature decrease, chlorophyll fluorescence monitored by a pulse amplitude modulated fluorometer diminished and did not recover even after rewarming to the initial temperature. Isolated chloroplasts were not directly affected by the rapid temperature decrease. Intracellular pH was monitored with a pH-dependent fluorescent dye. In palisade mesophyll cells damaged by rapid temperature decrease, the cytosolic pH decreased and the vacuolar membrane collapsed soon after a temperature decrease. In isolated chloroplasts, chlorophyll fluorescence declined when the pH of the medium was lowered. These results suggest that a rapid temperature decrease directly or indirectly affects the vacuolar membrane, resulting in a pH change in the cytosol that subsequently affects the chloroplasts in palisade mesophyll cells. We further confirmed that the same physiological damage occurs in other Gesneriaceae plants. These results strongly suggested that the vacuoles of palisade mesophyll cells collapsed during the initial phase of leaf injury.

  7. Physical properties of safflower grains. Part II: Volumetric shrinkage

    Directory of Open Access Journals (Sweden)

    Elton A. S. Martins

    Full Text Available ABSTRACT Agricultural products usually have their size reduced during the drying process. The quantification of the reduction in the dimensions is important for the development and optimization of equipment for the post-harvest of the product. The aim of the present study was to evaluate the effect of the variation in the moisture content during drying on the volumetric shrinkage of safflower grains and their respective axes. Safflower grains were harvested with an initial moisture content of approximately 0.445 decimal d.b. (dry basis and subjected to drying in an oven with forced air circulation at 40 °C, until the grains reached a final moisture content of 0.073 ± 0.008 decimal d.b. During drying, the contraction of the axes, unit volumetric shrinkage and volumetric shrinkage of the mass of safflower grains were determined at different moisture contents. Based on these results, it can be concluded that reducing the moisture content causes a reduction in the axes of safflower grains and, consequently, reductions in the unit volumetric shrinkage and volumetric shrinkage of the mass of approximately 16 and 13%, respectively, and both variables can be represented by the linear shrinkage model.

  8. Measurement of shrinkage and cracking in lyophilized amorphous cakes, part 3: hydrophobic vials and the question of adhesion. (United States)

    Ullrich, Sabine; Seyferth, Stefan; Lee, Geoffrey


    The importance of cake adhesion to the inside vial wall during lyophilization of amorphous trehalose cakes was determined by using hydrophobized vials. The degrees of cake shrinkage and cracking were determined independently by photographic imaging of the cake top surface in a dark cell. Additionally, measurements with microcomputed tomography were performed. Adhesion is found to be a determining factor in both cake shrinkage and cracking. The correlation between cake detachment from the vial inner wall and trehalose concentration indicates that adhesion of the frozen solute phase is a determining factor in shrinkage. The hydrophobized vials give reduced cracking at trehalose concentrations of up to 15%. The reduced wetting of the hydrophobized inside vial wall gives a planar cake topography with a uniform distribution of cracks within the cake. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  9. Microfluidic device for rapid solution exchange to study kinetics of cell physiology (United States)

    Hu, Howard; Honnatti, Meghana; Gillis, Kevin


    Exchanging the extracellular solution of the cell rapidly (less than 10ms) is an important requirement in study the kinetics of cell physiology. A microfluidic device is developed to exchange the solution around the cells as they flow through a junction at the intersection of two microfluidic channels. The solution exchange time is measured experimentally by fluorescently labeling the cell surface membranes with a styryl dye, FM1-43 or FM 2-10, and then observing the time course of cell fluorescence decay following the rapid drop in the extracellular concentration of the FM dye that occurs as the cell flows past the fluidic junction. A numerical model is developed to guide the experimental design of microfluidic device. In the model, the motion of a single cell through a fluid junction is simulated and the mixing process of the solutions is solved. The model also includes the kinetics of departitioning of FM dyes from the cell membrane. The departitioning time constants for the FM dyes are determined from fitting the measured data of the cell fluorescence decay. This departitioning kinetics is important as FM dyes are commonly used to label cell membranes for the purpose of measuring the release of neurotransmitter from synaptic vesicles via exocytosis and the subsequent reuptake of vesicular membrane by endocytosis.

  10. Rapid characterization of the biomechanical properties of drug-treated cells in a microfluidic device (United States)

    Zhang, Xiaofei; Chu, Henry K.; Zhang, Yang; Bai, Guohua; Wang, Kaiqun; Tan, Qiulin; Sun, Dong


    Cell mechanics is closely related to many cell functions. Recent studies have suggested that the deformability of cells can be an effective biomarker to indicate the onset and progression of diseases. In this paper, a microfluidic chip is designed for rapid characterization of the mechanics of drug-treated cells through stretching with dielectrophoresis (DEP) force. This chip was fabricated using PDMS and micro-electrodes were integrated and patterned on the ITO layer of the chip. Leukemia NB4 cells were considered and the effect of all-trans retinoic acid (ATRA) drug on NB4 cells were examined via the microfluidic chip. To induce a DEP force onto the cell, a relatively weak ac voltage was utilized to immobilize a cell at one side of the electrodes. The applied voltage was then increased to 3.5 V pp and the cell started to be stretched along the applied electric field lines. The elongation of the cell was observed using an optical microscope and the results showed that both types of cells were deformed by the induced DEP force. The strain of the NB4 cell without the drug treatment was recorded to be about 0.08 (time t = 180 s) and the drug-treated NB4 cell was about 0.21 (time t = 180 s), indicating a decrease in the stiffness after drug treatment. The elastic modulus of the cell was also evaluated and the modulus changed from 140 Pa to 41 Pa after drug treatment. This microfluidic chip can provide a simple and rapid platform for measuring the change in the biomechanical properties of cells and can potentially be used as the tool to determine the biomechanical effects of different drug treatments for drug discovery and development applications.

  11. A bacteriophage endolysin-based electrochemical impedance biosensor for the rapid detection of Listeria cells. (United States)

    Tolba, Mona; Ahmed, Minhaz Uddin; Tlili, Chaker; Eichenseher, Fritz; Loessner, Martin J; Zourob, Mohammed


    The objective of this study was to develop a biosensor using the cell wall binding domain (CBD) of bacteriophage-encoded peptidoglycan hydrolases (endolysin) immobilized on a gold screen printed electrode (SPE) and subsequent electrochemical impedance spectroscopy (EIS) for a rapid and specific detection of Listeria cells. The endolysin was amine-coupled to SPEs using EDC/NHS chemistry. The CBD-based electrode was used to capture and detect the Listeria innocua serovar 6b from pure culture and 2% artificially contaminated milk. In our study, the endolysin functionalized SPEs have been characterized using X-ray photoelectron spectroscopy (XPS). The integration of endolysin-based recognition for specific bacteria and EIS can be used for direct and rapid detection of Listeria cells with high specificity against non-Listeria cells with a limit of detection of 1.1 × 10(4) and 10(5) CFU mL(-1) in pure culture and 2% milk, respectively.

  12. A rapid and sensitive method for measuring N-acetylglucosaminidase activity in cultured cells.

    Directory of Open Access Journals (Sweden)

    Victor Mauri

    Full Text Available A rapid and sensitive method to quantitatively assess N-acetylglucosaminidase (NAG activity in cultured cells is highly desirable for both basic research and clinical studies. NAG activity is deficient in cells from patients with Mucopolysaccharidosis type IIIB (MPS IIIB due to mutations in NAGLU, the gene that encodes NAG. Currently available techniques for measuring NAG activity in patient-derived cell lines include chromogenic and fluorogenic assays and provide a biochemical method for the diagnosis of MPS IIIB. However, standard protocols require large amounts of cells, cell disruption by sonication or freeze-thawing, and normalization to the cellular protein content, resulting in an error-prone procedure that is material- and time-consuming and that produces highly variable results. Here we report a new procedure for measuring NAG activity in cultured cells. This procedure is based on the use of the fluorogenic NAG substrate, 4-Methylumbelliferyl-2-acetamido-2-deoxy-alpha-D-glucopyranoside (MUG, in a one-step cell assay that does not require cell disruption or post-assay normalization and that employs a low number of cells in 96-well plate format. We show that the NAG one-step cell assay greatly discriminates between wild-type and MPS IIIB patient-derived fibroblasts, thus providing a rapid method for the detection of deficiencies in NAG activity. We also show that the assay is sensitive to changes in NAG activity due to increases in NAGLU expression achieved by either overexpressing the transcription factor EB (TFEB, a master regulator of lysosomal function, or by inducing TFEB activation chemically. Because of its small format, rapidity, sensitivity and reproducibility, the NAG one-step cell assay is suitable for multiple procedures, including the high-throughput screening of chemical libraries to identify modulators of NAG expression, folding and activity, and the investigation of candidate molecules and constructs for applications in

  13. Rapidly acquired cytotoxicity of lymphoid cells from ice inoculated with allogeneic spleen cells. (United States)

    Slavina, E G; Karmanova, N V; Leipunskaya, I L; Zinzar, S N; Reinhöfer, J; Svet-Moldavsky, G J


    Spleen cells from C57BL/6J or CBA mice inoculated iv with spleen cells from BALB/c mice produced a strong nonspecific cytotoxic effect on target cells (mouse L-cells). Lymph node cells from CBA or C57BL/6J mice inoculated sc with BALB/c spleen cells also destroyed L-cells. Lymph node cells from mice inoculated with syngeneic spleen cells were not cytotoxic. The cytotoxic effect was observed ion of allogeneic but not syngeneic spleen cells. This effect was considerably reduced or completely suppressed after partial or total removal of plastic-adherent cells.

  14. Rapid Column-Free Enrichment of Mononuclear Cells from Solid Tissues (United States)

    Scoville, Steven D.; Keller, Karen A.; Cheng, Stephanie; Zhang, Michael; Zhang, Xiaoli; Caligiuri, Michael A.; Freud, Aharon G.


    We have developed a rapid negative selection method to enrich rare mononuclear cells from human tissues. Unwanted and antibody-tethered cells are selectively depleted during a Ficoll separation step, and there is no need for magnetic-based reagents and equipment. The new method is fast, customizable, inexpensive, remarkably efficient, and easy to perform, and per sample the overall cost is less than one-tenth the cost associated with a magnetic column-based method. PMID:26223896

  15. Rapid Evaluation of Power Degradation in Series Connection of Single Feeding Microsized Microbial Fuel Cells

    KAUST Repository

    Rojas, Jhonathan Prieto


    We have developed a sustainable, single feeding, microsized, air-cathode and membrane-free microbial fuel cells with a volume of 40 mu L each, which we have used for rapid evaluation of power generation and viability of a series array of three cells seeking higher voltage levels. Contrary to expectations, the achieved power density was modest (45 mWm(-3)), limited due to non-uniformities in assembly and the single-channel feeding system.

  16. Rapid and label-free separation of Burkitt's lymphoma cells from red blood cells by optically-induced electrokinetics.

    Directory of Open Access Journals (Sweden)

    Wenfeng Liang

    Full Text Available Early stage detection of lymphoma cells is invaluable for providing reliable prognosis to patients. However, the purity of lymphoma cells in extracted samples from human patients' marrow is typically low. To address this issue, we report here our work on using optically-induced dielectrophoresis (ODEP force to rapidly purify Raji cells' (a type of Burkitt's lymphoma cell sample from red blood cells (RBCs with a label-free process. This method utilizes dynamically moving virtual electrodes to induce negative ODEP force of varying magnitudes on the Raji cells and RBCs in an optically-induced electrokinetics (OEK chip. Polarization models for the two types of cells that reflect their discriminate electrical properties were established. Then, the cells' differential velocities caused by a specific ODEP force field were obtained by a finite element simulation model, thereby established the theoretical basis that the two types of cells could be separated using an ODEP force field. To ensure that the ODEP force dominated the separation process, a comparison of the ODEP force with other significant electrokinetics forces was conducted using numerical results. Furthermore, the performance of the ODEP-based approach for separating Raji cells from RBCs was experimentally investigated. The results showed that these two types of cells, with different concentration ratios, could be separated rapidly using externally-applied electrical field at a driven frequency of 50 kHz at 20 Vpp. In addition, we have found that in order to facilitate ODEP-based cell separation, Raji cells' adhesion to the OEK chip's substrate should be minimized. This paper also presents our experimental results of finding the appropriate bovine serum albumin concentration in an isotonic solution to reduce cell adhesion, while maintaining suitable medium conductivity for electrokinetics-based cell separation. In short, we have demonstrated that OEK technology could be a promising tool for

  17. Rapid and label-free separation of Burkitt's lymphoma cells from red blood cells by optically-induced electrokinetics. (United States)

    Liang, Wenfeng; Zhao, Yuliang; Liu, Lianqing; Wang, Yuechao; Dong, Zaili; Li, Wen Jung; Lee, Gwo-Bin; Xiao, Xiubin; Zhang, Weijing


    Early stage detection of lymphoma cells is invaluable for providing reliable prognosis to patients. However, the purity of lymphoma cells in extracted samples from human patients' marrow is typically low. To address this issue, we report here our work on using optically-induced dielectrophoresis (ODEP) force to rapidly purify Raji cells' (a type of Burkitt's lymphoma cell) sample from red blood cells (RBCs) with a label-free process. This method utilizes dynamically moving virtual electrodes to induce negative ODEP force of varying magnitudes on the Raji cells and RBCs in an optically-induced electrokinetics (OEK) chip. Polarization models for the two types of cells that reflect their discriminate electrical properties were established. Then, the cells' differential velocities caused by a specific ODEP force field were obtained by a finite element simulation model, thereby established the theoretical basis that the two types of cells could be separated using an ODEP force field. To ensure that the ODEP force dominated the separation process, a comparison of the ODEP force with other significant electrokinetics forces was conducted using numerical results. Furthermore, the performance of the ODEP-based approach for separating Raji cells from RBCs was experimentally investigated. The results showed that these two types of cells, with different concentration ratios, could be separated rapidly using externally-applied electrical field at a driven frequency of 50 kHz at 20 Vpp. In addition, we have found that in order to facilitate ODEP-based cell separation, Raji cells' adhesion to the OEK chip's substrate should be minimized. This paper also presents our experimental results of finding the appropriate bovine serum albumin concentration in an isotonic solution to reduce cell adhesion, while maintaining suitable medium conductivity for electrokinetics-based cell separation. In short, we have demonstrated that OEK technology could be a promising tool for efficient and

  18. Rapid reactivation of extralymphoid CD4 T cells during secondary infection.

    Directory of Open Access Journals (Sweden)

    Timothy J Chapman

    Full Text Available After infection, extralymphoid tissues are enriched with effector and memory T cells of a highly activated phenotype. The capacity for rapid effector cytokine response from extralymphoid tissue-memory T cells suggests these cells may perform a 'sentinel' function in the tissue. While it has been demonstrated that extralymphoid CD4+ T cells can directly respond to secondary infection, little is known about how rapidly this response is initiated, and how early activation of T cells in the tissue may affect the innate response to infection. Here we use a mouse model of secondary heterosubtypic influenza infection to show that CD4(+ T cells in the lung airways are reactivated within 24 hours of secondary challenge. Airway CD4(+ T cells initiate an inflammatory cytokine and chemokine program that both alters the composition of the early innate response and contributes to the reduction of viral titers in the lung. These results show that, unlike a primary infection, extralymphoid tissue-memory CD4(+ T cells respond alongside the innate response during secondary infection, thereby shaping the overall immune profile in the airways. These data provide new insights into the role of extralymphoid CD4(+ T cells during secondary immune responses.

  19. Easy and Rapid Detection of Mumps Virus by Live Fluorescent Visualization of Virus-Infected Cells. (United States)

    Takahashi, Tadanobu; Agarikuchi, Takashi; Kurebayashi, Yuuki; Shibahara, Nona; Suzuki, Chihiro; Kishikawa, Akiko; Fukushima, Keijo; Takano, Maiko; Suzuki, Fumie; Wada, Hirohisa; Otsubo, Tadamune; Ikeda, Kiyoshi; Minami, Akira; Suzuki, Takashi


    Mumps viruses show diverse cytopathic effects (CPEs) of infected cells and viral plaque formation (no CPE or no plaque formation in some cases) depending on the viral strain, highlighting the difficulty in mumps laboratory studies. In our previous study, a new sialidase substrate, 2-(benzothiazol-2-yl)-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac), was developed for visualization of sialidase activity. BTP3-Neu5Ac can easily and rapidly perform histochemical fluorescent visualization of influenza viruses and virus-infected cells without an antiviral antibody and cell fixation. In the present study, the potential utility of BTP3-Neu5Ac for rapid detection of mumps virus was demonstrated. BTP3-Neu5Ac could visualize dot-blotted mumps virus, virus-infected cells, and plaques (plaques should be called focuses due to staining of infected cells in this study), even if a CPE was not observed. Furthermore, virus cultivation was possible by direct pick-up from a fluorescent focus. In conventional methods, visible appearance of the CPE and focuses often requires more than 6 days after infection, but the new method with BTP3-Neu5Ac clearly visualized infected cells after 2 days and focuses after 4 days. The BTP3-Neu5Ac assay is a precise, easy, and rapid assay for confirmation and titration of mumps virus.

  20. Easy and Rapid Detection of Mumps Virus by Live Fluorescent Visualization of Virus-Infected Cells.

    Directory of Open Access Journals (Sweden)

    Tadanobu Takahashi

    Full Text Available Mumps viruses show diverse cytopathic effects (CPEs of infected cells and viral plaque formation (no CPE or no plaque formation in some cases depending on the viral strain, highlighting the difficulty in mumps laboratory studies. In our previous study, a new sialidase substrate, 2-(benzothiazol-2-yl-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac, was developed for visualization of sialidase activity. BTP3-Neu5Ac can easily and rapidly perform histochemical fluorescent visualization of influenza viruses and virus-infected cells without an antiviral antibody and cell fixation. In the present study, the potential utility of BTP3-Neu5Ac for rapid detection of mumps virus was demonstrated. BTP3-Neu5Ac could visualize dot-blotted mumps virus, virus-infected cells, and plaques (plaques should be called focuses due to staining of infected cells in this study, even if a CPE was not observed. Furthermore, virus cultivation was possible by direct pick-up from a fluorescent focus. In conventional methods, visible appearance of the CPE and focuses often requires more than 6 days after infection, but the new method with BTP3-Neu5Ac clearly visualized infected cells after 2 days and focuses after 4 days. The BTP3-Neu5Ac assay is a precise, easy, and rapid assay for confirmation and titration of mumps virus.

  1. Cost-effective and rapid blood analysis on a cell-phone. (United States)

    Zhu, Hongying; Sencan, Ikbal; Wong, Justin; Dimitrov, Stoyan; Tseng, Derek; Nagashima, Keita; Ozcan, Aydogan


    We demonstrate a compact and cost-effective imaging cytometry platform installed on a cell-phone for the measurement of the density of red and white blood cells as well as hemoglobin concentration in human blood samples. Fluorescent and bright-field images of blood samples are captured using separate optical attachments to the cell-phone and are rapidly processed through a custom-developed smart application running on the phone for counting of blood cells and determining hemoglobin density. We evaluated the performance of this cell-phone based blood analysis platform using anonymous human blood samples and achieved comparable results to a standard bench-top hematology analyser. Test results can either be stored on the cell-phone memory or be transmitted to a central server, providing remote diagnosis opportunities even in field settings.

  2. The acoustic sensor for rapid analysis of bacterial cells in the conductive suspensions. (United States)

    Borodina, I A; Zaitsev, B D; Guliy, O; Teplykh, A A; Shikhabudinov, A M


    The possibility of using the acoustic sensor on the basis of a two-channel delay line for rapid analysis of bacterial cells in the conductive suspensions was investigated. The dependencies of change in phase and insertion loss of output signal of the sensor on conductivity of buffer solution with various concentrations of cells due to a specific interaction "bacterial cells - mini-antibodies" for electrically open and electrically shorted channels of delay line were measured. It has been found that these changes have the most values for the electrically open channel. It has been also shown that the sensor rapidly responds to the specific interaction and the time stabilization of the phase and insertion loss of output signal is less than 10min. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. A Rapid Culture Technique Produces Functional Dendritic-Like Cells from Human Acute Myeloid Leukemia Cell Lines

    Directory of Open Access Journals (Sweden)

    Jian Ning


    Full Text Available Most anti-cancer immunotherapeutic strategies involving dendritic cells (DC as vaccines rely upon the adoptive transfer of DC loaded with exogenous tumour-peptides. This study utilized human acute myeloid leukemia (AML cells as progenitors from which functional dendritic-like antigen presenting cells (DLC were generated, that constitutively express tumour antigens for recognition by CD8+ T cells. DLC were generated from AML cell lines KG-1 and MUTZ-3 using rapid culture techniques and appropriate cytokines. DLC were evaluated for their cell-surface phenotype, antigen uptake and ability to stimulate allogeneic responder cell proliferation, and production of IFN-γ; compared with DC derived from normal human PBMC donors. KG-1 and MUTZ-3 DLC increased expression of CD80, CD83, CD86, and HLA-DR, and MUTZ-3 DLC downregulated CD14 and expressed CD1a. Importantly, both KG-1 and MUTZ-3-derived DLC promoted proliferation of allogeneic responder cells more efficiently than unmodified cells; neither cells incorporated FITC-labeled dextran, but both stimulated IFN-γ production from responding allogeneic CD8+ T cells. Control DC produced from PBMC using the FastDC culture also expressed high levels of critical cell surface ligands and demonstrated good APC function. This paper indicates that functional DLC can be cultured from the AML cell lines KG-1 and MUTZ-3, and FastDC culture generates functional KG-1 DLC.

  4. B-Cell Depletion Salvage Therapy in Rapidly Progressive Dermatomyositis Related Interstitial Lung Disease. (United States)

    Eissa, Khaled; Palomino, Jaime


    Interstitial lung disease (ILD) is a major cause of morbidity and mortality in patients with idiopathic inflammatory myopathies (IIM). Glucocorticoids are the initial standard treatment. However, many patients fail to respond and continue to progress despite treatment with high dose glucocorticoids. The efficacy of rituximab has been suggested in case reports and case series of refractory antisynthetase (AS) syndrome, but data on patients without auto-antibodies or with rapidly progressive ILD are scarce. We report a case of rapidly progressive dermatomyositis (DM) associated ILD treated successfully with B-cell depletion therapy.

  5. Geosynthetic clay liners shrinkage under simulated daily thermal cycles. (United States)

    Sarabadani, Hamid; Rayhani, Mohammad T


    Geosynthetic clay liners are used as part of composite liner systems in municipal solid waste landfills and other applications to restrict the escape of contaminants into the surrounding environment. This is attainable provided that the geosynthetic clay liner panels continuously cover the subsoil. Previous case histories, however, have shown that some geosynthetic clay liner panels are prone to significant shrinkage and separation when an overlying geomembrane is exposed to solar radiation. Experimental models were initiated to evaluate the potential shrinkage of different geosynthetic clay liner products placed over sand and clay subsoils, subjected to simulated daily thermal cycles (60°C for 8 hours and 22°C for 16 hours) modelling field conditions in which the liner is exposed to solar radiation. The variation of geosynthetic clay liner shrinkage was evaluated at specified times by a photogrammetry technique. The manufacturing techniques, the initial moisture content, and the aspect ratio (ratio of length to width) of the geosynthetic clay liner were found to considerably affect the shrinkage of geosynthetic clay liners. The particle size distribution of the subsoil and the associated suction at the geosynthetic clay liner-subsoil interface was also found to have significant effects on the shrinkage of the geosynthetic clay liner. © The Author(s) 2014.

  6. Influence of shrinkage-reducing admixture on drying shrinkage and mechanical properties of high-performance concrete

    Directory of Open Access Journals (Sweden)

    Nguyen Quangphu


    Full Text Available Abstract: High-performance concrete (HPC has specific performance advantages over conventional concrete in strength and durability. HPC mixtures are usually produced with water/binder mass ratios (mW/mB in the range of 0.2-0.4, so volume changes of concrete as a result of drying, chemical reactions, and temperature change cannot be avoided. For these reasons, shrinkage and cracking are frequent phenomena. It is necessary to add some types of admixture for reduction of shrinkage and cracking of HPC. This study used a shrinkage-reducing admixture (SRA for that purpose. Concrete was prepared with two different mW/mB (0.22 and 0.40 and four different mass fractions of SRA to binder (w(SRA = 0%, 1%, 2%, and 4%. The mineral admixtures used for concrete mixes were: 25% fly ash (FA and 25% slag by mass of binder for the mixture with mW/mB = 0.40, and 15% silica fume (SF and 25% FA for the mixture with mW/mB = 0.22. Tests were conducted on 24 prismatic specimens, and shrinkage strains were measured through 120 days of drying. Compressive strength, splitting strength, and static modulus of elasticity were also determined. The results show that the SRA effectively reduces some mechanical properties of HPC. The reductions in compressive strength, splitting tensile strength, and elastic modulus of the concrete were 7%-24%, 9%-19%, and 5%-12%, respectively, after 90 days, compared to concrete mixtures without SRA. SRA can also help reduce drying shrinkage of concrete. The shrinkage strains of HPC with SRA were only as high as 41% of the average free shrinkage of concrete without SRA after 120 days of drying.

  7. Cell-scale dynamic recycling and cortical flow of the actin–myosin cytoskeleton for rapid cell migration

    Directory of Open Access Journals (Sweden)

    Shigehiko Yumura


    Actin and myosin II play major roles in cell migration. Whereas pseudopod extension by actin polymerization has been intensively researched, less attention has been paid to how the rest of the actin cytoskeleton such as the actin cortex contributes to cell migration. In this study, cortical actin and myosin II filaments were simultaneously observed in migrating Dictyostelium cells under total internal reflection fluorescence microscopy. The cortical actin and myosin II filaments remained stationary with respect to the substratum as the cells advanced. However, fluorescence recovery after photobleaching experiments and direct observation of filaments showed that they rapidly turned over. When the cells were detached from the substratum, the actin and myosin filaments displayed a vigorous retrograde flow. Thus, when the cells migrate on the substratum, the cortical cytoskeleton firmly holds the substratum to generate the motive force instead. The present studies also demonstrate how myosin II localizes to the rear region of the migrating cells. The observed dynamic turnover of actin and myosin II filaments contributes to the recycling of their subunits across the whole cell and enables rapid reorganization of the cytoskeleton.

  8. Rapid Elimination of the Persistent Synergid through a Cell Fusion Mechanism

    KAUST Repository

    Maruyama, Daisuke


    In flowering plants, fertilization-dependent degeneration of the persistent synergid cell ensures one-on-one pairings of male and female gametes. Here, we report that the fusion of the persistent synergid cell and the endosperm selectively inactivates the persistent synergid cell in Arabidopsis thaliana. The synergid-endosperm fusion causes rapid dilution of pre-secreted pollen tube attractant in the persistent synergid cell and selective disorganization of the synergid nucleus during the endosperm proliferation, preventing attractions of excess number of pollen tubes (polytubey). The synergid-endosperm fusion is induced by fertilization of the central cell, while the egg cell fertilization predominantly activates ethylene signaling, an inducer of the synergid nuclear disorganization. Therefore, two female gametes (the egg and the central cell) control independent pathways yet coordinately accomplish the elimination of the persistent synergid cell by double fertilization. Two female gametes (the egg cell and the central cell) in flowering plants coordinately prevent attractions of excess number of pollen tubes via two mechanisms to inactivate persistent synergid cell. © 2015 Elsevier Inc.

  9. Rapid morphological oscillation of mitochondrion-rich cell in estuarine mudskipper following salinity changes. (United States)

    Sakamoto, T; Yokota, S; Ando, M


    Morphological changes in the chloride cells or mitochondrion-rich (MR) cells in the skin under the pectoral fin of the estuarine mudskipper (Periophthalmus modestus) were examined in relation to intertidal salinity oscillation in river mouth. MR cells were distinguished between those in contact with the water (cells labeled with both mitochondrial probe DASPEI and Concanavalin-A, an apical surface marker of MR cells) and those that are not (DASPEI-positive only). After transfer of the fish from seawater to freshwater, no difference in the total MR cell density was observed, but the subpopulation of MR cells that are Concanavalin-A-positive decreased dramatically within 30 min. After 6 hr in freshwater, the fish were returned to seawater; the number of Con-A-positive MR cells increased to the initial levels rapidly. Thus, in seawater, mudskippers seem to open the apical crypts of the MR cells to secrete salt; in freshwater, they close the crypt of the MR cells tentatively, and tolerate hypotonicity until the rising tide. This unique response of chloride cells may also be seen in gills of other estuarine species.

  10. Hyaluronic Acid-Serum Hydrogels Rapidly Restore Metabolism of Encapsulated Stem Cells and Promote Engraftment (United States)

    Chan, Angel T.; Karakas, Mehmet F.; Vakrou, Styliani; Afzal, Junaid; Rittenbach, Andrew; Lin, Xiaoping; Wahl, Richard L.; Pomper, Martin G.; Steenbergen, Charles J.; Tsui, Benjamin M.W.; Elisseeff, Jennifer H.; Abraham, M. Roselle


    Background Cell death due to anoikis, necrosis and cell egress from transplantation sites limits functional benefits of cellular cardiomyoplasty. Cell dissociation and suspension, which are a pre-requisite for most cell transplantation studies, lead to depression of cellular metabolism and anoikis, which contribute to low engraftment. Objective We tissue engineered scaffolds with the goal of rapidly restoring metabolism, promoting viability, proliferation and engraftment of encapsulated stem cells. Methods The carboxyl groups of HA were functionalized with N-hydroxysuccinimide (NHS) to yield HA succinimidyl succinate (HA-NHS) groups that react with free amine groups to form amide bonds. HA-NHS was cross-linked by serum to generate HA:Serum (HA:Ser) hydrogels. Physical properties of HA:Ser hydrogels were measured. Effect of encapsulating cardiosphere-derived cells (CDCs) in HA:Ser hydrogels on viability, proliferation, glucose uptake and metabolism was assessed in vitro. In vivo acute intra-myocardial cell retention of 18FDG-labeled CDCs encapsulated in HA:Ser hydrogels was quantified. Effect of CDC encapsulation in HA:Ser hydrogels on in vivo metabolism and engraftment at 7 days was assessed by serial, dual isotope SPECT-CT and bioluminescence imaging of CDCs expressing the Na-iodide symporter and firefly luciferase genes respectively. Effect of HA:Ser hydrogels +/− CDCs on cardiac function was assessed at 7 days & 28 days post-infarct. Results HA:Ser hydrogels are highly bio-adhesive, biodegradable, promote rapid cell adhesion, glucose uptake and restore bioenergetics of encapsulated cells within 1 h of encapsulation, both in vitro and in vivo. These metabolic scaffolds can be applied epicardially as a patch to beating hearts or injected intramyocardially. HA:Ser hydrogels markedly increase acute intramyocardial retention (~6 fold), promote in vivo viability, proliferation, engraftment of encapsulated stem cells and angiogenesis. Conclusion HA:Ser hydrogels

  11. Rapid recognition and functional analysis of membrane proteins on human cancer cells using atomic force microscopy. (United States)

    Li, Mi; Xiao, Xiubin; Liu, Lianqing; Xi, Ning; Wang, Yuechao


    Understanding the physicochemical properties of cell surface signalling molecules is important for us to uncover the underlying mechanisms that guide the cellular behaviors. Atomic force microscopy (AFM) has become a powerful tool for detecting the molecular interactions on individual cells with nanometer resolution. In this paper, AFM peak force tapping (PFT) imaging mode was applied to rapidly locate and visually map the CD20 molecules on human lymphoma cells using biochemically sensitive tips. First, avidin-biotin system was used to test the effectiveness of using PFT imaging mode to probe the specific molecular interactions. The adhesion images obtained on avidin-coated mica using biotin-tethered tips obviously showed the recognition spots which corresponded to the avidins in the simultaneously obtained topography images. The experiments confirmed the specificity and reproducibility of the recognition results. Then, the established procedure was applied to visualize the nanoscale organization of CD20s on the surface of human lymphoma Raji cells using rituximab (a monoclonal anti-CD20 antibody)-tethered tips. The experiments showed that the recognition spots in the adhesion images corresponded to the specific CD20-rituximab interactions. The cluster sizes of CD20s on lymphoma Raji cells were quantitatively analyzed from the recognition images. Finally, under the guidance of fluorescence recognition, the established procedure was applied to cancer cells from a clinical lymphoma patient. The results showed that there were significant differences between the adhesion images obtained on cancer cells and on normal cells (red blood cell). The CD20 distributions on ten cancer cells from the patient were quantified according to the adhesion images. The experimental results demonstrate the capability of applying PFT imaging to rapidly investigate the nanoscale biophysical properties of native membrane proteins on the cell surface, which is of potential significance in

  12. Rapid, efficient charging of lead-acid and nickel-zinc traction cells. [for electric vehicles (United States)

    Smithrick, J. J.


    Lead-acid and nickel-zinc traction cells were rapidly and efficiently charged using a high rate taped dc charge (HRTDC) method which could possibly be used for on-the-road service recharge of electric vehicles. The HRTDC method takes advantage of initial high cell charge acceptance and uses cell gassing rate and temperature as an indicator of charging efficiency. On the average, 300 amp-hour nickel-zinc traction cells were given a HRTDC to 78% of rated amp-hour capacity within 53 minutes at an amp-hour efficiency of 92% and an energy efficiency of 52%. Three-hundred amp-hour lead-acid traction cells were charged to 69% of rated amp-hour capacity within 46 minutes at an amp-hour efficiency of 91% with an energy efficiency of 64%.

  13. Linear Shrinkage Behaviour of Compacted Loam Masonry Blocks

    Directory of Open Access Journals (Sweden)



    Full Text Available Walls of wet loam, used in earthen houses, generally experience more shrinkage which results in cracks and less compressive strength. This paper presents a technique of producing loam masonry blocks that are compacted in drained state during casting process in order to minimize shrinkage. For this purpose, loam masonry blocks were cast and compacted at a pressure of 6 MPa and then dried in shade by covering them in plastic sheet. The results show that linear shrinkage of 2% occurred which is smaller when compared to un-compacted wet loam walls. This implies that the loam masonry blocks compacted in drained state is expected to perform better than un-compacted wet loam walls.

  14. Hydration of Portoguese cements, measurement and modelling of chemical shrinkage

    DEFF Research Database (Denmark)

    Maia, Lino; Geiker, Mette Rica; Figueiras, Joaquim A.


    Development of cement hydration was studied by measuring the chemical shrinkage of pastes. Five types of Portuguese Portland cement were used in cement pastes with . Chemical shrinkage was measured by gravimetry and dilatometry. In gravimeters results were recorded automatically during at least...... seven days, dilatometers were manually recorded during at least 56 days. The dispersion model was applied to fit chemical shrinkage results and to estimate the maximum (or ultimate) value for calculation of degree of hydration. Except for a pure Portland cement best fits were obtained by the general...... form of the dispersion model. The development of hydration varied between the investigated cements; based on the measured data the degree of hydration after 24 h hydration at 20 C varied between 40 and 50%. This should be taken into account when comparing properties of concrete made from the different...

  15. Improving the Incoherence of a Learned Dictionary via Rank Shrinkage. (United States)

    Ubaru, Shashanka; Seghouane, Abd-Krim; Saad, Yousef


    This letter considers the problem of dictionary learning for sparse signal representation whose atoms have low mutual coherence. To learn such dictionaries, at each step, we first update the dictionary using the method of optimal directions (MOD) and then apply a dictionary rank shrinkage step to decrease its mutual coherence. In the rank shrinkage step, we first compute a rank 1 decomposition of the column-normalized least squares estimate of the dictionary obtained from the MOD step. We then shrink the rank of this learned dictionary by transforming the problem of reducing the rank to a nonnegative garrotte estimation problem and solving it using a path-wise coordinate descent approach. We establish theoretical results that show that the rank shrinkage step included will reduce the coherence of the dictionary, which is further validated by experimental results. Numerical experiments illustrating the performance of the proposed algorithm in comparison to various other well-known dictionary learning algorithms are also presented.

  16. Limitation of Shrinkage Porosity in Aluminum Rotor Die Casting (United States)

    Kim, Young-Chan; Choi, Se-Weon; Kim, Cheol-Woo; Cho, Jae-Ik; Lee, Sung-Ho; Kang, Chang-Seog

    Aluminum rotor prone to have many casting defects especially large amount of air and shrinkage porosity, which caused eccentricity, loss and noise during motor operation. Many attempts have been made to develop methods of shrinkage porosity control, but still there are some problems to solve. In this research, the process of vacuum squeeze die casting is proposed for limitation of defects. The 6 pin point gated dies which were in capable of local squeeze at the end ring were used. Influences of filling patterns on HPDC were evaluated and the important process control parameters were high injection speed, squeeze length, venting and process conditions. By using local squeeze and vacuum during filling and solidification, air and shrinkage porosity were significantly reduced and the feeding efficiency at the upper end ring was improved 10%. As a result of controlling the defects, the dynamometer test showed improved motor efficiency by more than 4%.

  17. Shrinkage-stress kinetics of photopolymerised resin-composites (United States)

    Satterthwaite, Julian D.

    The use of directly-placed substances as restorative materials in teeth remains the technique of choice for preserving function and form in teeth that have cavities. The current aesthetic restorative materials of choice are resin-composite materials, although these undergo molecular densification during polymerisation, which has deleterious effects. Although shrinkage-strain is the cause, it is the shrinkage-stress effects that may be seen as being responsible for the problems with adhesive resin-based restorations that are encountered clinically, the bond may fail with separation of the material from the cavity wall, leading to marginal discolouration, pulpal irritation and subsequent necrosis, post operative sensitivity, recurrent caries and eventual failure of restorations. Other outcomes include cohesive fracture of enamel or cusps, cuspal movement (strain) and persistent pain. The aims of this research were to characterise the effects of variations in resin-composite formulation on shrinkage-strain and shrinkage-stress kinetics. In particular, the influence of the size and morphology of the dispersed phase was investigated through the study of experimental formulations. Polymerisation shrinkage-strain kinetics were assessed with the bonded-disk method. It was found that resin-composites with spherical filler particles had significantly lower shrinkage-strain compared to those with irregular filler particles. Additionally, shrinkage-strain was found to be dependent on the size of filler particle, and this trend was related, in part, to differences in the degree of conversion. The data were also used to calculate the activation energy for each material, and a relationship between this and filler particle size for the irregular fillers was demonstrated. A fixed-compliance cantilever beam instrument (Bioman) was used for characterisation of shrinkage-stress kinetics. Significant differences were identified between materials in relation to filler particle size and

  18. Optical scatter imaging: a microscopic modality for the rapid morphological assay of living cells (United States)

    Boustany, Nada N.


    Tumors derived from epithelial cells comprise the majority of human tumors and their growth results from the accumulation of multiple mutations affecting cellular processes critical for tissue homeostasis, including cell proliferation and cell death. To understand these processes and address the complexity of cancer cell function, multiple cellular responses to different experimental conditions and specific genetic mutations must be analyzed. Fundamental to this endeavor is the development of rapid cellular assays in genetically defined cells, and in particular, the development of optical imaging methods that allow dynamic observation and real-time monitoring of cellular processes. In this context, we are developing an optical scatter imaging technology that is intended to bridge the gap between light and electron microscopy by rapidly providing morphometric information about the relative size and shape of non-spherical organelles, with sub-wavelength resolution. Our goal is to complement current microscopy techniques used to study cells in-vitro, especially in long-term time-lapse studies of living cells, where exogenous labels can be toxic, and electron microscopy will destroy the sample. The optical measurements are based on Fourier spatial filtering in a standard microscope, and could ultimately be incorporated into existing high-throughput diagnostic platforms for cancer cell research and histopathology of neoplastic tissue arrays. Using an engineered epithelial cell model of tumor formation, we are currently studying how organelle structure and function are altered by defined genetic mutations affecting the propensity for cell death and oncogenic potential, and by environmental conditions promoting tumor growth. This talk will describe our optical scatter imaging technology and present results from our studies on apoptosis, and the function of BCL-2 family proteins.

  19. Rapid and serial quantification of adhesion forces of yeast and Mammalian cells.

    Directory of Open Access Journals (Sweden)

    Eva Potthoff

    Full Text Available Cell adhesion to surfaces represents the basis for niche colonization and survival. Here we establish serial quantification of adhesion forces of different cell types using a single probe. The pace of single-cell force-spectroscopy was accelerated to up to 200 yeast and 20 mammalian cells per probe when replacing the conventional cell trapping cantilever chemistry of atomic force microscopy by underpressure immobilization with fluidic force microscopy (FluidFM. In consequence, statistically relevant data could be recorded in a rapid manner, the spectrum of examinable cells was enlarged, and the cell physiology preserved until approached for force spectroscopy. Adhesion forces of Candida albicans increased from below 4 up to 16 nN at 37°C on hydrophobic surfaces, whereas a Δhgc1-mutant showed forces consistently below 4 nN. Monitoring adhesion of mammalian cells revealed mean adhesion forces of 600 nN of HeLa cells on fibronectin and were one order of magnitude higher than those observed for HEK cells.

  20. Advances in a rapidly emerging field of hair follicle stem cell research. (United States)

    Mokos, Zrinka Bukvić; Mosler, Elvira Lazić


    Human skin maintains the ability to regenerate during adulthood, as it constantly renews itself throughout adult life, and the hair follicle (HF) undergoes a perpetual cycle of growth and degeneration. The study of stem cells (SCs) in the epidermis and skin tissue engineering is a rapidly emerging field, where advances have been made in both basic and clinical research. Advances in basic science include the ability to assay SCs of the epidermis in vivo, identification of an independent interfollicular epidermal SC, and improved ability to analyze individual SCs divisions, as well as the recent hair organ regeneration via the bioengineered hair follicular unit transplantation (FUT) in mice. Advances in the clinic include recognition of the importance of SCs for wound repair and for gene therapy in inherited skin diseases, for example epidermolysis bullosa. The study of the HF stem cells (HFSCs) started by identification of epidermal SC in the HF bulge as quiescent "label retaining cells". The research of these cells emerged rapidly after the identification of bulge cell molecular markers, such as keratin 15 (K15) and CD34 in mice and CD200 in humans, which allowed the isolation and characterization of bulge cells from follicles. This paper provides an overview of the current knowledge on epidermal SCs in the HF describing their essential characteristics and the control of follicle SCs fate, their role in alopecia, as well as their use in tissue engineering.

  1. Self-compacting fine-grained concretes with compensated shrinkage

    Directory of Open Access Journals (Sweden)

    Alimov Lev


    Full Text Available This paper substantiates the efficiency of application of fine-grained concrete for erection of cast-in-place concrete and reinforced concrete structures of different purpose. On the basis of analysis of experimental research results it was established that the introduction of microfillers with expansion effect to composite binder allows not only improving the rheological properties of fine-grained concrete, but also decreasing of value of shrinkage strain and improving of concrete crack resistance and durability. The analysis of the results of industrial use of fine-grained concretes with compensated shrinkage is given.

  2. Rapid metabolism of exogenous angiotensin II by catecholaminergic neuronal cells in culture media. (United States)

    Basu, Urmi; Seravalli, Javier; Madayiputhiya, Nandakumar; Adamec, Jiri; Case, Adam J; Zimmerman, Matthew C


    Angiotensin II (AngII) acts on central neurons to increase neuronal firing and induce sympathoexcitation, which contribute to the pathogenesis of cardiovascular diseases including hypertension and heart failure. Numerous studies have examined the precise AngII-induced intraneuronal signaling mechanism in an attempt to identify new therapeutic targets for these diseases. Considering the technical challenges in studying specific intraneuronal signaling pathways in vivo, especially in the cardiovascular control brain regions, most studies have relied on neuronal cell culture models. However, there are numerous limitations in using cell culture models to study AngII intraneuronal signaling, including the lack of evidence indicating the stability of AngII in culture media. Herein, we tested the hypothesis that exogenous AngII is rapidly metabolized in neuronal cell culture media. Using liquid chromatography-tandem mass spectrometry, we measured levels of AngII and its metabolites, Ang III, Ang IV, and Ang-1-7, in neuronal cell culture media after administration of exogenous AngII (100 nmol/L) to a neuronal cell culture model (CATH.a neurons). AngII levels rapidly declined in the media, returning to near baseline levels within 3 h of administration. Additionally, levels of Ang III and Ang-1-7 acutely increased, while levels of Ang IV remained unchanged. Replenishing the media with exogenous AngII every 3 h for 24 h resulted in a consistent and significant increase in AngII levels for the duration of the treatment period. These data indicate that AngII is rapidly metabolized in neuronal cell culture media, and replenishing the media at least every 3 h is needed to sustain chronically elevated levels. © 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  3. Dexamethasone rapidly suppresses IL-33-stimulated mast cell function by blocking transcription factor activity. (United States)

    Paranjape, Anuya; Chernushevich, Oksana; Qayum, Amina Abdul; Spence, Andrew J; Taruselli, Marcela T; Abebayehu, Daniel; Barnstein, Brian O; McLeod, Jamie Josephine Avila; Baker, Bianca; Bajaj, Gurjas S; Chumanevich, Alena P; Oskeritzian, Carole A; Ryan, John J


    Mast cells are critical effectors of allergic disease and can be activated by IL-33, a proinflammatory member of the IL-1 cytokine family. IL-33 worsens the pathology of mast cell-mediated diseases, but therapies to antagonize IL-33 are still forthcoming. Because steroids are the mainstay of allergic disease treatment and are well known to suppress mast cell activation by other stimuli, we examined the effects of the steroid dexamethasone on IL-33-mediated mast cell function. We found that dexamethasone potently and rapidly suppressed cytokine production elicited by IL-33 from murine bone marrow-derived and peritoneal mast cells. IL-33 enhances IgE-mediated mast cell cytokine production, an activity that was also antagonized by dexamethasone. These effects were consistent in human mast cells. We additionally observed that IL-33 augmented migration of IgE-sensitized mast cells toward antigen. This enhancing effect was similarly reversed by dexamethasone. Simultaneous addition of dexamethasone with IL-33 had no effect on the phosphorylation of MAP kinases or NFκB p65 subunit; however, dexamethasone antagonized AP-1- and NFκB-mediated transcriptional activity. Intraperitoneal administration of dexamethasone completely abrogated IL-33-mediated peritoneal neutrophil recruitment and prevented plasma IL-6 elevation. These data demonstrate that steroid therapy may be an effective means of antagonizing the effects of IL-33 on mast cells in vitro and in vivo, acting partly by suppressing IL-33-induced NFκB and AP-1 activity. © Society for Leukocyte Biology.

  4. Rapid identification of cell-specific, internalizing RNA aptamers with bioinformatics analyses of a cell-based aptamer selection.

    Directory of Open Access Journals (Sweden)

    William H Thiel

    Full Text Available The broad applicability of RNA aptamers as cell-specific delivery tools for therapeutic reagents depends on the ability to identify aptamer sequences that selectively access the cytoplasm of distinct cell types. Towards this end, we have developed a novel approach that combines a cell-based selection method (cell-internalization SELEX with high-throughput sequencing (HTS and bioinformatics analyses to rapidly identify cell-specific, internalization-competent RNA aptamers.We demonstrate the utility of this approach by enriching for RNA aptamers capable of selective internalization into vascular smooth muscle cells (VSMCs. Several rounds of positive (VSMCs and negative (endothelial cells; ECs selection were performed to enrich for aptamer sequences that preferentially internalize into VSMCs. To identify candidate RNA aptamer sequences, HTS data from each round of selection were analyzed using bioinformatics methods: (1 metrics of selection enrichment; and (2 pairwise comparisons of sequence and structural similarity, termed edit and tree distance, respectively. Correlation analyses of experimentally validated aptamers or rounds revealed that the best cell-specific, internalizing aptamers are enriched as a result of the negative selection step performed against ECs.We describe a novel approach that combines cell-internalization SELEX with HTS and bioinformatics analysis to identify cell-specific, cell-internalizing RNA aptamers. Our data highlight the importance of performing a pre-clear step against a non-target cell in order to select for cell-specific aptamers. We expect the extended use of this approach to enable the identification of aptamers to a multitude of different cell types, thereby facilitating the broad development of targeted cell therapies.

  5. Rapid identification of cell-specific, internalizing RNA aptamers with bioinformatics analyses of a cell-based aptamer selection. (United States)

    Thiel, William H; Bair, Thomas; Peek, Andrew S; Liu, Xiuying; Dassie, Justin; Stockdale, Katie R; Behlke, Mark A; Miller, Francis J; Giangrande, Paloma H


    The broad applicability of RNA aptamers as cell-specific delivery tools for therapeutic reagents depends on the ability to identify aptamer sequences that selectively access the cytoplasm of distinct cell types. Towards this end, we have developed a novel approach that combines a cell-based selection method (cell-internalization SELEX) with high-throughput sequencing (HTS) and bioinformatics analyses to rapidly identify cell-specific, internalization-competent RNA aptamers. We demonstrate the utility of this approach by enriching for RNA aptamers capable of selective internalization into vascular smooth muscle cells (VSMCs). Several rounds of positive (VSMCs) and negative (endothelial cells; ECs) selection were performed to enrich for aptamer sequences that preferentially internalize into VSMCs. To identify candidate RNA aptamer sequences, HTS data from each round of selection were analyzed using bioinformatics methods: (1) metrics of selection enrichment; and (2) pairwise comparisons of sequence and structural similarity, termed edit and tree distance, respectively. Correlation analyses of experimentally validated aptamers or rounds revealed that the best cell-specific, internalizing aptamers are enriched as a result of the negative selection step performed against ECs. We describe a novel approach that combines cell-internalization SELEX with HTS and bioinformatics analysis to identify cell-specific, cell-internalizing RNA aptamers. Our data highlight the importance of performing a pre-clear step against a non-target cell in order to select for cell-specific aptamers. We expect the extended use of this approach to enable the identification of aptamers to a multitude of different cell types, thereby facilitating the broad development of targeted cell therapies.

  6. Rapid Fatal Outcome from Pulmonary Arteries Compression in Transitional Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Ioannis A. Voutsadakis


    Full Text Available Transitional cell carcinoma of the urinary bladder is a malignancy that metastasizes frequently to lymph nodes including the mediastinal lymph nodes. This occurrence may produce symptoms due to compression of adjacent structures such as the superior vena cava syndrome or dysphagia from esophageal compression. We report the case of a 59-year-old man with metastatic transitional cell carcinoma for whom mediastinal lymphadenopathy led to pulmonary artery compression and a rapidly fatal outcome. This rare occurrence has to be distinguished from pulmonary embolism, a much more frequent event in cancer patients, in order that proper and prompt treatment be initiated.

  7. Rapid NK-cell activation in chicken after infection with infectious bronchitis virus M41. (United States)

    Vervelde, L; Matthijs, M G R; van Haarlem, D A; de Wit, J J; Jansen, C A


    Natural killer (NK) cells are cytotoxic lymphocytes and play an important role in the early defence against viruses. In this study we focussed on NK cell and interferon (IFN) responses after infection with infectious bronchitis virus (IBV). Based on surface expression of CD107+, enhanced activation of lung NK cells was observed at 1 dpi, whereas in blood prolonged NK-cell activation was found. IFN-α and IFN-β mRNA and proteins were not rapidly induced whereas IFN-γ production in lung, measured by Elispot assay, increased over time at 2 and 4 dpi. In contrast, IFN-γ production in blood was highest at 1 dpi and decreased over time down to levels comparable to uninfected birds at 4 dpi. Collectively, infection with IBV-M41 resulted in activation of NK cells in the lung and blood and rapid production of IFN-γ and not IFN-α and IFN-β compared to uninfected birds. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Migratory dermal dendritic cells act as rapid sensors of protozoan parasites.

    Directory of Open Access Journals (Sweden)

    Lai Guan Ng


    Full Text Available Dendritic cells (DC, including those of the skin, act as sentinels for intruding microorganisms. In the epidermis, DC (termed Langerhans cells, LC are sessile and screen their microenvironment through occasional movements of their dendrites. The spatio-temporal orchestration of antigen encounter by dermal DC (DDC is not known. Since these cells are thought to be instrumental in the initiation of immune responses during infection, we investigated their behavior directly within their natural microenvironment using intravital two-photon microscopy. Surprisingly, we found that, under homeostatic conditions, DDC were highly motile, continuously crawling through the interstitial space in a Galpha(i protein-coupled receptor-dependent manner. However, within minutes after intradermal delivery of the protozoan parasite Leishmania major, DDC became immobile and incorporated multiple parasites into cytosolic vacuoles. Parasite uptake occurred through the extension of long, highly dynamic pseudopods capable of tracking and engulfing parasites. This was then followed by rapid dendrite retraction towards the cell body. DDC were proficient at discriminating between parasites and inert particles, and parasite uptake was independent of the presence of neutrophils. Together, our study has visualized the dynamics and microenvironmental context of parasite encounter by an innate immune cell subset during the initiation of the immune response. Our results uncover a unique migratory tissue surveillance program of DDC that ensures the rapid detection of pathogens.

  9. Rapid deterioration of preexisting renal insufficiency after autologous mesenchymal stem cell therapy

    Directory of Open Access Journals (Sweden)

    Jun-Seop Kim


    Full Text Available Administration of autologous mesenchymal stem cells (MSCs has been shown to improve renal function and histological findings in acute kidney injury (AKI models. However, its effects in chronic kidney disease (CKD are unclear, particularly in the clinical setting. Here, we report our experience with a CKD patient who was treated by intravenous infusion of autologous MSCs derived from adipose tissue in an unknown clinic outside of Korea. The renal function of the patient had been stable for several years before MSC administration. One week after the autologous MSC infusion, the preexisting renal insufficiency was rapidly aggravated without any other evidence of AKI. Hemodialysis was started 3 months after MSC administration. Renal biopsy findings at dialysis showed severe interstitial fibrosis and inflammatory cell infiltration, with a few cells expressing CD34 and CD117, 2 surface markers of stem cells. This case highlights the potential nephrotoxicity of autologous MSC therapy in CKD patients.

  10. Influence of shrinkage-reducing admixture on drying shrinkage and mechanical properties of high-performance concrete

    Directory of Open Access Journals (Sweden)

    Nguyen Quangphu


    Full Text Available High-performance concrete (HPC has specific performance advantages over conventional concrete in strength and durability. HPC mixtures are usually produced with water/binder mass ratios (mW/mB in the range of 0.2–0.4, so volume changes of concrete as a result of drying, chemical reactions, and temperature change cannot be avoided. For these reasons, shrinkage and cracking are frequent phenomena. It is necessary to add some types of admixture for reduction of shrinkage and cracking of HPC. This study used

  11. Rapid selection and proliferation of CD133+ cells from cancer cell lines: chemotherapeutic implications.

    Directory of Open Access Journals (Sweden)

    Sarah E Kelly


    Full Text Available Cancer stem cells (CSCs are considered a subset of the bulk tumor responsible for initiating and maintaining the disease. Several surface cellular markers have been recently used to identify CSCs. Among those is CD133, which is expressed by hematopoietic progenitor cells as well as embryonic stem cells and various cancers. We have recently isolated and cultured CD133 positive [CD133+] cells from various cancer cell lines using a NASA developed Hydrodynamic Focusing Bioreactor (HFB (Celdyne, Houston, TX. For comparison, another bioreactor, the rotary cell culture system (RCCS manufactured by Synthecon (Houston, TX was used. Both the HFB and the RCCS bioreactors simulate aspects of hypogravity. In our study, the HFB increased CD133+ cell growth from various cell lines compared to the RCCS vessel and to normal gravity control. We observed a +15-fold proliferation of the CD133+ cellular fraction with cancer cells that were cultured for 7-days at optimized conditions. The RCCS vessel instead yielded a (-4.8-fold decrease in the CD133+cellular fraction respect to the HFB after 7-days of culture. Interestingly, we also found that the hypogravity environment of the HFB greatly sensitized the CD133+ cancer cells, which are normally resistant to chemo treatment, to become susceptible to various chemotherapeutic agents, paving the way to less toxic and more effective chemotherapeutic treatment in patients. To be able to test the efficacy of cytotoxic agents in vitro prior to their use in clinical setting on cancer cells as well as on cancer stem cells may pave the way to more effective chemotherapeutic strategies in patients. This could be an important advancement in the therapeutic options of oncologic patients, allowing for more targeted and personalized chemotherapy regimens as well as for higher response rates.

  12. Drying and radial shrinkage characteristics and changes in color ...

    African Journals Online (AJOL)

    Drying and radial shrinkage characteristics and changes in color and shape of carrots tissues during air drying were studied. Slices dimensions were obtained by computer vision and the color was quantified by chroma, hue, whitening index and total carotenoids contents. The drying time became shorter of 1 h when ...

  13. Preliminary Investigation of Drying Shrinkage Cement Paste Specimens

    NARCIS (Netherlands)

    Jankovic, D.; Van Mier, J.G.M.


    In order to perform drying shrinkage observations in ESEM (Environmental Scanning Electron Microscope), a preparation procedure for cement paste samples is developed. Instead of casting standard prisms (40 x 40 x 160 mm3), cement paste samples were cast in specially designed moulds of 30 x 30 x 2

  14. Magnetic nanoparticles-induced anisotropic shrinkage of polymer emulsion droplets

    NARCIS (Netherlands)

    Liu, B; de Folter, J.W.J.; Möhwald, H.


    We here report magnetic nanoparticles (NPs)-induced buckling instability and anisotropic shrinkage behavior of polymer emulsion droplets. The oil-in-water emulsion is stabilized by the surfactant, and NPs are dispersed into the oil phase. The surface ligands (oleic acid and oleylamine) number of the

  15. Fast generation of computer-generated holograms using wavelet shrinkage. (United States)

    Shimobaba, Tomoyoshi; Ito, Tomoyoshi


    Computer-generated holograms (CGHs) are generated by superimposing complex amplitudes emitted from a number of object points. However, this superposition process remains very time-consuming even when using the latest computers. We propose a fast calculation algorithm for CGHs that uses a wavelet shrinkage method, eliminating small wavelet coefficient values to express approximated complex amplitudes using only a few representative wavelet coefficients.

  16. Fourier-transform infrared spectroscopy for rapid screening and live-cell monitoring: application to nanotoxicology

    Energy Technology Data Exchange (ETDEWEB)

    Sundaram, S. K.; Sacksteder, Colette A.; Weber, T. J.; Riley, Brian J.; Addleman, Raymond S.; Harrer, Bruce J.; Peterman, John W.


    A significant challenge to realize the full potential of nanotechnology for therapeutic and diagnostic applications is to understand and evaluate how live-cells interact with an external stimulus, e.g., a nanosized particle (NSP), and the toxicity and broad risk associated with these stimuli. NSPs are increasingly used in medicine with largely undetermined hazards in complex cell dynamics and environments. It is difficult to capture the complexity and dynamics of these interactions by following an omics-based approach exclusively, which are expensive and time-consuming. Additionally, this approach needs destructive sampling methods. Live-cell attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectrometry is well suited to provide noninvasive approach to provide rapid screening of cellular responses to potentially toxic NSPs or any stimuli. Herein we review the technical basis of the approach, the instrument configuration and interface with the biological media, and various effects that impact the data, data analysis, and toxicity. Our preliminary results on live-cell monitoring show promise for rapid screening the NSPs.

