Characterization of fluid flow by digital correlation of scattered light

NASA Technical Reports Server (NTRS)

Gilbert, John A.; Matthys, Donald R.

1989-01-01

The objective is to produce a physical system suitable for a space environment that can measure fluid velocities in a three-dimensional volume by the development of a particle correlation velocimetry technique. Experimental studies were conducted on a field test cell to demonstrate the suitability and accuracy of digital correlation techniques for measuring two-dimensional fluid flows. This objective was satisfied by: (1) the design of an appropriate illumination and detection system for making velocity measurements within a test cell; (2) the design and construction of a test cell; (3) the preliminary evaluations on fluid and seeding requirements; and (4) the performance of controlled tests using a multiple exposure correlation technique. This presentation is represented by viewgraphs with very little text.

Non-Flow-Through Fuel Cell System Test Results and Demonstration on the SCARAB Rover

NASA Technical Reports Server (NTRS)

Scheidegger, Brianne, T.; Burke, Kenneth A.; Jakupca, Ian J.

2012-01-01

This paper describes the results of the demonstration of a non-flow-through PEM fuel cell as part of a power system on the SCARAB rover. A 16-cell non-flow-through fuel cell stack from Infinity Fuel Cell and Hydrogen, Inc. was incorporated into a power system designed to act as a range extender by providing power to the rover s hotel loads. This work represents the first attempt at a ground demonstration of this new technology aboard a mobile test platform. Development and demonstration were supported by the Office of the Chief Technologist s Space Power Systems Project and the Advanced Exploration System Modular Power Systems Project.

Evaluation of Material Models within LS-DYNA(Registered TradeMark) for a Kevlar/Epoxy Composite Honeycomb

NASA Technical Reports Server (NTRS)

Polanco, Michael A.; Kellas, Sotiris; Jackson, Karen

2009-01-01

The performance of material models to simulate a novel composite honeycomb Deployable Energy Absorber (DEA) was evaluated using the nonlinear explicit dynamic finite element code LS-DYNA(Registered TradeMark). Prototypes of the DEA concept were manufactured using a Kevlar/Epoxy composite material in which the fibers are oriented at +/-45 degrees with respect to the loading axis. The development of the DEA has included laboratory tests at subcomponent and component levels such as three-point bend testing of single hexagonal cells, dynamic crush testing of single multi-cell components, and impact testing of a full-scale fuselage section fitted with a system of DEA components onto multi-terrain environments. Due to the thin nature of the cell walls, the DEA was modeled using shell elements. In an attempt to simulate the dynamic response of the DEA, it was first represented using *MAT_LAMINATED_COMPOSITE_FABRIC, or *MAT_58, in LS-DYNA. Values for each parameter within the material model were generated such that an in-plane isotropic configuration for the DEA material was assumed. Analytical predictions showed that the load-deflection behavior of a single-cell during three-point bending was within the range of test data, but predicted the DEA crush response to be very stiff. In addition, a *MAT_PIECEWISE_LINEAR_PLASTICITY, or *MAT_24, material model in LS-DYNA was developed, which represented the Kevlar/Epoxy composite as an isotropic elastic-plastic material with input from +/-45 degrees tensile coupon data. The predicted crush response matched that of the test and localized folding patterns of the DEA were captured under compression, but the model failed to predict the single-cell three-point bending response.

Hexamethylenebisacetamide (HMBA) is a growth factor for human, ovine and porcine thyroid cells.

PubMed

Fayet, G; Amphoux-Fazekas, T; Aouani, A; Hovsépian, S

1996-03-01

Hexamethylenebisacetamide (HMBA) provokes in murine erythroleukemia cells (MELC) a commitment to terminal differentiation leading to the activation of the expression of hemoglobin. HMBA has been tested also in other cells from colon cancer, melanoma or lung cancer. However it has not yet been tested in the thyroid. We demonstrate in this paper that HMBA in kinetics and concentration-response experiments increases the proliferation of human thyroid cells isolated from Graves'-Basedow patients. It also acts like a growth factor for ovine and porcine thyroid cells, respectively, from the OVNIS line and the ATHOS line. This molecule which is a differentiating factor in the MELC system and a growth factor in human thyroid cell cultures represents a potential to get human thyroid cell lines expressing specialized functions.

Pharmacology and toxicology of pahayokolide A, a bioactive metabolite from a freshwater species of Lyngbya isolated from the Florida Everglades

PubMed Central

Berry, John P.; Gantar, Miroslav; Gawley, Robert E.; Wang, Minglei; Rein, Kathleen S.

2008-01-01

The genus of filamentous cyanobacteria, Lyngbya, has been found to be a rich source of bioactive metabolites. However, identification of such compounds from Lyngbya has largely focused on a few marine representatives. Here, we report on the pharmacology and toxicology of pahayokolide A from a freshwater isolate, Lyngbya sp. strain 15−2, from the Florida Everglades. Specifically, we investigated inhibition of microbial representatives and mammalian cell lines, as well as toxicity of the compound to both invertebrate and vertebrate models. Pahayokolide A inhibited representatives of Bacillus, as well as the yeast, Saccharomyces cerevisiae. Interestingly, the compound also inhibited several representatives of green algae that were also isolated from the Everglades. Pahayokolide A was shown to inhibit a number of cancer cell lines over a range of concentrations (IC50 varied from 2.13 to 44.57 μM) depending on the cell-type. When tested against brine shrimp, pahayokolide was only marginally toxic at the highest concentrations tested (1 mg/mL). The compound was, however, acutely toxic to zebrafish embryos (LC50=2.15 μM). Possible biomedical and environmental health aspects of the pahayokolides remain to be investigated; however, the identification of bioactive metabolites such as these demonstrates the potential of the Florida Everglades as source of new toxins and drugs. PMID:15683832

Gelatine/PLLA sponge-like scaffolds: morphological and biological characterization.

PubMed

Lazzeri, Luigi; Cascone, Maria Grazia; Danti, Serena; Serino, Lorenzo Pio; Moscato, Stefania; Bernardini, Nunzia

2006-12-01

Biodegradable synthetic polymers such as poly(lactic acid) are widely used to prepare scaffolds for cell transplantation and tissue growth, using different techniques set up for the purpose. However the poor hydrophilicity of these polymers represents the main limitation to their use as scaffolds because it causes a low affinity for the cells. An effective way to solve this problem could be represented by the addition of biopolymers that are in general highly hydrophilic. The present work concerns porous biodegradable sponge-like systems based on poly(L-lactic acid) and gelatine. Morphology and porosity characteristics of the sponges were studied by scanning electron microscopy and mercury intrusion porosimetry respectively. Blood compatibility was investigated by bovine plasma fibrinogen adsorption test and platelet adhesion test. The cell culture method was used in order to evaluate the ability of the matrices to work as scaffolds for tissue regeneration. The obtained results indicate that the sponges have interesting porous characteristics, good blood compatibility and above all good ability to support cell adhesion and growth. In fact viable and metabolically active animal cells were found inside the sponges after 8 weeks in culture. On this basis the systems produced seem to be good candidates as scaffolds for tissue regeneration.

Synthesis and Evaluation of Cytotoxic Activity of Some Pyrroles and Fused Pyrroles.

PubMed

Fatahala, Samar S; Mohamed, Mosaad S; Youns, Mahmoud; Abd-El Hameed, Rania H

2017-01-01

Pyrrole derivatives represent a very interesting class as biologically active compounds. The objective of our study was to investigate the cytotoxic and apoptotic effects and antioxidant activity of the newly synthesized pyrrole derivatives. A series of novel pyrroles and fused pyrroles (tetrahydroindoles, pyrrolopyrimidines, pyrrolopyridines and pyrrolotriazines) were synthesized and characterized using IR, 1H NMR, 13C NMR, MS and elemental analysis techniques. The antiproliferative activity of our synthesized compounds and their modulatory effect apoptotic pathway were investigated. The effect on cellular proliferation and viability was monitored by resazurin assay. Apoptotic effect was evaluated by caspase glo 3/7 assay. Synthesized compounds are then tested for their anticancer activities against three different cell lines representing three different tumor types, namely; the HepG-2 (Human hepatocellular liver carcinoma cell line), the human MCF-7 cell line (breast cancer) and the pancreatic resistant Panc-1 cells. Compounds Ia-e, IIe, and IXc, d showed a promising anti-cancer activity on all tested cell lines. Antioxidant and wound healing invasion assays were examined for promising anticancer candidate compounds. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Microscopic or occult hematuria, when reflex testing is not good laboratory practice.

PubMed

Froom, Paul; Barak, Mira

2010-01-01

Consensus opinion suggests that hematuria found by dipstick and not confirmed on microscopic examination (<2 erythrocytes per high power field) signifies a false-positive reagent strip test result. Standard practice is to repeat the dipstick test several days later and if still positive to confirm by microscopic examination. If discordant results are obtained, experts recommend reflex testing for urinary myoglobin and hemoglobin concentrations. The question is whether or not this approach represents good laboratory practice. These recommendations are not evidence based. We conclude that the reference range for red blood cells on the reagent strip should be increased to 25x10(6) cells/L for young men, and 50x10(6) cells/L for the rest of the adult population, ranges consistent with flow cytometry reports. Confirmation reflex testing using tests that have inferior sensitivity, precision and probably accuracy is not recommended.

Evaluation and verification of epitaxial process sequence for silicon solar-cell production

NASA Technical Reports Server (NTRS)

Redfield, D.

1981-01-01

To achieve the program goals, 28 minimodules were fabricated and tested, using 600 cells made from three-inch-diameter wafers processed by the sequence chosen for this purpose. Of these 600 cells, half were made from epitaxially grown layers on potentially low-cost substrates. The other half were made from commercial semiconductor-grade (SG), single-crystal silicon wafers that served as controls. Cell processing was normally performed on mixed lots containing significant numbers of each of these two types of wafers. After evaluation of the performance of all cells, they were separated by types for incorporation into modules that were to be tested for electrical performance and response to environmental stress. A simplified flow chart displaying this scheme, for quantities representing half of the planned total to be processed, is presented.

EVA assembly of large space structure element

NASA Technical Reports Server (NTRS)

Bement, L. J.; Bush, H. G.; Heard, W. L., Jr.; Stokes, J. W., Jr.

1981-01-01

The results of a test program to assess the potential of manned extravehicular activity (EVA) assembly of erectable space trusses are described. Seventeen tests were conducted in which six "space-weight" columns were assembled into a regular tetrahedral cell by a team of two "space"-suited test subjects. This cell represents the fundamental "element" of a tetrahedral truss structure. The tests were conducted under simulated zero-gravity conditions. Both manual and simulated remote manipulator system modes were evaluated. Articulation limits of the pressure suit and zero gravity could be accommodated by work stations with foot restraints. The results of this study have confirmed that astronaut EVA assembly of large, erectable space structures is well within man's capabilities.

Amniotic fluid stem cells: a promising therapeutic resource for cell-based regenerative therapy.

PubMed

Antonucci, Ivana; Pantalone, Andrea; Tete, Stefano; Salini, Vincenzo; Borlongan, Cesar V; Hess, David; Stuppia, Liborio

2012-01-01

Stem cells have been proposed as a powerful tool in the treatment of several human diseases, both for their ability to represent a source of new cells to replace those lost due to tissue injuries or degenerative diseases, and for the ability of produce trophic molecules able to minimize damage and promote recovery in the injured tissue. Different cell types, such as embryonic, fetal or adult stem cells, human fetal tissues and genetically engineered cell lines, have been tested for their ability to replace damaged cells and to restore the tissue function after transplantation. Amniotic fluid -derived Stem cells (AFS) are considered a novel resource for cell transplantation therapy, due to their high renewal capacity, the "in vitro" expression of embryonic cell lineage markers, and the ability to differentiate in tissues derived from all the three embryonic layers. Moreover, AFS do not produce teratomas when transplanted into animals and are characterized by a low antigenicity, which could represent an advantage for cell transplantation or cell replacement therapy. The present review focuses on the biological features of AFS, and on their potential use in the treatment of pathological conditions such as ischemic brain injury and bone damages.

Comparison of in vitro methods for carboxylesterase activity determination in immortalized cells representative of the intestine, liver and kidney.

PubMed

Lamego, Joana; Ferreira, Pedro; Alves, Márcia; Matias, Ana; Simplício, Ana Luisa

2015-08-01

Herein we compare the fluorimetric determination of total and specific carboxylesterase activity in immortalized human derived living cells and in cell lysates. The cell lines chosen are representative of metabolism occurring in the intestine (Caco-2 and HT-29), kidney (HEK-293T) and liver (Hep G2). Caco-2 and HT-29, as cells prone to differentiation, were tested along the differentiation time. For evaluation of both methods when distinguishing activity of different carboxylesterases, HEK-293T transfected with the human carboxylestarase-2 (hCES2) were also tested. Application to Caco-2 or HT-29 cells demonstrated higher activity detected in cell lysates than in cell monolayers. The difference is most striking when comparing the methods at different stages of Caco-2 and HT-29 cell maturation, highlighting substrate accessibility as a limiting step in the in vivo hydrolysis rates (possibly limited by plasma and Endoplasmic Reticulum membrane permeability) with increasing relevance as the cells differentiate. Application to Hep G2 or to hCES2 transfected and non-transfected HEK-293T cells, demonstrated a tendency for higher sensitivity in living cell suspensions than that obtained with the cell lysates which indicates the importance of cell environment in the maintenance of enzyme activity. However, quantification of hCES2 activity relative to total esterase, or to total carboxylesterase activity, was not significantly different in any case. The results herein presented help to clarify which method is best suited for evaluation of carboxylesterase activity in vitro depending on the final goal of the study. Copyright © 2015 Elsevier Ltd. All rights reserved.

Density-based clustering analyses to identify heterogeneous cellular sub-populations

NASA Astrophysics Data System (ADS)

Heaster, Tiffany M.; Walsh, Alex J.; Landman, Bennett A.; Skala, Melissa C.

2017-02-01

Autofluorescence microscopy of NAD(P)H and FAD provides functional metabolic measurements at the single-cell level. Here, density-based clustering algorithms were applied to metabolic autofluorescence measurements to identify cell-level heterogeneity in tumor cell cultures. The performance of the density-based clustering algorithm, DENCLUE, was tested in samples with known heterogeneity (co-cultures of breast carcinoma lines). DENCLUE was found to better represent the distribution of cell clusters compared to Gaussian mixture modeling. Overall, DENCLUE is a promising approach to quantify cell-level heterogeneity, and could be used to understand single cell population dynamics in cancer progression and treatment.

Surface matching for correlation of virtual models: Theory and application

NASA Technical Reports Server (NTRS)

Caracciolo, Roberto; Fanton, Francesco; Gasparetto, Alessandro

1994-01-01

Virtual reality can enable a robot user to off line generate and test in a virtual environment a sequence of operations to be executed by the robot in an assembly cell. Virtual models of objects are to be correlated to the real entities they represent by means of a suitable transformation. A solution to the correlation problem, which is basically a problem of 3-dimensional adjusting, has been found exploiting the surface matching theory. An iterative algorithm has been developed, which matches the geometric surface representing the shape of the virtual model of an object, with a set of points measured on the surface in the real world. A peculiar feature of the algorithm is to work also if there is no one-to-one correspondence between the measured points and those representing the surface model. Furthermore the problem of avoiding convergence to local minima is solved, by defining a starting point of states ensuring convergence to the global minimum. The developed algorithm has been tested by simulation. Finally, this paper proposes a specific application, i.e., correlating a robot cell, equipped for biomedical use with its virtual representation.

Gelatine/PLLA sponge-like scaffolds: morphological and biological characterization.

PubMed

Lazzeri, Luigi; Cascone, Maria Grazia; Danti, Serena; Serino, Lorenzo Pio; Moscato, Stefania; Bernardini, Nunzia

2007-07-01

Biodegradable synthetic polymers such as poly(lactic acid) (PLA) are widely used to prepare scaffolds for cell transplantation and tissue growth, using different techniques set up for the purpose. However the poor hydrophilicity of these polymers represents the main limitation to their use as scaffolds because it causes a low affinity for the cells. An effective way to solve this problem could be represented by the addition of biopolymers that are in general highly hydrophilic. The present work concerns porous biodegradable sponge-like systems based on poly(L-lactic acid) (PLLA) and gelatine. Morphology and porosity characteristics of the sponges were studied by scanning electron microscopy and mercury intrusion porosimetry respectively. Blood compatibility was investigated by bovine plasma fibrinogen (BPF) adsorption test and platelet adhesion test (PAT). The cell culture method was used in order to evaluate the ability of the matrices to work as scaffolds for tissue regeneration. The obtained results indicate that the sponges have interesting porous characteristics, good blood compatibility and above all good ability to support cell adhesion and growth. In fact viable and metabolically active animal cells were found inside the sponges after 8 weeks in culture. On this basis the systems produced seem to be good candidates as scaffolds for tissue regeneration.

Real-Time linux dynamic clamp: a fast and flexible way to construct virtual ion channels in living cells.

PubMed

Dorval, A D; Christini, D J; White, J A

2001-10-01

We describe a system for real-time control of biological and other experiments. This device, based around the Real-Time Linux operating system, was tested specifically in the context of dynamic clamping, a demanding real-time task in which a computational system mimics the effects of nonlinear membrane conductances in living cells. The system is fast enough to represent dozens of nonlinear conductances in real time at clock rates well above 10 kHz. Conductances can be represented in deterministic form, or more accurately as discrete collections of stochastically gating ion channels. Tests were performed using a variety of complex models of nonlinear membrane mechanisms in excitable cells, including simulations of spatially extended excitable structures, and multiple interacting cells. Only in extreme cases does the computational load interfere with high-speed "hard" real-time processing (i.e., real-time processing that never falters). Freely available on the worldwide web, this experimental control system combines good performance. immense flexibility, low cost, and reasonable ease of use. It is easily adapted to any task involving real-time control, and excels in particular for applications requiring complex control algorithms that must operate at speeds over 1 kHz.

Characterization of the Murine Myeloid Precursor Cell Line MuMac-E8

PubMed Central

Fricke, Stephan; Riemschneider, Sina; Kohlschmidt, Janine; Hilger, Nadja; Fueldner, Christiane; Knauer, Jens; Sack, Ulrich; Emmrich, Frank; Lehmann, Jörg

2014-01-01

Starting point for the present work was the assumption that the cell line MuMac-E8 represents a murine cell population with stem cell properties. Preliminary studies already pointed to the expression of stem-cell associated markers and a self-regenerative potential of the cells. The cell line MuMac-E8 should be examined for their differential stage within stem cell hierarchy. MuMac-E8 cells were derived from a chimeric mouse model of arthritis. It could be shown that MuMac-E8 cells express mRNA of some genes associated with pluripotent stem cells (Nanog, Nucleostemin), of genes for hematopoietic markers (EPCR, Sca-1, CD11b, CD45), for the mesenchymal marker CD105 and of genes for the neural markers Pax-6 and Ezrin. In methylcellulose and May-Grünwald-Giemsa staining, hematopoietic colonies were obtained but the hematopoietic system of lethally irradiated mice could not be rescued. Osteogenic differentiation was not detectable. Thus, it became evident that MuMac-E8 represents not a stem cell line. However, MuMac-E8 cells expressed several myeloid surface markers (i.e. CD11b, F4/80, CD14, CD64), showed phagocytosis and is capable of producing nitric oxide. Thus, this cell line seems to be arrested an advanced stage of myeloid differentiation. Adherence data measured by impedance-based real-time cell analysis together with cell morphology data suggested that MuMac-E8 represents a new macrophage precursor cell line exhibiting weak adherence. This cell line is suitable as an in-vitro model for testing of macrophage functions. Moreover, it might be also useful for differentiation or reprogramming studies. PMID:25546418

Cosmos 2229 immunology study (Experiment K-8-07)

NASA Technical Reports Server (NTRS)

Sonnenfeld, Gerald

1993-01-01

The purpose of the current study was to further validate use of the rhesus monkey as a model for humans in future space flight testing. The areas of immunological importance examined in the Cosmos 2229 flight were represented by two sets of studies. The first set of studies determined the effect of space flight on the ability of bone marrow cells to respond to granulocyte/monocyte colony stimulating factor (GM-CSF). GM-CSF is an important regulator in the differentiation of bone marrow cells of both monocyte/macrophage and granulocyte lineages and any change in the ability of these cells to respond to GM-CSF can result in altered immune function. A second set of studies determined space flight effects on the expression of cell surface markers on both spleen and bone marrow cells. Immune cell markers included in this study were those for T-cell, B-cell, natural killer cell, and interleukin-2 populations. Variations from a normal cell population percentage, as represented by these markers, can be correlated with alterations in immunological function. Cells were stained with fluorescein-labelled antibodies directed against the appropriate antigens, and then analyzed using a flow cytometer.

Segments from red blood cell units should not be used for quality testing.

PubMed

Kurach, Jayme D R; Hansen, Adele L; Turner, Tracey R; Jenkins, Craig; Acker, Jason P

2014-02-01

Nondestructive testing of blood components could permit in-process quality control and reduce discards. Tubing segments, generated during red blood cell (RBC) component production, were tested to determine their suitability as a sample source for quality testing. Leukoreduced RBC components were produced from whole blood (WB) by two different methods: WB filtration and buffy coat (BC). Components and their corresponding segments were tested on Days 5 and 42 of hypothermic storage (HS) for spun hematocrit (Hct), hemoglobin (Hb) content, percentage hemolysis, hematologic indices, and adenosine triphosphate concentration to determine whether segment quality represents unit quality. Segment samples overestimated hemolysis on Days 5 and 42 of HS in both BC- and WB filtration-produced RBCs (p < 0.001 for all). Hct and Hb levels in the segments were also significantly different from the units at both time points for both production methods (p < 0.001 for all). Indeed, for all variables tested different results were obtained from segment and unit samples, and these differences were not consistent across production methods. The quality of samples from tubing segments is not representative of the quality of the corresponding RBC unit. Segments are not suitable surrogates with which to assess RBC quality. © 2013 American Association of Blood Banks.

Six consecutive false positive cases from cell-free fetal DNA testing in a single referring centre

PubMed Central

Dugo, Nella; Padula, Francesco; Mobili, Luisa; Brizzi, Cristiana; D’Emidio, Laura; Cignini, Pietro; Mesoraca, Alvaro; Bizzoco, Domenico; Cima, Antonella; Giorlandino, Claudio

2014-01-01

Introduction recent studies have proposed the introduction of cell-free fetal DNA testing (NIPT-Non Invasive Prenatal Testing) in routine clinical practice emphasizing its high sensibility and specificity. In any case, false positive and false negative findings may result from placental mosaicism, because cell-free fetal DNA originates mainly from placenta. Case we report six cases of women who underwent chorionic villus sampling (CVS) or amniocentesis to confirm the results from NIPT: two Turner syndromes, two Triple X, one Patau syndrome, one Edward syndrome. Results using classic cytogenetic analysis and, also, Array - Comparative Genomic Hybridization (Array CGH) the karyotype of all 5 fetuses was found to be normal. Conclusion results from NIPT must always be confirmed by invasive prenatal diagnosis. It is mandatory to inform the patient that the CVS and amniocentesis still represent the only form of prenatal diagnostic test available. PMID:25332757

Cytokeratin 5 positive cells represent a therapy resistant subpopulation in epithelial ovarian cancer

PubMed Central

Corr, Bradley R.; Finlay-Schultz, Jessica; Rosen, Rachel B.; Qamar, Lubna; Post, Miriam D.; Behbakht, Kian; Spillman, Monique A.; Sartorius, Carol A.

2015-01-01

Objective Cytokeratin 5 (CK5) is an epithelial cell marker implicated in stem and progenitor cell activity in glandular reproductive tissues and endocrine and chemotherapy resistance in estrogen receptor (ER)+ breast cancer. The goal of this study was to determine the prevalence of CK5 expression in ovarian cancer and the response of CK5+ cell populations to cisplatin therapy. Materials and Methods CK5 expression was evaluated in two ovarian tissue microarrays, representing 137 neoplasms, and six ovarian cancer cell lines. Cell lines were treated with IC50 cisplatin and the prevalence of CK5+ cells pre- and post-treatment determined. Proliferation of CK5+ vs. CK5− cell populations was determined using bromodeoxyuridine (BrdU) incorporation. Chemotherapy induced apoptosis in CK5+ vs. CK5− cells was measured using immunohistochemical staining for cleaved caspase-3. Results CK5 was expressed in 39.3% (42/107) of epithelial ovarian cancers with a range of 1-80% positive cells. Serous and endometrioid histologic subtypes had the highest percentage of CK5+ specimens. CK5 expression correlated with ER positivity (38/42 CK5+ tumors were also ER+). CK5 was expressed in 5/6 overall and 4/4 ER+ epithelial ovarian cancer cell lines ranging from 2.4-52.7% positive cells. CK5+ compared to CK5− cells were slower proliferating. The prevalence of CK5+ cells increased following 48 hour cisplatin treatment in 4/5 cell lines tested. CK5+ compared to CK5− ovarian cancer cells were more resistant to cisplatin induced apoptosis. Conclusions CK5 is expressed in a significant proportion of epithelial ovarian cancers and represents a slower proliferating, chemoresistant subpopulation that may warrant co-targeting in combination therapy. PMID:26495758

Alginate based 3D hydrogels as an in vitro co-culture model platform for the toxicity screening of new chemical entities.

PubMed

Lan, Shih-Feng; Starly, Binil

2011-10-01

Prediction of human response to potential therapeutic drugs is through conventional methods of in vitro cell culture assays and expensive in vivo animal testing. Alternatives to animal testing require sophisticated in vitro model systems that must replicate in vivo like function for reliable testing applications. Advancements in biomaterials have enabled the development of three-dimensional (3D) cell encapsulated hydrogels as in vitro drug screening tissue model systems. In this study, we have developed an in vitro platform to enable high density 3D culture of liver cells combined with a monolayer growth of target breast cancer cell line (MCF-7) in a static environment as a representative example of screening drug compounds for hepatotoxicity and drug efficacy. Alginate hydrogels encapsulated with serial cell densities of HepG2 cells (10(5)-10(8) cells/ml) are supported by a porous poly-carbonate disc platform and co-cultured with MCF-7 cells within standard cell culture plates during a 3 day study period. The clearance rates of drug transformation by HepG2 cells are measured using a coumarin based pro-drug. The platform was used to test for HepG2 cytotoxicity 50% (CT(50)) using commercially available drugs which further correlated well with published in vivo LD(50) values. The developed test platform allowed us to evaluate drug dose concentrations to predict hepatotoxicity and its effect on the target cells. The in vitro 3D co-culture platform provides a scalable and flexible approach to test multiple-cell types in a hybrid setting within standard cell culture plates which may open up novel 3D in vitro culture techniques to screen new chemical entity compounds. Copyright © 2011 Elsevier Inc. All rights reserved.

Method development for thermal analyses testing on Reillex HPQ resin using the advanced reactive system screening tool (ARSST)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Best, D.

Reillex™ HPQ resin was developed by Los Alamos Laboratory and Reilly Industries Inc. in an effort to increase safety and process efficiency during the recovery and purification of plutonium. Ionac™ A-641, another strong base macroporous anion exchange resin used in the nuclear industry, was known to undergo a runaway reaction in hot nitric acid solutions. Because of this, an extensive amount of thermal analyses testing on the Reillex™ HPQ resin in SRNL was performed in 1999-2001 prior to use. A report on the thermal stability qualification of the Reillex™ HPQ resin in 8M (35%) and 12M (53%) HNO 3 wasmore » reported in 2000. In 2001, the reactivity of Reillex™ HPQ resin in 14.4M (64%) HNO 3 was evaluated. In January of 2001, thermal stability scoping tests were performed on irradiated Reillex™ HPQ resin in 14.4M (64%) HNO 3 (as a worst case scenario) and the results sent to Fauske and Associates to calculate a rupture disk size for the HB-Line resin column. A technical report by Fauske and Associates was issued in February 2001 recommending a 2.0” vent line with a rupture disk set pressure of 60 psig. This calculation was based on ARSST thermal analyses scoping tests at SRNL in which 4 grams of dried resin and 6.0 grams of 64% nitric acid in a 10 gram test cell, produced a maximum pressure rate (dP/dt) of 720 psi/min (12 psi/sec) and a maximum temperature of 250 °C. In 2015, a new batch of Reillex™ HPQ resin was manufactured by Vertellus Industries. A test sample of the resin was sent to SRNL to perform acceptance and qualification thermal stability testing using the ARSST. During these tests, method development was performed to ensure that a representative resin to acid ratios were used while running the tests in the ARSST. Fauske and Associates recommended to either use a full test cell representative of the HB-Line column or a 10 gram sample in the test cell that was representative of the ratios of resin to nitric acid in the actual HB-Line column. An observation was made during method development testing that the 4 grams of resin to 6 grams of 64% acid ratio used in 2001 to determine the rupture disk size did not entirely wet the resin and was not representative of the actual HB-Line column. The results of this study raised questions as to whether the ratio of 4 grams of resin to 6 grams of 64% nitric acid recommended by Fauske and Associates in 2001 was the correct ratio.« less

Transgene vaccination using Ulex europaeus agglutinin I (UEA-1) for targeted mucosal immunization against HIV-1 envelope.

PubMed

Wang, Xinhai; Kochetkova, Irina; Haddad, Asmahan; Hoyt, Teri; Hone, David M; Pascual, David W

2005-05-31

Receptor-mediated gene transfer using an M cell ligand has been shown to be an efficient method for mucosal DNA immunization. To investigate further into alternative M cell ligands, the plant lectin, Ulex europaeus agglutinin I (UEA-1), was tested. UEA-1 binds to human intestinal Caco-2 cells, and these cells can be transfected with poly-l-lysine (PL)-conjugated UEA-1 for expression of reporter cDNAs. When tested in vivo, mice nasally immunized with UEA-1-PL complexed to plasmid encoding HIV-1 envelope showed elevated systemic and mucosal antibody responses, and these were supported by tissue antibody-forming cells. Likewise, elevated envelope-specific CTLs were induced. Thus, UEA-1 mediated DNA delivery represents an alternative mucosal formulation for inducing humoral and cellular immunity against HIV-1.

GAPD and tubulin are suitable internal controls for qPCR analysis of oral squamous cell carcinoma cell lines.

PubMed

Campos, M S; Rodini, C O; Pinto-Júnior, D S; Nunes, F D

2009-02-01

The selection of housekeeping genes is critical for gene expression studies. To address this issue, four candidate housekeeping genes, including several commonly used ones, were investigated in oral squamous cell carcinoma cell lines. A simple quantitative RT-PCR approach was employed by comparing relative expression of the four candidate genes within two cancerous cell lines (HN6 and HN31) and one noncancerous cell line (HaCaT) treated or not with EGF and TGF-beta1. Data were analyzed using ANOVA followed by the NormFinder software program. On this basis, stability of the candidate housekeeping genes was ranked and non statistical differences were found using ANOVA test. On the other hand, the NormFinder was able to show that GAPD and TUBB presented the less variable results, representing appropriated housekeeping genes for the samples and conditions analyzed. In conclusion, this study suggests that the GAPD and the TUBB represent adequate normalizers for gene profiling studies in OSCC cell lines, covering, respectively, high and low expression levels genes.

Assessment of the U937 cell line for the detection of contact allergens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Python, Francois; Goebel, Carsten; Aeby, Pierre

2007-04-15

The human myeloid cell line U937 was evaluated as an in vitro test system to identify contact sensitizers in order to develop alternatives to animal tests for the cosmetic industry. Specific culture conditions (i.e., presence of interleukin-4, IL-4) were applied to obtain a dendritic cell-like phenotype. In the described test protocol, these cells were exposed to test chemicals and then analyzed by flow cytometry for CD86 expression and by quantitative real-time reverse transcriptase-polymerase chain reaction for IL-1{beta} and IL-8 gene expressions. Eight sensitizers, three non-sensitizers and five oxidative hair dye precursors were examined after 24-, 48- and 72-h exposure times.more » Test item-specific modulations of the chosen activation markers (CD86, IL-1{beta} and IL-8) suggest that this U937 activation test could discriminate test items classified as contact sensitizers or non-sensitizers in the local lymph node assay in mice (LLNA). More specifically, a test item can be considered as a potential sensitizer when it significantly induced the upregulation of the expression of at least two markers. Using this approach, we could correctly evaluate the dendritic cell (DC) activation potential for 15 out of 16 tested chemicals. We conclude that the U937 activation test may represent an useful tool in a future in vitro test battery for predicting sensitizing properties of chemicals.« less

Sodium-sulfur battery flight experiment definition study

NASA Technical Reports Server (NTRS)

Chang, Rebecca; Minck, Robert

1990-01-01

Sodium-sulfur batteries are considered to be one of the most likely battery systems for space applications. Compared with the Ni-H2 or Ni-Co battery systems, Na-S batteries offer a mass reduction by a factor of 2 to 4, representing significant launch cost savings or increased payload mass capabilities. The Na-S battery operates at between 300 and 400 C, using liquid sodium and sulfur/polysulfide electrodes and solid ceramic electrolyte; the transport of the electrode materials to the surface of the electrolyte is through wicking/capillary forces. This paper describes five tests identified for the Na-S battery flight experiment definition study, which include the cell characterization test, the reactant distribution test, the current/temperature distribution test, the freeze/thaw test, and the multicell LEO test. A schematic diagram of Na-S cell is included.

Chimeric antigen receptor T-cell immunotherapy for glioblastoma: practical insights for neurosurgeons.

PubMed

Choi, Bryan D; Curry, William T; Carter, Bob S; Maus, Marcela V

2018-06-01

The prognosis for glioblastoma (GBM) remains exceedingly poor despite state-of-the-art multimodal therapy. Immunotherapy, particularly with cytotoxic T cells, represents a promising alternative. Perhaps the most prominent T-cell technology is the chimeric antigen receptor (CAR), which in 2017 received accelerated approval from the Food and Drug Administration for the treatment of hematological malignancies. Several CARs for GBM have been recently tested in clinical trials with exciting results. The authors review these clinical data and discuss areas of ongoing research.

Creation of Dystrophin Expressing Chimeric Cells of Myoblast Origin as a Novel Stem Cell Based Therapy for Duchenne Muscular Dystrophy.

PubMed

Siemionow, M; Cwykiel, J; Heydemann, A; Garcia-Martinez, J; Siemionow, K; Szilagyi, E

2018-04-01

Over the past decade different stem cell (SC) based approaches were tested to treat Duchenne Muscular Dystrophy (DMD), a lethal X-linked disorder caused by mutations in dystrophin gene. Despite research efforts, there is no curative therapy for DMD. Allogeneic SC therapies aim to restore dystrophin in the affected muscles; however, they are challenged by rejection and limited engraftment. Thus, there is a need to develop new more efficacious SC therapies. Chimeric Cells (CC), created via ex vivo fusion of donor and recipient cells, represent a promising therapeutic option for tissue regeneration and Vascularized Composite Allotransplantation (VCA) due to tolerogenic properties that eliminate the need for lifelong immunosuppression. This proof of concept study tested feasibility of myoblast fusion for Dystrophin Expressing. Chimeric Cell (DEC) therapy through in vitro characterization and in vivo assessment of engraftment, survival, and efficacy in the mdx mouse model of DMD. Murine DEC were created via ex vivo fusion of normal (snj) and dystrophin-deficient (mdx) myoblasts using polyethylene glycol. Efficacy of myoblast fusion was confirmed by flow cytometry and dystrophin immunostaining, while proliferative and myogenic differentiation capacity of DEC were assessed in vitro. Therapeutic effect after DEC transplant (0.5 × 10 6 ) into the gastrocnemius muscle (GM) of mdx mice was assessed by muscle functional tests. At 30 days post-transplant dystrophin expression in GM of injected mdx mice increased to 37.27 ± 12.1% and correlated with improvement of muscle strength and function. Our study confirmed feasibility and efficacy of DEC therapy and represents a novel SC based approach for treatment of muscular dystrophies.

DALMATIAN: An Algorithm for Automatic Cell Detection and Counting in 3D.

PubMed

Shuvaev, Sergey A; Lazutkin, Alexander A; Kedrov, Alexander V; Anokhin, Konstantin V; Enikolopov, Grigori N; Koulakov, Alexei A

2017-01-01

Current 3D imaging methods, including optical projection tomography, light-sheet microscopy, block-face imaging, and serial two photon tomography enable visualization of large samples of biological tissue. Large volumes of data obtained at high resolution require development of automatic image processing techniques, such as algorithms for automatic cell detection or, more generally, point-like object detection. Current approaches to automated cell detection suffer from difficulties originating from detection of particular cell types, cell populations of different brightness, non-uniformly stained, and overlapping cells. In this study, we present a set of algorithms for robust automatic cell detection in 3D. Our algorithms are suitable for, but not limited to, whole brain regions and individual brain sections. We used watershed procedure to split regional maxima representing overlapping cells. We developed a bootstrap Gaussian fit procedure to evaluate the statistical significance of detected cells. We compared cell detection quality of our algorithm and other software using 42 samples, representing 6 staining and imaging techniques. The results provided by our algorithm matched manual expert quantification with signal-to-noise dependent confidence, including samples with cells of different brightness, non-uniformly stained, and overlapping cells for whole brain regions and individual tissue sections. Our algorithm provided the best cell detection quality among tested free and commercial software.

Microvascularized 3D Breast Cancer Constructs for Drug Testing and Development

DTIC Science & Technology

2014-10-01

cells. Therefore, both bacterial cellulose and fibrin represent promising scaffolding materials for construction of the prevascularized, 3D co-culture...group consisting of: collagen, fibrin, elastin, keratin, Matrigel™, bacterial cellulose , HuBiogel™, and combinations thereof. 7. The 3D ...AWARD NUMBER: TITLE: Microvascularized 3D Breast Cancer Constructs for Drug Testing and Development PRINCIPAL INVESTIGATOR: Joel L. Berry

Alignment between the Science Curriculum and Assessment in Selected NY State Regents Exams

ERIC Educational Resources Information Center

Liu, Xiufeng; Fulmer, Gavin

2008-01-01

This article reports on an analysis of alignment between NY state core curricula and NY Regents tests in physics and chemistry. Both the curriculum and test were represented by a two dimensional table consisting of topics and cognitive demands. The cell values of the table were numbers of major understandings in the curriculum and points of test…

Oxidative stress resulting from exposure of a human salivary gland cells to paraoxon: an in vitro model for organophosphate oral exposure.

PubMed

Prins, John M; Chao, Chih-Kai; Jacobson, Saskia M; Thompson, Charles M; George, Kathleen M

2014-08-01

Organophosphate (OP) compounds are used as insecticides, acaricides, and chemical agents and share a common neurotoxic mechanism of action. The biochemical alterations leading to many of the deleterious effects have been studied in neuronal cell lines, however, non-neuronal toxic effects of OPs are far less well characterized in vitro, and specifically in cell lines representing oral routes of exposure. To address this void, the human salivary gland (HSG) cell line, representing likely interactions in the oral cavity, was exposed to the representative OP paraoxon (PX; O,O-diethyl-p-nitrophenoxy phosphate) over a range of concentrations (0.01-100 μM) and analyzed for cytotoxicity. PX induced cytotoxicity in HSG cells at most of the exposure concentrations as revealed by MTT assay, however, the release of LDH only occurred at the highest concentration of PX tested (100 μM) at 48 h. Slight increases in cellular ATP levels were measured in PX-exposed (10 μM) HSG cells at 24 h. Exposing HSG cells to 10 μM PX also led to an increase in DNA fragmentation prior to loss of cellular membrane integrity implicating reactive oxygen species (ROS) as a trigger of toxicity. The ROS genes gss, gstm2, gstt2 and sod2 were upregulated, and the presence of superoxide following 10 μM PX exposure was determined via dihydroethidium fluorescence studies further implicating PX-induced oxidative stress in HSG cells. Published by Elsevier Ltd.