  17. Rapid Turnover of Effector–Memory CD4+ T Cells in Healthy Humans (United States)

    Macallan, Derek C.; Wallace, Diana; Zhang, Yan; de Lara, Catherine; Worth, Andrew T.; Ghattas, Hala; Griffin, George E.; Beverley, Peter C.L.; Tough, David F.


    Memory T cells can be divided into central–memory (TCM) and effector–memory (TEM) cells, which differ in their functional properties. Although both subpopulations can persist long term, it is not known whether they are maintained by similar mechanisms. We used in vivo labeling with deuterated glucose to measure the turnover of CD4+ T cells in healthy humans. The CD45R0+CCR7− TEM subpopulation was shown to have a rapid proliferation rate of 4.7% per day compared with 1.5% per day for CD45R0+CCR7+ TCM cells; these values are equivalent to average intermitotic (doubling) times of 15 and 48 d, respectively. In contrast, the CD45RA+CCR7+ naive CD4+ T cell population was found to be much longer lived, being labeled at a rate of only 0.2% per day (corresponding to an intermitotic time of approximately 1 yr). These data indicate that human CD4+ TEM cells constitute a short-lived cell population that requires continuous replenishment in vivo. PMID:15249595

  18. Rapid in vitro derivation of endothelium directly from human cancer cells.

    Directory of Open Access Journals (Sweden)

    Jennifer D Elster

    Full Text Available The development of an independent blood supply by a tumor is essential for maintaining growth beyond a certain limited size and for providing a portal for metastatic dissemination. Host-derived endothelial cells (ECs residing in and compromising the tumor vasculature originate via distinct processes known as sprouting angiogenesis and vasculogenesis. More recently ECs originating directly from the tumor cells themselves have been described although the basis for this phenomenon remains poorly understood. Here we describe in vitro conditions that allow lung and ovarian cancer cells to undergo a rapid and efficient transition into ECs that are indistinguishable from those obtained in vivo. A variety of methods were used to establish that the acquired phenotypes and behaviors of these tumor-derived ECs (TDECs closely resemble those of authentic ECs. Xenografts arising from co-inoculated in vitro-derived TDECs and tumor cells were also more highly vascularized than control tumors; moreover, their blood vessels were on average larger and frequently contained admixtures of host-derived ECs and TDECs derived from the initial inoculum. These results demonstrate that cancer cells can be manipulated under well-defined in vitro conditions to initiate a tumor cell-to-EC transition that is largely cell-autonomous, highly efficient and closely mimics the in vivo process. These studies provide a suitable means by which to identify and perhaps modify the earliest steps in TDEC generation.

  19. Rapid Prototyping of Polymeric Nanopillars by 3D Direct Laser Writing for Controlling Cell Behavior. (United States)

    Buch-Månson, Nina; Spangenberg, Arnaud; Gomez, Laura Piedad Chia; Malval, Jean-Pierre; Soppera, Olivier; Martinez, Karen L


    Mammalian cells have been widely shown to respond to nano- and microtopography that mimics the extracellular matrix. Synthetic nano- and micron-sized structures are therefore of great interest in the field of tissue engineering, where polymers are particularly attractive due to excellent biocompatibility and versatile fabrication methods. Ordered arrays of polymeric pillars provide a controlled topographical environment to study and manipulate cells, but processing methods are typically either optimized for the nano- or microscale. Here, we demonstrate polymeric nanopillar (NP) fabrication using 3D direct laser writing (3D DLW), which offers a rapid prototyping across both size regimes. The NPs are interfaced with NIH3T3 cells and the effect of tuning geometrical parameters of the NP array is investigated. Cells are found to adhere on a wide range of geometries, but the interface depends on NP density and length. The Cell Interface with Nanostructure Arrays (CINA) model is successfully extended to predict the type of interface formed on different NP geometries, which is found to correlate with the efficiency of cell alignment along the NPs. The combination of the CINA model with the highly versatile 3D DLW fabrication thus holds the promise of improved design of polymeric NP arrays for controlling cell growth.

  20. Overcoming challenges to initiating cell therapy clinical trials in rapidly developing countries: India as a model. (United States)

    Viswanathan, Sowmya; Rao, Mahendra; Keating, Armand; Srivastava, Alok


    Increasingly, a number of rapidly developing countries, including India, China, Brazil, and others, are becoming global hot spots for the development of regenerative medicine applications, including stem cell-based therapies. Identifying and overcoming regulatory and translational research challenges and promoting scientific and ethical clinical trials with cells will help curb the growth of stem cell tourism for unproven therapies. It will also enable academic investigators, local regulators, and national and international biotechnology and biopharmaceutical companies to accelerate stem cell-based clinical research that could lead to effective innovative treatments in these regions. Using India as a model system and obtaining input from regulators, clinicians, academics, and industry representatives across the stem cell field in India, we reviewed the role of key agencies and processes involved in this field. We have identified areas that need attention and here provide solutions from other established and functioning models in the world to streamline and unify the regulatory and ethics approval processes for cell-based therapies. We also make recommendations to check the growth and functioning of clinics offering unproven treatments. Addressing these issues will remove considerable hurdles to both local and international investigators, accelerate the pace of research and development, and create a quality environment for reliable products to emerge. By doing so, these countries would have taken one important step to move to the forefront of stem cell-based therapeutics.

  1. Better shrinkage than Shrinky-Dinks. (United States)

    Nguyen, Diep; Taylor, Douglas; Qian, Kun; Norouzi, Nizilla; Rasmussen, Jerald; Botzet, Steve; Lehmann, Matt; Halverson, Kurt; Khine, Michelle


    Polyolefins are finding increased popularity in microfluidic applications due to their attractive mechanical, processing, and optical properties. While intricate features are typically realized in these thermoplastics by hot embossing and injection molding, such fabrication approaches are expensive and slow. Here, we apply our shrink-induced approach-first demonstrated with polystyrene 'Shrinky-Dink' sheets-to create micro- and nanostructures with cross-linked polyolefin thin films. These multi-layered films shrink by 95% and with greater uniformity than the Shrinky-Dinks. With such significant reduction in size, along with attractive material properties, such commodity films could find important applications in low cost microfluidic prototyping as well as in point-of-care diagnostics. In this technical note, we demonstrate the ability to rapidly and easily create unique microstructures, increase microarray feature density, and even induce self-assembled integrated metallic nanostructures with these shrink wrap films.

  2. Measurement of shrinkage and cracking in lyophilized amorphous cakes. Part I: final-product assessment. (United States)

    Ullrich, Sabine; Seyferth, Stefan; Lee, Geoffrey


    An experimental technique is presented to determine independently shrinkage and cracking in lyophilized amorphous cakes based on photographic imaging of their top surface. An inverse correlation between cake shrinkage and cracking during freeze-drying is seen. Shrinkage relaxes the drying tension and gives little cracking, whereas if shrinkage is restrained then more cracking occurs. A lower shrinkage and greater cracking with higher disaccharide concentration correlates with change in cake hardness and brittleness. Adhesion of the cake to the inside vial wall could not be identified as a determining factor for shrinkage. Shrinkage is non-uniform across the cake's surface and is manifested largely in the peripheral region. A correlation between shrinkage and w(g)' for different disaccharides suggests that drying tension develops as non-frozen water is lost from the porous solid after sublimation of the ice phase has exposed the solid/gas interface. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

  3. The Shrinkage Cracking Behavior in Reinforced Reactive Powder Concrete Walls

    Directory of Open Access Journals (Sweden)

    Samir A. Al-Mashhadi


    Full Text Available In this study, the reduced scale wall models were used (they are believed to resemble as much as possible the field conditions to study the shrinkage behavior of reactive powder concrete (RPC base restrained walls. Six base restrained RPC walls were casted in different length/height ratios of two ratios of steel fiber by volume in Summer. These walls were restrained by reinforced concrete bases to provide the continuous base restraint to the walls. The mechanical properties of reactive powder concrete investigated were; compressive strength between (75.3 – 140.1 MPa, splitting tensile strength between (5.7 – 13.9 MPa, flexural tensile strength (7.7 – 24.5 MPa, and static modulus of elasticity (32.7 – 47.1GPa. Based on the observations of this work, it was found that the cracks did not develop in the reduced scale of the reactive powder concrete (RPC walls restrained from movement at their bases for different L/H ratios (2, 5, and 10 and for two ratio of steel fiber (1% & 2% during 90 days period of drying conditions. Moreover, the shrinkage values increase toward the edges. Based on the results of this work, the increase in the maximum shrinkage values of walls with 1% steel fiber were (29%, 28%, 28% of the maximum shrinkage values of walls with 2% steel fiber of length/height ratios of (2, 5, and 10 respectively. The experimental observation in beam specimens showed that the free shrinkage, tensile strain capacity and elastic tensile strain capacity (at date of cracking of beams with 1% steel fiber were higher than the beams with 2% steel fiber by about (24%, (45% and (42% respectively

  4. Cure shrinkage effects in epoxy and polycyanate matrix composites

    Energy Technology Data Exchange (ETDEWEB)

    Spellman, G.P.


    A relatively new advanced composite matrix, polycyanate ester, was evaluated for cure shrinkage. The chemical cure shrinkage of composites is difficult to model but a number of clever experimental techniques are available to the investigator. In this work the method of curing a prepreg layup on top of a previously cured laminate of identical ply composition is utilized. The polymeric matrices used in advanced composites have been primarily epoxies and therefore a common system of this type, Fiberite 3501-6, was used as a base case material. Three polycyanate matrix systems were selected for the study. These are: Fiberite 954-2A, YLA RS-3, and Bryte Technology BTCy-1. The first three of these systems were unidirectional prepreg with carbon fiber reinforcement. The Bryte Technology material was reinforced with E-glass fabric. The technique used to evaluate cure shrinkage results in distortion of the flatness of an otherwise symmetric laminate. The first laminate is cured in a conventional fashion. An identical layup is cured on this first laminate. During the second cure all constituents are exposed to the same thermal cycles. However, only the new portion of the laminate will experience volumetric changes associate with matrix cure. The additional strain of cure shrinkage results in an unsymmetric distribution of residual stresses and an associated warpage of the laminate. The baseline material, Fiberite 3501-6, exhibited cure shrinkage that was in accordance with expectations. Cure strains were {minus}4.5E-04. The YLA RS-3 material had cure strains somewhat lower at {minus}3.2E-04. The Fiberite 954-2A cure strain was {minus}1.5E-04 that is 70% lower than the baseline material. The glass fabric material with the Bryte BTCy-1 matrix did not result in meaningful results because the processing methods were not fully compatible with the material.

  5. Simple and Rapid Bioink Jet Printing for Multiscale Cell Adhesion Islands. (United States)

    Mecozzi, Laura; Gennari, Oriella; Rega, Romina; Battista, Luigi; Ferraro, Pietro; Grilli, Simonetta


    A simple and rapid process for multiscale printing of bioinks with dot widths ranging from hundreds of microns down to 0.5 μm is presented. The process makes use of spontaneous surface charges generated pyroelectrically that are able to draw little daughter droplets directly from the free meniscus of a mother drop through jetting ("p-jet"), thus avoiding time-consuming and expensive fabrication of microstructured nozzles. Multiscale can be easily achieved by modulating the parameters of the p-jet process. Here, it is shown that the p-jet allows us to print well-defined adhesion islands where NIH-3T3 fibroblasts are constrained to live into cluster configurations ranging from 20 down to single cell level. The proposed fabrication approach can be useful for high-throughput studies on cell adhesion, cytoskeleton organization, and stem cell differentiation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Short communication: HIV type 1 escapes inactivation by saliva via rapid escape into oral epithelial cells. (United States)

    Dietrich, Elizabeth A; Gebhard, Kristin H; Fasching, Claudine E; Giacaman, Rodrigo A; Kappes, John C; Ross, Karen F; Herzberg, Mark C


    Saliva contains anti-HIV-1 factors, which show unclear efficacy in thwarting mucosal infection. When incubated in fresh, unfractionated whole saliva, infectious HIV-1 IIIb and BaL (X4- and R5-tropic, respectively) persisted from 4 to at least 30 min in a saliva concentration-dependent manner. In salivary supernatant for up to 6 h, both infectious HIV-1 strains "escaped" into immortalized oral epithelial cells; infectious BaL showed selectively enhanced escape in the presence of saliva. Fluorescently labeled HIV-1 virus-like particles entered oral epithelial cells within minutes of exposure. Using a previously unrecognized mechanism, therefore, strains of HIV-1 escape inactivation by saliva via rapid uptake into oral epithelial cells.

  7. TH-E-BRF-01: Exploiting Tumor Shrinkage in Split-Course Radiotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Unkelbach, J; Craft, D; Hong, T; Papp, D; Wolfgang, J; Bortfeld, T [Massachusetts General Hospital, Boston, MA (United States); Ramakrishnan, J [University of Wisconsin, Madison, Wisconsin (United States); Salari, E [Wichita State University, Wichita, KS (United States)


    Purpose: In split-course radiotherapy, a patient is treated in several stages separated by weeks or months. This regimen has been motivated by radiobiological considerations. However, using modern image-guidance, it also provides an approach to reduce normal tissue dose by exploiting tumor shrinkage. In this work, we consider the optimal design of split-course treatments, motivated by the clinical management of large liver tumors for which normal liver dose constraints prohibit the administration of an ablative radiation dose in a single treatment. Methods: We introduce a dynamic tumor model that incorporates three factors: radiation induced cell kill, tumor shrinkage, and tumor cell repopulation. The design of splitcourse radiotherapy is formulated as a mathematical optimization problem in which the total dose to the liver is minimized, subject to delivering the prescribed dose to the tumor. Based on the model, we gain insight into the optimal administration of radiation over time, i.e. the optimal treatment gaps and dose levels. Results: We analyze treatments consisting of two stages in detail. The analysis confirms the intuition that the second stage should be delivered just before the tumor size reaches a minimum and repopulation overcompensates shrinking. Furthermore, it was found that, for a large range of model parameters, approximately one third of the dose should be delivered in the first stage. The projected benefit of split-course treatments in terms of liver sparing depends on model assumptions. However, the model predicts large liver dose reductions by more than a factor of two for plausible model parameters. Conclusion: The analysis of the tumor model suggests that substantial reduction in normal tissue dose can be achieved by exploiting tumor shrinkage via an optimal design of multi-stage treatments. This suggests taking a fresh look at split-course radiotherapy for selected disease sites where substantial tumor regression translates into reduced

  8. Rapid telomere motions in live human cells analyzed by highly time-resolved microscopy

    Directory of Open Access Journals (Sweden)

    Wang Xueying


    Full Text Available Abstract Background Telomeres cap chromosome ends and protect the genome. We studied individual telomeres in live human cancer cells. In capturing telomere motions using quantitative imaging to acquire complete high-resolution three-dimensional datasets every second for 200 seconds, telomere dynamics were systematically analyzed. Results The motility of individual telomeres within the same cancer cell nucleus was widely heterogeneous. One class of internal heterochromatic regions of chromosomes analyzed moved more uniformly and showed less motion and heterogeneity than telomeres. The single telomere analyses in cancer cells revealed that shorter telomeres showed more motion, and the more rapid telomere motions were energy dependent. Experimentally increasing bulk telomere length dampened telomere motion. In contrast, telomere uncapping, but not a DNA damaging agent, methyl methanesulfonate, significantly increased telomere motion. Conclusion New methods for seconds-scale, four-dimensional, live cell microscopic imaging and data analysis, allowing systematic tracking of individual telomeres in live cells, have defined a previously undescribed form of telomere behavior in human cells, in which the degree of telomere motion was dependent upon telomere length and functionality.

  9. Random migration and signal integration promote rapid and robust T cell recruitment. (United States)

    Textor, Johannes; Henrickson, Sarah E; Mandl, Judith N; von Andrian, Ulrich H; Westermann, Jürgen; de Boer, Rob J; Beltman, Joost B


    To fight infections, rare T cells must quickly home to appropriate lymph nodes (LNs), and reliably localize the antigen (Ag) within them. The first challenge calls for rapid trafficking between LNs, whereas the second may require extensive search within each LN. Here we combine simulations and experimental data to investigate which features of random T cell migration within and between LNs allow meeting these two conflicting demands. Our model indicates that integrating signals from multiple random encounters with Ag-presenting cells permits reliable detection of even low-dose Ag, and predicts a kinetic feature of cognate T cell arrest in LNs that we confirm using intravital two-photon data. Furthermore, we obtain the most reliable retention if T cells transit through LNs stochastically, which may explain the long and widely distributed LN dwell times observed in vivo. Finally, we demonstrate that random migration, both between and within LNs, allows recruiting the majority of cognate precursors within a few days for various realistic infection scenarios. Thus, the combination of two-scale stochastic migration and signal integration is an efficient and robust strategy for T cell immune surveillance.

  10. Rapid spread of mouse mammary tumor virus in cultured human breast cells

    Directory of Open Access Journals (Sweden)

    Günzburg Walter H


    Full Text Available Abstract Background The role of mouse mammary tumor virus (MMTV as a causative agent in human breast carcinogenesis has recently been the subject of renewed interest. The proposed model is based on the detection of MMTV sequences in human breast cancer but not in healthy breast tissue. One of the main drawbacks to this model, however, was that until now human cells had not been demonstrated to sustain productive MMTV infection. Results Here, we show for the first time the rapid spread of mouse mammary tumor virus, MMTV(GR, in cultured human mammary cells (Hs578T, ultimately leading to the infection of every cell in culture. The replication of the virus was monitored by quantitative PCR, quantitative RT-PCR and immunofluorescence imaging. The infected human cells expressed, upon cultivation with dexamethasone, MMTV structural proteins and released spiked B-type virions, the infectivity of which could be neutralized by anti-MMTV antibody. Replication of the virus was efficiently blocked by an inhibitor of reverse transcription, 3'-azido-3'-deoxythymidine. The human origin of the infected cells was confirmed by determining a number of integration sites hosting the provirus, which were unequivocally identified as human sequences. Conclusion Taken together, our results show that human cells can support replication of mouse mammary tumor virus.

  11. Primary Cutaneous Peripheral T-Cell Lymphoma Not Otherwise Specified: A Rapidly Progressive Variant of Cutaneous T-Cell Lymphoma

    Directory of Open Access Journals (Sweden)

    Kimberly Aderhold


    Full Text Available Primary Cutaneous Peripheral T-Cell Lymphoma NOS (PTL-NOS is a rare, progressive, fatal dermatologic disease that presents with features similar to many common benign plaque-like skin conditions, making recognition of its distinguishing features critical for early diagnosis and treatment (Bolognia et al., 2008. A 78-year-old woman presented to ambulatory care with a single 5 cm nodule on her shoulder that had developed rapidly over 1-2 weeks. Examination was suspicious for malignancy and a biopsy was performed. Biopsy results demonstrated CD4 positivity, consistent with Mycosis Fungoides with coexpression of CD5, CD47, and CD7. Within three months her cancer had progressed into diffuse lesions spanning her entire body. As rapid progression is usually uncharacteristic of Mycosis Fungoides, her diagnosis was amended to PTL-NOS. Cutaneous T-Cell Lymphoma (CTCL should be suspected in patients with patches, plaques, erythroderma, or papules that persist or multiply despite conservative treatment. Singular biopsies are often nondiagnostic, requiring a high degree of suspicion if there is deviation from the anticipated clinical course. Multiple biopsies are often necessary to make the diagnosis. Physicians caring for patients with rapidly progressive, nonspecific dermatoses with features described above should keep more uncommon forms of CTCL in mind and refer for early biopsy.

  12. Monitoring and rapid quantification of total carotenoids in Rhodotorula glutinis cells using laser tweezers Raman spectroscopy. (United States)

    Tao, Zhanhua; Wang, Guiwen; Xu, Xiaodong; Yuan, Yufeng; Wang, Xue; Li, Yongqing


    Rhodotorula glutinis is known to accumulate large amounts of carotenoids under certain culture conditions, which have very important industrial applications. So far, the molecular mechanism of regulating carotenogenesis is still not well understood. To better understand the carotenogenesis process, it requires methods that can detect carotenogenesis rapidly and reliably in single live cells. In this paper, a method based on laser tweezers Raman spectroscopy (LTRS) was developed to directly detect carotenoids, as well as other important biological molecules in single live R. glutinis cells. The data showed that the accumulation of carotenoids and lipids occurred mainly in the late exponential and stationary phases when the cell growth was inhibited by nutrient limitation. Meanwhile, the carotenoid concentration changed together with the concentration of nucleic acids, which increased in the first phase and decreased in the last phase of the culture. These data demonstrate that LTRS is a rapid, convenient, and reliable method to study the carotenogenesis process in vivo. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  13. Gene-modified stem cells combined with rapid prototyping techniques: a novel strategy for periodontal regeneration. (United States)

    He, Huixia; Cao, Junkai; Wang, Dongsheng; Gu, Bing; Guo, Hong; Liu, Hongchen


    Periodontal disease, a worldwide prevalent chronic disease in adults, is characterized by the destruction of the periodontal supporting tissue including the cementum, periodontal ligament and alveolar bone. The regeneration of damaged periodontal tissue is the main goal of periodontal treatment. Because conventional periodontal treatments remain insufficient to attain complete and reliable periodontal regeneration, periodontal tissue engineering has emerged as a prospective alternative method for improving the regenerative capacity of periodontal tissue. However, the potential of periodontal regeneration seems to be limited by the understanding of the cellular and molecular events in the formation of periodontal tissue and by the insufficient collaboration of multi-disciplinary research that periodontal tissue engineering involves. In this paper, we first reviewed the recent advancements in stem cells, signaling factors, and scaffolds that relate to periodontal regeneration. Then we speculate that specific genes would improve regenerative capacity of these stem cells, which could differentiate into cementoblasts, osteoblasts and fibroblasts. In addition, the 3D scaffolds that mimic the different structure and physiologic functions of natural fibro-osseous tissue could be fabricated by rapid prototyping (RP) techniques. It was therefore hypothesized that gene-modified stem cells combined with rapid prototyping techniques would be a new strategy to promote more effective and efficient periodontal regeneration.

  14. Study of ‘real’ shrinkage by ESEM observations and digital image analysis

    NARCIS (Netherlands)

    Jankovic, D.


    Defining the 'real' shrinkage values of concrete is still a subject of much debate. In shrinkage experiments size effects are inherently present. Through an attempt to determine the real shrinkage of cement-based materials, these size effects have to be eliminated or at least reduced as much a

  15. Topology optimization of reinforced concrete structures considering control of shrinkage and strength failure

    DEFF Research Database (Denmark)

    Luo, Yangjun; Wang, Michael Yu; Zhou, Mingdong


    To take into account the shrinkage effect in the early stage of Reinforced Concrete (RC) design, an effective continuum topology optimization method is presented in this paper. Based on the power-law interpolation, shrinkage of concrete is numerically simulated by introducing an additional design...... to ensure the structural safety under the combined action of external loads and shrinkage....

  16. Effects of Type of Work and Age on Spinal Shrinkage | Ismaila ...

    African Journals Online (AJOL)

    spine. The present study was aimed at studying the effects of different types of work on spinal shrinkage. Moreover, the relationship between age and spinal shrinkage was also studied. The heights just before the start and after the close of work were measured in order to determine the spinal shrinkage. The study confirmed ...

  17. Parenteral nutrition rapidly reduces hepatic mononuclear cell numbers and lipopolysaccharide receptor expression on Kupffer cells in mice. (United States)

    Omata, Jiro; Fukatsu, Kazuhiko; Murakoshi, Satoshi; Noguchi, Midori; Moriya, Tomoyuki; Okamoto, Koichi; Saitoh, Daizoh; Yamamoto, Junji; Hase, Kazuo


    Parenteral nutrition (PN) reduces the number of hepatic mononuclear cell (MNCs) and impairs their function, resulting in poor survival after intraportal bacterial challenge in mice. Our recent animal study demonstrated resumption of enteral nutrition after PN to rapidly restore hepatic MNC numbers (in 12 hours) and lipopolysaccharide (LPS) receptor expression on Kupffer cells (in 48 hours). The present study examined the time courses of hepatic MNC number reductions and LPS receptor expression changes in mice receiving PN. Male mice (n = 49) from the Institute of Cancer Research were divided into chow (n = 8), PN0.5 (n = 8), PN1 (n = 8), PN2 (n = 9), PN3 (n = 9), and PN5 (n = 7) groups. The chow group was given chow with an intravenous saline infusion. The PN groups were fed parenterally for 0.5, 1, 2, 3, or 5 days following the chow-feeding courses. After 7 days of nutrition support, hepatic MNCs were isolated and counted. The expression of LPS receptors on Kupffer cells was analyzed by flow cytometry. Hepatic MNC numbers rapidly reached their lowest level in the PN0.5 and PN1 groups but were somewhat restored thereafter and remained stable after the third day, without significant differences between any 2 of the PN groups. CD14 and Toll-like receptor 4/MD-2 expressions both showed significant reductions in the PN1 group compared with the chow group and gradually decreased to their lowest levels in the PN5 group. PN administration rapidly reduces hepatic MNC numbers and LPS receptor expression on Kupffer cells.

  18. Rapidly induced, T-cell independent xenoantibody production is mediated by marginal zone B cells and requires help from NK cells. (United States)

    Li, Shengqiao; Yan, Yehong; Lin, Yuan; Bullens, Dominique M; Rutgeerts, Omer; Goebels, Jozef; Segers, Constant; Boon, Louis; Kasran, Ahmad; De Vos, Rita; Dewolf-Peeters, Christiane; Waer, Mark; Billiau, An D


    Xenoantibody production directed at a wide variety of T lymphocyte-dependent and T lymphocyte-independent xenoantigens remains the major immunologic obstacle for successful xenotransplantation. The B lymphocyte subpopulations and their helper factors, involved in T-cell-independent xenoantibody production are only partially understood, and their identification will contribute to the clinical applicability of xenotransplantation. Here we show, using models involving T-cell-deficient athymic recipient mice, that rapidly induced, T-cell-independent xenoantibody production is mediated by marginal zone B lymphocytes and requires help from natural killer (NK) cells. This collaboration neither required NK-cell-mediated IFN-gamma production, nor NK-cell-mediated cytolytic killing of xenogeneic target cells. The T-cell-independent IgM xenoantibody response could be partially suppressed by CD40L blockade.

  19. Rapid Detection of Cell-Free Mycobacterium tuberculosis DNA in Tuberculous Pleural Effusion. (United States)

    Che, Nanying; Yang, Xinting; Liu, Zichen; Li, Kun; Chen, Xiaoyou


    Tuberculous pleurisy is one of the most common types of extrapulmonary tuberculosis, but its diagnosis remains difficult. In this study, we report for the first time on the detection of cell-free Mycobacterium tuberculosis DNA in pleural effusion and an evaluation of a newly developed molecular assay for the detection of cell-free Mycobacterium tuberculosis DNA. A total of 78 patients with pleural effusion, 60 patients with tuberculous pleurisy, and 18 patients with alternative diseases were included in this study. Mycobacterial culture, the Xpert MTB/RIF assay, the adenosine deaminase assay, the T-SPOT.TB assay, and the cell-free Mycobacterium tuberculosis DNA assay were performed on all the pleural effusion samples. The cell-free Mycobacterium tuberculosis DNA assay and adenosine deaminase assay showed significantly higher sensitivities of 75.0% and 68.3%, respectively, than mycobacterial culture and the Xpert MTB/RIF assay, which had sensitivities of 26.7% and 20.0%, respectively (P Mycobacterium tuberculosis DNA assay detected as few as 1.25 copies of IS6110 per ml of pleural effusion and showed good accordance of the results between repeated tests (r = 0.978, P = 2.84 × 10-10). These data suggest that the cell-free Mycobacterium tuberculosis DNA assay is a rapid and accurate molecular test which provides direct evidence of Mycobacterium tuberculosis etiology. Copyright © 2017 American Society for Microbiology.

  20. A bioluminescent caspase-1 activity assay rapidly monitors inflammasome activation in cells. (United States)

    O'Brien, Martha; Moehring, Danielle; Muñoz-Planillo, Raúl; Núñez, Gabriel; Callaway, Justin; Ting, Jenny; Scurria, Mike; Ugo, Tim; Bernad, Laurent; Cali, James; Lazar, Dan


    Inflammasomes are protein complexes induced by diverse inflammatory stimuli that activate caspase-1, resulting in the processing and release of cytokines, IL-1β and IL-18, and pyroptosis, an immunogenic form of cell death. To provide a homogeneous method for detecting caspase-1 activity, we developed a bioluminescent, plate-based assay that combines a substrate, Z-WEHD-aminoluciferin, with a thermostable luciferase in an optimized lytic reagent added directly to cultured cells. Assay specificity for caspase-1 is conferred by inclusion of a proteasome inhibitor in the lytic reagent and by use of a caspase-1 inhibitor to confirm activity. This approach enables a specific and rapid determination of caspase-1 activation. Caspase-1 activity is stable in the reagent thereby providing assay convenience and flexibility. Using this assay system, caspase-1 activation has been determined in THP-1 cells following treatment with α-hemolysin, LPS, nigericin, gramicidin, MSU, R848, Pam3CSK4, and flagellin. Caspase-1 activation has also been demonstrated in treated J774A.1 mouse macrophages, bone marrow-derived macrophages (BMDMs) from mice, as well as in human primary monocytes. Caspase-1 activity was not detected in treated BMDMs derived from Casp1-/- mice, further confirming the specificity of the assay. Caspase-1 activity can be measured directly in cultured cells using the lytic reagent, or caspase-1 activity released into medium can be monitored by assay of transferred supernatant. The caspase-1 assay can be multiplexed with other assays to monitor additional parameters from the same cells, such as IL-1β release or cell death. The caspase-1 assay in combination with a sensitive real-time monitor of cell death allows one to accurately establish pyroptosis. This assay system provides a rapid, convenient, and flexible method to specifically and quantitatively monitor caspase-1 activation in cells in a plate-based format. This will allow a more efficient and effective

  1. Rapid Sequential in Situ Multiplexing with DNA Exchange Imaging in Neuronal Cells and Tissues. (United States)

    Wang, Yu; Woehrstein, Johannes B; Donoghue, Noah; Dai, Mingjie; Avendaño, Maier S; Schackmann, Ron C J; Zoeller, Jason J; Wang, Shan Shan H; Tillberg, Paul W; Park, Demian; Lapan, Sylvain W; Boyden, Edward S; Brugge, Joan S; Kaeser, Pascal S; Church, George M; Agasti, Sarit S; Jungmann, Ralf; Yin, Peng


    To decipher the molecular mechanisms of biological function, it is critical to map the molecular composition of individual cells or even more importantly tissue samples in the context of their biological environment in situ. Immunofluorescence (IF) provides specific labeling for molecular profiling. However, conventional IF methods have finite multiplexing capabilities due to spectral overlap of the fluorophores. Various sequential imaging methods have been developed to circumvent this spectral limit but are not widely adopted due to the common limitation of requiring multirounds of slow (typically over 2 h at room temperature to overnight at 4 °C in practice) immunostaining. We present here a practical and robust method, which we call DNA Exchange Imaging (DEI), for rapid in situ spectrally unlimited multiplexing. This technique overcomes speed restrictions by allowing for single-round immunostaining with DNA-barcoded antibodies, followed by rapid (less than 10 min) buffer exchange of fluorophore-bearing DNA imager strands. The programmability of DEI allows us to apply it to diverse microscopy platforms (with Exchange Confocal, Exchange-SIM, Exchange-STED, and Exchange-PAINT demonstrated here) at multiple desired resolution scales (from ∼300 nm down to sub-20 nm). We optimized and validated the use of DEI in complex biological samples, including primary neuron cultures and tissue sections. These results collectively suggest DNA exchange as a versatile, practical platform for rapid, highly multiplexed in situ imaging, potentially enabling new applications ranging from basic science, to drug discovery, and to clinical pathology.

  2. Use of bacteriophage cell wall-binding proteins for rapid diagnostics of Listeria. (United States)

    Schmelcher, Mathias; Loessner, Martin J


    Diagnostic protocols for food-borne bacterial pathogens such as Listeria need to be sensitive, specific, rapid, and inexpensive. Conventional culture methods are hampered by lengthy enrichment and incubation steps. Bacteriophage-derived high-affinity binding molecules (cell wall-binding domains, CBDs) specific for Listeria cells have recently been introduced as tools for detection and differentiation of this pathogen in foods. When coupled with magnetic separation, these proteins offer advantages in sensitivity and speed compared to the standard diagnostic methods. Furthermore, fusion of CBDs to differently colored fluorescent reporter proteins enables differentiation of Listeria strains in mixed cultures. This chapter provides protocols for detection of Listeria in food by CBD-based magnetic separation and subsequent multiplexed identification of strains of different serotypes with reporter-CBD fusion proteins.

  3. Transferrin-mediated rapid targeting, isolation, and detection of circulating tumor cells by multifunctional magneto-dendritic nanosystem. (United States)

    Banerjee, Shashwat S; Jalota-Badhwar, Archana; Satavalekar, Sneha D; Bhansali, Sujit G; Aher, Naval D; Mascarenhas, Russel R; Paul, Debjani; Sharma, Somesh; Khandare, Jayant J


    A multicomponent magneto-dendritic nanosystem (MDNS) is designed for rapid tumor cell targeting, isolation, and high-resolution imaging by a facile bioconjugation approach. The highly efficient and rapid-acting MDNS provides a convenient platform for simultaneous isolation and high-resolution imaging of tumor cells, potentially leading towards an early diagnosis of cancer. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Rapid Cell-Based Assay for Detection and Quantification of Active Staphylococcal Enterotoxin Type D. (United States)

    Rasooly, Reuven; Do, Paula M; Hernlem, Bradley J


    Food poisoning by Staphylococcus aureus is a result of ingestion of Staphylococcal enterotoxins (SEs) produced by this bacterium and is a major source of foodborne illness. Staphylococcal enterotoxin D (SED) is one of the predominant enterotoxins recovered in Staphylococcal food poisoning incidences, including a recent outbreak in Guam affecting 300 children. Current immunology methods for SED detection cannot distinguish between the biologically active form of the toxin, which poses a threat, from the inactive form, which poses no threat. In vivo bioassays that measure emetic activity in kitten and monkeys have been used, but these methods rely upon expensive procedures using live animals and raising ethical concerns. A rapid (5 h) quantitative bioluminescence assay, using a genetically engineered T-cell Jurkat cell line expressing luciferase under regulation of nuclear factor of activated T cells response elements, in combination with the lymphoblastoid B-cell line Raji for antigen presentation, was developed. In this assay, the detection limit of biologically active SED is 100 ng/mL, which is 10 times more sensitive than the splenocyte proliferation assay, and 105 times more sensitive than monkey or kitten bioassay. Pasteurization or repeated freeze-thaw cycles had no effect on SED activity, but reduction in SED activity was shown with heat treatment at 100°C for 5 min. It was also shown that milk exhibits a protective effect on SED. This bioluminescence assay may also be used to rapidly evaluate antibodies to SED for potential therapeutic application as a measurement of neutralizing biological effects of SED. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  5. A geometric atlas to predict lung tumor shrinkage for radiotherapy treatment planning (United States)

    Zhang, Pengpeng; Rimner, Andreas; Yorke, Ellen; Hu, Yu-Chi; Kuo, Licheng; Apte, Aditya; Lockney, Natalie; Jackson, Andrew; Mageras, Gig; Deasy, Joseph O.


    To develop a geometric atlas that can predict tumor shrinkage and guide treatment planning for non-small-cell lung cancer. To evaluate the impact of the shrinkage atlas on the ability of tumor dose escalation. The creation of a geometric atlas included twelve patients with lung cancer who underwent both planning CT and weekly CBCT for radiotherapy planning and delivery. The shrinkage pattern from the original pretreatment to the residual posttreatment tumor was modeled using a principal component analysis, and used for predicting the spatial distribution of the residual tumor. A predictive map was generated by unifying predictions from each individual patient in the atlas, followed by correction for the tumor’s surrounding tissue distribution. Sensitivity, specificity, and accuracy of the predictive model for classifying voxels inside the original gross tumor volume were evaluated. In addition, a retrospective study of predictive treatment planning (PTP) escalated dose to the predicted residual tumor while maintaining the same level of predicted complication rates for a clinical plan delivering uniform dose to the entire tumor. The effect of uncertainty on the predictive model’s ability to escalate dose was also evaluated. The sensitivity, specificity and accuracy of the predictive model were 0.73, 0.76, and 0.74, respectively. The area under the receiver operating characteristic curve for voxel classification was 0.87. The Dice coefficient and mean surface distance between the predicted and actual residual tumor averaged 0.75, and 1.6 mm, respectively. The PTP approach allowed elevation of PTV D95 and mean dose to the actual residual tumor by 6.5 Gy and 10.4 Gy, respectively, relative to the clinical uniform dose approach. A geometric atlas can provide useful information on the distribution of resistant tumors and effectively guide dose escalation to the tumor without compromising the organs at risk complications. The atlas can be further refined by using

  6. A simple and rapid flow cytometric method for detection of porcine cell surface markers. (United States)

    Stabel, T J; Bolin, S R; Pesch, B A; Rahner, T E


    The objective of this study was to develop a rapid and reliable method for flow cytometric analysis of porcine whole blood cells. Fifty-microliters of heparin- or EDTA-treated whole blood was added to wells of a round-bottom 96-well microtitration plate. Each well contained 10 microl of an appropriate dilution of four different antibodies (40 microl total; two primary monoclonal antibodies and two fluorescent-labeled secondary antibodies). For convenience, the antibody mixture could be added to plates 1-2 days prior to assay and stored at 4 degrees C. Once whole blood was added to wells, plates were mixed gently, placed in a sealed bag and incubated in the dark at room temperature for 20 min. Contents of wells were then transferred to polystyrene tubes containing 2 ml of 1.5% formalin in distilled water and mixed gently. Cells were fixed for a minimum of 30 min and then stored in the dark at 4 degrees C until analysis by flow cytometry. Analysis of cell samples may be done up to 3 days after fixation. Results indicate that the percentages of Class I, Class II, CD3, CD8, CD4, CD45, monocyte, gamma-delta T-cell populations, and total number of granulocytes identified using this method were comparable to standard values or to values obtained following separation of white blood cells from red blood cells. The percentage of labeled B-cells was lower than standard values. Total assay time from receipt of blood to acquisition of data by flow cytometry required less than 2 h. This modified assay was shown to be simple, reliable, and useful for screening large numbers of porcine samples in a minimal period of time.

  7. Acute Viral Respiratory Infection Rapidly Induces a CD8+ T Cell Exhaustion-like Phenotype. (United States)

    Erickson, John J; Lu, Pengcheng; Wen, Sherry; Hastings, Andrew K; Gilchuk, Pavlo; Joyce, Sebastian; Shyr, Yu; Williams, John V


    Acute viral infections typically generate functional effector CD8(+) T cells (TCD8) that aid in pathogen clearance. However, during acute viral lower respiratory infection, lung TCD8 are functionally impaired and do not optimally control viral replication. T cells also become unresponsive to Ag during chronic infections and cancer via signaling by inhibitory receptors such as programmed cell death-1 (PD-1). PD-1 also contributes to TCD8 impairment during viral lower respiratory infection, but how it regulates TCD8 impairment and the connection between this state and T cell exhaustion during chronic infections are unknown. In this study, we show that PD-1 operates in a cell-intrinsic manner to impair lung TCD8. In light of this, we compared global gene expression profiles of impaired epitope-specific lung TCD8 to functional spleen TCD8 in the same human metapneumovirus-infected mice. These two populations differentially regulate hundreds of genes, including the upregulation of numerous inhibitory receptors by lung TCD8. We then compared the gene expression of TCD8 during human metapneumovirus infection to those in acute or chronic lymphocytic choriomeningitis virus infection. We find that the immunophenotype of lung TCD8 more closely resembles T cell exhaustion late into chronic infection than do functional effector T cells arising early in acute infection. Finally, we demonstrate that trafficking to the infected lung alone is insufficient for TCD8 impairment or inhibitory receptor upregulation, but that viral Ag-induced TCR signaling is also required. Our results indicate that viral Ag in infected lungs rapidly induces an exhaustion-like state in lung TCD8 characterized by progressive functional impairment and upregulation of numerous inhibitory receptors. Copyright © 2015 by The American Association of Immunologists, Inc.

  8. A novel rapid and reproducible flow cytometric method for optimization of transfection efficiency in cells (United States)

    Homann, Stefanie; Hofmann, Christian; Gorin, Aleksandr M.; Nguyen, Huy Cong Xuan; Huynh, Diana; Hamid, Phillip; Maithel, Neil; Yacoubian, Vahe; Mu, Wenli; Kossyvakis, Athanasios; Sen Roy, Shubhendu; Yang, Otto Orlean


    Transfection is one of the most frequently used techniques in molecular biology that is also applicable for gene therapy studies in humans. One of the biggest challenges to investigate the protein function and interaction in gene therapy studies is to have reliable monospecific detection reagents, particularly antibodies, for all human gene products. Thus, a reliable method that can optimize transfection efficiency based on not only expression of the target protein of interest but also the uptake of the nucleic acid plasmid, can be an important tool in molecular biology. Here, we present a simple, rapid and robust flow cytometric method that can be used as a tool to optimize transfection efficiency at the single cell level while overcoming limitations of prior established methods that quantify transfection efficiency. By using optimized ratios of transfection reagent and a nucleic acid (DNA or RNA) vector directly labeled with a fluorochrome, this method can be used as a tool to simultaneously quantify cellular toxicity of different transfection reagents, the amount of nucleic acid plasmid that cells have taken up during transfection as well as the amount of the encoded expressed protein. Finally, we demonstrate that this method is reproducible, can be standardized and can reliably and rapidly quantify transfection efficiency, reducing assay costs and increasing throughput while increasing data robustness. PMID:28863132

  9. Peripheral blood mononuclear cell gene expression in healthy adults rapidly transported to high altitude

    Directory of Open Access Journals (Sweden)

    Herman NM


    Full Text Available Nicole M Herman,1 Diane E Grill,2 Paul J Anderson,1 Andrew D Miller,1 Jacob B Johnson,1 Kathy A O’Malley,1 Maile L Ceridon Richert,1 Bruce D Johnson1 1Department of Cardiovascular Diseases, 2Department of Biostatistics, Mayo Clinic Rochester, MN, USA Abstract: Although mechanisms of high altitude illness have been studied extensively, the processes behind the development of these conditions are still unclear. Few genome-wide studies on rapid exposure to high altitude have been performed. Each year, scientists and support workers are transferred by plane from McMurdo Station in Antarctica (sea level to the Amundsen-Scott South Pole Station at 2,835 meters. This uniform and rapid transfer to altitude provides a unique opportunity to study the effects of hypobaric hypoxia on gene expression that may help illustrate the body's adaptations to these conditions. We hypothesized that an extensive number of genes would change with rapid exposure to altitude and further expected that these genes would correspond to inflammatory pathways proposed as a mechanism in development of acute mountain sickness. Peripheral venous blood samples were drawn from 98 healthy subjects at sea level and again on day two at altitude. Microarray analysis was performed on these samples. In total, 1,118 probe sets with significant P-values and fold changes (90% upregulated were identified and entered into MetaCore™ software. Several pathways, including oxidative phosphorylation, cytoskeleton remodeling, and platelet aggregation, were significantly represented by the data set and all were upregulated. Many genes changed expression, and the vast majority of these increased. Increased metabolism in peripheral blood mononuclear cells suggests increased inflammatory activity. Keywords: peripheral blood mononuclear cells, microarray, gene expression, acute mountain sickness

  10. New System of Shrinkage Measurement through Cement Mortars Drying. (United States)

    Morón, Carlos; Saiz, Pablo; Ferrández, Daniel; García-Fuentevilla, Luisa


    Cement mortar is used as a conglomerate in the majority of construction work. There are multiple variants of cement according to the type of aggregate used in its fabrication. One of the major problems that occurs while working with this type of material is the excessive loss of moisture during cement hydration (setting and hardening), known as shrinkage, which provokes a great number of construction pathologies that are difficult to repair. In this way, the design of a new sensor able to measure the moisture loss of mortars at different age levels is useful to establish long-term predictions concerning mortar mass volume loss. The purpose of this research is the design and fabrication of a new capacitive sensor able to measure the moisture of mortars and to relate it with the shrinkage.

  11. Analysis of Shrinkage on Thick Plate Part using Genetic Algorithm

    Directory of Open Access Journals (Sweden)

    Najihah S.N.


    Full Text Available Injection moulding is the most widely used processes in manufacturing plastic products. Since the quality of injection improves plastic parts are mostly influenced by process conditions, the method to determine the optimum process conditions becomes the key to improving the part quality. This paper presents a systematic methodology to analyse the shrinkage of the thick plate part during the injection moulding process. Genetic Algorithm (GA method was proposed to optimise the process parameters that would result in optimal solutions of optimisation goals. Using the GA, the shrinkage of the thick plate part was improved by 39.1% in parallel direction and 17.21% in the normal direction of melt flow.

  12. The Process of Shrinkage as a Challenge to Urban Governance

    Directory of Open Access Journals (Sweden)

    Stryjakiewicz Tadeusz


    Full Text Available For many decades most researchers, planners and local authorities have been focusing almost exclusively on urban growth and its socio-economic and spatial consequences. However, in the current debate concerning the future of cities and regions in Europe the process of their shrinkage starts to attract more attention. In the conditions of a declining population, urban governance is an important challenge for local authorities, being usually much more difficult than during the periods of population growth.

  13. Spinal shrinkage in unloaded and loaded drop-jumping. (United States)

    Fowler, N E; Lees, A; Reilly, T


    Plyometric activities, engaging the muscle in a stretch-shortening cycle, are widely used in athletic training. One such plyometric exercise is drop-jumping, where the athlete drops from a raised platform and immediately on landing performs a maximal vertical jump. These actions are also performed with the athlete externally loaded by the addition of weights to provide greater resistance. Exercises which involve repeated impacts have been shown to give rise to a loss of stature (shrinkage) which can be measured by means of a sensitive stadiometer. This study examined the shrinkage induced by unloaded and loaded drop-jumping from a height of 26 cm. Eight male subjects, aged 20-24 years, performed the test protocol three times, at the same time of day on each occasion. Fifty drop-jumps from a height of 26 cm were performed with no additional load and with a load of 8.5 kg carried in a weighted vest. The third condition was a standing trial where the subject stood for 10 min (the time taken to perform the jumps) wearing the weighted vest. Stature was measured before exercise, immediately after exercise and after a 20 min standing recovery. On a separate occasion the regimen was performed and the vertical reaction force was measured using a Kistler force platform. The mean change in stature for the two jump conditions showed shrinkages of 0.62 (+/- 0.43) mm for unloaded and 2.14 (+/- 1.56) mm for the loaded (p < 0.05). The variance in shrinkage was greater in the loaded case compared to the unloaded condition (p < 0.05) indicating a wider range of jumping strategies.(ABSTRACT TRUNCATED AT 250 WORDS)


    Directory of Open Access Journals (Sweden)

    Agus Dwi Anggono


    Full Text Available Plastic injection merupakan proses manufactur untuk membuat produk dengan bahan dasar plastic atau dalam kesempatan ini polypropylene. Pada proses tersebut seringkali terjadi cacat produk seperti pengerutan, retak, dimensi tidak sesuai dan kerusakan saat produk keluar dari mould, sehingga banyak material yang terbuang percuma. Meskipun cacat produk tersebut dipengaruhi banyak factor, tetapi yang paling utama adalah masalah shrinkage, atau penyusutan material setelah terjadi pendinginan. Sangat penting untuk melakukan prediksi lebih awal terjadinya penyusutan setelah pendinginan untuk menghindari cacat produk. Dalam penelitian ini akan dilakukan prediksi shrinkage yang akan digunakan untuk material polypropylene dengan cara perhitungan standar. Pembuatan modeling dalam bentuk 3D (tiga dimensi injection molding baik cavity maupun corenya dengan menggunakan CATIA, kemudian dilakukan analisis dengan software MoldFlow untuk pembuatan mesh dan memberikan batasan panas pada komponen sehingga dapat diketahui mode penyusutannya. Analisis ini akan memberikan gambaran tentang distribusi panas pada mould dan memberikan tentang gambaran aliran fluida. Pada analisis tersebut dapat dilihat gejala terjadinya cacat produk, jika hal itu terjadi maka perlu dilakukan perubahan shrinkage, sampai diperoleh hasil analisis yang baik.

  15. Experimental Analysis on Shrinkage and Swelling in Ordinary Concrete

    Directory of Open Access Journals (Sweden)

    Barbara Kucharczyková


    Full Text Available The paper deals with the experimental determination of shrinkage development during concrete ageing. Three concrete mixtures were made. They differed in the amount of cement in the fresh mixture, 300, 350, and 400 kg/m3. In order to determine the influence of plasticiser on the progress of volume changes, another three concrete mixtures were prepared with plasticiser in the amount of 0.25% by cement mass. Measurements were performed with the goal of observing the influence of cement and plasticiser content on the overall development of volume changes in the concrete. Changes in length and mass losses of the concrete during ageing were measured simultaneously. The continuous measurement of concrete mass losses caused by drying of the specimen’s surface proved useful during the interpretation of results obtained from the concrete shrinkage measurement. During the first 24 hours of ageing, all the concrete mixtures exhibited swelling. Its magnitude and progress were influenced by cement, water, and plasticiser content. However, a loss of mass caused by water evaporation from the surface of the specimens was also recorded in this stage. The measured progress of shrinkage corresponded well to the progress of mass loss.

  16. Prediction of Prestressing Losses by Concrete Creep and Shrinkage

    Energy Technology Data Exchange (ETDEWEB)

    Woo, S.K. [Korea Electric Power Research Institute, Taejon (Korea)


    In this study, the personal-computer program was developed to predict prestressing losses of containment structures induced by concrete creep and shrinkage in nuclear power plants. Thie program is composed of three major parts in which are the pre-processor, calculation module and post-processor. Input data for this program are: material properties of concrete, rebar, liner and duct, test results of concrete creep and shrinkage, relative humidity, dimension of containment structures, and the number of prestressing tendon related in containment structures. To obtain better results, this program was designed to reflect the prestressing losses due to influence that occurred after prestressing of each tendon, thus it can predict prestressing losses and allowable prestressing forces of each tendon. As a case study, this program was applied to containment structures of Youngkwang 3 and 4 NPP's and analytical results were compared with test results in Inservice Inspection of containment structures. From this comparison, it was proved that this program could well predict prestressing losses by concrete creep and shrinkage. (author). 4 refs., 1 fig., 3 tabs.

  17. Controlled Shrinkage of Expanded Glass Particles in Metal Syntactic Foams. (United States)

    Al-Sahlani, Kadhim; Taherishargh, Mehdi; Kisi, Erich; Fiedler, Thomas


    Metal matrix syntactic foams have been fabricated via counter-gravity infiltration of a packed bed of recycled expanded glass particles (EG) with A356 aluminum alloy. Particle shrinkage was studied and has been utilized to increase the particles' strength and tailor the mechanical properties of the expanded glass/metal syntactic foam (EG-MSF). The crushing strength of particles could be doubled by shrinking them for 20 min at 700 °C. Owing to the low density of EG (0.20-0.26 g/cm³), the resulting foam exhibits a low density (1.03-1.19 g/cm³) that increases slightly due to particle shrinkage. Chemical and physical analyses of EG particles and the resulting foams were conducted. Furthermore, metal syntactic foam samples were tested in uni-axial compression tests. The stress-strain curves obtained exhibit three distinct regions: elastic deformation followed by a stress plateau and densification commencing at 70-80% macroscopic strain. Particle shrinkage increased the mechanical strength of the foam samples and their average plateau stress increased from 15.5 MPa to 26.7 MPa.

  18. Shrinkage of vitreous body caused by hydroxyl radical

    Energy Technology Data Exchange (ETDEWEB)

    Park, Myoung Joo; Shimada, Takashi; Matuo, Yoichirou; Akiyama, Yoko; Izumi, Yoshinobu; Nishijima, Shigehiro [Osaka University, Osaka (Japan)


    In the study, we examined the effect of hydroxyl radical generated by {gamma}-ray and UV irradiation on shrinkage of vitreous body. Change in gel ratio of vitreous body and change in the properties of its components (collagen, sodium hyaluronate) were analyzed. By comparing these results, the amount of hydroxyl radical, which induces the considerable shrinkage of vitreous body, was evaluated from theoretical calculation based on experimental condition and some reported kinetic parameters. It was concluded that the integrated amount of hydroxyl radical required to liquefy half of the vitreous body (vitreous body gel ratio = 50%) was estimated as 140 {mu}molg{sup -1} from {gamma}-ray irradiation experiment. Also, from UV irradiation experiment result, it was confirmed that the effect of hydroxyl radical is larger than that of other reactive species. The causes of shrinkage of vitreous body are supposed as follows, 1) decrease in viscosity by cleavage of glycoside bond in sodium hyaluronate, 2) leaching of collagen from vitreous body and 3) leaching of crosslinked products and scission products of collagen.

  19. Innate lymphoid cells: models of plasticity for immune homeostasis and rapid responsiveness in protection. (United States)

    Almeida, F F; Belz, G T


    Innate lymphoid cells (ILCs) have stormed onto the immune landscape as "newly discovered" cell types. These tissue-resident sentinels are enriched at mucosal surfaces and engage in complex cross talk with elements of the adaptive immune system and microenvironment to orchestrate immune homeostasis. Many parallels exist between innate cells and T cells leading to the initial partitioning of ILCs into rather rigid subsets that reflect their "adaptive-like" effector cytokines profiles. ILCs themselves, however, have unique attributes that are only just beginning to be elucidated. These features result in complementarity with, rather than complete duplication of, functions of the adaptive immune system. Key transcription factors determine the pathway of differentiation of progenitors towards an ILC1, ILC2, or ILC3 subset. Once formed, flexibility in the responses of these subsets to stimuli unexpectedly allows transdifferentation between the different subsets and the acquisition of altered phenotypes and function. This provides a mechanism for rapid innate immune responsiveness. Here, we discuss the models of differentiation for maintenance and activation of tissue-resident ILCs in maintaining immune homeostasis and protection.

  20. A microfluidic chip for direct and rapid trapping of white blood cells from whole blood (United States)

    Chen, Jingdong; Chen, Di; Yuan, Tao; Xie, Yao; Chen, Xiang


    Blood analysis plays a major role in medical and science applications and white blood cells (WBCs) are an important target of analysis. We proposed an integrated microfluidic chip for direct and rapid trapping WBCs from whole blood. The microfluidic chip consists of two basic functional units: a winding channel to mix and arrays of two-layer trapping structures to trap WBCs. Red blood cells (RBCs) were eliminated through moving the winding channel and then WBCs were trapped by the arrays of trapping structures. We fabricated the PDMS (polydimethylsiloxane) chip using soft lithography and determined the critical flow velocities of tartrazine and brilliant blue water mixing and whole blood and red blood cell lysis buffer mixing in the winding channel. They are 0.25 μl/min and 0.05 μl/min, respectively. The critical flow velocity of the whole blood and red blood cell lysis buffer is lower due to larger volume of the RBCs and higher kinematic viscosity of the whole blood. The time taken for complete lysis of whole blood was about 85 s under the flow velocity 0.05 μl/min. The RBCs were lysed completely by mixing and the WBCs were trapped by the trapping structures. The chip trapped about 2.0 × 103 from 3.3 × 103 WBCs. PMID:24404026

  1. Influence of length-to-diameter ratio on shrinkage of basalt fiber concrete (United States)

    Ruijie, MA; Yang, Jiansen; Liu, Yuan; Zheng, Xiaojun


    In order to study the shrinkage performance of basalt concrete, using the shrinkage rate as index, the work not only studied the influence of different length-to-diameter ratio (LDR) on plastic shrinkage and drying shrinkage of basalt fiber concrete, but also analyzed the action mechanism. The results show that when the fiber content is 0.1%, the LDR of 800 and 1200 take better effects on reducing plastic shrinkage, however the fiber content is 0.3%, that of LDR 600 is better. To improve drying shrinkage, the fiber of LDR 800 takes best effect. In the concrete structure, the adding basalt fibers form a uniform and chaotic supporting system, optimize the pore and the void structure of concrete, make the material further compacted, reduce the water loss, so as to decrease the shrinkage of concrete effectively.

  2. Evaluation of shrinkage and cracking in concrete of ring test by acoustic emission method (United States)

    Watanabe, Takeshi; Hashimoto, Chikanori


    Drying shrinkage of concrete is one of the typical problems related to reduce durability and defilation of concrete structures. Lime stone, expansive additive and low-heat Portland cement are used to reduce drying shrinkage in Japan. Drying shrinkage is commonly evaluated by methods of measurement for length change of mortar and concrete. In these methods, there is detected strain due to drying shrinkage of free body, although visible cracking does not occur. In this study, the ring test was employed to detect strain and age cracking of concrete. The acoustic emission (AE) method was adopted to detect micro cracking due to shrinkage. It was recognized that in concrete using lime stone, expansive additive and low-heat Portland cement are effective to decrease drying shrinkage and visible cracking. Micro cracking due to shrinkage of this concrete was detected and evaluated by the AE method.

  3. Active and energy-dependent rapid formation of cell aggregates in the thermophilic photosynthetic bacterium Chloroflexus aggregans. (United States)

    Hanada, Satoshi; Shimada, Keizo; Matsuura, Katsumi


    The thermophilic filamentous phototroph Chloroflexus aggregans was able to form a bacterial mat-like dense cell aggregate rapidly. The aggregate formation, which was observed in growing cells in a liquid medium in a bottle, occurred every time within 20-30 min after the cells were dispersed by shaking. The aggregation depended on the energy supplied by photosynthesis or respiration. Cells aggregated most rapidly under temperature and pH conditions that support maximum growth. The aggregation was also accelerated by the addition of 3-isobutyl-1-methylxanthine that inhibits cyclic 3',5'-AMP phosphodiesterase. Microscopic observation revealed that the bacterium has a fast gliding mobility (1-3 microm s(-1)). The distinctive cell aggregation of C. aggregans was due to this rapid gliding movement.

  4. The Warburg effect as an adaptation of cancer cells to rapid fluctuations in energy demand.

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    Tamir Epstein

    Full Text Available To maintain optimal fitness, a cell must balance the risk of inadequate energy reserve for response to a potentially fatal perturbation against the long-term cost of maintaining high concentrations of ATP to meet occasional spikes in demand. Here we apply a game theoretic approach to address the dynamics of energy production and expenditure in eukaryotic cells. Conventionally, glucose metabolism is viewed as a function of oxygen concentrations in which the more efficient oxidation of glucose to CO2 and H2O produces all or nearly all ATP except under hypoxic conditions when less efficient (2 ATP/ glucose vs. about 36ATP/glucose anaerobic metabolism of glucose to lactic acid provides an emergency backup. We propose an alternative in which energy production is governed by the complex temporal and spatial dynamics of intracellular ATP demand. In the short term, a cell must provide energy for constant baseline needs but also maintain capacity to rapidly respond to fluxes in demand particularly due to external perturbations on the cell membrane. Similarly, longer-term dynamics require a trade-off between the cost of maintaining high metabolic capacity to meet uncommon spikes in demand versus the risk of unsuccessfully responding to threats or opportunities. Here we develop a model and computationally explore the cell's optimal mix of glycolytic and oxidative capacity. We find the Warburg effect, high glycolytic metabolism even under normoxic conditions, is represents a metabolic strategy that allow cancer cells to optimally meet energy demands posed by stochastic or fluctuating tumor environments.

  5. Selective Rapid Eye Movement Sleep Deprivation Affects Cell Size and Number in Kitten Locus Coeruleus

    Directory of Open Access Journals (Sweden)

    James P Shaffery


    Full Text Available Cells in the locus coeruleus (LC constitute the sole source of norepinephrine (NE in the brain, and change their discharge rates according to vigilance state. In addition to its well established role in vigilance, NE affects synaptic plasticity in the postnatal critical period (CP of development. One form of CP synaptic plasticity affected by NE results from monocular occlusion, which leads to physiological and cytoarchitectural alterations in central visual areas. Selective suppression of rapid eye movement sleep (REMS in the CP kitten enhances the central effects of monocular occlusion. The mechanisms responsible for heightened cortical plasticity following REMS deprivation (REMSD remain undetermined. One possible mediator of an increase in plasticity is continuous NE outflow, which presumably persists during extended periods of REMSD. Tyrosine hydroxylase (TH is the rate-limiting enzyme in the synthesis of NE and serves as a marker for NE-producing cells. We selectively suppressed REMS in kittens for one week during the CP. The number and size of LC cells expressing immunoreactivity to tyrosine hydroxylase (TH-ir was assessed in age-matched REMS-deprived (RD-, treatment-control (TXC-, and home cage-reared (HCC animals. Sleep amounts and slow wave activity (SWA were also examined relative to baseline. Time spent in REMS during the study was lower in RD compared to TXC animals, and RD kittens increased SWA delta power in the latter half of the REMSD period. The estimated total number of TH-ir cells in LC was significantly lower in the RD- than in the TXC kittens and numerically lower than in HCC animals. The size of LC cells expressing TH-ir was greatest in the HCC group. They were significantly larger than the cells in the RD kittens. These data are consistent with a possible reduction in NE in forebrain areas, including visual cortex, caused by one week of REMSD.

  6. Influence of Surface Abrasion on Creep and Shrinkage of Railway Prestressed Concrete Sleepers (United States)

    Li, Dan; Ngamkhanong, Chayut; Kaewunruen, Sakdirat


    Ballasted railway track is very suitable for heavy-rail networks because of its many superior advantages in design, construction, short- and long-term maintenance, sustainability, and life-cycle cost. The sleeper, which supports rail and distributes loads from rail to ballast, is a very important component of rail track system. Prestressed concrete is very popular used in manufacturing sleepers. Therefore, improved knowledge about design techniques for prestressed concrete (PC) sleepers has been developed. However, the ballast angularity causes differential abrasions on the soffit or bottom surface of sleepers. Furthermore, in sharp curves and rapid gradient change, longitudinal and lateral dynamics of rails increase the likelihood of abrasions in concrete sleepers. This paper presents a comparative investigation using a variety of methods to evaluate creep and shrinkage effects in railway prestressed concrete sleepers. The outcome of this study will improve the material design, which is very critical to the durability of railway track components.

  7. Rapid Electrokinetic Isolation of Cancer-Related Circulating Cell-Free DNA Directly from Blood (United States)

    Sonnenberg, Avery; Marciniak, Jennifer Y.; Rassenti, Laura; Ghia, Emanuela M.; Skowronski, Elaine A.; Manouchehri, Sareh; McCanna, James; Widhopf, George F.; Kipps, Thomas J.; Heller, Michael J.


    BACKGROUND Circulating cell-free DNA (ccf-DNA) is becoming an important biomarker for cancer diagnostics and therapy monitoring. The isolation of ccf-DNA from plasma as a “liquid biopsy” may begin to replace more invasive tissue biopsies for the detection and analysis of cancer-related mutations. Conventional methods for the isolation of ccf-DNA from plasma are costly, time-consuming, and complex, preventing the use of ccf-DNA biomarkers for point-of-care diagnostics and limiting other biomedical research applications. METHODS We used an AC electrokinetic device to rapidly isolate ccf-DNA from 25 μL unprocessed blood. ccf-DNA from 15 chronic lymphocytic leukemia (CLL) patients and 3 healthy individuals was separated into dielectrophoretic (DEP) high-field regions, after which other blood components were removed by a fluidic wash. Concentrated ccf-DNA was detected by fluorescence and eluted for quantification,PCR,and DNA sequencing. The complete process, blood to PCR, required <10 min. ccf-DNA was amplified by PCR with immunoglobulin heavy chain variable region (IGHV)-specific primers to identify the unique IGHV gene expressed by the leukemic B-cell clone, and then sequenced. RESULTS PCR and DNA sequencing results obtained by DEP from 25 μL CLL blood matched results obtained by use of conventional methods for ccf-DNA isolation from 1 mL plasma and for genomic DNA isolation from CLL patient leukemic B cells isolated from 15–20 mL blood. CONCLUSIONS Rapid isolation of ccf-DNA directly from a drop of blood will advance disease-related biomarker research, accelerate the transition from tissue to liquid biopsies, and enable point-of-care diagnostic systems for patient monitoring. PMID:24270796

  8. Rapid electrokinetic isolation of cancer-related circulating cell-free DNA directly from blood. (United States)

    Sonnenberg, Avery; Marciniak, Jennifer Y; Rassenti, Laura; Ghia, Emanuela M; Skowronski, Elaine A; Manouchehri, Sareh; McCanna, James; Widhopf, George F; Kipps, Thomas J; Heller, Michael J


    Circulating cell-free DNA (ccf-DNA) is becoming an important biomarker for cancer diagnostics and therapy monitoring. The isolation of ccf-DNA from plasma as a "liquid biopsy" may begin to replace more invasive tissue biopsies for the detection and analysis of cancer-related mutations. Conventional methods for the isolation of ccf-DNA from plasma are costly, time-consuming, and complex, preventing the use of ccf-DNA biomarkers for point-of-care diagnostics and limiting other biomedical research applications. We used an AC electrokinetic device to rapidly isolate ccf-DNA from 25 μL unprocessed blood. ccf-DNA from 15 chronic lymphocytic leukemia (CLL) patients and 3 healthy individuals was separated into dielectrophoretic (DEP) high-field regions, after which other blood components were removed by a fluidic wash. Concentrated ccf-DNA was detected by fluorescence and eluted for quantification, PCR, and DNA sequencing. The complete process, blood to PCR, required B-cell clone, and then sequenced. PCR and DNA sequencing results obtained by DEP from 25 μL CLL blood matched results obtained by use of conventional methods for ccf-DNA isolation from 1 mL plasma and for genomic DNA isolation from CLL patient leukemic B cells isolated from 15-20 mL blood. Rapid isolation of ccf-DNA directly from a drop of blood will advance disease-related biomarker research, accelerate the transition from tissue to liquid biopsies, and enable point-of-care diagnostic systems for patient monitoring.

  9. Vibrational spectroscopy--a powerful tool for the rapid identification of microbial cells at the single-cell level. (United States)

    Harz, M; Rösch, P; Popp, J


    Rapid microbial detection and identification with a high grade of sensitivity and selectivity is a great and challenging issue in many fields, primarily in clinical diagnosis, pharmaceutical, or food processing technology. The tedious and time-consuming processes of current microbiological approaches call for faster ideally on-line identification techniques. The vibrational spectroscopic techniques IR absorption and Raman spectroscopy are noninvasive methods yielding molecular fingerprint information; thus, allowing for a fast and reliable analysis of complex biological systems such as bacterial or yeast cells. In this short review, we discuss recent vibrational spectroscopic advances in microbial identification of yeast and bacterial cells for bulk environment and single-cell analysis. IR absorption spectroscopy enables a bulk analysis whereas micro-Raman-spectroscopy with excitation in the near infrared or visible range has the potential for the analysis of single bacterial and yeast cells. The inherently weak Raman signal can be increased up to several orders of magnitude by applying Raman signal enhancement methods such as UV-resonance Raman spectroscopy with excitation in the deep UV region, surface enhanced Raman scattering, or tip-enhanced Raman scattering. Copyright 2008 International Society for Advancement of Cytometry

  10. Rapid Treatment of Leukostasis in Leukemic Mantle Cell Lymphoma Using Therapeutic Leukapheresis: A Case Report

    Directory of Open Access Journals (Sweden)

    Xuan Duc Nguyen


    Full Text Available We describe a case of severe leukocytosis caused by leukemic mantle cell lymphoma (MCL, complicated by leukostasis with myocardial infarction in which leukapheresis was used in the initial management. A 73-year-old male presented to the emergency department because of fatigue and thoracic pain. Blood count revealed 630 × 109/L WBC (white blood cells. The electrocardiogram showed ST-elevation with an increase of troponin and creatinine kinase. The diagnosis was ST-elevation myocardial infarction (STEMI induced and complicated by leukostasis. Immunophenotyping, morphology, cytogenetic and fluorescence-in-situ-hybridization analysis revealed the diagnosis of a blastoid variant of MCL. To remove leukocytes rapidly, leukapheresis was performed in the intensive care unit. Based on the differential blood count with 95% blasts, which were assigned to the lymphocyte population by the automatic hematology analyzer, leukapheresis procedures were then performed with the mononuclear cell standard program on the Spectra cell separator. The patient was treated with daily leukapheresis for 3 days. The WBC count decreased to 174 × 109/L after the third leukapheresis, with a 72% reduction. After the second apheresis, treatment with vincristine, cyclophosphamide, and prednisolone was started. The patient fully recovered in the further course of the treatment. To the best of our knowledge, this is the first report on blastoid MCL with leukostasis associated with a STEMI that was successfully treated by leukapheresis. Effective harvest of circulating lymphoma cells by leukapheresis requires adaptation of instrument settings based on the results of the differential blood count prior to apheresis.

  11. The influence of superabsorbent polymers on the autogenous shrinkage properties of cement pastes with supplementary cementitious materials

    DEFF Research Database (Denmark)

    Snoeck, D.; Jensen, Ole Mejlhede; De Belie, N.


    shrinkage was determined by manual and automated shrinkage measurements. Autogenous shrinkage was reduced in cement pastes with the supplementary cementitious materials versus Portland cement pastes. At later ages, the rate of autogenous shrinkage is higher due to the pozzolanic activity. Internal curing...

  12. Semi-automated, occupationally safe immunofluorescence microtip sensor for rapid detection of Mycobacterium cells in sputum.

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    Shinnosuke Inoue

    Full Text Available An occupationally safe (biosafe sputum liquefaction protocol was developed for use with a semi-automated antibody-based microtip immunofluorescence sensor. The protocol effectively liquefied sputum and inactivated microorganisms including Mycobacterium tuberculosis, while preserving the antibody-binding activity of Mycobacterium cell surface antigens. Sputum was treated with a synergistic chemical-thermal protocol that included moderate concentrations of NaOH and detergent at 60°C for 5 to 10 min. Samples spiked with M. tuberculosis complex cells showed approximately 10(6-fold inactivation of the pathogen after treatment. Antibody binding was retained post-treatment, as determined by analysis with a microtip immunosensor. The sensor correctly distinguished between Mycobacterium species and other cell types naturally present in biosafe-treated sputum, with a detection limit of 100 CFU/mL for M. tuberculosis, in a 30-minute sample-to-result process. The microtip device was also semi-automated and shown to be compatible with low-cost, LED-powered fluorescence microscopy. The device and biosafe sputum liquefaction method opens the door to rapid detection of tuberculosis in settings with limited laboratory infrastructure.

  13. Gentamicin rapidly inhibits mitochondrial metabolism in high-frequency cochlear outer hair cells.

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    Heather C Jensen-Smith

    Full Text Available Aminoglycosides (AG, including gentamicin (GM, are the most frequently used antibiotics in the world and are proposed to cause irreversible cochlear damage and hearing loss (HL in 1/4 of the patients receiving these life-saving drugs. Akin to the results of AG ototoxicity studies, high-frequency, basal turn outer hair cells (OHCs preferentially succumb to multiple HL pathologies while inner hair cells (IHCs are much more resilient. To determine if endogenous differences in IHC and OHC mitochondrial metabolism dictate differential sensitivities to AG-induced HL, IHC- and OHC-specific changes in mitochondrial reduced nicotinamide adenine dinucleotide (NADH fluorescence during acute (1 h GM treatment were compared. GM-mediated decreases in NADH fluorescence and succinate dehydrogenase activity were observed shortly after GM application. High-frequency basal turn OHCs were found to be metabolically biased to rapidly respond to alterations in their microenvironment including GM and elevated glucose exposures. These metabolic biases may predispose high-frequency OHCs to preferentially produce cell-damaging reactive oxygen species during traumatic challenge. Noise-induced and age-related HL pathologies share key characteristics with AG ototoxicity, including preferential OHC loss and reactive oxygen species production. Data from this report highlight the need to address the role of mitochondrial metabolism in regulating AG ototoxicity and the need to illuminate how fundamental differences in IHC and OHC metabolism may dictate differences in HC fate during multiple HL pathologies.

  14. Quality controls in cellular immunotherapies: rapid assessment of clinical grade dendritic cells by gene expression profiling. (United States)

    Castiello, Luciano; Sabatino, Marianna; Zhao, Yingdong; Tumaini, Barbara; Ren, Jiaqiang; Ping, Jin; Wang, Ena; Wood, Lauren V; Marincola, Francesco M; Puri, Raj K; Stroncek, David F


    Cell-based immunotherapies are among the most promising approaches for developing effective and targeted immune response. However, their clinical usefulness and the evaluation of their efficacy rely heavily on complex quality control assessment. Therefore, rapid systematic methods are urgently needed for the in-depth characterization of relevant factors affecting newly developed cell product consistency and the identification of reliable markers for quality control. Using dendritic cells (DCs) as a model, we present a strategy to comprehensively characterize manufactured cellular products in order to define factors affecting their variability, quality and function. After generating clinical grade human monocyte-derived mature DCs (mDCs), we tested by gene expression profiling the degrees of product consistency related to the manufacturing process and variability due to intra- and interdonor factors, and how each factor affects single gene variation. Then, by calculating for each gene an index of variation we selected candidate markers for identity testing, and defined a set of genes that may be useful comparability and potency markers. Subsequently, we confirmed the observed gene index of variation in a larger clinical data set. In conclusion, using high-throughput technology we developed a method for the characterization of cellular therapies and the discovery of novel candidate quality assurance markers.

  15. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis.

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    Nitzan Krinsky

    Full Text Available Cell-free protein synthesis (CFPS systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3 and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa. This system was able to produce 40-150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins.

  16. An automated robotic platform for rapid profiling oligosaccharide analysis of monoclonal antibodies directly from cell culture. (United States)

    Doherty, Margaret; Bones, Jonathan; McLoughlin, Niaobh; Telford, Jayne E; Harmon, Bryan; DeFelippis, Michael R; Rudd, Pauline M


    Oligosaccharides attached to Asn297 in each of the CH2 domains of monoclonal antibodies play an important role in antibody effector functions by modulating the affinity of interaction with Fc receptors displayed on cells of the innate immune system. Rapid, detailed, and quantitative N-glycan analysis is required at all stages of bioprocess development to ensure the safety and efficacy of the therapeutic. The high sample numbers generated during quality by design (QbD) and process analytical technology (PAT) create a demand for high-performance, high-throughput analytical technologies for comprehensive oligosaccharide analysis. We have developed an automated 96-well plate-based sample preparation platform for high-throughput N-glycan analysis using a liquid handling robotic system. Complete process automation includes monoclonal antibody (mAb) purification directly from bioreactor media, glycan release, fluorescent labeling, purification, and subsequent ultra-performance liquid chromatography (UPLC) analysis. The entire sample preparation and commencement of analysis is achieved within a 5-h timeframe. The automated sample preparation platform can easily be interfaced with other downstream analytical technologies, including mass spectrometry (MS) and capillary electrophoresis (CE), for rapid characterization of oligosaccharides present on therapeutic antibodies. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. Miniaturized Antimicrobial Susceptibility Test by Combining Concentration Gradient Generation and Rapid Cell Culturing

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    Samuel C. Kim


    Full Text Available Effective treatment of bacterial infection relies on timely diagnosis and proper prescription of antibiotic drugs. The antimicrobial susceptibility test (AST is one of the most crucial experimental procedures, providing the baseline information for choosing effective antibiotic agents and their dosages. Conventional methods, however, require long incubation times or significant instrumentation costs to obtain test results. We propose a lab-on-a-chip approach to perform AST in a simple, economic, and rapid manner. Our assay platform miniaturizes the standard broth microdilution method on a microfluidic device (20 × 20 mm that generates an antibiotic concentration gradient and delivers antibiotic-containing culture media to eight 30-nL chambers for cell culture. When tested with 20 μL samples of a model bacterial strain (E. coli ATCC 25922 treated with ampicillin or streptomycin, our method allows for the determination of minimum inhibitory concentrations consistent with the microdilution test in three hours, which is almost a factor of ten more rapid than the standard method.

  18. Glyphosate resistance in Ambrosia trifida: Part 1. Novel rapid cell death response to glyphosate. (United States)

    Van Horn, Christopher R; Moretti, Marcelo L; Robertson, Renae R; Segobye, Kabelo; Weller, Stephen C; Young, Bryan G; Johnson, William G; Schulz, Burkhard; Green, Amanda C; Jeffery, Taylor; Lespérance, Mackenzie A; Tardif, François J; Sikkema, Peter H; Hall, J Christopher; McLean, Michael D; Lawton, Mark B; Sammons, R Douglas; Wang, Dafu; Westra, Philip; Gaines, Todd A


    Glyphosate-resistant (GR) Ambrosia trifida is now present in the midwestern United States and in southwestern Ontario, Canada. Two distinct GR phenotypes are known, including a rapid response (GR RR) phenotype, which exhibits cell death within hours after treatment, and a non-rapid response (GR NRR) phenotype. The mechanisms of resistance in both GR RR and GR NRR remain unknown. Here, we present a description of the RR phenotype and an investigation of target-site mechanisms on multiple A. trifida accessions. Glyphosate resistance was confirmed in several accessions, and whole-plant levels of resistance ranged from 2.3- to 7.5-fold compared with glyphosate-susceptible (GS) accessions. The two GR phenotypes displayed similar levels of resistance, despite having dramatically different phenotypic responses to glyphosate. Glyphosate resistance was not associated with mutations in EPSPS sequence, increased EPSPS copy number, EPSPS quantity, or EPSPS activity. These encompassing results suggest that resistance to glyphosate in these GR RR A. trifida accessions is not conferred by a target-site resistance mechanism. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  19. Chitosan as coagulant on cyanobacteria in lake restoration management may cause rapid cell lysis. (United States)

    Mucci, Maíra; Noyma, Natalia Pessoa; de Magalhães, Leonardo; Miranda, Marcela; van Oosterhout, Frank; Guedes, Iamê Alves; Huszar, Vera L M; Marinho, Marcelo Manzi; Lürling, Miquel


    Combining coagulant and ballast to remove cyanobacteria from the water column is a promising restoration technique to mitigate cyanobacterial nuisance in surface waters. The organic, biodegradable polymer chitosan has been promoted as a coagulant and is viewed as non-toxic. In this study, we show that chitosan may rapidly compromise membrane integrity and kill certain cyanobacteria leading to release of cell contents in the water. A strain of Cylindrospermopsis raciborskii and one strain of Planktothrix agardhii were most sensitive. A 1.3 h exposure to a low dose of 0.5 mg l-1 chitosan already almost completely killed these cultures resulting in release of cell contents. After 24 h, reductions in PSII efficiencies of all cyanobacteria tested were observed. EC50 values varied from around 0.5 mg l-1 chitosan for the two sensitive strains, via about 5 mg l-1 chitosan for an Aphanizomenon flos-aquae strain, a toxic P. agardhii strain and two Anabaena cylindrica cultures, to more than 8 mg l-1 chitosan for a Microcystis aeruginosa strain and another A. flos-aquae strain. Differences in sensitivity to chitosan might be related to polymeric substances that surround cyanobacteria. Rapid lysis of toxic strains is likely and when chitosan flocking and sinking of cyanobacteria is considered in lake restoration, flocculation efficacy studies should be complemented with investigation on the effects of chitosan on the cyanobacteria assemblage being targeted. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  20. Experimental Study on Autogenous and Drying Shrinkage of Steel Fiber Reinforced Lightweight-Aggregate Concrete

    Directory of Open Access Journals (Sweden)

    Shunbo Zhao


    Full Text Available Steel fiber reinforced lightweight-aggregate concrete (SFRLAC has many advantages applied in structural engineering. In this paper, the autogenous shrinkage and drying shrinkage of SFRLAC for up to 270 days were measured, considering the effects of types of coarse and fine aggregates with the changes of water-to-binder ratio and volume fraction of steel fiber, respectively. The properties of mix workability, apparent density, and compressive strength of SFRLAC were also reported and discussed in relation to above factors. Test results show that the development of autogenous and drying shrinkage of SFRLAC was fast within 28 days and tended to be steady after 90 days. The development of autogenous shrinkage of SFRLAC reduced with the increasing water-to-binder ratio and by using the expanded shale with higher soundness and good water absorption, especially at early age within 28 days; the later drying shrinkage was reduced and the development of drying shrinkage was slowed down with the increasing volume fraction of steel fiber obviously; manufactured sand led to less autogenous shrinkage but greater drying shrinkage than fine lightweight aggregate in SFRLAC. The regularities of autogenous shrinkage and drying shrinkage of SFRLAC expressed as the series of hyperbola are analyzed.

  1. Rapid activation of Rac GTPase in living cells by force is independent of Src.

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    Yeh-Chuin Poh


    Full Text Available It is well known that mechanical forces are crucial in regulating functions of every tissue and organ in a human body. However, it remains unclear how mechanical forces are transduced into biochemical activities and biological responses at the cellular and molecular level. Using the magnetic twisting cytometry technique, we applied local mechanical stresses to living human airway smooth muscle cells with a magnetic bead bound to the cell surface via transmembrane adhesion molecule integrins. The temporal and spatial activation of Rac, a small guanosine triphosphatase, was quantified using a fluorescent resonance energy transfer (FRET method that measures changes in Rac activity in response to mechanical stresses by quantifying intensity ratios of ECFP (enhanced cyan fluorescent protein as a donor and YPet (a variant yellow fluorescent protein as an acceptor of the Rac biosensor. The applied stress induced rapid activation (less than 300 ms of Rac at the cell periphery. In contrast, platelet derived growth factor (PDGF induced Rac activation at a much later time (>30 sec. There was no stress-induced Rac activation when a mutant form of the Rac biosensor (RacN17 was transfected or when the magnetic bead was coated with transferrin or with poly-L-lysine. It is known that PDGF-induced Rac activation depends on Src activity. Surprisingly, pre-treatment of the cells with specific Src inhibitor PP1 or knocking-out Src gene had no effects on stress-induced Rac activation. In addition, eliminating lipid rafts through extraction of cholesterol from the plasma membrane did not prevent stress-induced Rac activation, suggesting a raft-independent mechanism in governing the Rac activation upon mechanical stimulation. Further evidence indicates that Rac activation by stress depends on the magnitudes of the applied stress and cytoskeletal integrity. Our results suggest that Rac activation by mechanical forces is rapid, direct and does not depend on Src

  2. Rapid labeling of intracellular His-tagged proteins in living cells (United States)

    Lai, Yau-Tsz; Chang, Yuen-Yan; Hu, Ligang; Yang, Ya; Chao, Ailun; Du, Zhi-Yan; Tanner, Julian A.; Chye, Mee-Len; Qian, Chengmin; Ng, Kwan-Ming; Li, Hongyan; Sun, Hongzhe


    Small molecule-based fluorescent probes have been used for real-time visualization of live cells and tracking of various cellular events with minimal perturbation on the cells being investigated. Given the wide utility of the (histidine)6-Ni2+-nitrilotriacetate (Ni-NTA) system in protein purification, there is significant interest in fluorescent Ni2+-NTA–based probes. Unfortunately, previous Ni-NTA–based probes suffer from poor membrane permeability and cannot label intracellular proteins. Here, we report the design and synthesis of, to our knowledge, the first membrane-permeable fluorescent probe Ni-NTA-AC via conjugation of NTA with fluorophore and arylazide followed by coordination with Ni2+ ions. The probe, driven by Ni2+-NTA, binds specifically to His-tags genetically fused to proteins and subsequently forms a covalent bond upon photoactivation of the arylazide, leading to a 13-fold fluorescence enhancement. The arylazide is indispensable not only for fluorescence enhancement, but also for strengthening the binding between the probe and proteins. Significantly, the Ni-NTA-AC probe can rapidly enter different types of cells, even plant tissues, to target His-tagged proteins. Using this probe, we visualized the subcellular localization of a DNA repair protein, Xeroderma pigmentosum group A (XPA122), which is known to be mainly enriched in the nucleus. We also demonstrated that the probe can image a genetically engineered His-tagged protein in plant tissues. This study thus offers a new opportunity for in situ visualization of large libraries of His-tagged proteins in various prokaryotic and eukaryotic cells. PMID:25713372

  3. Rapid adhesion of nerve cells to muscle fibers from adult rats is mediated by a sialic acid-binding receptor



    Single viable muscle fibers isolated from adult rats by collagenase digestion rapidly bind dissociated spinal neurons or PC-12 cells but not a variety of other cells tested. The adhesion process is calcium- independent, temperature-sensitive, and is not blocked by pretreating cells with inhibitors of energy metabolism or actin polymerization. Adhesion is mediated by a carbohydrate-binding protein and can be inhibited by N-acetylneuraminic acid or mucin, a glycoprotein with high sialic acids c...

  4. Polymerization shrinkage of flowable resin-based restorative materials. (United States)

    Stavridakis, Minos M; Dietschi, Didier; Krejci, Ivo


    This study measured the linear polymerization displacement and polymerization forces induced by polymerization shrinkage of a series of flowable resin-based restorative materials. The materials tested were 22 flowable resin-based restorative materials (Admira Flow, Aelite Flow, Aeliteflow LV, Aria, Crystal Essence, Definite Flow, Dyract Flow, Filtek Flow, FloRestore, Flow-it, Flow-Line, Freedom, Glacier, OmegaFlo, PermaFlo, Photo SC, Revolution 2, Star Flow, Synergy Flow, Tetric Flow, Ultraseal XT and Wave). Measurements for linear polymerization displacement and polymerization forces were performed using custom made measuring devices. Polymerization of the test materials was carried out for 60 seconds by means of a light curing unit, and each property was measured for 180 seconds from the start of curing in eight specimens for each material. Statistical evaluation of the data was performed with one-way analysis of variance (ANOVA), Tukey's Studentized Range (HSD) test (p=0.05) and simple linear regression. A wide range of values was recorded for linear polymerization displacement (26.61 to 80.74 microns) and polymerization forces (3.23 to 7.48 kilograms). Statistically significant differences among materials were found for both properties studied. Very few materials (Freedom, Glacier, and Photo SC) presented low values of linear polymerization displacement and polymerization forces (similar to hybrid resin composites), while the majority of materials presented very high values in both properties studied. Study of the shrinkage kinetics revealed the exponential growth process of both properties. The polymerization forces development exhibited a few seconds delay over linear polymerization displacement. Simple linear regression showed that the two polymerization shrinkage properties that were studied were not highly correlated (r2=0.59).

  5. Postoperative sensitivity associated with low shrinkage versus conventional composites

    Directory of Open Access Journals (Sweden)

    Ivanović Vladimir


    Full Text Available Introduction. Postoperative sensitivity in restorative dentistry can be related to preparation trauma, dentin adhesives’ ability to seal open dentinal tubules, deformation of restorations under occlusal stresses and microleakage. Objective. The study assessed possible reduction in postoperative sensitivity with low shrinkage compared to conventional composites using different bonding agents and the influence of the operator skill on the incidence of postoperative sensitivity. Methods. Nine hundred and sixty permanent premolars and molars with primary carious lesions from patients 21 to 40 years old were used. Cavities 2 to 3 mm deep and with margins in enamel were prepared by four operators. Two operators had five years (A and B and two had over 20 years (C and D of clinical experience. Teeth were divided into eight groups each contained 120 restorations: (1 Els®+James-2 (original formula, (2 Els®+James-2 (new formula, (3 Els®+Excite, (4 InTenSe®+James-2 (original formula, (5 InTenSe®+James-2 (new formula, (6 InTenSe®+Excite, (7 Tetric Ceram®+Excite, and (8 Point 4®+OptiBond Solo Plus. At 14 days postoperatively, two independent operators, who did not take part in the clinical procedure, assessed postoperative teeth sensitivity using special questionnaires. Data were analyzed using non-parametric chi-square, Mann-Whitney and ANOVA tests. Results. Group 8 showed significantly higher score than the other groups. Less postoperative sensitivity was reported with two low-shrinkage composites (groups 2, 3, and 5 but with no significant difference. There was no statistical difference between groups 1, 2, 3, 4, 5, 6 and 7. Operator A had the highest postoperative sensitivity score compared to the other three. Conclusion. Conventional composite material Point 4® with its bonding agent caused significantly more postoperative sensitivity than low shrinkage composites combined with different adhesives. Operator skill influenced the incidence of

  6. A rapid selection strategy for an anodophilic consortium for microbial fuel cells

    KAUST Repository

    Wang, Aijie


    A rapid selection method was developed to enrich for a stable and efficient anodophilic consortium (AC) for microbial fuel cells (MFCs). A biofilm sample from a microbial electrolysis cell was serially diluted up to 10-9 in anaerobic phosphate buffer solution and incubated in an Fe(III)-acetate medium, and an Fe(III)-reducing AC was obtained for dilutions up to 10-6. The activity of MFC inoculated with the enrichment AC was compared with those inoculated with original biofilm or activated sludge. The power densities and Coulombic efficiencies of the AC (226 mW/m2, 34%) were higher than those of the original biofilm (209 mW/m2, 23%) and activated sludge (192 mW/m2, 19%). The start-up period of the AC (60 h) was also shorter than those obtained with the other inocula (biofilm, 95 h; activated sludge, 300 h). This indicated that such a strategy is highly efficient for obtaining an anodophilic consortium for improving the performance of an MFC. © 2010 Elsevier Ltd.

  7. Cell-free expression of protein kinase a for rapid activity assays. (United States)

    Leippe, Donna M; Zhao, Kate Qin; Hsiao, Kevin; Slater, Michael R


    Functional protein analysis often calls for lengthy, laborious in vivo protein expression and purification, and can be complicated by the lack of stability of the purified protein. In this study, we demonstrate the feasibility of a simplified procedure for functional protein analysis on magnetic particles using cell-free protein synthesis of the catalytic subunit of human cAMP-dependent protein kinase as a HaloTag((R)) fusion protein. The cell-free protein synthesis systems provide quick access to the protein of interest, while the HaloTag technology provides efficient, covalent protein immobilization of the fusion protein, eliminating the need for further protein purification and minimizing storage-related stability issues. The immobilized cPKA fusion protein is assayed directly on magnetic beads and can be used in inhibitor analyses. The combination of rapid protein synthesis and capture technologies can greatly facilitate the process of protein expression and activity screening, and therefore, can become a valuable tool for functional proteomics studies.

  8. Cell-Free Expression of Protein Kinase a for Rapid Activity Assays

    Directory of Open Access Journals (Sweden)

    Donna M. Leippe


    Full Text Available Functional protein analysis often calls for lengthy, laborious in vivo protein expression and purification, and can be complicated by the lack of stability of the purified protein. In this study, we demonstrate the feasibility of a simplified procedure for functional protein analysis on magnetic particles using cell-free protein synthesis of the catalytic subunit of human cAMP-dependent protein kinase as a HaloTag ® fusion protein. The cell-free protein synthesis systems provide quick access to the protein of interest, while the HaloTag technology provides efficient, covalent protein immobilization of the fusion protein, eliminating the need for further protein purification and minimizing storage-related stability issues. The immobilized cPKA fusion protein is assayed directly on magnetic beads and can be used in inhibitor analyses. The combination of rapid protein synthesis and capture technologies can greatly facilitate the process of protein expression and activity screening, and therefore, can become a valuable tool for functional proteomics studies.

  9. Cell-Free Expression of Protein Kinase A for Rapid Activity Assays

    Directory of Open Access Journals (Sweden)

    Donna M. Leippe


    Full Text Available Functional protein analysis often calls for lengthy, laborious in vivo protein expression and purification, and can be complicated by the lack of stability of the purified protein. In this study, we demonstrate the feasibility of a simplified procedure for functional protein analysis on magnetic particles using cell-free protein synthesis of the catalytic subunit of human cAMP-dependent protein kinase as a HaloTag® fusion protein. The cell-free protein synthesis systems provide quick access to the protein of interest, while the HaloTag technology provides efficient, covalent protein immobilization of the fusion protein, eliminating the need for further protein purification and minimizing storage-related stability issues. The immobilized cPKA fusion protein is assayed directly on magnetic beads and can be used in inhibitor analyses. The combination of rapid protein synthesis and capture technologies can greatly facilitate the process of protein expression and activity screening, and therefore, can become a valuable tool for functional proteomics studies.

  10. Rapid fabricating technique for multi-layered human hepatic cell sheets by forceful contraction of the fibroblast monolayer.

    Directory of Open Access Journals (Sweden)

    Yusuke Sakai

    Full Text Available Cell sheet engineering is attracting attention from investigators in various fields, from basic research scientists to clinicians focused on regenerative medicine. However, hepatocytes have a limited proliferation potential in vitro, and it generally takes a several days to form a sheet morphology and multi-layered sheets. We herein report our rapid and efficient technique for generating multi-layered human hepatic cell (HepaRG® cell sheets using pre-cultured fibroblast monolayers derived from human skin (TIG-118 cells as a feeder layer on a temperature-responsive culture dish. Multi-layered TIG-118/HepaRG cell sheets with a thick morphology were harvested on day 4 of culturing HepaRG cells by forceful contraction of the TIG-118 cells, and the resulting sheet could be easily handled. In addition, the human albumin and alpha 1-antitrypsin synthesis activities of TIG-118/HepaRG cells were approximately 1.2 and 1.3 times higher than those of HepaRG cells, respectively. Therefore, this technique is considered to be a promising modality for rapidly fabricating multi-layered human hepatocyte sheets from cells with limited proliferation potential, and the engineered cell sheet could be used for cell transplantation with highly specific functions.

  11. Sparse electromagnetic imaging using nonlinear iterative shrinkage thresholding

    KAUST Repository

    Desmal, Abdulla


    A sparse nonlinear electromagnetic imaging scheme is proposed for reconstructing dielectric contrast of investigation domains from measured fields. The proposed approach constructs the optimization problem by introducing the sparsity constraint to the data misfit between the scattered fields expressed as a nonlinear function of the contrast and the measured fields and solves it using the nonlinear iterative shrinkage thresholding algorithm. The thresholding is applied to the result of every nonlinear Landweber iteration to enforce the sparsity constraint. Numerical results demonstrate the accuracy and efficiency of the proposed method in reconstructing sparse dielectric profiles.

  12. Essential Function of Dicer in Resolving DNA Damage in the Rapidly Dividing Cells of the Developing and Malignant Cerebellum

    Directory of Open Access Journals (Sweden)

    Vijay Swahari


    Full Text Available Maintenance of genomic integrity is critical during neurodevelopment, particularly in rapidly dividing cerebellar granule neuronal precursors that experience constitutive replication-associated DNA damage. As Dicer was recently recognized to have an unexpected function in the DNA damage response, we examined whether Dicer was important for preserving genomic integrity in the developing brain. We report that deletion of Dicer in the developing mouse cerebellum resulted in the accumulation of DNA damage leading to cerebellar progenitor degeneration, which was rescued with p53 deficiency; deletion of DGCR8 also resulted in similar DNA damage and cerebellar degeneration. Dicer deficiency also resulted in DNA damage and death in other rapidly dividing cells including embryonic stem cells and the malignant cerebellar progenitors in a mouse model of medulloblastoma. Together, these results identify an essential function of Dicer in resolving the spontaneous DNA damage that occurs during the rapid proliferation of developmental progenitors and malignant cells.

  13. Experimental Study on Forecasting Mathematical Model of Drying Shrinkage of Recycled Aggregate Concrete

    Directory of Open Access Journals (Sweden)

    Yuanchen Guo


    Full Text Available On the basis of basic law in AASHTO2007 model, the forecasting mathematical model of drying shrinkage of recycled aggregate concrete (RAC is established by regression analysis and experimental study. The research results show that (1 with the replacement rate of RCA increases, the drying shrinkage value of RAC increases; this trend is even more obvious in the early drying time. (2 The addition of fly ash can inhibit the drying shrinkage of RAC, but the effect is not very obvious. Specifically, the addition of fly ash will increase the shrinkage to some extent when the mixing amount is 20%. (3 The addition of expansive agent can obviously inhibit the shrinkage of RAC; the inhibition affection is better than that of fly ash. (4 The forecasting mathematical models of drying shrinkage of RAC established in this paper have high accuracy and rationality according to experiment validation and error analysis.

  14. Can superabsorbent polymers mitigate shrinkage in cementitious materials blended with supplementary cementitious materials?

    DEFF Research Database (Denmark)

    Snoeck, Didier; Jensen, Ole Mejlhede; De Belie, Nele


    A promising way to mitigate autogenous shrinkage in cementitious materials with a low water-to-binder ratio is internal curing by the use of superabsorbent polymers. Superabsorbent polymers are able to absorb multiple times their weight in water and can be applied as an internal water reservoir...... shrinkage in materials blended with fly ash or blast-furnace slag remain scarce, especially after one week of age. This paper focuses on the autogenous shrinkage by performing manual and automated shrinkage measurements up to one month of age. Without superabsorbent polymers, autogenous shrinkage...... was reduced in cement pastes with the supplementary cementitious materials versus Portland cement pastes. At later ages, the rate of autogenous shrinkage is higher due to the pozzolanic activity of the supplementary cementitious materials. Internal curing by means of superabsorbent polymers is successful...

  15. Development of shrinkage resistant microfibre-reinforced cement-based composites (United States)

    Hamedanimojarrad, P.; Adam, G.; Ray, A.; Thomas, P.; Vessalas, K.


    Different shrinkage types may cause serious durability dilemma on restrained concrete parts due to crack formation and propagation. Several classes of fibres are used by concrete industry in order to reduce crack size and crack number. In previous studies, most of these fibre types were found to be effective in reducing the number and sizes of the cracks, but not in shrinkage strain reduction. This study deals with the influence of a newly introduced type of polyethylene fibre on drying shrinkage reduction. The novel fibre is a polyethylene microfibre in a new geometry, which is proved to reduce the amount of total shrinkage in mortars. This special hydrophobic polyethylene microfibre also reduces moisture loss of mortar samples. The experimental results on short and long-term drying shrinkage as well as on several other properties are reported. The hydrophobic polyethylene microfibre showed promising improvement in shrinkage reduction even at very low concentrations (0.1% of cement weight).

  16. Rapid auxin-induced stimulation of cell wall synthesis in pea internodes

    Energy Technology Data Exchange (ETDEWEB)

    Kutschera, U.; Briggs, W.R.


    The effect of auxin (indole-3-acetic acid; IAA) on growth and incorporation of myo-(2-/sup 3/H(N)) inositol ((/sup 3/H)Ins) into noncellulosic polysacchharides in the cell walls of third internode sections from red light-grown pea seedlings (Pisum sativum L. cv. Alaska) was investigated. Intact section were incubated on (/sup 3/H)Ins for 4 hr to permit uptake of the tracer and then IAA was added. Growth started after a lag phase of 15 min under these conditions. The sections were removed from the tracer and separated into epidermis and cortical cylinder (cortex plus vascular tissue). In the epidermis, IAA-induced stimulation of (/sup 3/H)Ins incorporation started after a lag of 15 min. The amount of incorporation was 15% higher after 30 min and 24% higher after 2 hr than in the control. In the cortical cylinder, IAA-induced stimulation of (/sup 3/H)Ins incorporation started only approx. = 1 hr after adding IAA. The ionophore monensin (20 inhibited the IAA-induced growth by 95%. Under these conditions, the IAA-induced stimulation of (/sup 3/H)Ins incorporation and the IAA-induced increase in in vivo extensibility of the sections was almost completely inhibited, although oxygen uptake was unaffected. The authors suggest that wall synthesis (as represented by (/sup 3/H)Ins incorporation) and wall loosening (increase in in vivo extensibility) are related processes. The results support the hypothesis that IAA induces growth by rapid simulation of cell wall synthesis in the growth-limiting epidermal cell layer.

  17. A rapid and quantitative method to detect human circulating tumor cells in a preclinical animal model. (United States)

    Tu, Shih-Hsin; Hsieh, Yi-Chen; Huang, Li-Chi; Lin, Chun-Yu; Hsu, Kai-Wen; Hsieh, Wen-Shyang; Chi, Wei-Ming; Lee, Chia-Hwa


    As cancer metastasis is the deadliest aspect of cancer, causing 90% of human deaths, evaluating the molecular mechanisms underlying this process is the major interest to those in the drug development field. Both therapeutic target identification and proof-of-concept experimentation in anti-cancer drug development require appropriate animal models, such as xenograft tumor transplantation in transgenic and knockout mice. In the progression of cancer metastasis, circulating tumor cells (CTCs) are the most critical factor in determining the prognosis of cancer patients. Several studies have demonstrated that measuring CTC-specific markers in a clinical setting (e.g., flow cytometry) can provide a current status of cancer development in patients. However, this useful technique has rarely been applied in the real-time monitoring of CTCs in preclinical animal models. In this study, we designed a rapid and reliable detection method by combining a bioluminescent in vivo imaging system (IVIS) and quantitative polymerase chain reaction (QPCR)-based analysis to measure CTCs in animal blood. Using the IVIS Spectrum CT System with 3D-imaging on orthotropic-developed breast-tumor-bearing mice. In this manuscript, we established a quick and reliable method for measuring CTCs in a preclinical animal mode. The key to this technique is the use of specific human and mouse GUS primers on DNA/RNA of mouse peripheral blood under an absolute qPCR system. First, the high sensitivity of cancer cell detection on IVIS was presented by measuring the luciferase carried MDA-MB-231 cells from 5 to 5x10(11) cell numbers with great correlation (R(2) = 0.999). Next, the MDA-MB-231 cell numbers injected by tail vein and their IVIS radiance signals were strongly corrected with qPCR-calculated copy numbers (R(2) > 0.99). Furthermore, by applying an orthotropic implantation animal model, we successfully distinguished xenograft tumor-bearing mice and control mice with a significant difference (p < 0

  18. Experimental Study on Forecasting Mathematical Model of Drying Shrinkage of Recycled Aggregate Concrete


    Yuanchen Guo; Xue Wang


    On the basis of basic law in AASHTO2007 model, the forecasting mathematical model of drying shrinkage of recycled aggregate concrete (RAC) is established by regression analysis and experimental study. The research results show that (1) with the replacement rate of RCA increases, the drying shrinkage value of RAC increases; this trend is even more obvious in the early drying time. (2) The addition of fly ash can inhibit the drying shrinkage of RAC, but the effect is not very obvious. Specifica...

  19. Effect of steel fibers on plastic shrinkage cracking of normal and high strength concretes


    Özgür Eren; Khaled Marar


    Naturally concrete shrinks when it is subjected to a drying environment. If this shrinkage is restrained, tensile stresses develop and concrete may crack. Plastic shrinkage cracks are especially harmful on slabs. One of the methods to reduce the adverse effects of shrinkage cracking of concrete is by reinforcing concrete with short randomly distributed fibers. The main objective of this study was to investigate the effect of fiber volume and aspect ratio of hooked steel fibers on plastic shri...

  20. The influence of granulation on lightweight aggregate on early autogenous shrinkage of high strength concrete


    Maleš, Dijana


    Early autogenous shrinkage of high strength concretes is relatively large. It can be reduced by using internal water reservoirs. Lightweight aggregate was used for internal water reservoir. 12% of aggregate was replaced with pre-soaked lightweight aggregate in the concrete mixture. On the basis of experiments in the graduation thesis we studied the influence of granulation of lightweight aggregate on early autogenous shrinkage. The shrinkage was measured electronically during the first day. I...

  1. Early exposure to interleukin-21 limits rapidly generated anti-Epstein-Barr virus T-cell line differentiation. (United States)

    Orio, Julie; Carli, Cédric; Janelle, Valérie; Giroux, Martin; Taillefer, Julie; Goupil, Mathieu; Richaud, Manon; Roy, Denis-Claude; Delisle, Jean-Sébastien


    The adoptive transfer of ex vivo-expanded Epstein-Barr virus (EBV)-specific T-cell lines is an attractive strategy to treat EBV-related neoplasms. Current evidence suggests that for adoptive immunotherapy in general, clinical responses are superior if the transferred cells have not reached a late or terminal effector differentiation phenotype before infusion. The cytokine interleukin (IL)-21 has shown great promise at limiting late T-cell differentiation in vitro, but this remains to be demonstrated in anti-viral T-cell lines. We adapted a clinically validated protocol to rapidly generate EBV-specific T-cell lines in 12 to 14 days and tested whether the addition of IL-21 at the initiation of the culture would affect T-cell expansion and differentiation. We generated clinical-scale EBV-restricted T-cell line expansion with balanced T-cell subset ratios. The addition of IL-21 at the beginning of the culture decreased both T-cell expansion and effector memory T-cell accumulation, with a relative increase in less-differentiated T cells. Within CD4 T-cell subsets, exogenous IL-21 was notably associated with the cell surface expression of CD27 and high KLF2 transcript levels, further arguing for a role of IL-21 in the control of late T-cell differentiation. Our results show that IL-21 has profound effects on T-cell differentiation in a rapid T-cell line generation protocol and as such should be further explored as a novel approach to program anti-viral T cells with features associated with early differentiation and optimal therapeutic efficacy. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  2. Laboratory evaluation of selective in situ refractive cornea collagen shrinkage with continuous wave infrared laser combined with transepithelial collagen cross-linking: a novel refractive procedure


    Kanellopoulos AJ


    Anastasios John, KanellopoulosLaserVision gr Institute, Athens, Greece; New York University Medical School, New York City, NY, USABackground: This research comprised a laboratory evaluation of a novel refractive surgery technique involving sequential corneal subsurface shrinkage-driven reshaping using a continuous wave mid-infrared laser application followed by stiffening via rapid transepithelial higher fluence collagen cross-linking for shape persistence/longevity on cadaver corneas.Materia...

  3. Time-Dependent Behavior of Shrinkage Strain for Early Age Concrete Affected by Temperature Variation

    Directory of Open Access Journals (Sweden)

    Yu Qin


    Full Text Available Shrinkage has been proven to be an important property of early age concrete. The shrinkage strain leads to inherent engineering problems, such as cracking and loss of prestress. Atmospheric temperature is an important factor in shrinkage strain. However, current research does not provide much attention to the effect of atmospheric temperature on shrinkage of early age concrete. In this paper, a laboratory study was undertaken to present the time-dependent shrinkage of early age concrete under temperature variation. A newly developed Material Deformation Tester (MDT, which can simulate consecutive variation of atmospheric temperature, was used to collect the shrinkage strain of specimens and temperature data. A numerical model was established to describe the thermoelastic strain of a specimen. The results show that (1 there are several sharp shrinkages up to 600 μ for early age concrete in the first 3 days; (2 the absolute value of shrinkage strain is larger than thermal strain; and (3 the difference of shrinkage strain under temperature variation or constant temperature is up to 500 μ.

  4. Effect of resin-composite filler particle size and shape on shrinkage-stress. (United States)

    Satterthwaite, Julian D; Maisuria, Amit; Vogel, Karin; Watts, David C


    The aim of this study was to investigate the effect of variations in filler particle size and shape on the polymerization shrinkage-stress kinetics of resin-composites. A model series of 12 VLC resin-composites were studied. The particulate dispersed phase volume fraction was 56.7%: these filler particles were systematically graded in size, and further were either spherical or irregular. A Bioman instrument (cantilever beam method) was employed to determine the shrinkage-stress kinetics following 40s irradiation (600 mW/cm(2)) at 23°C (n=3). All data were captured for 60 min and the final shrinkage-stress calculated. Shrinkage-stress varied between 3.86 MPa (SD 0.14) for S3 (spherical filler particles of 500 nm) and 8.44 MPa (SD 0.41) for I1 (irregular filler particles of 450 nm). The shrinkage-stress values were generally lower for those composites with spherical filler particles than those with irregular filler particles. The differences in shrinkage-stress with filler particle size and shape were statistically significant (pparticles exhibit lower shrinkage-stress values compared to those with irregular filler particles. Shrinkage-stress and shrinkage-stress rate vary in a complex manner with variations in the size of the dispersed phase particles: a hypothesized explanation for the effect of filler particle size and shape is presented. Copyright © 2012 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

  5. Effectiveness of Fiber Reinforcement on the Mechanical Properties and Shrinkage Cracking of Recycled Fine Aggregate Concrete

    National Research Council Canada - National Science Library

    Jeongsoo Nam; Gyuyong Kim; Jaechul Yoo; Gyeongcheol Choe; Hongseop Kim; Hyeonggil Choi; Youngduck Kim


      This paper presents an experimental study conducted to investigate the effect of fiber reinforcement on the mechanical properties and shrinkage cracking of recycled fine aggregate concrete (RFAC...

  6. Design changes of device to investigation of alloys linear contraction and shrinkage stresses

    Directory of Open Access Journals (Sweden)

    J. Mutwil


    Full Text Available Some design changes in device elaborated by author to examination of linear contraction and shrinkage stresses progress of metals and alloys during– and after solidification have been described. The introduced changes have been focused on design of closing of shrinkage test rod mould. The introduced changes have been allowed to simplify a mounting procedure of thermocouples measuring a temperature of the shrinkage rod casting (in 6 points. Exemplary investigation results of linear contraction and shrinkage stresses development in Al-Si13.5% alloy have been presented.

  7. Investigation of Shrinkage Defect in Castings by Quantitative Ishikawa Diagram

    Directory of Open Access Journals (Sweden)

    Chokkalingam B.


    Full Text Available Metal casting process involves processes such as pattern making, moulding and melting etc. Casting defects occur due to combination of various processes even though efforts are taken to control them. The first step in the defect analysis is to identify the major casting defect among the many casting defects. Then the analysis is to be made to find the root cause of the particular defect. Moreover, it is especially difficult to identify the root causes of the defect. Therefore, a systematic method is required to identify the root cause of the defect among possible causes, consequently specific remedial measures have to be implemented to control them. This paper presents a systematic procedure to identify the root cause of shrinkage defect in an automobile body casting (SG 500/7 and control it by the application of Pareto chart and Ishikawa diagram. with quantitative Weightage. It was found that the root causes were larger volume section in the cope, insufficient feeding of riser and insufficient poured metal in the riser. The necessary remedial measures were taken and castings were reproduced. The shrinkage defect in the castings was completely eliminated.

  8. Impaired decision-making and brain shrinkage in alcoholism. (United States)

    Le Berre, A-P; Rauchs, G; La Joie, R; Mézenge, F; Boudehent, C; Vabret, F; Segobin, S; Viader, F; Allain, P; Eustache, F; Pitel, A-L; Beaunieux, H


    Alcohol-dependent individuals usually favor instant gratification of alcohol use and ignore its long-term negative consequences, reflecting impaired decision-making. According to the somatic marker hypothesis, decision-making abilities are subtended by an extended brain network. As chronic alcohol consumption is known to be associated with brain shrinkage in this network, the present study investigated relationships between brain shrinkage and decision-making impairments in alcohol-dependent individuals early in abstinence using voxel-based morphometry. Thirty patients performed the Iowa Gambling Task and underwent a magnetic resonance imaging investigation (1.5T). Decision-making performances and brain data were compared with those of age-matched healthy controls. In the alcoholic group, a multiple regression analysis was conducted with two predictors (gray matter [GM] volume and decision-making measure) and two covariates (number of withdrawals and duration of alcoholism). Compared with controls, alcoholics had impaired decision-making and widespread reduced gray matter volume, especially in regions involved in decision-making. The regression analysis revealed links between high GM volume in the ventromedial prefrontal cortex, dorsal anterior cingulate cortex and right hippocampal formation, and high decision-making scores (Palcoholism may result from impairment of both emotional and cognitive networks. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  9. Calcium fluxes cause nuclear shrinkage and the translocation of phospholipase C-delta1 into the nucleus. (United States)

    Okada, Masashi; Taguchi, Katsutoshi; Maekawa, Shohei; Fukami, Kiyoko; Yagisawa, Hitoshi


    Phospholipase C-delta1 (PLCdelta1) is the most fundamental form of the eukaryotic PLC and thought to play important roles in the regulation of cells. We previously reported that PLCdelta1 shuttles between the cytoplasm and nucleus, and an influx of Ca2+ triggers the nuclear import of PLCdelta1 via Ca2+-dependent interaction with importin beta1, although the physiological meaning of this is unclear. Here we have examined the distribution of PLCdelta1 using primary cultures of rat hippocampal neurons. Treatment of 7DIV neurons with ionomycin or thapsigargin caused the nuclear localization of PLCdelta1 as has been observed in other cell lines. Similar results were obtained with neurons treated with glutamate, suggesting that the nuclear localization of PLCdelta1 plays some roles in excitotoxicity associated with ischemic stress. Generally, cells undergoing ischemic or hypoxic cell death show nuclear shrinkage. We confirmed that a massive influx of Ca2+ caused similar results. Furthermore, overexpression of GFP-PLCdelta1 facilitated ionomycin-induced nuclear shrinkage in embryonic fibroblasts derived from PLCdelta1 gene-knockout mice (PLCdelta1KO-MEF). By contrast, an E341A mutant that cannot bind with importin beta1 and be imported into the nucleus by ionomycin and also lacks enzymatic activity did not cause nuclear shrinkage in PLCdelta1KO-MEF. Nuclear translocation and the PLC activity of PLCdelta1, therefore, may regulate the nuclear shape by controlling the nuclear scaffold during stress-induced cell death caused by high levels of Ca2+. 2010 Elsevier Ireland Ltd. All rights reserved.

  10. Reactive oxygen species regulatory mechanisms associated with rapid response of MC3T3-E1 cells for vibration stress

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Ling; Gan, Xueqi; Zhu, Zhuoli; Yang, Yang; He, Yuting; Yu, Haiyang, E-mail:


    Although many previous studies have shown that refractory period-dependent memory effect of vibration stress is anabolic for skeletal homeostasis, little is known about the rapid response of osteoblasts simply derived from vibration itself. In view of the potential role of reactive oxygen species (ROS) in mediating differentiated activity of osteoblasts, whether and how ROS regulates the rapid effect of vibration deserve to be demonstrated. Our findings indicated that MC3T3-E1 cells underwent decreased gene expression of Runx2, Col-I and ALP and impaired ALP activity accompanied by increased mitochondrial fission immediately after vibration loading. Moreover, we also revealed the involvement of ERK-Drp1 signal transduction in ROS regulatory mechanisms responsible for the rapid effect of vibration stress. - Highlights: • ROS contributed to the rapid response of MC3T3-E1 cells for vibration stress. • Imbalance of mitochondrial dynamics were linked to the LMHFV-derived rapid response. • The role of ERK-Drp1 signal pathway in the LMHFV-derived osteoblast rapid response.

  11. Global patent landscape of programmed cell death 1: implications of the rapid expansion. (United States)

    Kong, Xiangjun; Zhang, Qianru; Lai, Yunfeng; Hu, Hao; Chen, Xin; Hu, Yuanjia


    Inhibitors of programmed cell death 1 (PD-1) and its ligands are producing a paradigm shift in cancer treatment. The promising clinical outcomes and a multi-billion dollar market have prompted active research and development and resulted in relentless patent protection. However, the global patent landscape in this field remains unclear. Areas covered: The patent landscape encompassing global patenting activities and developing trends in the field is discussed based on a data set of 1287 patent families. Patenting activities have expanded rapidly in the past three years. Specific trends in relevant aspects are presented, including patent filing countries, patent ownership, co-patents, technical areas, and technological connections in terms of patent citation relationships. Expert opinion: Together with patenting momentum in recent years, fragmented ownership and dense technological connections of PD-1-related inventions raise the possibility of a patent thicket. The explosion of patent applications and complex citation relationships could also lead to considerable patent conflicts and disputes on overlapping intellectual property rights, in addition to existing legal uncertainties. Patent applicants in this field are encouraged to be aware of these concerns when developing valid patent strategies.

  12. Rapid identification of mRNA processing defects with a novel single-cell yeast reporter. (United States)

    Sorenson, Matthew R; Stevens, Scott W


    It has become increasingly evident that gene expression processes in eukaryotes involve communication and coordination between many complex, independent macromolecular machines. To query these processes and to explore the potential relationships between them in the budding yeast Saccharomyces cerevisiae, we designed a versatile reporter using multicolor high-throughput flow cytometry. Due to its design, this single reporter exhibits a distinctive signature for many defects in gene expression including transcription, histone modification, pre-mRNA splicing, mRNA export, nonsense-mediated decay, and mRNA degradation. Analysis of the reporter in 4967 nonessential yeast genes revealed striking phenotypic overlaps between chromatin remodeling, histone modification, and pre-mRNA splicing. Additionally, we developed a copper-inducible reporter, with which we demonstrate that 5-fluorouracil mimics the mRNA decay phenotype of cells lacking the 3'-5' exonuclease Rrp6p. Our reporter is capable of performing high-throughput, rapid, and large-scale screens to identify and characterize genetic and chemical perturbations of the major eukaryotic gene expression processes.