Report of the First International Consensus on Standardized Nomenclature of Antinuclear Antibody HEp-2 Cell Patterns 2014–2015

PubMed Central

Chan, Edward K. L.; Damoiseaux, Jan; Carballo, Orlando Gabriel; Conrad, Karsten; de Melo Cruvinel, Wilson; Francescantonio, Paulo Luiz Carvalho; Fritzler, Marvin J.; Garcia-De La Torre, Ignacio; Herold, Manfred; Mimori, Tsuneyo; Satoh, Minoru; von Mühlen, Carlos A.; Andrade, Luis E. C.

2015-01-01

During the 12th International Workshop on Autoantibodies and Autoimmunity held in Sao Paulo, Brazil, on August 28, 2014, a full day session was devoted to establishing a consensus on the nomenclature of staining patterns observed in the antinuclear antibody (ANA) indirect immunofluorescence test on HEp-2 cells. The current report summarizes the collective agreements with input from the host Brazilian and international communities that represented research, clinical, and diagnostic service laboratories. Patterns are categorized in three major groups (nuclear, cytoplasmic, and mitotic patterns) and each pattern has been defined and described in detail. The consensus nomenclature and representative patterns are made available online at the international consensus on antinuclear antibody pattern (ICAP) website (www.ANApatterns.org). To facilitate continuous improvement and input, specific comments on ICAP are encouraged and these will be discussed in subsequent ICAP meetings. The ultimate goal with the establishment of the ICAP is to promote harmonization and understanding of autoantibody test nomenclature, as well as interpretation guidelines for ANA testing, thereby optimizing usage in patient care. PMID:26347739

Environmental risk assessment of wastewaters from printed circuit board production: A multibiomarker approach using human cells.

PubMed

Gerić, Marko; Gajski, Goran; Oreščanin, Višnja; Domijan, Ana-Marija; Kollar, Robert; Garaj-Vrhovac, Vera

2017-02-01

Since the production of printed circuit boards (PCBs) generates wastewater contaminated with heavy metals and organic matter, PCB factories represent potential pollution sites. The wastewater toxicologically tested in this study contained several metals and the most abundant were copper and iron. At two exposure times tested (4 and 24 h) PCB wastewater (PCBW) proved to be cytotoxic (decreased cell viability) and genotoxic (increased comet assay tail intensity and tail moment) to human blood peripheral lymphocytes in vitro, and the oxidative stress parameter (malondialdehyde concentration) was also found to be higher. After application of combined treatment by waste base, ozone and waste sludge methods, concentrations of metals in purified PCBW were below the upper permitted levels and all tested toxicological parameters did not differ compared to the negative control. Taken together, similar methods could be implemented in PCB factories before discharging potentially toxic wastewater into the environment because purified PCBW does not represent a threat from the aspect of cytotoxicity and genotoxicity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cycle life test and failure model of nickel-hydrogen cells

NASA Technical Reports Server (NTRS)

Smithrick, J. J.

1983-01-01

Six ampere hour individual pressure vessel nickel hydrogen cells were charge/discharge cycled to failure. Failure as used here is defined to occur when the end of discharge voltage degraded to 0.9 volts. They were cycled under a low earth orbit cycle regime to a deep depth of discharge (80 percent of rated ampere hour capacity). Both cell designs were fabricated by the same manufacturer and represent current state of the art. A failure model was advanced which suggests both cell designs have inadequate volume tolerance characteristics. The limited existing data base at a deep depth of discharge (DOD) was expanded. Two cells of each design were cycled. One COMSAT cell failed at cycle 1712 and the other failed at cycle 1875. For the Air Force/Hughes cells, one cell failed at cycle 2250 and the other failed at cycle 2638. All cells, of both designs, failed due to low end of discharge voltage (0.9 volts). No cell failed due to electrical shorts. After cell failure, three different reconditioning tests (deep discharge, physical reorientation, and open circuit voltage stand) were conducted on all cells of each design. A fourth reconditioning test (electrolyte addition) was conducted on one cell of each design. In addition post cycle cell teardown and failure analysis were performed on the one cell of each design which did not have electrolyte added after failure.

Simulatng Sawtooth Mixers For Biofouling Mitigation

NASA Astrophysics Data System (ADS)

Waters, James; Balazs, Anna

2017-11-01

We demonstrate how a ridged surface can be used to generate vortices that will break up clusters of cells as they form. This offers an appealing avenue for fouling mitigation, as it relies on a physical mechanism without unintended environmental consequences. By adjusting the shape of these ridges, we can increase the effectiveness of the surface across a range of shear values. We represent such a system computationally using a hybrid of bulk fluid simulated via the lattice Boltzmann method, and deformable vesicles, representing cells, simulated via that lattice spring method. This simulation methodology allows us to rapidly implement and test different surface patterns, and explore how their parameters can most effectively deter the accumulation of biofilms.

Fuel cell technology program

NASA Technical Reports Server (NTRS)

1971-01-01

The results of a solid polymer electrolyte fuel cell development program are summarized. A base line design was defined, and materials and components of the base line configuration were fabricated and tested. Concepts representing base line capability extensions in the areas of life, power, specific weight and volume, versatility of operation, field maintenance, and thermal control were identified and evaluated. Liaison and coordination with space shuttle contractors resulted in the exchange of engineering data.

Evaluation of the In Vitro Pyrogen Test System Based on Proinflammatory Cytokine Release from Human Monocytes: Comparison with a Human Whole Blood Culture Test System and with the Rabbit Pyrogen Test

PubMed Central

Nakagawa, Yukari; Maeda, Hideko; Murai, Toshimi

2002-01-01

The reliability of an in vitro pyrogen test system based on proinflammatory cytokine release from human monocytic cells was assessed by comparison with a test system based on a human whole blood culture as well as with the conventional rabbit pyrogen test. The human cells used as the pyrogen indicator cells were newly selected by subcloning of a human monocytic cell line, Mono-Mac-6. The selected cells, named MM6-CA8, responded to various pyrogens, including endotoxin, peptidoglycan (PG), Staphylococcus aureus Cowan 1 (SAC), and poly(I ·  C), with a high sensitivity and produced proinflammatory cytokines, such as interleukin 1 (IL-1), IL-6, and tumor necrosis factor alpha. Among these cytokines, IL-6 was produced most sensitively in response to traces of the pyrogens and detected in the largest quantities in the culture medium. The cytokine-producing responses of MM6-CA8 cells correlated significantly with the responses of cultured human whole blood, which represents an ex vivo culture test system reproducing pyrogen-induced cytokine production in the human body. In terms of cytokine inducibility, the pyrogens were ranked in the order endotoxin > PG > poly (I · C) > SAC in both culture systems, a ranking which almost agreed with the ranking of their pyrogenicity as assessed by the rabbit pyrogen test. These results suggest that the in vitro responsiveness of MM6-CA8 cells to various pyrogens is highly relevant for human pyrogenic reactions. Therefore, the in vitro test system is useful and reliable for detecting the presence of materials that are pyrogenic for humans. PMID:11986265

EDIM-TKTL1/Apo10 Blood Test: An Innate Immune System Based Liquid Biopsy for the Early Detection, Characterization and Targeted Treatment of Cancer.

PubMed

Coy, Johannes F

2017-04-20

Epitope detection in monocytes (EDIM) represents a liquid biopsy exploiting the innate immune system. Activated monocytes (macrophages) phagocytose unwanted cells/cell fragments from the whole body including solid tissues. As they return to the blood, macrophages can be used for a non-invasive detection of biomarkers, thereby providing high sensitivity and specificity, because the intracellular presence of biomarkers is due to an innate immune response. Flow cytometry analysis of blood enables the detection of macrophages and phagocytosed intracellular biomarkers. In order to establish a pan-cancer test, biomarkers for two fundamental biophysical mechanisms have been exploited. The DNaseX/Apo10 protein epitope is a characteristic of tumor cells with abnormal apoptosis and proliferation. Transketolase-like 1 (TKTL1) is a marker for an anaerobic glucose metabolism (Warburg effect), which is concomitant with invasive growth/metastasis and resistant to radical and apoptosis inducing therapies. The detection of Apo10 and TKTL1 in blood macrophages allowed a sensitive (95.8%) and specific (97.3%) detection of prostate, breast and oral squamous cell carcinomas. Since TKTL1 represents a drugable target, the EDIM based detection of TKTL1 enables a targeted cancer therapy using the vitamin derivatives oxythiamine or benfo-oxythiamine.

Cancer Tissue Engineering: A Novel 3D Polystyrene Scaffold for In Vitro Isolation and Amplification of Lymphoma Cancer Cells from Heterogeneous Cell Mixtures

PubMed Central

Caicedo-Carvajal, Carlos E.; Liu, Qing; Remache, Yvonne; Goy, Andre; Suh, K. Stephen

2011-01-01

Isolation and amplification of primary lymphoma cells in vitro setting is technically and biologically challenging task. To optimize culture environment and mimic in vivo conditions, lymphoma cell lines were used as a test case and were grown in 3-dimension (3D) using a novel 3D tissue culture polystyrene scaffold with neonatal stromal cells to represent a lymphoma microenvironment. In this model, the cell proliferation was enhanced more than 200-fold or 20,000% neoplastic surplus in 7 days when less than 1% lymphoma cells were cocultured with 100-fold excess of neonatal stroma cells, representing 3.2-fold higher proliferative rate than 2D coculture model. The lymphoma cells grew and aggregated to form clusters during 3D coculture and did not maintained the parental phenotype to grow in single-cell suspension. The cluster size was over 5-fold bigger in the 3D coculture by day 4 than 2D coculture system and contained less than 0.00001% of neonatal fibroblast trace. This preliminary data indicate that novel 3D scaffold geometry and coculturing environment can be customized to amplify primary cancer cells from blood or tissues related to hematological cancer and subsequently used for personalized drug screening procedures. PMID:22073378

Environmental diversity as a surrogate for species representation.

PubMed

Beier, Paul; de Albuquerque, Fábio Suzart

2015-10-01

Because many species have not been described and most species ranges have not been mapped, conservation planners often use surrogates for conservation planning, but evidence for surrogate effectiveness is weak. Surrogates are well-mapped features such as soil types, landforms, occurrences of an easily observed taxon (discrete surrogates), and well-mapped environmental conditions (continuous surrogate). In the context of reserve selection, the idea is that a set of sites selected to span diversity in the surrogate will efficiently represent most species. Environmental diversity (ED) is a rarely used surrogate that selects sites to efficiently span multivariate ordination space. Because it selects across continuous environmental space, ED should perform better than discrete surrogates (which necessarily ignore within-bin and between-bin heterogeneity). Despite this theoretical advantage, ED appears to have performed poorly in previous tests of its ability to identify 50 × 50 km cells that represented vertebrates in Western Europe. Using an improved implementation of ED, we retested ED on Western European birds, mammals, reptiles, amphibians, and combined terrestrial vertebrates. We also tested ED on data sets for plants of Zimbabwe, birds of Spain, and birds of Arizona (United States). Sites selected using ED represented European mammals no better than randomly selected cells, but they represented species in the other 7 data sets with 20% to 84% effectiveness. This far exceeds the performance in previous tests of ED, and exceeds the performance of most discrete surrogates. We believe ED performed poorly in previous tests because those tests considered only a few candidate explanatory variables and used suboptimal forms of ED's selection algorithm. We suggest future work on ED focus on analyses at finer grain sizes more relevant to conservation decisions, explore the effect of selecting the explanatory variables most associated with species turnover, and investigate whether nonclimate abiotic variables can provide useful surrogates in an ED framework. © 2015 Society for Conservation Biology.

Dynamic Finite Element Predictions for Mars Sample Return Cellular Impact Test #4

NASA Technical Reports Server (NTRS)

Fasanella, Edwin L.; Billings, Marcus D.

2001-01-01

The nonlinear, transient dynamic finite element code, MSC.Dytran, was used to simulate an impact test of an energy absorbing Earth Entry Vehicle (EEV) that will impact without a parachute. EEVOs are designed to return materials from asteroids, comets, or planets for laboratory analysis on Earth. The EEV concept uses an energy absorbing cellular structure designed to contain and limit the acceleration of space exploration samples during Earth impact. The spherical shaped cellular structure is composed of solid hexagonal and pentagonal foam-filled cells with hybrid graphite-epoxy/Kevlar cell walls. Space samples fit inside a smaller sphere at the center of the EEVOs cellular structure. Pre-test analytical predictions were compared with the test results from a bungee accelerator. The model used to represent the foam and the proper failure criteria for the cell walls were critical in predicting the impact loads of the cellular structure. It was determined that a FOAM1 model for the foam and a 20% failure strain criteria for the cell walls gave an accurate prediction of the acceleration pulse for cellular impact.

Multiscale Analysis of Open-Cell Aluminum Foam for Impact Energy Absorption

NASA Astrophysics Data System (ADS)

Kim, Ji Hoon; Kim, Daeyong; Lee, Myoung-Gyu; Lee, Jong Kook

2016-09-01

The energy-absorbing characteristics of crash members in automotive collision play an important role in controlling the amount of damage to the passenger compartment. Aluminum foams have high strength-to-weight ratio and high deformability, thus good crashworthiness is expected while maintaining or even saving weights when foams are implemented in crash members. In order to investigate the effect of the open-cell aluminum foam fillers on impact performance and weight saving, a multiscale framework for evaluating the crashworthiness of aluminum foam-filled members is used. To circumvent the difficulties of mechanical tests on foams, a micromechanical model of the aluminum foam is constructed using the x-ray micro tomography and virtual tests are conducted for the micromechanical model to characterize the behavior of the foam. In the macroscale, the aluminum foam is represented by the crushable foam constitutive model, which is then incorporated into the impact test simulation of the foam-filled crash member. The multiscale foam-filled crash member model was validated for the high-speed impact test, which confirms that the material model characterized by the micromechanical approach represents the behavior of the open-cell foam under impact loading well. Finally, the crash member design for maximizing the energy absorption is discussed by investigating various designs from the foam-only structure to the hollow tube structure. It was found that the foam structure absorbs more energy than the hollow tube or foam-filled structure with the same weight.

Hepatitis B transmission by cell and tissue allografts: How safe is safe enough?

PubMed Central

Solves, Pilar; Mirabet, Vicente; Alvarez, Manuel

2014-01-01

More than 2 million human tissue transplants (bone, tendon, cartilage, skin, cornea, amniotic membrane, stem cells, heart valve, blood vessel, etc.), are performed worldwide every year. Cells and tissues are shared between countries which have different regulations and laboratory equipment and represent a risk of hepatitis B virus (HBV) transmission that has become a global safety concern. While the risk of transfusion-transmitted HBV infection from blood donations has been estimated, the rate of HBV transmission from donors to recipients of allografts is unknown and varies between different tissues. There are various important ways of reducing the transmission risk, but donor screening and donor testing are still the main factors for preventing HBV transmission. HBV detection is included in the routine screening tests for cell and tissue donors. The standard test for preventing transplant-transmitted hepatitis B is the hepatitis B surface antigen. The implementation of methods involving nucleic acid amplification and the new generation of reactives to detect viral antibodies or antigens with an immunoassay, has increased the sensitivity and the specificity of the screening tests. The objective of our research was to review the literature and critically analyse the different steps for avoiding HBV transmission in cell and tissue donors, focusing on the screening tests performed. PMID:24966613

Establishment of a cell-based wound healing assay for bio-relevant testing of wound therapeutics.

PubMed

Planz, Viktoria; Wang, Jing; Windbergs, Maike

Predictive in vitro testing of novel wound therapeutics requires adequate cell-based bio-assays. Such assays represent an integral part during preclinical development as pre-step before entering in vivo studies. Simple "scratch tests" based on defected skin cell monolayers exist, however these can solely be used for testing liquids, as cell monolayer destruction and excessive hydration limit their applicability for (semi-)solid systems like wound dressings. In this context, a cell-based wound healing assay is introduced for rapid and predictive testing of wound therapeutics independent of their physical state in a bio-relevant environment. A novel wound healing assay was established for bio-relevant and predictive testing of (semi-) solid wound therapeutics. The assay allows for physiologically relevant hydration of the tested wound therapeutics at the air-liquid interface and their removal without cell monolayer disruption. In a proof-of-concept study, the applicability and discriminative power could be demonstrated by examining unloaded and drug-loaded wound dressings with two different established wound healing actives (dexpanthenol and metyrapone) and their effect on skin cell behavior. The influence of the released drug on the cells´ healing behavior could successfully be monitored over time. Wound size assessment after 96h resulted in an eight fold smaller wound area for drug treated models compared to the ones treated with unloaded fibers and non-treated wounds. This assay provides valuable first insights towards the establishment of a valid screening and evaluation tool for preclinical wound therapeutic development from liquid to (semi-)solid systems to improve predictability in a simple, yet standardized way. Copyright © 2017 Elsevier Inc. All rights reserved.

Male reproductive development: gene expression profiling of maize anther and pollen ontogeny

PubMed Central

Ma, Jiong; Skibbe, David S; Fernandes, John; Walbot, Virginia

2008-01-01

Background During flowering, central anther cells switch from mitosis to meiosis, ultimately forming pollen containing haploid sperm. Four rings of surrounding somatic cells differentiate to support first meiosis and later pollen dispersal. Synchronous development of many anthers per tassel and within each anther facilitates dissection of carefully staged maize anthers for transcriptome profiling. Results Global gene expression profiles of 7 stages representing 29 days of anther development are analyzed using a 44 K oligonucleotide array querying approximately 80% of maize protein-coding genes. Mature haploid pollen containing just two cell types expresses 10,000 transcripts. Anthers contain 5 major cell types and express >24,000 transcript types: each anther stage expresses approximately 10,000 constitutive and approximately 10,000 or more transcripts restricted to one or a few stages. The lowest complexity is present during meiosis. Large suites of stage-specific and co-expressed genes are identified through Gene Ontology and clustering analyses as functional classes for pre-meiotic, meiotic, and post-meiotic anther development. MADS box and zinc finger transcription factors with constitutive and stage-limited expression are identified. Conclusions We propose that the extensive gene expression of anther cells and pollen represents the key test of maize genome fitness, permitting strong selection against deleterious alleles in diploid anthers and haploid pollen. Because flowering plants show a substantial bias for male-sterile compared to female-sterile mutations, we propose that this fitness test is general. Because both somatic and germinal cells are transcriptionally quiescent during meiosis, we hypothesize that successful completion of meiosis is required to trigger maturation of anther somatic cells. PMID:19099579

Preprototype independent air revitalization subsystem

NASA Technical Reports Server (NTRS)

Schubert, F. H.; Hallick, T. M.; Woods, R. R.

1982-01-01

The performance and maturity of a preprototype, three-person capacity, automatically controlled and monitored, self-contained independent air revitalization subsystem were evaluated. The subsystem maintains the cabin partial pressure of oxygen at 22 kPa (3.2 psia) and that of carbon dioxide at 400 Pa (3 mm Hg) over a wide range of cabin air relative humidity conditions. Consumption of water vapor by the water vapor electrolysis module also provides partial humidity control of the cabin environment. During operation, the average carbon dioxide removal efficiency at baseline conditions remained constant throughout the test at 84%. The average electrochemical depolarized concentrator cell voltage at the end of the parametric/endurance test was 0.41 V, representing a very slowly decreasing average cell voltage. The average water vapor electrolysis cell voltage increased only at a rate of 20 mu/h from the initial level of 1.67 V to the final level of 1.69 V at conclusion of the testing.

Evaluating β Diversity as a Surrogate for Species Representation at Fine Scale.

PubMed

Beier, Paul; Albuquerque, Fábio

2016-01-01

Species turnover or β diversity is a conceptually attractive surrogate for conservation planning. However, there has been only 1 attempt to determine how well sites selected to maximize β diversity represent species, and that test was done at a scale too coarse (2,500 km2 sites) to inform most conservation decisions. We used 8 plant datasets, 3 bird datasets, and 1 mammal dataset to evaluate whether sites selected to span β diversity will efficiently represent species at finer scale (sites sizes < 1 ha to 625 km2). We used ordinations to characterize dissimilarity in species assemblages (β diversity) among plots (inventory data) or among grid cells (atlas data). We then selected sites to maximize β diversity and used the Species Accumulation Index, SAI, to evaluate how efficiently the surrogate (selecting sites for maximum β diversity) represented species in the same taxon. Across all 12 datasets, sites selected for maximum β diversity represented species with a median efficiency of 24% (i.e., the surrogate was 24% more effective than random selection of sites), and an interquartile range of 4% to 41% efficiency. β diversity was a better surrogate for bird datasets than for plant datasets, and for atlas datasets with 10-km to 14-km grid cells than for atlas datasets with 25-km grid cells. We conclude that β diversity is more than a mere descriptor of how species are distributed on the landscape; in particular β diversity might be useful to maximize the complementarity of a set of sites. Because we tested only within-taxon surrogacy, our results do not prove that β diversity is useful for conservation planning. But our results do justify further investigation to identify the circumstances in which β diversity performs well, and to evaluate it as a cross-taxon surrogate.

Non-invasive prenatal testing using cell-free fetal DNA in maternal circulation.

PubMed

Liao, Gary J W; Gronowski, Ann M; Zhao, Zhen

2014-01-20

The identification of cell-free fetal DNA (cffDNA) in maternal circulation has made non-invasive prenatal testing (NIPT) possible. Maternal plasma cell free DNA is a mixture of maternal and fetal DNA, of which, fetal DNA represents a minor population in maternal plasma. Therefore, methods with high sensitivity and precision are required to detect and differentiate fetal DNA from the large background of maternal DNA. In recent years, technical advances in the molecular analysis of fetal DNA (e.g., digital PCR and massively parallel sequencing (MPS)) has enabled the successful implementation of noninvasive testing into clinical practice, such as fetal sex assessment, RhD genotyping, and fetal chromosomal aneuploidy detection.With the ability to decipher the entire fetal genome from maternal plasma DNA, we foresee that an increased number of non-invasive prenatal tests will be available for detecting many single-gene disorders in the near future. This review briefly summarizes the technical aspects of the NIPT and application of NIPT in clinical practice.

TEST OF A THEORETICAL COMMUTER EXPOSURE MODEL TO VEHICLE EXHAUST IN TRAFFIC

EPA Science Inventory

A theoretical model of commuter exposure is presented as a box or cell model with the automobile passenger compartment representing the microenvironment exposed to CO concentrations resulting from vehicle exhaust leaks and emissions from traffic. Equations which describe this sit...

Material Modeling of Stony Meteorites for Mechanical Properties

NASA Astrophysics Data System (ADS)

Agrawal, P.

2016-12-01

To assess the threat posed by an asteroid entering Earth's atmosphere, one must predict if, when, and how it fragments during entry. A comprehensive understanding of the asteroid material properties is needed to achieve this objective. At present, the meteorite material found on earth are the only objects (other than synthetic meteorites) from an entering asteroid that can be used as representative material and be tested inside a laboratory setting. Due to limited number of meteorites available for testing it is difficult to develop a material model that can be purely based on statistics from the test data. Therefore, we are developing computational models to determine the effective material properties of stony meteorites and in turn deduce the properties of asteroids. The internal structure of meteorites are very complex. They consists of several minerals that include the silica based materials such as Olivine, Pyroxene, Feldspar that are found in terrestrial rocks, as well as Fe-Ni based minerals such as Kamacite, Troilite and Taenite that are unique to meteorites. Each of these minerals have different densities and mechanical properties. In addition, the meteorites have different phases that can be summarized as chondrules, metal and matrix. The meteorites have varying degree of porosity and pre-cracked structure. In order to account for diverse petrology of the meteorites a unique methodology is developed the form of unit cell model. The unit cell is representative volume that accounts for diverse minerals, porosity, and matrix composition inside a meteorite. All the minerals and phases inside these unit cells are randomly distributed. Several hundreds of Monte-Carlo simulations are performed to generate the effective mechanical properties such as Young's Modulus and Poisson's Ratio of the unit cell. Stress-strain curves as well as strength estimates are generated based on the unit cell models. These estimates will used as material models for full scale modeling of atmospheric entry for asteroids. Terrestrial analogs such as Basalt and Gabbro are being used to validate the unit cell methodology. Structural tests are also being performed on some of the meteorites including Tamdakht and Mbole to validate the predictions from unit cell models.

Lower white blood cell counts in elite athletes training for highly aerobic sports.

PubMed

Horn, P L; Pyne, D B; Hopkins, W G; Barnes, C J

2010-11-01

White cell counts at rest might be lower in athletes participating in selected endurance-type sports. Here, we analysed blood tests of elite athletes collected over a 10-year period. Reference ranges were established for 14 female and 14 male sports involving 3,679 samples from 937 females and 4,654 samples from 1,310 males. Total white blood cell counts and counts of neutrophils, lymphocytes and monocytes were quantified. Each sport was scaled (1-5) for its perceived metabolic stress (aerobic-anaerobic) and mechanical stress (concentric-eccentric) by 13 sports physiologists. Substantially lower total white cell and neutrophil counts were observed in aerobic sports of cycling and triathlon (~16% of test results below the normal reference range) compared with team or skill-based sports such as water polo, cricket and volleyball. Mechanical stress of sports had less effect on the distribution of cell counts. The lower white cell counts in athletes in aerobic sports probably represent an adaptive response, not underlying pathology.

Investigation of Effective Material Properties of Stony Meteorites

NASA Technical Reports Server (NTRS)

Agrawal, Parul; Carlozzi, Alex; Bryson, Kathryn

2016-01-01

To assess the threat posed by an asteroid entering Earth's atmosphere, one must predict if, when, and how it fragments during entry. A comprehensive understanding of the Asteroid material properties is needed to achieve this objective. At present, the meteorite material found on Earth are the only objects from an entering asteroid that can be used as representative material and be tested inside a laboratory setting. Therefore, unit cell models are developed to determine the effective material properties of stony meteorites and in turn deduce the properties of asteroids. The unit cell is representative volume that accounts for diverse minerals, porosity, and matrix composition inside a meteorite. The various classes under investigation includes H-class, L-class, and LL-class chondrites. The effective mechanical properties such as Young's Modulus and Poisson's Ratio of the unit cell are calculated by performing several hundreds of Monte-Carlo simulations. Terrestrial analogs such as Basalt and Gabbro are being used to validate the unit cell methodology.

An Energy Model of Place Cell Network in Three Dimensional Space.

PubMed

Wang, Yihong; Xu, Xuying; Wang, Rubin

2018-01-01

Place cells are important elements in the spatial representation system of the brain. A considerable amount of experimental data and classical models are achieved in this area. However, an important question has not been addressed, which is how the three dimensional space is represented by the place cells. This question is preliminarily surveyed by energy coding method in this research. Energy coding method argues that neural information can be expressed by neural energy and it is convenient to model and compute for neural systems due to the global and linearly addable properties of neural energy. Nevertheless, the models of functional neural networks based on energy coding method have not been established. In this work, we construct a place cell network model to represent three dimensional space on an energy level. Then we define the place field and place field center and test the locating performance in three dimensional space. The results imply that the model successfully simulates the basic properties of place cells. The individual place cell obtains unique spatial selectivity. The place fields in three dimensional space vary in size and energy consumption. Furthermore, the locating error is limited to a certain level and the simulated place field agrees to the experimental results. In conclusion, this is an effective model to represent three dimensional space by energy method. The research verifies the energy efficiency principle of the brain during the neural coding for three dimensional spatial information. It is the first step to complete the three dimensional spatial representing system of the brain, and helps us further understand how the energy efficiency principle directs the locating, navigating, and path planning function of the brain.

Quantitative Biofractal Feedback Part II ’Devices, Scalability & Robust Control’

DTIC Science & Technology

2008-05-01

in the modelling of proton exchange membrane fuel cells ( PEMFC ) may work as a powerful tool in the development and widespread testing of alternative...energy sources in the next decade [9], where biofractal controllers will be used to control these complex systems. The dynamic model of PEMFC , is...dynamic response of the PEMFC . In the Iftukhar model, the fuel cell is represented by an equivalent circuit, whose components are identified with

Remedial Investigation Badger Army Ammunition Plant, Baraboo, Wisconsin. Volume 7. Appendices M Through R

DTIC Science & Technology

1991-01-01

cell transformation test with rat embryo cells (Price et al., 1978). An isomer, 1,1,2-trichloroethane, is carcinogenic in mice, inducing liver cancer and...pheochromocytomas in both sexes. Dichloroethanes, tetrachloroethanes and hexachloroethanes also produced liver cancer in mice and other types of...at this time, so the recommended criteria represents an E-6 estimated incrnental increase of cancer risk over a lifetime. Reference - 45 FR 79318 (11

Engineering kidney cells: reprogramming and directed differentiation to renal tissues.

PubMed

Kaminski, Michael M; Tosic, Jelena; Pichler, Roman; Arnold, Sebastian J; Lienkamp, Soeren S

2017-07-01

Growing knowledge of how cell identity is determined at the molecular level has enabled the generation of diverse tissue types, including renal cells from pluripotent or somatic cells. Recently, several in vitro protocols involving either directed differentiation or transcription-factor-based reprogramming to kidney cells have been established. Embryonic stem cells or induced pluripotent stem cells can be guided towards a kidney fate by exposing them to combinations of growth factors or small molecules. Here, renal development is recapitulated in vitro resulting in kidney cells or organoids that show striking similarities to mammalian embryonic nephrons. In addition, culture conditions are also defined that allow the expansion of renal progenitor cells in vitro. Another route towards the generation of kidney cells is direct reprogramming. Key transcription factors are used to directly impose renal cell identity on somatic cells, thus circumventing the pluripotent stage. This complementary approach to stem-cell-based differentiation has been demonstrated to generate renal tubule cells and nephron progenitors. In-vitro-generated renal cells offer new opportunities for modelling inherited and acquired renal diseases on a patient-specific genetic background. These cells represent a potential source for developing novel models for kidney diseases, drug screening and nephrotoxicity testing and might represent the first steps towards kidney cell replacement therapies. In this review, we summarize current approaches for the generation of renal cells in vitro and discuss the advantages of each approach and their potential applications.

Noninvasive prenatal testing using cell-free fetal DNA in maternal plasma.

PubMed

Dharajiya, Nilesh; Zwiefelhofer, Tricia; Guan, Xiaojun; Angkachatchai, Vach; Saldivar, Juan-Sebastian

2015-01-20

Noninvasive prenatal testing (NIPT) represents an outstanding example of how novel scientific discoveries can be quickly and successfully developed into hugely impactful clinical diagnostic tests. Since the introduction of NIPT to detect trisomy 21 in late 2011, the technology has rapidly advanced to analyze other autosomal and sex chromosome aneuploidies, and now includes the detection of subchromosomal deletion and duplication events. Here we provide a brief overview of how noninvasive prenatal testing using next-generation sequencing is performed. Copyright © 2015 John Wiley & Sons, Inc.

The 1988 Goddard Space Flight Center Battery Workshop

NASA Technical Reports Server (NTRS)

Yi, Thomas Y. (Editor)

1993-01-01

This document contains the proceedings of the 21st annual Battery Workshop held at Goddard Space Flight Center, Greenbelt, Maryland on November 1-3, 1988. The Workshop attendees included manufacturers, users, and government representatives interested in the latest developments in battery technology as they relate to high reliability operations and aerospace use. The subjects covered included battery testing methodologies and criteria, life testing of nickel-cadmium cells, testing and operation of nickel-hydrogen batteries in low earth orbit, and nickel-hydrogen technology issues and concerns.

Generation of insulin-producing human mesenchymal stem cells using recombinant adeno-associated virus.

PubMed

Kim, Jeong Hwan; Park, Si-Nae; Suh, Hwal

2007-02-28

The purpose of current experiment is the generation of insulin-producing human mesenchymal stem cells as therapeutic source for the cure of type 1 diabetes. Type 1 diabetes is generally caused by insulin deficiency accompanied by the destruction of islet beta-cells. In various trials for the treatment of type 1 diabetes, cell-based gene therapy using stem cells is considered as one of the most useful candidate for the treatment. In this experiment, human mesenchymal stem cells were transduced with AAV which is containing furin-cleavable human preproinsulin gene to generate insulin-producing cells as surrogate beta-cells for the type 1 diabetes therapy. In the rAAV production procedure, rAAV was generated by transfection of AD293 cells. Human mesenchymal stems cells were transduced using rAAV with a various multiplicity of infection. Transduction of recombinant AAV was also tested using beta-galactosidse expression. Cell viability was determined by using MTT assay to evaluate the toxicity of the transduction procedure. Expression and production of Insulin were tested using reverse transcriptase-polymerase chain reaction and immunocytochemistry. Secretion of human insulin and C-peptide from the cells was assayed using enzyme-linked immunosorbent assay. Production of insulin and C-peptide from the test group represented a higher increase compared to the control group. In this study, we examined generation of insulin-producing cells from mesenchymal stem cells by genetic engineering for diabetes therapy. This work might be valuable to the field of tissue engineering for diabetes treatment.

In vivo biocompatibility of boron nitride nanotubes: effects on stem cell biology and tissue regeneration in planarians.

PubMed

Salvetti, Alessandra; Rossi, Leonardo; Iacopetti, Paola; Li, Xia; Nitti, Simone; Pellegrino, Teresa; Mattoli, Virgilio; Golberg, Dmitri; Ciofani, Gianni

2015-07-01

Boron nitride nanotubes (BNNTs) represent an extremely interesting class of nanomaterials, and recent findings have suggested a number of applications in the biomedical field. Anyhow, extensive biocompatibility investigations are mandatory before any further advancement toward preclinical testing. Here, we report on the effects of multiwalled BNNTs in freshwater planarians, one of the best-characterized in vivo models for developmental biology and regeneration research. Obtained results indicate that BNNTs are biocompatible in the investigated model, since they do not induce oxidative DNA damage and apoptosis, and do not show adverse effects on planarian stem cell biology and on de novo tissue regeneration. In summary, collected findings represent another important step toward BNNT realistic applications in nanomedicine.

Solving a four-destination traveling salesman problem using Escherichia coli cells as biocomputers.

PubMed

Esau, Michael; Rozema, Mark; Zhang, Tuo Huang; Zeng, Dawson; Chiu, Stephanie; Kwan, Rachel; Moorhouse, Cadence; Murray, Cameron; Tseng, Nien-Tsu; Ridgway, Doug; Sauvageau, Dominic; Ellison, Michael

2014-12-19

The Traveling Salesman Problem involves finding the shortest possible route visiting all destinations on a map only once before returning to the point of origin. The present study demonstrates a strategy for solving Traveling Salesman Problems using modified E. coli cells as processors for massively parallel computing. Sequential, combinatorial DNA assembly was used to generate routes, in the form of plasmids made up of marker genes, each representing a path between destinations, and short connecting linkers, each representing a given destination. Upon growth of the population of modified E. coli, phenotypic selection was used to eliminate invalid routes, and statistical analysis was performed to successfully identify the optimal solution. The strategy was successfully employed to solve a four-destination test problem.

Volume-weighted particle-tracking method for solute-transport modeling; Implementation in MODFLOW–GWT

USGS Publications Warehouse

Winston, Richard B.; Konikow, Leonard F.; Hornberger, George Z.

2018-02-16

In the traditional method of characteristics for groundwater solute-transport models, advective transport is represented by moving particles that track concentration. This approach can lead to global mass-balance problems because in models of aquifers having complex boundary conditions and heterogeneous properties, particles can originate in cells having different pore volumes and (or) be introduced (or removed) at cells representing fluid sources (or sinks) of varying strengths. Use of volume-weighted particles means that each particle tracks solute mass. In source or sink cells, the changes in particle weights will match the volume of water added or removed through external fluxes. This enables the new method to conserve mass in source or sink cells as well as globally. This approach also leads to potential efficiencies by allowing the number of particles per cell to vary spatially—using more particles where concentration gradients are high and fewer where gradients are low. The approach also eliminates the need for the model user to have to distinguish between “weak” and “strong” fluid source (or sink) cells. The new model determines whether solute mass added by fluid sources in a cell should be represented by (1) new particles having weights representing appropriate fractions of the volume of water added by the source, or (2) distributing the solute mass added over all particles already in the source cell. The first option is more appropriate for the condition of a strong source; the latter option is more appropriate for a weak source. At sinks, decisions whether or not to remove a particle are replaced by a reduction in particle weight in proportion to the volume of water removed. A number of test cases demonstrate that the new method works well and conserves mass. The method is incorporated into a new version of the U.S. Geological Survey’s MODFLOW–GWT solute-transport model.

A Comparative Study of Multi-material Data Structures for Computational Physics Applications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garimella, Rao Veerabhadra; Robey, Robert W.

The data structures used to represent the multi-material state of a computational physics application can have a drastic impact on the performance of the application. We look at efficient data structures for sparse applications where there may be many materials, but only one or few in most computational cells. We develop simple performance models for use in selecting possible data structures and programming patterns. We verify the analytic models of performance through a small test program of the representative cases.

Speeding up pyrogenicity testing: Identification of suitable cell components and readout parameters for an accelerated monocyte activation test (MAT).

PubMed

Stoppelkamp, Sandra; Würschum, Noriana; Stang, Katharina; Löder, Jasmin; Avci-Adali, Meltem; Toliashvili, Leila; Schlensak, Christian; Wendel, Hans Peter; Fennrich, Stefan

2017-02-01

Pyrogen testing represents a crucial safety measure for parental drugs and medical devices, especially in direct contact with blood or liquor. The European Pharmacopoeia regulates these quality control measures for parenterals. Since 2010, the monocyte activation test (MAT) has been an accepted pyrogen test that can be performed with different human monocytic cell sources: whole blood, isolated monocytic cells or monocytic cell lines with IL1β, IL6, or TNFα as readout cytokines. In the present study, we examined the three different cell sources and cytokine readout parameters with the scope of accelerating the assay time. We could show that despite all cell types being able to detect pyrogens, primary cells were more sensitive than the monocytic cell line. Quantitative real-time PCR revealed IL6 mRNA transcripts having the largest change in Ct-values upon LPS-stimulation compared to IL1β and TNFα, but quantification was unreliable. IL6 protein secretion from whole blood or peripheral blood mononuclear cells (PBMC) was also best suited for an accelerated assay with a larger linear range and higher signal-to-noise ratios upon LPS-stimulation. The unique combination with propan-2-ol or a temperature increase could additionally increase the cytokine production for earlier detection in PBMC. The increased incubation temperature could finally increase not only responses to lipopolysaccharides (LPS) but also other pyrogens by up to 13-fold. Therefore, pyrogen detection can be accelerated considerably by using isolated primary blood cells with an increased incubation temperature and IL6 as readout. These results could expedite assay time and thus help to promote further acceptance of the MAT. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

7 CFR 58.140 - Field service.

Code of Federal Regulations, 2012 CFR

2012-01-01

... each producer whose milk tests positive for drug residue, exceeds the maximum somatic cell count level... milk and eliminate any potential causes of drug residues. A representative of the plant should routinely visit each producer as often as necessary to assist and encourage the production of high quality...

7 CFR 58.140 - Field service.

Code of Federal Regulations, 2011 CFR

2011-01-01

... each producer whose milk tests positive for drug residue, exceeds the maximum somatic cell count level... milk and eliminate any potential causes of drug residues. A representative of the plant should routinely visit each producer as often as necessary to assist and encourage the production of high quality...

7 CFR 58.140 - Field service.

Code of Federal Regulations, 2013 CFR

2013-01-01

... each producer whose milk tests positive for drug residue, exceeds the maximum somatic cell count level... milk and eliminate any potential causes of drug residues. A representative of the plant should routinely visit each producer as often as necessary to assist and encourage the production of high quality...

7 CFR 58.140 - Field service.

Code of Federal Regulations, 2010 CFR

2010-01-01

... each producer whose milk tests positive for drug residue, exceeds the maximum somatic cell count level... milk and eliminate any potential causes of drug residues. A representative of the plant should routinely visit each producer as often as necessary to assist and encourage the production of high quality...

7 CFR 58.140 - Field service.

Code of Federal Regulations, 2014 CFR

2014-01-01

... each producer whose milk tests positive for drug residue, exceeds the maximum somatic cell count level... milk and eliminate any potential causes of drug residues. A representative of the plant should routinely visit each producer as often as necessary to assist and encourage the production of high quality...

Cell and molecular biology of the spiny dogfish Squalus acanthias and little skate Leucoraja erinacea: insights from in vitro cultured cells.

PubMed

Barnes, D W

2012-04-01

Two of the most commonly used elasmobranch experimental model species are the spiny dogfish Squalus acanthias and the little skate Leucoraja erinacea. Comparative biology and genomics with these species have provided useful information in physiology, pharmacology, toxicology, immunology, evolutionary developmental biology and genetics. A wealth of information has been obtained using in vitro approaches to study isolated cells and tissues from these organisms under circumstances in which the extracellular environment can be controlled. In addition to classical work with primary cell cultures, continuously proliferating cell lines have been derived recently, representing the first cell lines from cartilaginous fishes. These lines have proved to be valuable tools with which to explore functional genomic and biological questions and to test hypotheses at the molecular level. In genomic experiments, complementary (c)DNA libraries have been constructed, and c. 8000 unique transcripts identified, with over 3000 representing previously unknown gene sequences. A sub-set of messenger (m)RNAs has been detected for which the 3' untranslated regions show elements that are remarkably well conserved evolutionarily, representing novel, potentially regulatory gene sequences. The cell culture systems provide physiologically valid tools to study functional roles of these sequences and other aspects of elasmobranch molecular cell biology and physiology. Information derived from the use of in vitro cell cultures is valuable in revealing gene diversity and information for genomic sequence assembly, as well as for identification of new genes and molecular markers, construction of gene-array probes and acquisition of full-length cDNA sequences. © 2012 The Author. Journal of Fish Biology © 2012 The Fisheries Society of the British Isles.

Alkaline regenerative fuel cell energy storage system for manned orbital satellites

NASA Technical Reports Server (NTRS)

Martin, R. E.; Gitlow, B.; Sheibley, D. W.

1982-01-01

It is pointed out that the alkaline regenerative fuel cell system represents a highly efficient, lightweight, reliable approach for providing energy storage in an orbiting satellite. In addition to its energy storage function, the system can supply hydrogen and oxygen for attitude control of the satellite and for life support. A summary is presented of the results to date obtained in connection with the NASA-sponsored fuel cell technology advancement program, giving particular attention to the requirements of the alkaline regenerative fuel cell and the low-earth mission. Attention is given to system design guidelines, weight considerations, gold-platinum cathode cell performance, matrix development, the electrolyte reservoir plate, and the cyclical load profile tests.

Testicular histology and germ cell cytology during spermatogenesis in the Mississippi map turtle, Graptemys pseudogeographica kohnii, from Northeast Arkansas.

PubMed

Lancaster, Kelsey; Trauth, Stanley E; Gribbins, Kevin M

2014-01-01

The testicular histology and cytology of spermatogenesis in Graptemys pseudogeographica kohnii were examined using specimens collected between July 1996 and May 2004 from counties in northeastern Arkansas. A histological examination of the testes and germ cell cytology indicates a postnuptial testicular cycle of spermatogenesis and a major fall spermiation event. The majority of the germ cell populations in May and June specimens are represented by resting spermatogonia, type A spermatogonia, type B spermatogonia, pre-leptotene spermatocytes, and numerous Sertoli cell nuclei near the basement membrane. The start of proliferation is evident as spermatogonia in metaphase are present near the basal lamina and many of these germ cells have entered meiosis in June seminiferous tubules. Major spermatogenic events occur in the June and July specimens and result in an increased height of the seminiferous epithelium and increased diameter of the seminiferous tubules. The germ cell population during this time is represented by spermatogonia (type A, B, and resting), hypertrophic cells, large populations of early primary spermatocytes, and early round spermatids. By September, the major germ cell population has progressed past meiosis with abundant round and early elongating spermatids dominating the seminiferous epithelium. October seminiferous epithelia are marked by a decreas in height and mature spermatozoa fill the luminal space. Round and elongating spermatids constitute the largest portion of the germ cell population. Following the spermiation event, the testes enter a period of quiescence that lasts till the next spermatogenic cycle, which begins in the subsequent spring. Based on the cytological development of the seminiferous tubules revealed by our study, Graptemys pseudogeographica kohnii demonstrates a temporal germ cell development strategy similar to other temperate reptiles. A single major generation of germ cells progresses through spermatogenesis each year resulting in a single spermiation event with sperm stored within the epididymis until the next spring mating season.

Testicular histology and germ cell cytology during spermatogenesis in the Mississippi map turtle, Graptemys pseudogeographica kohnii, from Northeast Arkansas

PubMed Central

Lancaster, Kelsey; Trauth, Stanley E; Gribbins, Kevin M

2014-01-01

The testicular histology and cytology of spermatogenesis in Graptemys pseudogeographica kohnii were examined using specimens collected between July 1996 and May 2004 from counties in northeastern Arkansas. A histological examination of the testes and germ cell cytology indicates a postnuptial testicular cycle of spermatogenesis and a major fall spermiation event. The majority of the germ cell populations in May and June specimens are represented by resting spermatogonia, type A spermatogonia, type B spermatogonia, pre-leptotene spermatocytes, and numerous Sertoli cell nuclei near the basement membrane. The start of proliferation is evident as spermatogonia in metaphase are present near the basal lamina and many of these germ cells have entered meiosis in June seminiferous tubules. Major spermatogenic events occur in the June and July specimens and result in an increased height of the seminiferous epithelium and increased diameter of the seminiferous tubules. The germ cell population during this time is represented by spermatogonia (type A, B, and resting), hypertrophic cells, large populations of early primary spermatocytes, and early round spermatids. By September, the major germ cell population has progressed past meiosis with abundant round and early elongating spermatids dominating the seminiferous epithelium. October seminiferous epithelia are marked by a decreas in height and mature spermatozoa fill the luminal space. Round and elongating spermatids constitute the largest portion of the germ cell population. Following the spermiation event, the testes enter a period of quiescence that lasts till the next spermatogenic cycle, which begins in the subsequent spring. Based on the cytological development of the seminiferous tubules revealed by our study, Graptemys pseudogeographica kohnii demonstrates a temporal germ cell development strategy similar to other temperate reptiles. A single major generation of germ cells progresses through spermatogenesis each year resulting in a single spermiation event with sperm stored within the epididymis until the next spring mating season. PMID:26413408

GMI-1070, a novel pan-selectin antagonist, reverses acute vascular occlusions in sickle cell mice

PubMed Central

Chang, Jungshan; Patton, John T.; Sarkar, Arun; Ernst, Beat

2010-01-01

Leukocyte adhesion in the microvasculature influences blood rheology and plays a key role in vaso-occlusive manifestations of sickle cell disease. Notably, polymorphonuclear neutrophils (PMNs) can capture circulating sickle red blood cells (sRBCs) in inflamed venules, leading to critical reduction in blood flow and vaso-occlusion. Recent studies have suggested that E-selectin expression by endothelial cells plays a key role by sending activating signals that lead to the activation of Mac-1 at the leading edge of PMNs, thereby allowing RBC capture. Thus, the inhibition of E-selectin may represent a valuable target in this disease. Here, we have tested the biologic properties of a novel synthetic pan-selectin inhibitor, GMI-1070, with in vitro assays and in a humanized model of sickle cell vaso-occlusion analyzed by intravital microscopy. We have found that GMI-1070 predominantly inhibited E-selectin–mediated adhesion and dramatically inhibited sRBC-leukocyte interactions, leading to improved microcirculatory blood flow and improved survival. These results suggest that GMI-1070 may represent a valuable novel therapeutic intervention for acute sickle cell crises that should be further evaluated in a clinical trial. PMID:20508165

Comparative Cytotoxicity Study of Silver Nanoparticles (AgNPs) in a Variety of Rainbow Trout Cell Lines (RTL-W1, RTH-149, RTG-2) and Primary Hepatocytes

PubMed Central

Connolly, Mona; Fernandez-Cruz, Maria-Luisa; Quesada-Garcia, Alba; Alte, Luis; Segner, Helmut; Navas, Jose M.

2015-01-01

Among all classes of nanomaterials, silver nanoparticles (AgNPs) have potentially an important ecotoxicological impact, especially in freshwater environments. Fish are particularly susceptible to the toxic effects of silver ions and, with knowledge gaps regarding the contribution of dissolution and unique particle effects to AgNP toxicity, they represent a group of vulnerable organisms. Using cell lines (RTL-W1, RTH-149, RTG-2) and primary hepatocytes of rainbow trout (Oncorhynchus mykiss) as in vitro test systems, we assessed the cytotoxicity of the representative AgNP, NM-300K, and AgNO3 as an Ag+ ion source. Lack of AgNP interference with the cytotoxicity assays (AlamarBlue, CFDA-AM, NRU assay) and their simultaneous application point to the compatibility and usefulness of such a battery of assays. The RTH-149 and RTL-W1 liver cell lines exhibited similar sensitivity as primary hepatocytes towards AgNP toxicity. Leibovitz’s L-15 culture medium composition (high amino acid content) had an important influence on the behaviour and toxicity of AgNPs towards the RTL-W1 cell line. The obtained results demonstrate that, with careful consideration, such an in vitro approach can provide valuable toxicological data to be used in an integrated testing strategy for NM-300K risk assessment. PMID:26006119

Evaluation and refinement of a field-portable drinking water toxicity sensor utilizing electric cell-substrate impedance sensing and a fluidic biochip.

PubMed

Widder, Mark W; Brennan, Linda M; Hanft, Elizabeth A; Schrock, Mary E; James, Ryan R; van der Schalie, William H

2015-07-01

The US Army's need for a reliable and field-portable drinking water toxicity sensor was the catalyst for the development and evaluation of an electric cell-substrate impedance sensing (ECIS) device. Water testing technologies currently available to soldiers in the field are analyte-specific and have limited capabilities to detect broad-based water toxicity. The ECIS sensor described here uses rainbow trout gill epithelial cells seeded on fluidic biochips to measure changes in impedance for the detection of possible chemical contamination of drinking water supplies. Chemicals selected for testing were chosen as representatives of a broad spectrum of toxic industrial compounds. Results of a US Environmental Protection Agency (USEPA)-sponsored evaluation of the field portable device were similar to previously published US Army testing results of a laboratory-based version of the same technology. Twelve of the 18 chemicals tested following USEPA Technology Testing and Evaluation Program procedures were detected by the ECIS sensor within 1 h at USEPA-derived human lethal concentrations. To simplify field-testing methods further, elimination of a procedural step that acclimated cells to serum-free media streamlined the test process with only a slight loss of chemical sensitivity. For field use, the ECIS sensor will be used in conjunction with an enzyme-based sensor that is responsive to carbamate and organophosphorus pesticides. Copyright © 2014 John Wiley & Sons, Ltd.

100 °C Thermal Stability of Printable Perovskite Solar Cells Using Porous Carbon Counter Electrodes.

PubMed

Baranwal, Ajay K; Kanaya, Shusaku; Peiris, T A Nirmal; Mizuta, Gai; Nishina, Tomoya; Kanda, Hiroyuki; Miyasaka, Tsutomu; Segawa, Hiroshi; Ito, Seigo

2016-09-22

Many efforts have been made towards improving perovskite (PVK) solar cell stability, but their thermal stability, particularly at 85 °C (IEC 61646 climate chamber tests), remains a challenge. Outdoors, the installed solar cell temperature can reach up to 85 °C, especially in desert regions, providing sufficient motivation to study the effect of temperature stress at or above this temperature (e.g., 100 °C) to confirm the commercial viability of PVK solar cells for industrial companies. In this work, a three-layer printable HTM-free CH 3 NH 3 PbI 3 PVK solar cell with a mesoporous carbon back contact and UV-curable sealant was fabricated and tested for thermal stability over 1500 h at 100 °C. Interestingly, the position of the UV-curing glue was found to drastically affect the device stability. The side-sealed cells show high PCE stability and represent a large step toward commercialization of next generation organic-inorganic lead halide PVK solar cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The use of FDTD in establishing in vitro experimentation conditions representative of lifelike cell phone radiation on the spermatozoa.

PubMed

Mouradi, Rand; Desai, Nisarg; Erdemir, Ahmet; Agarwal, Ashok

2012-01-01

Recent studies have shown that exposing human semen samples to cell phone radiation leads to a significant decline in sperm parameters. In daily living, a cell phone is usually kept in proximity to the groin, such as in a trouser pocket, separated from the testes by multiple layers of tissue. The aim of this study was to calculate the distance between cell phone and semen sample to set up an in vitro experiment that can mimic real life conditions (cell phone in trouser pocket separated by multiple tissue layers). For this reason, a computational model of scrotal tissues was designed by considering these separating layers, the results of which were used in a series of simulations using the Finite Difference Time Domain (FDTD) method. To provide an equivalent effect of multiple tissue layers, these results showed that the distance between a cell phone and semen sample should be 0.8 cm to 1.8 cm greater than the anticipated distance between a cell phone and the testes.

FEASIBILITY TESTING OF THE EDIX CELL CONCEPT FOR METAL RECOVERY FROM ACID MINE DRAINAGE. (R825549C025)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

78 FR 16514 - Secretary's Advisory Committee on Heritable Disorders in Newborns and Children; Notice of Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2013-03-15

... screening activities, technologies, policies, guidelines, and programs for effectively reducing morbidity... recommendations related to sickle cell trait testing; (2) a presentation on the Affordable Care Act and the impact... comments and present them through a single representative. No audiovisual presentations are permitted...

Plasma of argon enhances the adhesion of murine osteoblasts on different graft materials.

PubMed

Canullo, Luigi; Genova, Tullio; Naenni, Nadja; Nakajima, Yasushi; Masuda, Katsuhiko; Mussano, Federico

2018-04-25

plasma of argon treatment was demonstrated to increase material surface energy leading to stronger and faster interaction with cells. The aim of the present in vitro study was to test the effect of plasma treatment on different graft materials. synthetic hydroxyapatite (Mg-HA), biphasic calcium phosphate (BCP), cancellous and cortical xenogeneic bone matrices (CaBM, CoBM) were used representing commonly used classes of bone substitute materials. Fifty serially numbered disks with a 10mm-diameter from each graft material were randomly divided into two groups: Test group (argon plasma treatment) and Control group (absence of treatment). Cell morphology (using pre-osteoblastic murine cells) and protein adsorption were analyzed at all samples from both the test and control group. Differences between groups were analyzed using the Mann-Whitney test setting the level of significance at p<0.05. plasma treatment significantly increased the protein adsorption at all samples. Similarly, plasma treatment significantly increased cell adhesion in all groups. data confirmed that non-atmospheric plasma of argon treatment led to an increase of protein adsorption and cell adhesion in all groups of graft material to a similar extent. plasma of argon is able to improve the surface conditions of graft materials. Copyright © 2018 Elsevier GmbH. All rights reserved.

Nano-immunosafety: issues in assay validation

NASA Astrophysics Data System (ADS)

Boraschi, Diana; Oostingh, Gertie J.; Casals, Eudald; Italiani, Paola; Nelissen, Inge; Puntes, Victor F.; Duschl, Albert

2011-07-01

Assessing the safety of engineered nanomaterials for human health must include a thorough evaluation of their effects on the immune system, which is responsible for defending the integrity of our body from damage and disease. An array of robust and representative assays should be set up and validated, which could be predictive of the effects of nanomaterials on immune responses. In a trans-European collaborative work, in vitro assays have been developed to this end. In vitro tests have been preferred for their suitability to standardisation and easier applicability. Adapting classical assays to testing the immunotoxicological effects of nanoparticulate materials has raised a series of issues that needed to be appropriately addressed in order to ensure reliability of results. Besides the exquisitely immunological problem of selecting representative endpoints predictive of the risk of developing disease, assay results turned out to be significantly biased by artefactual interference of the nanomaterials or contaminating agents with the assay protocol. Having addressed such problems, a series of robust and representative assays have been developed that describe the effects of engineered nanoparticles on professional and non-professional human defence cells. Two of such assays are described here, one based on primary human monocytes and the other employing human lung epithelial cells transfected with a reporter gene.

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

PubMed

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

2016-01-01

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

Revealing structure and assembly cues for Arabidopsis root-inhabiting bacterial microbiota.

PubMed

Bulgarelli, Davide; Rott, Matthias; Schlaeppi, Klaus; Ver Loren van Themaat, Emiel; Ahmadinejad, Nahal; Assenza, Federica; Rauf, Philipp; Huettel, Bruno; Reinhardt, Richard; Schmelzer, Elmon; Peplies, Joerg; Gloeckner, Frank Oliver; Amann, Rudolf; Eickhorst, Thilo; Schulze-Lefert, Paul

2012-08-02

The plant root defines the interface between a multicellular eukaryote and soil, one of the richest microbial ecosystems on Earth. Notably, soil bacteria are able to multiply inside roots as benign endophytes and modulate plant growth and development, with implications ranging from enhanced crop productivity to phytoremediation. Endophytic colonization represents an apparent paradox of plant innate immunity because plant cells can detect an array of microbe-associated molecular patterns (also known as MAMPs) to initiate immune responses to terminate microbial multiplication. Several studies attempted to describe the structure of bacterial root endophytes; however, different sampling protocols and low-resolution profiling methods make it difficult to infer general principles. Here we describe methodology to characterize and compare soil- and root-inhabiting bacterial communities, which reveals not only a function for metabolically active plant cells but also for inert cell-wall features in the selection of soil bacteria for host colonization. We show that the roots of Arabidopsis thaliana, grown in different natural soils under controlled environmental conditions, are preferentially colonized by Proteobacteria, Bacteroidetes and Actinobacteria, and each bacterial phylum is represented by a dominating class or family. Soil type defines the composition of root-inhabiting bacterial communities and host genotype determines their ribotype profiles to a limited extent. The identification of soil-type-specific members within the root-inhabiting assemblies supports our conclusion that these represent soil-derived root endophytes. Surprisingly, plant cell-wall features of other tested plant species seem to provide a sufficient cue for the assembly of approximately 40% of the Arabidopsis bacterial root-inhabiting microbiota, with a bias for Betaproteobacteria. Thus, this root sub-community may not be Arabidopsis-specific but saprophytic bacteria that would naturally be found on any plant root or plant debris in the tested soils. By contrast, colonization of Arabidopsis roots by members of the Actinobacteria depends on other cues from metabolically active host cells.

Mast cells and basophils are essential for allergies: mechanisms of allergic inflammation and a proposed procedure for diagnosis

PubMed Central

He, Shao-heng; Zhang, Hui-yun; Zeng, Xiao-ning; Chen, Dong; Yang, Ping-chang

2013-01-01

The current definition of allergy is a group of IgE-mediated diseases. However, a large portion of patients with clinical manifestations of allergies do not exhibit elevated serum levels of IgE (sIgEs). In this article, three key factors, ie soluble allergens, sIgEs and mast cells or basophils, representing the causative factors, messengers and primary effector cells in allergic inflammation, respectively, were discussed. Based on current knowledge on allergic diseases, we propose that allergic diseases are a group of diseases mediated through activated mast cells and/or basophils in sensitive individuals, and allergic diseases include four subgroups: (1) IgE dependent; (2) other immunoglobulin dependent; (3) non-immunoglobulin mediated; (4) mixture of the first three subgroups. According to our proposed definition, pseudo-allergic-reactions, in which mast cell or basophil activation is not mediated via IgE, or to a lesser extent via IgG or IgM, should be non-IgE-mediated allergic diseases. Specific allergen challenge tests (SACTs) are gold standard tests for diagnosing allergies in vivo, but risky. The identification of surface membrane activation markers of mast cells and basophils (CD203c, CCR3, CD63, etc) has led to development of the basophil activation test (BAT), an in vitro specific allergen challenge test (SACT). Based on currently available laboratory allergy tests, we here propose a laboratory examination procedure for allergy. PMID:23974516

Studies on actively allergized cells

PubMed Central

Wilson, Anne B.; Kent, S. P.; Millar, Rose-Mary; McConnell, I.; Coombs, R. R. A.

1973-01-01

A proportion of the plasma cells in lymph nodes of allergized guinea-pigs and mice were found to possess membrane-bound receptors for antigen when tested for rosette-formation at 4° by the suspension-centrifugation technique. This was shown by staining rosetted cells with pyronin-methyl green. Rosette-forming cells of the guinea-pig were further examined by staining with fluorescein-conjugated antigen (rabbit Fab′) and by electron microscopy. By these techniques it was found that many plasma cells which contain antigen-specific intracellular antibodies do not form rosettes, and that cells of the plasmacytic series represented in the rosetted population are limited to plasmablasts and immature plasma cells. The lack of rosette-forming mature plasma cells suggests that a loss of receptors from the cell-membrane occurs as an accompaniment to maturation. ImagesFIG. 1FIG. 2FIG. 4 PMID:4717940

TRPM8 ion channels differentially modulate proliferation and cell cycle distribution of normal and cancer prostate cells.

PubMed

Valero, María Ll; Mello de Queiroz, Fernanda; Stühmer, Walter; Viana, Félix; Pardo, Luis A

2012-01-01

Overexpression of the cation-permeable channel TRPM8 in prostate cancers might represent a novel opportunity for their treatment. Inhibitors of TRPM8 reduce the growth of prostate cancer cells. We have used two recently described and highly specific blockers, AMTB and JNJ41876666, and RNAi to determine the relevance of TRPM8 expression in the proliferation of non-tumor and tumor cells. Inhibition of the expression or function of the channel reduces proliferation rates and proliferative fraction in all tumor cells tested, but not of non-tumor prostate cells. We observed no consistent acceleration of growth after stimulation of the channel with menthol or icilin, indicating that basal TRPM8 expression is enough to sustain growth of prostate cancer cells.

Tracking Internal Temperature and Structural Dynamics during Nail Penetration of Lithium-Ion Cells

DOE PAGES

Finegan, Donal P.; Tjaden, Bernhard; M. M. Heenan, Thomas; ...

2017-10-31

Mechanical abuse of lithium-ion batteries is widely used during testing to induce thermal runaway, characterize associated risks, and expose cell and module vulnerabilities. But, the repeatability of puncture or 'nail penetration' tests is a key issue as there is often a high degree of variability in the resulting thermal runaway process. Here, the failure mechanisms of 18650 cells punctured at different locations and orientations are characterized with respect to their internal structural degradation, and both their internal and surface temperature, all of which are monitored in real time. The initiation and propagation of thermal runaway is visualized via high-speed synchrotronmore » X-ray radiography at 2000 frames per second, and the surface and internal temperatures are recorded via infrared imaging and a thermocouple embedded in the tip of the penetrating nail, respectively. The influence of the nail, as well as how and where it penetrates the cell, on the initiation and propagation of thermal runaway is described and the suitability of this test method for representing in-field failures is discussed.« less

STS-47 PS Mohri uses Spacelab Japan microscope to study cells aboard OV-105

NASA Image and Video Library

1992-09-20

STS047-05-025 (12 - 20 Sept 1992) --- Payload specialist Mamoru Mohri, representing Japan's National Space Development Agency (NASDA), uses a microscope to produce photomicrographs of mammalian cells. The mammal cell structure experiment is one of a large number of tests that were performed during the eight-day Spacelab-J mission. On his back, Dr. Mohri totes a health monitoring experiment. The primary objective of the physiological monitoring system is to observe the health condition of the Japanese payload specialist so that good health can be maintained during and after the spaceflight.

Osteoblastic differentiating potential of dental pulp stem cells in vitro cultured on a chemically modified microrough titanium surface.

PubMed

DE Colli, Marianna; Radunovic, Milena; Zizzari, Vincenzo L; DI Giacomo, Viviana; DI Nisio, Chiara; Piattelli, Adriano; Calvo Guirado, José L; Zavan, Barbara; Cataldi, Amelia; Zara, Susi

2018-03-30

Titanium surface modification is critical for dental implant success. Our aim was to determine surfaces influence on dental pulp stem cells (DPSCs) viability and differentiation. Implants were divided into sandblasted/acid-etched (control) and sandblasted/acid-etched coated with calcium and magnesium ions (CaMg), supplied as composite (test). Proliferation was evaluated by MTT, differentiation checking osteoblastic gene expression, PGE2 secretion and matrix formation, inflammation by Interleukin 6 (IL-6) detection. MTT and IL-6 do not modify on test. A PGE2 increase on test is recorded. BMP2 is higher on test at early experimental points, Osterix and RUNX2 augment later. Alizarin-red S reveals higher matrix production on test. These results suggest that test surface is more osteoinductive, representing a start point for in vivo studies aiming at the construction of more biocompatible dental implants, whose integration and clinical performance are improved and some undesired effects, such as implant stability loss and further surgical procedures, are reduced.

Function of MYO7A in the Human RPE and the Validity of Shaker1 Mice as a Model for Usher Syndrome 1B

PubMed Central

Gibbs, Daniel; Diemer, Tanja; Khanobdee, Kornnika; Hu, Jane; Bok, Dean

2010-01-01

Purpose. To investigate the function of MYO7A in human RPE cells and to test the validity of using shaker1 RPE in preclinical studies on therapies for Usher syndrome 1B by comparing human and mouse cells. Methods. MYO7A was localized by immunofluorescence. Primary cultures of human and mouse RPE cells were used to measure melanosome motility and rod outer segment (ROS) phagocytosis and digestion. MYO7A was knocked down in the human RPE cells by RNAi to test for a mutant phenotype in melanosome motility. Results. The distribution of MYO7A in the RPE of human and mouse was found to be comparable, both in vivo and in primary cultures. Primary cultures of human RPE cells phagocytosed and digested ROSs with kinetics comparable to that of primary cultures of mouse RPE cells. Melanosome motility was also comparable, and, after RNAi knockdown, consisted of longer-range fast movements characteristic of melanosomes in shaker1 RPE. Conclusions. The localization and function of MYO7A in human RPE cells is comparable to that in mouse RPE cells. Although shaker1 retinas do not undergo degeneration, correction of mutant phenotypes in the shaker1 RPE represents a valid preclinical test for potential therapeutic treatments. PMID:19643958

LINDENS: A program for lineament length and density analysis*1

NASA Astrophysics Data System (ADS)

Casas, Antonio M.; Cortés, Angel L.; Maestro, Adolfo; Soriano, M. Asunción; Riaguas, Andres; Bernal, Javier

2000-11-01

Analysis of lineaments from satellite images normally includes the determination of their orientation and density. The spatial variation in the orientation and/or number of lineaments must be obtained by means of a network of cells, the lineaments included in each cell being analysed separately. The program presented in this work, LINDENS, allows the density of lineaments (number of lineaments per km 2 and length of lineaments per km 2) to be estimated. It also provides a tool for classifying the lineaments contained in different cells, so that their orientation can be represented in frequency histograms and/or rose diagrams. The input file must contain the planar coordinates of the beginning and end of each lineament. The density analysis is done by creating a network of square cells, and counting the number of lineaments that are contained within each cell, that have one of their ends within the cell or that cross-cut the cell boundary. The lengths of lineaments are then calculated. To obtain a representative density map the cell size must be fixed according to: (1) the average lineament length; (2) the distance between the lineaments; and (3) the boundaries of zones with low densities due to lithology or outcrop features. An example from the Neogene Duero Basin (Northern Spain) is provided to test the reliability of the density maps obtained with different cell sizes.

The 1987 Goddard Space Flight Center Battery Workshop

NASA Technical Reports Server (NTRS)

Morrow, George (Editor); Yi, Thomas Y. (Editor)

1993-01-01

This document contains the proceedings of the 20th annual Battery Workshop held at Goddard Space Flight Center, Greenbelt, Maryland on November 4-5, 1987. The workshop attendees included manufacturers, users, and government representatives interested in the latest developments in battery technology as they relate to high reliability operations and aerospace use. The subjects covered included lithium cell technology and safety improvements, nickel-cadmium electrode technology along with associated modifications, flight experience and life testing of nickel-cadmium cells, and nickel-hydrogen applications and technology.

High-rate Li-MnO2 cells for aerospace use

NASA Technical Reports Server (NTRS)

Becker-Kaiser, R.; Ruch, J.; Harms, H.-J.; Schmoede, P.; Welsh, J. R.; Vollmers, M.-J.; Pack, H.

1992-01-01

A series of comparative studies were undertaken on representative cells as objectively as possible in order to appreciate the respective advantages of the different systems. After reviewing the first test results our attention was soon focussed on the following four lithium systems: (1) Li-SOCl2; (2) Li-SO2; (3) Li-(CF(sub x))(sub n); and (4) Li-MnO2. This resulted in the decision in 1982 to adopt the Li-MnO2 system for high-rate applications.

The effect of compression on individual pressure vessel nickel/hydrogen components

NASA Technical Reports Server (NTRS)

Manzo, Michelle A.; Perez-Davis, Marla E.

1988-01-01

Compression tests were performed on representative Individual Pressure Vessel (IPV) Nickel/Hydrogen cell components in an effort to better understand the effects of force on component compression and the interactions of components under compression. It appears that the separator is the most easily compressed of all of the stack components. It will typically partially compress before any of the other components begin to compress. The compression characteristics of the cell components in assembly differed considerably from what would be predicted based on individual compression characteristics. Component interactions played a significant role in the stack response to compression. The results of the compression tests were factored into the design and selection of Belleville washers added to the cell stack to accommodate nickel electrode expansion while keeping the pressure on the stack within a reasonable range of the original preset.

Advanced solid electrolyte cell for CO2 and H2O electrolysis. [for extended duration manned spaceflights

NASA Technical Reports Server (NTRS)

Shumar, J. W.; Berger, T. A.

1978-01-01

A solid electrolyte cell with improved sealing characteristics was examined. A tube cell was designed, developed, fabricated, and tested. Design concepts incorporated in the tube cell to improve its sealing capability included minimizing the number of seals per cell and moving seals to lower temperature regions. The advanced tube cell design consists of one high temperature ceramic cement seal, one high temperature gasket seal, and three low temperature silicone elastomer seals. The two high temperature seals in the tube cell design represent a significant improvement over the ten high temperature precious metal seals required by the electrolyzer drum design. For the tube cell design the solid electrolyte was 8 mole percent yttria stabilized zirconium oxide slip cast into the shape of a tube with electrodes applied on the inside and outside surfaces.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Finegan, Donal P.; Tjaden, Bernhard; M. M. Heenan, Thomas

Mechanical abuse of lithium-ion batteries is widely used during testing to induce thermal runaway, characterize associated risks, and expose cell and module vulnerabilities. But, the repeatability of puncture or 'nail penetration' tests is a key issue as there is often a high degree of variability in the resulting thermal runaway process. Here, the failure mechanisms of 18650 cells punctured at different locations and orientations are characterized with respect to their internal structural degradation, and both their internal and surface temperature, all of which are monitored in real time. The initiation and propagation of thermal runaway is visualized via high-speed synchrotronmore » X-ray radiography at 2000 frames per second, and the surface and internal temperatures are recorded via infrared imaging and a thermocouple embedded in the tip of the penetrating nail, respectively. The influence of the nail, as well as how and where it penetrates the cell, on the initiation and propagation of thermal runaway is described and the suitability of this test method for representing in-field failures is discussed.« less

Advances in Plexcore active layer technology systems for organic photovoltaics: roof-top and accelerated lifetime analysis of high performance organic photovoltaic cells

NASA Astrophysics Data System (ADS)

Laird, Darin W.; Vaidya, Swanand; Li, Sergey; Mathai, Mathew; Woodworth, Brian; Sheina, Elena; Williams, Shawn; Hammond, Troy

2007-09-01

We report NREL-certified efficiencies and initial lifetime data for organic photovoltaic (OPV) cells based on Plexcore PV photoactive layer and Plexcore HTL-OPV hole transport layer technology. Plexcore PV-F3, a photoactive layer OPV ink, was certified in a single-layer OPV cell at the National Renewable Energy Laboratory (NREL) at 5.4%, which represents the highest official mark for a single-layer organic solar cell. We have fabricated and measured P3HT:PCBM solar cells with a peak efficiency of 4.4% and typical efficiencies of 3 - 4% (internal, NREL-calibrated measurement) with P3HT manufactured at Plextronics by the Grignard Metathesis (GRIM) method. Outdoor and accelerated lifetime testing of these devices is reported. Both Plexcore PV-F3 and P3HT:PCBM-based OPV cells exhibit >750 hours of outdoor roof-top, non-accelerated lifetime with less than 8% loss in initial efficiency for both active layer systems when exposed continuously to the climate of Western Pennsylvania. These devices are continuously being tested to date. Accelerated testing using a high-intensity (1000W) metal-halide lamp affords shorter lifetimes; however, the true acceleration factor is still to be determined.

Insulin-positive, Glut2-low cells present within mouse pancreas exhibit lineage plasticity and are enriched within extra-islet endocrine cell clusters.

PubMed

Beamish, Christine A; Strutt, Brenda J; Arany, Edith J; Hill, David J

2016-04-18

Regeneration of insulin-producing β-cells from resident pancreas progenitors requires an understanding of both progenitor identity and lineage plasticity. One model suggested that a rare β-cell sub-population within islets demonstrated multi-lineage plasticity. We hypothesized that β-cells from young mice (postnatal day 7, P7) exhibit such plasticity and used a model of islet dedifferentiation toward a ductal epithelial-cell phenotype to test this theory. RIPCre;Z/AP(+/+) mice were used to lineage trace the fate of β-cells during dedifferentiation culture by a human placental alkaline phosphatase (HPAP) reporter. There was a significant loss of HPAP-expressing β-cells in culture, but remaining HPAP(+) cells lost insulin expression while gaining expression of the epithelial duct cell marker cytokeratin-19 (Ck19). Flow cytometry and recovery of β-cell subpopulations from whole pancreas vs. islets suggest that the HPAP(+)Ck19(+) cells had derived from insulin-positive, glucose-transporter-2-low (Ins(+)Glut2(LO)) cells, representing 3.5% of all insulin-expressing cells. The majority of these cells were found outside of islets within clusters of <5 β-cells. These insulin(+)Glut2(LO) cells demonstrated a greater proliferation rate in vivo and in vitro as compared to insulin(+)Glut2(+) cells at P7, were retained into adulthood, and a subset differentiated into endocrine, ductal, and neural lineages, illustrating substantial plasticity. Results were confirmed using RIPCre;ROSA- eYFP mice. Quantitative PCR data indicated these cells possess an immature β-cell phenotype. These Ins(+)Glut2(LO) cells may represent a resident population of cells capable of forming new, functional β-cells, and which may be potentially exploited for regenerative therapies in the future.

Insulin-positive, Glut2-low cells present within mouse pancreas exhibit lineage plasticity and are enriched within extra-islet endocrine cell clusters

PubMed Central

Beamish, Christine A.; Strutt, Brenda J.; Arany, Edith J.; Hill, David J.

2016-01-01

ABSTRACT Regeneration of insulin-producing β-cells from resident pancreas progenitors requires an understanding of both progenitor identity and lineage plasticity. One model suggested that a rare β-cell sub-population within islets demonstrated multi-lineage plasticity. We hypothesized that β-cells from young mice (postnatal day 7, P7) exhibit such plasticity and used a model of islet dedifferentiation toward a ductal epithelial-cell phenotype to test this theory. RIPCre;Z/AP+/+ mice were used to lineage trace the fate of β-cells during dedifferentiation culture by a human placental alkaline phosphatase (HPAP) reporter. There was a significant loss of HPAP-expressing β-cells in culture, but remaining HPAP+ cells lost insulin expression while gaining expression of the epithelial duct cell marker cytokeratin-19 (Ck19). Flow cytometry and recovery of β-cell subpopulations from whole pancreas vs. islets suggest that the HPAP+Ck19+ cells had derived from insulin-positive, glucose-transporter-2-low (Ins+Glut2LO) cells, representing 3.5% of all insulin-expressing cells. The majority of these cells were found outside of islets within clusters of <5 β-cells. These insulin+Glut2LO cells demonstrated a greater proliferation rate in vivo and in vitro as compared to insulin+Glut2+ cells at P7, were retained into adulthood, and a subset differentiated into endocrine, ductal, and neural lineages, illustrating substantial plasticity. Results were confirmed using RIPCre;ROSA- eYFP mice. Quantitative PCR data indicated these cells possess an immature β-cell phenotype. These Ins+Glut2LO cells may represent a resident population of cells capable of forming new, functional β-cells, and which may be potentially exploited for regenerative therapies in the future. PMID:27010375

Stem cells in clinical trials for treatment of retinal degeneration.

PubMed

Klassen, Henry

2016-01-01

After decades of basic science research involving the testing of regenerative strategies in animal models of retinal degenerative diseases, a number of clinical trials are now underway, with additional trials set to begin shortly. These efforts will evaluate the safety and preliminary efficacy of cell-based products in the eyes of patients with a number of retinal conditions, notably including age-related macular degeneration, retinitis pigmentosa and Stargardt's disease. This review considers the scientific work and early trials with fetal cells and tissues that set the stage for the current clinical investigatory work, as well the trials themselves, specifically those either now completed, underway or close to initiation. The cells of interest include retinal pigment epithelial cells derived from embryonic stem or induced pluripotent stem cells, undifferentiated neural or retinal progenitors or cells from the vascular/bone marrow compartment or umbilical cord tissue. Degenerative diseases of the retina represent a popular target for emerging cell-based therapeutics and initial data from early stage clinical trials suggest that short-term safety objectives can be met in at least some cases. The question of efficacy will require additional time and testing to be adequately resolved.

Bilateral sertoli and interstitial cell tumours in abdominal testes of a goat with polled intersex syndrome (PIS).

PubMed

Canisso, I F; Coffee, L L; Ortved, K; Fubini, S L; Monteagudo, L V; Schlafer, D H; Gilbert, R O

2014-12-01

An 8-year-old, mixed breed, polled goat was presented for evaluation of male-like behaviour. Clinical findings included clitoromegaly, a heavily muscled neck, pronounced beard, and erect dorsal guard hairs, which are phenotypic characteristics commonly observed in intersex animals. Transrectal ultrasonography revealed the presence of two abdominal masses caudolateral to the uterine horns. Serum concentration of estradiol was elevated. Genetic evaluation was compatible with polled intersex syndrome defined by an XX karyotype without a Y chromosome or SRY gene. Based on gross and histologic evaluation, the abdominal masses were determined to be intra-abdominal testes, each of which was effaced by Sertoli cell and interstitial (Leydig) cell tumours. The Sertoli cell tumours (SCTs) represented two unique histologic patterns. Regardless of pattern, neoplastic Sertoli cells were consistently lipid laden and positive for vimentin. Interstitial cell tumours (ICTs) were negative for vimentin. Clinical and histopathologic findings suggest that prolonged exposure to steroids secreted by neoplastic Sertoli cells contributed to virilization. In addition, results from immunohistochemistry indicated that vimentin may be a valuable immunodiagnostic tool for differentiation between interstitial and Sertoli cell tumours in goats. © 2014 Blackwell Verlag GmbH.

Microarrays for the evaluation of cell-biomaterial surface interactions

NASA Astrophysics Data System (ADS)

Thissen, H.; Johnson, G.; McFarland, G.; Verbiest, B. C. H.; Gengenbach, T.; Voelcker, N. H.

2007-01-01

The evaluation of cell-material surface interactions is important for the design of novel biomaterials which are used in a variety of biomedical applications. While traditional in vitro test methods have routinely used samples of relatively large size, microarrays representing different biomaterials offer many advantages, including high throughput and reduced sample handling. Here, we describe the simultaneous cell-based testing of matrices of polymeric biomaterials, arrayed on glass slides with a low cell-attachment background coating. Arrays were constructed using a microarray robot at 6 fold redundancy with solid pins having a diameter of 375 μm. Printed solutions contained at least one monomer, an initiator and a bifunctional crosslinker. After subsequent UV polymerisation, the arrays were washed and characterised by X-ray photoelectron spectroscopy. Cell culture experiments were carried out over 24 hours using HeLa cells. After labelling with CellTracker ® Green for the final hour of incubation and subsequent fixation, the arrays were scanned. In addition, individual spots were also viewed by fluorescence microscopy. The evaluation of cell-surface interactions in high-throughput assays as demonstrated here is a key enabling technology for the effective development of future biomaterials.