  13. Rapid and sensitive detection of early esophageal squamous cell carcinoma with fluorescence probe targeting dipeptidylpeptidase IV (United States)

    Onoyama, Haruna; Kamiya, Mako; Kuriki, Yugo; Komatsu, Toru; Abe, Hiroyuki; Tsuji, Yosuke; Yagi, Koichi; Yamagata, Yukinori; Aikou, Susumu; Nishida, Masato; Mori, Kazuhiko; Yamashita, Hiroharu; Fujishiro, Mitsuhiro; Nomura, Sachiyo; Shimizu, Nobuyuki; Fukayama, Masashi; Koike, Kazuhiko; Urano, Yasuteru; Seto, Yasuyuki


    Early detection of esophageal squamous cell carcinoma (ESCC) is an important prognosticator, but is difficult to achieve by conventional endoscopy. Conventional lugol chromoendoscopy and equipment-based image-enhanced endoscopy, such as narrow-band imaging (NBI), have various practical limitations. Since fluorescence-based visualization is considered a promising approach, we aimed to develop an activatable fluorescence probe to visualize ESCCs. First, based on the fact that various aminopeptidase activities are elevated in cancer, we screened freshly resected specimens from patients with a series of aminopeptidase-activatable fluorescence probes. The results indicated that dipeptidylpeptidase IV (DPP-IV) is specifically activated in ESCCs, and would be a suitable molecular target for detection of esophageal cancer. Therefore, we designed, synthesized and characterized a series of DPP-IV-activatable fluorescence probes. When the selected probe was topically sprayed onto endoscopic submucosal dissection (ESD) or surgical specimens, tumors were visualized within 5 min, and when the probe was sprayed on biopsy samples, the sensitivity, specificity and accuracy reached 96.9%, 85.7% and 90.5%. We believe that DPP-IV-targeted activatable fluorescence probes are practically translatable as convenient tools for clinical application to enable rapid and accurate diagnosis of early esophageal cancer during endoscopic or surgical procedures. PMID:27245876

  14. Regulation of the efflux of putrescine and cadaverine from rapidly growing cultured RAW 264 cells by extracellular putrescine.


    Tjandrawinata, R R; Byus, C V


    Cultures of the macrophage-like RAW 264 cells were adapted to divide normally in a synthetic serum-supplemented culture medium lacking any polyamines and diamine oxidase activity. These rapidly dividing cells actively effluxed large amounts of putrescine and cadaverine, compared with the intracellular levels, into the culture medium. The efflux of putrescine was stimulated by the amino acid ornithine, whereas efflux of cadaverine was inhibited. Relatively low levels of spermidine and N1-acety...

  15. Rabbit antithymocyte globulin induces rapid expansion of effector memory CD8 T cells without accelerating acute graft versus host disease. (United States)

    Wittenbecher, Friedrich; Rieger, Kathrin; Dziubianau, Mikalai; Herholz, Anne; Mensen, Angela; Blau, Igor Wolfgang; Uharek, Lutz; Dörken, Bernd; Thiel, Andreas; Na, Il-Kang


    Rabbit antithymocyte globulin (Thymoglobulin(®)) is commonly used as graft-versus-host disease (GvHD) prophylaxis. Since we found similar total CD8 T cell numbers in patients with and without Thymoglobulin(®) therapy within the first six months after allogeneic hematopoietic stem cell transplantation, we have analyzed the reconstitution of the CD8 T cell compartment in detail. After T cell-depletion, higher and more sustained proliferative capacity of memory CD8 T cells resulted in their rapid expansion, whereas the fraction of naive CD8 T cells decreased. Importantly, this shift towards effector memory CD8 T cells did not accelerate the incidence of GvHD.

  16. Investigation of Near-Surface Defects Induced by Spike Rapid Thermal Annealing in c-SILICON Solar Cells (United States)

    Liu, Guodong; Ren, Pan; Zhang, Dayong; Wang, Weiping; Li, Jianfeng


    The defects induced by a spike rapid thermal annealing (RTA) process in crystalline silicon (c-Si) solar cells were investigated by the photoluminescence (PL) technique and the transmission electron microscopy (TEM), respectively. Dislocation defects were found to form in the near-surface junction region of the monocrystalline Si solar cell after a spike RTA process was performed at 1100∘C. Photo J-V characteristics were measured on the Si solar cell before and after the spike RTA treatments to reveal the effects of defects on the Si cell performances. In addition, the Silvaco device simulation program was used to study the effects of defects density on the cell performances by fitting the experimental data of RTA-treated cells. The results demonstrate that there was an obvious degradation in the Si solar cell performances when the defect density after the spike RTA treatment was above 1×1013cm-3.

  17. The effect of fibers on the loss of water by evaporation and shrinkage of concrete

    Directory of Open Access Journals (Sweden)

    N. M. P. Pillar

    Full Text Available Shrinkage is one of the least desirable attributes in concrete. Large areas of exposed concrete surfaces , such as in shotcrete tunnel linings, where it is practically impossible to make a moist cure, are highly susceptible to plastic shrinkage at early ages. The autogenous and drying shrinkage can lead to states of greater than threshold strength, causing fracture, mechanical damage and lack of durability of concrete structures. The addition of fibers can greatly reduce plastic shrinkage, but has limited effect in mitigating autogenous and drying shrinkage. To evaluate the performance of polypropylene and steel fibers to understand their effect on shrinkage of concrete, a study was carried out to relate the loss of water from the paste and the shrinkage during the first 28 days of age, and compare it with a control mix without fiber. The loss of water was obtained by the weight loss of the specimens at different ages, since the only component that could contribute for the loss of weight was the water lost by the paste of the concrete. And the paste itself is the only source of shrinkage. Uniaxial compressive tests from very early ages enabled the determination of time when plastic shrinkage ended. It was observed that the control concrete mix lost three times more water and developed plastic and drying shrinkage 60 % higher than the fiber reinforced concrete mixes. It was possible to demonstrate that the reduced loss of water caused by the incorporation of fibers is related to the mitigation of plastic shrinkage. It was observed that the fibers are effective to restrain the movement of water through the cement paste in the plastic state, however such effect is limited after concrete starts the hardening state.

  18. Development of an OP9 derived cell line as a robust model to rapidly study adipocyte differentiation.

    Directory of Open Access Journals (Sweden)

    Jacqueline M Lane

    Full Text Available One hallmark of obesity is adipocyte hypertrophy and hyperplasia. To gain novel insights into adipose biology and therapeutics, there is a pressing need for a robust, rapid, and informative cell model of adipocyte differentiation for potential RNAi and drug screens. Current models are prohibitive for drug and RNAi screens due to a slow differentiation time course and resistance to transfection. We asked if we could create a rapid, robust model of adipogenesis to potentially enable rapid functional and obesity therapeutic screens. We generated the clonal population OP9-K, which differentiates rapidly and reproducibly, and displays classic adipocyte morphology: rounded cell shape, lipid accumulation, and coalescence of lipids into a large droplet. We further validate the OP9-K cells as an adipocyte model system by microarray analysis of the differentiating transcriptome. OP9-K differentiates via known adipogenic pathways, involving the transcriptional activation and repression of common adipose markers Plin1, Gata2, C/Ebpα and C/Ebpβ and biological pathways, such as lipid metabolism, PPARγ signaling, and osteogenesis. We implemented a method to quantify lipid accumulation using automated microscopy and tested the ability of our model to detect alterations in lipid accumulation by reducing levels of the known master adipogenic regulator Pparγ. We further utilized our model to query the effects of a novel obesity therapeutic target, the transcription factor SPI1. We determine that reduction in levels of Spi1 leads to an increase in lipid accumulation. We demonstrate rapid, robust differentiation and efficient transfectability of the OP9-K cell model of adipogenesis. Together with our microscopy based lipid accumulation assay, adipogenesis assays can be achieved in just four days' time. The results of this study can contribute to the development of rapid screens with the potential to deepen our understanding of adipose biology and efficiently

  19. Concerted action of neuroepithelial basal shrinkage and active epithelial migration ensures efficient optic cup morphogenesis (United States)

    Sidhaye, Jaydeep; Norden, Caren


    Organ formation is a multi-scale event that involves changes at the intracellular, cellular and tissue level. Organogenesis often starts with the formation of characteristically shaped organ precursors. However, the cellular mechanisms driving organ precursor formation are often not clear. Here, using zebrafish, we investigate the epithelial rearrangements responsible for the development of the hemispherical retinal neuroepithelium (RNE), a part of the optic cup. We show that in addition to basal shrinkage of RNE cells, active migration of connected epithelial cells into the RNE is a crucial player in its formation. This cellular movement is driven by progressive cell-matrix contacts and actively translocates prospective RNE cells to their correct location before they adopt neuroepithelial fate. Failure of this migration during neuroepithelium formation leads to ectopic determination of RNE cells and consequently impairs optic cup formation. Overall, this study illustrates how spatiotemporal coordination between morphogenic movements and fate determination critically influences organogenesis. DOI: PMID:28372636

  20. Optimization of electrospun poly(N-isopropyl acrylamide) mats for the rapid reversible adhesion of mammalian cells. (United States)

    Cicotte, Kirsten N; Reed, Jamie A; Nguyen, Phuong Anh H; De Lora, Jacqueline A; Hedberg-Dirk, Elizabeth L; Canavan, Heather E


    Poly(N-isopropyl acrylamide) (pNIPAM) is a "smart" polymer that responds to changes in altering temperature near physiologically relevant temperatures, changing its relative hydrophobicity. Mammalian cells attach to pNIPAM at 37 °C and detach spontaneously as a confluent sheet when the temperature is shifted below the lower critical solution temperature (∼32 °C). A variety of methods have been used to create pNIPAM films, including plasma polymerization, self-assembled monolayers, and electron beam ionization. However, detachment of confluent cell sheets from these pNIPAM films can take well over an hour to achieve potentially impacting cellular behavior. In this work, pNIPAM mats were prepared via electrospinning (i.e., espNIPAM) by a previously described technique that the authors optimized for cell attachment and rapid cell detachment. Several electrospinning parameters were varied (needle gauge, collection time, and molecular weight of the polymer) to determine the optimum parameters. The espNIPAM mats were then characterized using Fourier-transform infrared, x-ray photoelectron spectroscopy, and scanning electron microscopy. The espNIPAM mats showing the most promise were seeded with mammalian cells from standard cell lines (MC3T3-E1) as well as cancerous tumor (EMT6) cells. Once confluent, the temperature of the cells and mats was changed to ∼25 °C, resulting in the extremely rapid swelling of the mats. The authors find that espNIPAM mats fabricated using small, dense fibers made of high molecular weight pNIPAM are extremely well-suited as a rapid release method for cell sheet harvesting.

  1. Study of Drying Shrinkage Cracking by Lattice Gas Automaton and Environmental Scanning Electron Microscope

    NARCIS (Netherlands)

    Van Mier, J.G.M.; Jankovic, D.


    Numerical modeling of moisture flow, drying shrinkage and crack phenomena in cement microstructure, by coupling a Lattice Gas Automaton and a Lattice Fracture Model, highlighted the importance of a shrinkage coefficient (?sh) as the most significant parameter for achieving realistic numerical

  2. Development and Performance Assessment of the High-Performance Shrinkage Reducing Agent for Concrete

    Directory of Open Access Journals (Sweden)

    Hyung Sub Han


    Full Text Available To develop a high-performance shrinkage reducing agent, this study investigated several shrinkage reducing materials and supplements for those materials. Fluidity and air content were satisfactory for the various shrinkage reducing materials. The decrease in viscosity was the lowest for glycol-based materials. The decrease in drying shrinkage was most prominent for mixtures containing glycol-based materials. In particular, mixtures containing G2 achieved a 40% decrease in the amount of drying shrinkage. Most shrinkage reducing materials had weaker level of compressive strength than that of the plain mixture. When 3% triethanolamine was used for early strength improvement, the strength was enhanced by 158% compared to that of the plain mixture on day 1; enhancement values were 135% on day 7 and 113% on day 28. To assess the performance of the developed high-performance shrinkage reducing agent and to determine the optimal amount, 2.0% shrinkage reducing agent was set as 40% of the value of the plain mixture. While the effect was more prominent at higher amounts, to prevent deterioration of the compressive strength and the other physical properties, the recommended amount is less than 2.0%.

  3. Hardness, density, and shrinkage characteristics of silk-oak from Hawaii (United States)

    R. L. Youngs


    Shrinkage, specific gravity, and hardness of two shipments of silk-oak (Grevillea robusta) from Hawaii were evaluated to provide basic information pertinent to the use of the wood for cabinet and furniture purposes. The wood resembles Hawaii-grown shamel ash (Fraxinus uhdei ) in the properties evaluated. Shrinkage compares well with that of black cherry, silver maple,...

  4. Modeling dental composite shrinkage by digital image correlation and finite element methods (United States)

    Chen, Terry Yuan-Fang; Huang, Pin-Sheng; Chuang, Shu-Fen


    Dental composites are light-curable resin-based materials with an inherent defect of polymerization shrinkage which may cause tooth deflection and debonding of restorations. This study aimed to combine digital image correlation (DIC) and finite element analysis (FEA) to model the shrinkage behaviors under different light curing regimens. Extracted human molars were prepared with proximal cavities for composite restorations, and then divided into three groups to receive different light curing protocols: regular intensity, low intensity, and step-curing consisting of low and high intensities. For each tooth, the composite fillings were consecutively placed under both unbonded and bonded conditions. At first, the shrinkage of the unbonded restorations was analyzed by DIC and adopted as the setting of FEA. The simulated shrinkage behaviors obtained from FEA were further validated by the measurements in the bonded cases. The results showed that different light curing regimens affected the shrinkage in unbonded restorations, with regular intensity showing the greatest shrinkage strain on the top surface. The shrinkage centers in the bonded cases were located closer to the cavity floor than those in the unbonded cases, and were less affected by curing regimens. The FEA results showed that the stress was modulated by the accumulated light energy density, while step-curing may alleviate the tensile stress along the cavity walls. In this study, DIC provides a complete description of the polymerization shrinkage behaviors of dental composites, which may facilitate the stress analysis in the numerical investigation.

  5. Effect of modulated photo-activation on polymerization shrinkage behavior of dental restorative resin composites

    NARCIS (Netherlands)

    Tauböck, T.T.; Feilzer, A.J.; Buchalla, W.; Kleverlaan, C.J.; Krejci, I.; Attin, T.


    This study investigated the influence of modulated photo-activation on axial polymerization shrinkage, shrinkage force, and hardening of light- and dual-curing resin-based composites. Three light-curing resin composites (SDR bulk-fill, Esthet X flow, and Esthet X HD) and one dual-curing material

  6. Shrinkage reduction of dental composites by addition of expandable zirconia filler

    DEFF Research Database (Denmark)

    Skovgaard, M.; Almdal, Kristoffer; Sørensen, Bent F.


    A problem with dental resin composites is the polymerization shrinkage, which makes the filling loosen from the tooth or induces crack formation. We have developed an expandable metastable tetragonal zirconia filler, which upon reaction with water, is able to counter the polymer shrinkage...

  7. Spontaneous tumour shrinkage in 1261 observed patients with sporadic vestibular schwannoma

    DEFF Research Database (Denmark)

    Huang, Xiaoshan; Caye-Thomasen, P; Stangerup, S-E


    To determine the rate of spontaneous tumour shrinkage in a group of patients with sporadic vestibular schwannoma managed with a 'wait and scan' approach.......To determine the rate of spontaneous tumour shrinkage in a group of patients with sporadic vestibular schwannoma managed with a 'wait and scan' approach....

  8. Analysis of gene set using shrinkage covariance matrix approach (United States)

    Karjanto, Suryaefiza; Aripin, Rasimah


    Microarray methodology has been exploited for different applications such as gene discovery and disease diagnosis. This technology is also used for quantitative and highly parallel measurements of gene expression. Recently, microarrays have been one of main interests of statisticians because they provide a perfect example of the paradigms of modern statistics. In this study, the alternative approach to estimate the covariance matrix has been proposed to solve the high dimensionality problem in microarrays. The extension of traditional Hotelling's T2 statistic is constructed for determining the significant gene sets across experimental conditions using shrinkage approach. Real data sets were used as illustrations to compare the performance of the proposed methods with other methods. The results across the methods are consistent, implying that this approach provides an alternative to existing techniques.

  9. Creep and shrinkage effects on integral abutment bridges (United States)

    Munuswamy, Sivakumar

    Integral abutment bridges provide bridge engineers an economical design alternative to traditional bridges with expansion joints owing to the benefits, arising from elimination of expensive joints installation and reduced maintenance cost. The superstructure for integral abutment bridges is cast integrally with abutments. Time-dependent effects of creep, shrinkage of concrete, relaxation of prestressing steel, temperature gradient, restraints provided by abutment foundation and backfill and statical indeterminacy of the structure introduce time-dependent variations in the redundant forces. An analytical model and numerical procedure to predict instantaneous linear behavior and non-linear time dependent long-term behavior of continuous composite superstructure are developed in which the redundant forces in the integral abutment bridges are derived considering the time-dependent effects. The redistributions of moments due to time-dependent effects have been considered in the analysis. The analysis includes nonlinearity due to cracking of the concrete, as well as the time-dependent deformations. American Concrete Institute (ACI) and American Association of State Highway and Transportation Officials (AASHTO) models for creep and shrinkage are considered in modeling the time dependent material behavior. The variations in the material property of the cross-section corresponding to the constituent materials are incorporated and age-adjusted effective modulus method with relaxation procedure is followed to include the creep behavior of concrete. The partial restraint provided by the abutment-pile-soil system is modeled using discrete spring stiffness as translational and rotational degrees of freedom. Numerical simulation of the behavior is carried out on continuous composite integral abutment bridges and the deformations and stresses due to time-dependent effects due to typical sustained loads are computed. The results from the analytical model are compared with the

  10. Rapid hyperfractionated radiotherapy. Clinical results in 178 advanced squamous cell carcinomas of the head and neck

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, T.D.; Demange, L.; Froissart, D.; Panis, X.; Loirette, M.


    The authors present a series of 178 patients with Stage III or IV squamous cell carcinoma of the head and neck treated by rapid irradiation using multiple and small fractions per day. An initial group of 91 patients (G1) received a total dose of 72 Gy in 80 sessions and 10 days, according to the following split course schedule: J1 to J5, 36 Gy in 40 sessions, eight daily fractions of .9 Gy separated by 2 hours; J6 to J20, rest period; J21 to J25, same as in J1 except that the spinal cord was shielded. This protocol was altered for the following 87 patients (G2) by lessening the total dose to 60 to 66 Gy and the number of fractions to 60. The rest period was lengthened to 4 weeks. All patients but five completed the whole program and the minimal follow-up period was 24 months. At the end of irradiation, 121 patients achieved a total remission, but local recurrences occurred in 56%. Moreover, acute intolerance was considered as severe in 34% of G1 patients, and included extensive mucosal necrosis and bleeding. Although this rate was significantly reduced in G2 patients, late complications were observed in 20 of the 25 survivors, and included trismus, cervical sclerosis, and recurrent laryngeal edema. The crude survival rate is 13% at 2 years. Although this study was not randomized, this particular type of accelerated and hyperfractionated combination of irradiation did not really improve the clinical results in advanced carcinoma of the head and neck. Other schedules and probably other tumors, less extended, should be tested.

  11. Endothelial Cells Are Susceptible to Rapid siRNA Transfection and Gene Silencing Ex Vivo (United States)

    Andersen, Nicholas D.; Chopra, Atish; Monahan, Thomas S.; Malek, Junaid Y.; Jain, Monica; Pradhan, Leena; Ferran, Christiane; LoGerfo, Frank W.


    BACKGROUND Endothelial gene silencing via small interfering RNA (siRNA) transfection represents a promising strategy for the control of vascular disease. Here, we demonstrate endothelial gene silencing in human saphenous vein using three rapid siRNA transfection techniques amenable for use in the operating room. MATERIALS AND METHODS Control siRNA, Cy5 siRNA, or siRNA targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or endothelial specific nitric oxide synthase (eNOS) were applied to surplus human saphenous vein for 10 minutes by (i) soaking, (ii) applying 300 mmHg hyperbaric pressure, or (iii) 120 mmHg luminal distending pressure. Transfected vein segments were maintained in organ culture. siRNA delivery and gene silencing were assessed by tissue layer using confocal microscopy and immunohistochemistry. RESULTS Distending pressure transfection yielded the highest levels of endothelial siRNA delivery (22% pixels fluorescing) and gene silencing (60% GAPDH knockdown, 55% eNOS knockdown) as compared to hyperbaric (12% pixels fluorescing, 36% GAPDH knockdown, 30% eNOS knockdown) or non-pressurized transfections (10% pixels fluorescing, 30% GAPDH knockdown, 25% eNOS knockdown). Cumulative endothelial siRNA delivery (16% pixels fluorescing) and gene silencing (46% GAPDH knockdown) exceeded levels achieved in the media/adventitia (8% pixels fluorescing, 24% GAPDH knockdown) across all transfection methods. CONCLUSION Endothelial gene silencing is possible within the timeframe and conditions of surgical application without the use of transfection reagents. The high sensitivity of endothelial cells to siRNA transfection marks the endothelium as a promising target of gene therapy in vascular disease. PMID:20801607

  12. Parametric study and shrinkage modelling of natural rubber sheet drying using COMSOL multiphysics (United States)

    Ajani, C.; Kumar, A.; Curcio, S.; Tekasakul, P.


    Natural rubber is one of the major exporting cash crops in Thailand. Shrinkage in natural rubber sheet during drying creates uneven stresses in the rubber, hampers the quality and the water activity predisposes it to microbial activation. Hence, the effect of drying parameters on shrinkage has been considered in this work. The finite element concept coupled with the arbitrary Lagrangian-Eulerian (ALE) method was used to solve the two-dimensional coupled physics in the rubber sheet drying chamber and account for the shrinkage effect. The spatial domain (drying chamber), material domain (rubber sheet) was analysed. An isotropic linear elastic model was assumed for the rubber sheet for analysis. Three Case studies of different velocity, temperature, relative humidity and shrinkage coefficient were considered in the numerical study using COMSOL Multiphysics. It is concluded that increase in the operating parameters increases the shrinkage of the rubber. Therefore, rubber should dry at relatively lower operating parameters to improve its quality.

  13. Analysis of the effect of shrinkage on macrosegregation in alloy solidification (United States)

    Krane, Matthew John M.; Incropera, Frank P.


    Numerical calculations based on a continuum model are used to examine the effects of solidification shrinkage on the redistribution of solute in a Pb-19.2 pct Sn mixture which is convectively cooled at a sidewall. For each of three different cooling rates, separate calculations are performed for shrinkage and buoyancy-induced flows, as well as for the combined influence of shrinkage and buoyancy effects. The calculations reveal that flow and macrosegregation patterns are more strongly influenced by buoyancy effects over a wide range of solidification rates. Although extremely large solidification rates yield small regions near the chilled wall in which shrinkage-induced flows control the redis-tribution of solute, the overall effect on macrosegregation is small relative to that associated with buoyancy. Scaling analysis of the governing equations produces reference shrinkage and buoyancy velocities which can be used to extend the current numerical results to other binary systems.

  14. Effects of prepolymerized particle size and polymerization kinetics on volumetric shrinkage of dental modeling resins. (United States)

    Kwon, Tae-Yub; Ha, Jung-Yun; Chun, Ju-Na; Son, Jun Sik; Kim, Kyo-Han


    Dental modeling resins have been developed for use in areas where highly precise resin structures are needed. The manufacturers claim that these polymethyl methacrylate/methyl methacrylate (PMMA/MMA) resins show little or no shrinkage after polymerization. This study examined the polymerization shrinkage of five dental modeling resins as well as one temporary PMMA/MMA resin (control). The morphology and the particle size of the prepolymerized PMMA powders were investigated by scanning electron microscopy and laser diffraction particle size analysis, respectively. Linear polymerization shrinkage strains of the resins were monitored for 20 minutes using a custom-made linometer, and the final values (at 20 minutes) were converted into volumetric shrinkages. The final volumetric shrinkage values for the modeling resins were statistically similar (P > 0.05) or significantly larger (P resin and were related to the polymerization kinetics (P resin structures rather than the use of dental modeling resins.

  15. Rapid selection of escape mutants by the first CD8 T cell responses in acute HIV-1 infection

    Energy Technology Data Exchange (ETDEWEB)

    Korber, Bette Tina Marie [Los Alamos National Laboratory


    The recent failure of a vaccine that primes T cell responses to control primary HIV-1 infection has raised doubts about the role of CD8+ T cells in early HIV-1 infection. We studied four patients who were identified shortly after HIV-1 infection and before seroconversion. In each patient there was very rapid selection of multiple HIV-1 escape mutants in the transmitted virus by CD8 T cells, including examples of complete fixation of non-synonymous substitutions within 2 weeks. Sequencing by single genome amplification suggested that the high rate of virus replication in acute infection gave a selective advantage to virus molecules that contained simultaneous and gained sequential T cell escape mutations. These observations show that whilst early HIV-1 specific CD8 T cells can act against virus, rapid escape means that these T cell responses are unlikely to benefit the patient and may in part explain why current HIV-1 T cell vaccines may not be protective.

  16. Photoresist shrinkage effects in 16 nm node extreme ultraviolet (EUV) photoresist targets (United States)

    Bunday, Benjamin; Montgomery, Cecilia; Montgomery, Warren; Cepler, Aron


    Photoresist shrinkage (i.e., line slimming) is an important systematic uncertainty source in critical dimension-scanning electron microscope (CD-SEM) metrology of lithographic features [1] [2] [3] [4] [5]. It influences both the precision and the accuracy of CD-SEM measurements, while locally damaging the sample. Minimization or elimination of shrinkage is desirable, yet elusive. This error source will be a factor in CD-SEM metrology on polymer materials in EUV lithography. Recent work has demonstrated improved understanding of the trends in the shrinkage response depending on electron beam and target parameters in static measurements [2] [3] [4] [5] [6]. Some research has highlighted a second mode of shrinkage that is apparent over time and progresses as a function of time between consecutive measurements, a form of "dynamic shrinkage" that appears to be activated by electron beam, in which the activated feature perpetually and logarithmically shrinks [7] [8]. Another work has demonstrated that as pitches continue to get smaller with resulting reductions in spaces between lines, charging may emerge as an additional, competing, unpredictable error source for CD-SEM metrology on dense photoresist features, an issue that is predicted to become more common as these spaces become more confined [9]. In this work, we explore the static shrinkage behaviors of various EUV photoresists into the 16 nm half-pitch node, with samples generated using the advanced EUV lithography capable of generating such tight pitches [10]. Dynamic shrinkage behavior was explored on these materials last year [15]. The static shrinkage behaviors will be validated to show compliance with the SEMATECH shrinkage model [5] [6] on small EUV resist features. Using the results of the model fits, a simulation study will predict the shrinkage trends at future nodes. Further studies will confirm whether or not charging phenomena are observable, and the beginning of a charging simulation study will be

  17. Aerosol particle shrinkage event phenomenology in a South European suburban area during 2009-2015 (United States)

    Alonso-Blanco, E.; Gómez-Moreno, F. J.; Núñez, L.; Pujadas, M.; Cusack, M.; Artíñano, B.


    A high number of aerosol particle shrinkage cases (70) have been identified and analyzed from an extensive and representative database of aerosol size distributions obtained between 2009 and 2015 at an urban background site in Madrid (Spain). A descriptive classification based on the process from which the shrinkage began is proposed according which shrinkage events were divided into three groups: (1) NPF + shrinkage (NPF + S) events, (2) aerosol particle growth process + shrinkage (G + S) events, and (3) pure shrinkage (S) events. The largest number of shrinkages corresponded to the S-type followed by NPF + S, while the G + S events were the least frequent group recorded. Duration of shrinkages varied widely from 0.75 to 8.5 h and SR from -1.0 to -11.1 nm h-1. These processes typically occurred in the afternoon, around 18:00 UTC, caused by two situations: i) a wind speed increase usually associated with a change in the wind direction (over 60% of the observations) and ii) the reduction of photochemical activity at the end of the day. All shrinkages were detected during the warm period, mainly between May and August, when local meteorological conditions (high solar irradiance and temperature and low relative humidity), atmospheric processes (high photochemical activity) and availability of aerosol-forming precursors were favorable for their development. As a consequence of these processes, the particles concentration corresponding to the Aitken mode decreased into the nucleation mode. The accumulation mode did not undergo significant changes during these processes. In some cases, a dilution of the particulate content in the ambient air was observed. This work, goes further than others works dealing with aerosol particles shrinkages, as it incorporates as a main novelty a classification methodology for studying these processes. Moreover, compared to other studies, it is supported by a high and representative number of observations. Thus, this study contributes to

  18. α6 Integrin (α6high/Transferrin Receptor (CD71low Keratinocyte Stem Cells Are More Potent for Generating Reconstructed Skin Epidermis Than Rapid Adherent Cells

    Directory of Open Access Journals (Sweden)

    Elodie Metral


    Full Text Available The epidermis basal layer is composed of two keratinocyte populations: Keratinocyte Stem cells (KSC and Transitory Amplifying (TA cells that arise from KSC division. Unfortunately, no specific marker exists to differ between KSC and TA cells. Here, we aimed at comparing two different methods that pretended to isolate these two populations: (i the rapid adhesion method on coated substrate and (ii the flow cytometry method, which is based on the difference in cell surface expressions of the α6 integrin and transferrin receptor (CD71. Then, we compared different parameters that are known to discriminate KSC and TA populations. Interestingly, we showed that both methods allow enrichment in stem cells. However, cell sorting by flow cytometry (α6high/CD71low phenotype leads to a better enrichment of KSC since the colony forming efficiency is five times increased versus total cell suspension, whereas it is only 1.4 times for the adhesion method. Moreover, α6high/CD71low cells give rise to a thicker pluristratified epithelium with lower seeding density and display a low Ki67 positive cells number, showing that they have reached the balance between proliferation and differentiation. We clearly demonstrated that cells isolated by a rapid adherent method are not the same population as KSC isolated by flow cytometry following α6high/CD71low phenotype.

  19. Effect of temperature and humidity on post-gel shrinkage, cusp deformation, bond strength and shrinkage stress - Construction of a chamber to simulate the oral environment. (United States)

    Bicalho, Aline Aredes; de Souza, Silas Júnior Boaventura; de Rosatto, Camila Maria Peres; Tantbirojn, Daranee; Versluis, Antheunis; Soares, Carlos José


    Evaluate the effect of environment on post-gel shrinkage (Shr), cuspal strains (CS), microtensile bond strength (μTBS), elastic modulus (E) and shrinkage stress in molars with large class II restorations. Sixty human molars received standardized Class II mesio-oclusal-distal cavity preparations. Restorations were made with two composites (CHA, Charisma Diamond, Heraus Kulzer and IPS Empress Direct, Ivoclar-Vivadent) using three environment conditions (22°C/50% humidity, 37°C/50% humidity and 37°C/90% humidity) simulated in custom developed chamber. Shr was measured using the strain gauge technique (n=10). CS was measured using strain gauges. Half of the teeth (n=5) were used to assess the elastic modulus (E) and Knoop hardness (KHN) at different depths using microhardness indentation. The other half (n=5) was used to measure the μTBS. The composites and environment conditions were simulated in a two-dimensional finite element analysis of a tooth restoration. Polymerization shrinkage was modeled using Shr data. The Shr, CS, μTBS, KHN and E data were statistically analyzed using two-way ANOVA and Tukey test (significance level: 0.05). Both composites had similar Shr, CS, μTBS and shrinkage stress. CHA had higher elastic modulus than IPS. Increasing temperature and humidity significantly increased Shr, CS and shrinkage stress. μTBS were similar for groups with lower humidity, irrespective of temperature, and higher with higher humidity. E and KHN were constant through the depth for CHA. E and KHN values were affected by environment only for IPS, mainly deeper in the cavity. Shrinkage stress at dentin/composite interface had high inverse correlation with μTBS. Shrinkage stress in enamel had high correlation with CS. Increasing temperature and humidity caused higher post-gel shrinkage and cusp deformation with higher shrinkage stresses in the tooth structure and tooth/restoration interface for both composites tested. The chamber developed for simulating the

  20. Hydrosoluble, UV-crosslinkable and injectable chitosan for patterned cell-laden microgel and rapid transdermal curing hydrogel in vivo. (United States)

    Li, Baoqiang; Wang, Lei; Xu, Feng; Gang, Xiaomin; Demirci, Utkan; Wei, Daqing; Li, Ying; Feng, Yujie; Jia, Dechang; Zhou, Yu


    Natural and biodegradable chitosan with unique amino groups has found widespread applications in tissue engineering and drug delivery. However, its applications have been limited by the poor solubility of native chitosan in neutral pH solution, which subsequently fails to achieve cell-laden hydrogel at physiological pH. To address this, we incorporated UV crosslinking ability in chitosan, allowing fabrication of patterned cell-laden and rapid transdermal curing hydrogel in vivo. The hydrosoluble, UV crosslinkable and injectable N-methacryloyl chitosan (N-MAC) was synthesized via single-step chemoselective N-acylation reaction, which simultaneously endowed chitosan with well solubility in neutral pH solution, UV crosslinkable ability and injectability. The solubility of N-MAC in neutral pH solution increased 2.21-fold with substitution degree increasing from 10.9% to 28.4%. The N-MAC allowed fabrication of cell-laden microgels with on-demand patterns via photolithography, and the cell viability in N-MAC hydrogel maintained 96.3 ± 1.3% N-MAC allowed rapid transdermal curing hydrogel in vivo within 60s through minimally invasive clinical surgery. Histological analysis revealed that low-dose UV irradiation hardly induced skin injury and acute inflammatory response disappeared after 7 days. N-MAC would allow rapid, robust and cost-effective fabrication of patterned cell-laden polysaccharide microgels with unique amino groups serving as building blocks for tissue engineering and rapid transdermal curing hydrogel in vivo for localized and sustained protein delivery. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

  1. Rapid establishment of a HEK 293 cell line expressing FVIII-BDD using AAV site-specific integration plasmids. (United States)

    Liu, Xiaomei; Ping, Han; Zhang, Chun


    Stable human cell lines have gradually become the preferred system for large scale production of recombinant proteins for clinical applications because of their capacity of proper protein post-translational modification and low immunogenicity. However, human cell line development technologies are commonly based on random genome integration of protein expressing genes. It is required to screen large numbers of cell clones to identify stable high producer cell clones and the cell line development process usually takes 6 to 12 months. Adeno-associated virus type 2 (AAV2) Rep protein is known to induce rAAV DNA integration into a specific site (AAVS1) of the human chromosome 19 and integrated transgenes can stably express proteins. We take advantage of this AAV unique feature to develop a rapid protocol to clone a stable recombinant protein expression human cell line. We have constructed two plasmids. One plasmid, pSVAV2, contains the AAV rep gene for the synthesis of integrase; the second plasmid, pTRP5GFPFVIII-BDD, contains B-domain-deleted factor VIII (FVIII-BDD) and GFP gene flanked by AAV ITRs. Human embryonic kidney (HEK) 293 cells were co-transfected with the two plasmids and the cells were screened by green fluorescence to establish the recombinant FVIII-BDD cell line. PCR analysis showed that the FVIII-BDD gene has been integrated into the AAVS1 site of human chromosome 19. The FVIII-BDD protein secreted into the extracellular media exhibited coagulant activity. We developed a method of rapid establishment of human HEK 293 cell line expressing recombinant FVIII-BDD protein with AAV site-specific integration plasmids.

  2. Transcriptome Signatures Reveal Rapid Induction of Immune-Responsive Genes in Human Memory CD8(+) T Cells. (United States)

    Yang, Cheng; Khanniche, Asma; DiSpirito, Joanna R; Ji, Ping; Wang, Shujun; Wang, Ying; Shen, Hao


    Memory T cells (TM) play a prominent role in protection and auto-immunity due to their ability to mount a more effective response than naïve T cells (TN). However, the molecular mechanisms underlying enhanced functionality of TM are not well defined, particularly in human TM. We examined the global gene expression profiles of human CD8(+) TN and TM before and after stimulation. There were 1,284, 1,373 and 1,629 differentially expressed genes between TN and TM at 0 hr, 4 hr and 24 hr after stimulation, respectively, with more genes expressed to higher levels in TM. Genes rapidly up-regulated in TN cells were largely involved in nitrogen, nucleoside and amino acid metabolisms. In contrast, those in CD8(+) TM were significantly enriched for immune-response-associated processes, including cytokine production, lymphocyte activation and chemotaxis. Multiple cytokines were rapidly up-regulated in TM cells, including effector cytokines known to be produced by CD8(+) T cells and important for their functions, as well as regulatory cytokines, both pro- and anti-inflammatory, that are not typically produced by CD8(+) T cells. These results provide new insights into molecular mechanisms that contribute to the enhanced functionality of human CD8(+) TM and their prominent role in protection and auto-immunity.

  3. Rapid fabrication of microfluidic polymer electrolyte membrane fuel cell in PDMS by surface patterning of perfluorinated ion-exchange resin

    Energy Technology Data Exchange (ETDEWEB)

    Song, Yong-Ak; Han, Jongyoon [Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139 (United States); Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139 (United States); Batista, Candy [Roxbury Community College, 1234 Columbus Ave., Roxbury Crossing, MA 02120 (United States); Sarpeshkar, Rahul [Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139 (United States)


    In this paper we demonstrate a simple and rapid fabrication method for a microfluidic polymer electrolyte membrane (PEM) fuel cell using polydimethylsiloxane (PDMS), which has become the de facto standard material in BioMEMS. Instead of integrating a Nafion sheet film between two layers of a PDMS device in a traditional ''sandwich format,'' we pattern a perfluorinated ion-exchange resin such as a Nafion resin on a glass substrate using a reversibly bonded PDMS microchannel to generate an ion-selective membrane between the fuel-cell electrodes. After this patterning step, the assembly of the microfluidic fuel cell is accomplished by simple oxygen plasma bonding between the PDMS chip and the glass substrate. In an example implementation, the planar PEM microfluidic fuel cell generates an open circuit voltage of 600-800 mV and delivers a maximum current output of nearly 4 {mu}A. To enhance the power output of the fuel cell we utilize self-assembled colloidal arrays as a support matrix for the Nafion resin. Such arrays allow us to increase the thickness of the ion-selective membrane to 20 {mu}m and increase the current output by 166%. Our novel fabrication method enables rapid prototyping of microfluidic fuel cells to study various ion-exchange resins for the polymer electrolyte membrane. Our work will facilitate the development of miniature, implantable, on-chip power sources for biomedical applications. (author)

  4. Omcg1 is critically required for mitosis in rapidly dividing mouse intestinal progenitors and embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Teddy Léguillier


    Recent studies have shown that factors involved in transcription-coupled mRNA processing are important for the maintenance of genome integrity. How these processes are linked and regulated in vivo remains largely unknown. In this study, we addressed in the mouse model the function of Omcg1, which has been shown to participate in co-transcriptional processes, including splicing and transcription-coupled repair. Using inducible mouse models, we found that Omcg1 is most critically required in intestinal progenitors. In absence of OMCG1, proliferating intestinal epithelial cells underwent abnormal mitosis followed by apoptotic cell death. As a consequence, the crypt proliferative compartment of the small intestine was quickly and totally abrogated leading to the rapid death of the mice. Lack of OMCG1 in embryonic stem cells led to a similar cellular phenotype, with multiple mitotic defects and rapid cell death. We showed that mutant intestinal progenitors and embryonic stem cells exhibited a reduced cell cycle arrest following irradiation, suggesting that mitotic defects may be consecutive to M phase entry with unrepaired DNA damages. These findings unravel a crucial role for pre-mRNA processing in the homeostasis of the small intestine and point to a major role of OMCG1 in the maintenance of genome integrity.

  5. Somatically Hypermutated Plasmodium-Specific IgM(+) Memory B Cells Are Rapid, Plastic, Early Responders upon Malaria Rechallenge. (United States)

    Krishnamurty, Akshay T; Thouvenel, Christopher D; Portugal, Silvia; Keitany, Gladys J; Kim, Karen S; Holder, Anthony; Crompton, Peter D; Rawlings, David J; Pepper, Marion


    Humoral immunity consists of pre-existing antibodies expressed by long-lived plasma cells and rapidly reactive memory B cells (MBC). Recent studies of MBC development and function after protein immunization have uncovered significant MBC heterogeneity. To clarify functional roles for distinct MBC subsets during malaria infection, we generated tetramers that identify Plasmodium-specific MBCs in both humans and mice. Long-lived murine Plasmodium-specific MBCs consisted of three populations: somatically hypermutated immunoglobulin M(+) (IgM(+)) and IgG(+) MBC subsets and an unmutated IgD(+) MBC population. Rechallenge experiments revealed that high affinity, somatically hypermutated Plasmodium-specific IgM(+) MBCs proliferated and gave rise to antibody-secreting cells that dominated the early secondary response to parasite rechallenge. IgM(+) MBCs also gave rise to T cell-dependent IgM(+) and IgG(+)B220(+)CD138(+) plasmablasts or T cell-independent B220(-)CD138(+) IgM(+) plasma cells. Thus, even in competition with IgG(+) MBCs, IgM(+) MBCs are rapid, plastic, early responders to a secondary Plasmodium rechallenge and should be targeted by vaccine strategies. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Evaluation of polymerization shrinkage, polymerization shrinkage stress, wear resistance, and compressive strength of a silorane-based composite: A finite element analysis study

    Directory of Open Access Journals (Sweden)

    Suresh Mitthra


    Full Text Available Background: Understanding the mechanical properties is important in predicting the clinical behavior of composites. Finite element analysis (FEA evaluates properties of materials replicating clinical scenario. Aim: This study evaluated polymerization shrinkage and stress, wear resistance (WR, and compressive strength (CS of silorane in comparison with two methacrylate resins. Settings and Design: This study design was a numerical study using FEA. Materials and Methods: Three-dimensional (3D models of maxillary premolar with Class I cavities (2 mm depth, 4 mm length, and 2.5 mm width created and restored with silorane, nanohybrid, and microhybrid; Groups I, II, and III, respectively. Loads of 200–600 N were applied. Polymerization shrinkage was first determined by displacement produced in the X, Y, and Z planes. Maximum stress distribution due to shrinkage was calculated using AN SYS software. 3D cube models of composite resins were simulated with varying filler particle size. Similar loads were applied. WR and compressive stress were calculated: K W L/H and load/cross-sectional area, respectively. Statistical analysis done using one-way ANOVA, Kruskal–Wallis, and Tukey's honestly significant difference test (P < 0.05. Results: Polymerization shrinkage (0.99% and shrinkage stress (233.21 Mpa of silorane were less compared to microhybrid (2.14% and 472.43 Mpa and nanohybrid (2.32% and 464.88 Mpa. Silorane (7.92×/1011 μm/mm3 and nanohybrid (7.79×/1011 showed superior WR than microhybrid (1.113×/1017. There was no significant difference in compressive stress among the groups. Conclusion: Silorane exhibited less polymerization shrinkage and shrinkage stress compared to methacrylates. Silorane and nanohybrid showed greater WR compared to microhybrid. CS of all groups was similar.

  7. The caudal regeneration blastema is an accumulation of rapidly proliferating stem cells in the flatworm Macrostomum lignano. (United States)

    Egger, Bernhard; Gschwentner, Robert; Hess, Michael W; Nimeth, Katharina T; Adamski, Zbigniew; Willems, Maxime; Rieger, Reinhard; Salvenmoser, Willi


    Macrostomum lignano is a small free-living flatworm capable of regenerating all body parts posterior of the pharynx and anterior to the brain. We quantified the cellular composition of the caudal-most body region, the tail plate, and investigated regeneration of the tail plate in vivo and in semithin sections labeled with bromodeoxyuridine, a marker for stem cells (neoblasts) in S-phase. The tail plate accomodates the male genital apparatus and consists of about 3,100 cells, about half of which are epidermal cells. A distinct regeneration blastema, characterized by a local accumulation of rapidly proliferating neoblasts and consisting of about 420 cells (excluding epidermal cells), was formed 24 hours after amputation. Differentiated cells in the blastema were observed two days after amputation (with about 920 blastema cells), while the male genital apparatus required four to five days for full differentiation. At all time points, mitoses were found within the blastema. At the place of organ differentiation, neoblasts did not replicate or divide. After three days, the blastema was made of about 1420 cells and gradually transformed into organ primordia, while the proliferation rate decreased. The cell number of the tail plate, including about 960 epidermal cells, was restored to 75% at this time point. Regeneration after artificial amputation of the tail plate of adult specimens of Macrostomum lignano involves wound healing and the formation of a regeneration blastema. Neoblasts undergo extensive proliferation within the blastema. Proliferation patterns of S-phase neoblasts indicate that neoblasts are either determined to follow a specific cell fate not before, but after going through S-phase, or that they can be redetermined after S-phase. In pulse-chase experiments, dispersed distribution of label suggests that S-phase labeled progenitor cells of the male genital apparatus undergo further proliferation before differentiation, in contrast to progenitor cells of

  8. Rapid generation of mature hepatocyte-like cells from human induced pluripotent stem cells by an efficient three-step protocol. (United States)

    Chen, Yu-Fan; Tseng, Chien-Yu; Wang, Hsei-Wei; Kuo, Hung-Chih; Yang, Vincent W; Lee, Oscar K


    Liver transplantation is the only definitive treatment for end-stage cirrhosis and fulminant liver failure, but the lack of available donor livers is a major obstacle to liver transplantation. Recently, induced pluripotent stem cells (iPSCs) derived from the reprogramming of somatic fibroblasts, have been shown to resemble embryonic stem (ES) cells in that they have pluripotent properties and the potential to differentiate into all cell lineages in vitro, including hepatocytes. Thus, iPSCs could serve as a favorable cell source for a wide range of applications, including drug toxicity testing, cell transplantation, and patient-specific disease modeling. Here, we describe an efficient and rapid three-step protocol that is able to rapidly generate hepatocyte-like cells from human iPSCs. This occurs because the endodermal induction step allows for more efficient and definitive endoderm cell formation. We show that hepatocyte growth factor (HGF), which synergizes with activin A and Wnt3a, elevates the expression of the endodermal marker Foxa2 (forkhead box a2) by 39.3% compared to when HGF is absent (14.2%) during the endodermal induction step. In addition, iPSC-derived hepatocytes had a similar gene expression profile to mature hepatocytes. Importantly, the hepatocyte-like cells exhibited cytochrome P450 3A4 (CYP3A4) enzyme activity, secreted urea, uptake of low-density lipoprotein (LDL), and possessed the ability to store glycogen. Moreover, the hepatocyte-like cells rescued lethal fulminant hepatic failure in a nonobese diabetic severe combined immunodeficient mouse model. We have established a rapid and efficient differentiation protocol that is able to generate functional hepatocyte-like cells from human iPSCs. This may offer an alternative option for treatment of liver diseases. Copyright © 2011 American Association for the Study of Liver Diseases.

  9. All-in-one nanowire-decorated multifunctional membrane for rapid cell lysis and direct DNA isolation.

    KAUST Repository

    So, Hongyun


    This paper describes a handheld device that uses an all-in-one membrane for continuous mechanical cell lysis and rapid DNA isolation without the assistance of power sources, lysis reagents, and routine centrifugation. This nanowire-decorated multifunctional membrane was fabricated to isolate DNA by selective adsorption to silica surface immediately after disruption of nucleus membranes by ultrasharp tips of nanowires for a rapid cell lysis, and it can be directly assembled with commercial syringe filter holders. The membrane was fabricated by photoelectrochemical etching to create microchannel arrays followed by hydrothermal synthesis of nanowires and deposition of silica. The proposed membrane successfully purifies high-quality DNA within 5 min, whereas a commercial purification kit needs more than an hour.

  10. Rapid Antibiotic Susceptibility Testing of Uropathogenic E. coli by Tracking Submicron Scale Motion of Single Bacterial Cells. (United States)

    Syal, Karan; Shen, Simon; Yang, Yunze; Wang, Shaopeng; Haydel, Shelley E; Tao, Nongjian


    To combat antibiotic resistance, a rapid antibiotic susceptibility testing (AST) technology that can identify resistant infections at disease onset is required. Current clinical AST technologies take 1-3 days, which is often too slow for accurate treatment. Here we demonstrate a rapid AST method by tracking sub-μm scale bacterial motion with an optical imaging and tracking technique. We apply the method to clinically relevant bacterial pathogens, Escherichia coli O157: H7 and uropathogenic E. coli (UPEC) loosely tethered to a glass surface. By analyzing dose-dependent sub-μm motion changes in a population of bacterial cells, we obtain the minimum bactericidal concentration within 2 h using human urine samples spiked with UPEC. We validate the AST method using the standard culture-based AST methods. In addition to population studies, the method allows single cell analysis, which can identify subpopulations of resistance strains within a sample.

  11. Rapid effects of 17beta-estradiol on epithelial TRPV6 Ca2+ channel in human T84 colonic cells.

    LENUS (Irish Health Repository)

    Irnaten, Mustapha


    The control of calcium homeostasis is essential for cell survival and is of crucial importance for several physiological functions. The discovery of the epithelial calcium channel Transient Receptor Potential Vaniloid (TRPV6) in intestine has uncovered important Ca(2+) absorptive pathways involved in the regulation of whole body Ca(2+) homeostasis. The role of steroid hormone 17beta-estradiol (E(2)), in [Ca(2+)](i) regulation involving TRPV6 has been only limited at the protein expression levels in over-expressing heterologous systems. In the present study, using a combination of calcium-imaging, whole-cell patch-clamp techniques and siRNA technology to specifically knockdown TRPV6 protein expression, we were able to (i) show that TRPV6 is natively, rather than exogenously, expressed at mRNA and protein levels in human T84 colonic cells, (ii) characterize functional TRPV6 channels and (iii) demonstrate, for the first time, the rapid effects of E(2) in [Ca(2+)](i) regulation involving directly TRPV6 channels in T84 cells. Treatment with E(2) rapidly (<5 min) enhanced [Ca(2+)](i) and this increase was partially but significantly prevented when cells were pre-treated with ruthenium red and completely abolished in cells treated with siRNA specifically targeting TRPV6 protein expression. These results indicate that when cells are stimulated by E(2), Ca(2+) enters the cell through TRPV6 channels. TRPV6 channels in T84 cells contribute to the Ca(2+) entry\\/signalling pathway that is sensitive to 17beta-estradiol.

  12. Rapid prototyping of nano- and micro-patterned substrates for the control of cell neuritogenesis by topographic and chemical cues

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Ajay V.; Gailite, Lasma; Vyas, Varun [European School of Molecular Medicine (SEMM), IFOM-IEO Campus, Via Adamello 16, I-20139 Milano (Italy); CIMAINA and Dipartimento di Fisica, Universita di Milano, via Celoria 16, I-20133 Milano (Italy); Lenardi, Cristina, E-mail: [CIMAINA and Dipartimento di Scienze Molecolari Applicate ai Biosistemi, Universita di Milano, via Trentacoste 2, I-20134 Milano (Italy); Fondazione Filarete, viale Ortles 22/4, I-20139 Milano (Italy); Forti, Stefania [CIMAINA and Dipartimento di Fisica, Universita di Milano, via Celoria 16, I-20133 Milano (Italy); Matteoli, Michela [Dipartimento di Farmacologia, Chemioterapia e Tossicologia Medica, Universita di Milano, via Vanvitelli 32, I-20139 Milano (Italy); Fondazione Filarete, viale Ortles 22/4, I-20139 Milano (Italy); Milani, Paolo, E-mail: [CIMAINA and Dipartimento di Fisica, Universita di Milano, via Celoria 16, I-20133 Milano (Italy); Fondazione Filarete, viale Ortles 22/4, I-20139 Milano (Italy)


    Rapid prototyping of titania substrates with micro and nanofeatures is obtained by combining nanosphere lithography with supersonic cluster beam deposition on protein-functionalized glass supports. The proliferation and differentiation of PC12 cells were studied on these substrates. The facile control and modification of the substrate structure at the micro- and nanoscale allowed us to characterize the role of functional and structural features on neuritogenesis and to control this phenomenon by identifying the optimal topography.

  13. A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix. (United States)

    Hellewell, Andrew L; Rosini, Silvia; Adams, Josephine C


    The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation. Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

  14. Influence of fly ash fineness on water requirement and shrinkage of blended cement mortars

    Directory of Open Access Journals (Sweden)

    Vanissorn Vimonsatit


    Full Text Available In this paper, the influence of fly ash fineness on water requirement and shrinkage of blended cement mortar was studied. The results indicate that the water requirement and shrinkage characteristic of the blended cement mortar are dependent on fly ash fineness and replacement level. The use of coarse fly ash slightly reduces the water requirement but greatly reduced the drying and the autogenous shrinkage of the blended cement mortars and the reduction is more with an increase in the fly ash replacement level. The finer fly ashes further reduce the water requirement, but increase the drying and the autogenous shrinkages as compared with coarser fly ash. The incorporation of superplasticizer drastically reduces the water requirement, but the effect on the drying and autogenous shrinkages of the normal Portland cement mortar is small. However, for the fly ash mortar, the use of superplasticizer results in a decrease in drying shrinkage and in a substantial increase in the autogenous shrinkage particularly for the fine fly ash at a high replacement level.

  15. Influence of shade of adhesive resin cement on its polymerization shrinkage

    Directory of Open Access Journals (Sweden)

    Ender Akan


    Full Text Available Resin cements have the ability to bond to the tooth structure and to several indirect restorative materials and they are available in different transparency and shades. However, polymerization shrinkage remains to be a problem that leads to gap formation at the margins of restoration, and accordingly microleakage may occur. The purpose of this study was to investigate the influence of the shade of adhesive resin cement on its polymerization shrinkage. Five shades (Group 1: universal; Group 2: brown; Group 3: clear; Group 4: opaque white; Group 5: opaque yellow of a dual-cure composite cement system (Clearfil Esthetic Cement, Kuraray, Tokyo, Japan were studied with 10 specimens of each shade. Volumetric shrinkages of the composite cements of these five shades were evaluated using a video-imaging device (Acu-Vol; Bisco, Inc. by measuring the volumes before and after polymerization. Different shades displayed different volumetric shrinkages. Group 5 (2.62% and Group 2 (3.96% displayed the lowest and highest shrinkage percentages, respectively, with their respective shrinkage values being significantly different from those of the other groups (p < 0.05. The obtained results indicate that the shade of resin cement does affect its degree of polymerization shrinkage.

  16. Investigations of linear contraction and shrinkage stresses development in hypereutectic al-si binary alloys

    Directory of Open Access Journals (Sweden)

    J. Mutwil


    Full Text Available Shrinkage phenomena during solidification and cooling of hypereutectic aluminium-silicon alloys (AlSi18, AlSi21 have been examined. A vertical shrinkage rod casting with circular cross-section (constant or fixed: tapered has been used as a test sample. Two type of experiments have been conducted: 1 on development of the test sample linear dimension changes (linear expansion/contraction, 2 on development of shrinkage stresses in the test sample. By the linear contraction experiments the linear dimension changes of the test sample and the metal test mould as well a temperature in six points of the test sample have been registered. By shrinkage stresses examination a shrinkage tension force and linear dimension changes of the test sample as well a temperature in three points of the test sample have been registered. Registered time dependences of the test bar and the test mould linear dimension changes have shown, that so-called pre-shrinkage extension has been mainly by mould thermal extension caused. The investigation results have shown that both: the linear contraction as well as the shrinkage stresses development are evident dependent on metal temperature in a warmest region the sample (thermal centre.

  17. Influence of shrinkage porosity on fatigue performance of iron castings and life estimation method

    Directory of Open Access Journals (Sweden)

    Wei Liu


    Full Text Available Shrinkage porosity exists more or less in heavy castings, and it plays an important role in the fatigue behavior of cast materials. In this study, fatigue tests were carried out on the QT400-18 cast iron specimens containing random degrees of shrinkage porosity defect. Experimental results showed that the order of magnitude of life scattered from 103 to 106 cycles when the shrinkage percentage ranged from 0.67% to 5.91%. SEM analyses were carried out on the shrinkage porosity region. The inter-granular discontinuous, micro cracks and inclusions interfered with the fatigue sliding or hindering process. The slip in shrinkage porosity region was not as orderly as the ordinary continuous medium. The shrinkage porosity area on fracture surface (SPAFS and alternating stress intensity factor (ASIF were applied to evaluate the tendency of residual life distribution; their relationship was fitted by negative exponent functions. Based on the intermediate variable of ASIF, a fatigue life prediction model of nodular cast iron containing shrinkage porosity defects was established. The modeling prediction was in agreement with the experimental results.

  18. Creep and shrinkage of high performance lightweight concrete: A multi-scale investigation (United States)

    Lopez, Mauricio

    This multi-scale investigation aimed to provide new knowledge and understanding of creep and shrinkage of high performance lightweight concrete (HPLC) by assessing prestress losses in HPLC prestressed members in a large-scale study; by quantifying the effect of the constituent materials and external conditions on creep and shrinkage in a medium-scale study; and by improving the fundamental understanding of creep and shrinkage in a small-scale study. Creep plus shrinkage prestress losses were between two and eight times lower than those estimated for the design standards and approximately 50% of those measured in similar strength normal weight high performance concrete girders. The lower creep and shrinkage exhibited by HPLC was found to be caused by a synergy between the pre-soaked lightweight aggregate and the low water-to-cementitious material ratio matrix. That is, the water contained in the lightweight aggregate contributes to enhance hydration by providing an internal moist curing. The water in the aggregate also contributes to maintain a high internal relative humidity which reduces or eliminates autogenous shrinkage. This higher internal relative humidity also reduces creep by preventing load-induced water migration. Finally, lightweight aggregate exhibits a better elastic compatibility with the paste than normal weight aggregate. This improved elastic matching and the enhanced hydration are believed to reduce peak deformations at the ITZ which further decreases creep and shrinkage.

  19. Polymerisation shrinkage versus layer thickness of a dentine bonding resin: Method development

    Directory of Open Access Journals (Sweden)

    Jafarzadeh T


    Full Text Available Dentine bonding systems are usually unfilled, and so their shrinkage may be significant. High"nshrinkage may cause internal stress at the interface between resin-composite restoration and the dentine"nsubstrate. Failure of the adhesive interface may be observed due to the interna! stress. The aims of this"nstudy were:"nA To obtain a suitable method for measuring the kinetics of polymerisation shrinkage in unfilled resm at different thicknesses, particularly for thin films."nB Consideraing the effect of thickness on shrinkage."nScotchbond Multipurpose (3M adhesive bond resin was used. To overcome the particular challenges presented by thin films, a filled-ring measurement procedure was used. Also, a non-contact laser analogue displacement sensor system was developed and applied to measure polymerisation shrinkage. Regression analysis was performed on a complete data set. Non-linear regression analysis established a logarithmic relationship between polymerisation shrinkage and layer thickness. The method applied in this study was found to be sensitive and accurate procedure for determining photo-polymerisation shrinkage of thin films. Polymerisation shrinkage increased with logarithmic of the adhesive thickness.

  20. Compositional Changes for Reduction of Polymerisation-Induced Shrinkage in Holographic Photopolymers

    Directory of Open Access Journals (Sweden)

    D. Cody


    Full Text Available Polymerisation-induced shrinkage is one of the main reasons why many photopolymer materials are not used for certain applications including holographic optical elements and holographic data storage. Here, two compositional changes for the reduction of shrinkage in an acrylamide-based photopolymer are reported. A holographic interferometric technique was used to study changes in the dynamics of the shrinkage processes occurring in the modified photopolymer during holographic recording in real time. Firstly, the effect of the replacement of the acrylamide monomer in the photopolymer composition with a larger monomer molecule, diacetone acrylamide, on polymerisation-induced shrinkage has been studied. A reduction in relative shrinkage of 10–15% is obtained using this compositional change. The second method tested for shrinkage reduction involved the incorporation of BEA-type zeolite nanoparticles in the acrylamide-based photopolymer. A reduction in relative shrinkage of 13% was observed for acrylamide photopolymer layers doped with 2.5% wt. BEA zeolites in comparison to the undoped photopolymer.

  1. Measurement of Shrinkage and Cracking in Lyophilized Amorphous Cakes. Part II: Kinetics. (United States)

    Ullrich, Sabine; Seyferth, Stefan; Lee, Geoffrey


    Measurement of the kinetic development of shrinkage and cracking of an amorphous trehalose cake as they take place during lyophilization. A novel technique has been developed which monitors a vial in situ during the freeze-drying cycle. The 2-dimensional degrees of shrinkage and cracking in its top surface are determined quantitatively using a digital camera and evaluated using AxioVision. Shrinkage and cracking develop largely already during programmed primary drying and are coupled. For trehalose, sucrose and maltose no clear correlation between shrinkage and wg' is found. There is no dependence of cake rim detachment from the vial inner surface on the trehalose concentration. Cake adhesion is therefore likely not the only determining factor for detachment and shrinkage. If shrinkage can occur during primary drying, then this relaxes the drying tension produced by desorption of non-frozen water out of the amorphous structure left behind as the sublimation front passes through a volume element, and causes little or no cracking. If shrinkage is restrained, then the drying tension is relaxed by cracking of the brittle cake.

  2. Rapid progression of mediastinal tumor within a few days: A case report of T cell lymphoblastic lymphoma

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, Tae Ran; Lee, Young Kyung; Jun, Hyun Jung; Jung, Eun Ah; Son, Jin Sung [Seoul Medical Center, Seoul (Korea, Republic of)


    T-cell lymphoblastic lymphoma is a highly aggressive tumor derived from lymphocyte of the thymus, which accounts for 2% of non-Hodgkin's lymphoma. The disease occurs most commonly in adolescent and young adult males. It often results in respiratory emergency because of high proliferation rate. In this case, we confirmed the rapid progression of T-cell lymphoblastic lymphoma through the chest CT scan with one week interval. Three days of empirical chemotherapy resulted in substantial reduction of mediastinal mass, pleural thickening and pleural effusion.

  3. Rapid and Sensitive Detection of Breast Cancer Cells in Patient Blood with Nuclease-Activated Probe Technology

    Directory of Open Access Journals (Sweden)

    Sven Kruspe


    Full Text Available A challenge for circulating tumor cell (CTC-based diagnostics is the development of simple and inexpensive methods that reliably detect the diverse cells that make up CTCs. CTC-derived nucleases are one category of proteins that could be exploited to meet this challenge. Advantages of nucleases as CTC biomarkers include: (1 their elevated expression in many cancer cells, including cells implicated in metastasis that have undergone epithelial-to-mesenchymal transition; and (2 their enzymatic activity, which can be exploited for signal amplification in detection methods. Here, we describe a diagnostic assay based on quenched fluorescent nucleic acid probes that detect breast cancer CTCs via their nuclease activity. This assay exhibited robust performance in distinguishing breast cancer patients from healthy controls, and it is rapid, inexpensive, and easy to implement in most clinical labs. Given its broad applicability, this technology has the potential to have a substantive impact on the diagnosis and treatment of many cancers.

  4. Rapid Conversion from Carbohydrates to Large-Scale Carbon Quantum Dots for All-Weather Solar Cells. (United States)

    Tang, Qunwei; Zhu, Wanlu; He, Benlin; Yang, Peizhi


    A great challenge for state-of-the-art solar cells is to generate electricity in all weather. We present here the rapid conversion of carbon quantum dots (CQDs) from carbohydrates (including glucose, maltol, sucrose) for an all-weather solar cell, which comprises a CQD-sensitized mesoscopic titanium dioxide/long-persistence phosphor (m-TiO 2 /LPP) photoanode, a I - /I 3 - redox electrolyte, and a platinum counter electrode. In virtue of the light storing and luminescent behaviors of LPP phosphors, the generated all-weather solar cells can not only convert sunlight into electricity on sunny days but persistently realize electricity output in all dark-light conditions. The maximized photoelectric conversion efficiency is as high as 15.1% for so-called all-weather CQD solar cells in dark conditions.

  5. Precise spatio-temporal control of rapid optogenetic cell ablation with mem-KillerRed in Zebrafish. (United States)

    Buckley, C; Carvalho, M T; Young, L K; Rider, S A; McFadden, C; Berlage, C; Verdon, R F; Taylor, J M; Girkin, J M; Mullins, J J


    The ability to kill individual or groups of cells in vivo is important for studying cellular processes and their physiological function. Cell-specific genetically encoded photosensitizing proteins, such as KillerRed, permit spatiotemporal optogenetic ablation with low-power laser light. We report dramatically improved resolution and speed of cell targeting in the zebrafish kidney through the use of a selective plane illumination microscope (SPIM). Furthermore, through the novel incorporation of a Bessel beam into the SPIM imaging arm, we were able to improve on targeting speed and precision. The low diffraction of the Bessel beam coupled with the ability to tightly focus it through a high NA lens allowed precise, rapid targeting of subsets of cells at anatomical depth in live, developing zebrafish kidneys. We demonstrate that these specific targeting strategies significantly increase the speed of optoablation as well as fish survival.

  6. Control of drying shrinkage in magnesium silicate hydrate (M-S-H) gel mortars


    Zhang, T; Liang, X; Li, C; Lorin, M; Li, Y; Vandeperre, LJM; Cheeseman, CR


    ? 2016 Elsevier Ltd. All rights reserved.Magnesium silicate hydrate (M-S-H) gel can be formed by the reaction of MgO with amorphous silica in the presence of sodium hexametaphosphate (Na-HMP). Typical pastes contain 40% MgO and 60% SF and have a w/c ratio of 0.5, but these exhibit shrinkage cracking on drying. The shrinkage characteristics of M-S-H mortar samples containing different additions of sand have been studied using dilatometry. The drying shrinkage was found to decrease with increas...

  7. Glucose Evokes Rapid Ca2+ and Cyclic AMP Signals by Activating the Cell-Surface Glucose-Sensing Receptor in Pancreatic β-Cells (United States)

    Nakagawa, Yuko; Nagasawa, Masahiro; Medina, Johan; Kojima, Itaru


    Glucose is a primary stimulator of insulin secretion in pancreatic β-cells. High concentration of glucose has been thought to exert its action solely through its metabolism. In this regard, we have recently reported that glucose also activates a cell-surface glucose-sensing receptor and facilitates its own metabolism. In the present study, we investigated whether glucose activates the glucose-sensing receptor and elicits receptor-mediated rapid actions. In MIN6 cells and isolated mouse β-cells, glucose induced triphasic changes in cytoplasmic Ca2+ concentration ([Ca2+]c); glucose evoked an immediate elevation of [Ca2+]c, which was followed by a decrease in [Ca2+]c, and after a certain lag period it induced large oscillatory elevations of [Ca2+]c. Initial rapid peak and subsequent reduction of [Ca2+]c were independent of glucose metabolism and reproduced by a nonmetabolizable glucose analogue. These signals were also blocked by an inhibitor of T1R3, a subunit of the glucose-sensing receptor, and by deletion of the T1R3 gene. Besides Ca2+, glucose also induced an immediate and sustained elevation of intracellular cAMP ([cAMP]c). The elevation of [cAMP]c was blocked by transduction of the dominant-negative Gs, and deletion of the T1R3 gene. These results indicate that glucose induces rapid changes in [Ca2+]c and [cAMP]c by activating the cell-surface glucose-sensing receptor. Hence, glucose generates rapid intracellular signals by activating the cell-surface receptor. PMID:26630567

  8. Rapid generation of mitochondrial superoxide induces mitochondrion-dependent but caspase-independent cell death in hippocampal neuronal cells that morphologically resembles necroptosis

    Energy Technology Data Exchange (ETDEWEB)

    Fukui, Masayuki; Choi, Hye Joung; Zhu, Bao Ting, E-mail:


    Studies in recent years have revealed that excess mitochondrial superoxide production is an important etiological factor in neurodegenerative diseases, resulting from oxidative modifications of cellular lipids, proteins, and nucleic acids. Hence, it is important to understand the mechanism by which mitochondrial oxidative stress causes neuronal death. In this study, the immortalized mouse hippocampal neuronal cells (HT22) in culture were used as a model and they were exposed to menadione (also known as vitamin K{sub 3}) to increase intracellular superoxide production. We found that menadione causes preferential accumulation of superoxide in the mitochondria of these cells, along with the rapid development of mitochondrial dysfunction and cellular ATP depletion. Neuronal death induced by menadione is independent of the activation of the MAPK signaling pathways and caspases. The lack of caspase activation is due to the rapid depletion of cellular ATP. It was observed that two ATP-independent mitochondrial nucleases, namely, AIF and Endo G, are released following menadione exposure. Silencing of their expression using specific siRNAs results in transient suppression (for ∼ 12 h) of mitochondrial superoxide-induced neuronal death. While suppression of the mitochondrial superoxide dismutase expression markedly sensitizes neuronal cells to mitochondrial superoxide-induced cytotoxicity, its over-expression confers strong protection. Collectively, these findings showed that many of the observed features associated with mitochondrial superoxide-induced cell death, including caspase independency, rapid depletion of ATP level, mitochondrial release of AIF and Endo G, and mitochondrial swelling, are distinctly different from those of apoptosis; instead they resemble some of the known features of necroptosis. -- Highlights: ► Menadione causes mitochondrial superoxide accumulation and injury. ► Menadione-induced cell death is caspase-independent, due to rapid depletion of

  9. Shrinkage/swelling of compacted clayey loose and dense soils (United States)

    Nowamooz, Hossein; Masrouri, Farimah


    This Note presents an experimental study performed on expansive compacted loose and dense samples using osmotic oedometers. Several successive wetting and drying cycles were applied in a suction range between 0 and 8 MPa under different values of constant net vertical stress (15, 30, and 60 kPa). During the suction cycles, the dense samples showed cumulative swelling strains, while the loose samples showed volumetric shrinkage accumulation. At the end of the suction cycles, the volumetric strains converged to an equilibrium stage that indicated elastic behavior of the swelling soil for any further hydraulic variations. At this stage, the compression curves for the studied soil at the different imposed suctions (0, 2, and 8 MPa) converged towards the saturated state curve for the high applied vertical stresses. We defined this pressure as the saturation stress(P). The compression curves provided sufficient data to examine the soil mechanical behavior at the equilibrium stage. To cite this article: H. Nowamooz, F. Masrouri, C. R. Mecanique 337 (2009).

  10. Shrinkage, abrasion, erosion and sorption of clay plasters

    Directory of Open Access Journals (Sweden)

    Minke, G.


    Full Text Available At the Buildung Research Institute (FEB, Faculty of Architecture, University of Kassel, Germany, in the last years several hundred tests were made to study the characteristics of different loam mortars in respect of their linear shrinkage, absorption of humidity and their resistance against abrasion and erosion. In order to get data about abrasion and erosion new test methods and special apparatusses were developed. The mortars tested, chosen from the market, showed extremely varying test results.

    En el Laboratorio de Construcciones Experimentales (FEB de la Facultad de Arquitectura, Universidad de Kassel, Alemania, fueron testeados cientos de diferentes pruebas de revoque de barro para estudiar su contracción durante el secado, su absorción de humedad y su resistencia contra abrasión, erosión y absorción. Para recibir datos sobre abrasión y erosión, nuevas aparatos y metodos fueron desarrollados. Los resultados de los revoques comprados en el mercado muestran gran diferencias en los valores.

  11. Polymerization Shrinkage and Flexural Modulus of Flowable Dental Composites

    Directory of Open Access Journals (Sweden)

    Janaína Cavalcanti Xavier


    Full Text Available Linear polymerization shrinkage (LPS, flexural strength (FS and modulus of elasticity (ME of low-viscosity resin composites (Admira Flow™, Grandio Flow™/VOCO; Filtek Z350 Flow™/3M ESPE; Tetric Flow™/Ivoclar-Vivadent was evaluated using a well-established conventional micro-hybrid composite as a standard (Filtek Z250™/3M ESPE. For the measurement of LPS, composites were applied to a cylindrical metallic mould and polymerized (n = 8. The gap formed at the resin/mould interface was observed using SEM (1500×. For FS and ME, specimens were prepared according to the ISO 4049 specifications (n = 10. Statistical analysis of the data was performed with one-way ANOVA and the Tukey test. The conventional resin presented significantly lower LPS associated with high FS and ME, but only the ME values of the conventional resin differed significantly from the low-viscosity composites. The relationship between ME and LPS of low-viscosity resin composites when used as restorative material is a critical factor in contraction stress relief and marginal leakage.

  12. T cells play an essential role in anti-F1 mediated rapid protection against bubonic plague. (United States)

    Levy, Yinon; Flashner, Yehuda; Tidhar, Avital; Zauberman, Ayelet; Aftalion, Moshe; Lazar, Shirley; Gur, David; Shafferman, Avigdor; Mamroud, Emanuelle


    Plague, which is initiated by Yersinia pestis infection, is a fatal disease that progresses rapidly and leads to high mortality rates if not treated. Antibiotics are an effective plague therapy, but antibiotic-resistant Y. pestis strains have been reported and therefore alternative countermeasures are needed. In the present study, we assessed the potential of an F1 plus LcrV-based vaccine to provide protection shortly pre- or post-exposure to a lethal Y. pestis infection. Mice vaccinated up to one day before or even several hours after subcutaneous challenge were effectively protected. Mice immunized one or three days pre-challenge were protected even though their anti-F1 and anti-LcrV titers were below detection levels at the day of challenge. Moreover, using B-cell deficient μMT mice, we found that rapidly induced protective immunity requires the integrity of the humoral immune system. Analysis of the individual contributions of vaccine components to protection revealed that rF1 is responsible for the observed rapid antibody-mediated immunity. Applying anti-F1 passive therapy in the mouse model of bubonic plague demonstrated that anti-F1 F(ab')(2) can delay mortality, but it cannot provide long-lasting protection, as do intact anti-F1 molecules. Fc-dependent immune components, such as the complement system and (to a lesser extent) neutrophils, were found to contribute to mouse survival. Interestingly, T cells but not B cells were found to be essential for the recovery of infected animals following passive anti-F1 mediated therapy. These data extend our understanding of the immune mechanisms required for the development of a rapid and effective post-exposure therapy against plague. Copyright © 2011 Elsevier Ltd. All rights reserved.

  13. Single-cell pH imaging and detection for pH profiling and label-free rapid identification of cancer-cells. (United States)

    Hou, Hui; Zhao, Yangyang; Li, Chuanping; Wang, Minmin; Xu, Xiaolong; Jin, Yongdong


    Single-cell pH-sensing and accurate detection and label-free fast identification of cancer-cells are two long-standing pursuits in cell and life science, as intracellular pH plays a crucial role in many cellular events and fates, while the latter is vital for early cancer theranostics. Numerous methods based on functionalized nanoparticles and fluorescence probes have been developed for cell pH-sensing, but are often hindered for single-cell studies by their main drawbacks of complicated probe preparation and labeling, low sensitivity and poor reproducibility. Here we report a simple and reliable method for single-cell pH imaging and sensing by innovative combined use of UV-Vis microspectroscopy and common pH indicators. Accurate and sensitive pH detection on single-cell or sub-cell level with good reproducibility is achieved by the method, which enables facile single-cell pH profiling and label-free rapid identification of cancer-cells (due to distinguishable intracellular pH levels) for early cancer diagnosis, and may open a new avenue for pH-related single-cell studies.

  14. A rapid method for assessment of natural killer cell function after multiple receptor crosslinking. (United States)

    Al-Hubeshy, Z B; Coleman, A; Nelson, M; Goodier, M R


    NK cell function is regulated by the integration of signals from activating and inhibitory receptors. We developed an assay to study the effect of co-crosslinking NK cell receptors in pair-wise combinations without the need to purify NK cells. Monoclonal antibodies recognising inhibitory and activating receptors were coated to flat bottomed tissue culture plates and degranulation was measured within unfractionated, freshly isolated resting or cytokine activated peripheral blood mononuclear cells by flow cytometric analysis of CD107a expression. Measured degranulation responses were NK cell specific, since no expression of CD107a was induced in gated T cells. We detected enhancement of degranulation in response to combinations of antibodies against activating NK cell receptors, including CD16, NKG2D, NKp30 and NKp46 compared to each antibody when combined with an isotype matched control antibody. Co-crosslinking of NKG2A resulted in the inhibition of degranulation measured in response to anti-NKp30 or anti-NKp46 alone in both resting or cytokine pre-activated NK cells, but had no effect on CD16 or NKG2D mediated responses. Interferon gamma production was assayed by intracellular cytokine staining and in cell culture supernatants after receptor crosslinking. No IFN-γ could be detected from resting NK cells after receptor crosslinking whereas the pattern of IFN-γ production in cytokine pre-activated NK cells reflected that observed for degranulation. We conclude that this assay is suitable for the analysis of the impact of NK cell receptor co-crosslinking on multiple NK cell functions and has the potential for application to pathologic conditions where limited numbers of cells are available for study. Published by Elsevier B.V.

  15. Rapid effects of 17beta-estradiol on TRPV5 epithelial Ca2+ channels in rat renal cells.

    LENUS (Irish Health Repository)

    Irnaten, Mustapha


    The renal distal tubules and collecting ducts play a key role in the control of electrolyte and fluid homeostasis. The discovery of highly calcium selective channels, Transient Receptor Potential Vanilloid 5 (TRPV5) of the TRP superfamily, has clarified the nature of the calcium entry channels. It has been proposed that this channel mediates the critical Ca(2+) entry step in transcellular Ca(2+) re-absorption in the kidney. The regulation of transmembrane Ca(2+) flux through TRPV5 is of particular importance for whole body calcium homeostasis.In this study, we provide evidence that the TRPV5 channel is present in rat cortical collecting duct (RCCD(2)) cells at mRNA and protein levels. We demonstrate that 17beta-estradiol (E(2)) is involved in the regulation of Ca(2+) influx in these cells via the epithelial Ca(2+) channels TRPV5. By combining whole-cell patch-clamp and Ca(2+)-imaging techniques, we have characterized the electrophysiological properties of the TRPV5 channel and showed that treatment with 20-50nM E(2) rapidly (<5min) induced a transient increase in inward whole-cell currents and intracellular Ca(2+) via TRPV5 channels. This rise was significantly prevented when cells were pre-treated with ruthenium red and completely abolished in cells treated with siRNA specifically targeting TRPV5.These data demonstrate for the first time, a novel rapid modulation of endogenously expressed TRPV5 channels by E(2) in kidney cells. Furthermore, the results suggest calcitropic effects of E(2). The results are discussed in relation to present concepts of non-genomic actions of E(2) in Ca(2+) homeostasis.

  16. Rapid Endolysosomal Escape and Controlled Intracellular Trafficking of Cell Surface Mimetic Quantum-Dots-Anchored Peptides and Glycopeptides. (United States)

    Tan, Roger S; Naruchi, Kentaro; Amano, Maho; Hinou, Hiroshi; Nishimura, Shin-Ichiro


    A novel strategy for the development of a high performance nanoparticules platform was established by means of cell surface mimetic quantum-dots (QDs)-anchored peptides/glycopeptides, which was developed as a model system for nanoparticle-based drug delivery (NDD) vehicles with defined functions helping the specific intracellular trafficking after initial endocytosis. In this paper, we proposed a standardized protocol for the preparation of multifunctional QDs that allows for efficient cellular uptake and rapid escaping from the endolysosomal system and subsequent cytoplasmic molecular delivery to the target cellular compartment. Chemoselective ligation of the ketone-functionalized hexahistidine derivative facilitated both efficient endocytic entry and rapid endolysosomal escape of the aminooxy/phosphorylcholine self-assembled monolayer-coated QDs (AO/PCSAM-QDs) to the cytosol in various cell lines such as human normal and cancer cells, while modifications of these QDs with cell-penetrating arginine-rich peptides showed poor cellular uptake and induced self-aggregation of AO/PCSAM-QDs. Combined use of hexahistidylated AO/PCSAM-QDs with serglycine-like glycopeptides, namely synthetic proteoglycan initiators (PGIs), elicited the entry and controlled intracellular trafficking, Golgi localization, and also excretion of these nanoparticles, which suggested that the present approach would provide an ideal platform for the design of high performance NDD systems.

  17. Cell-on-hydrogel platform made of agar and alginate for rapid, low-cost, multidimensional test of antimicrobial susceptibility. (United States)

    Sun, Han; Liu, Zhengzhi; Hu, Chong; Ren, Kangning


    Antimicrobial resistance (AMR) is a rapidly increasing threat to the effective treatment of infectious diseases worldwide. The two major remedies include: (1) using narrow-spectrum antibiotics based on rapid diagnosis; and (2) developing new antibiotics. A key part of both remedies is the antimicrobial susceptibility test (AST). However, the current standard ASTs that monitor colony formation are costly and time-consuming and the new strategies proposed are not yet practical to be implemented. Herein, we report a strategy to fabricate whole-hydrogel microfluidic chips using alginate-doped agar. This agar-based microfabrication makes it possible to prepare inexpensive hydrogel devices, and allows a seamless link between microfluidics and conventional agar-based cell culture. Different from common microfluidic systems, in our system the cells are cultured on top of the device, similar to normal agar plate culture; on the other hand, the microfluidic channels inside the hydrogel allow precise generation of linear gradient of drugs, thus giving a better performance than the conventional disk diffusion method. Cells in this system are not exposed to any shear flow, which allows the reliable tracking of individual cells and AST results to be obtained within 2-3 hours. Furthermore, our system could test the synergistic effect of drugs through two-dimensional gradient generation. Finally, the platform could be directly implemented to new drug discovery and other applications wherein a fast, cost-efficient method for studying the response of microorganisms upon drug administration is desirable.

  18. Controlling human corneal stromal stem cell contraction to mediate rapid cell and matrix organization of real architecture for 3-dimensional tissue equivalents. (United States)

    Mukhey, Dev; Phillips, James B; Daniels, Julie T; Kureshi, Alvena K


    The architecture of the human corneal stroma consists of a highly organized extracellular matrix (ECM) interspersed with keratocytes. Their progenitor cells; corneal stromal stem cells (CSSC) are located at the periphery, in the limbal stroma. A highly organized corneal ECM is critical for effective transmission of light but this structure may be compromised during injury or disease, resulting in loss of vision. Re-creating normal organization in engineered tissue equivalents for transplantation often involves lengthy culture times that are inappropriate for clinical use or utilisation of synthetic substrates that bring complications such as corneal melting. CSSC have great therapeutic potential owing to their ability to reorganize a disorganized matrix, restoring transparency in scarred corneas. We examined CSSC contractile behavior to assess whether this property could be exploited to rapidly generate cell and ECM organization in Real Architecture For 3D Tissues (RAFT) tissue equivalents (TE) for transplantation. Free-floating collagen gels were characterized to assess contractile behavior of CSSC and establish optimum cell density and culture times. To mediate cell and collagen organization, tethered collagen gels seeded with CSSC were cultured and subsequently stabilized with the RAFT process. We demonstrated rapid creation of biomimetic RAFT TE with tunable structural properties. These displayed three distinct regions of varying degrees of cellular and collagen organization. Interestingly, increased organization coincided with a dramatic loss of PAX6 expression in CSSC, indicating rapid differentiation into keratocytes. The organized RAFT TE system could be a useful bioengineering tool to rapidly create an organized ECM while simultaneously controlling cell phenotype. For the first time, we have demonstrated that human CSSC exhibit the phenomenon of cellular self-alignment in tethered collagen gels. We found this mediated rapid co-alignment of collagen fibrils

  19. Lenvatinib induces early tumor shrinkage in patients with advanced thyroid carcinoma. (United States)

    Masaki, Chie; Sugino, Kiminori; Saito, Naoko; Saito, Yoshiyuki; Tanaka, Tomoaki; Ogimi, Yuna; Maeda, Tetsuyo; Osaku, Tadatoshi; Akaishi, Junko; Hames, Kiyomi Y; Tomoda, Chisato; Suzuki, Akifumi; Matsuzu, Kenichi; Uruno, Takashi; Ohkuwa, Keiko; Kitagawa, Wataru; Nagahama, Mitsuji; Takami, Hiroshi; Ito, Koichi


    Although advanced thyroid carcinoma patients who cannot be cured by conventional therapy have lacked effective treatment, multitargeted tyrosine kinase inhibitors have recently become available. Phase 3 trials of lenvatinib showed a median time to objective response of 2 (95 % confidence interval (CI) 1.9-3.5) months, demonstrating that shrinks tumors rapidly. The phenomenon of immediate tumor shrink is known as early tumor shrinkage (ETS) which is related to clinical outcome in other malignancies. However, precisely when within 8 weeks lenvatinib starts to affect tumors remains unclear. In tumors near the carotid arteries, trachea, or esophagus, a rapid therapeutic effect can induce fistula formation or arterial bleeding. To prevent such treatment-emergent serious adverse events (SAE), early imaging evaluation seems to be very important. In this study, the point in time when lenvatinib started to shrink tumors was retrospectively investigated. The subjects were 16 patients who started lenvatinib administration between May and August 2015. Tumor size was evaluated by computed tomography (CT) scans frequently within the first 8 weeks according to the Response Evaluation Criteria In Solid Tumors (RECIST) guideline. Initial tumor response was defined as ≥ 10% tumor reduction. Serum thyroglobulin (Tg) level was monitored in 8 differentiated thyroid carcinoma (DTC) without TgAb patients. At the first evaluation, 13 patients (83.3 %) showed tumor reduction and that decreased with time. Thirteen patients (83.3 %) showed >10 % tumor reduction within 8 weeks. In all DTC patients, serum Tg level was markedly decreased. In conclusion, lenvatinib immediately shrinks tumors, the so-called ETS phenomenon. Therefore, careful attention should be paid to fistula formation from the early phase.

  20. Rapid preparation and single-cell analysis of concentrated blood smears using a high-throughput blood cell separator and a microfabricated grid film. (United States)

    You, Dongwon; Oh, Sein; Kim, Byeongyeon; Hahn, Young Ki; Choi, Sungyoung


    Cytological examination of peripheral white blood cells inhomogeneously distributed on a blood smear is currently limited by the low abundance and random sampling of the target cells. To address the challenges, we present a new approach to prepare and analyze concentrated blood smears by rapidly enriching white blood cells up to 32-fold with 92% recovery on average at a high throughput (1mL/min) using a deterministic migration-based separator and by systematically analyzing a large number of the cells distributed over a blood slide using a microfabricated grid film. We anticipate that our approach will improve the clinical utility of blood smear tests, while offering the capability to detect rare cell populations. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. A rapid chemical method for lysing Arabidopsis cells for protein analysis


    Takano Tetsuo; Liu Shenkui; Tsugama Daisuke


    Abstract Background Protein extraction is a frequent procedure in biological research. For preparation of plant cell extracts, plant materials usually have to be ground and homogenized to physically break the robust cell wall, but this step is laborious and time-consuming when a large number of samples are handled at once. Results We developed a chemical method for lysing Arabidopsis cells without grinding. In this method, plants are boiled for just 10 minutes in a solution containing a Ca2+ ...

  2. Rapid Selection in Modified BHK-21 Cells of a Foot-and-Mouth Disease Virus Variant Showing Alterations in Cell Tropism (United States)

    Escarmís, Cristina; Carrillo, Elisa C.; Ferrer, Marcela; Arriaza, Juan F. García; Lopez, Nora; Tami, Cecilia; Verdaguer, Nuria; Domingo, Esteban; Franze-Fernández, Maria T.


    With persistent foot-and-mouth disease virus (FMDV) in BHK-21 cells, there is coevolution of the cells and the resident virus; the virulence of the virus for the parental BHK-21 cells is gradually increased, and the cells become partially resistant to FMDV. Here we report that variants of FMDV C3Arg/85 were selected in a single infection of partially resistant BHK-21 cells (termed BHK-Rb cells). Indirect immunofluorescence showed that the BHK-Rb cell population was heterogeneous with regard to susceptibility to C3Arg/85 infection. Infection of BHK-Rb cells with C3Arg/85 resulted in an early phase of partial cytopathology which was followed at 6 to 10 days postinfection by the shedding of mutant FMDVs, termed C3-Rb. The selected C3-Rb variants showed increased virulence for BHK-21 cells, were able to overcome the resistance of modified BHK-21 cells to infection, and had acquired the ability to bind heparin and to infect wild-type Chinese hamster ovary (CHO) cells. A comparison of the genomic sequences of the parental and modified viruses revealed only two amino acid differences, located at the surface of the particle, at the fivefold axis of the viral capsid (Asp-9→Ala in VP3 and either Gly-110→Arg or His-108→Arg in VP1). The same phenotypic and genotypic modifications occurred in a highly reproducible manner; they were seen in a number of independent infections of BHK-Rb cells with viral preparation C3Arg/85 or with clones derived from it. Neither amino acid substitutions in other structural or nonstructural proteins nor nucleotide substitutions in regulatory regions were found. These results prove that infection of partially permissive cells can promote the rapid selection of virus variants that show alterations in cell tropism and are highly virulent for the same cells. PMID:9811758

  3. Sudden shrinkage of free rectus abdominis musculocutaneous flap 15 years after maxilla reconstruction

    Directory of Open Access Journals (Sweden)

    Yasushi Mochizuki


    Full Text Available A 60-year-old male displayed sudden shrinkage of a left free rectus abdominis musculocutaneous flap, which had been grafted to his left maxilla 15 years previously. No post-reconstructive irradiation had been performed, and no late occlusion of the vascular anastomosis, local infection, recurrence of the maxillary cancer, or body weight loss was observed. However, the shrinkage amounted to approximately 50%. This is considerably more than previously reported cases of shrinkage of various free flaps, which ranged between 10% and 25%. The resultant depression was successfully augmented with a right free deep inferior epigastric artery perforator flap. The residual fat volume of the previously grafted shrunken flap was revealed to be compatible with that of the newly harvested contralateral perforator flap. Thus, the volume of the previously grafted flap may reflect the status of the intact contralateral donor site, although the mechanism of sudden flap shrinkage is unclear.

  4. Report D : self-consolidating concrete (SCC) for infrastructure elements - creep, shrinkage and abrasion resistance. (United States)


    Concrete specimens were fabricated for shrinkage, creep, and abrasion resistance : testing. Variations of self-consolidating concrete (SCC) and conventional concrete were : all tested. The results were compared to previous similar testing programs an...

  5. Strength and Drying Shrinkage of Alkali-Activated Slag Paste and Mortar

    Directory of Open Access Journals (Sweden)

    Mao-chieh Chi


    Full Text Available The aim of this study is to investigate the strengths and drying shrinkage of alkali-activated slag paste and mortar. Compressive strength, tensile strength, and drying shrinkage of alkali-activated slag paste and mortar were measured with various liquid/slag ratios, sand/slag ratios, curing ages, and curing temperatures. Experimental results show that the higher compressive strength and tensile strength have been observed in the higher curing temperature. At the age of 56 days, AAS mortars show higher compressive strength than Portland cement mortars and AAS mortars with liquid/slag ratio of 0.54 have the highest tensile strength in all AAS mortars. In addition, AAS pastes of the drying shrinkage are higher than AAS mortars. Meanwhile, higher drying shrinkage was observed in AAS mortars than that observed comparable Portland cement mortars.

  6. Drying shrinkage problems in high-plastic clay soils in Oklahoma. (United States)


    Longitudinal cracking in pavements due to drying shrinkage of high-plastic subgrade soils has been a major : problem in Oklahoma. Annual maintenance to seal and repair these distress problems costs significant amount of : money to the state. The long...

  7. Validation Tests of Prediction Modules of Shrinkage Defects in Cast Iron Sample

    Directory of Open Access Journals (Sweden)

    Hajkowski J.


    Full Text Available The paper presents the results of experimental-simulation tests of expansion-shrinkage phenomena occurring in cast iron castings. The tests were based on the standard test for inspecting the tendency of steel-carbon alloys to create compacted discontinuities of the pipe shrinkage type. The cast alloy was a high-silicone ductile iron of GJS - 600 - 10 grade. The validation regarding correctness of prognoses of the shrinkage defects was applied mostly to the simulation code (system NovaFlow & Solid CV (NFS CV. The obtained results were referred to the results obtained using the Procast system (macro- and micromodel. The analysis of sensitivity of the modules responsible for predicting the shrinkage discontinuities on selected pre-processing parameters was performed, focusing mostly on critical fractions concerning the feeding flows (mass and capillary and variation of initial temperature of the alloy in the mould and heat transfer coefficient (HTC on the casting - chill interface.

  8. Development of high shrinkage polyethylene terephthalate (PET) shape memory polymer tendons for concrete crack closure (United States)

    Teall, Oliver; Pilegis, Martins; Sweeney, John; Gough, Tim; Thompson, Glen; Jefferson, Anthony; Lark, Robert; Gardner, Diane


    The shrinkage force exerted by restrained shape memory polymers (SMPs) can potentially be used to close cracks in structural concrete. This paper describes the physical processing and experimental work undertaken to develop high shrinkage die-drawn polyethylene terephthalate (PET) SMP tendons for use within a crack closure system. The extrusion and die-drawing procedure used to manufacture a series of PET tendon samples is described. The results from a set of restrained shrinkage tests, undertaken at differing activation temperatures, are also presented along with the mechanical properties of the most promising samples. The stress developed within the tendons is found to be related to the activation temperature, the cross-sectional area and to the draw rate used during manufacture. Comparisons with commercially-available PET strip samples used in previous research are made, demonstrating an increase in restrained shrinkage stress by a factor of two for manufactured PET filament samples.

  9. Concrete pavement mixture design and analysis (MDA) : factors influencing drying shrinkage. (United States)


    This literature review focuses on factors influencing drying shrinkage of concrete. Although the factors are normally interrelated, they : can be categorized into three groups: paste quantity, paste quality, and other factors.

  10. Numerical Simulation on Open Wellbore Shrinkage and Casing Equivalent Stress in Bedded Salt Rock Stratum

    Directory of Open Access Journals (Sweden)

    Jianjun Liu


    Full Text Available Most salt rock has interbed of mudstone in China. Owing to the enormous difference of mechanical properties between the mudstone interbed and salt rock, the stress-strain and creep behaviors of salt rock are significantly influenced by neighboring mudstone interbed. In order to identify the rules of wellbore shrinkage and casings equivalent stress in bedded salt rock stratum, three-dimensional finite difference models were established. The effects of thickness and elasticity modulus of mudstone interbed on the open wellbore shrinkage and equivalent stress of casing after cementing operation were studied, respectively. The results indicate that the shrinkage of open wellbore and equivalent stress of casings decreases with the increase of mudstone interbed thickness. The increasing of elasticity modulus will reduce the shrinkage of open wellbore and casing equivalent stress. Research results can provide the scientific basis for the design of mud density and casing strength.

  11. Numerical simulation on open wellbore shrinkage and casing equivalent stress in bedded salt rock stratum. (United States)

    Liu, Jianjun; Zhang, Linzhi; Zhao, Jinzhou


    Most salt rock has interbed of mudstone in China. Owing to the enormous difference of mechanical properties between the mudstone interbed and salt rock, the stress-strain and creep behaviors of salt rock are significantly influenced by neighboring mudstone interbed. In order to identify the rules of wellbore shrinkage and casings equivalent stress in bedded salt rock stratum, three-dimensional finite difference models were established. The effects of thickness and elasticity modulus of mudstone interbed on the open wellbore shrinkage and equivalent stress of casing after cementing operation were studied, respectively. The results indicate that the shrinkage of open wellbore and equivalent stress of casings decreases with the increase of mudstone interbed thickness. The increasing of elasticity modulus will reduce the shrinkage of open wellbore and casing equivalent stress. Research results can provide the scientific basis for the design of mud density and casing strength.

  12. Rapid generation of NY-ESO-1-specific CD4(+) THELPER1 cells for adoptive T-cell therapy. (United States)

    Kayser, Simone; Boβ, Cristina; Feucht, Judith; Witte, Kai-Erik; Scheu, Alexander; Bülow, Hans-Jörg; Joachim, Stefanie; Stevanović, Stefan; Schumm, Michael; Rittig, Susanne M; Lang, Peter; Röcken, Martin; Handgretinger, Rupert; Feuchtinger, Tobias


    Tumor-associated antigens such as NY-ESO-1 are expressed in a variety of solid tumors but absent in mature healthy tissues with the exception of germline cells. The immune system anti-cancer attack is mediated by cell lysis or induction of growth arrest through paralysis of tumor cells, the latter of which can be achieved by tumor-specific CD4(+), IFNγ-producing THelper type 1 (TH1) cells. Translation of these immune-mediated mechanisms into clinical application has been limited by availability of immune effectors, as well as the need for complex in vitro protocols and regulatory hurdles. Here, we report a procedure to generate cancer-testis antigen NY-ESO-1-targeting CD4(+) TH1 cells in vitro for cancer immunotherapy in the clinic. After in vitro sensitization by stimulating T cells with protein-spanning, overlapping peptide pools of NY-ESO-1 in combination with IL-7 and low dose IL-2, antigen-specific T cells were isolated using IFNγ capture technique and subsequently expanded with IL-2, IL-7 and IL-15. Large numbers of NY-ESO-1-specific CD4(+) T cells with a TH1 cytokine profile and lower numbers of cytokine-secreting CD8(+) T cells could be generated from healthy donors with a high specificity and expansion potential. Manufactured CD4(+) T cells showed strong specific TH1-responses with IFNγ(+), TNFα(+), IL-2(+) and induced cell cycle arrest and apoptosis in tumor cells. The protocol is GMP-grade and approved by the regulatory authorities. The tumor-antigen specific CD4(+) TH1 lymphocytes can be adoptively transferred as a T-cell therapy to boost anticancer immunity and this novel cancer treatment approach is applicable to both T cells from healthy allogeneic donors as well as to autologous T cells derived from cancer patients.

  13. Rapid generation of NY-ESO-1-specific CD4+ THELPER1 cells for adoptive T-cell therapy (United States)

    Kayser, Simone; Boβ, Cristina; Feucht, Judith; Witte, Kai-Erik; Scheu, Alexander; Bülow, Hans-Jörg; Joachim, Stefanie; Stevanović, Stefan; Schumm, Michael; Rittig, Susanne M; Lang, Peter; Röcken, Martin; Handgretinger, Rupert; Feuchtinger, Tobias


    Tumor-associated antigens such as NY-ESO-1 are expressed in a variety of solid tumors but absent in mature healthy tissues with the exception of germline cells. The immune system anti-cancer attack is mediated by cell lysis or induction of growth arrest through paralysis of tumor cells, the latter of which can be achieved by tumor-specific CD4+, IFNγ-producing THelper type 1 (TH1) cells. Translation of these immune-mediated mechanisms into clinical application has been limited by availability of immune effectors, as well as the need for complex in vitro protocols and regulatory hurdles. Here, we report a procedure to generate cancer-testis antigen NY-ESO-1-targeting CD4+ TH1 cells in vitro for cancer immunotherapy in the clinic. After in vitro sensitization by stimulating T cells with protein-spanning, overlapping peptide pools of NY-ESO-1 in combination with IL-7 and low dose IL-2, antigen-specific T cells were isolated using IFNγ capture technique and subsequently expanded with IL-2, IL-7 and IL-15. Large numbers of NY-ESO-1-specific CD4+ T cells with a TH1 cytokine profile and lower numbers of cytokine-secreting CD8+ T cells could be generated from healthy donors with a high specificity and expansion potential. Manufactured CD4+ T cells showed strong specific TH1-responses with IFNγ+, TNFα+, IL-2+ and induced cell cycle arrest and apoptosis in tumor cells. The protocol is GMP-grade and approved by the regulatory authorities. The tumor-antigen specific CD4+ TH1 lymphocytes can be adoptively transferred as a T-cell therapy to boost anticancer immunity and this novel cancer treatment approach is applicable to both T cells from healthy allogeneic donors as well as to autologous T cells derived from cancer patients. PMID:26155389

  14. Development of a Rapid Cell-free Method for Cytotoxicity Assessment of Vapor Phase of Cigarette Smoke

    Directory of Open Access Journals (Sweden)

    Cahours X


    Full Text Available Currently, several in vitro tests are widely used to measure toxicological properties of mainstream smoke (Neutral Red Uptake Assay, Micronucleus assay, Ames Test. These tests are necessary to assess cytotoxicity, genotoxicity, and mutagenicity, but are time consuming. This is essentially due to the preparation and the handling of cells. It is difficult to use these in vitro tests as screening method for product testing and development. For a better assessment of the cytotoxicity of the vapor phase, a rapid cell-free method has been developed. This paper describes a capillary electrophoresis cell-free method, based on the depletion of an anti-oxidant L-gamma-glutamyl-L-cysteinylglycine (GSH, applied to an aliquot of vapor phase phosphate buffered saline (PBS-trapped cigarette smoke (as recommended for in vitro testing. The correlation between this method and the survival/viability test (Neutral Red cytotoxicity is excellent (coefficient of correlation (r = 0.99.

  15. Rapid diagnosis of M.tuberculosis meningitis by enumeration of cerebrospinal fluid antigen-specific T cells (United States)

    Thomas, MM; Hinks, TSC; Raghuraman, S; Ramalingam, N; Ernst, M; Nau, R; Lange, C; Kösters, K; Gnanamuthu, C; John, GT; Marshall, B; Lalvani, A


    SUMMARY Setting Hospital in-patients with suspected tuberculous meningitis predominantly in India. Objective To determine whether interferon-γ-secreting Mycobacterium tuberculosis-antigen-specific T cells are present in the cerebrospinal fluid of patients with tuberculous meningitis, and to evaluate the feasibility of cerebrospinal fluid enzyme-linked immunospot for the diagnosis of active tuberculous meningitis. Design Prospective, blinded, hospital-based study. Results The overnight enzyme-linked immunospot assay detected Mycobacterium tuberculosis-antigen-specific interferon-γ-secreting T cells in cerebrospinal fluid from 9 out of 10 prospectively recruited patients with tuberculous meningitis, and 0 out of 7 control patients with meningitis of other aetiology. This corresponds to a diagnostic sensitivity of 90% (95%CI 56-100%) and specificity of 100% (95%CI 59-100%). Conclusion This pilot study demonstrates proof-of-principle for a new T cell-based diagnostic test for tuberculous meningitis which is rapid, sensitive and specific. PMID:18492332

  16. The Rapid Detection of Single Bacterial Cells by Deep UV Micro Raman Spectroscopy. (United States)


    developed for the purpose of rapid bacterial detection. Techniques include mass spectroscopy and its various combinations with chromatography and pyrolysis...Methods: Chromatography and Mass Spectroscopy", Plenum Press, N.Y. 1990. 6. P.J.H. Jackman in "Methods in Microbiology", Vol. 19, eds., R.R., Colwell and R...4847198 issued July 11, 1989. 5. "Ultraviolet Resonance Raman Spectra of Bacteria, Bacterial Spores, Protoplasts and Calcium Dipicolinate", R

  17. Low concentration thresholds of plasma membranes for rapid energy-independent translocation of a cell-penetrating peptide. (United States)

    Watkins, Catherine L; Schmaljohann, Dirk; Futaki, Shiroh; Jones, Arwyn T


    The exact mechanisms by which cell-penetrating peptides such as oligo-arginines and penetratin cross biological membranes has yet to be elucidated, but this is required if they are to reach their full potential as cellular delivery vectors. In the present study, qualitative and quantitative analysis of the influence of temperature, peptide concentration and plasma membrane cholesterol on the uptake and subcellular distribution of the model cell-penetrating peptide octa-arginine was performed in a number of suspension and adherent cell lines. When experiments were performed on ice, the peptide at 2 microM extracellular concentration efficiently entered and uniformly labelled the cytoplasm of all the suspension cells studied, but a 10-fold higher concentration was required to observe similar results in adherent cells. At 37 degrees C and at higher peptide concentrations, time-lapse microscopy experiments showed that the peptide rapidly penetrated the entire plasma membrane of suspension cells, with no evidence of a requirement for nucleation zones to promote this effect. Cholesterol depletion with methyl-beta-cyclodextrin enhanced translocation of octa-arginine across the plasma membrane of suspension cells at 37 degrees C, but decreased overall peptide accumulation. Under the same conditions in adherent cells this agent had no effect on peptide uptake or distribution. Cholesterol depletion increased the overall accumulation of the peptide at 4 degrees C in KG1a cells, but this effect could be reversed by re-addition of cholesterol as methyl-beta-cyclodextrin-cholesterol complexes. The results highlight the relatively high porosity of the plasma membrane of suspension cells to this peptide, especially at low temperatures, suggesting that this feature could be exploited for delivering bioactive entities.

  18. Vesicle formation in the membrane of onion cells (Allium cepa) during rapid osmotic dehydration. (United States)

    Assani, Akym; Moundanga, Sylvie; Beney, Laurent; Gervais, Patrick


    Optimization of osmotic dehydration in different plant cells has been investigated through the variation of parameters such as the nature of the sugar used, the concentration of osmotic solutions and the processing time. In micro-organisms such as the yeast, Saccharomyces cerevisiae, the exposure of a cell to a slow increase in osmotic pressure preserves cell viability after rehydration, while sudden dehydration involves a lower rate of cell viability, which could be due to membrane vesiculation. The aim of this work is to study cytoplasmic vesicle formation in onion epidermal cells (Allium cepa) as a function of the kinetics of osmotic pressure variation in the external medium. Onion epidermal cells were submitted either to an osmotic shock or to a progressive osmotic shift from an osmotic pressure of 2 to 24 MPa to induce plasmolysis. After 30 min in the treatment solution, deplasmolysis was carried out. Cells were observed by microscopy during the whole cycle of dehydration-rehydration. The application of an osmotic shock to onion cells, from an initial osmotic pressure of 2 MPa to a final one of 24 MPa for osmotic shift, from an initial osmotic pressure of 2 MPa to a final one of 24 MPa for 30 min, no vesicles were observed. Additionally, the absence of Hechtian strand connections led to the bursting of vesicles in the case of the osmotic shock. It is concluded that the kinetics of osmotic dehydration strongly influence vesicle formation in onion cells, and that Hechtian strand connections between protoplasts and exocytotic vesicles are a prerequisite for successful deplasmolysis. These results suggest that a decrease in the area-to-volume ratio of a cell could cause cell death following an osmotic shock.

  19. NF-κB is activated in CD4+ iNKT cells by sickle cell disease and mediates rapid induction of adenosine A2A receptors.

    Directory of Open Access Journals (Sweden)

    Gene Lin

    Full Text Available Reperfusion injury following tissue ischemia occurs as a consequence of vaso-occlusion that is initiated by activation of invariant natural killer T (iNKT cells. Sickle cell disease (SDC results in widely disseminated microvascular ischemia and reperfusion injury as a result of vaso-occlusion by rigid and adhesive sickle red blood cells. In mice, iNKT cell activation requires NF-κB signaling and can be inhibited by the activation of anti-inflammatory adenosine A2A receptors (A2ARs. Human iNKT cells are divided into subsets of CD4+ and CD4- cells. In this study we found that human CD4+ iNKT cells, but not CD4- cells undergo rapid NF-κB activation (phosphorylation of NF-κB on p65 and induction of A2ARs (detected with a monoclonal antibody 7F6-G5-A2 during SCD painful vaso-occlusive crises. These findings indicate that SCD primarily activates the CD4+ subset of iNKT cells. Activation of NF-κB and induction of A2ARs is concordant, i.e. only CD4+ iNKT cells with activated NF-κB expressed high levels of A2ARs. iNKT cells that are not activated during pVOC express low levels of A2AR immunoreactivity. These finding suggest that A2AR transcription may be induced in CD4+ iNKT cells as a result of NF-κB activation in SCD. In order to test this hypothesis further we examined cultured human iNKT cells. In cultured cells, blockade of NF-κB with Bay 11-7082 or IKK inhibitor VII prevented rapid induction of A2AR mRNA and protein upon iNKT activation. In conclusion, NF-κB-mediated induction of A2ARs in iNKT cells may serve as a counter-regulatory mechanism to limit the extent and duration of inflammatory immune responses. As activated iNKT cells express high levels of A2ARs following their activation, they may become highly sensitive to inhibition by A2AR agonists.

  20. Pore Structure and Influence of Recycled Aggregate Concrete on Drying Shrinkage


    Guo, Yuanchen; Qian, Jueshi; Wang, Xue


    Pore structure plays an important role in the drying shrinkage of recycled aggregate concrete (RAC). High-precision mercury intrusion and water evaporation were utilized to study the pore structure of RAC, which has a different replacement rate of recycled concrete aggregate (RCA), and to analyze its influence on drying shrinkage. Finally, a fractal-dimension calculation model was established based on the principles of mercury intrusion and fractal-geometry theory. Calculations were performed...

  1. Experimental drying shrinkage of hardened cement pastes as a function of relative humidity

    DEFF Research Database (Denmark)

    Hansen, Kurt Kielsgaard; Baroghel, V.B.


    The results of an experimental study concerning drying shrinkage measured as a function of relative humidity on thin specimens of mature hardened cement pastes are presented. The results obtained at two laboratories are compared.......The results of an experimental study concerning drying shrinkage measured as a function of relative humidity on thin specimens of mature hardened cement pastes are presented. The results obtained at two laboratories are compared....

  2. Influence of Aggregate Wettability with Different Lithology Aggregates on Concrete Drying Shrinkage


    Yuanchen Guo; Jueshi Qian; Xue Wang; Zhengyi Yan; Huadong Zhong


    The correlation of the wettability of different lithology aggregates and the drying shrinkage of concrete materials is studied, and some influential factors such as wettability and wetting angle are analyzed. A mercury porosimeter is used to measure the porosities of different lithology aggregates accurately, and the pore size ranges that significantly affect the drying shrinkage of different lithology aggregate concretes are confirmed. The pore distribution curve of the different coarse aggr...

  3. Effects of Prepolymerized Particle Size and Polymerization Kinetics on Volumetric Shrinkage of Dental Modeling Resins


    Kwon, Tae-Yub; Ha, Jung-Yun; Chun, Ju-Na; Son, Jun Sik; Kim, Kyo-Han


    Dental modeling resins have been developed for use in areas where highly precise resin structures are needed. The manufacturers claim that these polymethyl methacrylate/methyl methacrylate (PMMA/MMA) resins show little or no shrinkage after polymerization. This study examined the polymerization shrinkage of five dental modeling resins as well as one temporary PMMA/MMA resin (control). The morphology and the particle size of the prepolymerized PMMA powders were investigated by scanning electro...

  4. Urban shrinkage, local housing markets and the role of voluntary community organisations

    DEFF Research Database (Denmark)

    Larsen, Jacob Norvig

    Since the beginning of the crisis in 2007-08 urban shrinkage has hit a large number of Danish municipalities, towns and villages outside the two major metropolitan areas in the country .Abandoned homes, plunging property prices and out-migration are among the major symptoms. As a consequence...... and voluntary organizations to strengthen civil society and counteract urban shrinkage. The concluding section includes some policy recommendation....

  5. Astrocytic mechanisms explaining neural-activity-induced shrinkage of extraneuronal space

    DEFF Research Database (Denmark)

    Østby, Ivar; Øyehaug, Leiv; Einevoll, Gaute T


    Neuronal stimulation causes approximately 30% shrinkage of the extracellular space (ECS) between neurons and surrounding astrocytes in grey and white matter under experimental conditions. Despite its possible implications for a proper understanding of basic aspects of potassium clearance and astr......Neuronal stimulation causes approximately 30% shrinkage of the extracellular space (ECS) between neurons and surrounding astrocytes in grey and white matter under experimental conditions. Despite its possible implications for a proper understanding of basic aspects of potassium clearance...

  6. Case series: Rapidly growing squamous cell carcinoma after cutaneous surgical intervention

    Directory of Open Access Journals (Sweden)

    Abdullah Ibrahim


    This case series explores the various mechanisms of de-novo squamous cell carcinoma development in areas of cutaneous surgical intervention, including graft harvest. It also provides recommendations regarding the necessary precautions to avoid implantation of squamous cell carcinoma into distant sites. Lastly it highlights the importance of surveillance for any suspicious lesions arising from areas of previous cutaneous surgical intervention.

  7. Rapid NK-cell activation in chicken after infection with infectious bronchitis virus M41

    NARCIS (Netherlands)

    Vervelde, L.; Matthijs, M.G.R.; van Haarlem, D.A.; de Wit, Sjaak; Jansen, C.A.


    Natural killer (NK) cells are cytotoxic lymphocytes and play an important role in the early defence against viruses. In this study we focussed on NK cell and interferon (IFN) responses after infection with infectious bronchitis virus (IBV). Based on surface expression of CD107+, enhanced activation

  8. Digital image analysis of radial shrinkage of fresh spruce (Picea abies L.) wood. (United States)

    Hansmann, Christian; Konnerth, Johannes; Rosner, Sabine


    Contact-free digital image analysis was performed of the radial shrinkage of fresh, fully saturated small spruce wood beams. An experimental test set-up was developed to ensure constant distance from the charge-coupled device camera to the sample surface as well as constant climate and light conditions during the whole experiment. Dimensional changes were observed immediately after the drying process began. An unexpected distinct effect could be observed which could not be explained by drying surface layers only. After a fast initial radial shrinkage a slowing down of the dimensional changes occurred at high mean moisture contents. A complete interruption of any dimensional changes followed. Finally, a recovery from shrinkage was even observed. It is assumed that strong negative pressure occurred in the fully saturated capillaries owing to dehydration which led to additional dimensional changes. As a consequence, the break of the water column and aeration in these capillaries finally resulted in a recovery period in the shrinkage rate due to the pressure release. After this effect, the dehydration was characterized by a phase of fast and almost linear shrinkage due to drying surface layers. Finally, the shrinkage slowed down to zero when reaching equilibrium moisture content.

  9. Effect of shrinkage porosity on mechanical properties of ferritic ductile iron

    Directory of Open Access Journals (Sweden)

    Wang Zehua


    Full Text Available Casting defects could largely affect the mechanical properties of casting products. A number of test pieces made of ductile iron (EN-GJS-400-18-LT with different levels of shrinkage porosity were prepared and then tensile and fatigue tests were performed to investigate the impact of shrinkage porosity on their mechanical properties. The results showed that the tensile strength decreases linearly with increasing of the shrinkage porosity. The tensile elongation decreases sharply with the increase of the shrinkage porosity mainly due to the non-uniform plastic deformation. The fatigue life also dramatically declines with increasing of the porosity and follows a power law relationship with the area percentage of porosity. The existence of the shrinkage porosity made the fatigue fracture complex. The shrinkage pores, especially those close to the surface usually became the crack initiation sites. For test pieces with less porosity, the fatigue fracture was clearly composed of crack initiation, propagation, and overloading. While for samples with high level of porosity, multiple crack initiation sites were observed.

  10. Effect on effective diffusion coefficients and investigation of shrinkage during osmotic dehydration of apricot

    Energy Technology Data Exchange (ETDEWEB)

    Togrul, Inci Turk; Ispir, Ayse [Firat University, Engineering Faculty, Department of Chemical Engineering, 23279 Elazig (Turkey)


    This article represents the results of the variation in density and shrinkage of apricots during its osmotic dehydration. Shrinkage was investigated by means of dimensionless volume, diameter and length. Various osmotic agents such as sucrose, glucose, fructose, maltodextrin and sorbitol were used. It was found that the shrinkage of apricots could be well correlated with the moisture content of the sample during osmotic dehydration. The relationship between dimensionless parameters and moisture content was investigated by using eight non-linear models for each osmotic agent. It was find that the following proposed model can be confidently use for explaining the effect of shrinkage during osmotic dehydration of apricots.V/V{sub 0},D/D{sub 0},L/L{sub 0},{rho}/{rho}{sub 0}=a+b. exp (cX)+d. exp (e.X{sup f})In addition, the osmotic dehydration kinetics of apricots with and without shrinkage was studied. The effective diffusivities calculated from the diffusional model with and without shrinkage varied from 10.342 x 10{sup -9} m{sup 2}/s to 5.139 x 10{sup -9} and from 1.755 x 10{sup -10} and 0.767 x 10{sup -10} m{sup 2}/s, respectively. (author)

  11. The effect of mucosal cuff shrinkage around dental implants during healing abutment replacement. (United States)

    Nissan, J; Zenziper, E; Rosner, O; Kolerman, R; Chaushu, L; Chaushu, G


    Soft tissue shrinkage during the course of restoring dental implants may result in biological and prosthodontic difficulties. This study was conducted to measure the continuous shrinkage of the mucosal cuff around dental implants following the removal of the healing abutment up to 60 s. Individuals treated with implant-supported fixed partial dentures were included. Implant data--location, type, length, diameter and healing abutments' dimensions--were recorded. Mucosal cuff shrinkage, following removal of the healing abutments, was measured in bucco-lingual direction at four time points--immediately after 20, 40 and 60 s. anova was used to for statistical analysis. Eighty-seven patients (49 women and 38 men) with a total of 311 implants were evaluated (120 maxilla; 191 mandible; 291 posterior segments; 20 anterior segments). Two-hundred and five (66%) implants displayed thick and 106 (34%) thin gingival biotype. Time was the sole statistically significant parameter affecting mucosal cuff shrinkage around dental implants (P < 0.001). From time 0 to 20, 40 and 60 s, the mean diameter changed from 4.1 to 4.07, 3.4 and 2.81 mm, respectively. The shrinkage was 1%, 17% and 31%, respectively. The gingival biotype had no statistically significant influence on mucosal cuff shrinkage (P = 0.672). Time required replacing a healing abutment with a prosthetic element should be minimised (up to 20/40 s), to avoid pain, discomfort and misfit. © 2015 John Wiley & Sons Ltd.

  12. A Portable Cell Maintenance System for Rapid Toxicity Monitoring Final Report CRADA No. TC-02081-04

    Energy Technology Data Exchange (ETDEWEB)

    Kane, S. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Zhou, P. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)


    The Phase I STTR research project was targeted at meeting the objectives and requirements stated in STTR solicitation A04-T028 for a Portable Cell Maintenance System for Rapid Toxicity Monitoring. In accordance with the requirements for STTR programs, collaboration was formed between a small business, Kionix, Inc., and The Regents of the University of California, Lawrence Livermore National Laboratory (LLNL). The collaboration included CytoDiscovery, Inc. (CDI) which, in collaboration with Kionix, provided access to membrane chip technology and provided program support and coordination. The objective of the overall program (excerpted from the original solicitation) was: “To develop a small, portable cell maintenance system for the transport, storage, and monitoring of viable vertebrate cells and tissues.” The goal of the Phase I project was to demonstrate the feasibility of achieving the program objectives utilizing a system comprised of a small-size, microfluidic chip-based cell maintenance cartridge (CMC) and a portable cell maintenance system (CMS) capable of housing a minimum of four CMCs. The system was designed to be capable of optimally maintaining multiple vertebrate cell types while supporting a wide variety of cellular assays.