A Comprehensive Tutorial on In Vitro Characterization of New Photosensitizers for Photodynamic Antitumor Therapy and Photodynamic Inactivation of Microorganisms

PubMed Central

Maisch, Tim; Berneburg, Mark; Plaetzer, Kristjan

2013-01-01

In vitro research performed on eukaryotic or prokaryotic cell cultures usually represents the initial step for characterization of a novel photosensitizer (PS) intended for application in photodynamic therapy (PDT) of cancer or photodynamic inactivation (PDI) of microorganisms. Although many experimental steps of PS testing make use of the wide spectrum of methods readily employed in cell biology, special aspects of working with photoactive substances, such as the autofluorescence of the PS molecule or the requirement of light protection, need to be considered when performing in vitro experiments in PDT/PDI. This tutorial represents a comprehensive collection of operative instructions, by which, based on photochemical and photophysical properties of a PS, its uptake into cells, the intracellular localization and photodynamic action in both tumor cells and microorganisms novel photoactive molecules may be characterized for their suitability for PDT/PDI. Furthermore, it shall stimulate the efforts to expand the convincing benefits of photodynamic therapy and photodynamic inactivation within both established and new fields of applications and motivate scientists of all disciplines to get involved in photodynamic research. PMID:23762860

The 1977 Goddard Space Flight Center Battery Workshop

NASA Technical Reports Server (NTRS)

1977-01-01

The papers presented were derived from transcripts taken at the Tenth Annual Battery Workshop held at the Goddard Space Flight Center, November 15-17, 1977. The Workshop was attended by manufacturers, users, and government representatives interested in the latest results of testing, analysis, and development of the sealed nickel cadmium cell system. The purpose of the Workshop was to share flight and test experience, stimulate discussion on problem areas, and to review the latest technology improvements.

Hyperforin inhibits cell proliferation and differentiation in mouse embryonic stem cells.

PubMed

Nakamura, K; Aizawa, K; Yamauchi, J; Tanoue, A

2013-10-01

Hyperforin, a phloroglucinol derivative of St. John's Wort, has been identified as the major molecule responsible for this plant's products anti-depressant effects. It can be expected that exposure to St. John's Wort during pregnancy occurs with some frequency although embryotoxic or teratogenic effects of St. John's Wort and hyperforin have not yet been experimentally examined in detail. In this study, to determine any embryotoxic effects of hyperforin, we have attempted to determine whether hyperforin affects growth and survival processes of employing mouse embryonic stem (mES) cells (representing embryonic tissue) and fibroblasts (representing adult tissues). We used a modified embryonic stem cell test, which has been validated as an in vitro developmental toxicity protocol, mES cells, to assess embryotoxic potential of chemicals under investigation. We have identified that high concentrations of hyperforin inhibited mouse ES cell population growth and induced apoptosis in fibroblasts. Under our cell culture conditions, ES cells mainly differentiated into cardiomyocytes, although various other cell types were also produced. In this condition, hyperforin affected ES cell differentiation into cardiomyocytes in a dose-dependent manner. Analysis of tissue-specific marker expression also revealed that hyperforin at high concentrations partially inhibited ES cell differentiation into mesodermal and endodermal lineages. Hyperforin is currently used in the clinic as a safe and effective antidepressant. Our data indicate that at typical dosages it has only a low risk of embryotoxicity; ingestion of large amounts of hyperforin by pregnant women, however, may pose embryotoxic and teratogenic risks. © 2013 John Wiley & Sons Ltd.

Low Intensity Low Temperature (LILT) Measurements and Coefficients on New Photovoltaic Structures

NASA Technical Reports Server (NTRS)

Scheiman, David A.; Jenkins, Phillip P.; Brinker, David J.; Appelbaum, Joseph

1995-01-01

Past NASA missions to Mars, Jupiter and the outer planets were powered by radioisotope thermal generators (RTGs). Although these devices proved to be reliable, their high cost and highly toxic radioactive heat source has made them far less desirable for future planetary missions. This has resulted in a renewed search for alternate energy sources, some of them being photovoltaics (PV) and thermophotovoltaics (TPV). Both of these alternate energy sources convert light/thermal energy directly into electricity. In order to create a viable PV data base for planetary mission planners and cell designers, we have compiled low intensity low temperature (LILT) I-V data on single junction and multi-junction high efficiency solar cells. The cells tested here represent the latest photovoltaic technology. Using this LILT data to calculate Short Circuit Current (I(sub sc)), Open Circuit Voltage (V(sub os)), and Fill Factor (FF) as a function of temperature and intensity, an accurate prediction of cell performance under the AM0 spectrum can be determined. When combined with QUantum efficiency at Low Temperature (QULT) data, one can further enhance the data by adding spectral variations to the measurements. This paper presents an overview of LILT measurements and is only intended to be used as a guideline for material selection and performance predictions. As single junction and multi-junction cell technologies emerge, new test data must be collected. Cell materials included are Si, GaAs/Ge, GaInP/GaAs/GaAs, InP, InGaAs/InP, InP/InGaAs/InP, and GaInP. Temperatures range down to as low as -180 C and intensities range from 1 sun down to 0.02 suns. The coefficients presented in this paper represent experimental results and are intended to provide the user with approximate numbers.

Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation system.

PubMed

Buntru, Matthias; Vogel, Simon; Spiegel, Holger; Schillberg, Stefan

2014-05-03

Cell-free protein synthesis is a rapid and efficient method for the production of recombinant proteins. Usage of prokaryotic cell-free extracts often leads to non-functional proteins. Eukaryotic counterparts such as wheat germ extract (WGE) and rabbit reticulocyte lysate (RLL) may improve solubility and promote the correct folding of eukaryotic multi-domain proteins that are difficult to express in bacteria. However, the preparation of WGEs is complex and time-consuming, whereas RLLs suffer from low yields. Here we report the development of a novel cell-free system based on tobacco Bright Yellow 2 (BY-2) cells harvested in the exponential growth phase. The highly-productive BY-2 lysate (BYL) can be prepared quickly within 4-5 h, compared to 4-5 d for WGE. The efficiency of the BYL was tested using three model proteins: enhanced yellow fluorescent protein (eYFP) and two versions of luciferase. The added mRNA was optimized by testing different 5' and 3' untranslated regions (UTRs). The protein yield in batch and dialysis reactions using BYL was much higher than that of a commercial Promega WGE preparation, achieving a maximum yield of 80 μg/mL of eYFP and 100 μg/mL of luciferase, compared to only 45 μg/mL of eYFP and 35 μg/mL of luciferase in WGEs. In dialysis reactions, the BYL yielded about 400 μg/mL eYFP, representing up to 50% more of the target protein than the Promega WGE, and equivalent to the amount using 5Prime WGE system. Due to the high yield and the short preparation time the BYL represents a remarkable improvement over current eukaryotic cell-free systems.

A synthetic lethal screen identifies ATR-inhibition as a novel therapeutic approach for POLD1-deficient cancers

PubMed Central

Hocke, Sandra; Guo, Yang; Job, Albert; Orth, Michael; Ziesch, Andreas; Lauber, Kirsten; De Toni, Enrico N; Gress, Thomas M.; Herbst, Andreas; Göke, Burkhard; Gallmeier, Eike

2016-01-01

The phosphoinositide 3-kinase-related kinase ATR represents a central checkpoint regulator and mediator of DNA-repair. Its inhibition selectively eliminates certain subsets of cancer cells in various tumor types, but the underlying genetic determinants remain enigmatic. Here, we applied a synthetic lethal screen directed against 288 DNA-repair genes using the well-defined ATR knock-in model of DLD1 colorectal cancer cells to identify potential DNA-repair defects mediating these effects. We identified a set of DNA-repair proteins, whose knockdown selectively killed ATR-deficient cancer cells. From this set, we further investigated the profound synthetic lethal interaction between ATR and POLD1. ATR-dependent POLD1 knockdown-induced cell killing was reproducible pharmacologically in POLD1-depleted DLD1 cells and a panel of other colorectal cancer cell lines by using chemical inhibitors of ATR or its major effector kinase CHK1. Mechanistically, POLD1 depletion in ATR-deficient cells caused caspase-dependent apoptosis without preceding cell cycle arrest and increased DNA-damage along with impaired DNA-repair. Our data could have clinical implications regarding tumor genotype-based cancer therapy, as inactivating POLD1 mutations have recently been identified in small subsets of colorectal and endometrial cancers. POLD1 deficiency might thus represent a predictive marker for treatment response towards ATR- or CHK1-inhibitors that are currently tested in clinical trials. PMID:26755646

Low-Coherence Reflectometry for Refractive Index Measurements of Cells in Micro-Capillaries.

PubMed

Carpignano, Francesca; Rigamonti, Giulia; Mazzini, Giuliano; Merlo, Sabina

2016-10-11

The refractive index of cells provides insights into their composition, organization and function. Moreover, a good knowledge of the cell refractive index would allow an improvement of optical cytometric and diagnostic systems. Although interferometric techniques undoubtedly represent a good solution for quantifying optical path variation, obtaining the refractive index of a population of cells non-invasively remains challenging because of the variability in the geometrical thickness of the sample. In this paper, we demonstrate the use of infrared low-coherence reflectometry for non-invasively quantifying the average refractive index of cell populations gently confined in rectangular glass micro-capillaries. A suspension of human red blood cells in plasma is tested as a reference. As a use example, we apply this technique to estimate the average refractive index of cell populations belonging to epithelial and hematological families.

Low-Coherence Reflectometry for Refractive Index Measurements of Cells in Micro-Capillaries

PubMed Central

Carpignano, Francesca; Rigamonti, Giulia; Mazzini, Giuliano; Merlo, Sabina

2016-01-01

The refractive index of cells provides insights into their composition, organization and function. Moreover, a good knowledge of the cell refractive index would allow an improvement of optical cytometric and diagnostic systems. Although interferometric techniques undoubtedly represent a good solution for quantifying optical path variation, obtaining the refractive index of a population of cells non-invasively remains challenging because of the variability in the geometrical thickness of the sample. In this paper, we demonstrate the use of infrared low-coherence reflectometry for non-invasively quantifying the average refractive index of cell populations gently confined in rectangular glass micro-capillaries. A suspension of human red blood cells in plasma is tested as a reference. As a use example, we apply this technique to estimate the average refractive index of cell populations belonging to epithelial and hematological families. PMID:27727172

Simulating the Response of a Composite Honeycomb Energy Absorber. Part 1; Dynamic Crushing of Components and Multi-Terrain Impacts

NASA Technical Reports Server (NTRS)

Jackson, Karen E.; Fasanella, Edwin L.; Polanco, Michael A.

2012-01-01

This paper describes the experimental and analytical evaluation of an externally deployable composite honeycomb structure that is designed to attenuate impact energy during helicopter crashes. The concept, designated the Deployable Energy Absorber (DEA), utilizes an expandable Kevlar (Registered Trademark) honeycomb to dissipate kinetic energy through crushing. The DEA incorporates a unique flexible hinge design that allows the honeycomb to be packaged and stowed until needed for deployment. Experimental evaluation of the DEA included dynamic crush tests of multi-cell components and vertical drop tests of a composite fuselage section, retrofitted with DEA blocks, onto multi-terrain. Finite element models of the test articles were developed and simulations were performed using the transient dynamic code, LSDYNA (Registered Trademark). In each simulation, the DEA was represented using shell elements assigned two different material properties: Mat 24, an isotropic piecewise linear plasticity model, and Mat 58, a continuum damage mechanics model used to represent laminated composite fabrics. DEA model development and test-analysis comparisons are presented.

The influence of biomechanical properties and cannabinoids on tumor invasion.

PubMed

Hohmann, Tim; Grabiec, Urszula; Ghadban, Chalid; Feese, Kerstin; Dehghani, Faramarz

2017-01-02

Cannabinoids are known to have an anti-tumorous effect, but the underlying mechanisms are only sparsely understood. Mechanical characteristics of tumor cells represent a promising marker to distinguish between tumor cells and the healthy tissue. We tested the hypothesis whether cannabinoids influence the tumor cell specific mechanical and migratory properties and if these factors are a prognostic marker for the invasiveness of tumor cells. 3 different glioblastoma cell lines were treated with cannabinoids and changes of mechanical and migratory properties of single cells were measured using atomic force microscopy and time lapse imaging. The invasiveness of cell lines was determined using a co-culture model with organotypic hippocampal slice cultures. We found that cannabinoids are capable of influencing migratory and mechanical properties in a cell line specific manner. A network analysis revealed a correlation between a "generalized stiffness" and the invasiveness for all tumor cell lines after 3 and 4 d of invasion time: r 3d = -0.88 [-0.52;-0.97]; r 4d = -0.90 [-0.59;-0.98]. Here we could show that a "generalized stiffness" is a profound marker for the invasiveness of a tumor cell population in our model and thus might be of high clinical relevance for drug testing. Additionally cannabinoids were shown to be of potential use for therapeutic approaches of glioblastoma.

Experimental Results of Thin-Film Photovoltaic Cells in a Low Density LEO Plasma Environment: Ground Tests

NASA Technical Reports Server (NTRS)

Galofaro, Joel T.; Vayner, Boris V.

2006-01-01

Plasma ground testing results, conducted at the Glenn Research Center (GRC) National Plasma Interaction (N-PI) Facility, are presented for a number of thin-film photovoltaic cells. The cells represent a mix of promising new technologies identified by the Air Force Research Laboratory (AFRL) under the CYGNUS Space Science Technology Experiment (SSTE-4) Program. The current ground tests are aimed at characterizing the performance and survivability of thin film technologies in the harsh low earth orbital space environment where they will be flown. Measurements of parasitic current loss, charging/dielectric breakdown of cover-slide coatings and arcing threshold tests are performed for each individual cell. These measurements are followed by a series of experiments designed to test for catastrophic arc failure mechanisms. A special type of power supply, called a solar array simulator (SAS) with adjustable voltage and current limits on the supply s output, is employed to bias two adjacent cells at a predetermined voltage and current. The bias voltage is incrementally ramped up until a sustained arc results. Sustained arcs are precursors to catastrophic arc failure where the arc current rises to a maximum value for long timescales often ranging between 30 to 100 sec times. Normal arcs by comparison, are short lived events with a timescale between 10 to 30 sec. Sustained arcs lead to pyrolization with extreme cell damage and have been shown to cause the loss of entire array strings in solar arrays. The collected data will be used to evaluate the suitability of thin-film photovoltaic technologies for future space operations.

Kupffer Cell Isolation for Nanoparticle Toxicity Testing

PubMed Central

Bourgognon, Maxime; Klippstein, Rebecca; Al-Jamal, Khuloud T.

2015-01-01

The large majority of in vitro nanotoxicological studies have used immortalized cell lines for their practicality. However, results from nanoparticle toxicity testing in immortalized cell lines or primary cells have shown discrepancies, highlighting the need to extend the use of primary cells for in vitro assays. This protocol describes the isolation of mouse liver macrophages, named Kupffer cells, and their use to study nanoparticle toxicity. Kupffer cells are the most abundant macrophage population in the body and constitute part of the reticulo-endothelial system (RES), responsible for the capture of circulating nanoparticles. The Kupffer cell isolation method reported here is based on a 2-step perfusion method followed by purification on density gradient. The method, based on collagenase digestion and density centrifugation, is adapted from the original protocol developed by Smedsrød et al. designed for rat liver cell isolation and provides high yield (up to 14 x 106 cells per mouse) and high purity (>95%) of Kupffer cells. This isolation method does not require sophisticated or expensive equipment and therefore represents an ideal compromise between complexity and cell yield. The use of heavier mice (35-45 g) improves the yield of the isolation method but also facilitates remarkably the procedure of portal vein cannulation. The toxicity of functionalized carbon nanotubes f-CNTs was measured in this model by the modified LDH assay. This method assesses cell viability by measuring the lack of structural integrity of Kupffer cell membrane after incubation with f-CNTs. Toxicity induced by f-CNTs can be measured consistently using this assay, highlighting that isolated Kupffer cells are useful for nanoparticle toxicity testing. The overall understanding of nanotoxicology could benefit from such models, making the nanoparticle selection for clinical translation more efficient. PMID:26327223

Clinical translation and regulatory aspects of CAR/TCR-based adoptive cell therapies-the German Cancer Consortium approach.

PubMed

Krackhardt, Angela M; Anliker, Brigitte; Hildebrandt, Martin; Bachmann, Michael; Eichmüller, Stefan B; Nettelbeck, Dirk M; Renner, Matthias; Uharek, Lutz; Willimsky, Gerald; Schmitt, Michael; Wels, Winfried S; Schüssler-Lenz, Martina

2018-04-01

Adoptive transfer of T cells genetically modified by TCRs or CARs represents a highly attractive novel therapeutic strategy to treat malignant diseases. Various approaches for the development of such gene therapy medicinal products (GTMPs) have been initiated by scientists in recent years. To date, however, the number of clinical trials commenced in Germany and Europe is still low. Several hurdles may contribute to the delay in clinical translation of these therapeutic innovations including the significant complexity of manufacture and non-clinical testing of these novel medicinal products, the limited knowledge about the intricate regulatory requirements of the academic developers as well as limitations of funds for clinical testing. A suitable good manufacturing practice (GMP) environment is a key prerequisite and platform for the development, validation, and manufacture of such cell-based therapies, but may also represent a bottleneck for clinical translation. The German Cancer Consortium (DKTK) and the Paul-Ehrlich-Institut (PEI) have initiated joint efforts of researchers and regulators to facilitate and advance early phase, academia-driven clinical trials. Starting with a workshop held in 2016, stakeholders from academia and regulatory authorities in Germany have entered into continuing discussions on a diversity of scientific, manufacturing, and regulatory aspects, as well as the benefits and risks of clinical application of CAR/TCR-based cell therapies. This review summarizes the current state of discussions of this cooperative approach providing a basis for further policy-making and suitable modification of processes.

Cabozantinib Affects Osteosarcoma Growth Through A Direct Effect On Tumor Cells and Modifications In Bone Microenvironment.

PubMed

Fioramonti, M; Fausti, V; Pantano, F; Iuliani, M; Ribelli, G; Lotti, F; Pignochino, Y; Grignani, G; Santini, D; Tonini, G; Vincenzi, B

2018-03-08

Osteosarcoma (OS) is the most common primary malignant tumor of the bone. Due to its high heterogeneity and to survival signals from bone microenvironment, OS can resist to standard treatments, therefore novel therapies are needed. c-MET oncogene, a tyrosine-kinase receptor, plays a crucial role in OS initiation and progression. The present study aimed to evaluate the effect of c-MET inhibitor cabozantinib (CBZ) on OS both directly and through its action on bone microenvironment. We tested different doses of CBZ in in vitro models of OS alone or in co-culture with bone cells in order to reproduce OS-tumor microenvironment interactions. CBZ is able to decrease proliferation and migration of OS cells, inhibiting ERK and AKT signaling pathways. Furthermore, CBZ leads to the inhibition of the proliferation of OS cells expressing receptor activator of nuclear factor κB (RANK), due to its effect on bone microenvironment, where it causes an overproduction of osteoprotegerin and a decrease of production of RANK ligand by osteoblasts. Overall, our data demonstrate that CBZ might represent a new potential treatment against OS, affecting both OS cells and their microenvironment. In this scenario, RANK expression in OS cells could represent a predictive factor of better response to CBZ treatment.

Thermal Cycling of Mir Cooperative Solar Array (MCSA) Test Panels

NASA Technical Reports Server (NTRS)

Hoffman, David J.; Scheiman, David A.

1997-01-01

The Mir Cooperative Solar Array (MCSA) project was a joint US/Russian effort to build a photovoltaic (PV) solar array and deliver it to the Russian space station Mir. The MCSA is currently being used to increase the electrical power on Mir and provide PV array performance data in support of Phase 1 of the International Space Station (ISS), which will use arrays based on the same solar cells used in the MCSA. The US supplied the photovoltaic power modules (PPMs) and provided technical and programmatic oversight while Russia provided the array support structures and deployment mechanism and built and tested the array. In order to ensure that there would be no problems with the interface between US and Russian hardware, an accelerated thermal life cycle test was performed at NASA Lewis Research Center on two representative samples of the MCSA. Over an eight-month period (August 1994 - March 1995), two 15-cell MCSA solar array 'mini' panel test articles were simultaneously put through 24,000 thermal cycles (+80 C to -100 C), equivalent to four years on-orbit. The test objectives, facility, procedure and results are described in this paper. Post-test inspection and evaluation revealed no significant degradation in the structural integrity of the test articles and no electrical degradation, not including one cell damaged early as an artifact of the test and removed from consideration. The interesting nature of the performance degradation caused by this one cell, which only occurred at elevated temperatures, is discussed. As a result of this test, changes were made to improve some aspects of the solar cell coupon-to-support frame interface on the flight unit. It was concluded from the results that the integration of the US solar cell modules with the Russian support structure would be able to withstand at least 24,000 thermal cycles (4 years on-orbit).

Degradation mechanisms and accelerated testing in PEM fuel cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Borup, Rodney L; Mukundan, Rangachary

2010-01-01

The durability of PEM fuel cells is a major barrier to the commercialization of these systems for stationary and transportation power applications. Although there has been recent progress in improving durability, further improvements are needed to meet the commercialization targets. Past improvements have largely been made possible because of the fundamental understanding of the underlying degradation mechanisms. By investigating component and cell degradation modes; defining the fundamental degradation mechanisms of components and component interactions new materials can be designed to improve durability. Various factors have been shown to affect the useful life of PEM fuel cells. Other issues arise frommore » component optimization. Operational conditions (such as impurities in either the fuel and oxidant stream), cell environment, temperature (including subfreezing exposure), pressure, current, voltage, etc.; or transient versus continuous operation, including start-up and shutdown procedures, represent other factors that can affect cell performance and durability. The need for Accelerated Stress Tests (ASTs) can be quickly understood given the target lives for fuel cell systems: 5000 hours ({approx} 7 months) for automotive, and 40,000 hrs ({approx} 4.6 years) for stationary systems. Thus testing methods that enable more rapid screening of individual components to determine their durability characteristics, such as off-line environmental testing, are needed for evaluating new component durability in a reasonable turn-around time. This allows proposed improvements in a component to be evaluated rapidly and independently, subsequently allowing rapid advancement in PEM fuel cell durability. These tests are also crucial to developers in order to make sure that they do not sacrifice durability while making improvements in costs (e.g. lower platinum group metal [PGM] loading) and performance (e.g. thinner membrane or a GDL with better water management properties). To achieve a deeper understanding and improve PEM fuel cell durability LANL is conducting research to better define fuel cell component degradation mechanisms and correlate AST measurements to component in 'real-world' situations.« less

Applying the cell-based coagulation model in the management of critical bleeding.

PubMed

Ho, K M; Pavey, W

2017-03-01

The cell-based coagulation model was proposed 15 years ago, yet has not been applied commonly in the management of critical bleeding. Nevertheless, this alternative model may better explain the physiological basis of current coagulation management during critical bleeding. In this article we describe the limitations of the traditional coagulation protein cascade and standard coagulation tests, and explain the potential advantages of applying the cell-based model in current coagulation management strategies. The cell-based coagulation model builds on the traditional coagulation model and explains many recent clinical observations and research findings related to critical bleeding unexplained by the traditional model, including the encouraging results of using empirical 1:1:1 fresh frozen plasma:platelets:red blood cells transfusion strategy, and the use of viscoelastic and platelet function tests in patients with critical bleeding. From a practical perspective, applying the cell-based coagulation model also explains why new direct oral anticoagulants are effective systemic anticoagulants even without affecting activated partial thromboplastin time or the International Normalized Ratio in a dose-related fashion. The cell-based coagulation model represents the most cohesive scientific framework on which we can understand and manage coagulation during critical bleeding.

Long-term storage of nickel-hydrogen cells

NASA Technical Reports Server (NTRS)

Vaidyanathan, Hari

1987-01-01

Representative samples of nickel hydrogen cells for the INTELSAT program were used to evaluate the effects of prolonged storage under passive conditions such as open circuit discharged at 0 C, room temperature, and -20 C, and under quasidynamic conditions such as top-off charge and trickle charge. Cell capacity declines when cells are stored open-circuit discharged at room temperature, and a second plateau occurs in the discharge curve. Capacity loss was 47 percent for a cell with hydrogen precharge and 24.5 percent for one with no hydrogen precharge. Capacity recovery was observed following top-off charge storage of cells which had exhibited faded capacity as a result of passive storage at room temperature. Cells stored either at -20 C or on trickle charge maintained their capacity. At 0 C storage, the capacity of all three cells under tests was greater than 55 Ah (which exceeds the required minimum of 44 Ah) after 7 months.

Cinnamoyl compounds as simple molecules that inhibit p300 histone acetyltransferase.

PubMed

Costi, Roberta; Di Santo, Roberto; Artico, Marino; Miele, Gaetano; Valentini, Paola; Novellino, Ettore; Cereseto, Anna

2007-04-19

Cinnamoly compounds 1a-c and 2a-d were designed, synthesized, and in vitro tested as p300 inhibitors. At different degrees, all tested compounds were proven to inactivate p300, particularly, derivative 2c was the most active inhibitor, also showing high specificity for p300 as compared to other histone acetyltransferases. Most notably, 2c showed anti-acetylase activity in mammalian cells. These compounds represent a new class of synthetic inhibitors of p300, characterized by simple chemical structures.

Synthetic battery cycling

NASA Technical Reports Server (NTRS)

Thaller, L. H.

1981-01-01

The use of interactive computer graphics is suggested as an aid in battery system development. Mathematical representations of simplistic but fully representative functions of many electrochemical concepts of current practical interest will permit battery level charge and discharge phenomena to be analyzed in a qualitative manner prior to the assembly and testing of actual hardware. This technique is a useful addition to the variety of tools available to the battery system designer as he bridges the gap between interesting single cell life test data and reliable energy storage subsystems.

Thrombo-ischaemic pinnal necrosis associated with fenbendazole treatment in a dog.

PubMed

Nuttall, T J; Burrow, R; Fraser, I; Kipar, A

2005-05-01

An 11-week-old, female West Highland white terrier was presented with necrosis of the distal third of both pinnae. Haematology, biochemistry and urinalysis, Coombs test, antinuclear antibody and cold autoagglutinin antibody tests were normal. A drug reaction to fenbendazole was diagnosed. The necrotic ear tips were surgically removed. Histopathology revealed extensive coagulative necrosis of the epidermis and superficial to mid-dermis, a moderate interstitial neutrophilic infiltrate and complete thrombotic occlusion and necrosis of blood vessels. There was also endothelial cell activation and proliferation with endothelial cell cushions protruding into the vascular lumen. Immunohistochemistry for factor VIII-related antigen confirmed endothelial cell involvement. This case represents an unusual, drug-induced, thrombo-ischaemic necrosis of the pinnae. It is also, to the authors' knowledge, the first report of fenbendazole sensitivity in a dog. The histopathology is similar to previous cases of proliferative thrombovascular pinnal necrosis, suggesting that drug reactions should be considered in this condition.

Polyphenol-rich beverages enhance zinc uptake and metallothionein expression in Caco-2 cells.

PubMed

Sreenivasulu, Kilari; Raghu, Pullakhandam; Nair, K Madhavan

2010-05-01

The effect of red wine (RW), red grape juice (RGJ), green tea (GT), and representative polyphenols on Caco-2 cell (65)Zn uptake was explored. RW, RGJ, and GT enhanced the uptake of zinc from rice matrix. Fractionation of RW revealed that enhancing activity of zinc uptake was exclusively resided in the polyphenol fraction. Among the polyphenols tested, only tannic acid and quercitin stimulated the uptake of zinc while others did not influence the uptake. In tune with these results, only tannic acid and quercitin competed with zinquin (a zinc selective fluorophore) for zinc in vitro. Although all the polyphenols tested appear to enhance the expression of metallothionein (MT), the induction was higher with tannic acid, quercitin, and RW extract. Furthermore, phytic acid abrogated the tannic acid-induced MT expression. These results suggest that polyphenol-rich beverages, tannic acid, and quercitin bind and stimulate the zinc uptake and MT expression in Caco-2 cells.

Description and field analysis of a coupled ground-water/surface-water flow model (MODFLOW/BRANCH) with modifications for structures and wetlands in southern Dade County, Florida

USGS Publications Warehouse

Swain, E.D.; Howie, Barbara; Dixon, Joann

1996-01-01

A coupled surface-water model (BRANCH) and ground-water model (MODFLOW) model were tested to simulate the interacting wetlands/surface-water/ ground-water system of southern Dade County. Several options created for the MODFLOW ground- ground-water model were used in representing this field situation. The primary option is the MODBRANCH interfacing software, which allows leakage to be accounted for between the MODFLOW ground-water model and the BRANCH dynamic model for simulation of flow in an interconnected network of open channels. A modification to an existing software routine, which is referred to as BCF2, allows cells in MODFLOW to rewet when dry--a requirement in representing the seasonal wetlands in Dade County. A companion to BCF2 is the modified evapotranspiration routine EVT2. The EVT2 routine changes the cells where evapotranspiration occurs, depending on which cells are wet. The Streamlink package represents direct connections between the canals and wetlands at locations where canals open directly into overland flow. Within the BRANCH model, the capability to represent the numerous hydraulic structures, gated spillways, gated culverts, and pumps was added. The application of these modifications to model surface-water/ground-water interactions in southern Dade County demonstrated the usefulness of the coupled MODFLOW/BRANCH model. Ground-water and surface-water flows are both simulated with dynamic models. Flow exchange between models, intermittent wetting and drying, evapotranspiration, and hydraulic structure operations are all represented appropriately. Comparison was made with a simulation using the RIV1 package instead of MODBRANCH to represent the canals. RIV1 represents the canals by user-defined stages, and computes leakage to the aquifer. Greater accuracy in reproducing measured ground- water heads was achieved with MODBRANCH, which also computes dynamic flow conditions in the canals, unlike RIV1. The surface-water integrated flow and transport two-dimensional model (SWIFT2D) was also applied to the southeastern coastal wetlands for comparison with the wetlands flow approximation made in MODFLOW. MODFLOW simulates the wetlands as a highly conductive upper layer of the aquifer, whereas SWIFT2D solves the hydrodynamic equations. Comparison in this limited test demonstrated no specific advantage for either method of representation. However, much additional testing on a wider variety of geometric and hydraulic situations, such as in areas with greater tidal or other dynamic forcing effects, is needed to make definite conclusions. A submodel of the existing southern Dade County model schematization was used to examine water-delivery alternatives proposed by the U.S. Army Corps of Engineers. For this application, the coupled MODFLOW/BRANCH model was used as a design tool. A new canal and several pumps to be tested to maintain lower water levels in a residential area (while water levels in the Everglades are raised) were added to the model schematization. The pumps were assumed to have infinite supply capacity in the model so that their maximum pumping rates during the simulation could be used to determine pump sizes.

Normal Taste Acceptance and Preference of PANX1 Knockout Mice.

PubMed

Tordoff, Michael G; Aleman, Tiffany R; Ellis, Hillary T; Ohmoto, Makoto; Matsumoto, Ichiro; Shestopalov, Val I; Mitchell, Claire H; Foskett, J Kevin; Poole, Rachel L

2015-09-01

Taste compounds detected by G protein-coupled receptors on the apical surface of Type 2 taste cells initiate an intracellular molecular cascade culminating in the release of ATP. It has been suggested that this ATP release is accomplished by pannexin 1 (PANX1). However, we report here that PANX1 knockout mice do not differ from wild-type controls in response to representative taste solutions, measured using 5-s brief-access tests or 48-h two-bottle choice tests. This implies that PANX1 is unnecessary for taste detection and consequently that ATP release from Type 2 taste cells does not require PANX1. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Characterizing the Radiation Survivability of Space Solar Cell Technologies for Heliospheric Missions

NASA Astrophysics Data System (ADS)

Lee, J. H.; Walker, D.; Mann, C. J.; Yue, Y.; Nocerino, J. C.; Smith, B. S.; Mulligan, T.

2016-12-01

Space solar cells are responsible for powering the majority of heliospheric space missions. This paper will discuss methods for characterizing space solar cell technologies for on-orbit operations that rely on a series of laboratory tests that include measuring the solar cells' beginning of life performance under simulated (e.g. AM0 or air mass zero) sunlight over different operating temperatures and observing their end of life performance following exposure to laboratory-generated charged particle radiation (protons and electrons). The Aerospace Corporation operates a proton implanter as well as electron gun facilities and collaborates with external radiation effects facilities to expose space solar cells or other space technologies to representative space radiation environments (i.e. heliosphere or magnetosphere of Earth or other planets), with goals of characterizing how the technologies perform over an anticipated space mission timeline and, through the application of precision diagnostic capabilities, understanding what part of the solar cell is impacted by varying space radiation environments. More recently, Aerospace has been hosting solar cell flight tests on its previously-flown CubeSat avionics bus, providing opportunities to compare the laboratory tests to on-orbit observations. We hope through discussion of the lessons learned and methods we use to characterize how solar cells perform after space radiation exposure that similar methodology could be adopted by others to improve the state of knowledge on the survivability of other space technologies required for future space missions.

Effects of chemical and physical agents on recombination events in cells of the germ line of male and female Drosophila melanogaster.

PubMed

Würgler, F E

1991-01-01

Genotoxic agents can induce mutations as well as recombination in the genetic material. The fruit fly Drosophila melanogaster was one of the first assay systems to test physical and chemical agents for recombinogenic effects. Such effects can be observed in cells of the germ line as well as in somatic cells. At present information is available on 54 agents, among them 48 chemicals that have been tested in cells of the germ line of males and/or females. Effects on meiotic recombination in female germ cells cannot simply be classified as positive or negative since for a number of agents, depending on the chromosome region studied, recombination frequencies may be increased, unaffected or decreased. The male germ line of D. melanogaster represents a unique situation because meiotic recombination does not occur. Among 25 agents tested in male germ cells 24 did induce male recombination, among them alkylating, intercalating and cross-linking agents, direct-acting ones as well as compounds needing metabolic activation. With several compounds the frequency of induced recombination is highest in the heterochromatic regions near the centromeres. In brood pattern analyses, e.g., after exposure of adult males to ionizing radiation, the first appearance of crossover progeny is indicative of the sampling of exposed spermatocytes. In premeiotic cells of the male and the female germ line mitotic recombination can occur. Upon clonal expansion of the recombinant cells, clusters of identical crossovers can be observed.

Micro-macroscopic coupled modeling of batteries and fuel cells. 2: Application to nickel-cadmium and nickel-metal hydride cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gu, W.B.; Wang, C.Y.; Liaw, B.Y.

1998-10-01

The micro-macroscopic coupled model developed in a companion paper is applied to predict the discharge and charge behaviors of nickel-cadmium (Ni-Cd) and nickel-metal hydride (Ni-MH) cells. The model integrates important microscopic phenomena such as proton or hydrogen diffusion and conduction of electrons in active materials into the macroscopic calculations of species and charge transfer. Simulation results for a full Ni-Cd cell and single MH electrode are presented and validated against the pseudo two-dimensional numerical model in the literature. In good agreement with the previous results, the present family of models is computationally more efficient and is particularly suitable for simulationsmore » of complex test conditions, such as the dynamic stress test and pulse charging for electric vehicles. In addition, a mathematical model for full Ni-MH cells is presented and sample simulations are performed for discharge and recharge with oxygen generation and recombination taken into account. These gas reactions represent an important mechanism for battery overcharge in the electric vehicle application.« less

NMP22 BladderChek Test: point-of-care technology with life- and money-saving potential.

PubMed

Tomera, Kevin M

2004-11-01

A new, relatively obscure tumor marker assay, the NMP22 BladderChek Test (Matritech, Inc.), represents a paradigm shift in the diagnosis and management of urinary bladder cancer (transitional cell carcinoma). Specifically, BladderChek should be employed every time a cystoscopy is performed, with corresponding changes in the diagnostic protocol and the guidelines of the American Urological Association for the diagnosis and management of bladder cancer. Currently, cystoscopy is the reference standard and NMP22 BladderChek Test in combination with cystoscopy improves the performance of cystoscopy. At every stage of disease, BladderChek provides a higher sensitivity for the detection of bladder cancer than cytology, which now represents the adjunctive standard of care. Moreover, BladderChek is four-times more sensitive than cytology and is available at half the cost. Early detection of bladder cancer improves prognosis, quality of life and survival. BladderChek may be analogous to the prostate-specific antigen test and eventually expand beyond the urologic setting into the primary care setting for the testing of high-risk patients characterized by smoking history, occupational exposures or age.

Pros and cons of fish skin cells in culture: long-term full skin and short-term scale cell culture from rainbow trout, Oncorhynchus mykiss.

PubMed

Rakers, Sebastian; Klinger, Matthias; Kruse, Charli; Gebert, Marina

2011-12-01

Here, we report the establishment of a permanent skin cell culture from rainbow trout (Oncorhynchus mykiss). The cells of the fish skin cell culture could be propagated over 60 passages so far. Furthermore, we show for the first time that it is possible to integrate freshly harvested rainbow trout scales into this new fish skin cell culture. We further demonstrated that epithelial cells derived from the scales survived in the artificial micro-environment of surrounding fibroblast-like cells. Also, antibody staining indicated that both cell types proliferated and started to build connections with the other cell type. It seems that it is possible to generate an 'artificial skin' with two different cell types. This could lead to the development of a three-dimensional test system, which might be a better in vitro representative of fish skin in vivo than individual skin cell lines. Copyright © 2011 Elsevier GmbH. All rights reserved.

Purpose-driven biomaterials research in liver-tissue engineering.

PubMed

Ananthanarayanan, Abhishek; Narmada, Balakrishnan Chakrapani; Mo, Xuejun; McMillian, Michael; Yu, Hanry

2011-03-01

Bottom-up engineering of microscale tissue ("microtissue") constructs to recapitulate partially the complex structure-function relationships of liver parenchyma has been realized through the development of sophisticated biomaterial scaffolds, liver-cell sources, and in vitro culture techniques. With regard to in vivo applications, the long-lived stem/progenitor cell constructs can improve cell engraftment, whereas the short-lived, but highly functional hepatocyte constructs stimulate host liver regeneration. With regard to in vitro applications, microtissue constructs are being adapted or custom-engineered into cell-based assays for testing acute, chronic and idiosyncratic toxicities of drugs or pathogens. Systems-level methods and computational models that represent quantitative relationships between biomaterial scaffolds, cells and microtissue constructs will further enable their rational design for optimal integration into specific biomedical applications. Copyright © 2010 Elsevier Ltd. All rights reserved.

Invasion of vascular cells in vitro by Porphyromonas endodontalis.

PubMed

Dorn, B R; Harris, L J; Wujick, C T; Vertucci, F J; Progulske-Fox, A

2002-04-01

The objective of this study was to determine whether laboratory strains and clinical isolates of microorganisms associated with root canal infections can invade primary cultures of cardiovascular cells. Quantitative levels of bacterial invasion of human coronary artery endothelial cells (HCAEC) and coronary artery smooth muscle cells (CASMC) were measured using a standard antibiotic protection assay. Transmission electron microscopy was used to confirm and visualize internalization within the vascular cells. Of the laboratory and clinical strains tested, only P. endodontalis ATCC 35406 was invasive in an antibiotic protection assay using HCAEC and CASMC. Invasion of P. endodontalis ATCC 35406 was confirmed by transmission electron microscopy. Certain microorganisms associated with endodontic infections are invasive. If bacterial invasion of the vasculature contributes to the pathogenesis of cardiovascular disease, then microorganisms in the pulp chamber represent potential pathogens.

Study of fuel cell powerplant with heat recovery

NASA Technical Reports Server (NTRS)

King, J. M.; Grasso, A. P.; Clausi, J. V.

1975-01-01

It was shown that heat can be recovered from fuel cell power plants by replacing the air-cooled heat exchangers in present designs with units which transfer the heat to the integrated utility system. Energy availability for a 40-kW power plant was studied and showed that the total usable energy at rated power represents 84 percent of the fuel lower heating value. The effects of design variables on heat availability proved to be small. Design requirements were established for the heat recovery heat exchangers, including measurement of the characteristics of two candidate fuel cell coolants after exposure to fuel cell operating conditions. A heat exchanger test program was defined to assess fouling and other characteristics of fuel cell heat exchangers needed to confirm heat exchanger designs for heat recovery.