  13. Highly purified CD34+ cells isolated using magnetically activated cell selection provide rapid engraftment following high-dose chemotherapy in breast cancer patients. (United States)

    Richel, D J; Johnsen, H E; Canon, J; Guillaume, T; Schaafsma, M R; Schenkeveld, C; Hansen, S W; McNiece, I; Gringeri, A J; Briddell, R; Ewen, C; Davies, R; Freeman, J; Miltenyi, S; Symann, M


    The primary objective of this study was to evaluate the safety of infusion of CD34+ cells, selected using a clinical scale magnetically activated cell sorting device, assessed by time to hematological engraftment and incidence of adverse events. Secondary objectives included evaluation of device performance in terms of purity and recovery of the CD34+ cell product. Breast cancer patients suitable for transplantation received cyclophosphamide and filgrastim for mobilisation, followed by three leukaphereses. The products of the first two leukaphereses underwent CD34+ cell selection. The product of the third leukapheresis was cryopreserved unmanipulated. Following high-dose cyclophosphamide, thiotepa and carboplatin, selected CD34+ cells were infused. In 54 patients who received selected cells only, the median time to platelet recovery and neutrophil recovery was 11 days (range 5-51) and 9 days (range 5-51), respectively. There were no adverse events associated with infusion of selected cells. A total of 126 leukapheresis samples was available before and after selection for central CD34+ analysis. The median purity was 96.1% (27.4-99.4) and the median recovery was 52. 3% (15.2-146.3). These data show that cells selected using magnetically activated cell selection provide safe and rapid engraftment after high-dose therapy. Bone Marrow Transplantation (2000) 25, 243-249.

  14. A rapid, efficient, and economic device and method for the isolation and purification of mouse islet cells.

    Directory of Open Access Journals (Sweden)

    Yin Zongyi

    Full Text Available Rapid, efficient, and economic method for the isolation and purification of islets has been pursued by numerous islet-related researchers. In this study, we compared the advantages and disadvantages of our developed patented method with those of commonly used conventional methods (Ficoll-400, 1077, and handpicking methods. Cell viability was assayed using Trypan blue, cell purity and yield were assayed using diphenylthiocarbazone, and islet function was assayed using acridine orange/ethidium bromide staining and enzyme-linked immunosorbent assay-glucose stimulation testing 4 days after cultivation. The results showed that our islet isolation and purification method required 12 ± 3 min, which was significantly shorter than the time required in Ficoll-400, 1077, and HPU groups (34 ± 3, 41 ± 4, and 30 ± 4 min, respectively; P 1000 islets. In summary, the MCT method is a rapid, efficient, and economic method for isolating and purifying murine islet cell clumps. This method overcomes some of the shortcomings of conventional methods, showing a relatively higher quality and yield of islets within a shorter duration at a lower cost. Therefore, the current method provides researchers with an alternative option for islet isolation and should be widely generalized.

  15. A rapid, efficient, and economic device and method for the isolation and purification of mouse islet cells. (United States)

    Zongyi, Yin; Funian, Zou; Hao, Li; Ying, Cheng; Jialin, Zhang; Baifeng, Li


    Rapid, efficient, and economic method for the isolation and purification of islets has been pursued by numerous islet-related researchers. In this study, we compared the advantages and disadvantages of our developed patented method with those of commonly used conventional methods (Ficoll-400, 1077, and handpicking methods). Cell viability was assayed using Trypan blue, cell purity and yield were assayed using diphenylthiocarbazone, and islet function was assayed using acridine orange/ethidium bromide staining and enzyme-linked immunosorbent assay-glucose stimulation testing 4 days after cultivation. The results showed that our islet isolation and purification method required 12 ± 3 min, which was significantly shorter than the time required in Ficoll-400, 1077, and HPU groups (34 ± 3, 41 ± 4, and 30 ± 4 min, respectively; P 1000 islets). In summary, the MCT method is a rapid, efficient, and economic method for isolating and purifying murine islet cell clumps. This method overcomes some of the shortcomings of conventional methods, showing a relatively higher quality and yield of islets within a shorter duration at a lower cost. Therefore, the current method provides researchers with an alternative option for islet isolation and should be widely generalized.

  16. Rapid Genome-wide Recruitment of RNA Polymerase II Drives Transcription, Splicing, and Translation Events during T Cell Responses

    Directory of Open Access Journals (Sweden)

    Kathrin Davari


    Full Text Available Activation of immune cells results in rapid functional changes, but how such fast changes are accomplished remains enigmatic. By combining time courses of 4sU-seq, RNA-seq, ribosome profiling (RP, and RNA polymerase II (RNA Pol II ChIP-seq during T cell activation, we illustrate genome-wide temporal dynamics for ∼10,000 genes. This approach reveals not only immediate-early and posttranscriptionally regulated genes but also coupled changes in transcription and translation for >90% of genes. Recruitment, rather than release of paused RNA Pol II, primarily mediates transcriptional changes. This coincides with a genome-wide temporary slowdown in cotranscriptional splicing, even for polyadenylated mRNAs that are localized at the chromatin. Subsequent splicing optimization correlates with increasing Ser-2 phosphorylation of the RNA Pol II carboxy-terminal domain (CTD and activation of the positive transcription elongation factor (pTEFb. Thus, rapid de novo recruitment of RNA Pol II dictates the course of events during T cell activation, particularly transcription, splicing, and consequently translation.

  17. A combination of four cell types for rapid detection of enteroviruses in clinical specimens. (United States)

    Dagan, R; Menegus, M A


    Isolation in cell culture is currently the most sensitive and reliable way to demonstrate enterovirus (EV) in clinical specimens. During July-October 1982 and 1983, we studied the impact of adding two new cell lines, Buffalo green monkey kidney (BGM) and human rhabdomyosarcoma (RD), to the more traditional cell combination used for EV isolation, human embryonic lung (HEL) and primary cynomolgus monkey kidney (CMK) cells; 2,558 specimens were studied: 632 fecal, 677 respiratory, 537 CSF, and 712 blood. An EV was isolated from 417 (16%); of these, 77 (18%) were positive only in BGM or RD; 35% (146/417) of the specimens were positive in BGM, RD, or both, at least one day earlier than in the traditional cells. BGM cells were helpful in isolation of group B coxsackieviruses (CB): 99% of 121 positive specimens were detected in BGM vs 73% in CMK and 23% in HEL; 72% of the CB isolates were detected by day 2 in BGM vs 48% in CMK and 0% in HEL. RD cells were helpful in the isolation of echoviruses: 59% of the 189 positive specimens were detected in RD vs 67% in HEL and 58% in CMK. RD was the only positive cell type in 28/189 (15%) positive specimens; 31% of the echovirus isolates were detected by day 2 in RD, vs 20% in HEL and 19% in CMK. Using the cell types described, we provided the clinician with results in 42% of the EV-positive specimens by day 2 after inoculation and in 61% by day 4.

  18. The caudal regeneration blastema is an accumulation of rapidly proliferating stem cells in the flatworm Macrostomum lignano

    Directory of Open Access Journals (Sweden)

    Adamski Zbigniew


    Full Text Available Abstract Background Macrostomum lignano is a small free-living flatworm capable of regenerating all body parts posterior of the pharynx and anterior to the brain. We quantified the cellular composition of the caudal-most body region, the tail plate, and investigated regeneration of the tail plate in vivo and in semithin sections labeled with bromodeoxyuridine, a marker for stem cells (neoblasts in S-phase. Results The tail plate accomodates the male genital apparatus and consists of about 3,100 cells, about half of which are epidermal cells. A distinct regeneration blastema, characterized by a local accumulation of rapidly proliferating neoblasts and consisting of about 420 cells (excluding epidermal cells, was formed 24 hours after amputation. Differentiated cells in the blastema were observed two days after amputation (with about 920 blastema cells, while the male genital apparatus required four to five days for full differentiation. At all time points, mitoses were found within the blastema. At the place of organ differentiation, neoblasts did not replicate or divide. After three days, the blastema was made of about 1420 cells and gradually transformed into organ primordia, while the proliferation rate decreased. The cell number of the tail plate, including about 960 epidermal cells, was restored to 75% at this time point. Conclusion Regeneration after artificial amputation of the tail plate of adult specimens of Macrostomum lignano involves wound healing and the formation of a regeneration blastema. Neoblasts undergo extensive proliferation within the blastema. Proliferation patterns of S-phase neoblasts indicate that neoblasts are either determined to follow a specific cell fate not before, but after going through S-phase, or that they can be redetermined after S-phase. In pulse-chase experiments, dispersed distribution of label suggests that S-phase labeled progenitor cells of the male genital apparatus undergo further proliferation before

  19. Rapid and correct identification of intestinal Bacteroides spp. with chromosomal DNA probes by whole-cell dot blot hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Morotomi, M.; Ohno, T.; Mutai, M.


    A dot blot hybridization procedure with /sup 32/P-labeled whole chromosomal DNA of the type strains as probes was developed as a rapid and simple method for identification of intestinal Bacteroides species. Bacterial cells were fixed onto membrane filters by slight suction, treated with 0.5 N NaOH, and hybridized with these probes. Of 65 Bacteroides strains isolated from 19 human fecal specimens, which were identified as B. fragilis, B. thetaiotaomicron, B. ovatus, B. caccae, B. uniformis, B. stercoris, B. vulgatus, B. distasonis, and B. merdae by conventional phenotypic characterization, 62 (95%) were correctly identified with this hybridization procedure.

  20. A new aggregation-induced emission fluorescent probe for rapid detection of nitroreductase and its application in living cells (United States)

    Xu, Gaoping; Tang, Yonghe; Ma, Yanyan; Xu, An; Lin, Weiying


    The biological activity of nitroreductase (NTR) is closely related to biological hypoxia status in organisms. The development of effective methods for monitoring the activity of NTR is of great significance for medical diagnosis and tumor research. Toward this goal, we have developed a new aggregation-induced emission (AIE) fluorescence NTR probe TPE-HY used the tetraphenylethene as the fluorophore, and used the nitro group as the NTR recognition site. The probe TPE-HY has many excellent properties, including rapid response, AIE characteristics, high sensitivity and selectivity, and low cytotoxicity. Importantly, the probe TPE-HY is successfully applied to monitor endogenous NTR in living HeLa cells.

  1. Tetracycline rapidly reaches all the constituent cells of uropathogenic Escherichia coli biofilms (United States)

    Stone, G.; Wood, P.; Dixon, L.; Keyhan, M.; Matin, A.; Demain, A. L. (Principal Investigator)


    We have developed a method for visualizing Escherichia coli cells that are exposed to tetracycline in a biofilm, based on a previous report that liposomes containing the E. coli TetR(B) protein fluoresce when exposed to this antibiotic. By our method, cells devoid of TetR(B) also exhibited tetracycline-dependent fluorescence. At 50 microg of tetracycline ml(-1), planktonic cells of a uropathogenic E. coli (UPEC) strain developed maximal fluorescence after 7.5 to 10 min of exposure. A similar behavior was exhibited by cells in a 24- or 48-h UPEC biofilm, as examined by confocal laser microscopy, regardless of whether they lined empty spaces or occupied densely packed regions. Further, a comparison of phase-contrast and fluorescent images of corresponding biofilm zones showed that all the cells fluoresced. Thus, all the biofilm cells were exposed to tetracycline and there were no pockets within the biofilm where the antibiotic failed to reach. It also appeared unlikely that niches of reduced exposure to the antibiotic existed within the biofilms.

  2. Rapid and unambiguous detection of DNase I hypersensitive site in rare population of cells.

    Directory of Open Access Journals (Sweden)

    Wei-Ping Zeng

    Full Text Available DNase I hypersensitive (DHS sites are important for understanding cis regulation of gene expression. However, existing methods for detecting DHS sites in small numbers of cells can lead to ambiguous results. Here we describe a simple new method, in which DNA fragments with ends generated by DNase I digestion are isolated and used as templates for two PCR reactions. In the first PCR, primers are derived from sequences up- and down-stream of the DHS site. If the DHS site exists in the cells, the first PCR will not produce PCR products due to the cuts of the templates by DNase I between the primer sequences. In the second PCR, one primer is derived from sequence outside the DHS site and the other from the adaptor. This will produce a smear of PCR products of different sizes due to cuts by DNase I at different positions at the DHS site. With this design, we detected a DHS site at the CD4 gene in two CD4 T cell populations using as few as 2×10(4 cells. We further validated this method by detecting a DHS site of the IL-4 gene that is specifically present in type 2 but not type 1 T helper cells. Overall, this method overcomes the interference by genomic DNA not cut by DNase I at the DHS site, thereby offering unambiguous detection of DHS sites in the cells.

  3. Rapid single-step induction of functional neurons from human pluripotent stem cells. (United States)

    Zhang, Yingsha; Pak, Changhui; Han, Yan; Ahlenius, Henrik; Zhang, Zhenjie; Chanda, Soham; Marro, Samuele; Patzke, Christopher; Acuna, Claudio; Covy, Jason; Xu, Wei; Yang, Nan; Danko, Tamas; Chen, Lu; Wernig, Marius; Südhof, Thomas C


    Available methods for differentiating human embryonic stem cells (ESCs) and induced pluripotent cells (iPSCs) into neurons are often cumbersome, slow, and variable. Alternatively, human fibroblasts can be directly converted into induced neuronal (iN) cells. However, with present techniques conversion is inefficient, synapse formation is limited, and only small amounts of neurons can be generated. Here, we show that human ESCs and iPSCs can be converted into functional iN cells with nearly 100% yield and purity in less than 2 weeks by forced expression of a single transcription factor. The resulting ES-iN or iPS-iN cells exhibit quantitatively reproducible properties independent of the cell line of origin, form mature pre- and postsynaptic specializations, and integrate into existing synaptic networks when transplanted into mouse brain. As illustrated by selected examples, our approach enables large-scale studies of human neurons for questions such as analyses of human diseases, examination of human-specific genes, and drug screening. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Rapid Isolation of Monoclonal Antibodies Specific for Cell Surface Differentiation Antigens (United States)

    Barclay, Stephen L.; Smith, Alan M.


    Two immunization procedures were compared for their ability to yield monoclonal antibodies that react with plasma membrane-bound differentiation antigens of Dictyostelium. In the first method, hybridomas prepared from BALB/c mice immunized with aggregating amoebae produced monoclonal antibodies that recognized antigens present on both growing and aggregating Dictyostelium amoebae. None of the monoclonal antibodies reacted with only the injected aggregation-stage cell type. In contrast, monoclonal antibodies that reacted with differentiation antigens were easily obtained by primary immunization of BALB/c mice with living aggregation-stage cells, followed by secondary immunization with a preparation of plasma membrane from aggregating cells or intact aggregating cells mixed with polyclonal BALB/c antiserum raised against undifferentiated cells. By this method, approximately 20% of all anti-Dictyostelium monoclonal antibodies obtained in a fusion are specific for differentiation antigens. The properties and developmental regulation of several of these antigens are described. The possible uses of this immunological method to detect unique determinants on other kinds of cells and the likely immune mechanisms that make it successful are discussed.

  5. Herpes simplex virus type 2 induces rapid cell death and functional impairment of murine dendritic cells in vitro

    NARCIS (Netherlands)

    Jones, CA; Fernandez, M; Herc, K; Bosnjak, L; Miranda-Saksena, M; Boadle, RA; Cunningham, A


    Dendritic cells (DC) are critical for stimulation of naive T cells. Little is known about the effect of herpes simplex virus type 2 (HSV-2) infection on DC structure or function or if the observed effects of HSV-1 on human DC are reproduced in murine DC. Here, we demonstrate that by 12 h

  6. Calcium influx rescues adenylate cyclase-hemolysin from rapid cell membrane removal and enables phagocyte permeabilization by toxin pores.

    Directory of Open Access Journals (Sweden)

    Radovan Fiser

    Full Text Available Bordetella adenylate cyclase toxin-hemolysin (CyaA penetrates the cytoplasmic membrane of phagocytes and employs two distinct conformers to exert its multiple activities. One conformer forms cation-selective pores that permeabilize phagocyte membrane for efflux of cytosolic potassium. The other conformer conducts extracellular calcium ions across cytoplasmic membrane of cells, relocates into lipid rafts, translocates the adenylate cyclase enzyme (AC domain into cells and converts cytosolic ATP to cAMP. We show that the calcium-conducting activity of CyaA controls the path and kinetics of endocytic removal of toxin pores from phagocyte membrane. The enzymatically inactive but calcium-conducting CyaA-AC⁻ toxoid was endocytosed via a clathrin-dependent pathway. In contrast, a doubly mutated (E570K+E581P toxoid, unable to conduct Ca²⁺ into cells, was rapidly internalized by membrane macropinocytosis, unless rescued by Ca²⁺ influx promoted in trans by ionomycin or intact toxoid. Moreover, a fully pore-forming CyaA-ΔAC hemolysin failed to permeabilize phagocytes, unless endocytic removal of its pores from cell membrane was decelerated through Ca²⁺ influx promoted by molecules locked in a Ca²⁺-conducting conformation by the 3D1 antibody. Inhibition of endocytosis also enabled the native B. pertussis-produced CyaA to induce lysis of J774A.1 macrophages at concentrations starting from 100 ng/ml. Hence, by mediating calcium influx into cells, the translocating conformer of CyaA controls the removal of bystander toxin pores from phagocyte membrane. This triggers a positive feedback loop of exacerbated cell permeabilization, where the efflux of cellular potassium yields further decreased toxin pore removal from cell membrane and this further enhances cell permeabilization and potassium efflux.

  7. Measurement of proliferation and disappearance of rapid turnover cell populations in human studies using deuterium-labeled glucose. (United States)

    Macallan, Derek C; Asquith, Becca; Zhang, Yan; de Lara, Catherine; Ghattas, Hala; Defoiche, Julien; Beverley, Peter C L


    Cell proliferation may be measured in vivo by quantifying DNA synthesis with isotopically labeled deoxyribonucleotide precursors. Deuterium-labeled glucose is one such precursor which, because it achieves high levels of enrichment for a short period, is well suited to the study of rapidly dividing cells, in contrast to the longer term labeling achieved with heavy water ((2)H(2)O). As deuterium is non-radioactive and glucose can be readily administered, this approach is suitable for clinical studies. It has been widely applied to investigate human lymphocyte proliferation, but solid tissue samples may also be analyzed. Rate, duration and route (intravenous or oral) of [6,6-(2)H(2)]-glucose administration should be adapted to the target cell of interest. For lymphocytes, cell separation is best achieved by fluorescence activated cell sorting (FACS), although magnetic bead separation is an alternative. DNA is then extracted, hydrolyzed enzymatically and analyzed by gas chromatography mass spectrometry (GC/MS). Appropriate mathematical modeling is critical to interpretation. Typical time requirements are as follows: labeling, 10-24 h; sampling, approximately 3 weeks; DNA extraction/derivatization, 2-3 d; and GC/MS analysis, approximately 2 d.

  8. Human Adipose-Derived Stem Cells on Rapid Prototyped Three-Dimensional Hydroxyapatite/Beta-Tricalcium Phosphate Scaffold. (United States)

    Canciani, Elena; Dellavia, Claudia; Ferreira, Lorena Maria; Giannasi, Chiara; Carmagnola, Daniela; Carrassi, Antonio; Brini, Anna Teresa


    In the study, we assess a rapid prototyped scaffold composed of 30/70 hydroxyapatite (HA) and beta-tricalcium-phosphate (β-TCP) loaded with human adipose-derived stem cells (hASCs) to determine cell proliferation, differentiation toward osteogenic lineage, adhesion and penetration on/into the scaffold.In this in vitro study, hASCs isolated from fat tissue discarded after plastic surgery were expanded, characterized, and then loaded onto the scaffold. Cells were tested for: viability assay (Alamar Blue at days 3, 7 and Live/Dead at day 32), differentiation index (alkaline phosphatase activity at day 14), scaffold adhesion (standard error of the mean analysis at days 5 and 18), and penetration (ground sections at day 32).All the hASC populations displayed stemness markers and the ability to differentiate toward adipogenic and osteogenic lineages.Cellular vitality increased between 3 and 7 days, and no inhibitory effect by HA/β-TCP was observed. Under osteogenic stimuli, scaffold increased alkaline phosphatase activity of +243% compared with undifferentiated samples. Human adipose-derived stem cells adhered on HA/β-TCP surface through citoplasmatic extensions that occupied the macropores and built networks among them. Human adipose derived stem cells were observed in the core of HA/β-TCP. The current combination of hASCs and HA/β-TCP scaffold provided encouraging results. If authors' data will be confirmed in preclinical models, the present engineering approach could represent an interesting tool in treating large bone defects.

  9. A portable cell-based optical detection device for rapid detection of Listeria and Bacillus toxins (United States)

    Banerjee, Pratik; Banada, Padmapriya P.; Rickus, Jenna L.; Morgan, Mark T.; Bhunia, Arun K.


    A mammalian cell-based optical biosensor was built to detect pathogenic Listeria and Bacillus species. This sensor measures the ability of the pathogens to infect and induce cytotoxicity on hybrid lymphocyte cell line (Ped-2E9) resulting in the release of alkaline phosphatase (ALP) that can be detected optically using a portable spectrophotometer. The Ped-2E9 cells were encapsulated in collagen gel matrices and grown in 48-well plates or in specially designed filtration tube units. Toxin preparations or bacterial cells were introduced and ALP release was assayed after 3-5 h. Pathogenic L. monocytogenes strains or the listeriolysin toxins preparation showed cytotoxicity ranging from 55% - 92%. Toxin preparations (~20 μg/ml) from B. cereus strains showed 24 - 98% cytotoxicity. In contrast, a non-pathogenic L. innocua (F4247) and a B. substilis induced only 2% and 8% cytotoxicity, respectively. This cell-based detection device demonstrates its ability to detect the presence of pathogenic Listeria and Bacillus species and can potentially be used onsite for food safety or in biosecurity application.

  10. A rapid crosstalk of human gammadelta T cells and monocytes drives the acute inflammation in bacterial infections.

    Directory of Open Access Journals (Sweden)

    Matthias Eberl


    Full Text Available Vgamma9/Vdelta2 T cells are a minor subset of T cells in human blood and differ from other T cells by their immediate responsiveness to microbes. We previously demonstrated that the primary target for Vgamma9/Vdelta2 T cells is (E-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP, an essential metabolite produced by a large range of pathogens. Here we wished to study the consequence of this unique responsiveness in microbial infection. The majority of peripheral Vgamma9/Vdelta2 T cells shares migration properties with circulating monocytes, which explains the presence of these two distinct blood cell types in the inflammatory infiltrate at sites of infection and suggests that they synergize in anti-microbial immune responses. Our present findings demonstrate a rapid and HMB-PP-dependent crosstalk between Vgamma9/Vdelta2 T cells and autologous monocytes that results in the immediate production of inflammatory mediators including the cytokines interleukin (IL-6, interferon (IFN-gamma, tumor necrosis factor (TNF-alpha, and oncostatin M (OSM; the chemokines CCL2, CXCL8, and CXCL10; and TNF-related apoptosis-inducing ligand (TRAIL. Moreover, under these co-culture conditions monocytes differentiate within 18 hours into inflammatory dendritic cells (DCs with antigen-presenting functions. Addition of further microbial stimuli (lipopolysaccharide, peptidoglycan induces CCR7 and enables these inflammatory DCs to trigger the generation of CD4(+ effector alphabeta T cells expressing IFN-gamma and/or IL-17. Importantly, our in vitro model replicates the responsiveness to microbes of effluent cells from peritoneal dialysis (PD patients and translates directly to episodes of acute PD-associated bacterial peritonitis, where Vgamma9/Vdelta2 T cell numbers and soluble inflammatory mediators are elevated in patients infected with HMB-PP-producing pathogens. Collectively, these findings suggest a direct link between invading pathogens, microbe

  11. Dendritic cells from the human female reproductive tract rapidly capture and respond to HIV. (United States)

    Rodriguez-Garcia, M; Shen, Z; Barr, F D; Boesch, A W; Ackerman, M E; Kappes, J C; Ochsenbauer, C; Wira, C R


    Dendritic cells (DCs) throughout the female reproductive tract (FRT) were examined for phenotype, HIV capture ability and innate anti-HIV responses. Two main CD11c + DC subsets were identified: CD11b + and CD11b low DCs. CD11b + CD14 + DCs were the most abundant throughout the tract. A majority of CD11c + CD14 + cells corresponded to CD1c + myeloid DCs, whereas the rest lacked CD1c and CD163 expression (macrophage marker) and may represent monocyte-derived cells. In addition, we identified CD103 + DCs, located exclusively in the endometrium, whereas DC-SIGN + DCs were broadly distributed throughout the FRT. Following exposure to GFP-labeled HIV particles, CD14 + DC-SIGN + as well as CD14 + DC-SIGN - cells captured virus, with ∼30% of these cells representing CD1c + myeloid DCs. CD103 + DCs lacked HIV capture ability. Exposure of FRT DCs to HIV induced secretion of CCL2, CCR5 ligands, interleukin (IL)-8, elafin, and secretory leukocyte peptidase inhibitor (SLPI) within 3 h of exposure, whereas classical pro-inflammatory molecules did not change and interferon-α2 and IL-10 were undetectable. Furthermore, elafin and SLPI upregulation, but not CCL5, were suppressed by estradiol pre-treatment. Our results suggest that specific DC subsets in the FRT have the potential for capture and dissemination of HIV, exert antiviral responses and likely contribute to the recruitment of HIV-target cells through the secretion of innate immune molecules.

  12. Polymer based biosensor for rapid electrochemical detection of virus infection of human cells

    DEFF Research Database (Denmark)

    Kiilerich-Pedersen, Katrine; Poulsen, Claus R.; Jain, Titoo


    The demand in the field of medical diagnostics for simple, cost efficient and disposable devices is growing. Here, we present a label free, all-polymer electrochemical biosensor for detection of acute viral disease. The dynamics of a viral infection in human cell culture was investigated in a micro...... fluidic system on conductive polymer PEDOT:TsO microelectrodes by electrochemical impedance spectroscopy and video time lapse microscopy.Employing this sensitive, real time electrochemical technique, we could measure the immediate cell response to cytomegalovirus, and detect an infection within 3h, which...

  13. Measurement of rapid membrane permeation in cell suspensions by application of a generalized capillary method

    DEFF Research Database (Denmark)

    Klösgen, Beate; Schönert, Hansjürgen; Deuticke, Bernhard


    the diffusion process of a solute in a composite system was derived using a series-parallel-pathway model with explicit consideration of the diffusion pathways inside and between the cells. This renders the technique insensitive to unstirred layer effects. Any single cell population of known size distribution...... may be investigated. High permeabilities (above 5 · 10-3 cm/s) can be measured with the greatest precision, but lower permeabilities, down to a limit of about 5 · 10-4 cm/s, may also be determined by the method. Measurements in erythrocyte suspensions have been made using non...... of suspensions of membrane vesicles....

  14. Shrinkage of the scleral canal during cupping reversal in children. (United States)

    Mochizuki, Hideki; Lesley, Ashley G; Brandt, James D


    The mechanism of cupping reversal seen after lowering intraocular pressure (IOP) in pediatric glaucoma is unknown. Theories include forward movement of the lamina cribrosa or shrinkage of a stretched scleral canal. Our study aimed to quantify changes in optic disc size occurring in children who had undergone glaucoma surgery. Retrospective, single-center, observational case series. Children undergoing incisional surgery for pediatric glaucoma at the University of California, Davis. The electronic charts of all patients with pediatric glaucoma were reviewed for the presence of RetCam digital optic nerve photographs (Clarity Medical Systems, Pleasanton, CA). Cases in which the photographs (baseline and follow-up after surgical intervention) were of sufficient quality were analyzed. The optic disc margin was outlined manually using ImageJ software. Inter-session changes in magnification were accounted for by drawing a control polygon joining 4 or 5 fixed landmarks (e.g., vessel crossings) to include a second larger area containing the optic nerve. The optic disc area (in pixels adjusted with the control polygon) was compared between baseline and follow-up images. Change in disc area between baseline and follow-up after surgery. We identified 29 eyes for which baseline and follow-up images were available for analysis. Fifteen eyes of 9 children showed clinically obvious cupping reversal. Fourteen eyes of 12 children showed no cupping reversal. Disc area decreased by 6.8% (95% confidence interval [CI], -10.0 to -3.3) in the obvious reversal group and increased by 4.3% (95% CI, +1.0 to +7.6) in the no reversal group after surgery (P cupping reversal is clinically apparent after successful IOP-lowering surgery for congenital glaucoma, the scleral canal shrinks in area. In contrast, when cupping reversal is not observed, the scleral ring continues to enlarge, indicating ongoing stress on the optic nerve. Clinically obvious cupping reversal is less frequently observed in

  15. Formation of shrinkage porosity during solidification of steel: Numerical simulation and experimental validation (United States)

    Riedler, M.; Michelic, S.; Bernhard, C.


    The phase transformations in solidification of steel are accompanied by shrinkage and sudden changes in the solubility of alloying elements, resulting in negative side effects as micro- and macrosegregation and the formation of gas and shrinkage porosities. This paper deals with the numerical and experimental simulation of the formation of shrinkage porosity during the solidification of steel. First the physical basics for the mechanism of shrinkage pore formation will be discussed. The main reason for this type of porosity is the restraint of fluid flow in the mushy zone which leads to a pressure drop. The pressure decreases from the dendrite tip to the root. When the pressure falls below a critical value, a pore can form. The second part of the paper deals with different approaches for the prediction of the formation of shrinkage porosity. The most common one according to these models is the usage of a simple criterion function, like the Niyama criterion. For the computation of the porosity criterion the thermal gradient, cooling rate and solidification rate must be known, easily to determine from numerical simulation. More complex simulation tools like ProCAST include higher sophisticated models, which allow further calculations of the shrinkage cavity. Finally, the different approaches will be applied to a benchmark laboratory experiment. The presented results deal with an ingot casting experiment under variation of taper. The dominant influence of mould taper on the formation of shrinkage porosities can both be demonstrated by the lab experiment as well as numerical simulations. These results serve for the optimization of all ingot layouts for lab castings at the Chair of Ferrous Metallurgy.

  16. Water storage change estimation from in situ shrinkage measurements of clay soils

    Directory of Open Access Journals (Sweden)

    B. te Brake


    Full Text Available The objective of this study is to assess the applicability of clay soil elevation change measurements to estimate soil water storage changes, using a simplified approach. We measured moisture contents in aggregates by EC-5 sensors, and in multiple aggregate and inter-aggregate spaces (bulk soil by CS616 sensors. In a long dry period, the assumption of constant isotropic shrinkage proved invalid and a soil moisture dependant geometry factor was applied. The relative overestimation made by assuming constant isotropic shrinkage in the linear (basic shrinkage phase was 26.4% (17.5 mm for the actively shrinking layer between 0 and 60 cm. Aggregate-scale water storage and volume change revealed a linear relation for layers ≥ 30 cm depth. The range of basic shrinkage in the bulk soil was limited by delayed drying of deep soil layers, and maximum water loss in the structural shrinkage phase was 40% of total water loss in the 0–60 cm layer, and over 60% in deeper layers. In the dry period, fitted slopes of the ΔV–ΔW relationship ranged from 0.41 to 0.56 (EC-5 and 0.42 to 0.55 (CS616. Under a dynamic drying and wetting regime, slopes ranged from 0.21 to 0.38 (EC-5 and 0.22 to 0.36 (CS616. Alternating shrinkage and incomplete swelling resulted in limited volume change relative to water storage change. The slope of the ΔV–ΔW relationship depended on the drying regime, measurement scale and combined effect of different soil layers. Therefore, solely relying on surface level elevation changes to infer soil water storage changes will lead to large underestimations. Recent and future developments might provide a basis for application of shrinkage relations to field situations, but in situ observations will be required to do so.

  17. Rapid and label-free cell detection by metal-cluster-decorated carbon nanotube biosensors. (United States)

    Ishikawa, Fumiaki N; Stauffer, Beth; Caron, David A; Zhou, Chongwu


    In this paper, the use of carbon nanotube biosensors toward alga cell detection was examined. The biosensor devices were fabricated on complete 4 in. wafers by first growing carbon nanotubes (CNTs) and then depositing metal electrodes using a shadow mask. In addition, we decorated the biosensors with metal-clusters resulted in enhancing the sensitivity by 2000-folds and has enabled the detection of streptavidin down to 10 pM concentration. This sensitivity enhancement was attributed to activation of CNT channels due to formation of Schottky junctions between CNTs and metal-clusters. Real-time cell detection has been successfully carried out using the CNT biosensors for two kinds of alga related to brown tides: Aureococcus anophagefferens and BT3. Functionalization of the CNT biosensors with the monoclonal antibody for A. anophagefferens has led to detection at a concentration of 10(4) cells/ml, with sensitivity lower than 10(4) cells/ml projected based on the signal-to-noise ratio of the sensors. Further functionalization with tween 20 led to suppression of non-specific binding of BT3 and enabled label-free and selective detection of A. anophagefferens. These nanobiosensors may find potential applications for environmental monitoring and disease diagnosis.

  18. Integrated Systems for Rapid Point of Care (PoC) Blood Cell Analysis

    NARCIS (Netherlands)

    Van Berkel, C.; Gwyer, J.D.; Deane, S.; Hollis, V.; Holloway, J.; Green, N.; Morgan, H.M.


    Counting the different subpopulations of cells in a fingerprick of human blood is important for a number of clinical Point of Care applications. It is a challenge to demonstrate the integration of sample preparation and detection techniques in a single platform. In this article we review the

  19. Rapid desensitization of the histamine H2 receptor on the human monocytic cell line U937

    NARCIS (Netherlands)

    Smit, M J; Leurs, R; Shukrula, S R; Bast, A; Timmerman, H


    In the present study we have subjected the histamine H2 receptor on the monocytic cell line U937 to a thorough pharmacological characterization using a series of selective histamine H1, H2 and H3 receptor agonists and antagonists. Recent reports have demonstrated the existence of a histamine H2

  20. Tissue-resident adult stem cell populations of rapidly self-renewing organs

    NARCIS (Netherlands)

    Barker, N.; Bartfeld, S.; Clevers, H.


    The epithelial lining of the intestine, stomach, and skin is continuously exposed to environmental assault, imposing a requirement for regular self-renewal. Resident adult stem cell populations drive this renewal, and much effort has been invested in revealing their identity. Reliable adult stem

  1. Rapid detection of radiation-induced chromosomal aberrations in lymphocytes and hematopoietic progenitor cells by mFISH

    Energy Technology Data Exchange (ETDEWEB)

    Greulich, K.M.; Rhein, A.P.; Brueckner, M.; Molls, M. [Department of Radiation Oncology, Technical University of Munich, Ismaninger Strasse 22, D-81675 Munich (Germany); Kreja, L. [Institute for Occupational, Social and Environmental Medicine, University of Ulm, Ulm (Germany); Heinze, B. [Department of Medical Genetics, University of Ulm, Ulm (Germany); Weier, H.-U.G. [Life Sciences Division, E.O. Lawrence Berkeley National Laboratory, Berkeley, CA (United States); Fuchs, P. [Vysis GmbH, Bergisch-Gladbach (Germany)


    Structural chromosome aberrations (SCAs) are sensitive indicators of a preceding exposure of the hematopoietic system to ionizing radiation. Cytogenetic investigations have therefore become routine tools for an assessment of absorbed radiation doses and their biological effects after occupational exposure or radiation accidents. Due to its speed and ease of use, fluorescence in situ hybridization (FISH) with whole chromosome painting (WCP) probes has become a method of choice to visualize SCAs. Until recently, this technique was limited to a rather small number of chromosomes, which could be tested simultaneously. As a result, only a fraction of the structural aberrations present in a sample could be detected and the overall dose effect had to be calculated by extrapolation. The recent introduction of two genome-wide screening techniques in tumor research, i.e., Spectral Karyotyping (SKY) and multicolor FISH (mFISH) now allows the detection of translocations involving any two non-homologous chromosomes. The present study was prompted by our desire to bring the power of mFISH to bear for the rapid identification of radiation-induced SCAs. We chose two model systems to investigate the utility of mFISH: lymphocytes that were exposed in vitro to 3 Gy photons and single hematopoietic progenitor cell colonies isolated from a Chernobyl victim 9 years after in vivo exposure to 5.4 Sv. In lymphocytes, we found up to 15 different chromosomes involved in rearrangements indicating complex radiation effects. Stable aberrations detected in hematopoietic cell colonies, on the other hand, showed involvement of up to three different chromosomes. These results demonstrated that mFISH is a rapid and powerful approach to detect and characterize radiation-induced SCAs in the hemopoietic system. The application of mFISH is expected to result in a more detailed and, thus, more informative picture of radiation effects. Eventually, this technique will allow researchers to rapidly delineate

  2. Hypercytotoxicity and rapid loss of NKp44+ innate lymphoid cells during acute SIV infection.

    Directory of Open Access Journals (Sweden)

    Haiying Li


    Full Text Available HIV/SIV infections break down the integrity of the gastrointestinal mucosa and lead to chronic immune activation and associated disease progression. Innate lymphoid cells (ILCs, distinguishable by high expression of NKp44 and RORγt, play key roles in mucosal defense and homeostasis, but are depleted from gastrointestinal (GI tract large bowel during chronic SIV infection. However, less is known about the kinetics of ILC loss, or if it occurs systemically. In acute SIV infection, we found a massive, up to 8-fold, loss of NKp44+ILCs in all mucosae as early as day 6 post-infection, which was sustained through chronic disease. Interestingly, no loss of ILCs was observed in mucosa-draining lymph nodes. In contrast, classical NK cells were not depleted either from gut or draining lymph nodes. Both ILCs and NK cells exhibited significantly increased levels of apoptosis as measured by increased Annexin-V expression, but while classical NK cells also showed increased proliferation, ILCs did not. Interestingly, ILCs, which are normally noncytolytic, dramatically upregulated cytotoxic functions in acute and chronic infection and acquired a polyfunctional phenotype secreting IFN-γ, MIP1-β, and TNF-α, but decreased production of the prototypical cytokine, IL-17. Classical NK cells had less dramatic functional change, but upregulated perforin expression and increased cytotoxic potential. Finally, we show that numerical and functional loss of ILCs was due to increased apoptosis and ROR γt suppression induced by inflammatory cytokines in the gut milieu. Herein we demonstrate the first evidence for acute, systemic, and permanent loss of mucosal ILCs during SIV infection associated with reduction of IL-17. The massive reduction of ILCs involves apoptosis without compensatory de novo development/proliferation, but the full mechanism of depletion and the impact of functional change so early in infection remain unclear.

  3. Rapid Biosynthesis of Silver Nanoparticles Using Pepino (Solanum muricatum Leaf Extract and Their Cytotoxicity on HeLa Cells

    Directory of Open Access Journals (Sweden)

    Mónica Gorbe


    Full Text Available Within nanotechnology, gold and silver nanostructures have unique physical, chemical, and electronic properties [1,2], which make them suitable for a number of applications. Moreover, biosynthetic methods are considered to be a safer alternative to conventional physicochemical procedures for both the environmental and biomedical applications, due to their eco-friendly nature and the avoidance of toxic chemicals in the synthesis. For this reason, employing bio routes in the synthesis of functionalized silver nanoparticles (FAgNP have gained importance recently in this field. In the present study, we report the rapid synthesis of FAgNP through the extract of pepino (Solanum muricatum leaves and employing microwave oven irradiation. The core-shell globular morphology and characterization of the different shaped and sized FAgNP, with a core of 20–50 nm of diameter is established using the UV-Visible spectroscopy (UV-vis, field emission scanning electron microscopy (FESEM, transmission electron microscopy (TEM and Zeta potential and dynamic light scanning (DLS studies. Moreover, cytotoxic studies employing HeLa (human cervix carcinoma cells were undertaken to understand FAgNP interactions with cells. HeLa cells showed significant dose dependent antiproliferative activity in the presence of FAgNP at relatively low concentrations. The calculated IC50 value was 37.5 µg/mL, similar to others obtained for FAgNPs against HeLa cells.

  4. Rapidly developed squamous cell carcinoma after laser therapy used to treat chemical burn wound: a case report. (United States)

    Cho, Hyung-Rok; Kwon, Soon-Sung; Chung, Seum; Kie, Jeong-Hae


    In chronic wounds, especially burn scars, malignant tumors can arise. However, it is rare for a subacute burn injury to change to a malignant lesion within one month. Moreover, a case of squamous cell carcinoma arising from HeNe laser therapy after a chemical burn has never been reported. In this report, we examine a rare case of squamous cell carcinoma arising from HeNe laser therapy after a chemical burn. Because pathologic investigations were made from the first operation, both early detection of the squamous cell carcinoma and consideration of the HeNe laser therapy as a risk factor for the skin cancer were possible. The cancer was completely removed and reconstruction of the defect was successfully achieved in a timely manner. Although there has as yet been no reported case of squamous cell carcinoma induced by laser therapy, it is important for clinicians to recognize both the possibility of laser-induced cancer and the rapid change of cancer, so they can provide appropriate and timely treatment.

  5. Development of a rapid culture method to induce adipocyte differentiation of human bone marrow-derived mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Ninomiya, Yuichi [Translational Research Center, Saitama International Medical, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1298 (Japan); Sugahara-Yamashita, Yzumi; Nakachi, Yutaka; Tokuzawa, Yoshimi; Okazaki, Yasushi [Division of Functional Genomics and Systems Medicine, Research Center for Genomic Medicine, Saitama Medical University, Saitama 350-1241 (Japan); Nishiyama, Masahiko, E-mail: [Translational Research Center, Saitama International Medical, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1298 (Japan)


    Human mesenchymal stem cells (hMSCs) derived from bone marrow are multipotent stem cells that can regenerate mesenchymal tissues such as adipose, bone or muscle. It is thought that hMSCs can be utilized as a cell resource for tissue engineering and as human models to study cell differentiation mechanisms, such as adipogenesis, osteoblastogenesis and so on. Since it takes 2-3 weeks for hMSCs to differentiate into adipocytes using conventional culture methods, the development of methods to induce faster differentiation into adipocytes is required. In this study we optimized the culture conditions for adipocyte induction to achieve a shorter cultivation time for the induction of adipocyte differentiation in bone marrow-derived hMSCs. Briefly, we used a cocktail of dexamethasone, insulin, methylisobutylxanthine (DIM) plus a peroxisome proliferator-activated receptor {gamma} agonist, rosiglitazone (DIMRo) as a new adipogenic differentiation medium. We successfully shortened the period of cultivation to 7-8 days from 2-3 weeks. We also found that rosiglitazone alone was unable to induce adipocyte differentiation from hMSCs in vitro. However, rosiglitazone appears to enhance hMSC adipogenesis in the presence of other hormones and/or compounds, such as DIM. Furthermore, the inhibitory activity of TGF-{beta}1 on adipogenesis could be investigated using DIMRo-treated hMSCs. We conclude that our rapid new culture method is very useful in measuring the effect of molecules that affect adipogenesis in hMSCs.

  6. Evaluation of a rapid, quantitative real-time PCR method for enumeration of pathogenic Candida cells in water (United States)

    Brinkman, Nichole E.; Haugland, Richard A.; Wymer, Larry J.; Byappanahalli, Muruleedhara N.; Whitman, Richard L.; Vesper, Stephen J.


    Quantitative PCR (QPCR) technology, incorporating fluorigenic 5′ nuclease (TaqMan) chemistry, was utilized for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. lusitaniae) in water. Known numbers of target cells were added to distilled and tap water samples, filtered, and disrupted directly on the membranes for recovery of DNA for QPCR analysis. The assay's sensitivities were between one and three cells per filter. The accuracy of the cell estimates was between 50 and 200% of their true value (95% confidence level). In similar tests with surface water samples, the presence of PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts, with resultant reductions in sensitivity but generally not in quantitative accuracy. Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QPCR results and colony counts of the corresponding target species. Positive correlations were also seen between the cell quantities of the target Candida species detected in these analyses and colony counts of Enterococcus organisms. With a combined sample processing and analysis time of less than 4 h, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic Candida species from drinking and recreational waters and may have applications in the detection of fecal pollution.

  7. Rapid detection of herpes simplex virus in clinical specimens with human embryonic lung fibroblast and primary rabbit kidney cell cultures. (United States)

    Callihan, D R; Menegus, M A


    The performance of a culture system for isolation of herpes simplex virus, consisting of one tube each of human embryonic lung fibroblasts and primary rabbit kidney cells, was evaluated. Cultures were incubated at 37 degrees C on a roller drum and observed daily for characteristic cytopathic effect for 5 days. During 1982, a positive isolation rate of 28.1% was seen among 3,154 specimens submitted. Cultures from genital sources were positive more frequently from males (43.8%) than from females (25.5%). Oral lesion cultures were positive as often from males (34.6%) as from females (38.4%). Although detection of herpes simplex virus occurred significantly earlier in rabbit kidney cells on days 1 and 2 of incubation, by day 3 the number of positive cultures was nearly the same in both cell types. By day 4 of incubation, 99.5% of the positive cultures were detected. These results demonstrate that cell culture can be a rapid and sensitive method for detecting herpes simplex virus.

  8. Rapid deterioration of preexisting renal insufficiency after autologous mesenchymal stem cell therapy


    Jun-Seop Kim; Jong-Hak Lee; Owen Kwon; Jang-Hee Cho; Ji-Young Choi; Sun-Hee Park; Chan-Duck Kim; Yong-Jin Kim; Yong-Lim Kim


    Administration of autologous mesenchymal stem cells (MSCs) has been shown to improve renal function and histological findings in acute kidney injury (AKI) models. However, its effects in chronic kidney disease (CKD) are unclear, particularly in the clinical setting. Here, we report our experience with a CKD patient who was treated by intravenous infusion of autologous MSCs derived from adipose tissue in an unknown clinic outside of Korea. The renal function of the patient had been stable for ...

  9. Expandable and Rapidly Differentiating Human Induced Neural Stem Cell Lines for Multiple Tissue Engineering Applications

    Directory of Open Access Journals (Sweden)

    Dana M. Cairns


    Full Text Available Limited availability of human neurons poses a significant barrier to progress in biological and preclinical studies of the human nervous system. Current stem cell-based approaches of neuron generation are still hindered by prolonged culture requirements, protocol complexity, and variability in neuronal differentiation. Here we establish stable human induced neural stem cell (hiNSC lines through the direct reprogramming of neonatal fibroblasts and adult adipose-derived stem cells. These hiNSCs can be passaged indefinitely and cryopreserved as colonies. Independently of media composition, hiNSCs robustly differentiate into TUJ1-positive neurons within 4 days, making them ideal for innervated co-cultures. In vivo, hiNSCs migrate, engraft, and contribute to both central and peripheral nervous systems. Lastly, we demonstrate utility of hiNSCs in a 3D human brain model. This method provides a valuable interdisciplinary tool that could be used to develop drug screening applications as well as patient-specific disease models related to disorders of innervation and the brain.

  10. Rapid and sustained CD4(+) T-cell-independent immunity from adenovirus-encoded vaccine antigens

    DEFF Research Database (Denmark)

    Holst, Peter J; Bartholdy, Christina; Buus, Anette Stryhn


    Many novel vaccine strategies rely on recombinant viral vectors for antigen delivery, and adenovirus vectors have emerged among the most potent of these. In this report, we have compared the immune response induced through priming with adenovirus vector-encoded full-length viral protein to that e......Many novel vaccine strategies rely on recombinant viral vectors for antigen delivery, and adenovirus vectors have emerged among the most potent of these. In this report, we have compared the immune response induced through priming with adenovirus vector-encoded full-length viral protein...... to that elicited with an adenovirus-encoded minimal epitope covalently linked to beta(2)-microglobulin. We demonstrate that the beta(2)-microglobulin-linked epitope induced an accelerated and augmented CD8(+) T-cell response. Furthermore, the immunity conferred by vaccination with beta(2)-microglobulin...... in the absence of CD4(+) T-cell help were sustained in the long term and able to expand and control a secondary challenge with LCMV. Our results demonstrate that modifications to the antigen used in adenovirus vaccines may be used to improve the induced T-cell response. Such a strategy for CD4(+) T...

  11. A system for the rapid detection of bacterial contamination in cell-based therapeutica (United States)

    Bolwien, Carsten; Erhardt, Christian; Sulz, Gerd; Thielecke, Hagen; Johann, Robert; Pudlas, Marieke; Mertsching, Heike; Koch, Steffen


    Monitoring the sterility of cell or tissue cultures is of major concern, particularly in the fields of regenerative medicine and tissue engineering when implanting cells into the human body. Our sterility-control system is based on a Raman micro-spectrometer and is able to perform fast sterility testing on microliters of liquid samples. In conventional sterility control, samples are incubated for weeks to proliferate the contaminants to concentrations above the detection limit of conventional analysis. By contrast, our system filters particles from the liquid sample. The filter chip fabricated in microsystem technology comprises a silicon nitride membrane with millions of sub-micrometer holes to retain particles of critical sizes and is embedded in a microfluidic cell specially suited for concomitant microscopic observation. After filtration, identification is carried out on the single particle level: image processing detects possible contaminants and prepares them for Raman spectroscopic analysis. A custom-built Raman-spectrometer-attachment coupled to the commercial microscope uses 532nm or 785nm Raman excitation and records spectra up to 3400cm-1. In the final step, the recorded spectrum of a single particle is compared to an extensive library of GMP-relevant organisms, and classification is carried out based on a support vector machine.

  12. Differentially pumped spray deposition as a rapid screening tool for organic and perovskite solar cells. (United States)

    Jung, Yen-Sook; Hwang, Kyeongil; Scholes, Fiona H; Watkins, Scott E; Kim, Dong-Yu; Vak, Doojin


    We report a spray deposition technique as a screening tool for solution processed solar cells. A dual-feed spray nozzle is introduced to deposit donor and acceptor materials separately and to form blended films on substrates in situ. Using a differential pump system with a motorised spray nozzle, the effect of film thickness, solution flow rates and the blend ratio of donor and acceptor materials on device performance can be found in a single experiment. Using this method, polymer solar cells based on poly(3-hexylthiophene) (P3HT):(6,6)-phenyl C61 butyric acid methyl ester (PC61BM) are fabricated with numerous combinations of thicknesses and blend ratios. Results obtained from this technique show that the optimum ratio of materials is consistent with previously reported values confirming this technique is a very useful and effective screening method. This high throughput screening method is also used in a single-feed configuration. In the single-feed mode, methylammonium iodide solution is deposited on lead iodide films to create a photoactive layer of perovskite solar cells. Devices featuring a perovskite layer fabricated by this spray process demonstrated a power conversion efficiencies of up to 7.9%.

  13. Rapid engraftment by peripheral blood progenitor cells mobilized by recombinant human stem cell factor and recombinant human granulocyte colony-stimulating factor in nonhuman primates. (United States)

    Andrews, R G; Briddell, R A; Knitter, G H; Rowley, S D; Appelbaum, F R; McNiece, I K


    We have previously shown that administration of low-dose recombinant human stem cell factor (rhSCF) plus recombinant human granulocyte colony-stimulating factor (rhG-CSF) to baboons mobilizes greater numbers of progenitor cells in the blood than does administration of rhG-CSF alone. The purpose of the present study was to determine whether marrow repopulating cells are present in the blood of nonhuman primates administered low-dose rhSCF plus rhG-CSF, and if present, whether these cells engraft lethally irradiated recipients as rapidly as blood cells mobilized by treatment with rhG-CSF alone. One group of baboons was administered low-dose rhSCF (25 micrograms/kg/d) plus rhG-CSF (100 micrograms/kg/d) while a second group received rhG-CSF alone (100 micrograms/kg/d). Each animal underwent a single 2-hour leukapheresis occurring the day when the number of progenitor cells per volume of blood was maximal. For baboons administered low-dose rhSCF plus rhG-CSF, the leukapheresis products contained 1.8-fold more mononuclear cells and 14.0-fold more progenitor cells compared to the leukapheresis products from animals treated with rhG-CSF alone. All animals successfully engrafted after transplantation of cryopreserved autologous blood cells. In animals transplanted with low-dose rhSCF plus rhG-CSF mobilized blood cells, we observed a time to a platelet count of > 20,000 was 8 days +/- 0, to a white blood cell count (WBC) of > 1,000 was 11 +/- 1 days, and to an absolute neutrophil count (ANC) of > 500 was 12 +/- 1 days. These results compared with 42 +/- 12, 16 +/- 1, and 24 +/- 4 days to achieve platelets > 20,000, WBC > 1,000, and ANC > 500, respectively, for baboons transplanted with rhG-CSF mobilized blood cells. Animals transplanted with low-dose rhSCF plus rhG-CSF mobilized blood cells had blood counts equivalent to pretransplant values within 3 weeks after transplant. The results suggest that the combination of low-dose rhSCF plus rhG-CSF mobilizes greater numbers of

  14. Rapid identification and enumeration of Saccharomyces cerevisiae cells in wine by real-time PCR. (United States)

    Martorell, P; Querol, A; Fernández-Espinar, M T


    Despite the beneficial role of Saccharomyces cerevisiae in the food industry for food and beverage production, it is able to cause spoilage in wines. We have developed a real-time PCR method to directly detect and quantify this yeast species in wine samples to provide winemakers with a rapid and sensitive method to detect and prevent wine spoilage. Specific primers were designed for S. cerevisiae using the sequence information obtained from a cloned random amplified polymorphic DNA band that differentiated S. cerevisiae from its sibling species Saccharomyces bayanus, Saccharomyces pastorianus, and Saccharomyces paradoxus. The specificity of the primers was demonstrated for typical wine spoilage yeast species. The method was useful for estimating the level of S. cerevisiae directly in sweet wines and red wines without preenrichment when yeast is present in concentrations as low as 3.8 and 5 CFU per ml. This detection limit is in the same order as that obtained from glucose-peptone-yeast growth medium (GPY). Moreover, it was possible to quantify S. cerevisiae in artificially contaminated samples accurately. Limits for accurate quantification in wine were established, from 3.8 x 10(5) to 3.8 CFU/ml in sweet wine and from 5 x 10(6) to 50 CFU/ml in red wine.

  15. Understanding the Capsanthin Tails in Regulating the Hydrophilic-Lipophilic Balance of Carbon Dots for a Rapid Crossing Cell Membrane. (United States)

    Chen, Jing; Zhang, Xiang; Zhang, Ye; Wang, Wei; Li, Shuya; Wang, Yucai; Hu, Mengyue; Liu, Li; Bi, Hong


    Here we use natural Chinese paprika to prepare a new kind of amphiphilic carbon dot (A-Dot) that exhibits bright, multicolored fluorescence and contains hydrophilic groups as well as lipophilic capsanthin tails on the surface. It is found that the capsanthin tails in a phospholipid-like structure can promote cell internalization of the A-Dots via crossing cell membranes rapidly in an energy-independent fashion. Compared to highly hydrophilic carbon dots (H-Dots), a control sample prepared from the microwave thermolysis of citric acid and ethylenediamine, our synthesized A-Dots can be taken up by CHO, HeLa, and HFF cells more easily. More importantly, we develop a method to calibrate the hydrophilic-lipophilic balance (HLB) values of various kinds of carbon dots (C-Dots). HLB values of A-Dots and H-Dots are determined to be 6.4 and 18.4, respectively. Moreover, we discover that the cellular uptake efficiency of C-Dots is closely related to their HLBs, and the C-Dots with an HLB value of around 6.4 cross the cell membrane easier and faster. As we regulate the HLB value of the A-Dots from 6.4 to 15.3 by removing the capsanthin tails from their surfaces via alkali refluxing, it is found that the refluxed A-Dots can hardly cross HeLa cell membranes. Our work is an essential step toward understanding the importance of regulating the HLB values as well as the surface polarity of the C-Dots for their practical use in bioimaging and also provides a simple but effective way to judge whether the C-Dots in hand are appropriate for cell imaging.

  16. Antioxidants cause rapid expansion of human adipose-derived mesenchymal stem cells via CDK and CDK inhibitor regulation (United States)


    Background Antioxidants have been shown to enhance the proliferation of adipose-derived mesenchymal stem cells (ADMSCs) in vitro, although the detailed mechanism(s) and potential side effects are not fully understood. In this study, human ADMSCs cultured in ImF-A medium supplemented with antioxidants (N-acetyl-l-cysteine and ascorbic acid-2-phosphate) and fibroblast growth factor 2 (FGF-2) were compared with ADMSCs cultured with FGF-2 alone (ImF) or with FGF-2 under 5% pO2 conditions (ImF-H). Results During log-phase growth, exposure to ImF-A resulted in a higher percentage of ADMSCs in the S phase of the cell cycle and a smaller percentage in G0/G1 phase. This resulted in a significantly reduced cell-doubling time and increased number of cells in the antioxidant-supplemented cultures compared with those supplemented with FGF-2 alone, an approximately 225% higher cell density after 7 days. Western blotting showed that the levels of the CDK inhibitors p21 and p27 decreased after ImF-A treatment, whereas CDK2, CDK4, and CDC2 levels clearly increased. In addition, ImF-A resulted in significant reduction in the expression of CD29, CD90, and CD105, whereas relative telomere length, osteogenesis, adipogenesis, and chondrogenesis were enhanced. The results were similar for ADMSCs treated with antioxidants and those under hypoxic conditions. Conclusion Antioxidant treatment promotes entry of ADMSCs into the S phase by suppressing cyclin-dependent kinase inhibitors and results in rapid cell proliferation similar to that observed under hypoxic conditions. PMID:23915242

  17. Rapid production of a H₉ N₂ influenza vaccine from MDCK cells for protecting chicken against influenza virus infection. (United States)

    Ren, Zhenghua; Lu, Zhongzheng; Wang, Lei; Huo, Zeren; Cui, Jianhua; Zheng, Tingting; Dai, Qing; Chen, Cuiling; Qin, Mengying; Chen, Meihua; Yang, Rirong


    H9N2 subtype avian influenza viruses are widespread in domestic poultry, and vaccination remains the most effective way to protect the chicken population from avian influenza pandemics. Currently, egg-based H9N2 influenza vaccine production has several disadvantages and mammalian MDCK cells are being investigated as candidates for influenza vaccine production. However, little research has been conducted on low pathogenic avian influenza viruses (LPAIV) such as H9N2 replicating in mammalian cells using microcarrier beads in a bioreactor. In this study, we present a systematic analysis of a safe H9N2 influenza vaccine derived from MDCK cells for protecting chickens against influenza virus infection. In 2008, we isolated two novel H9N2 influenza viruses from chickens raised in southern China, and these H9N2 viruses were adapted to MDCK cells. The H9N2 virus was produced in MDCK cells in a scalable bioreactor, purified, inactivated, and investigated for use as a vaccine. The MDCK-derived H9N2 vaccine was able to induce high titers of neutralizing antibodies in chickens of different ages. Histopathological examination, direct immunofluorescence, HI assay, CD4(+)/CD8(+) ratio test, and cytokine evaluation indicated that the MDCK-derived H9N2 vaccine evoked a rapid and effective immune response to protect chickens from influenza infection. High titers of H9N2-specific antibodies were maintained in chickens for 5 months, and the MDCK-derived H9N2 vaccine had no effects on chicken growth. The use of MDCK cells in bioreactors for LPAIV vaccine production is an attractive option to prevent outbreaks of LPAIV in poultry.

  18. Comparison of two rapid assays for Clostridium difficile Common antigen and a C difficile toxin A/B assay with the cell culture neutralization assay. (United States)

    Reller, Megan E; Alcabasa, Romina C; Lema, Clara A; Carroll, Karen C


    We compared 3 rapid assays for Clostridium difficile with a cell culture cytotoxicity neutralization assay (CCNA). Of 600 stool samples, 46 were positive for toxigenic C difficile. Both rapid common antigen assays were highly sensitive (91.3%-100%) and, therefore, were appropriate screening tests. The rapid toxin assay had poor sensitivity (61%) but excellent specificity (99.3%). Testing stools for glutamate dehydrogenase (step 1) and those positive with a rapid toxin assay (step 2) would correctly classify 81% of submitted specimens within 2 hours, including during periods of limited staffing (evenings, nights, and weekends). CCNA could then be used as a third step to test rapid toxin-negative samples, thereby providing a final result for the remaining 19% of samples by 48 to 72 hours. The use of rapid assays as outlined could enhance timely diagnosis of C difficile.

  19. Drying Shrinkage Behaviour of Fibre Reinforced Concrete Incorporating Polyvinyl Alcohol Fibres and Fly Ash

    Directory of Open Access Journals (Sweden)

    Amin Noushini


    Full Text Available The current study assesses the drying shrinkage behaviour of polyvinyl alcohol fibre reinforced concrete (PVA-FRC containing short-length (6 mm and long-length (12 mm uncoated monofilament PVA fibres at 0.125%, 0.25%, 0.375%, and 0.5% volumetric fractions. Fly ash is also used as a partial replacement of Portland cement in all mixes. PVA-FRC mixes have been compared to length change of control concrete (devoid of fibres at 3 storage intervals: early-age (0–7 days, short-term (0–28 days, and long-term (28–112 days intervals. The shrinkage results of FRC and control concrete up to 112 days indicated that all PVA-FRC mixes exhibited higher drying shrinkage than control. The shrinkage exhibited by PVA-FRC mixes ranged from 449 to 480 microstrain, where this value was only 427 microstrain in the case of control. In addition, the longer fibres exhibited higher mass loss, thus potentially contributing to higher shrinkage.

  20. Effect of limestone and granite coarse aggregate on drying shrinkage of a concrete

    Directory of Open Access Journals (Sweden)

    Woyciechowski Piotr


    Full Text Available In this paper the effect of limestone and granite aggregate on drying shrinkage of a concrete is investigated. Mixes containing limestone and granite with different Dmax and cement paste volume were tested. A two level, full factorial experiment with three independent factors was conducted which allowed not only to determine the influence of every single factor itself but also potential interactions between them. Measurements of shrinkage were performed up to 98 days after demolding on the beams (10×10×50 cm, in accordance with the procedure from polish standard PN-84/B-06714/23 (Amsler’s method. The multivariate analysis of variances (MANOVA was used for the statistical analysis of the results. The obtained results confirm that the cement paste volume has a decisive influence on a drying shrinkage of a concrete. However statistical analysis showed that with the assumed variability, either the Dmax or type of coarse aggregate has no significant influence on drying shrinkage, still those parameters may potentially be crucial in some cases. A statistically significant interaction between Dmax and type of aggregate has been found. Furthermore the results of drying shrinkage measurements were compared with estimation results from Bażant-Baweja B3 Model and Eurocode 2. The comparison shows that B3 Model provided more accurate results than Eurocode 2.

  1. Modified creep and shrinkage prediction model B3 for serviceability limit state analysis of composite slabs (United States)

    Gholamhoseini, Alireza


    Relatively little research has been reported on the time-dependent in-service behavior of composite concrete slabs with profiled steel decking as permanent formwork and little guidance is available for calculating long-term deflections. The drying shrinkage profile through the thickness of a composite slab is greatly affected by the impermeable steel deck at the slab soffit, and this has only recently been quantified. This paper presents the results of long-term laboratory tests on composite slabs subjected to both drying shrinkage and sustained loads. Based on laboratory measurements, a design model for the shrinkage strain profile through the thickness of a slab is proposed. The design model is based on some modifications to an existing creep and shrinkage prediction model B3. In addition, an analytical model is developed to calculate the time-dependent deflection of composite slabs taking into account the time-dependent effects of creep and shrinkage. The calculated deflections are shown to be in good agreement with the experimental measurements.

  2. Pore Structure and Influence of Recycled Aggregate Concrete on Drying Shrinkage

    Directory of Open Access Journals (Sweden)

    Yuanchen Guo


    Full Text Available Pore structure plays an important role in the drying shrinkage of recycled aggregate concrete (RAC. High-precision mercury intrusion and water evaporation were utilized to study the pore structure of RAC, which has a different replacement rate of recycled concrete aggregate (RCA, and to analyze its influence on drying shrinkage. Finally, a fractal-dimension calculation model was established based on the principles of mercury intrusion and fractal-geometry theory. Calculations were performed to study the pore-structure fractal dimension of RAC. Results show the following. (1 With the increase in RCA content, the drying shrinkage values increase gradually. (2 Pores with the greatest impact on concrete shrinkage are those whose sizes ranging from 2.5 nm to 50 nm and from 50 nm to 100 nm. In the above two ranges, the proportions of RAC are greater than those of RC0 (natural aggregate concrete, NAC, which is the main reason the shrinkage values of RAC are greater than those of NAC. (3 The pore structure of RAC has good fractal feature, and the addition of RCA increases the complexity of the pore surface of concrete.

  3. Shrinkage modeling of concrete reinforced by palm fibres in hot dry environments (United States)

    Akchiche, Hamida; Kriker, Abdelouahed


    The cement materials, such as concrete and conventional mortar present very little resistance to traction and cracking, these hydraulic materials which induces large withdrawals on materials and cracks in structures. The hot dry environments such as: the Saharan regions of Algeria, Indeed, concrete structures in these regions are very fragile, and present high shrinkage. Strengthening of these materials by fibers can provide technical solutions for improving the mechanical performance. The aim of this study is firstly, to reduce the shrinkage of conventional concrete with its reinforcement with date palm fibers. In fact, Algeria has an extraordinary resources in natural fibers (from Palm, Abaca, Hemp) but without valorization in practical areas, especially in building materials. Secondly, to model the shrinkage behavior of concrete was reinforced by date palm fibers. In the literature, several models for still fiber concrete were founded but few are offers for natural fiber concretes. To do so, a still fiber concretes model of YOUNG - CHERN was used. According to the results, a reduction of shrinkage with reinforcement by date palm fibers was showed. A good ability of molding of shrinkage of date palm reinforced concrete with YOUNG - CHERN Modified model was obtained. In fact, a good correlation between experimental data and the model data was recorded.

  4. Some Issues of Shrinkage-Reducing Admixtures Application in Alkali-Activated Slag Systems. (United States)

    Bílek, Vlastimil; Kalina, Lukáš; Novotný, Radoslav; Tkacz, Jakub; Pařízek, Ladislav


    Significant drying shrinkage is one of the main limitations for the wider utilization of alkali-activated slag (AAS). Few previous works revealed that it is possible to reduce AAS drying shrinkage by the use of shrinkage-reducing admixtures (SRAs). However, these studies were mainly focused on SRA based on polypropylene glycol, while as it is shown in this paper, the behavior of SRA based on 2-methyl-2,4-pentanediol can be significantly different. While 0.25% and 0.50% had only a minor effect on the AAS properties, 1.0% of this SRA reduced the drying shrinkage of waterglass-activated slag mortar by more than 80%, but it greatly reduced early strengths simultaneously. This feature was further studied by isothermal calorimetry, mercury intrusion porosimetry (MIP) and scanning electron microscopy (SEM). Calorimetric experiments showed that 1% of SRA modified the second peak of the pre-induction period and delayed the maximum of the main hydration peak by several days, which corresponds well with observed strength development as well as with the MIP and SEM results. These observations proved the certain incompatibility of SRA with the studied AAS system, because the drying shrinkage reduction was induced by the strong retardation of hydration, resulting in a coarsening of the pore structure rather than the proper function of the SRA.

  5. Effect of steel fibers on plastic shrinkage cracking of normal and high strength concretes

    Directory of Open Access Journals (Sweden)

    Özgür Eren


    Full Text Available Naturally concrete shrinks when it is subjected to a drying environment. If this shrinkage is restrained, tensile stresses develop and concrete may crack. Plastic shrinkage cracks are especially harmful on slabs. One of the methods to reduce the adverse effects of shrinkage cracking of concrete is by reinforcing concrete with short randomly distributed fibers. The main objective of this study was to investigate the effect of fiber volume and aspect ratio of hooked steel fibers on plastic shrinkage cracking behavior together with some other properties of concrete. In this research two different compressive strength levels namely 56 and 73 MPa were studied. Concretes were produced by adding steel fibers of 3 different volumes of 3 different aspect ratios. From this research study, it is observed that steel fibers can significantly reduce plastic shrinkage cracking behavior of concretes. On the other hand, it was observed that these steel fibers can adversely affect some other properties of concrete during fresh and hardened states.

  6. A shrinkage method for testing the Hardy-Weinberg equilibrium in case-control studies. (United States)

    Zang, Yong; Yuan, Ying


    Testing for the Hardy-Weinberg equilibrium (HWE) is often used as an initial step for checking the quality of genotyping. When testing the HWE for case-control data, the impact of a potential genetic association between the marker and the disease must be controlled for otherwise the results may be biased. Li and Li [2008] proposed a likelihood ratio test (LRT) that accounts for this potential genetic association and it is more powerful than the commonly used control-only χ² test. However, the LRT is not efficient when the marker is independent of the disease, and also requires numerical optimization to calculate the test statistic. In this article, we propose a novel shrinkage test for assessing the HWE. The proposed shrinkage test yields higher statistical power than the LRT when the marker is independent of or weakly associated with the disease, and converges to the LRT when the marker is strongly associated with the disease. In addition, the proposed shrinkage test has a closed form and can be easily used to test the HWE for large datasets that result from genome-wide association studies. We compare the performance of the shrinkage test with existing methods using simulation studies, and apply the shrinkage test to a genome-wide association dataset for Alzheimer's disease. © 2013 WILEY PERIODICALS, INC.

  7. A generalized DEMATEL theory with a shrinkage coefficient for an indirect relation matrix

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    Liu Hsiang-Chuan


    Full Text Available In this paper, a novel decision-making trial and evaluation laboratory (DEMATEL theory with a shrinkage coefficient of indirect relation matrix is proposed, and a useful validity index, called Liu’s validity index, is also proposed for evaluating the performance of any DEMATEL model. If the shrinkage coefficient of an indirect relation matrix is equal to 1, then this new theory is identical to the traditional theory; in other words, it is a generalization of the traditional theory. Furthermore, the indirect relation is always considerably greater than the direct one in traditional DEMATEL theory, which is unreasonable and unfair because it overemphasizes the influence of the indirect relation. We prove in this paper that if the shrinkage coefficient is equal to 0.5, then the indirect relation is less than its direct relation. Because the shrinkage coefficient belongs to [0.5, 1], according to Liu’s validity index, we can find a more appropriate shrinkage coefficient to obtain a more efficient DEMATEL method. Some crucial properties of this new theory are discussed, and a simple example is provided to illustrate the advantages of the proposed theory.

  8. Effects of Prepolymerized Particle Size and Polymerization Kinetics on Volumetric Shrinkage of Dental Modeling Resins

    Directory of Open Access Journals (Sweden)

    Tae-Yub Kwon


    Full Text Available Dental modeling resins have been developed for use in areas where highly precise resin structures are needed. The manufacturers claim that these polymethyl methacrylate/methyl methacrylate (PMMA/MMA resins show little or no shrinkage after polymerization. This study examined the polymerization shrinkage of five dental modeling resins as well as one temporary PMMA/MMA resin (control. The morphology and the particle size of the prepolymerized PMMA powders were investigated by scanning electron microscopy and laser diffraction particle size analysis, respectively. Linear polymerization shrinkage strains of the resins were monitored for 20 minutes using a custom-made linometer, and the final values (at 20 minutes were converted into volumetric shrinkages. The final volumetric shrinkage values for the modeling resins were statistically similar (P>0.05 or significantly larger (P<0.05 than that of the control resin and were related to the polymerization kinetics (P<0.05 rather than the PMMA bead size (P=0.335. Therefore, the optimal control of the polymerization kinetics seems to be more important for producing high-precision resin structures rather than the use of dental modeling resins.

  9. Measurement of linear polymerization shrinkage in light cure Ideal Makoo composite resin

    Directory of Open Access Journals (Sweden)

    Ghavam M.


    Full Text Available "nAbstract: Polymerization shrinkage of light cure composite resins causes many complications in conservative and esthetic restorations. The objective of this in-vitro study was to evaluate the polymerization shrinkage, degree of conversion and the amount of filler in IDM and tetric ceram composites. Ten disk shaped, uncured specimens (8mm×1.547mm of each composite were placed on glass slide in the center of the metal attached to it. Then specimens were light cured for 60s from underneath. After 30 minutes, the thickness of specimens, using a micrometer and the percent of the polymerization shrinkage of each sample were measured. Statistical analysis was carried out by t-test (P<0.05. Also the degree of conversion of specimens was evaluated with FTIR and the mineral filler content was measured by burning in electric oven. Polymerization shrinkage in IDM and tetric ceram was not significantly different. Degree of conversion and mineral filler content in tetric ceram was greater than that of IDM. "nIt is assumed that the low degree of conversion in IDM is due to its chemical composition and filler content. Also, the similarity in linear polymerization shrinkage between IDM and tetric ceram may be caused by the low degree of conversion in IDM.

  10. Precise Cross-Sectional Measurement of Photoresist Shrinkage Caused by Electron Beam Irradiation (United States)

    Ohashi, Takeyoshi; Sekiguchi, Tomoko; Yamaguchi, Atsuko; Tanaka, Junichi; Kawada, Hiroki


    A mechanism of the photoresist shrinkage induced by electron-beam (EB) irradiation was studied in detail. A precise cross-sectional profile of a photoresist pattern is obtained by a scanning transmission electron microscope (STEM) after HfO2 atomic layer deposition on a sample. Photoresist lines and spaces formed on either of bottom anti-reflective coating (BARC) layer or spin-on-glass (SOG) layer were exposed to EB at a much higher dose than a practical dose (to accelerate shrinkage intentionally). The obtained STEM images of the patterns before and after EB irradiation showed that EB irradiation causes necking of the pattern profile as well as linewidth slimming. In addition, it was suggested that the BARC layer shrinkage results in the elastic deformation of the pattern profile, whereas the SOG layer does not shrink. Furthermore, EB irradiation only to the lines or spaces was performed. The EB irradiation to spaces caused sidewall shrinkage and necking of the pattern profile, although no electron was irradiated directly into the pattern. This result is considered to be due to the electrons scattered from the spaces to the pattern sidewall. Finally, a Monte Carlo simulation showed that the distribution of the deposited energy on the pattern surface corresponds to the change of the pattern shape. Consequently, our study clarifies the importance of the effect of elastic shape change and the impact of the electrons scattered from the underlying layer to the sidewall on photoresist shrinkage.

  11. Shrinkage Behaviour of Fibre Reinforced Concrete with Recycled Tyre Polymer Fibres

    Directory of Open Access Journals (Sweden)

    Marijana Serdar


    Full Text Available Different types of fibres are often used in concrete to prevent microcracking due to shrinkage, and polypropylene fibres are among the most often used ones. If not prevented, microcracks can lead to the development of larger cracks as drying shrinkage occurs, enabling penetration of aggressive substances from the environment and reducing durability of concrete structures. The hypothesis of the present research is that polypropylene fibres, used in concrete for controlling formation of microcracks due to shrinkage, can be replaced with recycled polymer fibres obtained from end-of-life tyres. To test the hypothesis, concrete mixtures containing polypropylene fibres and recycled tyre polymer fibres were prepared and tested. Experimental programme focused on autogenous, free, and restrained shrinkage. It was shown that PP fibres can be substituted with higher amount of recycled tyre polymer fibres obtaining concrete with similar shrinkage behaviour. The results indicate promising possibilities of using recycled tyre polymer fibres in concrete products. At the same time, such applications would contribute to solving the problem of waste tyre disposal.

  12. Shrinkage Characteristics of Experimental Polymer Containing Composites under Controlled Light Curing Modes

    Directory of Open Access Journals (Sweden)

    Alain Pefferkorn


    Full Text Available The adsorption of polymethylmethacrylate polymer of different molecular weight at the aerosil/ethyleneglycol- or 1,3 butanediol-dimethacrylate interfaces was determined to provide microstructured networks. Their structural characteristics were determined to be controlled by the amount of polymer initially supplied to the system. The sediment (the settled phase characteristics, determined as a function of the polymer concentration and the rate of the polymerization shrinkage determined for composite resins, obtained by extrusion of the sediment after centrifugation, were found to be correlated. The specific role of the adsorbed polymer was found to be differently perturbed with the supplementary supply of dimethacrylate based monomer additives. Particularly, the bisphenol A dimethacrylate that generated crystals within the sediment was found to impede the shrinkage along the crystal lateral faces and strongly limit the shrinkage along its basal faces. Addition of ethyleneglycol- or polyethylene-glycoldimethacrylate monomers was determined to modify the sedimentation characteristics of the aerosil suspension and the shrinkage properties of the composites. Finally, the effects of stepwise light curing methods with prolonged lighting-off periods were investigated and found to modify the development and the final values of the composite shrinkage.

  13. Some Issues of Shrinkage-Reducing Admixtures Application in Alkali-Activated Slag Systems (United States)

    Bílek, Vlastimil; Kalina, Lukáš; Novotný, Radoslav; Tkacz, Jakub; Pařízek, Ladislav


    Significant drying shrinkage is one of the main limitations for the wider utilization of alkali-activated slag (AAS). Few previous works revealed that it is possible to reduce AAS drying shrinkage by the use of shrinkage-reducing admixtures (SRAs). However, these studies were mainly focused on SRA based on polypropylene glycol, while as it is shown in this paper, the behavior of SRA based on 2-methyl-2,4-pentanediol can be significantly different. While 0.25% and 0.50% had only a minor effect on the AAS properties, 1.0% of this SRA reduced the drying shrinkage of waterglass-activated slag mortar by more than 80%, but it greatly reduced early strengths simultaneously. This feature was further studied by isothermal calorimetry, mercury intrusion porosimetry (MIP) and scanning electron microscopy (SEM). Calorimetric experiments showed that 1% of SRA modified the second peak of the pre-induction period and delayed the maximum of the main hydration peak by several days, which corresponds well with observed strength development as well as with the MIP and SEM results. These observations proved the certain incompatibility of SRA with the studied AAS system, because the drying shrinkage reduction was induced by the strong retardation of hydration, resulting in a coarsening of the pore structure rather than the proper function of the SRA. PMID:28773584

  14. Rapid green synthesis of ZnO nanoparticles using a hydroelectric cell without an electrolyte (United States)

    Shah, Jyoti; Kumar Kotnala, Ravinder


    In this study, zinc oxide (ZnO) nanoparticles were synthesized using a novel environmentally friendly hydroelectric cell without an electrolyte or external current source. The hydroelectric cell comprised a nanoporous Li substituted magnesium ferrite pellet in contact with two electrodes, with zinc as the anode and silver as an inert cathode. The surface unsaturated cations and oxygen vacancies in the nanoporous ferrite dissociated water molecules into hydronium and hydroxide ions when the hydroelectric cell was dipped into deionized water. Hydroxide ions migrated toward the zinc electrode to form zinc hydroxide and the hydronium ions were evolved as H2 gas at the silver electrode. The zinc hydroxide collected as anode mud was converted into ZnO nanoparticles by heating at 250 °C. Structural analysis using Raman spectroscopy indicated the good crystallinity of the ZnO nanoparticles according to the presence of a high intensity E2-(high) mode. The nanoparticle size distribution was 5-20 nm according to high resolution transmission electron microscopy. An indirect band gap of 2.75 eV was determined based on the Tauc plot, which indicated the existence of an interstitial cation level in ZnO. Near band edge and blue emissions were detected in photoluminescence spectral studies. The blue emissions obtained from the ZnO nanoparticles could potentially have applications in blue lasers and LEDs. The ZnO nanoparticles synthesized using this method had a high dielectric constant value of 5 at a frequency of 1 MHz, which could be useful for fabricating nano-oscillators. This facile, clean, and cost-effective method obtained a significant yield of 0.017 g for ZnO nanoparticles without applying an external current source.

  15. Rapid prototyping of microbial cell factories via genome-scale engineering. (United States)

    Si, Tong; Xiao, Han; Zhao, Huimin


    Advances in reading, writing and editing genetic materials have greatly expanded our ability to reprogram biological systems at the resolution of a single nucleotide and on the scale of a whole genome. Such capacity has greatly accelerated the cycles of design, build and test to engineer microbes for efficient synthesis of fuels, chemicals and drugs. In this review, we summarize the emerging technologies that have been applied, or are potentially useful for genome-scale engineering in microbial systems. We will focus on the development of high-throughput methodologies, which may accelerate the prototyping of microbial cell factories. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. AQP4e-Based Orthogonal Arrays Regulate Rapid Cell Volume Changes in Astrocytes. (United States)

    Lisjak, Marjeta; Potokar, Maja; Rituper, Boštjan; Jorgačevski, Jernej; Zorec, Robert


    Water channel aquaporin 4 (AQP4) plays a key role in the regulation of water homeostasis in the brain. It is predominantly expressed in astrocytes at the blood-brain and blood-liquor interfaces. Although several AQP4 isoforms have been identified in the mammalian brain, two, AQP4a (M1) and AQP4c (M23), have been confirmed to cluster into plasma membrane supramolecular structures, termed orthogonal arrays of particles (OAPs) and to enhance water transport through the plasma membrane. However, the role of the newly described water-conductive mammalian isoform AQP4e is unknown. Here, the dynamics of AQP4e aggregation into OAPs and its role in the regulation of astrocyte water homeostasis have been studied. Using super-resolution structured illumination, atomic force, and confocal microscopies, the results revealed that, in female rat astrocytes, AQP4e isoform colocalizes with OAPs, affecting its structural dynamics. In hypoosmotic conditions, which elicit cell edema, OAP formation was considerably enhanced by overexpressed AQP4e. Moreover, the kinetics of the cell swelling and of the regulatory volume decrease was faster in astrocytes overexpressing AQP4e compared with untransfected controls. Furthermore, the increase in maximal cell volume elicited by hypoosmotic stimulation was significantly smaller in AQP4e-overexpressing astrocytes. For the first time, this study demonstrates an active role of AQP4e in the regulation of OAP structural dynamics and in water homeostasis.SIGNIFICANCE STATEMENT Water channel aquaporin 4 (AQP4) plays a key role in the regulation of water homeostasis in the brain. To date, only AQP4a and AQP4c isoforms have been confirmed to enhance water transport through plasmalemma and to cluster into orthogonal arrays of particles (OAPs). We here studied the dynamics, aggregation, and role in the regulation of astrocyte water homeostasis of the newly described water-conductive mammalian isoform AQP4e. Our main findings are as follows: brain edema

  17. Rapid changes in cell physiology as a result of acute thermal stress house sparrows, Passer domesticus. (United States)

    Jimenez, Ana G; Williams, Joseph B


    Given that our climate is rapidly changing, Physiological Ecologists have the critical task of identifying characteristics of species that make them either resilient or susceptible to changes in their natural air temperature regime. Because climate change models suggest that heat events will become more common, and in some places more extreme, it is important to consider how extreme heat events might affect the physiology of a species. The implications of more frequent heat wave events for birds have only recently begun to be addressed, however, the impact of these events on the cellular physiology of a species is difficult to assess. We have developed a novel approach using dermal fibroblasts to explore how short-term thermal stress at the whole animal level might affect cellular rates of metabolism. House sparrows, Passer domesticus were separated into a "control group" and a "heat shocked" group, the latter acclimated to 43°C for 24h. We determined the plasticity of cellular thermal responses by assigning a "recovery group" that was heat shocked as above, but then returned to room temperature for 24h. Primary dermal fibroblasts were grown from skin of all treatment groups and the pectoralis muscle was collected. We found that glycolysis (ECAR) and oxygen consumption rates (OCR), measured using a Seahorse XF 96 analyzer, were significantly higher in the fibroblasts from the heat shocked group of House sparrows compared with their control counterparts. Additionally, muscle fiber diameters decreased and, in turn, Na(+)-K(+)-ATPase maximal activity in the muscle significantly increased in heat shocked sparrows compared with birds in the control group. All of these physiological alterations due to short-term heat exposure were reversible within 24h of recovery at room temperature. These results show that acute exposure to heat stress significantly alters the cellular physiology of sparrows, but that this species is plastic enough to recover from such a thermal

  18. In situ investigation of the shrinkage of photopolymerized micro/nanostructures: the effect of the drying process. (United States)

    Sun, Quan; Ueno, Kosei; Misawa, Hiroaki


    We report on experimental study of the shrinkage of photopolymerized micro/nanostructures fabricated by femtosecond direct laser writing in organic-inorganic resists. Blueshift of the stop-band positions of fabricated photonic crystals during the drying process, which follows the development and rinsing stages, indicates that the drying process plays an important role in the formation of the shrinkage. It is further confirmed that the shrinkage almost completely occurs during the drying process by in situ optical monitoring the structures. These findings will help to better understand, control, and even positively utilize the shrinkage in the applications of the photopolymerization-based direct laser writing technique.

  19. Conjugation of diisocyanate side chains to dimethacrylate reduces polymerization shrinkage and increases the hardness of composite resins

    Directory of Open Access Journals (Sweden)

    Yih-Dean Jan


    Conclusion: Conjugation of diisocyanate side chains to dimethacrylate represents an effective means of reducing polymerization shrinkage and increasing the surface hardness of dental composite resins.

  20. An Experimental Study on Shrinkage Strains of Normal-and High-Strength Concrete-Filled Frp Tubes (United States)

    Vincent, Thomas; Ozbakkaloglu, Togay


    It is now well established that concrete-filled fiber reinforced polymer (FRP) tubes (CFFTs) are an attractive construction technique for new columns, however studies examining concrete shrinkage in CFFTs remain limited. Concrete shrinkage may pose a concern for CFFTs, as in these members the curing of concrete takes place inside the FRP tube. This paper reports the findings from an experimental study on concrete shrinkage strain measurements for CFFTs manufactured with normal- and high-strength concrete (NSC and HSC). A total of 6 aramid FRP (AFRP)-confined concrete specimens with circular cross-sections were manufactured, with 3 specimens each manufactured using NSC and HSC. The specimens were instrumented with surface and embedded strain gauges to monitor shrinkage development of exposed concrete and concrete sealed inside the CFFTs, respectively. All specimens were cylinders with a 152 mm diameter and 305 mm height, and their unconfined concrete strengths were 44.8 or 83.2 MPa. Analysis of the shrinkage measurements from concrete sealed inside the CFFTs revealed that embedment depth and concrete compressive strength only had minor influences on recorded shrinkage strains. However, an analysis of shrinkage measurements from the exposed concrete surface revealed that higher amounts of shrinkage can occur in HSC. Finally, it was observed that shrinkage strains are significantly higher for concrete exposed at the surface compared to concrete sealed inside the CFFTs.

  1. Rapid and specific detection of cell-derived microvesicles using a magnetoresistive biochip. (United States)

    Cherré, Solène; Fernandes, Elisabete; Germano, José; Dias, Tomás; Cardoso, Susana; Piedade, Moisés S; Rozlosnik, Noemi; Oliveira, Marta I; Freitas, Paulo P


    Microvesicles (MVs) are a promising source of diagnostic biomarkers which have gained a wide interest in the biomedical and biosensing field. They can be interpreted as a "fingerprint" of various diseases. Nonetheless, MVs implementation into clinical settings has been hampered by the lack of technologies to accurately characterize, detect and quantify them. Here, we report the specific sensing and quantification of MVs from endothelial cells using a portable magnetoresistive (MR) biochip platform, in less than one hour and within physiologically relevant concentrations (1 × 10(8) MVs per ml). MVs were isolated from both endothelial and epithelial cells undergoing apoptosis, and characterized by atomic force microscopy (AFM) and nanoparticle tracking analysis (NTA), which revealed similar MV sizes. Importantly, our results showed that the two distinct MV populations could be discriminated with the MR biochip platform, with over a 5-fold capture efficiency of endothelial MVs in comparison to the control (epithelial MVs). Also, unspecific binding of MVs to BSA was less than 1% of the specific signal. The detection strategy was based on a sandwich immunoassay, where MVs were labelled with magnetic nanoparticles (MNPs) functionalized with Annexin V and then captured by anti-CD31 antibodies previously immobilized on the surface of the sensor. Results suggest that this approach allows the detection of specific MVs from complex samples such as serum, and highlight the potential of this technology to become a suitable tool for MVs detection as a complementary method of diagnosis.

  2. Rapid detection of defects in fuel-cell electrodes using infrared reactive-flow-through technique (United States)

    Das, Prodip K.; Weber, Adam Z.; Bender, Guido; Manak, Austin; Bittinat, Daniel; Herring, Andrew M.; Ulsh, Michael


    As fuel cells become more prominent, new manufacturing and production methods will need to be developed to deal efficiently and effectively with increased demand. One necessary component of this industrial growth is the accurate measurement of the variability in the manufacturing process. In this study, we present a diagnostic system that combines infrared thermography with a reactive-flow-through technique to detect catalyst-loading defects in fuel-cell gas-diffusion electrodes accurately with high spatial and temporal resolutions. Experimental results are compared with model predictions of thermal response with good agreement. Data analysis, operating-condition impacts, and detection limits are explored using both experiments and simulation. Overall, the results demonstrate the potential of this technique to measure defects on the millimeter length scale with temporal resolutions appropriate for use on a web-line. Thus we present the first development stage of a next-generation non-destructive diagnostic tool, which may be amenable to eventual use on roll-to-roll manufacturing lines.

  3. An easy, rapid method to isolate RPE cell protein from the mouse eye. (United States)

    Wei, Hong; Xun, Zixian; Granado, Herta; Wu, Angela; Handa, James T


    The retinal pigment epithelium (RPE) is essential for maintaining the health of the neural retina. RPE cell dysfunction plays a critical role in many common blinding diseases including age-related macular degeneration (AMD), diabetic retinopathy, retinal dystrophies. Mouse models of ocular disease are commonly used to study these blinding diseases. Since isolating the RPE from the choroid has been challenging, most techniques separate the RPE from the retina, but not the choroid. As a result, the protein signature actually represents a heterogeneous population of cells that may not accurately represent the RPE response. Herein, we describe a method for separating proteins from the RPE that is free from retinal and choroidal contamination. After removing the anterior segment and retina from enucleated mouse eyes, protein from the RPE was extracted separately from the choroid by incubating the posterior eyecup with a protein lysis buffer for 10 min. Western blot analysis identified RPE65, an RPE specific protein in the RPE lysates, but not in choroidal lysates. The RPE lysates were devoid of rhodopsin and collagen VI, which are abundant in the retina and choroid, respectively. This technique will be very helpful for measuring the protein signal from the RPE without retinal or choroidal contamination. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Rapid detection of Clostridium difficile via magnetic bead aggregation in cost-effective polyester microdevices with cell phone image analysis. (United States)

    DuVall, Jacquelyn A; Cabaniss, Scott T; Angotti, Morgan L; Moore, John H; Abhyankar, Mayuresh; Shukla, Nishant; Mills, Daniel L; Kessel, Bryan G; Garner, Gavin T; Swami, Nathan S; Landers, James P


    Pathogen detection has traditionally been accomplished by utilizing methods such as cell culture, immunoassays, and nucleic acid amplification tests; however, these methods are not easily implemented in resource-limited settings because special equipment for detection and thermal cycling is often required. In this study, we present a magnetic bead aggregation assay coupled to an inexpensive microfluidic fabrication technique that allows for cell phone detection and analysis of a notable pathogen in less than one hour. Detection is achieved through the use of a custom-built system that allows for fluid flow control via centrifugal force, as well as manipulation of magnetic beads with an adjustable rotating magnetic field. Cell phone image capture and analysis is housed in a 3D-printed case with LED backlighting and a lid-mounted Android phone. A custom-written application (app.) is employed to interrogate images for the extent of aggregation present following loop-mediated isothermal amplification (LAMP) coupled to product-inhibited bead aggregation (PiBA) for detection of target sequences. Clostridium difficile is a pathogen of increasing interest due to its causative role in intestinal infections following antibiotic treatment, and was therefore chosen as the pathogen of interest in the present study to demonstrate the rapid, cost-effective, and sequence-specific detection capabilities of the microfluidic platform described herein.

  5. Bisphenol A induces a rapid activation of Erk1/2 through GPR30 in human breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Dong, S. [Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566 (Japan); Institute of Urban Environment, Chinese Academy of Sciences, Xiamen (China); Terasaka, S. [Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566 (Japan); Kiyama, R., E-mail: kiyama.r@aist.go.j [Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566 (Japan)


    Bisphenol A (BPA) has been considered as an endocrine disruptor due to its ability to interact with estrogen receptors (ERs). While G protein-coupled receptor 30 (GPR30) is a novel estrogen receptor, its role in BPA-induced activation of Erk1/2 remains unknown. Human breast cancer cell lines, MCF-7, MDA-MB-231 and SKBR3, were used as experimental models to discriminate between ERs-dependent, putative ERs-independent and/or GPR30-associated effects. BPA induced a rapid activation of Erk1/2 in both ER{alpha}/{beta}-positive and negative breast cancer cells, and this effect was not blocked with an ER antagonist, ICI 182,780. A small interfering RNA assay revealed that the expression of GPR30 was necessary for BPA-induced activation of Erk1/2 and transcriptional regulation of c-fos. In addition, BPA regulates the expression of c-fos likely through an AP1-mediated pathway. As a conclusion, GPR30 plays an important role in the BPA-induced activation of Erk1/2 in a manner distinguishable from that in ER{alpha}-mediated signaling. - We showed here that the mechanism by which BPA induces the activation of Erk1/2 is distinguishable from the mechanism of ER{alpha}-mediated signaling in human breast cancer cells.

  6. Extranodal NK/T-cell lymphoma, nasal type, manifesting as rapidly progressive dementia without any mass or enhancing brain lesion. (United States)

    Shimatani, Yoshimitsu; Nakano, Yuta; Tsuyama, Naoko; Murayama, Shigeo; Oki, Ryosuke; Miyamoto, Ryosuke; Murakami, Nagahisa; Fujita, Koji; Watanabe, Syunsuke; Uehara, Hisanori; Abe, Takashi; Nodera, Hiroyuki; Kawarai, Toshitaka; Izumi, Yuishin; Kaji, Ryuji


    Among the many potential etiologies for rapidly progressive dementia (RPD), primary central nervous system extranodal NK/T-cell lymphoma, nasal-type (ENKL) is a rare entity. We present the first reported case of autopsy-proven RPD due to ENKL without any mass or enhancing lesion of the brain. A 54-year-old immunocompetent man presented with RPD, myoclonus and ataxia. The mini-mental state examination (MMSE) score was 22/30. His brain MRI revealed progressive brain atrophy without gadolinium enhancement or mass lesion. Five months after the initial evaluation, cognitive impairment further worsened with an MMSE score of 3/30. At the advanced stage, lumbar MRI showed swollen cauda equina with gadolinium enhancement. The number of Epstein-Barr virus (EBV) DNA in cerebrospinal fluid had gradually increased. Twelve months after onset, the patient died of respiratory failure. Pathological findings revealed that lymphoma cells had diffusely invaded the meninges, parenchyma of the brain, spinal cord and cauda equina. Cells were positive for CD3, CD56 and EBV-encoded small RNAs and negative for CD20. No evidence of malignancy was identified in the visceral organs. This report indicates that ENKL should be recognized as one of the rare causes of RPD. Early testing for EBV-DNA in cerebrospinal fluid and imaging of cauda equina would be useful diagnostic tools. © 2016 Japanese Society of Neuropathology.

  7. Candida albicans and Candida parapsilosis rapidly up-regulate galectin-3 secretion by human gingival epithelial cells. (United States)

    Tamai, Riyoko; Kiyoura, Yusuke


    Galectin-3 is a β-galactoside-binding C-type lectin that plays an important role in innate immunity. The purpose of this study was to determine whether Candida albicans and Candida parapsilosis up-regulate galectin-3 secretion by human gingival epithelial cells and gingival fibroblasts. Ca9-22, a human gingival epithelial cell line, and human gingival fibroblasts were incubated in the presence or absence of C. albicans or C. parapsilosis without serum. Levels of secreted human galectin-3 in culture supernatants were measured by enzyme-linked immunosorbent assay. We also pretreated Ca9-22 cells with cytochalasin D (an actin polymerization inhibitor), ALLN (a calpain inhibitor) and LY294002 [a phosphatidylinositol-3 kinase (PI3K) inhibitor] to determine whether the up-regulation of galectin-3 secretion was mediated by cytoskeletal changes, protease activity, or PI3K signaling. Galectin-3 secretion was significantly and rapidly up-regulated by live C. albicans and C. parapsilosis, as well as heat-killed C. albicans. In addition, cytochalasin D, LY294002 and ALLN did not inhibit the up-regulation in galectin-3 secretion. These results suggest that both live and heat-killed C. albicans and C. parapsilosis may increase the activity of the innate immune system and invasion by other microorganisms via up-regulation of galectin-3 secretion.

  8. A rapid and sensitive assay for detection of replication-competent adenoviruses by a combination of microcarrier cell culture and quantitative PCR

    NARCIS (Netherlands)

    Schalk, Johanna A. C.; de Vries, Claudette G. J. C. A.; Orzechowski, Tom J. H.; Rots, Marianne G.


    The development of a rapid and sensitive assay for detection of replication-competent adenoviruses (RCAs) is described. This RCA assay consists of an incubation step of 4 days of adenoviral vectors on A549 cells in a microcarrier cell culture system followed by detection of amplified RCAs by

  9. An enzyme-activatable probe with a self-immolative linker for rapid and sensitive alkaline phosphatase detection and cell imaging through a cascade reaction. (United States)

    Zhang, Hongmei; Xu, Chenglong; Liu, Jie; Li, Xiaohong; Guo, Lin; Li, Xinming


    We report the design and synthesis of a novel probe (1) for ALP assay by incorporating a self-immolative linker between a phosphate moiety and resorufin. Because of its good biocompatibility and rapid cell internalization, this probe also exhibited great potential for real-time monitoring of endogenous phosphatase activity in living cells.

  10. Optical Aptamer Probes of Fluorescent Imaging to Rapid Monitoring of Circulating Tumor Cell

    Directory of Open Access Journals (Sweden)

    Ji Yeon Hwang


    Full Text Available Fluorescence detecting of exogenous EpCAM (epithelial cell adhesion molecule or muc1 (mucin1 expression correlated to cancer metastasis using nanoparticles provides pivotal information on CTC (circulating tumor cell occurrence in a noninvasive tool. In this study, we study a new skill to detect extracellular EpCAM/muc1 using quantum dot-based aptamer beacon (QD-EpCAM/muc1 ALB (aptamer linker beacon. The QD-EpCAM/muc1 ALB was designed using QDs (quantum dots and probe. The EpCAM/muc1-targeting aptamer contains a Ep-CAM/muc1 binding sequence and BHQ1 (black hole quencher 1 or BHQ2 (black hole quencher2. In the absence of target EpCAM/muc1, the QD-EpCAM/muc1 ALB forms a partial duplex loop-like aptamer beacon and remained in quenched state because the BHQ1/2 quenches the fluorescence signal-on of the QD-EpCAM/muc1 ALB. The binding of EpCAM/muc1 of CTC to the EpCAM/muc1 binding aptamer sequence of the EpCAM/muc1-targeting oligonucleotide triggered the dissociation of the BHQ1/2 quencher and subsequent signal-on of a green/red fluorescence signal. Furthermore, acute inflammation was stimulated by trigger such as caerulein in vivo, which resulted in increased fluorescent signal of the cy5.5-EpCAM/muc1 ALB during cancer metastasis due to exogenous expression of EpCAM/muc1 in Panc02-implanted mouse model.

  11. Cobalamin C Deficiency Shows a Rapidly Progressing Maculopathy With Severe Photoreceptor and Ganglion Cell Loss (United States)

    Bonafede, Lucas; Ficicioglu, Can H.; Serrano, Leona; Han, Grace; Morgan, Jessica I. W.; Mills, Monte D.; Forbes, Brian J.; Davidson, Stefanie L.; Binenbaum, Gil; Kaplan, Paige B.; Nichols, Charles W.; Verloo, Patrick; Leroy, Bart P.; Maguire, Albert M.; Aleman, Tomas S.


    Purpose To describe in detail the retinal structure and function of a group of patients with cobalamin C (cblC) disease. Methods Patients (n = 11, age 4 months to 15 years) with cblC disease (9/11, early onset) diagnosed by newborn screening underwent complete ophthalmic examinations, fundus photography, near-infrared reflectance imaging, and spectral-domain optical coherence tomography (SD-OCT). Electroretinograms (ERGs) were performed in a subset of patients. Results Patients carried homozygous or compound heterozygote mutations in the methylmalonic aciduria and homocystinuria type C (MMACHC) gene. Late-onset patients had a normal exam. All early-onset patients showed a maculopathy; older subjects had a retina-wide degeneration (n = 4; >7 years of age). In general, retinal changes were first observed before 1 year of age and progressed within months to a well-established maculopathy. Pseudocolobomas were documented in three patients. Measurable visual acuities ranged from 20/200 to 20/540. Nystagmus was present in 8/11 patients; 5/6 patients had normal ERGs; 1/6 had reduced rod-mediated responses. Spectral-domain OCT showed macular thinning, with severe ganglion cell layer (GCL) and outer nuclear layer (ONL) loss. Inner retinal thickening was observed in areas of total GCL/ONL loss. A normal lamination pattern in the peripapillary nasal retina was often seen despite severe central and/or retina-wide disease. Conclusions Patients with early-onset cblC and MMACHC mutations showed an early-onset, unusually fast-progressing maculopathy with severe central ONL and GCL loss. An abnormally thickened inner retina supports a remodeling response to both photoreceptor and ganglion cell degeneration and/or an interference with normal development in early-onset cblC. PMID:26658511

  12. Prediction and improvement of shrinkage porosity in TiAl based alloy

    Directory of Open Access Journals (Sweden)

    Gao Yong


    Full Text Available The present research has developed a novel investment casting process for ingot production of TiAl alloys through forming a small vertical temperature gradient on the mold. The advantage of this process is to guarantee that the castings solidify sequentially from bottom to top. The analysis of numerical simulation and experimental results showed that the shrinkage porosity of Ti-47Al-2Cr-2Nb alloy was significantly improved by forming a vertical temperature gradient of 3 ℃/mm on the mold, while the increase of pouring temperature and pressure on the molten alloys had no apparent effect on the reduction of shrinkage porosity. The critical value of the Niyama criterion that can reliably predict the shrinkage porosity in Ti-47Al-2Cr-2Nb alloy was identified by the comparison of experimental and simulated results.

  13. Significance of Shrinkage Induced Clamping Pressure in Fiber-Matrix Bonding in Cementitious Composite Materials

    DEFF Research Database (Denmark)

    Stang, Henrik


    inhomogeneity embedded in a matrix consisting of acementitious material undergoing shrinkage during hydration(autogenous shrinkage). Furthermore, the paperpresents the analysis necessary to perform an interpretation of the experimental results and which allows for thedetermination of the clamping pressure......The present paper accesses the significance of shrinkage inducedclamping pressure in fiber/matrix bonding mechanisms incementitious composite materials. The paper contains a description of an experimental setup whichallows mbox{measurement} of the clamping pressure which develops on anelastic...... acting on any elastic inhomogeneityembedded in the same cementitious matrix material. Fiber-shaped inhomogeneities are of special interest in cementitious composite material systems andresults are presented for the development of clamping pressure on three typical fiber types in two typical cementpastes...

  14. Numerical simulation of early-age shrinkage effects on RC member deflections and cracking development

    Directory of Open Access Journals (Sweden)

    P. Bernardi


    Full Text Available Shrinkage effects on short-term behavior of reinforced concrete elements are often neglected both in design code provisions and in numerical simulations. However, it is known that their influence on serviceability performance can be significant, especially in case of lightly-reinforced beams. As a matter of fact, the restraint provided by the reinforcement on concrete determines a reduction of the cracking load of the structural element, as well as an increase of its deflection. This paper deals with the modeling of early-age shrinkage effects in the field of smeared crack approaches. To this aim, an existing non-linear constitutive relation for cracked reinforced concrete elements is extended herein to include early-age concrete shrinkage. Careful verifications of the model are carried out by comparing numerical results with significant experimental data reported in technical literature, providing a good agreement both in terms of global and local behavior.

  15. Shrinkage Analysis on Thick Plate Part using Response Surface Methodology (RSM

    Directory of Open Access Journals (Sweden)

    Isafiq M.


    Full Text Available The work reported herein is about an analysis on the quality (shrinkage on a thick plate part using Response Surface Methodology (RSM. Previous researches showed that the most influential factor affecting the shrinkage on moulded parts are mould and melt temperature. Autodesk Moldflow Insight software was used for the analysis, while specifications of Nessei NEX 1000 injection moulding machine and P20 mould material were incorporated in this study on top of Acrylonitrile Butadiene Styrene (ABS as a moulded thermoplastic material. Mould temperature, melt temperature, packing pressure and packing time were selected as variable parameters. The results show that the shrinkage have improved 42.48% and 14.41% in parallel and normal directions respectively after the optimisation process.

  16. A paper-based microbial fuel cell array for rapid and high-throughput screening of electricity-producing bacteria. (United States)

    Choi, Gihoon; Hassett, Daniel J; Choi, Seokheun


    There is a large global effort to improve microbial fuel cell (MFC) techniques and advance their translational potential toward practical, real-world applications. Significant boosts in MFC performance can be achieved with the development of new techniques in synthetic biology that can regulate microbial metabolic pathways or control their gene expression. For these new directions, a high-throughput and rapid screening tool for microbial biopower production is needed. In this work, a 48-well, paper-based sensing platform was developed for the high-throughput and rapid characterization of the electricity-producing capability of microbes. 48 spatially distinct wells of a sensor array were prepared by patterning 48 hydrophilic reservoirs on paper with hydrophobic wax boundaries. This paper-based platform exploited the ability of paper to quickly wick fluid and promoted bacterial attachment to the anode pads, resulting in instant current generation upon loading of the bacterial inoculum. We validated the utility of our MFC array by studying how strategic genetic modifications impacted the electrochemical activity of various Pseudomonas aeruginosa mutant strains. Within just 20 minutes, we successfully determined the electricity generation capacity of eight isogenic mutants of P. aeruginosa. These efforts demonstrate that our MFC array displays highly comparable performance characteristics and identifies genes in P. aeruginosa that can trigger a higher power density.

  17. Permeability Enhancement in Fine-Grained Sediments by Chemically Induced Clay Fabric Shrinkage

    Energy Technology Data Exchange (ETDEWEB)

    Wijesinghe, A M; Kansa, E J; Viani, B E; Blake, R G; Roberts, J J; Huber, R D


    ethanol in the unaltered sediment?'' To this end, we performed a crack propagation experiment under confining stress on an initially water-saturated bentonite clay layer that was exposed to pure ethanol on one surface and water on the other. We measured the rate of transport of ethanol across the clay layer and found that crack breakthrough across the clay layer was accompanied by a very large sudden increase in solvent flow through the layer. Although an experimental artifact prevented measurement of the exact breakthrough time, visual evidence indicates that the clay layer cracked rapidly in this experiment. Direct evidence of the cracks provided by X-ray tomographic images provide clear proof that an extensive array of cracks was created by the ethanol-induced shrinkage. Calculations based on measured fluid pressure and flow rates, and published permeabilities of unaltered calcium bentonite clays, show that the effective permeability of the clay layer increased by a factor of 10{sup 9}-10{sup 12}. Estimates of effective permeability based on crack dimensions measured from the X-ray tomographic images support these findings. Further detailed experiments and analyses are needed to develop a predictive theory and a quantitative design capability for this process. We have begun work on securing the necessary support for these studies, with the ultimate objective of developing and deploying this novel concept as a practical remediation technology in the field.

  18. Diversity shrinkage: Cross-validating pareto-optimal weights to enhance diversity via hiring practices. (United States)

    Song, Q Chelsea; Wee, Serena; Newman, Daniel A


    To reduce adverse impact potential and improve diversity outcomes from personnel selection, one promising technique is De Corte, Lievens, and Sackett's (2007) Pareto-optimal weighting strategy. De Corte et al.'s strategy has been demonstrated on (a) a composite of cognitive and noncognitive (e.g., personality) tests (De Corte, Lievens, & Sackett, 2008) and (b) a composite of specific cognitive ability subtests (Wee, Newman, & Joseph, 2014). Both studies illustrated how Pareto-weighting (in contrast to unit weighting) could lead to substantial improvement in diversity outcomes (i.e., diversity improvement), sometimes more than doubling the number of job offers for minority applicants. The current work addresses a key limitation of the technique-the possibility of shrinkage, especially diversity shrinkage, in the Pareto-optimal solutions. Using Monte Carlo simulations, sample size and predictor combinations were varied and cross-validated Pareto-optimal solutions were obtained. Although diversity shrinkage was sizable for a composite of cognitive and noncognitive predictors when sample size was at or below 500, diversity shrinkage was typically negligible for a composite of specific cognitive subtest predictors when sample size was at least 100. Diversity shrinkage was larger when the Pareto-optimal solution suggested substantial diversity improvement. When sample size was at least 100, cross-validated Pareto-optimal weights typically outperformed unit weights-suggesting that diversity improvement is often possible, despite diversity shrinkage. Implications for Pareto-optimal weighting, adverse impact, sample size of validation studies, and optimizing the diversity-job performance tradeoff are discussed. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

  19. Influence of resin cement polymerization shrinkage on stresses in porcelain crowns. (United States)

    May, Liliana G; Kelly, J Robert


    The aim of this study was to analyze the influence of polymerization shrinkage of the cement layer on stresses within feldspathic ceramic crowns, using experimentally validated FEA models for (1) increasing occlusal cement thickness; and, (2) bonded versus non-bonded ceramic-cement interfaces. 2-D axial symmetric models simulated stylized feldspathic crowns (1.5mm occlusal thickness) cemented with resin-cement layers of 50-500μm on dentin preparations, being loaded (500N) or not. Ceramic-cement interface was either bonded or not. Cement was bonded to the dentin in all models. Maximum axial shrinkage of 0%, 1%, 2%, 3%, 4% and 4.65% were simulated. The first principal stresses developing in the cementation surface at the center and at the occluso-axial line-angle of the crown were registered. Polymerization shrinkage of the cement increased tensile stresses in the ceramic, especially in loaded non-bonded crowns for thicker cement layers. Stresses in loaded non-bonded crowns increased as much as 87% when cement shrinkage increased from 0% to 4.65% (100-187MPa), for a 500μm-thick cement. Increasing polymerization shrinkage strain raised the tensile stresses, especially at the internal occlusal-axial line-angle, for bonded crowns. Changes in the polymerization shrinkage strain (from 0% to 4.65%) have little effect on the tensile stresses generated at the cementation surface of the ceramic crowns, when the occlusal cement thickness is thin (approx. 50μm for bonded crowns). However, as the cement becomes thicker stresses within the ceramic become significant. Copyright © 2013 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

  20. Conventional and high-intensity halogen light effects on polymerization shrinkage of orthodontic adhesives. (United States)

    Sener, Yagmur; Uysal, Tancan; Basciftci, Faruk Ayhan; Demir, Abdullah; Botsali, Murat Selim


    The objectives of this study were to compare the polymerization shrinkage of three orthodontic adhesives. In addition we wanted to determine the effectiveness of the high-intensity quartz tungsten halogen (HQTH) in curing orthodontic adhesives on polymerization shrinkage with that of the quartz tungsten halogen (QTH). A total of 120 glass ring molds were prepared using a low-speed saw. The internal surface of the glass rings were roughened and etched. Adhesive pastes were placed into the glass molds, which were sandwiched between two glass slides. Samples were divided into six groups according to the combination of three orthodontic adhesives (Kurasper F, Light Bond, and Transbond XT) and two light intensities. One half of each 40 samples of three adhesive pastes was polymerized for 20 seconds by a QTH (Hilux 350), and the other half was polymerized for 10 seconds by a HQTH (Optilux 501). The volumetric polymerization shrinkage for each system was measured through the specific density method modified by Puckett and Smith. Statistical analysis was performed using two-way analysis of variance for intergroup comparisons. The HQTH-curing unit resulted in a more polymerization shrinkage than did the QTH for all investigated adhesives. However, no statistically significant differences were found. The highest shrinkage was observed for Light Bond cured with HQTH (1.59 +/- 0.82%), and the lowest value was observed for Transbond XT cured with QTH (1.23 +/- 0.60%). There are no significant differences in polymerization shrinkage of the three investigated orthodontic adhesives when polymerized with a QTH or a HQTH.

  1. Precise measurement of photoresist cross-sectional shape change caused by SEM-induced shrinkage (United States)

    Ohashi, Takeyoshi; Sekiguchi, Tomoko; Yamaguchi, Atsuko; Tanaka, Junichi; Kawada, Hiroki


    The mechanism of photoresist shrinkage induced by electron-beam (EB) irradiation was studied. A precise cross-sectional profile of a photoresist pattern was obtained by a scanning transmission electron microscope (STEM) after atomic layer deposition of HfO2 on the sample patterns. Photoresist lines and spaces fabricated with positive-tone development and negative-tone development were exposed to an EB with much higher dose than a practical dose (to accelerate shrinkage intentionally). The obtained STEM images of the patterns before and after EB irradiation show that the shrinkage of the negative-tone-developed patterns is smaller than that of the positive-tone-developed patterns. This observation is explained by the fact that negative-tone-developed photoresist molecules do not contain protection groups, whose volatilization caused by EB irradiation is one of the origins of shrinkage. Another finding is that the EB irradiation causes top-rounding and necking of the pattern profile as well as linewidth slimming. The rounding of the pattern top profile suggests that the pattern's shape was elastically deformed. In addition, EB irradiation only onto the spaces caused sidewall shrinkage and a necking profile, although no electrons were irradiated directly onto the pattern. These phenomena are considered to be due to the electrons scattered from the spaces to the pattern sidewall. Finally, a Monte Carlo simulation of electron scattering showed that the distribution of the deposited EB energy on the pattern surface corresponds to the above-described change in pattern shape. Consequently, these observations and simulation results clarify the importance of the effect of elastic shape change and the impact of the electrons scattered from the underlying layer onto the sidewall in the mechanism of photoresist shrinkage.

  2. Regional brain shrinkage over two years: individual differences and effects of pro-inflammatory genetic polymorphisms. (United States)

    Persson, N; Ghisletta, P; Dahle, C L; Bender, A R; Yang, Y; Yuan, P; Daugherty, A M; Raz, N


    We examined regional changes in brain volume in healthy adults (N=167, age 19-79years at baseline; N=90 at follow-up) over approximately two years. With latent change score models, we evaluated mean change and individual differences in rates of change in 10 anatomically-defined and manually-traced regions of interest (ROIs): lateral prefrontal cortex (LPFC), orbital frontal cortex (OF), prefrontal white matter (PFw), hippocampus (Hc), parahippocampal gyrus (PhG), caudate nucleus (Cd), putamen (Pt), insula (In), cerebellar hemispheres (CbH), and primary visual cortex (VC). Significant mean shrinkage was observed in the Hc, CbH, In, OF, and PhG, and individual differences in change were noted in all regions, except the OF. Pro-inflammatory genetic variants modified shrinkage in PhG and CbH. Carriers of two T alleles of interleukin-1β (IL-1β C-511T, rs16944) and a T allele of methylenetetrahydrofolate reductase (MTHFR C677T, rs1801133) polymorphisms showed increased PhG shrinkage. No effects of a pro-inflammatory polymorphism for C-reactive protein (CRP-286C>A>T, rs3091244) or apolipoprotein (APOE) ε4 allele were noted. These results replicate the pattern of brain shrinkage observed in previous studies, with a notable exception of the LPFC, thus casting doubt on the unique importance of prefrontal cortex in aging. Larger baseline volumes of CbH and In were associated with increased shrinkage, in conflict with the brain reserve hypothesis. Contrary to previous reports, we observed no significant linear effects of age and hypertension on regional brain shrinkage. Our findings warrant further investigation of the effects of neuroinflammation on structural brain change throughout the lifespan. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Substance P induces rapid and transient membrane blebbing in U373MG cells in a p21-activated kinase-dependent manner.

    Directory of Open Access Journals (Sweden)

    John Meshki

    Full Text Available U373MG astrocytoma cells endogenously express the full-length neurokinin 1 receptor (NK1R. Substance P (SP, the natural ligand for NK1R, triggers rapid and transient membrane blebbing and we report that these morphological changes have different dynamics and intracellular signaling as compared to the changes that we have previously described in HEK293-NK1R cells. In both cell lines, the SP-induced morphological changes are Gq-independent, and they require the Rho, Rho-associated coiled-coil kinase (ROCK signaling pathway. Using confocal microscopy we have demonstrated that tubulin is phosphorylated subsequent to cell stimulation with SP and that tubulin accumulates inside the blebs. Colchicine, a tubulin polymerization inhibitor, blocked SP-induced blebbing in U373MG but not in HEK293-NK1R cells. Although p21-activated kinase (PAK is expressed in both cell lines, SP induced rapid phosphorylation of PAK in U373MG, but failed to phosphorylate PAK in HEK293-NK1R cells. The cell-permeable Rho inhibitor C3 transferase inhibited SP-induced PAK phosphorylation, but the ROCK inhibitor Y27632 had no effect on PAK phosphorylation, suggesting that Rho activates PAK in a ROCK-independent manner. Our study demonstrates that SP triggers rapid changes in cell morphology mediated by distinct intracellular signaling mechanisms in U373MG versus HEK293-NK1R cells.