Ultrasonically targeted delivery into endothelial and smooth muscle cells in ex vivo arteries

PubMed Central

Hallow, Daniel M.; Mahajan, Anuj D.; Prausnitz, Mark R.

2007-01-01

This study tested the hypothesis that ultrasound can target intracellular uptake of drugs into vascular endothelial cells (ECs) at low to intermediate energy and into smooth muscle cells (SMCs) at high energy. Ultrasound-enhanced delivery has been shown to enhance and target intracellular drug and gene delivery in the vasculature to treat cardiovascular disease, but quantitative studies of the delivery process are lacking. Viable ex vivo porcine carotid arteries were placed in a solution containing a model drug, TO-PRO®-1, and Optison® microbubbles. Arteries were exposed to ultrasound at 1.1 MHz and acoustic energies of 5.0, 66, or 630 J/cm2. Using confocal microscopy and fluorescent labeling of cells, the artery endothelium and media were imaged to determine the localization and to quantify intracellular uptake and cell death. At low to intermediate ultrasound energy, ultrasound was shown to target intracellular delivery into viable cells that represented 9 – 24% of exposed ECs. These conditions also typically caused 7 – 25% EC death. At high energy, intracellular delivery was targeted to SMCs, which was associated with denuding or death of proximal ECs. This work represents the first known in-depth study to evaluate intracellular uptake into cells in tissue. We conclude that significant intracellular uptake of molecules can be targeted into ECs and SMCs by ultrasound-enhanced delivery suggesting possible applications for treatment of cardivascular diseases and dysfunctions. PMID:17291619

Quantification of Slackia and Eggerthella spp. in Human Feces and Adhesion of Representatives Strains to Caco-2 Cells

PubMed Central

Cho, Gyu-Sung; Ritzmann, Felix; Eckstein, Marie; Huch, Melanie; Briviba, Karlis; Behsnilian, Diana; Neve, Horst; Franz, Charles M. A. P.

2016-01-01

Eggerthella and Slackia spp. are gut associated bacteria that have been suggested to play roles in host lipid and xenobiotic metabolism. A quantitative PCR method for the selective enumeration of bacteria belonging to either the genus Eggerthella or Slackia was developed in order to establish the numbers of these bacteria occurring in human feces. The primers developed for selective amplification of these genera were tested first in conventional PCR to test for their specificity. Representative species of Eggerthella and Slackia, as well as closely related genera of the Coriobacteriia, were included in the investigation. The selected primers were shown to be capable of specific amplification of species of the genera Eggerthella and Slackia, but not all species of the genera may be amplified by the respective primers. Their use in qPCR experiments to assess the levels of Slackia equolifaciens and Eggerthella lenta in the feces of 19 human volunteers showed they occurred at mean counts of 7 × 105 and 3.1 × 105 CFU/g for Eggerthella spp. and Slackia spp., respectively. Electron microscopy investigations showed that while E. lenta cells exhibited slender and very regular shaped rods, Slackia cells showed a remarkably pleomorphic phenotype. Both species did not appear to have fimbriae or pili. Some S. equolifaciens cells showed a characteristic “ribbon” of presumably extracellular material around the cells, particularly at the areas of cell division. The two species also differed markedly in their adhesion behavior to Caco-2 cells in cell culture, as E. lenta DSMZ 15644 showed a high adhesion capacity of 74.2% adherence of the bacterial cells added to Caco-2 cells, while S. equolifaciens DSM 24851T on the other hand showed only low adhesion capability, as 6.1% of bacterial cells remained bound. Speculatively, this may imply that the ecological compartments where these bacteria reside in the gut may be different, i.e., E. lenta may be associated more with the gut wall, while Slackia may be free living in the lumen. PMID:27242689

Antiretroviral treatment scale-up among persons living with HIV in Kenya: results from a nationally representative survey.

PubMed

Odhiambo, Jacob O; Kellogg, Timothy A; Kim, Andrea A; Ng'ang'a, Lucy; Mukui, Irene; Umuro, Mamo; Mohammed, Ibrahim; De Cock, Kevin M; Kimanga, Davies O; Schwarcz, Sandra

2014-05-01

In 2007, 29% of HIV-infected Kenyans in need of antiretroviral therapy (ART), based on an immunologic criterion of CD4 ≤350 cells per microliter, were receiving ART. Since then, substantial treatment scale-up has occurred in the country. We analyzed data from the second Kenya AIDS Indicator Survey (KAIS 2012) to assess progress of treatment scale-up in Kenya. KAIS 2012 was a nationally representative survey of persons aged 18 months to 64 years that collected information on HIV status, care, and treatment. ART eligibility was defined based on 2 standards: (1) 2011 Kenya eligibility criteria for ART initiation: CD4 ≤350 cells per microliter or co-infection with active tuberculosis and (2) 2013 World Health Organization (WHO) eligibility criteria for ART initiation: CD4 ≤500 cells per microliter, co-infection with active tuberculosis, currently pregnant or breastfeeding, and infected partners in serodiscordant relationships. Blood specimens were tested for HIV antibodies and HIV-positive specimens tested for CD4 cell counts. Among 13,720 adults and adolescents aged 15-64 years, 11,626 provided a blood sample, and 648 were HIV infected. Overall, 58.8% [95% confidence interval (CI): 52.0 to 65.5) were eligible for treatment using the 2011 Kenya eligibility criteria and 77.4% (95% CI: 72.4 to 82.4) using the 2013 WHO eligibility criteria. Coverage of ART was 60.5% (95% CI: 50.8 to 70.2) using the 2011 Kenya eligibility criteria and 45.9% (95% CI: 37.7 to 54.2) using the 2013 WHO eligibility criteria. ART coverage has increased from 29% in 2007 to 61% in 2012. If Kenya adopts the 2013 WHO guidelines for ART initiation, need for ART increases by an additional 19 percentage points and current coverage decreases by an additional 15 percentage points, representing an additional 214,000 persons who will need to be reached.

Molecular Basis of 9G4 B Cell Autoreactivity in Human Systemic Lupus Erythematosus

PubMed Central

Richardson, Christopher; Chida, Asiya Seema; Adlowitz, Diana; Silver, Lin; Fox, Erin; Jenks, Scott A.; Palmer, Elise; Wang, Youliang; Heimburg-Molinaro, Jamie; Li, Quan-Zhen; Mohan, Chandra; Cummings, Richard; Tipton, Christopher

2013-01-01

9G4+ IgG Abs expand in systemic lupus erythematosus (SLE) in a disease-specific fashion and react with different lupus Ags including B cell Ags and apoptotic cells. Their shared use of VH4-34 represents a unique system to understand the molecular basis of lupus autoreactivity. In this study, a large panel of recombinant 9G4+ mAbs from single naive and memory cells was generated and tested against B cells, apoptotic cells, and other Ags. Mutagenesis eliminated the framework-1 hydrophobic patch (HP) responsible for the 9G4 idiotype. The expression of the HP in unselected VH4-34 cells was assessed by deep sequencing. We found that 9G4 Abs recognize several Ags following two distinct structural patterns. B cell binding is dependent on the HP, whereas anti-nuclear Abs, apoptotic cells, and dsDNA binding are HP independent and correlate with positively charged H chain third CDR. The majority of mutated VH4-34 memory cells retain the HP, thereby suggesting selection by Ags that require this germline structure. Our findings show that the germline-encoded HP is compulsory for the anti–B cell reactivity largely associated with 9G4 Abs in SLE but is not required for reactivity against apoptotic cells, dsDNA, chromatin, anti-nuclear Abs, or cardiolipin. Given that the lupus memory compartment contains a majority of HP+ VH4-34 cells but decreased B cell reactivity, additional HP-dependent Ags must participate in the selection of this compartment. This study represents the first analysis, to our knowledge, of VH-restricted autoreactive B cells specifically expanded in SLE and provides the foundation to understand the antigenic forces at play in this disease. PMID:24108696

Predicting skin sensitization potential and inter-laboratory reproducibility of a human Cell Line Activation Test (h-CLAT) in the European Cosmetics Association (COLIPA) ring trials.

PubMed

Sakaguchi, Hitoshi; Ryan, Cindy; Ovigne, Jean-Marc; Schroeder, Klaus R; Ashikaga, Takao

2010-09-01

Regulatory policies in Europe prohibited the testing of cosmetic ingredients in animals for a number of toxicological endpoints. Currently no validated non-animal test methods exist for skin sensitization. Evaluation of changes in cell surface marker expression in dendritic cell (DC)-surrogate cell lines represents one non-animal approach. The human Cell Line Activation Test (h-CLAT) examines the level of CD86 and CD54 expression on the surface of THP-1 cells, a human monocytic leukemia cell line, following 24h of chemical exposure. To examine protocol transferability, between-lab reproducibility, and predictive capacity, the h-CLAT has been evaluated by five independent laboratories in several ring trials (RTs) coordinated by the European Cosmetics Association (COLIPA). The results of the first and second RTs demonstrated that the protocol was transferable and basically had good between-lab reproducibility and predictivity, but there were some false negative data. To improve performance, protocol and prediction model were modified. Using the modified prediction model in the first and second RT, accuracy was improved. However, about 15% of the outcomes were not correctly identified, which exposes some of the limitations of the assay. For the chemicals evaluated, the limitation may due to chemical being a weak allergen or having low solubility (ex. alpha-hexylcinnamaldehyde). The third RT evaluated the modified prediction model and satisfactory results were obtained. From the RT data, the feasibility of utilizing cell lines as surrogate DC in development of in vitro skin sensitization methods shows promise. The data also support initiating formal pre-validation of the h-CLAT in order to fully understand the capabilities and limitations of the assay. Copyright 2010 Elsevier Ltd. All rights reserved.

The preservation of living cells with biocompatible microparticles

NASA Astrophysics Data System (ADS)

Yang, Jing; Zhu, Yingnan; Xu, Tong; Pan, Chao; Cai, Nana; Huang, He; Zhang, Lei

2016-07-01

Biomedical applications of living cells have rapidly expanded in many fields such as toxic detection, drug screening, and regenerative medicine, etc. Efficient methods to support cell survival and maintain activity in vitro have become increasingly important. However, traditional cryopreservation for living cell-based applications is limited by several problems. Here, we report that magnetic hydrogel microparticles can physically assemble into a 3D environment for efficient cell preservation in physiological conditions, avoiding any chemical reactions that would damage the cells. Two representative cell lines (loosely and firmly adherent) were tested to evaluate the versatility of this method. The results showed that cell longevity was significantly extended to at least 15 days, while the control cell samples without microparticles quickly died within 3 days. Moreover, after preservation, cells can be easily retrieved by applying a magnet to separate the magnetic particles. This strategy can also inhibit cell over-proliferation while avoiding the use of temperature extremes or toxic cryoprotectants that are essential in cryopreservation.

Dependence of Impedance of Embedded Single Cells on Cellular Behaviour.

PubMed

Cho, Sungbo; Castellarnau, Marc; Samitier, Josep; Thielecke, Hagen

2008-02-21

Non-invasive single cell analyses are increasingly required for the medicaldiagnostics of test substances or the development of drugs and therapies on the single celllevel. For the non-invasive characterisation of cells, impedance spectroscopy whichprovides the frequency dependent electrical properties has been used. Recently,microfludic systems have been investigated to manipulate the single cells and tocharacterise the electrical properties of embedded cells. In this article, the impedance ofpartially embedded single cells dependent on the cellular behaviour was investigated byusing the microcapillary. An analytical equation was derived to relate the impedance ofembedded cells with respect to the morphological and physiological change ofextracellular interface. The capillary system with impedance measurement showed afeasibility to monitor the impedance change of embedded single cells caused bymorphological and physiological change of cell during the addition of DMSO. By fittingthe derived equation to the measured impedance of cell embedded at different negativepressure levels, it was able to extrapolate the equivalent gap and gap conductivity betweenthe cell and capillary wall representing the cellular behaviour.

Prevalidation of a model for predicting acute neutropenia by colony forming unit granulocyte/macrophage (CFU-GM) assay.

PubMed

Pessina, A; Albella, B; Bueren, J; Brantom, P; Casati, S; Gribaldo, L; Croera, C; Gagliardi, G; Foti, P; Parchment, R; Parent-Massin, D; Sibiril, Y; Van Den Heuvel, R

2001-12-01

This report describes an international prevalidation study conducted to optimise the Standard Operating Procedure (SOP) for detecting myelosuppressive agents by CFU-GM assay and to study a model for predicting (by means of this in vitro hematopoietic assay) the acute xenobiotic exposure levels that cause maximum tolerated decreases in absolute neutrophil counts (ANC). In the first phase of the study (Protocol Refinement), two SOPs were assessed, by using two cell culture media (Test A, containing GM-CSF; and Test B, containing G-CSF, GM-CSF, IL-3, IL-6 and SCF), and the two tests were applied to cells from both human (bone marrow and umbilical cord blood) and mouse (bone marrow) CFU-GM. In the second phase (Protocol Transfer), the SOPs were transferred to four laboratories to verify the linearity of the assay response and its interlaboratory reproducibility. After a further phase (Protocol Performance), dedicated to a training set of six anticancer drugs (adriamycin, flavopindol, morpholino-doxorubicin, pyrazoloacridine, taxol and topotecan), a model for predicting neutropenia was verified. Results showed that the assay is linear under SOP conditions, and that the in vitro endpoints used by the clinical prediction model of neutropenia are highly reproducible within and between laboratories. Valid tests represented 95% of all tests attempted. The 90% inhibitory concentration values (IC(90)) from Test A and Test B accurately predicted the human maximum tolerated dose (MTD) for five of six and for four of six myelosuppressive anticancer drugs, respectively, that were selected as prototype xenobiotics. As expected, both tests failed to accurately predict the human MTD of a drug that is a likely protoxicant. It is concluded that Test A offers significant cost advantages compared to Test B, without any loss of performance or predictive accuracy. On the basis of these results, we proposed a formal Phase II validation study using the Test A SOP for 16-18 additional xenobiotics that represent the spectrum of haematotoxic potential.

Two is better than one: advances in pathogen-boosted immunotherapy and adoptive T-cell therapy.

PubMed

Xin, Gang; Schauder, David M; Zander, Ryan; Cui, Weiguo

2017-09-01

The recent tremendous successes in clinical trials take cancer immunotherapy into a new era and have attracted major attention from both academia and industry. Among the variety of immunotherapy strategies developed to boost patients' own immune systems to fight against malignant cells, the pathogen-based and adoptive cell transfer therapies have shown the most promise for treating multiple types of cancer. Pathogen-based therapies could either break the immune tolerance to enhance the effectiveness of cancer vaccines or directly infect and kill cancer cells. Adoptive cell transfer can induce a strong durable antitumor response, with recent advances including engineering dual specificity into T cells to recognize multiple antigens and improving the metabolic fitness of transferred cells. In this review, we focus on the recent prospects in these two areas and summarize some ongoing studies that represent potential advancements for anticancer immunotherapy, including testing combinations of these two strategies.

The effect of fibrin on cultured vascular endothelial cells.

PubMed

Kadish, J L; Butterfield, C E; Folkman, J

1979-01-01

The normal cobblestone monolayer architecture of cultured vascular endothelium becomes rapidly disorganized after contact of the cell layer with a fibrin clot. The cells of a confluent endothelial monolayer separate into individual migratory cells in 4--6 hr after contact with fibrin. The effect is reversible in that removal of the fibrin clot results in resumption of the normal morphology within about 2 hr. No other cell type tested exhibits the same change in organization when exposed to fibrin. A similar morphological change in endothelium does occur after the cell layer is overlaid with a collagen fibril gel but a gel of methylcellulose has no effect. It is proposed that the change in behavior of endothelial cells in response to contact with fibrin may represent a cellular component of fibrinolysis. The implications of this finding for the pathophysiology of disease states involving intravascular fibrin deposition are discussed.

Preliminary Low Temperature Electron Irradiation of Triple Junction Solar Cells

NASA Technical Reports Server (NTRS)

Stella, Paul M.; Mueller, Robert L.; Scrivner, Roy L.; Helizon, Roger S.

2007-01-01

For many years extending solar power missions far from the sun has been a challenge not only due to the rapid falloff in solar intensity (intensity varies as inverse square of solar distance) but also because some of the solar cells in an array may exhibit a LILT (low intensity low temperature) degradation that reduces array performance. Recent LILT tests performed on commercial triple junction solar cells have shown that high performance can be obtained at solar distances as great as approx. 5 AU1. As a result, their use for missions going far from the sun has become very attractive. One additional question that remains is whether the radiation damage experienced by solar cells under low temperature conditions will be more severe than when measured during room temperature radiation tests where thermal annealing may take place. This is especially pertinent to missions such as the New Frontiers mission Juno, which will experience cell irradiation from the trapped electron environment at Jupiter. Recent testing2 has shown that low temperature proton irradiation (10 MeV) produces cell degradation results similar to room temperature irradiations and that thermal annealing does not play a factor. Although it is suggestive to propose the same would be observed for low temperature electron irradiations, this has not been verified. JPL has routinely performed radiation testing on commercial solar cells and has also performed LILT testing to characterize cell performance under far sun operating conditions. This research activity was intended to combine the features of both capabilities to investigate the possibility of any room temperature annealing that might influence the measured radiation damage. Although it was not possible to maintain the test cells at a constant low temperature between irradiation and electrical measurements, it was possible to obtain measurements with the cell temperature kept well below room temperature. A fluence of 1E15 1MeV electrons was selected as representative of a moderately high dose that might be expected for a solar powered mission. Fluences much greater than this would require large increases in array area and mass, compromising the ability of PV to compete with non-solar alternatives.

Low pulse energy Nd:YAG laser irradiation exerts a biostimulative effect on different cells of the oral microenvironment: "an in vitro study".

PubMed

Chellini, Flaminia; Sassoli, Chiara; Nosi, Daniele; Deledda, Cristiana; Tonelli, Paolo; Zecchi-Orlandini, Sandra; Formigli, Lucia; Giannelli, Marco

2010-08-01

Dental lasers represent a promising therapeutic tool in the treatment of periodontal and peri-implant diseases. However, their clinical application remains still limited. Here, we investigated the potential biostimulatory effect of low pulse energy neodymium:yttrium-aluminum-garnet (Nd:YAG) laser irradiation on different cells representative of the oral microenvironment and elucidated the underlying molecular mechanisms. Saos-2 osteoblasts, H-end endothelial cells, and NIH/3T3 fibroblasts pre-treated or not with photosensitizing dye methylene blue (MB), were irradiated with low pulse energy (20 mJ) and high repetition rate (50-70 Hz) Nd:YAG laser, and evaluated for cell viability and proliferation as well as for the expression of specific differentiation markers by confocal immunofluorescence and real-time RT-PCR. Changes in intracellular Ca(2+) levels after laser exposure were also evaluated in living osteoblasts. Nd:YAG laser irradiation did not affect cell viability in all the tested cell types, even when combined with pre-treatment with MB, and efficiently stimulated cell growth in the non-sensitized osteoblasts. Moreover, a significant induction in the expression of osteopontin, ALP, and Runx2 in osteoblasts, type I collagen in fibroblasts, and vinculin in endothelial cells could be observed in the irradiated cells. Pre-treatment with MB negatively affected cell differentiation in the unstimulated and laser-stimulated cells. Notably, laser irradiation also caused an increase in the intracellular Ca(2+) in osteoblasts through the activation of TRPC1 ion channels. Moreover, the pharmacologic or genetic inhibition of these channels strongly attenuated laser-induced osteopontin expression, suggesting a role for the laser-mediated Ca(2+) influx in regulating osteoblast differentiation. Low pulse energy and high repetition rate Nd:YAG laser irradiation may exert a biostimulative effect on different cells representative of the oral microenvironment, particularly osteoblasts. Pre-treatment with MB prior to irradiation hampers this effect and limits the potential clinical application of photosensitizing dyes in dental practice. (c) 2010 Wiley-Liss, Inc.

Evaluation of the Human Host Range of Bovine and Porcine Viruses that may Contaminate Bovine Serum and Porcine Trypsin Used in the Manufacture of Biological Products

PubMed Central

Marcus-Sekura, Carol; Richardson, James C.; Harston, Rebecca K.; Sane, Nandini; Sheets, Rebecca L.

2011-01-01

Current U.S. requirements for testing cell substrates used in production of human biological products for contamination with bovine and porcine viruses are U.S. Department of Agriculture (USDA) 9CFR tests for bovine serum or porcine trypsin. 9CFR requires testing of bovine serum for seven specific viruses in six families (immunofluorescence) and at least 2 additional families non-specifically (cytopathicity and hemadsorption). 9CFR testing of porcine trypsin is for porcine parvovirus. Recent contaminations suggest these tests may not be sufficient. Assay sensitivity was not the issue for these contaminations that were caused by viruses/virus families not represented in the 9CFR screen. A detailed literature search was undertaken to determine which viruses that infect cattle or swine or bovine or porcine cells in culture also have human host range [ability to infect humans or human cells in culture] and to predict their detection by the currently used 9CFR procedures. There are more viruses of potential risk to biological products manufactured using bovine or porcine raw materials than are likely to be detected by 9CFR testing procedures; even within families, not all members would necessarily be detected. Testing gaps and alternative methodologies should be evaluated to continue to ensure safe, high quality human biologicals. PMID:22000165

Epithelial toxicity of alkylglycoside surfactants.

PubMed

Vllasaliu, Driton; Shubber, Saif; Fowler, Robyn; Garnett, Martin; Alexander, Cameron; Stolnik, Snow

2013-01-01

Alkylglycoside surfactants have been proposed as drug delivery excipients with the potential to enhance mucosal drug absorption of therapeutic macromolecules. Previous work reported their drug absorption-promoting potential by demonstrating that several compounds within this class of surfactants improve mucosal absorption of peptides, proteins and other macromolecules. However, detailed investigation of their toxicity has not been conducted. Using Calu-3 epithelial cell layers as a model of the airway mucosa, and liposomes as models of cell membranes, this work investigates the cytotoxicity of dodecylmaltoside, tridecylmaltoside and tetradecylmaltoside, as representative alkylglycosides. A combination of different toxicity assays and other tests indicating cell membrane disruption were used to assess cytotoxicity. The alkylglycosides tested induced a dramatic reduction in cell viability, cell membrane and liposome-disruptive effects, as well as abrogation of transepithelial electrical resistance that did not recover completely. Importantly, these phenomena were noted at concentrations markedly lower than those typically used in the literature studies demonstrating the absorption-enhancing properties of alkylglycosides. This work therefore demonstrates that alkylglycosides exhibit significant toxicity towards airway epithelial cells, most likely resulting from a membrane-damaging effect, highlighting a need for further evaluation of their safety as absorption-enhancing excipients. Copyright © 2012 Wiley Periodicals, Inc.

Analyzing Study of Path loss Propagation Models in Wireless Communications at 0.8 GHz

NASA Astrophysics Data System (ADS)

Kadhim Hoomod, Haider; Al-Mejibli, Intisar; Issa Jabboory, Abbas

2018-05-01

The paths loss propagation model is an important tool in wireless network planning, allowing network planner to optimize the cell towers distribution and meet expected service level requirements. However, each type of path loss propagation model is designed to predict path loss in a particular environment that may be inaccurate in other different environment. In this research different propagation models (Hata Model, ICC-33 Model, Ericson Model and Coast-231 Model) have been analyzed and compared based on the measured data. The measured data represent signal strength of two cell towers placed in two different environments which obtained by a drive test of them. First one in AL-Habebea represents an urban environment (high-density region) and the second in AL-Hindea district represents a rural environment (low-density region) with operating frequency 0.8 GHz. The results of performing the analysis and comparison conclude that Hata model and Ericsson model shows small deviation from real measurements in urban environment and Hata model generally gives better prediction in the rural environment.

Correlations Between Life-Detection Techniques and Implications for Sampling Site Selection in Planetary Analog Missions

NASA Astrophysics Data System (ADS)

Gentry, Diana M.; Amador, Elena S.; Cable, Morgan L.; Chaudry, Nosheen; Cullen, Thomas; Jacobsen, Malene B.; Murukesan, Gayathri; Schwieterman, Edward W.; Stevens, Adam H.; Stockton, Amanda; Tan, George; Yin, Chang; Cullen, David C.; Geppert, Wolf

2017-10-01

We conducted an analog sampling expedition under simulated mission constraints to areas dominated by basaltic tephra of the Eldfell and Fimmvörðuháls lava fields (Iceland). Sites were selected to be "homogeneous" at a coarse remote sensing resolution (10-100 m) in apparent color, morphology, moisture, and grain size, with best-effort realism in numbers of locations and replicates. Three different biomarker assays (counting of nucleic-acid-stained cells via fluorescent microscopy, a luciferin/luciferase assay for adenosine triphosphate, and quantitative polymerase chain reaction (qPCR) to detect DNA associated with bacteria, archaea, and fungi) were characterized at four nested spatial scales (1 m, 10 m, 100 m, and >1 km) by using five common metrics for sample site representativeness (sample mean variance, group F tests, pairwise t tests, and the distribution-free rank sum H and u tests). Correlations between all assays were characterized with Spearman's rank test. The bioluminescence assay showed the most variance across the sites, followed by qPCR for bacterial and archaeal DNA; these results could not be considered representative at the finest resolution tested (1 m). Cell concentration and fungal DNA also had significant local variation, but they were homogeneous over scales of >1 km. These results show that the selection of life detection assays and the number, distribution, and location of sampling sites in a low biomass environment with limited a priori characterization can yield both contrasting and complementary results, and that their interdependence must be given due consideration to maximize science return in future biomarker sampling expeditions.

A thermodynamic assay to test pharmacological chaperones for Fabry disease.

PubMed

Andreotti, Giuseppina; Citro, Valentina; Correra, Antonella; Cubellis, Maria Vittoria

2014-03-01

The majority of the disease-causing mutations affect protein stability, but not functional sites and are amenable, in principle, to be treated with pharmacological chaperones. These drugs enhance the thermodynamic stability of their targets. Fabry disease, a disorder caused by mutations in the gene encoding lysosomal alpha-galactosidase, represents an excellent model system to develop experimental protocols to test the efficiency of such drugs. The stability of lysosomal alpha-galactosidase under different conditions was studied by urea-induced unfolding followed by limited proteolysis and Western blotting. We measured the concentration of urea needed to obtain half-maximal unfolding because this parameter represents an objective indicator of protein stability. Urea-induced unfolding is a versatile technique that can be adapted to cell extracts containing tiny amounts of wild-type or mutant proteins. It allows testing of protein stability as a function of pH, in the presence or in the absence of drugs. Results are not influenced by the method used to express the protein in transfected cells. Scarce and dispersed populations pose a problem for the clinical trial of drugs for rare diseases. This is particularly true for pharmacological chaperones that must be tested on each mutation associated with a given disease. Diverse in vitro tests are needed. We used a method based on chemically induced unfolding as a tool to assess whether a particular Fabry mutation is responsive to pharmacological chaperones, but, by no means is our protocol limited to this disease. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Dynamic Finite Element Predictions for Mars Sample Return Cellular Impact Test #4

NASA Technical Reports Server (NTRS)

Fasanella, Edwin L.; Billings, Marcus D.

2001-01-01

The nonlinear finite element program MSC.Dytran was used to predict the impact pulse for (he drop test of an energy absorbing cellular structure. This pre-test simulation was performed to aid in the design of an energy absorbing concept for a highly reliable passive Earth Entry Vehicle (EEV) that will directly impact the Earth without a parachute. In addition, a goal of the simulation was to bound the acceleration pulse produced and delivered to the simulated space cargo container. EEV's are designed to return materials from asteroids, comets, or planets for laboratory analysis on Earth. The EEV concept uses an energy absorbing cellular structure designed to contain and limit the acceleration of space exploration samples during Earth impact. The spherical shaped cellular structure is composed of solid hexagonal and pentagonal foam-filled cells with hybrid graphite-epoxy/Kevlar cell walls. Space samples fit inside a smaller sphere at the enter of the EEV's cellular structure. The material models and failure criteria were varied to determine their effect on the resulting acceleration pulse. Pre-test analytical predictions using MSC.Dytran were compared with the test results obtained from impact test #4 using bungee accelerator located at the NASA Langley Research Center Impact Dynamics Research Facility. The material model used to represent the foam and the proper failure criteria for the cell walls were critical in predicting the impact loads of the cellular structure. It was determined that a FOAMI model for the foam and a 20% failure strain criteria for the cell walls gave an accurate prediction of the acceleration pulse for drop test #4.

Niche players

PubMed Central

Seandel, Marco; Falciatori, Ilaria; Shmelkov, Sergey V.; Kim, Jiyeon; James, Daylon; Rafii, Shahin

2010-01-01

The undifferentiated spermatogonia of adult mouse testes are composed of both true stem cells and committed progenitors. It is unclear what normally prevents these adult germ cells from manifesting multipotency. The critical elements of the spermatogonial stem cell niche, while poorly understood, are thought to be composed of Sertoli cells with several other somatic cell types in close proximity. We recently discovered a novel orphan G-protein coupled receptor (GPR125) that is restricted to undifferentiated spermatogonia within the testis. GPR125 expression was maintained when the progenitor cells were extracted from the in vivo niche and propagated under growth conditions that recapitulate key elements of the niche. Such conditions preserved the ability of the cells to generate multipotent derivatives, known as multipotent adult spermatogonial derived progenitor cells (MASCs). Upon differentiation, the latter produced a variety tissues including functional endothelium, illustrating the potential applications of such cells. Thus, GPR125 represents a novel target for purifying adult stem and progenitors from tissues, with the goal of developing autologous multipotent cell lines. PMID:18256534

Absence of micronucleus formation in CHO-K1 cells cultivated in platelet lysate enriched medium.

PubMed

Bernardi, Martina; Adami, Valentina; Albiero, Elena; Madeo, Domenico; Rodeghiero, Francesco; Astori, Giuseppe

2014-03-01

Human platelet lysate (PL) represents an effective substitute of fetal bovine serum (FBS) for mesenchymal stromal cell (MSC) cultivation. Compared to FBS, PL favors MSC proliferation significantly shortening the population doubling time and avoiding the risks related to the use of animal derivatives. Growth factors contained in the platelets are released upon platelet disruption following freezing/thawing cycles or as we have recently described by using ultrasound. We have investigated whether the increased cell proliferation achieved by using PL could induce mitotic stress and whether the potential formation of free radicals during PL production by ultrasound could cause chromosomal instability in mammalian cells. We have applied an image analysis assisted high content screening (HCS) in vitro micronucleus assay in the Chinese Hamster Ovarian K1 (CHO-K1) rodent mammalian cell line. PL was produced by sonication; for the micronucleus assay, CHO-K1 cells were exposed to increasing concentrations of PL. Cytokinesis was blocked by cytochalasin B, nuclei were stained with bisbenzimide and images were acquired and analyzed automatically using an HCS system, both with a 20× and a 10× objective. Our results suggest that growth stimulus induced by the use of PL did not significantly increase micronucleus formation in CHO-K1 cells compared to negative control. Micronucleus testing in conjunction with HCS could represent a valid tool to evaluate the safety of ancillary materials used in the production of cell-based medicinal products. Copyright © 2013 Elsevier GmbH. All rights reserved.

Digital PCR Improves Mutation Analysis in Pancreas Fine Needle Aspiration Biopsy Specimens.

PubMed

Sho, Shonan; Court, Colin M; Kim, Stephen; Braxton, David R; Hou, Shuang; Muthusamy, V Raman; Watson, Rabindra R; Sedarat, Alireza; Tseng, Hsian-Rong; Tomlinson, James S

2017-01-01

Applications of precision oncology strategies rely on accurate tumor genotyping from clinically available specimens. Fine needle aspirations (FNA) are frequently obtained in cancer management and often represent the only source of tumor tissues for patients with metastatic or locally advanced diseases. However, FNAs obtained from pancreas ductal adenocarcinoma (PDAC) are often limited in cellularity and/or tumor cell purity, precluding accurate tumor genotyping in many cases. Digital PCR (dPCR) is a technology with exceptional sensitivity and low DNA template requirement, characteristics that are necessary for analyzing PDAC FNA samples. In the current study, we sought to evaluate dPCR as a mutation analysis tool for pancreas FNA specimens. To this end, we analyzed alterations in the KRAS gene in pancreas FNAs using dPCR. The sensitivity of dPCR mutation analysis was first determined using serial dilution cell spiking studies. Single-cell laser-microdissection (LMD) was then utilized to identify the minimal number of tumor cells needed for mutation detection. Lastly, dPCR mutation analysis was performed on 44 pancreas FNAs (34 formalin-fixed paraffin-embedded (FFPE) and 10 fresh (non-fixed)), including samples highly limited in cellularity (100 cells) and tumor cell purity (1%). We found dPCR to detect mutations with allele frequencies as low as 0.17%. Additionally, a single tumor cell could be detected within an abundance of normal cells. Using clinical FNA samples, dPCR mutation analysis was successful in all preoperative FNA biopsies tested, and its accuracy was confirmed via comparison with resected tumor specimens. Moreover, dPCR revealed additional KRAS mutations representing minor subclones within a tumor that were not detected by the current clinical gold standard method of Sanger sequencing. In conclusion, dPCR performs sensitive and accurate mutation analysis in pancreas FNAs, detecting not only the dominant mutation subtype, but also the additional rare mutation subtypes representing tumor heterogeneity.

Digital PCR Improves Mutation Analysis in Pancreas Fine Needle Aspiration Biopsy Specimens

PubMed Central

Court, Colin M.; Kim, Stephen; Braxton, David R.; Hou, Shuang; Muthusamy, V. Raman; Watson, Rabindra R.; Sedarat, Alireza; Tseng, Hsian-Rong; Tomlinson, James S.

2017-01-01

Applications of precision oncology strategies rely on accurate tumor genotyping from clinically available specimens. Fine needle aspirations (FNA) are frequently obtained in cancer management and often represent the only source of tumor tissues for patients with metastatic or locally advanced diseases. However, FNAs obtained from pancreas ductal adenocarcinoma (PDAC) are often limited in cellularity and/or tumor cell purity, precluding accurate tumor genotyping in many cases. Digital PCR (dPCR) is a technology with exceptional sensitivity and low DNA template requirement, characteristics that are necessary for analyzing PDAC FNA samples. In the current study, we sought to evaluate dPCR as a mutation analysis tool for pancreas FNA specimens. To this end, we analyzed alterations in the KRAS gene in pancreas FNAs using dPCR. The sensitivity of dPCR mutation analysis was first determined using serial dilution cell spiking studies. Single-cell laser-microdissection (LMD) was then utilized to identify the minimal number of tumor cells needed for mutation detection. Lastly, dPCR mutation analysis was performed on 44 pancreas FNAs (34 formalin-fixed paraffin-embedded (FFPE) and 10 fresh (non-fixed)), including samples highly limited in cellularity (100 cells) and tumor cell purity (1%). We found dPCR to detect mutations with allele frequencies as low as 0.17%. Additionally, a single tumor cell could be detected within an abundance of normal cells. Using clinical FNA samples, dPCR mutation analysis was successful in all preoperative FNA biopsies tested, and its accuracy was confirmed via comparison with resected tumor specimens. Moreover, dPCR revealed additional KRAS mutations representing minor subclones within a tumor that were not detected by the current clinical gold standard method of Sanger sequencing. In conclusion, dPCR performs sensitive and accurate mutation analysis in pancreas FNAs, detecting not only the dominant mutation subtype, but also the additional rare mutation subtypes representing tumor heterogeneity. PMID:28125707

In vitro antitumor actions of extracts from endemic plant Helichrysum zivojinii

PubMed Central

2013-01-01

Background The aim of this research was to determine the intensity and mechanisms of the cytotoxic actions of five extracts isolated from the endemic plant species Helichrysum zivojinii Černjavski & Soška (family Asteraceae) against specific cancer cell lines. In order to evaluate the sensitivity of normal immunocompetent cells implicated in the antitumor immune response, the cytotoxicity of extracts was also tested against healthy peripheral blood mononuclear cells (PBMC). Methods The aerial parts of the plants were air-dried, powdered, and successively extracted with solvents of increasing polarity to obtain hexane, dichloromethane, ethyl-acetate, n-butanol and methanol extracts. The cytotoxic activities of the extracts against human cervix adenocarcinoma HeLa, human melanoma Fem-x, human myelogenous leukemia K562, human breast adenocarcinoma MDA-MB-361 cells and PBMC were evaluated by the MTT test. The mode of HeLa cell death was investigated by morphological analysis. Changes in the cell cycle of HeLa cells treated with the extracts were analyzed by flow cytometry. The apoptotic mechanisms induced by the tested extracts were determined using specific caspase inhibitors. Results The investigated Helichrysum zivojinii extracts exerted selective dose-dependent cytotoxic actions against selected cancer cell lines and healthy immunocompetent PBMC stimulated to proliferate, while the cytotoxic actions exerted on unstimulated PBMC were less pronounced. The tested extracts exhibited considerably stronger cytotoxic activities towards HeLa, Fem-x and K562 cells in comparison to resting and stimulated PBMC. It is worth noting that the cytotoxicity of the extracts was weaker against unstimulated PBMC in comparison to stimulated PBMC. Furthermore, each of the five extracts induced apoptosis in HeLa cells, through the activation of both intrinsic and extrinsic signaling pathways. Conclusion Extracts obtained from the endemic plant Helichrysum zivojinii may represent an important source of novel potential antitumor agents due to their pronounced and selective cytotoxic actions towards malignant cells. PMID:23414290

In vitro antitumor actions of extracts from endemic plant Helichrysum zivojinii.

PubMed

Matić, Ivana Z; Aljančić, Ivana; Žižak, Željko; Vajs, Vlatka; Jadranin, Milka; Milosavljević, Slobodan; Juranić, Zorica D

2013-02-18

The aim of this research was to determine the intensity and mechanisms of the cytotoxic actions of five extracts isolated from the endemic plant species Helichrysum zivojinii Černjavski & Soška (family Asteraceae) against specific cancer cell lines. In order to evaluate the sensitivity of normal immunocompetent cells implicated in the antitumor immune response, the cytotoxicity of extracts was also tested against healthy peripheral blood mononuclear cells (PBMC). The aerial parts of the plants were air-dried, powdered, and successively extracted with solvents of increasing polarity to obtain hexane, dichloromethane, ethyl-acetate, n-butanol and methanol extracts. The cytotoxic activities of the extracts against human cervix adenocarcinoma HeLa, human melanoma Fem-x, human myelogenous leukemia K562, human breast adenocarcinoma MDA-MB-361 cells and PBMC were evaluated by the MTT test. The mode of HeLa cell death was investigated by morphological analysis. Changes in the cell cycle of HeLa cells treated with the extracts were analyzed by flow cytometry. The apoptotic mechanisms induced by the tested extracts were determined using specific caspase inhibitors. The investigated Helichrysum zivojinii extracts exerted selective dose-dependent cytotoxic actions against selected cancer cell lines and healthy immunocompetent PBMC stimulated to proliferate, while the cytotoxic actions exerted on unstimulated PBMC were less pronounced. The tested extracts exhibited considerably stronger cytotoxic activities towards HeLa, Fem-x and K562 cells in comparison to resting and stimulated PBMC. It is worth noting that the cytotoxicity of the extracts was weaker against unstimulated PBMC in comparison to stimulated PBMC. Furthermore, each of the five extracts induced apoptosis in HeLa cells, through the activation of both intrinsic and extrinsic signaling pathways. Extracts obtained from the endemic plant Helichrysum zivojinii may represent an important source of novel potential antitumor agents due to their pronounced and selective cytotoxic actions towards malignant cells.

"RCL-Pooling Assay": A Simplified Method for the Detection of Replication-Competent Lentiviruses in Vector Batches Using Sequential Pooling.

PubMed

Corre, Guillaume; Dessainte, Michel; Marteau, Jean-Brice; Dalle, Bruno; Fenard, David; Galy, Anne

2016-02-01

Nonreplicative recombinant HIV-1-derived lentiviral vectors (LV) are increasingly used in gene therapy of various genetic diseases, infectious diseases, and cancer. Before they are used in humans, preparations of LV must undergo extensive quality control testing. In particular, testing of LV must demonstrate the absence of replication-competent lentiviruses (RCL) with suitable methods, on representative fractions of vector batches. Current methods based on cell culture are challenging because high titers of vector batches translate into high volumes of cell culture to be tested in RCL assays. As vector batch size and titers are continuously increasing because of the improvement of production and purification methods, it became necessary for us to modify the current RCL assay based on the detection of p24 in cultures of indicator cells. Here, we propose a practical optimization of this method using a pairwise pooling strategy enabling easier testing of higher vector inoculum volumes. These modifications significantly decrease material handling and operator time, leading to a cost-effective method, while maintaining optimal sensibility of the RCL testing. This optimized "RCL-pooling assay" ameliorates the feasibility of the quality control of large-scale batches of clinical-grade LV while maintaining the same sensitivity.

A microfluidic platform for 3-dimensional cell culture and cell-based assays.

PubMed

Kim, Minseok S; Yeon, Ju Hun; Park, Je-Kyun

2007-02-01

This paper reports a novel microfluidic platform introducing peptide hydrogel to make biocompatible microenvironment as well as realizing in situ cell-based assays. Collagen composite, OPLA and Puramatrix scaffolds are compared to select good environment for human hepatocellular carcinoma cells (HepG2) by albumin measurement. The selected biocompatible self-assembling peptide hydrogel, Puramatrix, is hydrodynamically focused in the middle of main channel of a microfluidic device, and at the same time the cells are 3-dimensionally immobilized and encapsulated without any additional surface treatment. HepG2 cells have been 3-dimensionally cultured in a poly(dimethylsiloxane) (PDMS) microfluidic device for 4 days. The cells cultured in micro peptide scaffold are compared with those cultured by conventional petri dish in morphology and the rate of albumin secretion. By injection of different reagents into either side of the peptide scaffold, the microfluidic device also forms a linear concentration gradient profile across the peptide scaffold due to molecular diffusion. Based on this characteristic, toxicity tests are performed by Triton X-100. As the higher toxicant concentration gradient forms, the wider dead zone of cells in the peptide scaffold represents. This microfluidic platform facilitates in vivo-like 3-dimensional microenvironment, and have a potential for the applications of reliable cell-based screening and assays including cytotoxicity test, real-time cell viability monitoring, and continuous dose-response assay.

The influence of biomechanical properties and cannabinoids on tumor invasion

PubMed Central

Hohmann, Tim; Grabiec, Urszula; Ghadban, Chalid; Feese, Kerstin; Dehghani, Faramarz

2017-01-01

ABSTRACT Background: Cannabinoids are known to have an anti-tumorous effect, but the underlying mechanisms are only sparsely understood. Mechanical characteristics of tumor cells represent a promising marker to distinguish between tumor cells and the healthy tissue. We tested the hypothesis whether cannabinoids influence the tumor cell specific mechanical and migratory properties and if these factors are a prognostic marker for the invasiveness of tumor cells. Methods: 3 different glioblastoma cell lines were treated with cannabinoids and changes of mechanical and migratory properties of single cells were measured using atomic force microscopy and time lapse imaging. The invasiveness of cell lines was determined using a co-culture model with organotypic hippocampal slice cultures. Results: We found that cannabinoids are capable of influencing migratory and mechanical properties in a cell line specific manner. A network analysis revealed a correlation between a “generalized stiffness” and the invasiveness for all tumor cell lines after 3 and 4 d of invasion time: r3d = −0.88 [−0.52;−0.97]; r4d = −0.90 [−0.59;−0.98]. Conclusions: Here we could show that a “generalized stiffness” is a profound marker for the invasiveness of a tumor cell population in our model and thus might be of high clinical relevance for drug testing. Additionally cannabinoids were shown to be of potential use for therapeutic approaches of glioblastoma. PMID:27149140

The price of performance: a cost and performance analysis of the implementation of cell-free fetal DNA testing for Down syndrome in Ontario, Canada.

PubMed

Okun, N; Teitelbaum, M; Huang, T; Dewa, C S; Hoch, J S

2014-04-01

To examine the cost and performance implications of introducing cell-free fetal DNA (cffDNA) testing within modeled scenarios in a publicly funded Canadian provincial Down syndrome (DS) prenatal screening program. Two clinical algorithms were created: the first to represent the current screening program and the second to represent one that incorporates cffDNA testing. From these algorithms, eight distinct scenarios were modeled to examine: (1) the current program (no cffDNA), (2) the current program with first trimester screening (FTS) as the nuchal translucency-based primary screen (no cffDNA), (3) a program substituting current screening with primary cffDNA, (4) contingent cffDNA with current FTS performance, (5) contingent cffDNA at a fixed price to result in overall cost neutrality,(6) contingent cffDNA with an improved detection rate (DR) of FTS, (7) contingent cffDNA with higher uptake of FTS, and (8) contingent cffDNA with optimized FTS (higher uptake and improved DR). This modeling study demonstrates that introducing contingent cffDNA testing improves performance by increasing the number of cases of DS detected prenatally, and reducing the number of amniocenteses performed and concomitant iatrogenic pregnancy loss of pregnancies not affected by DS. Costs are modestly increased, although the cost per case of DS detected is decreased with contingent cffDNA testing. Contingent models of cffDNA testing can improve overall screening performance while maintaining the provision of an 11- to 13-week scan. Costs are modestly increased, but cost per prenatally detected case of DS is decreased. © 2013 John Wiley & Sons, Ltd.

Human organ-on-a-chip BioMEMS devices for testing new diagnostic and therapeutic strategies

NASA Astrophysics Data System (ADS)

Leary, James F.; Key, Jaehong; Vidi, Pierre-Alexandre; Cooper, Christy L.; Kole, Ayeeshik; Reece, Lisa M.; Lelièvre, Sophie A.

2013-03-01

MEMS human "organs-on-a-chip" can be used to create model human organ systems for developing new diagnostic and therapeutic strategies. They represent a promising new strategy for rapid testing of new diagnostic and therapeutic approaches without the need for involving risks to human subjects. We are developing multicomponent, superparamagnetic and fluorescent nanoparticles as X-ray and MRI contrast agents for noninvasive multimodal imaging and for antibody- or peptide-targeted drug delivery to tumor and precancerous cells inside these artificial organ MEMS devices. Magnetic fields can be used to move the nanoparticles "upstream" to find their target cells in an organs-on-achip model of human ductal breast cancer. Theoretically, unbound nanoparticles can then be removed by reversing the magnetic field to give a greatly enhanced image of tumor cells within these artificial organ structures. Using branched PDMS microchannels and 3D tissue engineering of normal and malignant human breast cancer cells inside those MEMS channels, we can mimic the early stages of human ductal breast cancer with the goal to improve the sensitivity and resolution of mammography and MRI of very small tumors and test new strategies for treatments. Nanomedical systems can easily be imaged by multicolor confocal microscopy inside the artificial organs to test targeting and therapeutic responses including the differential viability of normal and tumor cells during treatments. Currently we are using 2-dimensional MEMS structures, but these studies can be extended to more complex 3D structures using new 3D printing technologies.

Acidic conditions induce the suppression of CD86 and CD54 expression in THP-1 cells.

PubMed

Mitachi, Takafumi; Mezaki, Minori; Yamashita, Kunihiko; Itagaki, Hiroshi

2018-01-01

To evaluate the sensitization potential of chemicals in cosmetics, using non-animal methods, a number of in vitro safety tests have been designed. Current assays are based on the expression of cell surface markers, such as CD86 and CD54, which are associated with the activation of dendritic cells, in skin sensitization tests. However, these markers are influenced by culture conditions through activating danger signals. In this study, we investigated the relationship between extracellular pH and the expression of the skin sensitization test human cell line activation test (h-CLAT) markers CD86 and CD54. We measured expression levels after THP-1 cells were exposed to representative contact allergens, i.e., 2,4-dinitrochlorobenzene and imidazolidinyl urea, under acidic conditions. These conditions were set by exposure to hydrochloric acid, lactic acid, and citric acid. An acidic extracellular pH (6-7) suppressed the augmentation of CD86 and CD54 levels by the sensitizer. Additionally, when the CD86/CD54 expression levels were suppressed, a reduction in the intracellular pH was confirmed. Furthermore, we observed that Na + /H + exchanger 1 (NHE-1), a protein that contributes to the regulation of extracellular/intracellular pH, is involved in CD86 and CD54 expression. These findings suggest that the extracellular/intracellular pH has substantial effects on in vitro skin sensitization markers and should be considered in evaluations of the safety of mixtures and commercial products in the future.

Renal pathophysiologic role of cortical tubular inclusion bodies.

PubMed

Radi, Zaher A; Stewart, Zachary S; Grzemski, Felicity A; Bobrowski, Walter F

2013-01-01

Renal tubular inclusion bodies are rarely associated with drug administration. The authors describe the finding of renal cortical tubular intranuclear and intracytoplasmic inclusion bodies associated with the oral administration of a norepinephrine/serotonin reuptake inhibitor (NSRI) test article in Sprague-Dawley (SD) rats. Rats were given an NSRI daily for 4 weeks, and kidney histopathologic, ultrastructural pathology, and immunohistochemical examinations were performed. Round eosinophilic intranuclear inclusion bodies were observed histologically in the tubular epithelial cells of the renal cortex in male and female SD rats given the NSRI compound. No evidence of degeneration or necrosis was noted in the inclusion-containing renal cells. By ultrastructural pathology, inclusion bodies consisted of finely granular, amorphous, and uniformly stained nonmembrane-bound material. By immunohistochemistry, inclusion bodies stained positive for d-amino acid oxidase (DAO) protein. In addition, similar inclusion bodies were noted in the cytoplasmic tubular epithelial compartment by ultrastructural and immunohistochemical examination.  This is the first description of these renal inclusion bodies after an NSRI test article administration in SD rats. Such drug-induced renal inclusion bodies are rat-specific, do not represent an expression of nephrotoxicity, represent altered metabolism of d-amino acids, and are not relevant to human safety risk assessment.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao, Xiugong, E-mail: xiugong.gao@fda.hhs.gov; Sprando, Robert L.; Yourick, Jeffrey J.

Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72 h after exposuremore » to 0.25 mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment. - Highlights: • Studied genomic changes in mouse embryonic stem cells upon thalidomide exposure • Identified gene expression changes that may represent thalidomide embryotoxicity • The toxicogenomic changes coincide well with known thalidomide clinical outcomes. • The mouse embryonic stem cell model is suitable for developmental toxicity testing. • The model has the potential for high-throughput screening of a multitude of compounds.« less

Analyzing thematic maps and mapping for accuracy

USGS Publications Warehouse

Rosenfield, G.H.

1982-01-01

Two problems which exist while attempting to test the accuracy of thematic maps and mapping are: (1) evaluating the accuracy of thematic content, and (2) evaluating the effects of the variables on thematic mapping. Statistical analysis techniques are applicable to both these problems and include techniques for sampling the data and determining their accuracy. In addition, techniques for hypothesis testing, or inferential statistics, are used when comparing the effects of variables. A comprehensive and valid accuracy test of a classification project, such as thematic mapping from remotely sensed data, includes the following components of statistical analysis: (1) sample design, including the sample distribution, sample size, size of the sample unit, and sampling procedure; and (2) accuracy estimation, including estimation of the variance and confidence limits. Careful consideration must be given to the minimum sample size necessary to validate the accuracy of a given. classification category. The results of an accuracy test are presented in a contingency table sometimes called a classification error matrix. Usually the rows represent the interpretation, and the columns represent the verification. The diagonal elements represent the correct classifications. The remaining elements of the rows represent errors by commission, and the remaining elements of the columns represent the errors of omission. For tests of hypothesis that compare variables, the general practice has been to use only the diagonal elements from several related classification error matrices. These data are arranged in the form of another contingency table. The columns of the table represent the different variables being compared, such as different scales of mapping. The rows represent the blocking characteristics, such as the various categories of classification. The values in the cells of the tables might be the counts of correct classification or the binomial proportions of these counts divided by either the row totals or the column totals from the original classification error matrices. In hypothesis testing, when the results of tests of multiple sample cases prove to be significant, some form of statistical test must be used to separate any results that differ significantly from the others. In the past, many analyses of the data in this error matrix were made by comparing the relative magnitudes of the percentage of correct classifications, for either individual categories, the entire map or both. More rigorous analyses have used data transformations and (or) two-way classification analysis of variance. A more sophisticated step of data analysis techniques would be to use the entire classification error matrices using the methods of discrete multivariate analysis or of multiviariate analysis of variance.

Artificial Symmetry-Breaking for Morphogenetic Engineering Bacterial Colonies.

PubMed

Nuñez, Isaac N; Matute, Tamara F; Del Valle, Ilenne D; Kan, Anton; Choksi, Atri; Endy, Drew; Haseloff, Jim; Rudge, Timothy J; Federici, Fernan

2017-02-17

Morphogenetic engineering is an emerging field that explores the design and implementation of self-organized patterns, morphologies, and architectures in systems composed of multiple agents such as cells and swarm robots. Synthetic biology, on the other hand, aims to develop tools and formalisms that increase reproducibility, tractability, and efficiency in the engineering of biological systems. We seek to apply synthetic biology approaches to the engineering of morphologies in multicellular systems. Here, we describe the engineering of two mechanisms, symmetry-breaking and domain-specific cell regulation, as elementary functions for the prototyping of morphogenetic instructions in bacterial colonies. The former represents an artificial patterning mechanism based on plasmid segregation while the latter plays the role of artificial cell differentiation by spatial colocalization of ubiquitous and segregated components. This separation of patterning from actuation facilitates the design-build-test-improve engineering cycle. We created computational modules for CellModeller representing these basic functions and used it to guide the design process and explore the design space in silico. We applied these tools to encode spatially structured functions such as metabolic complementation, RNAPT7 gene expression, and CRISPRi/Cas9 regulation. Finally, as a proof of concept, we used CRISPRi/Cas technology to regulate cell growth by controlling methionine synthesis. These mechanisms start from single cells enabling the study of morphogenetic principles and the engineering of novel population scale structures from the bottom up.

Extensive and systematic rewiring of histone post-translational modifications in cancer model systems.

PubMed

Noberini, Roberta; Osti, Daniela; Miccolo, Claudia; Richichi, Cristina; Lupia, Michela; Corleone, Giacomo; Hong, Sung-Pil; Colombo, Piergiuseppe; Pollo, Bianca; Fornasari, Lorenzo; Pruneri, Giancarlo; Magnani, Luca; Cavallaro, Ugo; Chiocca, Susanna; Minucci, Saverio; Pelicci, Giuliana; Bonaldi, Tiziana

2018-05-04

Histone post-translational modifications (PTMs) generate a complex combinatorial code that regulates gene expression and nuclear functions, and whose deregulation has been documented in different types of cancers. Therefore, the availability of relevant culture models that can be manipulated and that retain the epigenetic features of the tissue of origin is absolutely crucial for studying the epigenetic mechanisms underlying cancer and testing epigenetic drugs. In this study, we took advantage of quantitative mass spectrometry to comprehensively profile histone PTMs in patient tumor tissues, primary cultures and cell lines from three representative tumor models, breast cancer, glioblastoma and ovarian cancer, revealing an extensive and systematic rewiring of histone marks in cell culture conditions, which includes a decrease of H3K27me2/me3, H3K79me1/me2 and H3K9ac/K14ac, and an increase of H3K36me1/me2. While some changes occur in short-term primary cultures, most of them are instead time-dependent and appear only in long-term cultures. Remarkably, such changes mostly revert in cell line- and primary cell-derived in vivo xenograft models. Taken together, these results support the use of xenografts as the most representative models of in vivo epigenetic processes, suggesting caution when using cultured cells, in particular cell lines and long-term primary cultures, for epigenetic investigations.

Extensive and systematic rewiring of histone post-translational modifications in cancer model systems

PubMed Central

Noberini, Roberta; Osti, Daniela; Miccolo, Claudia; Richichi, Cristina; Lupia, Michela; Corleone, Giacomo; Hong, Sung-Pil; Colombo, Piergiuseppe; Pollo, Bianca; Fornasari, Lorenzo; Pruneri, Giancarlo; Magnani, Luca; Cavallaro, Ugo; Chiocca, Susanna; Minucci, Saverio; Pelicci, Giuliana; Bonaldi, Tiziana

2018-01-01

Abstract Histone post-translational modifications (PTMs) generate a complex combinatorial code that regulates gene expression and nuclear functions, and whose deregulation has been documented in different types of cancers. Therefore, the availability of relevant culture models that can be manipulated and that retain the epigenetic features of the tissue of origin is absolutely crucial for studying the epigenetic mechanisms underlying cancer and testing epigenetic drugs. In this study, we took advantage of quantitative mass spectrometry to comprehensively profile histone PTMs in patient tumor tissues, primary cultures and cell lines from three representative tumor models, breast cancer, glioblastoma and ovarian cancer, revealing an extensive and systematic rewiring of histone marks in cell culture conditions, which includes a decrease of H3K27me2/me3, H3K79me1/me2 and H3K9ac/K14ac, and an increase of H3K36me1/me2. While some changes occur in short-term primary cultures, most of them are instead time-dependent and appear only in long-term cultures. Remarkably, such changes mostly revert in cell line- and primary cell-derived in vivo xenograft models. Taken together, these results support the use of xenografts as the most representative models of in vivo epigenetic processes, suggesting caution when using cultured cells, in particular cell lines and long-term primary cultures, for epigenetic investigations. PMID:29618087

Human dental pulp pluripotent-like stem cells promote wound healing and muscle regeneration.

PubMed

Martínez-Sarrà, Ester; Montori, Sheyla; Gil-Recio, Carlos; Núñez-Toldrà, Raquel; Costamagna, Domiziana; Rotini, Alessio; Atari, Maher; Luttun, Aernout; Sampaolesi, Maurilio

2017-07-27

Dental pulp represents an easily accessible autologous source of adult stem cells. A subset of these cells, named dental pulp pluripotent-like stem cells (DPPSC), shows high plasticity and can undergo multiple population doublings, making DPPSC an appealing tool for tissue repair or maintenance. DPPSC were harvested from the dental pulp of third molars extracted from young patients. Growth factors released by DPPSC were analysed using antibody arrays. Cells were cultured in specific differentiation media and their endothelial, smooth and skeletal muscle differentiation potential was evaluated. The therapeutic potential of DPPSC was tested in a wound healing mouse model and in two genetic mouse models of muscular dystrophy (Scid/mdx and Sgcb-null Rag2-null γc-null). DPPSC secreted several growth factors involved in angiogenesis and extracellular matrix deposition and improved vascularisation in all three murine models. Moreover, DPPSC stimulated re-epithelialisation and ameliorated collagen deposition and organisation in healing wounds. In dystrophic mice, DPPSC engrafted in the skeletal muscle of both dystrophic murine models and showed integration in muscular fibres and vessels. In addition, DPPSC treatment resulted in reduced fibrosis and collagen content, larger cross-sectional area of type II fast-glycolytic fibres and infiltration of higher numbers of proangiogenic CD206 + macrophages. Overall, DPPSC represent a potential source of stem cells to enhance the wound healing process and slow down dystrophic muscle degeneration.

RCA SATCOM Battery in Orbit Performance Update and Accelerated Life Test Results

NASA Technical Reports Server (NTRS)

Gaston, S. J.; Schiffer, S. F.

1984-01-01

No significant degradation of nickel cadmium battery performance in SATCOM F1 and F2 after almost 8 and 7-3/4 years in orbit was shown. Battery minimum discharge voltage data are presented for these spacecraft. In addition, 2 groups of nickel cadmium cells which are representative of those in orbit are undergoing real time eclipse-reduced suntime cycling in the laboratory. These groups of cells, which are being cycled at a maximum of 53% and 62% depth of discharge (based on actual capacity), have completed 14 and 15 eclipse seasons, respectively. Data for these groups of cells are presented and are compared with the in-orbit battery data.

Analysis of stratocumulus cloud fields using LANDSAT imagery: Size distributions and spatial separations

NASA Technical Reports Server (NTRS)

Welch, R. M.; Sengupta, S. K.; Chen, D. W.

1990-01-01

Stratocumulus cloud fields in the FIRE IFO region are analyzed using LANDSAT Thematic Mapper imagery. Structural properties such as cloud cell size distribution, cell horizontal aspect ratio, fractional coverage and fractal dimension are determined. It is found that stratocumulus cloud number densities are represented by a power law. Cell horizontal aspect ratio has a tendency to increase at large cell sizes, and cells are bi-fractal in nature. Using LANDSAT Multispectral Scanner imagery for twelve selected stratocumulus scenes acquired during previous years, similar structural characteristics are obtained. Cloud field spatial organization also is analyzed. Nearest-neighbor spacings are fit with a number of functions, with Weibull and Gamma distributions providing the best fits. Poisson tests show that the spatial separations are not random. Second order statistics are used to examine clustering.

Generating compact classifier systems using a simple artificial immune system.

PubMed

Leung, Kevin; Cheong, France; Cheong, Christopher

2007-10-01

Current artificial immune system (AIS) classifiers have two major problems: 1) their populations of B-cells can grow to huge proportions, and 2) optimizing one B-cell (part of the classifier) at a time does not necessarily guarantee that the B-cell pool (the whole classifier) will be optimized. In this paper, the design of a new AIS algorithm and classifier system called simple AIS is described. It is different from traditional AIS classifiers in that it takes only one B-cell, instead of a B-cell pool, to represent the classifier. This approach ensures global optimization of the whole system, and in addition, no population control mechanism is needed. The classifier was tested on seven benchmark data sets using different classification techniques and was found to be very competitive when compared to other classifiers.

Client views and attitudes to non-invasive prenatal diagnosis for sickle cell disease, thalassaemia and cystic fibrosis.

PubMed

Hill, Melissa; Compton, Cecilia; Karunaratna, Madhavi; Lewis, Celine; Chitty, Lyn

2014-12-01

In the near future the availability of non-invasive prenatal diagnosis (NIPD) for single gene disorders will change the prenatal diagnosis options available to couples who are carriers of conditions such as cystic fibrosis, sickle cell disorder and thalassaemia. Client opinions about NIPD are needed to inform the implementation of NIPD for single gene disorders. This qualitative study used two focus groups (n = 12) and one-to-one interviews (n = 16) with carriers and support group representatives of sickle cell disease, thalassaemia and cystic fibrosis. Discussions were digitally recorded, transcribed verbatim and analysed using thematic analysis. Opinions about NIPD were very positive and participants valued the opportunity to have safe and early testing. Uptake of prenatal testing is likely to increase as women who had previously declined invasive testing expressed interest in having NIPD. Participant concerns about NIPD centred on the need for accuracy to be high to be used for subsequent decision making about termination of pregnancy. Participants also raised concerns that less thought may be given to having a blood test compared to an invasive test and that the perceived ease of a blood test may bring increased pressure to have testing. Participants thought NIPD should be offered through existing specialist services to ensure appropriate genetic counseling and support. Maintaining all testing options is important as some people may prefer invasive testing over NIPD if invasive testing was more accurate or if invasive testing could give information about other conditions such as Down syndrome.

Xenobiotic metabolism capacities of human skin in comparison with a 3D epidermis model and keratinocyte-based cell culture as in vitro alternatives for chemical testing: activating enzymes (Phase I).

PubMed

Götz, Christine; Pfeiffer, Roland; Tigges, Julia; Blatz, Veronika; Jäckh, Christine; Freytag, Eva-Maria; Fabian, Eric; Landsiedel, Robert; Merk, Hans F; Krutmann, Jean; Edwards, Robert J; Pease, Camilla; Goebel, Carsten; Hewitt, Nicola; Fritsche, Ellen

2012-05-01

Skin is important for the absorption and metabolism of exposed chemicals such as cosmetics or pharmaceuticals. The Seventh Amendment to the EU Cosmetics Directive prohibits the use of animals for cosmetic testing for certain endpoints, such as genotoxicity; therefore, there is an urgent need to understand the xenobiotic metabolizing capacities of human skin and to compare these activities with reconstructed 3D skin models developed to replace animal testing. We have measured Phase I enzyme activities of cytochrome P450 (CYP) and cyclooxygenase (COX) in ex vivo human skin, the 3D skin model EpiDerm™ (EPI-200), immortalized keratinocyte-based cell lines and primary normal human epidermal keratinocytes. Our data demonstrate that basal CYP enzyme activities are very low in whole human skin and EPI-200 as well as keratinocytes. In addition, activities in monolayer cells differed from organotypic tissues after induction. COX activity was similar in skin, EPI-200 and NHEK cells, but was significantly lower in immortalized keratinocytes. Hence, the 3D model EPI-200 might represent a more suitable model for dermatotoxicological studies. Altogether, these data help to better understand skin metabolism and expand the knowledge of in vitro alternatives used for dermatotoxicity testing. © 2012 John Wiley & Sons A/S.

Demonstration of Advanced C/SiC Cooled Ramp

NASA Technical Reports Server (NTRS)

Bouquet, Clement; Laithier, Frederic; Lawrence, Timothy; Eckel, Andrew; Munafo, Paul M. (Technical Monitor)

2002-01-01

Under a NASA contract, SPS is evaluating its C/SiC to metal brazing technique for the development of light, composite, actively cooled panels. The program first consisted of defining a system applicable to the X-33 nozzle ramp. SPS then performed evaluation tests for tube, composite, and braze material selection, and for the adaptation of braze process parameters to the parts geometry. SPS is presently manufacturing a 250x60 millimeter squared specimen, including 10 metallic tubes, which will be cycled in the NASA/GRC-CELL-22 test bed under engine representative conditions.

Molecular Pharmacology of Malignant Pleural Mesothelioma: Challenges and Perspectives From Preclinical and Clinical Studies.

PubMed

Thellung, Stefano; Favoni, Roberto E; Würth, Roberto; Nizzari, Mario; Pattarozzi, Alessandra; Daga, Antonio; Florio, Tullio; Barbieri, Federica

2016-01-01

Malignant pleural mesothelioma (MPM) is one of the deadliest and most heterogeneous tumors, highly refractory to multimodal therapeutic approach, including surgery, chemo- and radiotherapy. Preclinical and clinical studies exploring the efficacy of drugs targeting tyrosine kinases, angiogenesis and histone deacetylases, did not fulfil the expected clinical benefits. Thus, novel molecular targets should be identified from a definite knowledge of the unique biology and most relevant transduction pathways of MPM cells. Cancer stem cells (CSCs) are a subset of malignant precursors responsible for initiation, progression, resistance to cytotoxic drugs, recurrence and metastatic diffusion of tumor cells. CSCs are putative driving factors for MPM development and contribute to its clinical and biological heterogeneity; hence, targeted eradication of CSCs represents an ineludible goal to counteract MPM aggressiveness. In this context, innovative preclinical models could be exploited to identify novel intracellular pathway inhibitors able to target CSC viability. Novel drug targets have been identified among key factors responsible for the oncogenic transformation of mesothelial cells, often directly induced by asbestos. These include mitogenic and anti-apoptotic signaling that may also be activated by autocrine and paracrine cytokine pathways controlling cell plasticity. Both signaling pathways affecting proto-oncogene and transcription factor expression, or genetic and epigenetic alterations, such as mutations in cell cycle genes and silencing of tumor suppressor genes, represent promising disease-specific targets. In this review we describe current knowledge of MPM cell biology, focusing on potential targets to be tested in pharmacological studies, and highlighting results and challenges of clinical translation.

Real time blood testing using quantitative phase imaging.

PubMed

Pham, Hoa V; Bhaduri, Basanta; Tangella, Krishnarao; Best-Popescu, Catherine; Popescu, Gabriel

2013-01-01

We demonstrate a real-time blood testing system that can provide remote diagnosis with minimal human intervention in economically challenged areas. Our instrument combines novel advances in label-free optical imaging with parallel computing. Specifically, we use quantitative phase imaging for extracting red blood cell morphology with nanoscale sensitivity and NVIDIA's CUDA programming language to perform real time cellular-level analysis. While the blood smear is translated through focus, our system is able to segment and analyze all the cells in the one megapixel field of view, at a rate of 40 frames/s. The variety of diagnostic parameters measured from each cell (e.g., surface area, sphericity, and minimum cylindrical diameter) are currently not available with current state of the art clinical instruments. In addition, we show that our instrument correctly recovers the red blood cell volume distribution, as evidenced by the excellent agreement with the cell counter results obtained on normal patients and those with microcytic and macrocytic anemia. The final data outputted by our instrument represent arrays of numbers associated with these morphological parameters and not images. Thus, the memory necessary to store these data is of the order of kilobytes, which allows for their remote transmission via, for example, the cellular network. We envision that such a system will dramatically increase access for blood testing and furthermore, may pave the way to digital hematology.

Vessel-associated stem cells from skeletal muscle: From biology to future uses in cell therapy.

PubMed

Sancricca, Cristina; Mirabella, Massimiliano; Gliubizzi, Carla; Broccolini, Aldobrando; Gidaro, Teresa; Morosetti, Roberta

2010-06-26

Over the last years, the existence of different stem cells with myogenic potential has been widely investigated. Besides the classical skeletal muscle progenitors represented by satellite cells, numerous multipotent and embryologically unrelated progenitors with a potential role in muscle differentiation and repair have been identified. In order to conceive a therapeutic approach for degenerative muscle disorders, it is of primary importance to identify an ideal stem cell endowed with all the features for a possible use in vivo. Among all emerging populations, vessel-associated stem cells are a novel and promising class of multipotent progenitors of mesodermal origin and with high myogenic potential which seem to best fit all the requirements for a possible cell therapy. In vitro and in vivostudies have already tested the effectiveness and safety of vessel-associated stem cells in animal models. This leads to the concrete possibility in the future to start pilot human clinical trials, hopefully opening the way to a turning point in the treatment of genetic and acquired muscle disorders.

Axial tomography in live cell laser microscopy

NASA Astrophysics Data System (ADS)

Richter, Verena; Bruns, Sarah; Bruns, Thomas; Weber, Petra; Wagner, Michael; Cremer, Christoph; Schneckenburger, Herbert

2017-09-01

Single cell microscopy in a three-dimensional (3-D) environment is reported. Cells are grown in an agarose culture gel, located within microcapillaries and observed from different sides after adaptation of an innovative device for sample rotation. Thus, z-stacks can be recorded by confocal microscopy in different directions and used for illustration in 3-D. This gives additional information, since cells or organelles that appear superimposed in one direction, may be well resolved in another one. The method is tested and validated with single cells expressing a membrane or a mitochondrially associated green fluorescent protein, or cells accumulating fluorescent quantum dots. In addition, axial tomography supports measurements of cellular uptake and distribution of the anticancer drug doxorubicin in the nucleus (2 to 6 h after incubation) or the cytoplasm (24 h). This paper discusses that upon cell rotation an enhanced optical resolution in lateral direction compared to axial direction can be utilized to obtain an improved effective 3-D resolution, which represents an important step toward super-resolution microscopy of living cells.

T-cell receptor transfer into human T cells with ecotropic retroviral vectors.

PubMed

Koste, L; Beissert, T; Hoff, H; Pretsch, L; Türeci, Ö; Sahin, U

2014-05-01

Adoptive T-cell transfer for cancer immunotherapy requires genetic modification of T cells with recombinant T-cell receptors (TCRs). Amphotropic retroviral vectors (RVs) used for TCR transduction for this purpose are considered safe in principle. Despite this, TCR-coding and packaging vectors could theoretically recombine to produce replication competent vectors (RCVs), and transduced T-cell preparations must be proven free of RCV. To eliminate the need for RCV testing, we transduced human T cells with ecotropic RVs so potential RCV would be non-infectious for human cells. We show that transfection of synthetic messenger RNA encoding murine cationic amino-acid transporter 1 (mCAT-1), the receptor for murine retroviruses, enables efficient transient ecotropic transduction of human T cells. mCAT-1-dependent transduction was more efficient than amphotropic transduction performed in parallel, and preferentially targeted naive T cells. Moreover, we demonstrate that ecotropic TCR transduction results in antigen-specific restimulation of primary human T cells. Thus, ecotropic RVs represent a versatile, safe and potent tool to prepare T cells for the adoptive transfer.

Dependence of Impedance of Embedded Single Cells on Cellular Behaviour

PubMed Central

Cho, Sungbo; Castellarnau, Marc; Samitier, Josep; Thielecke, Hagen

2008-01-01

Non-invasive single cell analyses are increasingly required for the medical diagnostics of test substances or the development of drugs and therapies on the single cell level. For the non-invasive characterisation of cells, impedance spectroscopy which provides the frequency dependent electrical properties has been used. Recently, microfludic systems have been investigated to manipulate the single cells and to characterise the electrical properties of embedded cells. In this article, the impedance of partially embedded single cells dependent on the cellular behaviour was investigated by using the microcapillary. An analytical equation was derived to relate the impedance of embedded cells with respect to the morphological and physiological change of extracellular interface. The capillary system with impedance measurement showed a feasibility to monitor the impedance change of embedded single cells caused by morphological and physiological change of cell during the addition of DMSO. By fitting the derived equation to the measured impedance of cell embedded at different negative pressure levels, it was able to extrapolate the equivalent gap and gap conductivity between the cell and capillary wall representing the cellular behaviour. PMID:27879760

Human immunotoxicologic markers of chemical exposures: preliminary validation studies.

PubMed

Wartenberg, D; Laskin, D; Kipen, H

1993-01-01

The circulating cells of the immune system are sensitive to environmental contaminants, and effects are often manifested as changes in the cell surface differentiation antigens of affected populations of cells, particularly lymphocytes. In this investigation, we explore the likelihood that variation in the expression of the surface markers of immune cells can be used as an index of exposure to toxic chemicals. We recruited 38 healthy New Jersey men to study pesticides effects: 19 orchard farmers (high exposure); 13 berry farmers (low exposure); and 6 hardware store owners (no exposure). Immunophenotyping was performed assaying the following cell surface antigens: CD2, CD4, CD8, CD14, CD20, CD26, CD29, CD45R, CD56, and PMN. Data were analyzed using univariate and multivariate methods. There were no significant differences among the groups with respect to routine medical histories, physical examinations, or routine laboratory parameters. No striking differences between groups were seen in univariate tests. Multivariate tests suggested some differences among groups and limited ability to correctly classify individuals based on immunophenotyping results. Immunophenotyping represents a fruitful area of research for improved exposure classification. Work is needed both on mechanistic understanding of the patterns observed and on the statistical interpretation of these patterns.

Modeling cell adhesion and proliferation: a cellular-automata based approach.

PubMed

Vivas, J; Garzón-Alvarado, D; Cerrolaza, M

Cell adhesion is a process that involves the interaction between the cell membrane and another surface, either a cell or a substrate. Unlike experimental tests, computer models can simulate processes and study the result of experiments in a shorter time and lower costs. One of the tools used to simulate biological processes is the cellular automata, which is a dynamic system that is discrete both in space and time. This work describes a computer model based on cellular automata for the adhesion process and cell proliferation to predict the behavior of a cell population in suspension and adhered to a substrate. The values of the simulated system were obtained through experimental tests on fibroblast monolayer cultures. The results allow us to estimate the cells settling time in culture as well as the adhesion and proliferation time. The change in the cells morphology as the adhesion over the contact surface progress was also observed. The formation of the initial link between cell and the substrate of the adhesion was observed after 100 min where the cell on the substrate retains its spherical morphology during the simulation. The cellular automata model developed is, however, a simplified representation of the steps in the adhesion process and the subsequent proliferation. A combined framework of experimental and computational simulation based on cellular automata was proposed to represent the fibroblast adhesion on substrates and changes in a macro-scale observed in the cell during the adhesion process. The approach showed to be simple and efficient.

Influence of the treatment of Listeria monocytogenes and Salmonella enterica serovar Typhimurium with citral on the efficacy of various antibiotics.

PubMed

Zanini, Surama F; Silva-Angulo, Angela B; Rosenthal, Amauri; Aliaga, Dolores Rodrigo; Martínez, Antonio

2014-04-01

The main goal of this work was to study the bacterial adaptive responses to antibiotics induced by sublethal concentration of citral on first-and second-generation cells of Listeria monocytogenes serovar 4b (CECT 4032) and Salmonella enterica serovar Typhimurium (CECT 443). The first-generation cells were not pretreated with citral, while the second-generation cells were obtained from cells previously exposed to citral during 5 h. The trials were conducted at 37°C. The presence of citral in the culture medium and the antibiotic strips resulted in a reduced minimum inhibitory concentration (MIC) for the first-generation cells of Listeria monocytogenes serovar 4b and Salmonella Typhimurium. This result was observed for almost all the antibiotics, compared with the same microorganisms of the control group (without citral), which could represent an additive effect. For Listeria serovar 4b, the second-generation cells of the test group maintained the same susceptibility to antibiotics compared with cells in the control group and in the test group of the first generation. The second-generation cells of the control group indicated that the Salmonella Typhimurium maintained the same sensitivity to the antibiotics tested compared with the first generation of this group, except in the case of erythromycin, which exhibited an increased MIC value. With respect to the second-generation cells of Salmonella Typhimurium, the presence of citral determined a decrease in the antibiotic susceptibility for almost all of the antibiotics, except colistin, compared with the first-generation of the test group, which can be seen by increase of MIC values. In conclusion, the presence of citral in the culture medium of Listeria 4b and Salmonella Typhimurium increased the antibiotic susceptibility of the first generations, while we observed an increase in antibiotic resistance in the second generation of Salmonella Typhimurium.

Turbine Engine Loads Simulator (TELS) Radiographic Systems Requirements Study

DTIC Science & Technology

1977-06-01

cell and viewed from the rear. Locations of the rotor case pinch points are illustrated in Figures C-4 through C-! 9. Table C-Ill represents the test...radiation is also attentuated by air, therefore we can write: pb2 03 EXP(~a b) ( El4 ) W = [ K---~-T X1 ] Let U = T = 1, P = 0.01 rads, #a = 3 x 10 -5 cm

Impedances of electrochemically impregnated nickel electrodes as functions of potential, KOH concentration, and impregnation method

NASA Technical Reports Server (NTRS)

Reid, Margaret A.

1989-01-01

Impedances of fifteen electrodes form each of the four U.S. manufactures were measured at 0.200 V vs. the Hg/HgO reference electrode. This corresponds to a voltage of 1.145 for a Ni/H2 cell. Measurements were also made of a representative sample of these at 0.44 V. At the higher voltage, the impedances were small and very similar, but at the lower voltage there were major differences between manufacturers. Electrodes from the same manufacturers showed only small differences. The impedances of electrodes from two manufacturers were considerably different in 26 percent KOH from those in 31 percent KOH. These preliminary results seen to correlate with the limited data from earlier life testing of cells from these manufacturers. The impedances of cells being tested for Space Station Freedom are being followed, and more impendance measurements of electrodes are being performed as functions of manufacturer, voltage, electrolyte concentration, and cycle history in hopes of finding better correlations of impedance with life.

Impact of yeast starter formulations on the production of volatile compounds during wine fermentation.

PubMed

Romano, Patrizia; Pietrafesa, Rocchina; Romaniello, Rossana; Zambuto, Marianna; Calabretti, Antonella; Capece, Angela

2015-01-01

The most diffused starter formulation in winemaking is actually represented by active dry yeast (ADY). Spray-drying has been reported as an appropriate preservation method for yeast and other micro-organisms. Despite the numerous advantages of this method, the high air temperatures used can negatively affect cell viability and the fermentative performance of dried cells. In the present study, 11 wine S. cerevisiae strains (both indigenous and commercial) were submitted to spray-drying; different process conditions were tested in order to select the conditions allowing the highest strain survival. The strains exhibited high variability for tolerance to spray-drying treatment. Selected strains were tested in fermentation at laboratory scale in different formulations (free fresh cells, free dried cells, immobilized fresh cells and immobilized dried cells), in order to assess the influence of starter formulation on fermentative fitness of strains and aromatic quality of wine. The analysis of volatile fraction in the experimental wines produced by selected strains in different formulations allowed identification of > 50 aromatic compounds (alcohols, esters, ketones, aldehydes and terpenes). The results obtained showed that the starter formulation significantly influenced the content of volatile compounds. In particular, the wines obtained by strains in dried forms (as both free and immobilized cells) contained higher numbers of volatile compounds than wines obtained from fresh cells. Copyright © 2014 John Wiley & Sons, Ltd.