  4. Microbial fuel cell biosensor for rapid assessment of assimilable organic carbon under marine conditions. (United States)

    Quek, Soon Bee; Cheng, Liang; Cord-Ruwisch, Ralf


    The development of an assimilable organic carbon (AOC) detecting marine microbial fuel cell (MFC) biosensor inoculated with microorganisms from marine sediment was successful within 36 days. This established marine MFC was tested as an AOC biosensor and reproducible microbiologically produced electrical signals in response to defined acetate concentration were achieved. The dependency of the biosensor sensitivity on the potential of the electron-accepting electrode (anode) was investigated. A linear correlation (R(2) > 0.98) between electrochemical signals (change in anodic potential and peak current) and acetate concentration ranging from 0 to 150 μM (0-3600 μg/L of AOC) was achieved. However, the present biosensor indicated a different-linear relation at somewhat elevated acetate concentration ranging from 150 to 450 μM (3600-10,800 μg/L of AOC). This high concentration of acetate addition could be measured by coulombic measurement (cumulative charges) with a linear correlation. For the acetate concentration detected in this study, the sensor recovery time could be controlled within 100 min. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Cake shrinkage during freeze drying: a combined experimental and theoretical study. (United States)

    Rambhatla, S; Obert, J P; Luthra, S; Bhugra, C; Pikal, Michael J


    The aim of this study is to investigate, by experimental studies and theoretical analysis, the phenomenon of cake shrinkage during the lyophilization process and to investigate the effect of shelf temperature during primary drying and secondary drying on the degree of cake shrinkage. Freeze-drying experiments were performed using 5% w/v sucrose where the drying protocols were altered in order to produce differing product temperature profiles. Resistance data during freeze drying were evaluated by the Manometric Temperature Measurement (MTM) method. Theoretical simulation of the freeze-drying process was performed using the Passage Freeze-Drying software. The difference between the glass transition temperature and the product temperature (Tg-T) obtained from the theoretical analysis was calculated and used for correlation with experimental shrinkage data. The Brunauer, Emmeth, Teller (BET) Specific Surface Area (SSA) Analysis was used as a method to quantify the degree of shrinkage. Samples were also analyzed for pore volume by mercury porosimetry. The SSA analysis on the freeze-dried samples showed an increase in SSA when samples were freeze dried at a lower shelf temperature during primary drying and at a slower ramp rate during secondary drying. The trend in surface area values was consistent with that obtained for pore size values. However, differences obtained among the various samples are small and cake diameter measurements showed that there was approximately 17% shrinkage even in the sample freeze dried at temperatures well below the Tg' and Tg. Variations in process and product temperature only accounted for an additional 2%-3% shrinkage. Resistance data obtained at various primary drying shelf temperatures showed a good correlation with surface area. The Tg-T behavior of the freeze-dried samples showed that a slow ramp rate of 0.1 degrees C/min during secondary drying maintains a product well below the Tg at all times and a higher ramp rate gives negative

  6. The effect of mold surface topography on plastic parat in-process shrinkage in injection molding

    DEFF Research Database (Denmark)

    Arlø, Uffe Rolf; Hansen, Hans Nørgaard; Kjær, Erik Michael


    An experimental study of the effect of mold surface roughness on in-process in-flow linear part shrinkage in injection molding has been carried out. The investigation is based on an experimental two-cavity tool, where the cavities have different surface topographies, but are otherwise identical....... The study has been carried out for typical commercial polystyrene and polypropylene grades. The relationship between mold surface topography and linear shrinkage has been investigated with an experimental two-cavity mold producing simple rectangular parts with the nominal dimensions 1 x 25 x 50 mm (see...

  7. Sparse contrast-source inversion using linear-shrinkage-enhanced inexact Newton method

    KAUST Repository

    Desmal, Abdulla


    A contrast-source inversion scheme is proposed for microwave imaging of domains with sparse content. The scheme uses inexact Newton and linear shrinkage methods to account for the nonlinearity and ill-posedness of the electromagnetic inverse scattering problem, respectively. Thresholded shrinkage iterations are accelerated using a preconditioning technique. Additionally, during Newton iterations, the weight of the penalty term is reduced consistently with the quadratic convergence of the Newton method to increase accuracy and efficiency. Numerical results demonstrate the applicability of the proposed method.

  8. Novel photosensitizers trigger rapid death of malignant human cells and rodent tumor transplants via lipid photodamage and membrane permeabilization.

    Directory of Open Access Journals (Sweden)

    Mikhail M Moisenovich

    Full Text Available BACKGROUND: Apoptotic cascades may frequently be impaired in tumor cells; therefore, the approaches to circumvent these obstacles emerge as important therapeutic modalities. METHODOLOGY/PRINCIPAL FINDINGS: Our novel derivatives of chlorin e(6, that is, its amide (compound 2 and boronated amide (compound 5 evoked no dark toxicity and demonstrated a significantly higher photosensitizing efficacy than chlorin e(6 against transplanted aggressive tumors such as B16 melanoma and M-1 sarcoma. Compound 5 showed superior therapeutic potency. Illumination with red light of mammalian tumor cells loaded with 0.1 µM of 5 caused rapid (within the initial minutes necrosis as determined by propidium iodide staining. The laser confocal microscopy-assisted analysis of cell death revealed the following order of events: prior to illumination, 5 accumulated in Golgi cysternae, endoplasmic reticulum and in some (but not all lysosomes. In response to light, the reactive oxygen species burst was concomitant with the drop of mitochondrial transmembrane electric potential, the dramatic changes of mitochondrial shape and the loss of integrity of mitochondria and lysosomes. Within 3-4 min post illumination, the plasma membrane became permeable for propidium iodide. Compounds 2 and 5 were one order of magnitude more potent than chlorin e(6 in photodamage of artificial liposomes monitored in a dye release assay. The latter effect depended on the content of non-saturated lipids; in liposomes consisting of saturated lipids no photodamage was detectable. The increased therapeutic efficacy of 5 compared with 2 was attributed to a striking difference in the ability of these photosensitizers to permeate through hydrophobic membrane interior as evidenced by measurements of voltage jump-induced relaxation of transmembrane current on planar lipid bilayers. CONCLUSIONS/SIGNIFICANCE: The multimembrane photodestruction and cell necrosis induced by photoactivation of 2 and 5 are

  9. Rapid mobilization of hematopoietic progenitors by AMD3100 and catecholamines is mediated by CXCR4-dependent SDF-1 release from bone marrow stromal cells (United States)

    Kalinkovich, Alexander; Itkin, Tomer; Ludin, Aya; Kao, Wei-Ming; Battista, Michela; Tesio, Melania; Kollet, Orit; Cohen, Neta Netzer; Margalit, Raanan; Buss, Eike C.; Baleux, Francoise; Oishi, Shinya; Fujii, Nobutaka; Larochelle, Andre; Dunbar, Cynthia E.; Broxmeyer, Hal E.; Frenette, Paul S.; Lapidot, Tsvee


    Steady-state egress of hematopoietic progenitor cells can be rapidly amplified by mobilizing agents such as AMD3100, the mechanism, however, is poorly understood. We report that AMD3100 increased the homeostatic release of the chemokine SDF-1 to the circulation in mice and non-human primates. Neutralizing antibodies against CXCR4 or SDF-1 inhibited both steady-state and AMD3100-induced SDF-1 release and reduced egress of murine progenitor cells over mature leukocytes. Intra-bone injection of biotinylated SDF-1 also enhanced release of this chemokine and murine progenitor cell mobilization. AMD3100 directly induced SDF-1 release from CXCR4+ human bone marrow osteoblasts and endothelial cells and activated uPA in a CXCR4/JNK-dependent manner. Additionally, ROS inhibition reduced AMD3100-induced SDF-1 release, activation of circulating uPA and mobilization of progenitor cells. Norepinephrine treatment, mimicking acute stress, rapidly increased SDF-1 release and progenitor cell mobilization, while β2-adrenergic antagonist inhibited both steady-state and AMD3100-induced SDF-1 release and progenitor cell mobilization in mice. In conclusion, this study reveals that SDF-1 release from bone marrow stromal cells to the circulation emerges as a pivotal mechanism essential for steady state egress and rapid mobilization of hematopoietic progenitor cells, but not mature leukocytes. PMID:21494253

  10. Rapid establishment of the European Bank for induced Pluripotent Stem Cells (EBiSC) - the Hot Start experience. (United States)

    De Sousa, Paul A; Steeg, Rachel; Wachter, Elisabeth; Bruce, Kevin; King, Jason; Hoeve, Marieke; Khadun, Shalinee; McConnachie, George; Holder, Julie; Kurtz, Andreas; Seltmann, Stefanie; Dewender, Johannes; Reimann, Sascha; Stacey, Glyn; O'Shea, Orla; Chapman, Charlotte; Healy, Lyn; Zimmermann, Heiko; Bolton, Bryan; Rawat, Trisha; Atkin, Isobel; Veiga, Anna; Kuebler, Bernd; Serano, Blanca Miranda; Saric, Tomo; Hescheler, Jürgen; Brüstle, Oliver; Peitz, Michael; Thiele, Cornelia; Geijsen, Niels; Holst, Bjørn; Clausen, Christian; Lako, Majlinda; Armstrong, Lyle; Gupta, Shailesh K; Kvist, Alexander J; Hicks, Ryan; Jonebring, Anna; Brolén, Gabriella; Ebneth, Andreas; Cabrera-Socorro, Alfredo; Foerch, Patrik; Geraerts, Martine; Stummann, Tina C; Harmon, Shawn; George, Carol; Streeter, Ian; Clarke, Laura; Parkinson, Helen; Harrison, Peter W; Faulconbridge, Adam; Cherubin, Luca; Burdett, Tony; Trigueros, Cesar; Patel, Minal J; Lucas, Christa; Hardy, Barry; Predan, Rok; Dokler, Joh; Brajnik, Maja; Keminer, Oliver; Pless, Ole; Gribbon, Philip; Claussen, Carsten; Ringwald, Annette; Kreisel, Beate; Courtney, Aidan; Allsopp, Timothy E


    A fast track "Hot Start" process was implemented to launch the European Bank for Induced Pluripotent Stem Cells (EBiSC) to provide early release of a range of established control and disease linked human induced pluripotent stem cell (hiPSC) lines. Established practice amongst consortium members was surveyed to arrive at harmonised and publically accessible Standard Operations Procedures (SOPs) for tissue procurement, bio-sample tracking, iPSC expansion, cryopreservation, qualification and distribution to the research community. These were implemented to create a quality managed foundational collection of lines and associated data made available for distribution. Here we report on the successful outcome of this experience and work flow for banking and facilitating access to an otherwise disparate European resource, with lessons to benefit the international research community. ETOC: The report focuses on the EBiSC experience of rapidly establishing an operational capacity to procure, bank and distribute a foundational collection of established hiPSC lines. It validates the feasibility and defines the challenges of harnessing and integrating the capability and productivity of centres across Europe using commonly available resources currently in the field. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  11. Microfabricated Renewable Beads-Trapping/Releasing Flow Cell for Rapid Antigen-Antibody Reaction in Chemiluminescent Immunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Zhifeng; Shao, Guocheng; Wang, Jun; Lu, Donglai; Wang, Wanjun; Lin, Yuehe


    A filter pillar-array microstructure was coupled with a pneumatic micro-valve to fabricate a reusable miniaturized beads-trapping/releasing flow cell, in which trapping and releasing beads can be conveniently realized by switching the micro-valve. This miniaturized device was suitable to construct automatic fluidic system for “renewable surface analysis”. The renewable surface strategy based on pneumatic micro-valve enabled capture of beads in beads chamber prior to each assay, and release of the used beads after the assay. Chemiluminescent competitive immunoassay of 3,5,6-trichloropyridinol (TCP) was performed as a model to demonstrate the application potential of this reusable miniaturized flow cell. The whole fluidic assay process including beads trapping, immuno-binding, beads washing, beads releasing and signal collection could be completed in 10 min. Immunoassay of TCP using this miniaturized device showed a linear range of 0.20-70 ng/mL with a limit of detection of 0.080 ng/mL. The device had been successfully used for detection of TCP spiked in rat serum with average recovery of 97%. This investigation provides a rapid, sensitive, reusable, low-cost and automatic miniaturized device for solid-phase biochemical analysis for various purposes.

  12. Strategies for rapidly mapping proviral integration sites and assessing cardiogenic potential of nascent human induced pluripotent stem cell clones. (United States)

    Dambrot, Cheryl; Buermans, Henk P J; Varga, Eszter; Kosmidis, Georgios; Langenberg, Karin; Casini, Simona; Elliott, David A; Dinnyes, Andras; Atsma, Douwe E; Mummery, Christine L; Braam, Stefan R; Davis, Richard P


    Recent methodological advances have improved the ease and efficiency of generating human induced pluripotent stem cells (hiPSCs), but this now typically results in a greater number of hiPSC clones being derived than can be wholly characterized. It is therefore imperative that methods are developed which facilitate rapid selection of hiPSC clones most suited for the downstream research aims. Here we describe a combination of procedures enabling the simultaneous screening of multiple clones to determine their genomic integrity as well as their cardiac differentiation potential within two weeks of the putative reprogrammed colonies initially appearing. By coupling splinkerette-PCR with Ion Torrent sequencing, we could ascertain the number and map the proviral integration sites in lentiviral-reprogrammed hiPSCs. In parallel, we developed an effective cardiac differentiation protocol that generated functional cardiomyocytes within 10 days without requiring line-specific optimization for any of the six independent human pluripotent stem cell lines tested. Finally, to demonstrate the scalable potential of these procedures, we picked 20 nascent iPSC clones and performed these independent assays concurrently. Before the clones required passaging, we were able to identify clones with a single integrated copy of the reprogramming vector and robust cardiac differentiation potential for further analysis. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Rapid fabrication of mesoporous TiO2 thin films by pulsed fibre laser for dye sensitized solar cells (United States)

    Hadi, Aseel; Alhabradi, Mansour; Chen, Qian; Liu, Hong; Guo, Wei; Curioni, Michele; Cernik, Robert; Liu, Zhu


    In this paper we demonstrate for the first time that a fibre laser with a wavelength of 1070 nm and a pulse width of milliseconds can be applied to generate mesoporous nanocrystalline (nc) TiO2 thin films on ITO coated glass in ambient atmosphere, by complete vaporisation of organic binder and inter-connection of TiO2 nanoparticles, without thermally damaging the ITO layer and the glass substrate. The fabrication of the mesoporous TiO2 thin films was achieved by stationary laser beam irradiation of 1 min. The dye sensitized solar cell (DSSC) with the laser-sintered TiO2 photoanode reached higher power conversion efficiency (PCE) of 3.20% for the TiO2 film thickness of 6 μm compared with 2.99% for the furnace-sintered. Electrochemical impedance spectroscopy studies revealed that the laser sintering under the optimised condition effectively decreased charge transfer resistance and increased electron lifetime of the TiO2 thin films. The use of the fibre laser with over 40% wall-plug efficiency offers an economically-feasible, industrial viable solution to the major challenge of rapid fabrication of large scale, mass production of mesoporous metal oxide thin film based solar energy systems, potentially for perovskite and monolithic tandem solar cells, in the future.

  14. Rapid generation of hydrogen peroxide contributes to the complex cell death induction by the angucycline antibiotic landomycin E. (United States)

    Panchuk, Rostyslav R; Lehka, Lilya V; Terenzi, Alessio; Matselyukh, Bohdan P; Rohr, Jürgen; Jha, Amit K; Downey, Theresa; Kril, Iryna J; Herbacek, Irene; van Schoonhoven, Sushilla; Heffeter, Petra; Stoika, Rostyslav S; Berger, Walter


    Landomycin E (LE) is an angucycline antibiotic produced by Streptomyces globisporus. Previously, we have shown a broad anticancer activity of LE which is, in contrast to the structurally related and clinically used anthracycline doxorubicin (Dx), only mildly affected by multidrug resistance-mediated drug efflux. In the present study, cellular and molecular mechanisms underlying the anticancer activity of landomycin E towards Jurkat T-cell leukemia cells were dissected focusing on the involvement of radical oxygen species (ROS). LE-induced apoptosis distinctly differed in several aspects from the one induced by Dx. Rapid generation of both extracellular and cell-derived hydrogen peroxide already at one hour drug exposure was observed in case of LE but not found before 24h for Dx. In contrast, Dx but not LE induced production of superoxide radicals. Mitochondrial damage, as revealed by JC-1 staining, was weakly enhanced already at 3h LE treatment and increased significantly with time. Accordingly, activation of the intrinsic apoptosis pathway initiator caspase-9 was not detectable before 12h exposure. In contrast, cleavage of the down-stream caspase substrate PARP-1 was clearly induced already at the three hour time point. Out of all caspases tested, only activation of effector caspase-7 was induced at this early time points paralleling the LE-induced oxidative burst. Accordingly, this massive cleavage of caspase-7 at early time points was inhibitable by the radical scavenger N-acetylcysteine (NAC). Additionally, only simultaneous inhibition of multiple caspases reduced LE-induced apoptosis. Specific scavengers of both H2O2 and OH(•) effectively decreased LE-induced ROS production, but only partially inhibited LE-induced apoptosis. In contrast, NAC efficiently blocked both parameters. Summarizing, rapid H2O2 generation and a complex caspase activation pattern contribute to the antileukemic effects of LE. As superoxide generation is considered as the main

  15. Use of rice husk ash for mitigating the autogenous shrinkage of cement pastes at low water cement ratio

    NARCIS (Netherlands)

    Huang, H.; Ye, G.; Fehling, Ekkehard; Middendorf, Bernhard; Thiemicke, Jenny


    It is well recognized that the high risk of early age micro-crack of HPC/UHPC is attributed to the large magnitude of early age autogenous shrinkage caused by self-desiccation in binder hydration. Over the years, several methods have been proposed to mitigate autogenous shrinkage based on internal

  16. Synthetic virus-like particles target dendritic cell lipid rafts for rapid endocytosis primarily but not exclusively by macropinocytosis.

    Directory of Open Access Journals (Sweden)

    Rajni Sharma

    Full Text Available DC employ several endocytic routes for processing antigens, driving forward adaptive immunity. Recent advances in synthetic biology have created small (20-30 nm virus-like particles based on lipopeptides containing a virus-derived coiled coil sequence coupled to synthetic B- and T-cell epitope mimetics. These self-assembling SVLP efficiently induce adaptive immunity without requirement for adjuvant. We hypothesized that the characteristics of DC interaction with SVLP would elaborate on the roles of cell membrane and intracellular compartments in the handling of a virus-like entity known for its efficacy as a vaccine. DC rapidly bind SVLP within min, co-localised with CTB and CD9, but not caveolin-1. In contrast, internalisation is a relatively slow process, delivering SVLP into the cell periphery where they are maintained for a number of hrs in association with microtubules. Although there is early association with clathrin, this is no longer seen after 10 min. Association with EEA-1(+ early endosomes is also early, but proteolytic processing appears slow, the SVLP-vesicles remaining peripheral. Association with transferrin occurs rarely, and only in the periphery, possibly signifying translocation of some SVLP for delivery to B-lymphocytes. Most SVLP co-localise with high molecular weight dextran. Uptake of both is impaired with mature DC, but there remains a residual uptake of SVLP. These results imply that DC use multiple endocytic routes for SVLP uptake, dominated by caveolin-independent, lipid raft-mediated macropinocytosis. With most SVLP-containing vesicles being retained in the periphery, not always interacting with early endosomes, this relates to slow proteolytic degradation and antigen retention by DC. The present characterization allows for a definition of how DC handle virus-like particles showing efficacious immunogenicity, elements valuable for novel vaccine design in the future.

  17. Thymic irradiation inhibits the rapid recovery of TH1 but not TH2-like functions of CD4+ T cells after total lymphoid irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Bass, H.; Adkins, B.; Strober, S. (Department of Medicine, Stanford University School of Medicine, CA (United States))


    Four to six weeks after total lymphoid irradiation (TLI), there is a selective deficit in the CD4+ T cells which secrete IL-2, proliferate in the MLR, and induce GVHD (Th1-like functions). A similar deficit in CD4+ T cells which secrete IL-4 and help antibody responses (Th2-like functions) is not observed. In the present study, shielding of the thymus with lead during TLI increased the Th1-like functions of CD4+ cells. Mice without thymus shields showed a marked selective reduction in the medullary stromal cells identified with the monoclonal antibody, MD1, and the severe reduction was prevented with thymus shields. Thus, shielding the thymus prevents the depletion of thymic medullary stromal cells and allows for a rapid recovery of Th1-like functions in the mouse spleen after TLI. Th2-like functions recover rapidly after TLI whether or not the thymus is irradiated.

  18. Experimental evaluation and simulation of volumetric shrinkage and warpage on polymeric composite reinforced with short natural fibers (United States)

    Santos, Jonnathan D.; Fajardo, Jorge I.; Cuji, Alvaro R.; García, Jaime A.; Garzón, Luis E.; López, Luis M.


    A polymeric natural fiber-reinforced composite is developed by extrusion and injection molding process. The shrinkage and warpage of high-density polyethylene reinforced with short natural fibers of Guadua angustifolia Kunth are analyzed by experimental measurements and computer simulations. Autodesk Moldflow® and Solid Works® are employed to simulate both volumetric shrinkage and warpage of injected parts at different configurations: 0 wt.%, 20 wt.%, 30 wt.% and 40 wt.% reinforcing on shrinkage and warpage behavior of polymer composite. Become evident the restrictive effect of reinforcing on the volumetric shrinkage and warpage of injected parts. The results indicate that volumetric shrinkage of natural composite is reduced up to 58% with fiber increasing, whereas the warpage shows a reduction form 79% to 86% with major fiber content. These results suggest that it is a highly beneficial use of natural fibers to improve the assembly properties of polymeric natural fiber-reinforced composites.

  19. Taguchi design and flower pollination algorithm application to optimize the shrinkage of triaxial porcelain containing palm oil fuel ash (United States)

    Zainudin, A.; Sia, C. K.; Ong, P.; Narong, O. L. C.; Nor, N. H. M.


    In the preparation of triaxial porcelain from Palm Oil Fuel Ash (POFA), a new parameter variable must be determined. The parameters involved are the particle size of POFA, percentage of POFA in triaxial porcelain composition, moulding pressure, sintering temperature and soaking time. Meanwhile, the shrinkage is the dependent variable. The optimization process was investigated using a hybrid Taguchi design and flower pollination algorithm (FPA). The interaction model of shrinkage was derived from regression analysis and found that the shrinkage is highly dependent on the sintering temperature followed by POFA composition, moulding pressure, POFA particle size and soaking time. The interaction between sintering temperature and soaking time highly affects the shrinkage. From the FPA process, targeted shrinkage approaching zero values were predicted for 142 μm particle sizes of POFA, 22.5 wt% of POFA, 3.4 tonne moulding pressure, 948.5 °C sintering temperature and 264 minutes soaking time.

  20. Linear shrinkage test: justification for its reintroduction as a standard South African test method

    CSIR Research Space (South Africa)

    Sampson, LR


    Full Text Available Several problems with the linear shrinkage test specified in Method A4 of the THM 1 1979 were addressed as part of this investigation in an effort to improve the alleged poor reproducibility of the test and justify its reintroduction into THM 1. A...

  1. Fabrication and characterization of self-folding thermoplastic sheets using unbalanced thermal shrinkage. (United States)

    Danielson, Christian; Mehrnezhad, Ali; YekrangSafakar, Ashkan; Park, Kidong


    Self-folding or micro-origami technologies are actively investigated as a novel manufacturing process to fabricate three-dimensional macro/micro-structures. In this paper, we present a simple process to produce a self-folding structure with a biaxially oriented polystyrene sheet (BOPS) or Shrinky Dinks. A BOPS sheet is known to shrink to one-third of its original size in plane, when it is heated above 160 °C. A grid pattern is engraved on one side of the BOPS film with a laser engraver to decrease the thermal shrinkage of the engraved side. The thermal shrinkage of the non-engraved side remains the same and this unbalanced thermal shrinkage causes folding of the structure as the structure shrinks at high temperature. We investigated the self-folding mechanism and characterized how the grid geometry, the grid size, and the power of the laser engraver affect the bending curvature. The developed fabrication process to locally modulate thermomechanical properties of the material by engraving the grid pattern and the demonstrated design methodology to harness the unbalanced thermal shrinkage can be applied to develop complicated self-folding macro/micro structures.

  2. Analysis of the shrinkage at the thick plate part using response surface methodology (United States)

    Hatta, N. M.; Azlan, M. Z.; Shayfull, Z.; Roselina, S.; Nasir, S. M.


    Injection moulding is well known for its manufacturing process especially in producing plastic products. To measure the final product quality, there are lots of precautions to be taken into such as parameters setting at the initial stage of the process. Sometimes, if these parameters were set up wrongly, defects may be occurred and one of the well-known defects in the injection moulding process is a shrinkage. To overcome this problem, a maximisation at the precaution stage by making an optimal adjustment on the parameter setting need to be done and this paper focuses on analysing the shrinkage by optimising the parameter at thick plate part with the help of Response Surface Methodology (RSM) and ANOVA analysis. From the previous study, the outstanding parameter gained from the optimisation method in minimising the shrinkage at the moulded part was packing pressure. Therefore, with the reference from the previous literature, packing pressure was selected as the parameter setting for this study with other three parameters which are melt temperature, cooling time and mould temperature. The analysis of the process was obtained from the simulation by Autodesk Moldflow Insight (AMI) software and the material used for moulded part was Acrylonitrile Butadiene Styrene (ABS). The analysis and result were obtained and it found that the shrinkage can be minimised and the significant parameters were found as packing pressure, mould temperature and melt temperature.

  3. A quantitative analysis of mineral loss and shrinkage of in vitro demineralized human root surfaces. (United States)

    Ten Cate, J M; Nyvad, B; Van de Plassche-Simons, Y M; Fejerskov, O


    Demineralization of dentin specimens proceeds at a faster rate than that of enamel. Although this is generally accepted, a quantification of the rate of formation of root lesions is hampered by the shrinkage of the lesions when these are dried prior to microradiographic analysis. This leads to a significant underestimation of the lesion depth and total mineral loss. The aim of this paper was to quantitate the rate of mineral loss during root lesion formation in vitro and to determine the shrinkage of root specimens as a result of drying. Unerupted roots of human teeth were subjected to a demineralizing system of 0.1 mol/L lactate buffer (pH = 4.8) with 0.2 mmol/L methanehydroxydiphosphonate during four, 11, 22, and 44 days. The root lesions were assessed by quantitative microradiography. The demineralizing solutions were analyzed to determine the amounts of root tissue dissolved. A comparison of these two sets of data showed that, with the demineralizing system used, root lesions may shrink up to 62%. Fixation of the specimens in fixative did not affect this shrinkage. Chemical analysis showed that mineral loss proceeded linearly with time. From the data-sets of this study, a model was developed to compensate for the shrinkage in the dentin specimens. In this way, it was possible to calculate the lesion depth at four demineralization times as being 130, 220, 320, and 530 microns, respectively. These values were in agreement with a microscopic determination of the lesion depth.

  4. A modelling study of drying shrinkage damage in concrete repair systems

    NARCIS (Netherlands)

    Lukovic, M.; Savija, B.; Schlangen, E.; Ye, G.; van Breugel, K.


    Differential shrinkage between repair material and concrete substrate is considered to be the main cause of premature failure of repair systems (Martinola, Sadouki et al. 2001, Beushausen and Alexander 2007). Magnitude of induced stresses depends on many factors, for example the amount of restraint,

  5. Combined Use of Shrinkage Reducing Admixture and CaO in Cement Based Materials (United States)

    Tittarelli, Francesca; Giosuè, Chiara; Monosi, Saveria


    The combined addition of a Shrinkage-Reducing Admixture (SRA) with a CaO-based expansive agent (CaO) has been found to have a synergistic effect to improve the dimensional stability of cement based materials. In this work, aimed to further investigate the effect, mortar and self-compacting concrete specimens were prepared either without admixtures, as reference, or with SRA alone and/or CaO. Their performance was compared in terms of compressive strength and free shrinkage measurements. Results showed that the synergistic effect in reducing shrinkage is confirmed in the specimens manufactured with SRA and CaO. In order to clarify this phenomenon, the effect of SRA on the hydration of CaO as well as cement was evaluated through different techniques. The obtained results show that SRA induces a finer microstructure of the CaO hydration products and a retarding effect on the microstructure development of cement based materials. A more deformable mortar or concrete, due to the delay in microstructure development by SRA, coupled with a finer microstructure of CaO hydration products could allow higher early expansion, which might contribute in contrasting better the successive drying shrinkage.

  6. Effectiveness of Fiber Reinforcement on the Mechanical Properties and Shrinkage Cracking of Recycled Fine Aggregate Concrete. (United States)

    Nam, Jeongsoo; Kim, Gyuyong; Yoo, Jaechul; Choe, Gyeongcheol; Kim, Hongseop; Choi, Hyeonggil; Kim, Youngduck


    This paper presents an experimental study conducted to investigate the effect of fiber reinforcement on the mechanical properties and shrinkage cracking of recycled fine aggregate concrete (RFAC) with two types of fiber-polyvinyl alcohol (PVA) and nylon. A small fiber volume fraction, such as 0.05% or 0.1%, in RFAC with polyvinyl alcohol or nylon fibers was used for optimum efficiency in minimum quantity. Additionally, to make a comparative evaluation of the mechanical properties and shrinkage cracking, we examined natural fine aggregate concrete as well. The test results revealed that the addition of fibers and fine aggregates plays an important role in improving the mechanical performance of the investigated concrete specimens as well as controlling their cracking behavior. The mechanical properties such as compressive strength, splitting tensile strength, and flexural strength of fiber-reinforced RFAC were slightly better than those of non-fiber-reinforced RFAC. The shrinkage cracking behavior was examined using plat-ring-type and slab-type tests. The fiber-reinforced RFAC showed a greater reduction in the surface cracks than non-fiber-reinforced concrete. The addition of fibers at a small volume fraction in RFAC is more effective for drying shrinkage cracks than for improving mechanical performance.

  7. Specimen size effect in the volumetric shrinkage of cancellous bone measured at two levels of dehydration. (United States)

    Lievers, W Brent; Lee, Victoria; Arsenault, Simon M; Waldman, Stephen D; Pilkey, A Keith


    Water is commonly removed from bone to study its effect on mechanical behaviour; however, dehydration also alters the bone structure. To make matters worse, measuring structural changes in cancellous bone is complicated by a number of factors. Therefore, the goals of this study were to address these issues by (1) comparing Archimedes' method and a helium pycnometer as methods for measuring cancellous bone volume; (2) measuring the apparent dimensional and volumetric tissue shrinkage of cancellous bone at two levels of dehydration; and, (3) identifying whether a size effect exists in cancellous bone shrinkage. Cylindrical specimens (3, 5 and 8.3 mm diameters) of cancellous bone were taken from the distal bovine femur. The apparent dimensions of each cylindrical specimen were measured in a fully hydrated state (HYD), after drying at room temperature (AIR), and after oven drying at 105 degrees C (OVEN). Tissue volume measurements for those three hydration states were obtained using both a helium pycnometer and Archimedes' method. Aluminium foams, which mimic the cancellous structure, were used as controls. The results suggest that the helium pycnometer and Archimedes' method yield identical results in the HYD and AIR states, but that Archimedes' method under-predicts the nominal OVEN volume by incorporating the collagen-apatite porosity. A distinct size effect on volumetric shrinkage is observed (pshrinkage (2% and 7%) at the two dehydration levels is much smaller than the measured volumetric tissue shrinkage (16% and 29%), which results in a reduced dehydrated bone volume fraction.

  8. Short-term effects of radiofrequency shrinkage treatment for anterior cruciate ligament relaxation on proprioception. (United States)

    Wei, Min; Liu, Yujie; Li, Zhongli; Wang, Zhigang


    Radiofrequency (RF) shrinkage is used in anterior cruciate ligament (ACL) reconstruction. The present study investigated the therapeutic effects of RF on ACL relaxation and the probable influencing factors. Patients with ACL relaxation were included. Participants were randomly divided into two groups: a treatment group, in which patients were treated with RF shrinkage (RF group); a control group, in which patients received conventional surgical treatment. Thermal shrinkage was performed on ACL using an ArthroCare® CAPSure® wand. Lysholm scores, proprioceptive testing and Tegner activity scores were evaluated before and after treatment (at 6 and 12 months). A total of 38 patients were included. The mean ± SD Lysholm score of those in the RF group at 12 months' post-treatment was significantly higher than in controls. The angle of deviation of the knee joint in RF group was significantly larger than in the control group at 6 months' post-treatment. RF shrinkage treatment for ACL laxity could improve knee scores, and may affect proprioception and recovery of activity after surgery.

  9. Shrinkage-reducing admixtures and early-age desiccation in cement pastes and mortars

    DEFF Research Database (Denmark)

    Bentz, D. P.; Geiker, Mette Rica; Hansen, Kurt Kielsgaard


    Fundamental studies of the early-age desiccation of cement-based materials with and without a shrinkage-reducing admixture (SRA) have been performed. Studies have been conducted under both sealed and drying conditions. Physical measurements include mass loss, surface tension, X-ray absorption...

  10. A 3D Lattice Modelling Study of Drying Shrinkage Damage in Concrete Repair Systems

    NARCIS (Netherlands)

    Lukovic, M.; Savija, B.; Schlangen, H.E.J.G.; Ye, G.; van Breugel, K.


    Differential shrinkage between repair material and concrete substrate is considered to be the main cause of premature failure of repair systems. The magnitude of induced stresses depends on many factors, for example the degree of restraint, moisture gradients caused by curing and drying conditions,

  11. Prediction of Shrinkage Porosity Defect in Sand Casting Process of LM25 (United States)

    Rathod, Hardik; Dhulia, Jay K.; Maniar, Nirav P.


    In the present worldwide and aggressive environment, foundry commercial enterprises need to perform productively with least number of rejections and create casting parts in shortest lead time. It has become extremely difficult for foundry industries to meet demands of defects free casting and meet strict delivery schedules. The process of casting solidification is complex in nature. Prediction of shrinkage defect in metal casting is one of the critical concern in foundries and is one of the potential research areas in casting. Due to increasing pressure to improve quality and to reduce cost, it is very essential to upgrade the level of current methodology used in foundries. In the present research work, prediction methodology of shrinkage porosity defect in sand casting process of LM25 using experimentation and ANSYS is proposed. The objectives successfully achieved are prediction of shrinkage porosity distribution in Al-Si casting and determining effectiveness of investigated function for predicting shrinkage porosity by correlating results of simulating studies to those obtained experimentally. The real-time application of the research reflects from the fact that experimentation is performed on 9 different Y junctions at foundry industry and practical data obtained from experimentation are used for simulation.

  12. Setting shrinkage strains of chemical-cured glass ionomer-based ...

    African Journals Online (AJOL)

    Shrinkage strains are exhibited by the current formulations of chemical-cured dental restorative systems. In resin-modified glass ionomer systems, these have been linked to filler contents, types and quantity of monomer. Post-gelation rigid contraction that follows onset of cure leading to marginal defects is a clinically ...

  13. Direct voxel-based comparisons between grey matter shrinkage and glucose hypometabolism in chronic alcoholism. (United States)

    Ritz, Ludivine; Segobin, Shailendra; Lannuzel, Coralie; Boudehent, Céline; Vabret, François; Eustache, Francis; Beaunieux, Hélène; Pitel, Anne L


    Alcoholism is associated with widespread brain structural abnormalities affecting mainly the frontocerebellar and the Papez's circuits. Brain glucose metabolism has received limited attention, and few studies used regions of interest approach and showed reduced global brain metabolism predominantly in the frontal and parietal lobes. Even though these studies have examined the relationship between grey matter shrinkage and hypometabolism, none has performed a direct voxel-by-voxel comparison between the degrees of structural and metabolic abnormalities. Seventeen alcoholic patients and 16 control subjects underwent both structural magnetic resonance imaging and (18)F-2-fluoro-deoxy-glucose-positron emission tomography examinations. Structural abnormalities and hypometabolism were examined in alcoholic patients compared with control subjects using two-sample t-tests. Then, these two patterns of brain damage were directly compared with a paired t-test. Compared to controls, alcoholic patients had grey matter shrinkage and hypometabolism in the fronto-cerebellar circuit and several nodes of Papez's circuit. The direct comparison revealed greater shrinkage than hypometabolism in the cerebellum, cingulate cortex, thalamus and hippocampus and parahippocampal gyrus. Conversely, hypometabolism was more severe than shrinkage in the dorsolateral, premotor and parietal cortices. The distinct profiles of abnormalities found within the Papez's circuit, the fronto-cerebellar circuit and the parietal gyrus in chronic alcoholism suggest the involvement of different pathological mechanisms. © The Author(s) 2015.

  14. Effectiveness of Fiber Reinforcement on the Mechanical Properties and Shrinkage Cracking of Recycled Fine Aggregate Concrete (United States)

    Nam, Jeongsoo; Kim, Gyuyong; Yoo, Jaechul; Choe, Gyeongcheol; Kim, Hongseop; Choi, Hyeonggil; Kim, Youngduck


    This paper presents an experimental study conducted to investigate the effect of fiber reinforcement on the mechanical properties and shrinkage cracking of recycled fine aggregate concrete (RFAC) with two types of fiber—polyvinyl alcohol (PVA) and nylon. A small fiber volume fraction, such as 0.05% or 0.1%, in RFAC with polyvinyl alcohol or nylon fibers was used for optimum efficiency in minimum quantity. Additionally, to make a comparative evaluation of the mechanical properties and shrinkage cracking, we examined natural fine aggregate concrete as well. The test results revealed that the addition of fibers and fine aggregates plays an important role in improving the mechanical performance of the investigated concrete specimens as well as controlling their cracking behavior. The mechanical properties such as compressive strength, splitting tensile strength, and flexural strength of fiber-reinforced RFAC were slightly better than those of non-fiber-reinforced RFAC. The shrinkage cracking behavior was examined using plat-ring-type and slab-type tests. The fiber-reinforced RFAC showed a greater reduction in the surface cracks than non-fiber-reinforced concrete. The addition of fibers at a small volume fraction in RFAC is more effective for drying shrinkage cracks than for improving mechanical performance. PMID:28773256

  15. Ultra high performance concrete made with rice husk ash for reduced autogenous shrinkage

    NARCIS (Netherlands)

    Van Breugel, K.; Van Tuan, N.


    Ultra High Strength Concrete (UHPC) is generally made with low w/c mixtures and by adding silica fume. Low w/c mixtures, however, exhibit high autogenous shrinkage, while a high amount of silica fume increases the price of these mixtures. For designing ultra high strength mixtures with low

  16. Quantification of shrinkage microcracking in young mortar with fluorescence light microscopy and ESEM

    NARCIS (Netherlands)

    Bisschop, J.; Van Mier, J.C.M.


    In this paper a method is described to quantify shrinkage microcracking in young mortar by means of crack mapping. Visualisation of the microcracks is realised with two techniques: Fluorescence Light Microscopy (FLM) and Environmental Scanning Electron Microscopy (ESEM). The preliminary results

  17. SEM-induced shrinkage and site-selective modification of single-crystal silicon nanopores (United States)

    Chen, Qi; Wang, Yifan; Deng, Tao; Liu, Zewen


    Solid-state nanopores with feature sizes around 5 nm play a critical role in bio-sensing fields, especially in single molecule detection and sequencing of DNA, RNA and proteins. In this paper we present a systematic study on shrinkage and site-selective modification of single-crystal silicon nanopores with a conventional scanning electron microscope (SEM). Square nanopores with measurable sizes as small as 8 nm × 8 nm and rectangle nanopores with feature sizes (the smaller one between length and width) down to 5 nm have been obtained, using the SEM-induced shrinkage technique. The analysis of energy dispersive x-ray spectroscopy and the recovery of the pore size and morphology reveal that the grown material along with the edge of the nanopore is the result of deposition of hydrocarbon compounds, without structural damage during the shrinking process. A simplified model for pore shrinkage has been developed based on observation of the cross-sectional morphology of the shrunk nanopore. The main factors impacting on the task of controllably shrinking the nanopores, such as the accelerating voltage, spot size, scanned area of e-beam, and the initial pore size have been discussed. It is found that single-crystal silicon nanopores shrink linearly with time under localized irradiation by SEM e-beam in all cases, and the pore shrinkage rate is inversely proportional to the initial equivalent diameter of the pore under the same e-beam conditions.

  18. The evolution of shrinkage strain of pet-mortar composite eco ...

    African Journals Online (AJOL)

    Concretes and mortars are subjected to several kinds of shrinkage strains which represent the volumic variations resulting from the cement hydration and are governed by various physical and chemical aspects. The use of polyethylene terephthalate PET plastic wastes which are available in quantity and within low cost in ...

  19. Reducing of on Polymerization Shrinkage by Application of UV Curable Dental Restorative Composites

    Directory of Open Access Journals (Sweden)

    Świderska Jolanta


    Full Text Available This manuscript describes dental compositions contain in-organic fillers, multifunctional methacrylates and photoinitiators. The main problem by application and UV curing process is the shrinkage of photoreactive dental materials during and after UV curing process. Total shrinkage of UV curable dental composites is a phenomenon of polymerization shrinkage, typical behavior for multifunctional methacrylates during polymerization process. The important factors by curing of dental composites are: kind and concentration of used methacrylates, their functionality, double bond concentration, kind and concentration of added photoinitiator and UV dose. They are investigated UV-curable dental compositions based on 2,2-bis-[4-(2-hydroxy-3-methacryxloyloxypropylphenyl]propane (Bis-GMA and containing such multifunctional monomers as 1,3-butanediol dimethacrylate (1,3-BDDMA, diethylene glycol dimethacrylate (DEGDMA, tetraethylene glycol dimethacrylate (T3EGDMA, trimethylolpropane trimethacrylate (TMPTMA, polyethylene glycol 200 dimethacrylate (PEG200DA. Reduction of polymerization shrinkage of dental compositions is at the moment a major problem by dental technology.

  20. Influence of rare earths on shrinkage porosity in thin walled ductile cast iron

    DEFF Research Database (Denmark)

    Pedersen, Karl Martin; Tiedje, Niels Skat


    Ductile cast iron has been cast in test bars with thickness from 2 to 10 mm. The rare earth elements La and Ce have been added to some of the castings to evaluate their influence on microstructure and shrinkage tendency. Both La and Ce increased the graphite nodule count, especially for thickness...

  1. Measurement of shrinkage and cracking in lyophilized amorphous cakes. Part IV: Effects of freezing protocol. (United States)

    Ullrich, Sabine; Seyferth, Stefan; Lee, Geoffrey


    The shrinkage and cracking of pure trehalose cakes during lyophilization has been determined quantitatively using different protocols for the freezing step. The influences of shelf cooling rate and of a two-step freezing protocol with holding and annealing phases were investigated. A small change in the shelf cooling rate from 0.4°C to 0.2°C per minute produced surprisingly large increases in shrinkage and reductions in cracking over all trehalose concentrations up to 30% w/v. The two-step freezing protocol also increased shrinkage and reduced cracking in the final-product cakes, especially at trehalose concentrations ≥ 15% and with large vial fill height. A combination of two-step freezing with use of TopLyo vials produced less than 1.5% cracking even at high trehalose concentrations and large fill height. The results give further confirmation of the causal linkage of shrinkage and cracking during lyophilization, and also illustrate how cracking can be greatly reduced by manipulating the freezing protocol. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Mitigation strategies for early-age shrinkage cracking in bridge decks. (United States)


    Early-age shrinkage cracking has been observed in many concrete bridge decks in Washington State and elsewhere around the U.S. The cracking increases the effects of freeze-thaw damage, spalling, and corrosion of steel reinforcement, thus resulting in...

  3. Autogenous shrinkage of Ducorit S5R ASTM C 1698-09 test method

    DEFF Research Database (Denmark)

    Damkilde, Lars

    The report deals with experimental measurement of autogenous shrinkage of Ducorit S5R according to the test method ASTM C 1698-09. This test method measures the bulk strain of a sealed cementitious specimen, at constant temperature and not subjected to external forces, from the time of final...

  4. Porous stainless steel hollow fibers with shrinkage-controlled small radial dimensions

    NARCIS (Netherlands)

    Luiten-Olieman, Maria W.J.; Raaijmakers, Michiel; Raaijmakers, Michiel J.T.; Winnubst, Aloysius J.A.; Wessling, Matthias; Nijmeijer, Arian; Benes, Nieck Edwin


    A method is presented for the preparation of thin (∼250 μm) porous stainless steel hollow fiber membranes based on dry–wet spinning of a particle-loaded polymer solution followed by heat treatment. Extraordinarily small radial dimensions were achieved by controlled shrinkage during thermal

  5. The Influence of Water Sorption of Dental Light-Cured Composites on Shrinkage Stress

    Directory of Open Access Journals (Sweden)

    Kinga Bociong


    Full Text Available The contraction stress generated during the photopolymerization of resin dental composites is the major disadvantage. The water sorption in the oral environment should counteract the contraction stress. The purpose was to evaluate the influence of the water sorption of composite materials on polymerization shrinkage stress generated at the restoration-tooth interface. The following materials were tested: Filtek Ultimate, Gradia Direct LoFlo, Heliomolar Flow, Tetric EvoCeram, Tetric EvoCeram Bulk Fill, Tetric EvoFlow, Tetric EvoFlow Bulk Fill, X-tra Base, Venus BulkFil, and Ceram.X One. The shrinkage stress was measured immediately after curing and after: 0.5 h, 24 h, 72 h, 96 h, 168 h, 240 h, 336 h, 504 h, 672 h, and 1344 h by means of photoelastic study. Moreover, water sorption and solubility were evaluated. Material samples were weighted on scale in time intervals to measure the water absorbency and the dynamic of this process. The tested materials during polymerization generated shrinkage stresses ranging from 6.3 MPa to 12.5 MPa. Upon water conditioning (56 days, the decrease in shrinkage strain (not less than 48% was observed. The decrease in value stress in time is material-dependent.

  6. Astrocytic mechanisms explaining neural-activity-induced shrinkage of extraneuronal space.

    Directory of Open Access Journals (Sweden)

    Ivar Østby


    Full Text Available Neuronal stimulation causes approximately 30% shrinkage of the extracellular space (ECS between neurons and surrounding astrocytes in grey and white matter under experimental conditions. Despite its possible implications for a proper understanding of basic aspects of potassium clearance and astrocyte function, the phenomenon remains unexplained. Here we present a dynamic model that accounts for current experimental data related to the shrinkage phenomenon in wild-type as well as in gene knockout individuals. We find that neuronal release of potassium and uptake of sodium during stimulation, astrocyte uptake of potassium, sodium, and chloride in passive channels, action of the Na/K/ATPase pump, and osmotically driven transport of water through the astrocyte membrane together seem sufficient for generating ECS shrinkage as such. However, when taking into account ECS and astrocyte ion concentrations observed in connection with neuronal stimulation, the actions of the Na(+/K(+/Cl(- (NKCC1 and the Na(+/HCO(3 (- (NBC cotransporters appear to be critical determinants for achieving observed quantitative levels of ECS shrinkage. Considering the current state of knowledge, the model framework appears sufficiently detailed and constrained to guide future key experiments and pave the way for more comprehensive astroglia-neuron interaction models for normal as well as pathophysiological situations.

  7. Measurement with corrugated tubes of early-age autogenous shrinkage of cement-based material

    DEFF Research Database (Denmark)

    Tian, Qian; Jensen, Ole Mejlhede


    The use of a special corrugated mould enables transformation of volume strain into horizontal, linear strain measurement in the fluid stage. This allows continuous measurement of the autogenous shrinkage of cement-based materials since casting, and also effectively eliminates unwanted influence...

  8. Characterizetion of Flexural Strength, Warpage and Shrinkage of Polypropylene-Nanoclay-Nanocomposites Blend with Gigantochloa Scortechinii. (United States)

    Khamis, S. Z.; Othman, M. H.; Hasan, S.; Ibrahim, M. H. I.


    This paper presents the characterization of Flexural Strength, Warpage and Shrinkage of reinforcement gigantochloa scortechinii fibre. The content of fiber were fixed at 0 wt.%, 3 wt.% and 6 wt.% in uniform increased. The selected injection moulding processing conditions were packing pressure, melt temperature, screw speed and filling time. The quality factors that need to be improved upon the characterization were flexural strength, warpage and shrinkage. This research started by drying the Gigantochloa Scortechinii fibres at 120°C. After that, 3 wt.% of the fibres were mixed with 81 wt.% of polypropylene, 15 wt.% of polypropylene grafted maleic anhydride (compatibilizer) and 1 wt. % of nanoclay. Samples with 6 wt.% of fibers were also prepared for comparison purpose. The mixing process was performed by using Brabender Lab-Compounder KETSE 20/40 and the pallets were produced using used Brabender® pelletizer with diameters of 1 to 4 mm. The optimisation process was accomplished by adopting the Taguchi L9 orthogonal array method. According to the results, for 0 wt.% GS, the flexural strength is 30.0082 MPa, the warpage is 0.0030000 mm and the shrinkage is 0.0003830 mm at packing pressure 40%, melt temperature 165°C, filled time 2 seconds and screw speed 35%. For the result 3 wt.% GS, the flexural strength is 32.2477 MPa, the warpage is 0.006670 mm and the shrinkage is 0.0003830 mm at packing pressure is 35%, melt temperature 165°C, filled times is 1 seconds and screw speed is 30%. While for the 6 wt.% GS, the results of the flexural strength is 36.9084 MPa, the warpage is 0.0066700 mm and the shrinkage is 0.0003830 mm at packing pressure is 35%, melt temperature 165°C, filled time is 2 seconds and screw speed is 30%. The existence of Gigantochloa Scortechinii fibre was also proven to effect significantly towards flexural strength with 6% increasing value ordering from 0 wt.% GS to 6 wt.% GS. while, the warpage value increasing from 0.003000 mm to 0.00667 mm and

  9. Escherichia coli α-hemolysin triggers shrinkage of erythrocytes via K(Ca)3.1 and TMEM16A channels with subsequent phosphatidylserine exposure

    DEFF Research Database (Denmark)

    Skals, Marianne Gerberg; Jensen, Uffe Birk; Ousingsawat, Jiraporn


    -34 prevent the shrinkage and potentiate hemolysis produced by HlyA. Notably, the recently described Ca(2+)-activated Cl(-) channel TMEM16A contributes substantially to HlyA-induced cell volume reduction. Erythrocytes isolated from TMEM16A(-/-) mice showed significantly attenuated crenation......, which depends on Ca(2+)-activated efflux of K(+) via K(Ca)3.1 and Cl(-) via TMEM16A, with subsequent phosphatidylserine exposure. This mechanism might potentially allow HlyA-damaged erythrocytes to be removed from the bloodstream by macrophages and thereby reduce the risk of intravascular hemolysis....

  10. A rapid and efficient polyethylenimine-based transfection method to prepare lentiviral or retroviral vectors: useful for making iPS cells and transduction of primary cells. (United States)

    Yang, Shaozhe; Shi, Haijun; Chu, Xinran; Zhou, Xiaoling; Sun, Pingnan


    To improve the efficiency, reproducibility and consistency of the PEI-based transfection method that is often used in preparation of recombinant lentiviral or retroviral vectors. The contributions to transfection efficiency of multi-factors including concentration of PEI or DNA, dilution buffer for PEI/DNA, manner to prepare PEI/DNA complexes, influence of serum, incubation time for PEI/DNA complexes, and transfection time were studied. Gentle mixing during the preparation of PEI/DNA transfection complexes is critical for a high transfection efficiency. PEI could be stored at room temperature or 4 °C, and most importantly, multigelation should be avoided. The transfection efficiency of the PEI-based new method in different types of cells, such as 293T, Cos-7, HeLa, HepG2, Hep3B, Huh7 and L02, was also higher than that of the previous method. After optimization, the titer of our lentiviral system or retroviral system produced by PEI-based new method was about 10- or 3-times greater than that produced by PEI-based previous method, respectively. We provide a rapid and efficient PEI-based method for preparation of recombinant lentiviral or retroviral vectors which is useful for making iPS cells as well as transduction of primary cell cultures.

  11. Rapid reduction of MDCK cell cholesterol by methyl-beta-cyclodextrin alters steady state transepithelial electrical resistance. (United States)

    Francis, S A; Kelly, J M; McCormack, J; Rogers, R A; Lai, J; Schneeberger, E E; Lynch, R D


    The role of plasma membrane lipids in regulating the passage of ions and other solutes through the paracellular pathway remains controversial. In this study we explore the contribution of cholesterol (CH) in maintaining the barrier function of an epithelial cell line using the CH-solubilizing agent methyl beta-cyclodextrin (MBCD) to stimulate CH efflux. Inclusion of 20 mM MBCD in both apical and basolateral media reduced CH levels by 70-80% with no significant effect on cell viability. Most of that decrease occurred during the first 30 min of incubation. Recovery of CH content to initial values was nearly complete 22 h after removal of MBCD. Within 30 min of adding MBCD to the culture medium, transepithelial electrical resistance (TER) increased, reaching maximum values 30-40% above controls. This early rise in TER occurred when MBCD was added to either side of the monolayer. The later rapid decline in TER was observed only when MBCD bathed the basolateral surface from which, coincidentally, CH efflux was most rapid. Freeze fracture replicas and transmission electron microscopy of monolayers exposed to MBCD for only 30 min revealed no increase in either the average tight junction (TJ) strand number or the dimensions of the lateral intercellular space. There was a statistically significant increase in the number of TJ particles associated with the E fracture face at this time. This raises the interesting possibility that during CH efflux there is a change in the interaction between TJ particles and underlying cytoskeletal elements. There was no change in staining for occludin and ZO-1. After exposing the basolateral surface to MBCD for 2 h, TER fell below control levels. The accompanying increase in mannitol flux suggests strongly that the decrease in TER resulted from an increase in the permeability of the paracellular and not the transcellular pathway. A decrease in immuno-staining for occludin and ZO-1 at TJs, a striking accumulation of actin at tri

  12. Rapid engraftment without significant graft-versus-host disease after allogeneic transplantation of CD34+ selected cells from peripheral blood. (United States)

    Urbano-Ispizua, A; Rozman, C; Martínez, C; Marín, P; Briones, J; Rovira, M; Féliz, P; Viguria, M C; Merino, A; Sierra, J; Mazzara, R; Carreras, E; Montserrat, E


    have relapsed, and one of them is again in hematologic and cytogenetic remission after infusion of the donor lymphocytes. Two patients died in remission: one on day +109 of pulmonary aspergillosis and the other on day +251 of metastasic relapse of a previous breast cancer. Sixteen of the 20 patients are alive in remission after a median follow-up of 7.5 months (range, 2 to 22). In conclusion, despite the small number of patients and limited follow-up, it appears that this method allows a high CD34+ cell recovery from G-CSF mobilized PBPC and is associated with rapid engraftment without significant GVHD, and with low transplant related mortality.

  13. Predicting shrinkage and warpage in injection molding: Towards automatized mold design (United States)

    Zwicke, Florian; Behr, Marek; Elgeti, Stefanie


    It is an inevitable part of any plastics molding process that the material undergoes some shrinkage during solidification. Mainly due to unavoidable inhomogeneities in the cooling process, the overall shrinkage cannot be assumed as homogeneous in all volumetric directions. The direct consequence is warpage. The accurate prediction of such shrinkage and warpage effects has been the subject of a considerable amount of research, but it is important to note that this behavior depends greatly on the type of material that is used as well as the process details. Without limiting ourselves to any specific properties of certain materials or process designs, we aim to develop a method for the automatized design of a mold cavity that will produce correctly shaped moldings after solidification. Essentially, this can be stated as a shape optimization problem, where the cavity shape is optimized to fulfill some objective function that measures defects in the molding shape. In order to be able to develop and evaluate such a method, we first require simulation methods for the diffierent steps involved in the injection molding process that can represent the phenomena responsible for shrinkage and warpage ina sufficiently accurate manner. As a starting point, we consider the solidification of purely amorphous materials. In this case, the material slowly transitions from fluid-like to solid-like behavior as it cools down. This behavior is modeled using adjusted viscoelastic material models. Once the material has passed a certain temperature threshold during cooling, any viscous effects are neglected and the behavior is assumed to be fully elastic. Non-linear elastic laws are used to predict shrinkage and warpage that occur after this point. We will present the current state of these simulation methods and show some first approaches towards optimizing the mold cavity shape based on these methods.

  14. Control of polymerization shrinkage and stress in nanogel-modified monomer and composite materials. (United States)

    Moraes, Rafael R; Garcia, Jeffrey W; Barros, Matthew D; Lewis, Steven H; Pfeifer, Carmem S; Liu, JianCheng; Stansbury, Jeffrey W


    This study demonstrates the effects of nano-scale prepolymer particles as additives to model dental monomer and composite formulations. Discrete nanogel particles were prepared by solution photopolymerization of isobornyl methacrylate and urethane dimethacrylate in the presence of a chain transfer agent, which also provided a means to attach reactive groups to the prepolymer. Nanogel was added to triethylene glycol dimethacrylate (TEGDMA) in increments between 5 and 40 wt% with resin viscosity, reaction kinetics, shrinkage, mechanical properties, stress and optical properties evaluated. Maximum loading of barium glass filler was determined as a function of nanogel content and composites with varied nanogel content but uniform filler loading were compared in terms of consistency, conversion, shrinkage and mechanical properties. High conversion, high molecular weight internally crosslinked and cyclized nanogel prepolymer was efficiently prepared and redispersed into TEGDMA with an exponential rise in viscosity accompanying nanogel content. Nanogel addition at any level produced no deleterious effects on reaction kinetics, conversion or mechanical properties, as long as reactive nanogels were used. A reduction in polymerization shrinkage and stress was achieved in proportion to nanogel content. Even at high nanogel concentrations, the maximum loading of glass filler was only marginally reduced relative to the control and high strength composite materials with low shrinkage were obtained. The use of reactive nanogels offers a versatile platform from which resin and composite handling properties can be adjusted while the polymerization shrinkage and stress development that challenge the adhesive bonding of dental restoratives are controllably reduced. Copyright © 2011 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

  15. Measurement, growth types and shrinkage of newly formed aerosol particles at an urban research platform (United States)

    Salma, Imre; Németh, Zoltán; Weidinger, Tamás; Kovács, Boldizsár; Kristóf, Gergely


    Budapest platform for Aerosol Research and Training (BpART) was created for advancing long-term on-line atmospheric measurements and intensive aerosol sample collection campaigns in Budapest. A joint study including atmospheric chemistry or physics, meteorology, and fluid dynamics on several-year-long data sets obtained at the platform confirmed that the location represents a well-mixed, average atmospheric environment for the city centre. The air streamlines indicated that the host and neighbouring buildings together with the natural orography play an important role in the near-field dispersion processes. Details and features of the airflow structure were derived, and they can be readily utilised for further interpretations. An experimental method to determine particle diffusion losses in the differential mobility particle sizer (DMPS) system of the BpART facility was proposed. It is based on CPC-CPC (condensation particle counter) and DMPS-CPC comparisons. Growth types of nucleated particles observed in 4 years of measurements were presented and discussed specifically for cities. Arch-shaped size distribution surface plots consisting of a growth phase followed by a shrinkage phase were characterised separately since they supply information on nucleated particles. They were observed in 4.5 % of quantifiable nucleation events. The shrinkage phase took 1 h 34 min in general, and the mean shrinkage rate with standard deviation was -3.8 ± 1.0 nm h-1. The shrinkage of particles was mostly linked to changes in local atmospheric conditions, especially in global radiation and the gas-phase H2SO4 concentration through its proxy, or to atmospheric mixing in few cases. Some indirect results indicate that variations in the formation and growth rates of nucleated particles during their atmospheric transport could be a driving force of shrinkage for particles of very small sizes and on specific occasions.

  16. Critical role of perforin-dependent CD8+ T cell immunity for rapid protective vaccination