A New Bone Substitute Developed from 3D-Prints of Polylactide (PLA) Loaded with Collagen I: An In Vitro Study.

PubMed

Ritz, Ulrike; Gerke, Rebekka; Götz, Hermann; Stein, Stefan; Rommens, Pol Maria

2017-11-29

Although a lot of research has been performed, large segmental bone defects caused by trauma, infection, bone tumors or revision surgeries still represent big challenges for trauma surgeons. New and innovative bone substitutes are needed. Three-dimensional (3D) printing is a novel procedure to create 3D porous scaffolds that can be used for bone tissue engineering. In the present study, solid discs as well as porous cage-like 3D prints made of polylactide (PLA) are coated or filled with collagen, respectively, and tested for biocompatibility and endotoxin contamination. Microscopic analyses as well as proliferation assays were performed using various cell types on PLA discs. Stromal-derived factor (SDF-1) release from cages filled with collagen was analyzed and the effect on endothelial cells tested. This study confirms the biocompatibility of PLA and demonstrates an endotoxin contamination clearly below the FDA (Food and Drug Administration) limit. Cells of various cell types (osteoblasts, osteoblast-like cells, fibroblasts and endothelial cells) grow, spread and proliferate on PLA-printed discs. PLA cages loaded with SDF-1 collagen display a steady SDF-1 release, support cell growth of endothelial cells and induce neo-vessel formation. These results demonstrate the potential for PLA scaffolds printed with an inexpensive desktop printer in medical applications, for example, in bone tissue engineering.

A phenotypic screening approach to identify anticancer compounds derived from marine fungi.

PubMed

Ellinger, Bernhard; Silber, Johanna; Prashar, Anjali; Landskron, Johannes; Weber, Jonas; Rehermann, Sarah; Müller, Franz-Josef; Smith, Stephen; Wrigley, Stephen; Taskén, Kjetil; Gribbon, Philip; Labes, Antje; Imhoff, Johannes F

2014-04-01

This study covers the isolation, testing, and identification of natural products with anticancer properties. Secondary metabolites were isolated from fungal strains originating from a variety of marine habitats. Strain culture protocols were optimized with respect to growth media composition and fermentation conditions. From these producers, isolated compounds were screened for their effect on the viability and proliferation of a subset of the NCI60 panel of cancer cell lines. Active compounds of interest were identified and selected for detailed assessments and structural elucidation using nuclear magnetic resonance. This revealed the majority of fungal-derived compounds represented known anticancer chemotypes, confirming the integrity of the process and the ability to identify suitable compounds. Examination of effects of selected compounds on cancer-associated cell signaling pathways used phospho flow cytometry in combination with 3D fluorescent cell barcoding. In parallel, the study addressed the logistical aspects of maintaining multiple cancer cell lines in culture simultaneously. A potential solution involving microbead-based cell culture was investigated (BioLevitator, Hamilton). Selected cell lines were cultured in microbead and 2D methods and cell viability tests showed comparable compound inhibition in both methods (R2=0.95). In a further technology assessment, an image-based assay system was investigated for its utility as a possible complement to ATP-based detection for quantifying cell growth and viability in a label-free manner.

A New Bone Substitute Developed from 3D-Prints of Polylactide (PLA) Loaded with Collagen I: An In Vitro Study

PubMed Central

Gerke, Rebekka; Götz, Hermann; Rommens, Pol Maria

2017-01-01

Although a lot of research has been performed, large segmental bone defects caused by trauma, infection, bone tumors or revision surgeries still represent big challenges for trauma surgeons. New and innovative bone substitutes are needed. Three-dimensional (3D) printing is a novel procedure to create 3D porous scaffolds that can be used for bone tissue engineering. In the present study, solid discs as well as porous cage-like 3D prints made of polylactide (PLA) are coated or filled with collagen, respectively, and tested for biocompatibility and endotoxin contamination. Microscopic analyses as well as proliferation assays were performed using various cell types on PLA discs. Stromal-derived factor (SDF-1) release from cages filled with collagen was analyzed and the effect on endothelial cells tested. This study confirms the biocompatibility of PLA and demonstrates an endotoxin contamination clearly below the FDA (Food and Drug Administration) limit. Cells of various cell types (osteoblasts, osteoblast-like cells, fibroblasts and endothelial cells) grow, spread and proliferate on PLA-printed discs. PLA cages loaded with SDF-1 collagen display a steady SDF-1 release, support cell growth of endothelial cells and induce neo-vessel formation. These results demonstrate the potential for PLA scaffolds printed with an inexpensive desktop printer in medical applications, for example, in bone tissue engineering. PMID:29186036

Cytokine networking of innate immunity cells: a potential target of therapy.

PubMed

Striz, Ilja; Brabcova, Eva; Kolesar, Libor; Sekerkova, Alena

2014-05-01

Innate immune cells, particularly macrophages and epithelial cells, play a key role in multiple layers of immune responses. Alarmins and pro-inflammatory cytokines from the IL (interleukin)-1 and TNF (tumour necrosis factor) families initiate the cascade of events by inducing chemokine release from bystander cells and by the up-regulation of adhesion molecules required for transendothelial trafficking of immune cells. Furthermore, innate cytokines produced by dendritic cells, macrophages, epithelial cells and innate lymphoid cells seem to play a critical role in polarization of helper T-cell cytokine profiles into specific subsets of Th1/Th2/Th17 effector cells or regulatory T-cells. Lastly, the innate immune system down-regulates effector mechanisms and restores homoeostasis in injured tissue via cytokines from the IL-10 and TGF (transforming growth factor) families mainly released from macrophages, preferentially the M2 subset, which have a capacity to induce regulatory T-cells, inhibit the production of pro-inflammatory cytokines and induce healing of the tissue by regulating extracellular matrix protein deposition and angiogenesis. Cytokines produced by innate immune cells represent an attractive target for therapeutic intervention, and multiple molecules are currently being tested clinically in patients with inflammatory bowel disease, rheumatoid arthritis, systemic diseases, autoinflammatory syndromes, fibrosing processes or malignancies. In addition to the already widely used blockers of TNFα and the tested inhibitors of IL-1 and IL-6, multiple therapeutic molecules are currently in clinical trials targeting TNF-related molecules [APRIL (a proliferation-inducing ligand) and BAFF (B-cell-activating factor belonging to the TNF family)], chemokine receptors, IL-17, TGFβ and other cytokines.

Testing of SNS-032 in a Panel of Human Neuroblastoma Cell Lines with Acquired Resistance to a Broad Range of Drugs.

PubMed

Löschmann, Nadine; Michaelis, Martin; Rothweiler, Florian; Zehner, Richard; Cinatl, Jaroslav; Voges, Yvonne; Sharifi, Mohsen; Riecken, Kristoffer; Meyer, Jochen; von Deimling, Andreas; Fichtner, Iduna; Ghafourian, Taravat; Westermann, Frank; Cinatl, Jindrich

2013-12-01

Novel treatment options are needed for the successful therapy of patients with high-risk neuroblastoma. Here, we investigated the cyclin-dependent kinase (CDK) inhibitor SNS-032 in a panel of 109 neuroblastoma cell lines consisting of 19 parental cell lines and 90 sublines with acquired resistance to 14 different anticancer drugs. Seventy-three percent of the investigated neuroblastoma cell lines and all four investigated primary tumor samples displayed concentrations that reduce cell viability by 50% in the range of the therapeutic plasma levels reported for SNS-032 (<754 nM). Sixty-two percent of the cell lines and two of the primary samples displayed concentrations that reduce cell viability by 90% in this concentration range. SNS-032 also impaired the growth of the multidrug-resistant cisplatin-adapted UKF-NB-3 subline UKF-NB-3(r)CDDP(1000) in mice. ABCB1 expression (but not ABCG2 expression) conferred resistance to SNS-032. The antineuroblastoma effects of SNS-032 did not depend on functional p53. The antineuroblastoma mechanism of SNS-032 included CDK7 and CDK9 inhibition-mediated suppression of RNA synthesis and subsequent depletion of antiapoptotic proteins with a fast turnover rate including X-linked inhibitor of apoptosis (XIAP), myeloid cell leukemia sequence 1 (Mcl-1), baculoviral IAP repeat containing 2 (BIRC2; cIAP-1), and survivin. In conclusion, CDK7 and CDK9 represent promising drug targets and SNS-032 represents a potential treatment option for neuroblastoma including therapy-refractory cases.

Testing of SNS-032 in a Panel of Human Neuroblastoma Cell Lines with Acquired Resistance to a Broad Range of Drugs12

PubMed Central

Löschmann, Nadine; Michaelis, Martin; Rothweiler, Florian; Zehner, Richard; Cinatl, Jaroslav; Voges, Yvonne; Sharifi, Mohsen; Riecken, Kristoffer; Meyer, Jochen; von Deimling, Andreas; Fichtner, Iduna; Ghafourian, Taravat; Westermann, Frank; Cinatl, Jindrich

2013-01-01

Novel treatment options are needed for the successful therapy of patients with high-risk neuroblastoma. Here, we investigated the cyclin-dependent kinase (CDK) inhibitor SNS-032 in a panel of 109 neuroblastoma cell lines consisting of 19 parental cell lines and 90 sublines with acquired resistance to 14 different anticancer drugs. Seventy-three percent of the investigated neuroblastoma cell lines and all four investigated primary tumor samples displayed concentrations that reduce cell viability by 50% in the range of the therapeutic plasma levels reported for SNS-032 (<754 nM). Sixty-two percent of the cell lines and two of the primary samples displayed concentrations that reduce cell viability by 90% in this concentration range. SNS-032 also impaired the growth of the multidrug-resistant cisplatin-adapted UKF-NB-3 subline UKF-NB-3rCDDP1000 in mice. ABCB1 expression (but not ABCG2 expression) conferred resistance to SNS-032. The antineuroblastoma effects of SNS-032 did not depend on functional p53. The antineuroblastoma mechanism of SNS-032 included CDK7 and CDK9 inhibition-mediated suppression of RNA synthesis and subsequent depletion of antiapoptotic proteins with a fast turnover rate including X-linked inhibitor of apoptosis (XIAP), myeloid cell leukemia sequence 1 (Mcl-1), baculoviral IAP repeat containing 2 (BIRC2; cIAP-1), and survivin. In conclusion, CDK7 and CDK9 represent promising drug targets and SNS-032 represents a potential treatment option for neuroblastoma including therapy-refractory cases. PMID:24466371

Comparison and optimization of machine learning methods for automated classification of circulating tumor cells.

PubMed

Lannin, Timothy B; Thege, Fredrik I; Kirby, Brian J

2016-10-01

Advances in rare cell capture technology have made possible the interrogation of circulating tumor cells (CTCs) captured from whole patient blood. However, locating captured cells in the device by manual counting bottlenecks data processing by being tedious (hours per sample) and compromises the results by being inconsistent and prone to user bias. Some recent work has been done to automate the cell location and classification process to address these problems, employing image processing and machine learning (ML) algorithms to locate and classify cells in fluorescent microscope images. However, the type of machine learning method used is a part of the design space that has not been thoroughly explored. Thus, we have trained four ML algorithms on three different datasets. The trained ML algorithms locate and classify thousands of possible cells in a few minutes rather than a few hours, representing an order of magnitude increase in processing speed. Furthermore, some algorithms have a significantly (P < 0.05) higher area under the receiver operating characteristic curve than do other algorithms. Additionally, significant (P < 0.05) losses to performance occur when training on cell lines and testing on CTCs (and vice versa), indicating the need to train on a system that is representative of future unlabeled data. Optimal algorithm selection depends on the peculiarities of the individual dataset, indicating the need of a careful comparison and optimization of algorithms for individual image classification tasks. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Correlations Between Life-Detection Techniques and Implications for Sampling Site Selection in Planetary Analog Missions.

PubMed

Gentry, Diana M; Amador, Elena S; Cable, Morgan L; Chaudry, Nosheen; Cullen, Thomas; Jacobsen, Malene B; Murukesan, Gayathri; Schwieterman, Edward W; Stevens, Adam H; Stockton, Amanda; Tan, George; Yin, Chang; Cullen, David C; Geppert, Wolf

2017-10-01

We conducted an analog sampling expedition under simulated mission constraints to areas dominated by basaltic tephra of the Eldfell and Fimmvörðuháls lava fields (Iceland). Sites were selected to be "homogeneous" at a coarse remote sensing resolution (10-100 m) in apparent color, morphology, moisture, and grain size, with best-effort realism in numbers of locations and replicates. Three different biomarker assays (counting of nucleic-acid-stained cells via fluorescent microscopy, a luciferin/luciferase assay for adenosine triphosphate, and quantitative polymerase chain reaction (qPCR) to detect DNA associated with bacteria, archaea, and fungi) were characterized at four nested spatial scales (1 m, 10 m, 100 m, and >1 km) by using five common metrics for sample site representativeness (sample mean variance, group F tests, pairwise t tests, and the distribution-free rank sum H and u tests). Correlations between all assays were characterized with Spearman's rank test. The bioluminescence assay showed the most variance across the sites, followed by qPCR for bacterial and archaeal DNA; these results could not be considered representative at the finest resolution tested (1 m). Cell concentration and fungal DNA also had significant local variation, but they were homogeneous over scales of >1 km. These results show that the selection of life detection assays and the number, distribution, and location of sampling sites in a low biomass environment with limited a priori characterization can yield both contrasting and complementary results, and that their interdependence must be given due consideration to maximize science return in future biomarker sampling expeditions. Key Words: Astrobiology-Biodiversity-Microbiology-Iceland-Planetary exploration-Mars mission simulation-Biomarker. Astrobiology 17, 1009-1021.

Fluorescent Immortalized Human Adipose Derived Stromal Cells (hASCs-TS/GFP+) for Studying Cell Drug Delivery Mediated by Microvesicles.

PubMed

Cocce, Valentina; Balducci, Luigi; Falchetti, Maria L; Pascucci, Luisa; Ciusani, Emilio; Brini, Anna T; Sisto, Francesca; Piovani, Giovanna; Alessandri, Giulio; Parati, Eugenio; Cabeza, Laura; Pessina, Augusto

2017-11-24

A new tool for the drug delivery is based on the use of Mesenchymal Stromal Cells (MSCs) loaded in vitro with anti-cancer drugs. Unfortunately, the restricted lifespan of MSCs represents a significant limitation to produce them in high amounts and for long time studies. Immortalized MSCs from adipose tissue (hASCs) have been generated as good source of cells with stable features. These cells could improve the development of standardized procedures for both in vitro and preclinical studies. Furthermore they facilitate procedures for preparing large amounts of secretome containing microvesicles (MVs). We used human adipose tissue derived MSCs immortalized with hTERT+SV40 (TS) genes and transfected with GFP (hASCs-TS/GFP+). This line was investigated for its ability to uptake and release anticancer drugs. Microvesicles associated to paclitaxel (MVs/PTX) were isolated, quantified, and tested on pancreatic cancer cells. The line hASCs-TS/GFP+ maintained the main mesenchymal characters and was able to uptake and release, in active form, both paclitaxel and gemcitabine. From paclitaxel loaded hASCs-TS/GFP+ cells were isolated microvesicles in sufficient amount to inhibit "in vitro" the proliferation of pancreatic tumor cells. Our study suggests that human immortalized MSCs could be used for a large scale production of cells for mediated drug delivery. Moreover, the secretion of drug-associated MVs could represent a new way for producing new drug formulation by "biogenesis". In the context of the "advanced cell therapy procedure", the MVs/PTX production would use less resource and time and it could possibly contribute to simplification of GMP procedures. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

IUTA: a tool for effectively detecting differential isoform usage from RNA-Seq data.

PubMed

Niu, Liang; Huang, Weichun; Umbach, David M; Li, Leping

2014-10-06

Most genes in mammals generate several transcript isoforms that differ in stability and translational efficiency through alternative splicing. Such alternative splicing can be tissue- and developmental stage-specific, and such specificity is sometimes associated with disease. Thus, detecting differential isoform usage for a gene between tissues or cell lines/types (differences in the fraction of total expression of a gene represented by the expression of each of its isoforms) is potentially important for cell and developmental biology. We present a new method IUTA that is designed to test each gene in the genome for differential isoform usage between two groups of samples. IUTA also estimates isoform usage for each gene in each sample as well as averaged across samples within each group. IUTA is the first method to formulate the testing problem as testing for equal means of two probability distributions under the Aitchison geometry, which is widely recognized as the most appropriate geometry for compositional data (vectors that contain the relative amount of each component comprising the whole). Evaluation using simulated data showed that IUTA was able to provide test results for many more genes than was Cuffdiff2 (version 2.2.0, released in Mar. 2014), and IUTA performed better than Cuffdiff2 for the limited number of genes that Cuffdiff2 did analyze. When applied to actual mouse RNA-Seq datasets from six tissues, IUTA identified 2,073 significant genes with clear patterns of differential isoform usage between a pair of tissues. IUTA is implemented as an R package and is available at http://www.niehs.nih.gov/research/resources/software/biostatistics/iuta/index.cfm. Both simulation and real-data results suggest that IUTA accurately detects differential isoform usage. We believe that our analysis of RNA-seq data from six mouse tissues represents the first comprehensive characterization of isoform usage in these tissues. IUTA will be a valuable resource for those who study the roles of alternative transcripts in cell development and disease.

Inhibition of human papillomavirus expression using DNAzymes.

PubMed

Benítez-Hess, María Luisa; Reyes-Gutiérrez, Pablo; Alvarez-Salas, Luis Marat

2011-01-01

Deoxyribozymes (DXZs) are catalytic oligodeoxynucleotides capable of performing diverse functions including the specific cleavage of a target RNA. These molecules represent a new type of therapeutic oligonucleotides combining the efficiency of ribozymes and the intracellular endurance and simplicity of modified antisense oligonucleotides. Commonly used DXZs include the 8-17 and 10-23 motifs, which have been engineered to destroy disease-associated genes with remarkable efficiency. Targeting DXZs to disease-associated transcripts requires extensive biochemical testing to establish target RNA accessibility, catalytic efficiency, and nuclease sensibility. The usage of modified nucleotides to render nuclease-resistance DXZs must be counterweighted against deleterious consequences on catalytic activity. Further intracellular testing is required to establish the effect of microenvironmental conditions on DXZ activity and off-target issues. Application of modified DXZs to cervical cancer results in specific growth inhibition, cell death, and apoptosis. Thus, DXZs represent a highly effective antisense moiety with minimal secondary effects.

Intracellular targeting of CD44+ cells with self-assembling, protein only nanoparticles.

PubMed

Pesarrodona, Mireia; Ferrer-Miralles, Neus; Unzueta, Ugutz; Gener, Petra; Tatkiewicz, Witold; Abasolo, Ibane; Ratera, Imma; Veciana, Jaume; Schwartz, Simó; Villaverde, Antonio; Vazquez, Esther

2014-10-01

CD44 is a multifunctional cell surface protein involved in proliferation and differentiation, angiogenesis and signaling. The expression of CD44 is up-regulated in several types of human tumors and particularly in cancer stem cells, representing an appealing target for drug delivery in the treatment of cancer. We have explored here several protein ligands of CD44 for the construction of self-assembling modular proteins designed to bind and internalize target cells. Among five tested ligands, two of them (A5G27 and FNI/II/V) drive the formation of protein-only, ring-shaped nanoparticles of about 14 nm that efficiently bind and penetrate CD44(+) cells by an endosomal route. The potential of these newly designed nanoparticles is evaluated regarding the need of biocompatible nanostructured materials for drug delivery in CD44-linked conditions. Copyright © 2014 Elsevier B.V. All rights reserved.

Cell-selfish modes of evolution and mutations directed after transcriptional bypass.

PubMed

Holmquist, Gerald P

2002-12-29

During transcription, prokaryotic and eukaryotic RNA polymerases bypass and misread (transcriptional mutagenesis) several classes of DNA lesions. For example, misreading of 8-OH-dG generates mRNAs containing G to T transversions. After translation, if the mutant protein briefly allowed the cell a growth-DNA replication advantage, then precocious DNA replication would bypass that unrepaired 8-OH-dG and misinsert dA opposite the directing DNA lesion with a higher probability than would be experienced for 8-OH-G lesions at other positions in otherwise identical neighboring cells. Such retromutations would have been tested for their imparted growth advantage as mRNA before they became heritable DNA mutations. The logical properties of a mode of evolution that utilizes directed-retromutagenesis were compared one by one with those of the standard neo-Darwinian mode. The retromutagenesis mode, while minimizing mutational load, is cell-selfish; fitness is for an immediate growth advantage rather than future reproductive potential. In prokaryotes, an evolutionary mode that involves standard Darwinian fitness testing of novel alleles in the genetic background of origin followed by clonal expansion also favors cell-selfish allele combinations when linkage disequilibrium is practiced. For metazoa and plants to have evolved organized tissues, cell-selfish modes of evolution represent systems-poisons that must be totally suppressed. The feedback loops that allow evolution to be cell-serving in prokaryotes are actively blocked in eukaryotes by traits that restrict fitness to future reproductive potential. These traits include (i) delay of fitness testing until after the mutation is made permanently heritable, (ii) diploidy to further delay fitness testing, (iii) segregation of somatic lines from germ lines, (iv) testing of novel alleles against randomized allele combinations constructed by obligate sex, and (v) obligate genetic death to insure that that the most basic systems unit of selfish allele combinatorial uniqueness is the species instead of the cell. The analyses indicate that modes of evolution in addition to our neo-Darwinian one could have existed utilizing known molecular mechanisms. The evolution of multicellularity was as much the discarding of old cell-selfish habits as the acquisition of new altruistic ones.

Accurate prediction of acute fish toxicity of fragrance chemicals with the RTgill-W1 cell assay.

PubMed

Natsch, Andreas; Laue, Heike; Haupt, Tina; von Niederhäusern, Valentin; Sanders, Gordon

2018-03-01

Testing for acute fish toxicity is an integral part of the environmental safety assessment of chemicals. A true replacement of primary fish tissue was recently proposed using cell viability in a fish gill cell line (RTgill-W1) as a means of predicting acute toxicity, showing good predictivity on 35 chemicals. To promote regulatory acceptance, the predictivity and applicability domain of novel tests need to be carefully evaluated on chemicals with existing high-quality in vivo data. We applied the RTgill-W1 cell assay to 38 fragrance chemicals with a wide range of both physicochemical properties and median lethal concentration (LC50) values and representing a diverse range of chemistries. A strong correlation (R 2  = 0.90-0.94) between the logarithmic in vivo LC50 values, based on fish mortality, and the logarithmic in vitro median effect concentration (EC50) values based on cell viability was observed. A leave-one-out analysis illustrates a median under-/overprediction from in vitro EC50 values to in vivo LC50 values by a factor of 1.5. This assay offers a simple, accurate, and reliable alternative to in vivo acute fish toxicity testing for chemicals, presumably acting mainly by a narcotic mode of action. Furthermore, the present study provides validation of the predictivity of the RTgill-W1 assay on a completely independent set of chemicals that had not been previously tested and indicates that fragrance chemicals are clearly within the applicability domain. Environ Toxicol Chem 2018;37:931-941. © 2017 SETAC. © 2017 SETAC.

Role of p38 MAPK in the selective release of IL-8 induced by chemical allergen in naive THp-1 cells.

PubMed

Mitjans, Montserrat; Viviani, Barbara; Lucchi, Laura; Galli, Corrado L; Marinovich, Marina; Corsini, Emanuela

2008-03-01

At present, the assessment of the allergenic potential of chemicals is carried out using animal models. Over the last decade, several in vitro methods mainly using primary dendritic cells have been proposed to identify the potential of chemicals to induce skin sensitization to meet current animal welfare and public opinions. The major limitations of such tests are the donor-to-donor variability, the low levels in the source, and a possible shortage of human sources. The aim of the present investigation was to establish an in vitro test to identify chemical allergens using the human promyelocytic cell line THP-1 in order to avoid some of these difficulties. We investigated whether the chemokine interleukin-8 or CXCL8 (IL-8) production could provide a methodology for the detection of both respiratory and contact allergens. THP-1 cells were exposed to contact allergens (cinnamaldehyde, dinitrochlorobenzene, nickel sulfate, penicillin G, p-phenylenediamine, tetramethylthiuram disulfide), to respiratory allergens (ammonium hexachloroplatinate, diphenylmethane diisocyanate, trimellitic anhydride) and to irritants (salicylic acid, phenol, sodium lauryl sulphate). Following 48 h of incubation, the release of IL-8 was evaluated by sandwich ELISA. IL-8 production was significantly increased after stimulation with all allergens tested, with the exception of trimellitic anhydride, whereas irritants exposure failed to induce IL-8 release. The lack of IL-8 production by trimellitic anhydride can be explained by the rapid hydrolysis of this chemical in water to trimellitic acid, which is not an allergen. In contrast to IL-8 release, CD54 and CD86 expression did not provide a sensitive method failing to correctly identify approximately 30% of the tested compounds. Although CD86 appears to be a more sensitive marker than CD54 when discriminating allergens from irritants neither of these markers provided robust methodology. We also investigated if a common activation pathway in allergen-induced IL-8 production involving p38 mitogen-activated protein kinase could be identified. By Western blot analysis we could indeed demonstrate p38 activation by all chemical allergens tested and, using the selective p38 MAPK inhibitor SB203580, a significant modulation of allergen-induced IL-8 release could be achieved in all cases. Our data suggests that production of IL-8 by naïve THP-1 cells may represent a promising in vitro model for the screening of potential chemical allergens and activation of p38 MAPK represents a common pathway triggered by allergens.

Blockade of Androgen Markers Using a Novel Betasitosterol, Thioctic Acid and Carnitine-containing Compound in Prostate and Hair Follicle Cell-based Assays.

PubMed

Chen, Li; Wang, Jiaolong; Mouser, Glen; Li, Yan Chun; Marcovici, Geno

2016-06-01

Androgenetic alopecia (AGA) affects approximately 70% of men and 40% of women in an age-dependent manner and is partially mediated by androgen hormones. Benign prostatic hyperplasia (BPH) similarly affects 50% of the male population, rising by 10% each decade. Finasteride inhibits 5-alpha reductase (5AR) and is used to treat both disorders, despite offering limited clinical benefits accompanied by significant adverse side effects. Building on our previous work demonstrating the efficacy of naturally derived 5AR inhibitors (such as stigmasterol and beta sitosterol), we hypothesize that targeting 5AR as well as inflammatory pathways may yield improved efficacy in AGA and BPH. Here we address these dual pathomechanisms by examining the potency of a novel composition using in vitro assays of representative cell lines for AGA (hair follicle dermal papilla cells) and BPH (LNCaP prostate cells), respectively. Exposure of cells to the novel test composition down-regulated mRNA expression profiles characteristic of both disease processes, which outperformed finasteride. Changes in mRNA expression were corroborated at the protein level as assessed by western blotting. These studies provide proof of concept that novel, naturally derived compositions simultaneously targeting 5AR and inflammatory mediators may represent a rational approach to treating AGA and BPH. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Human CD30+ B cells represent a unique subset related to Hodgkin lymphoma cells.

PubMed

Weniger, Marc A; Tiacci, Enrico; Schneider, Stefanie; Arnolds, Judith; Rüschenbaum, Sabrina; Duppach, Janine; Seifert, Marc; Döring, Claudia; Hansmann, Martin-Leo; Küppers, Ralf

2018-06-11

Very few B cells in germinal centers (GCs) and extrafollicular (EF) regions of lymph nodes express CD30. Their specific features and relationship to CD30-expressing Hodgkin and Reed/Sternberg (HRS) cells of Hodgkin lymphoma are unclear but highly relevant, because numerous patients with lymphoma are currently treated with an anti-CD30 immunotoxin. We performed a comprehensive analysis of human CD30+ B cells. Phenotypic and IgV gene analyses indicated that CD30+ GC B lymphocytes represent typical GC B cells, and that CD30+ EF B cells are mostly post-GC B cells. The transcriptomes of CD30+ GC and EF B cells largely overlapped, sharing a strong MYC signature, but were strikingly different from conventional GC B cells and memory B and plasma cells, respectively. CD30+ GC B cells represent MYC+ centrocytes redifferentiating into centroblasts; CD30+ EF B cells represent active, proliferating memory B cells. HRS cells shared typical transcriptome patterns with CD30+ B cells, suggesting that they originate from these lymphocytes or acquire their characteristic features during lymphomagenesis. By comparing HRS to normal CD30+ B cells we redefined aberrant and disease-specific features of HRS cells. A remarkable downregulation of genes regulating genomic stability and cytokinesis in HRS cells may explain their genomic instability and multinuclearity.

Changes on protein expression associated with salinity tolerance in Brassica cell cultures.

PubMed

Martín, J P; Elavummoottil, O C; Moreno, M L

1993-09-01

The synthesis of proteins from salt-tolerant Brassica oleracea L. var. botrytis L. subvar. cauliflora (Gars.) DC. (cauliflower) cell cultures is modified in relation to controls in several features. There are nine newly induced polypeptides in tolerant cultures (absent in control conditions). Some of them are only present under low salt levels (85 mM NaCl). Another group seems to be representative of moderate and high salt levels (170 and 255 mM NaCl), and a third group is present in all the salt conditions tested. On the other hand, the synthesis of most of the polypeptides present in control conditions is modified in salt-tolerant cultures by increasing, decreasing or stopping their synthesis in any of the tested conditions. The relationship between these changes in Brassica and other plant systems is discussed.

Clonal populations of amniotic cells by dilution and direct plating: evidence for hidden diversity.

PubMed

Wilson, Patricia G; Devkota, Lorna; Payne, Tiffany; Crisp, Laddie; Winter, Allison; Wang, Zhan

2012-01-01

Fetal cells are widely considered a superior cell source for regenerative medicine; fetal cells show higher proliferative capacity and have undergone fewer replicative cycles that could generate spontaneous mutations. Fetal cells in amniotic fluid were among the first normal primary cells to be cultured ex vivo, but the undefined composition of amniotic fluid has hindered advance for regenerative applications. We first developed a highly efficient method to generate clonal populations by dilution of amniocentesis samples in media and direct plating without intervening refrigeration, centrifugation, or exposure of cells to the paracrine effects in mixed cell cultures. More than 40 clonal populations were recovered from 4 amniocentesis samples and representative clones were characterized by flow cytometry, conventional assays for differentiation potential, immunofluorescence imaging, and transcript analysis. The results revealed previously unreported diversity among stromal and epithelial cell types and identified unique cell types that could be lost or undetected in mixed cell populations. The differentiation potential of amniotic cells proved to be uncoupled from expression of definitive cell surface or cytoplasmic markers for stromal and epithelial cells. Evidence for diversity among stromal and epithelial cells in amniotic fluid bears on interpretations applied to molecular and functional tests of amniotic cell populations.

Highly sensitive fetal goat tongue cell line for detection and isolation of foot-and-mouth disease virus.

PubMed

Brehm, K E; Ferris, N P; Lenk, M; Riebe, R; Haas, B

2009-10-01

A fetal goat cell line (ZZ-R 127) supplied by the Collection of Cell Lines in Veterinary Medicine of the Friedrich Loeffler Institute was examined for susceptibility to infection by foot-and-mouth disease (FMD) virus (FMDV) and by two other viruses causing clinically indistinguishable vesicular conditions, namely, the viruses of swine vesicular disease and vesicular stomatitis. Primary bovine thyroid (BTY) cells are generally the most sensitive cell culture system for FMDV detection but are problematic to produce, particularly for laboratories that infrequently perform FMD diagnostic tests and for those in countries where FMD is endemic that face problems in sourcing thyroid glands from FMD-negative calves. Strains representing all seven serotypes of FMDV could be isolated in ZZ-R 127 cells with a sensitivity that was considerably higher than that of established cell lines and within 0.5 log of that for BTY cells. The ZZ-R 127 cell line was found to be a sensitive, rapid, and convenient tool for the isolation of FMDV and a useful alternative to BTY cells for FMD diagnosis.

An aza-macrocycle containing maltolic side-arms (maltonis) as potential drug against human pediatric sarcomas

PubMed Central

2014-01-01

Background Identification of new drugs against paediatric sarcomas represents an urgent clinical need that mainly relies on public investments due to the rarity of these diseases. In this paper we evaluated the in vitro and in vivo efficacy of a new maltol derived molecule (maltonis), belonging to the family of molecules named hydroxypyrones. Methods Maltonis was screened for its ability to induce structural alteration of DNA molecules in comparison to another maltolic molecule (malten). In vitro antitumour efficacy was tested using a panel of sarcoma cell lines, representative of Ewing sarcoma, osteosarcoma and rhabdomyosarcoma, the three most common paediatric sarcomas, and in normal human mesenchymal primary cell cultures. In vivo efficacy was tested against TC-71 Ewing sarcoma xenografts. Results Maltonis, a soluble maltol-derived synthetic molecule, was able to alter the DNA structure, inhibit proliferation and induce apoptotic cell death in paediatric sarcoma cells, either sensitive or resistant to some conventional chemotherapeutic drugs, such as doxorubicin and cisplatin. In addition, maltonis was able to induce: i) p21, p15 and Gadd45a mRNA upregulation; ii) Bcl-2, survivin, CDK6 and CDK8 down-regulation; iii) formation of γ-H2AX nuclear foci; iv) cleavage of PARP and Caspase 3. Two independent in vivo experiments demonstrated the tolerability and efficacy of maltonis in the inhibition of tumour growth. Finally maltonis was not extruded by ABCB1, one of the major determinants of chemotherapy failure, nor appeared to be a substrate of the glutathione-related detoxification system. Conclusions Considering that treatment of poorly responsive patients still suffers for the paucity of agents able to revert chemoresistance, maltonis may be considered for the future development of new therapeutic approaches for refractory metastatic patients. PMID:24575739

Cell invasion of poultry-associated Salmonella enterica serovar Enteritidis isolates is associated with pathogenicity, motility and proteins secreted by the type III secretion system

PubMed Central

Zhou, Xiaohui; Addwebi, Tarek; Davis, Margaret A.; Orfe, Lisa; Call, Douglas R.; Guard, Jean; Besser, Thomas E.

2011-01-01

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major cause of food-borne gastroenteritis in humans worldwide. Poultry and poultry products are considered the major vehicles of transmission to humans. Using cell invasiveness as a surrogate marker for pathogenicity, we tested the invasiveness of 53 poultry-associated isolates of S. Enteritidis in a well-differentiated intestinal epithelial cell model (Caco-2). The method allowed classification of the isolates into low (n = 7), medium (n = 18) and high (n = 30) invasiveness categories. Cell invasiveness of the isolates did not correlate with the presence of the virulence-associated gene spvB or the ability of the isolates to form biofilms. Testing of representative isolates with high and low invasiveness in a mouse model revealed that the former were more invasive in vivo and caused more and earlier mortalities, whereas the latter were significantly less invasive in vivo, causing few or no mortalities. Further characterization of representative isolates with low and high invasiveness showed that most of the isolates with low invasiveness had impaired motility and impaired secretion of either flagella-associated proteins (FlgK, FljB and FlgL) or type III secretion system (TTSS)-secreted proteins (SipA and SipD) encoded on Salmonella pathogenicity island-1. In addition, isolates with low invasiveness had impaired ability to invade and/or survive within chicken macrophages. These data suggest that not all isolates of S. Enteritidis recovered from poultry may be equally pathogenic, and that the pathogenicity of S. Enteritidis isolates is associated, in part, with both motility and secretion of TTSS effector proteins. PMID:21292746

Highly efficient nonprecious metal catalyst prepared with metal–organic framework in a continuous carbon nanofibrous network

PubMed Central

Shui, Jianglan; Chen, Chen; Grabstanowicz, Lauren; Zhao, Dan; Liu, Di-Jia

2015-01-01

Fuel cell vehicles, the only all-electric technology with a demonstrated >300 miles per fill travel range, use Pt as the electrode catalyst. The high price of Pt creates a major cost barrier for large-scale implementation of polymer electrolyte membrane fuel cells. Nonprecious metal catalysts (NPMCs) represent attractive low-cost alternatives. However, a significantly lower turnover frequency at the individual catalytic site renders the traditional carbon-supported NPMCs inadequate in reaching the desired performance afforded by Pt. Unconventional catalyst design aiming at maximizing the active site density at much improved mass and charge transports is essential for the next-generation NPMC. We report here a method of preparing highly efficient, nanofibrous NPMC for cathodic oxygen reduction reaction by electrospinning a polymer solution containing ferrous organometallics and zeolitic imidazolate framework followed by thermal activation. The catalyst offers a carbon nanonetwork architecture made of microporous nanofibers decorated by uniformly distributed high-density active sites. In a single-cell test, the membrane electrode containing such a catalyst delivered unprecedented volumetric activities of 3.3 A⋅cm−3 at 0.9 V or 450 A⋅cm−3 extrapolated at 0.8 V, representing the highest reported value in the literature. Improved fuel cell durability was also observed. PMID:26261338

Highly efficient nonprecious metal catalyst prepared with metal–organic framework in a continuous carbon nanofibrous network

DOE PAGES

Shui, Jianglan; Chen, Chen; Grabstanowicz, Lauren; ...

2015-08-25

Fuel cell vehicles, the only all-electric technology with a demonstrated >300 miles per fill travel range, use Pt as the electrode catalyst. The high price of Pt creates a major cost barrier for large-scale implementation of polymer electrolyte membrane fuel cells. Nonprecious metal catalysts (NPMCs) represent attractive low-cost alternatives. However, a significantly lower turnover frequency at the individual catalytic site renders the traditional carbon-supported NPMCs inadequate in reaching the desired performance afforded by Pt. Unconventional catalyst design aiming at maximizing the active site density at much improved mass and charge transports is essential for the next-generation NPMC. We report heremore » a method of preparing highly efficient, nanofibrous NPMC for cathodic oxygen reduction reaction by electrospinning a polymer solution containing ferrous organometallics and zeolitic imidazolate framework followed by thermal activation. The catalyst offers a carbon nanonetwork architecture made of microporous nanofibers decorated by uniformly distributed high-density active sites. In a single-cell test, the membrane electrode containing such a catalyst delivered unprecedented volumetric activities of 3.3 A∙cm -3 at 0.9 V or 450 A∙cm -3 extrapolated at 0.8 V, representing the highest reported value in the literature. Improved fuel cell durability was also observed.« less

Highly efficient nonprecious metal catalyst prepared with metal-organic framework in a continuous carbon nanofibrous network.

PubMed

Shui, Jianglan; Chen, Chen; Grabstanowicz, Lauren; Zhao, Dan; Liu, Di-Jia

2015-08-25

Fuel cell vehicles, the only all-electric technology with a demonstrated >300 miles per fill travel range, use Pt as the electrode catalyst. The high price of Pt creates a major cost barrier for large-scale implementation of polymer electrolyte membrane fuel cells. Nonprecious metal catalysts (NPMCs) represent attractive low-cost alternatives. However, a significantly lower turnover frequency at the individual catalytic site renders the traditional carbon-supported NPMCs inadequate in reaching the desired performance afforded by Pt. Unconventional catalyst design aiming at maximizing the active site density at much improved mass and charge transports is essential for the next-generation NPMC. We report here a method of preparing highly efficient, nanofibrous NPMC for cathodic oxygen reduction reaction by electrospinning a polymer solution containing ferrous organometallics and zeolitic imidazolate framework followed by thermal activation. The catalyst offers a carbon nanonetwork architecture made of microporous nanofibers decorated by uniformly distributed high-density active sites. In a single-cell test, the membrane electrode containing such a catalyst delivered unprecedented volumetric activities of 3.3 A ⋅ cm(-3) at 0.9 V or 450 A ⋅ cm(-3) extrapolated at 0.8 V, representing the highest reported value in the literature. Improved fuel cell durability was also observed.

In vitro toxicity testing with microplate cell cultures: Impact of cell binding.

PubMed

Gülden, Michael; Schreiner, Jeannine; Seibert, Hasso

2015-06-05

In vitro generated data on toxic potencies are generally based on nominal concentrations. However, cellular and extracellular binding and elimination processes may reduce the available free fraction of a compound. Then, nominal effective concentrations do not represent appropriate measures of toxic exposure in vitro and underestimate toxic potencies. In this study it was investigated whether cell binding can affect the availability of chemicals in microplate based toxicity assays. To this end the cytotoxicity of compounds like mercury chloride, digitonin and alcohol ethoxylates, accumulated by cells via different modes, was investigated in 96-well microplate cultures with varying concentrations of Balb/c 3T3 cells. The median effective nominal concentrations of all but one of the tested compounds depended linearly from the cell concentration. Applying a previously developed equilibrium distribution model cell concentration-independent median effective extracellular concentrations and cell burdens, respectively, could be calculated. The compounds were accumulated by the cells with bioconcentration factors, BCF, between 480 and ≥ 25,000. Cell binding of the alcohol ethoxylates was correlated with their lipophilicity. The results show that significant cell binding can occur even at the small cell volume fractions (∼ 1 × 10(-5) to 3 × 10(-3) L/L) encountered in microplate assays. To what extent cell binding affects the bioavailability depends on the BCF and the cell volume fraction. EC50 measurements in the presence of at least two different cell concentrations allow for excluding or detecting significant cell binding and for determining more appropriate measures of toxic exposure in vitro like median effective extracellular (free) concentrations or cell burdens. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

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

PubMed

Bausinger, Julia; Speit, Günter

2014-11-01

The comet assay is widely used in human biomonitoring to measure DNA damage in whole blood or isolated peripheral blood mononuclear cells (PBMC) as a marker of exposure to genotoxic agents. Cytogenetic assays with phytohemagglutinin (PHA)-stimulated cultured T lymphocytes are also frequently performed in human biomonitoring. Cytogenetic effects (micronuclei, chromosome aberrations, sister chromatid exchanges) may be induced in vivo but also occur ex vivo during the cultivation of lymphocytes as a consequence of DNA damage present in lymphocytes at the time of sampling. To better understand whether DNA damage measured by the comet assay in PBMC is representative for DNA damage in T cells, we comparatively investigated DNA damage and its repair in PBMC and T cells obtained by immunomagnetic cell sorting. PBMC cultures and T cell cultures were exposed to mutagens with different modes of genotoxic action and DNA damage was measured by the comet assay after the end of a 2h exposure and after 18h post-incubation. The mutagens tested were methyl methanesulfonate (MMS), (±)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), 4-nitroquinoline-1-oxide (4NQO), styrene oxide and potassium bromate. MMS and potassium bromate were also tested by the modified comet assay with formamido pyrimidine glycosylase (FPG) protein. The results indicate that the mutagens tested induce DNA damage in PBMC and T cells in the same range of concentrations and removal of induced DNA lesions occurs to a comparable extent. Based on these results, we conclude that the comet assay with PBMC is suited to predict DNA damage and its removal in T cells. Copyright © 2014 Elsevier B.V. All rights reserved.

CRISPR/Cas9-generated p47phox-deficient cell line for Chronic Granulomatous Disease gene therapy vector development.

PubMed

Wrona, Dominik; Siler, Ulrich; Reichenbach, Janine

2017-03-13

Development of gene therapy vectors requires cellular models reflecting the genetic background of a disease thus allowing for robust preclinical vector testing. For human p47 phox -deficient chronic granulomatous disease (CGD) vector testing we generated a cellular model using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to introduce a GT-dinucleotide deletion (ΔGT) mutation in p47 phox encoding NCF1 gene in the human acute myeloid leukemia PLB-985 cell line. CGD is a group of hereditary immunodeficiencies characterized by impaired respiratory burst activity in phagocytes due to a defective phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In Western countries autosomal-recessive p47 phox -subunit deficiency represents the second largest CGD patient cohort with unique genetics, as the vast majority of p47 phox CGD patients carries ΔGT deletion in exon two of the NCF1 gene. The established PLB-985 NCF1 ΔGT cell line reflects the most frequent form of p47 phox -deficient CGD genetically and functionally. It can be differentiated to granulocytes efficiently, what creates an attractive alternative to currently used iPSC models for rapid testing of novel gene therapy approaches.

Synthesis and biological evaluation of new coumarins bearing 2,4-diaminothiazole-5-carbonyl moiety.

PubMed

Ayati, Adileh; Oghabi Bakhshaiesh, Tayebeh; Moghimi, Setareh; Esmaeili, Rezvan; Majidzadeh-A, Keivan; Safavi, Maliheh; Firoozpour, Loghman; Emami, Saeed; Foroumadi, Alireza

2018-06-07

A series of new coumarin-containing compounds 3a-l and 4a-c was designed and synthesized based on the chalcone-type 4-amino-5-cinnamoylthiazole scaffold 2, and screened for their in vitro anticancer and antioxidant activities. Representatively, the 2-thiomorpholinothiazole derivative 3k with IC 50 values of 7.5-16.9 μg/ml demonstrated good cytotoxic effects against tested cell lines MCF-7, HepG2 and SW480. Further investigation by flow cytometric analysis confirmed that this compound induces apoptotic cell death in MCF-7 cells and cause G1-phase arrest in the cell cycle. Moreover, most of compounds had intrinsic potential for radical scavenging activity and ferric-reducing power as investigated by DPPH and FRAP assays. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Photoacoustic sensor for medical diagnostics

NASA Astrophysics Data System (ADS)

Wolff, Marcus; Groninga, Hinrich G.; Harde, Hermann

2004-03-01

The development of new optical sensor technologies has a major impact on the progress of diagnostic methods. Of the permanently increasing number of non-invasive breath tests, the 13C-Urea Breath Test (UBT) for the detection of Helicobacter pylori is the most prominent. However, many recent developments, like the detection of cancer by breath test, go beyond gastroenterological applications. We present a new detection scheme for breath analysis that employs an especially compact and simple set-up. Photoacoustic Spectroscopy (PAS) represents an offset-free technique that allows for short absorption paths and small sample cells. Using a single-frequency diode laser and taking advantage of acoustical resonances of the sample cell, we performed extremely sensitive and selective measurements. The smart data processing method contributes to the extraordinary sensitivity and selectivity as well. Also, the reasonable acquisition cost and low operational cost make this detection scheme attractive for many biomedical applications. The experimental set-up and data processing method, together with exemplary isotope-selective measurements on carbon dioxide, are presented.

Chitosan-thioglycolic acid as a versatile antimicrobial agent.

PubMed

Geisberger, Georg; Gyenge, Emina Besic; Hinger, Doris; Käch, Andres; Maake, Caroline; Patzke, Greta R

2013-04-08

As functionalized chitosans hold great potential for the development of effective and broad-spectrum antibiotics, representative chitosan derivatives were tested for antimicrobial activity in neutral media: trimethyl chitosan (TMC), carboxy-methyl chitosan (CMC), and chitosan-thioglycolic acid (TGA; medium molecular weight: MMW-TGA; low molecular weight: LMW-TGA). Colony forming assays indicated that LMW-TGA displayed superior antimicrobial activity over the other derivatives tested: a 30 min incubation killed 100% Streptococcus sobrinus (Gram-positive bacteria) and reduced colony counts by 99.99% in Neisseria subflava (Gram-negative bacteria) and 99.97% in Candida albicans (fungi). To elucidate LMW-TGA effects at the cellular level, microscopic studies were performed. Use of fluorescein isothiocyanate (FITC)-labeled chitosan derivates in confocal microscopy showed that LMW-TGA attaches to microbial cell walls, while transmission electron microscopy indicated that this derivative severely affects cell wall integrity and intracellular ultrastructure in all species tested. We therefore propose LMW-TGA as a promising and effective broad-band antimicrobial compound.

Non-invasive prenatal diagnosis (NIPD) for single gene disorders: cost analysis of NIPD and invasive testing pathways.

PubMed

Verhoef, Talitha I; Hill, Melissa; Drury, Suzanne; Mason, Sarah; Jenkins, Lucy; Morris, Stephen; Chitty, Lyn S

2016-07-01

Evaluate the costs of offering non-invasive prenatal diagnosis (NIPD) for single gene disorders compared to traditional invasive testing to inform NIPD implementation into clinical practice. Total costs of diagnosis using NIPD or invasive testing pathways were compared for a representative set of single gene disorders. For autosomal dominant conditions, where NIPD molecular techniques are straightforward, NIPD cost £314 less than invasive testing. NIPD for autosomal recessive and X-linked conditions requires more complicated technical approaches and total costs were more than invasive testing, e.g. NIPD for spinal muscular atrophy was £1090 more than invasive testing. Impact of test uptake on costs was assessed using sickle cell disorder as an example. Anticipated high uptake of NIPD resulted in an incremental cost of NIPD over invasive testing of £48 635 per 100 pregnancies at risk of sickle cell disorder. Total costs of NIPD are dependent upon the complexity of the testing technique required. Anticipated increased demand for testing may have economic implications for prenatal diagnostic services. Ethical issues requiring further consideration are highlighted including directing resources to NIPD when used for information only and restricting access to safe tests if it is not cost-effective to develop NIPD for rare conditions. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Human herpesvirus 8-associated lymphoma mimicking cutaneous anaplastic large T-cell lymphoma in a patient with human immunodeficiency virus infection.

PubMed

Li, Meng-Fang; Hsiao, Cheng-Hsiang; Chen, Yi-Lin; Huang, Wen-Ya; Lee, Yi-Hsuan; Huang, Hsien-Neng; Lien, Huang-Chun

2012-02-01

Primary effusion lymphoma, a human herpesvirus 8 (HHV8)-associated lymphoma, is uncommon, and it is usually seen in human immunodeficiency virus (HIV)-infected patients. It presents as a body cavity-based lymphomatous effusion, but several cases of the so-called solid primary effusion lymphoma presenting as solid tumors without associated lymphomatous effusion have been reported. They have similar clinical, histopathological and immunophenotypical features. Most of them have a B-cell genotype. This suggests the solid variant may represent a clinicopathological spectrum of primary effusion lymphoma. We report a case of HHV8-associated lymphoma histopathologically and immunophenotypically mimicking cutaneous anaplastic large cell lymphoma. The patient was a 31-year-old HIV-seropositive man presenting with skin nodules over his right thigh. Biopsy of the nodules showed anaplastic large cells infiltrating the dermis. These malignant cells strongly expressed CD3, CD30 and CD43. Cutaneous anaplastic large T-cell lymphoma was initially diagnosed, but further tests, including immunoreactivity for HHV8 protein and clonal rearrangements of immunoglobulin genes, confirmed the diagnosis of HHV8-associated B-cell lymphoma with aberrant T-cell marker expression. This case provides an example of solid primary effusion lymphoma mimicking cutaneous anaplastic large T-cell lymphoma and highlights the importance of HHV8 immunohistochemistry and molecular tests in the diagnosis of HHV8-associated lymphoma with a cutaneous presentation. Copyright © 2011 John Wiley & Sons A/S.

Using the peptide BP100 as a cell-penetrating tool for the chemical engineering of actin filaments within living plant cells.

PubMed

Eggenberger, Kai; Mink, Christian; Wadhwani, Parvesh; Ulrich, Anne S; Nick, Peter

2011-01-03

The delivery of externally applied macromolecules or nanoparticles into living cells still represents a critically limiting step before the full capabilities of chemical engineering can be explored. Molecular transporters such as cell-penetrating peptides, peptoids, and other mimetics can be used to carry cargo across the cellular membrane, but it is still difficult to find suitable sequences that operate efficiently for any particular type of cell. Here we report that BP100 (KKLFKKILKYL-amide), originally designed as an antimicrobial peptide against plant pathogens, can be employed as a fast and efficient cell-penetrating agent to transport fluorescent test cargoes into the cytosol of walled plant cells. The uptake of BP100 proceeds slightly more slowly than the endocytosis of fluorescent dextranes, but BP100 accumulates more efficiently and to much higher levels (by an order of magnitude). The entry of BP100 can be efficiently blocked by latrunculin B; this suggests that actin filaments are essential to the uptake mechanism. To test whether this novel transporter can also be used to deliver functional cargoes, we designed a fusion construct of BP100 with the actin-binding Lifeact peptide (MGVADLIKKFESISKEE). We demonstrated that the short BP100 could transport the attached 17-residue sequence quickly and efficiently into tobacco cells. The Lifeact construct retained its functionality as it successfully labeled the actin bundles that tether the nucleus in the cell center.

Tailoring microfluidic systems for organ-like cell culture applications using multiphysics simulations

NASA Astrophysics Data System (ADS)

Hagmeyer, Britta; Schütte, Julia; Böttger, Jan; Gebhardt, Rolf; Stelzle, Martin

2013-03-01

Replacing animal testing with in vitro cocultures of human cells is a long-term goal in pre-clinical drug tests used to gain reliable insight into drug-induced cell toxicity. However, current state-of-the-art 2D or 3D cell cultures aiming at mimicking human organs in vitro still lack organ-like morphology and perfusion and thus organ-like functions. To this end, microfluidic systems enable construction of cell culture devices which can be designed to more closely resemble the smallest functional unit of organs. Multiphysics simulations represent a powerful tool to study the various relevant physical phenomena and their impact on functionality inside microfluidic structures. This is particularly useful as it allows for assessment of system functions already during the design stage prior to actual chip fabrication. In the HepaChip®, dielectrophoretic forces are used to assemble human hepatocytes and human endothelial cells in liver sinusoid-like structures. Numerical simulations of flow distribution, shear stress, electrical fields and heat dissipation inside the cell assembly chambers as well as surface wetting and surface tension effects during filling of the microchannel network supported the design of this human-liver-on-chip microfluidic system for cell culture applications. Based on the device design resulting thereof, a prototype chip was injection-moulded in COP (cyclic olefin polymer). Functional hepatocyte and endothelial cell cocultures were established inside the HepaChip® showing excellent metabolic and secretory performance.

Hyaluronic acid in complexes with surfactants: The efficient tool for reduction of the cytotoxic effect of surfactants on human cell types.

PubMed

Sauerová, Pavla; Pilgrová, Tereza; Pekař, Miloslav; Hubálek Kalbáčová, Marie

2017-10-01

The cationic surfactants carbethoxypendecinium bromide (Septonex) and cetyltrimethylammonium bromide (CTAB) are known to be harmful for certain cell types (bacteria, fungi, mammal cells, etc.). Colloidal complexes of these surfactants with negatively-charged hyaluronic acid (HyA) were prepared for potential drug and/or universal delivery applications. The complexes were tested for their cytotoxic effect on different human cell types - osteoblasts, keratinocytes and fibroblasts. Both the CTAB-HyA and Septonex-HyA complexes were found to reduce the cytotoxicity induced by surfactants alone concerning all the tested concentrations. Moreover, we suggested the limits of HyA protection provided by the surfactant-HyA complexes, e.g. the importance of the amount of HyA applied. We also determined the specific sensitivity of different cell types to surfactant treatment. Keratinocytes were more sensitive to CTAB, while osteoblasts and fibroblasts were more sensitive to Septonex. Moreover, it was indirectly shown that CTAB combines lethal toxicity with cell metabolism induction, while Septonex predominantly causes lethal toxicity concerning fibroblasts. This comprehensive study of the effect of surfactant-HyA complexes on various human cell types revealed that HyA represents a useful CTAB or Septonex cytotoxic effect modulator at diverse levels. Potential applications for these complexes include drug and/or nucleic acid delivery systems, diagnostic dye carriers and cosmetics production. Copyright © 2017 Elsevier B.V. All rights reserved.

The effect of Nullomer-derived peptides 9R, 9S1R and 124R on the NCI-60 panel and normal cell lines.

PubMed

Alileche, Abdelkrim; Hampikian, Greg

2017-08-09

Nullomer peptides are the smallest sequences absent from databases of natural proteins. We first began compiling a list of absent 5-amino acid strings in 2006 (1). We report here the effects of Nullomer-derived peptides 9R, 9S1R and 124R on the NCI-60 panel, derived from human cancers of 9 organs (kidney, ovary, skin melanoma, lung, brain, lung, colon, prostate and the hematopoietic system), and four normal cell lines (endothelial HUVEC, skin fibroblasts BJ, colon epithelial FHC and normal prostate RWPE-1). NCI-60 cancer cell panel and four normal cell lines were cultured in vitro in RPMI1640 supplemented with 10% Hyclone fetal bovine serum and exposed for 48 h to 5 μM, 25 μM and 50 μM of peptides 9R, 9S1R and 124R. Viability was assessed by CCK-8 assay. For peptide ATP depletion effects, one cell line representing each organ in the NCI-60 panel, and four normal cell lines were exposed to 50 μM of peptides 9R, 9S1R and 124R for 3 h. The ATP content was assessed in whole cells, and their supernatants. Peptides 9S1R and 9R are respectively lethal to 95 and 81.6% of the 60 cancer cell lines tested. Control peptide 124R has no effect on the growth of these cells. Especially interesting the fact that peptides 9R and 9S1R are capable of killing drug-resistant and hormone-resistant cell lines, and even cancer stem cells. Peptides 9R and 9S1R have a broader activity spectrum than many cancer drugs in current use, can completely deplete cellular ATP within 3 h, and are less toxic to 3 of the 4 normal cell lines tested than they are to several cancers. Nullomer peptides 9R and 9S1R have a large broad lethal effect on cancer cell lines derived from nine organs represented in the NCI-60 panel. This broad activity crosses many of the categorical divisions used in the general classification of cancers: solid vs liquid cancers, drug sensitive vs drug resistant, hormone sensitive vs hormone resistant, cytokine sensitive vs cytokine non sensitive, slow growing vs rapid growing, differentiated vs dedifferentiated cancers. Furthermore peptides 9R and 9S1R are lethal to cancer stem cells and breast canrcinosarcoma.

Safety: Special Effects of Thermal Runaway Chapter Heading for Encyclopedia of Electrochemical Power Sources (PREPRINT)

DTIC Science & Technology

2007-11-09

been following developments related to the recent lithium ion battery recalls and is preparing itself for revising its battery safety standard...manufacturer (OEM) Critical Components Committee. In October 2006, the IPC Lithium Ion Battery Subcommittee, that represents both the major...cover process requirements, quality control and assurance for lithium ion battery cells. Electric and Hybrid Electric Vehicle Power Source Testing In

Can currently available non-animal methods detect pre and ...

EPA Pesticide Factsheets

Predictive testing to identify and characterise substances for their skin sensitisation potential has historically been based on animal tests such as the Local Lymph Node Assay (LLNA). In recent years, regulations in the cosmetics and chemicals sectors has provided a strong impetus to develop and evaluate non-animal alternative methods. The AOP for skin sensitisation provides a framework to anchor non-animal test methods to key events in the pathway to help identify what tests can be combined together to generate the potency information required for risk assessment. The 3 test methods that have undergone extensive development and validation are the direct peptide reactivity assay (DPRA), the KeratinoSensTM and the human Cell Line Activation Test (h-CLAT). Whilst these methods have been shown to perform relatively well in predicting LLNA results (accuracy ~ 80%), a particular concern that has been raised is their ability to predict chemicals that need to be activated to act as sensitisers (either abiotically on the skin (pre-hapten) or metabolically in the skin (pro-hapten)). The DPRA is a cell free system whereas the other two methods make use of cells that do not fully represent the in vivo metabolic situation. Based on previously published datasets of LLNA data, it has been found that approximately 25% of sensitisers are pre- and/or pro-haptens. This study reviewed an EURL ECVAM dataset of 127 substances for which information was available in the LLNA and the

Experimental Characterization and Micromechanical Modeling of Woven Carbon/Copper Composites

NASA Technical Reports Server (NTRS)

Bednarcyk, Brett A.; Pauly, Christopher C.; Pindera, Marek-Jerzy

1997-01-01

The results of an extensive experimental characterization and a preliminary analytical modeling effort for the elastoplastic mechanical behavior of 8-harness satin weave carbon/copper (C/Cu) composites are presented. Previous experimental and modeling investigations of woven composites are discussed, as is the evolution of, and motivation for, the continuing research on C/Cu composites. Experimental results of monotonic and cyclic tension, compression, and Iosipescu shear tests, and combined tension-compression tests, are presented. With regard to the test results, emphasis is placed on the effect of strain gauge size and placement, the effect of alloying the copper matrix to improve fiber-matrix bonding, yield surface characterization, and failure mechanisms. The analytical methodology used in this investigation consists of an extension of the three-dimensional generalized method of cells (GMC-3D) micromechanics model, developed by Aboudi (1994), to include inhomogeneity and plasticity effects on the subcell level. The extension of the model allows prediction of the elastoplastic mechanical response of woven composites, as represented by a true repeating unit cell for the woven composite. The model is used to examine the effects of refining the representative geometry of the composite, altering the composite overall fiber volume fraction, changing the size and placement of the strain gauge with respect to the composite's reinforcement weave, and including porosity within the infiltrated fiber yarns on the in-plane elastoplastic tensile, compressive, and shear response of 8-harness satin C/Cu. The model predictions are also compared with the appropriate monotonic experimental results.

Development of an artificial neural network model for risk assessment of skin sensitization using human cell line activation test, direct peptide reactivity assay, KeratinoSens™ and in silico structure alert parameter.

PubMed

Hirota, Morihiko; Ashikaga, Takao; Kouzuki, Hirokazu

2018-04-01

It is important to predict the potential of cosmetic ingredients to cause skin sensitization, and in accordance with the European Union cosmetic directive for the replacement of animal tests, several in vitro tests based on the adverse outcome pathway have been developed for hazard identification, such as the direct peptide reactivity assay, KeratinoSens™ and the human cell line activation test. Here, we describe the development of an artificial neural network (ANN) prediction model for skin sensitization risk assessment based on the integrated testing strategy concept, using direct peptide reactivity assay, KeratinoSens™, human cell line activation test and an in silico or structure alert parameter. We first investigated the relationship between published murine local lymph node assay EC3 values, which represent skin sensitization potency, and in vitro test results using a panel of about 134 chemicals for which all the required data were available. Predictions based on ANN analysis using combinations of parameters from all three in vitro tests showed a good correlation with local lymph node assay EC3 values. However, when the ANN model was applied to a testing set of 28 chemicals that had not been included in the training set, predicted EC3s were overestimated for some chemicals. Incorporation of an additional in silico or structure alert descriptor (obtained with TIMES-M or Toxtree software) in the ANN model improved the results. Our findings suggest that the ANN model based on the integrated testing strategy concept could be useful for evaluating the skin sensitization potential. Copyright © 2017 John Wiley & Sons, Ltd.

Phytoplankton and bacterial community structures and their interaction during red-tide phenomena

NASA Astrophysics Data System (ADS)

Ismail, Mona Mohamed; Ibrahim, Hassan Abd Allah

2017-09-01

Phytoplankton and bacteria diversity were studied before, during and after red tide phenomena during spring season 2015 in the Eastern Harbour (E.H.) of Alexandria, Egypt. Fifty five species of phytoplankton were identified and represented different distinct classes "Bacillariophyceae; Dinophyceae, Chlorophyceae, Cyanophyceae and Eugelenophyceae". Also, Diatom formed the most dominant group. The average number of the phytoplankton density varied from 4.8 × 104 to 1.1 × 106 cell l-1 during the study period and Skeletonema costatum was the agent causing the red tide. The existence percentages of bacteria ranged from 2.6 to 17.9% on all media tested. The bacterial isolates on the nutrient agar medium represented the highest existence with a total percentage of 43.6%, followed by MSA medium (25.7%), while the lowest percentage was for the AA medium at 7.8%. However, twelve isolates were selected as representative for bacterial community during study interval. Based on the morphological, biochemical, physiological and enzymatic characteristics, the bacterial strains were described. Depending on the 16S rDNA gene sequence, three common antagonists were aligned as: Vibrio toranzoniae strain Vb 10.8, Ruegeria pelagia strain NBRC 102038 and Psychrobacter adeliensis strain DSM 15333. The interaction between these bacteria and S. costatum was studied. The growth of S. costatum was significantly lower whenever each bacterium was present as compared to axenic culture. More specifically, 30% (v/v) of the all tested bacteria showed the strongest algicidal activities, as all S. costatum cells were killed after two days. 10% of R. pelagia and P. adeliensis also showed significant algicidal activities within six days.

Proton pump inhibitor-induced tumour cell death by inhibition of a detoxification mechanism.

PubMed

Fais, S

2010-05-01

This review presents a possible new approach against cancer, as represented by inhibition of proton pumps, a mechanism used by tumour cells to avoid intracellular accumulation of toxic substances. Proton pump inhibitors (PPIs) belong to a family of pro-drugs that are currently used in the treatment of peptic diseases needing acidity to be activated. PPIs target the acidic tumour mass, where they are metabolized, thus blocking proton traffic. Proton pump inhibition triggers a rapid cell death as a result of intracellular acidification, caspase activation and early accumulation of reactive oxygen species into tumour cells. As a whole, the devastating effect of PPIs on tumour cells suggest the triggering of a fatal cell toxification. Many human tumours, including melanoma, osteosarcoma, lymphomas and various adenocarcinomas are responsive to PPIs. This appears highly conceivable, in as much as almost all human tumours are acidic and express high levels of proton pumps. Paradoxically, metastatic tumours appear to be more responsive to PPIs being more acidic than the majority of primary tumours. However, two clinical trials test the effectiveness of PPIs in chemosensitizing melanoma and osteosarcoma patients. Indeed, tumour acidity represents a very potent mechanism of chemoresistance. A majority of cytotoxic agents, being weak bases, are quickly protonated outside and do not enter the cells, thus preventing drugs to reach specific cellular targets. Clinical data will provide the proof of concept on the use of PPIs as a new class of antitumour agent with a very low level of systemic toxicity as compared with standard chemotherapeutic agents.

Intercode comparison of gyrokinetic global electromagnetic modes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Görler, T., E-mail: tobias.goerler@ipp.mpg.de; Tronko, N.; Hornsby, W. A.

Aiming to fill a corresponding lack of sophisticated test cases for global electromagnetic gyrokinetic codes, a new hierarchical benchmark is proposed. Starting from established test sets with adiabatic electrons, fully gyrokinetic electrons, and electrostatic fluctuations are taken into account before finally studying the global electromagnetic micro-instabilities. Results from up to five codes involving representatives from different numerical approaches as particle-in-cell methods, Eulerian and Semi-Lagrangian are shown. By means of spectrally resolved growth rates and frequencies and mode structure comparisons, agreement can be confirmed on ion-gyro-radius scales, thus providing confidence in the correct implementation of the underlying equations.

Antibiotic Sensitivity of Micrococcus radiodurans

PubMed Central

Hawiger, J.; Jeljaszewicz, J.

1967-01-01

A wild-type strain of Micrococcus radiodurans and its nonpigmented mutant W1 were tested for sensitivity to 10 antibiotics selected from the standpoint of their mechanism of action. Representatives of groups of antibiotics inhibiting deoxyribonucleic acid (DNA) synthesis, DNA-dependent ribonucleic acid synthesis, protein synthesis, and cell wall synthesis were selected. M. radiodurans and its mutant exhibited full susceptibility to all antibiotics tested (mitomycin C, actinomycin D, chloramphenicol, dihydrostreptomycin, erythromycin, neomycin, kanamycin, benzylpenicillin, bacitracin, and vancomycin), the degree of susceptibility being of the same order as that of a standard strain of Staphylococcus aureus 209 P, with the exception of dihydrostreptomycin. PMID:4166078

Collection of quantitative chemical release field data.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Demirgian, J.; Macha, S.; Loyola Univ.

1999-01-01

Detection and quantitation of chemicals in the environment requires Fourier-transform infrared (FTIR) instruments that are properly calibrated and tested. This calibration and testing requires field testing using matrices that are representative of actual instrument use conditions. Three methods commonly used for developing calibration files and training sets in the field are a closed optical cell or chamber, a large-scale chemical release, and a small-scale chemical release. There is no best method. The advantages and limitations of each method should be considered in evaluating field results. Proper calibration characterizes the sensitivity of an instrument, its ability to detect a component inmore » different matrices, and the quantitative accuracy and precision of the results.« less

Exclusion from spheroid formation identifies loss of essential cell-cell adhesion molecules in colon cancer cells.

PubMed

Stadler, Mira; Scherzer, Martin; Walter, Stefanie; Holzner, Silvio; Pudelko, Karoline; Riedl, Angelika; Unger, Christine; Kramer, Nina; Weil, Beatrix; Neesen, Jürgen; Hengstschläger, Markus; Dolznig, Helmut

2018-01-18

Many cell lines derived from solid cancers can form spheroids, which recapitulate tumor cell clusters and are more representative of the in vivo situation than 2D cultures. During spheroid formation, a small proportion of a variety of different colon cancer cell lines did not integrate into the sphere and lost cell-cell adhesion properties. An enrichment protocol was developed to augment the proportion of these cells to 100% purity. The basis for the separation of spheroids from non-spheroid forming (NSF) cells is simple gravity-sedimentation. This protocol gives rise to sub-populations of colon cancer cells with stable loss of cell-cell adhesion. SW620 cells lacked E-cadherin, DLD-1 cells lost α-catenin and HCT116 cells lacked P-cadherin in the NSF state. Knockdown of these molecules in the corresponding spheroid-forming cells demonstrated that loss of the respective proteins were indeed responsible for the NSF phenotypes. Loss of the spheroid forming phenotype was associated with increased migration and invasion properties in all cell lines tested. Hence, we identified critical molecules involved in spheroid formation in different cancer cell lines. We present here a simple, powerful and broadly applicable method to generate new sublines of tumor cell lines to study loss of cell-cell adhesion in cancer progression.

Boosting Natural Killer Cell-Based Immunotherapy with Anticancer Drugs: a Perspective.

PubMed

Cifaldi, Loredana; Locatelli, Franco; Marasco, Emiliano; Moretta, Lorenzo; Pistoia, Vito

2017-12-01

Natural killer (NK) cells efficiently recognize and kill tumor cells through several mechanisms including the expression of ligands for NK cell-activating receptors on target cells. Different clinical trials indicate that NK cell-based immunotherapy represents a promising antitumor treatment. However, tumors develop immune-evasion strategies, including downregulation of ligands for NK cell-activating receptors, that can negatively affect antitumor activity of NK cells, which either reside endogenously, or are adoptively transferred. Thus, restoration of the expression of NK cell-activating ligands on tumor cells represents a strategic therapeutic goal. As discussed here, various anticancer drugs can fulfill this task via different mechanisms. We envision that the combination of selected chemotherapeutic agents with NK cell adoptive transfer may represent a novel strategy for cancer immunotherapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Antiproliferative activity of pristimerin isolated from Maytenus ilicifolia (Celastraceae) in human HL-60 cells.

PubMed

Costa, Patricia Marçal da; Ferreira, Paulo Michel Pinheiro; Bolzani, Vanderlan da Silva; Furlan, Maysa; de Freitas Formenton Macedo Dos Santos, Vânia Aparecida; Corsino, Joaquim; de Moraes, Manoel Odorico; Costa-Lotufo, Letícia Veras; Montenegro, Raquel Carvalho; Pessoa, Cláudia

2008-06-01

Pristimerin has been shown to be cytotoxic to several cancer cell lines. In the present work, the cytotoxicity of pristimerin was evaluated in human tumor cell lines and in human peripheral blood mononuclear cells (PBMC). This work also examined the effects of pristimerin (0.4; 0.8 and 1.7 microM) in HL-60 cells, after 6, 12 and 24h of exposure. Pristimerin reduced the number of viable cells and increased number of non-viable cells in a concentration-dependent manner by tripan blue test showing morphological changes consistent with apoptosis. Nevertheless, pristimerin was not selective to cancer cells, since it inhibited PBMC proliferation with an IC50 of 0.88 microM. DNA synthesis inhibition assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation in HL-60 cells was 70% and 83% for the concentrations of 0.4 and 0.8 microM, respectively. Pristimerin (10 and 20 microM) was not able to inhibit topoisomerase I. In AO/EB (acridine orange/ethidium bromide) staining, all tested concentrations reduced the number of HL-60 viable cells, with the occurrence of necrosis and apoptosis in a concentration-dependent manner, results in agreement with trypan blue exclusion findings. The analysis of membrane integrity and internucleosomal DNA fragmentation by flow cytometry in the presence of pristimerin indicated that treated cells underwent apoptosis. The present data point to the importance of pristimerin as representative of an emerging class of potential anticancer chemicals, exhibiting an antiproliferative effect by inhibiting DNA synthesis and triggering apoptosis.

Perisinusoidal cell hypertrophy in a patient with acquired immunodeficiency syndrome.

PubMed

Kossaifi, T; Dupon, M; Le Bail, B; Lacut, Y; Balabaud, C; Bioulac-Sage, P

1990-08-01

A 33-year-old heterosexual white man underwent a liver biopsy for determination of mild elevation of aminotransferase levels (aspartate aminotransferase, two times; alanine aminotransferase, three times). The patient had acquired immunodeficiency syndrome (stage IVC2) with tuberculosis of the lymph nodes. Antibody to hepatitis B surface antigen and antibody to hepatitis B core antigen were positive. Syphillis tests were positive. Liver architecture was normal; sinusoids were dilated with perisinusoidal, centrilobular, and portal fibrosis. On a 1-micron-thick section and under electron microscopy, perisinusoidal cells appeared to be massively loaded with lipids, while endothelial cells contained numerous dense bodies. Some hepatocytes presented evidence of cell damage. Sinusoids were infiltrated by an increased number of lymphocytes and macrophages. This patient who had recently been treated for tuberculosis was not taking extra vitamin A. He had no disease so far reported as being associated with perisinusoidal cell hypertrophy. This case and others are evidence that acquired immunodeficiency syndrome represents another cause of perisinusoidal cell hypertrophy in which there is no documented hypervitaminosis A.

The translational potential of human induced pluripotent stem cells for clinical neurology : The translational potential of hiPSCs in neurology.

PubMed

Devine, Helen; Patani, Rickie

2017-04-01

The induced pluripotent state represents a decade-old Nobel prize-winning discovery. Human-induced pluripotent stem cells (hiPSCs) are generated by the nuclear reprogramming of any somatic cell using a variety of established but evolving methods. This approach offers medical science unparalleled experimental opportunity to model an individual patient's disease "in a dish." HiPSCs permit developmentally rationalized directed differentiation into any cell type, which express donor cell mutation(s) at pathophysiological levels and thus hold considerable potential for disease modeling, drug discovery, and potentially cell-based therapies. This review will focus on the translational potential of hiPSCs in clinical neurology and the importance of integrating this approach with complementary model systems to increase the translational yield of preclinical testing for the benefit of patients. This strategy is particularly important given the expected increase in prevalence of neurodegenerative disease, which poses a major burden to global health over the coming decades.

ATM Quality of Service Parameters at 45 Mbps Using a Satellite Emulator: Laboratory Measurements

NASA Technical Reports Server (NTRS)

Ivancic, William D.; Bobinsky, Eric A.

1997-01-01

Results of 45-Mbps DS3 intermediate-frequency loopback measurements of asynchronous transfer mode (ATM) quality of service parameters (cell error ratio and cell loss ratio) are presented. These tests, which were conducted at the NASA Lewis Research Center in support of satellite-ATM interoperability research, represent initial efforts to quantify the minimum parameters for stringent ATM applications, such as MPEG-1 and MPEG-2 video transmission. Portions of these results were originally presented to the International Telecommunications Union's ITU-R Working Party 4B in February 1996 in support of their Draft Preliminary Recommendation on the Transmission of ATM Traffic via Satellite.

Onboard Hydrogen/Helium Sensors in Support of the Global Technical Regulation: An Assessment of Performance in Fuel Cell Electric Vehicle Crash Tests

DOE Office of Scientific and Technical Information (OSTI.GOV)

Post, M. B.; Burgess, R.; Rivkin, C.

2012-09-01

Automobile manufacturers in North America, Europe, and Asia project a 2015 release of commercial hydrogen fuel cell powered light-duty road vehicles. These vehicles will be for general consumer applications, albeit initially in select markets but with much broader market penetration expected by 2025. To assure international harmony, North American, European, and Asian regulatory representatives are striving to base respective national regulations on an international safety standard, the Global Technical Regulation (GTR), Hydrogen Fueled Vehicle, which is part of an international agreement pertaining to wheeled vehicles and equipment for wheeled vehicles.

Associative Pattern Recognition In Analog VLSI Circuits

NASA Technical Reports Server (NTRS)

Tawel, Raoul

1995-01-01

Winner-take-all circuit selects best-match stored pattern. Prototype cascadable very-large-scale integrated (VLSI) circuit chips built and tested to demonstrate concept of electronic associative pattern recognition. Based on low-power, sub-threshold analog complementary oxide/semiconductor (CMOS) VLSI circuitry, each chip can store 128 sets (vectors) of 16 analog values (vector components), vectors representing known patterns as diverse as spectra, histograms, graphs, or brightnesses of pixels in images. Chips exploit parallel nature of vector quantization architecture to implement highly parallel processing in relatively simple computational cells. Through collective action, cells classify input pattern in fraction of microsecond while consuming power of few microwatts.

Preliminary Evaluation of the Field and Laboratory Emission Cell (FLEC) for Sampling Attribution Signatures from Building Materials

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harvey, Scott D.; He, Lijian; Wahl, Jon H.

2012-08-30

This study provides a preliminary evaluation of the Field and Laboratory Emission Cell (FLEC) for its suitability for sampling building materials for toxic compounds and their associated impurities and residues that might remain after a terrorist chemical attack. Chemical warfare (CW) agents and toxic industrial chemicals were represented by a range of test probes that included CW surrogates. The test probes encompassed the acid-base properties, volatilities, and polarities of the expected chemical agents and residual compounds. Results indicated that dissipation of the test probes depended heavily on the underlying material. Near complete dissipation of almost all test probes occurred frommore » galvanized stainless steel within 3.0 hrs, whereas far stronger retention with concomitant slower release was observed for vinyl composition floor tiles. The test probes displayed immediated permanence on Teflon. FLEC sampling was further evaluated by profiling residues remaining after the evaporation of 2-chloroethyl ethyl sulfide, a sulfur mustard simulant. This study lays the groundwork for the eventual goal of applying this sampling approach for collection of forensic attribution signatures that remain after a terrorist chemical attack.« less

Review: Occult hepatitis C virus infection: still remains a controversy.

PubMed

Vidimliski, Pavlina Dzekova; Nikolov, Igor; Geshkovska, Nadica Matevska; Dimovski, Aleksandar; Rostaing, Lionel; Sikole, Aleksandar

2014-09-01

Occult hepatitis C virus (HCV) infection is characterized by the presence of HCV RNA in the liver cells or peripheral blood mononuclear cells of the patients whose serum samples test negative for HCV RNA, with or without presence of HCV antibodies. The present study reviews the existing literature on the persistence of occult hepatitis C virus infection, with description of the clinical characteristics and methods for identification of occult hepatitis C. Occult hepatitis C virus infection was detected in patients with abnormal results of liver function tests of unknown origin, with HCV antibodies and HCV RNA negativity in serum, and also in patients with spontaneous or treatment-induced recovery from hepatitis C. The viral replication in the liver cells and/or peripheral blood mononuclear cells was present in all clinical presentations of occult hepatitis C. The peripheral blood mononuclear cells represent an extra-hepatic site of HCV replication. The reason why HCV RNA was not detectable in the serum of patients with occult hepatitis C, could be the low number of circulating viral particles not detectable by the diagnostic tests with low sensitivity. It is uncertain whether occult hepatitis C is a different clinical entity or just a form of chronic hepatitis C virus infection. Data accumulated over the last decade demonstrated that an effective approach to the diagnosis of HCV infection would be the implementation of more sensitive HCV RNA diagnostic assays, and also, examination of the presence of viral particles in the cells of the immune system. © 2014 Wiley Periodicals, Inc.

Spermatogenesis in Platynereis massiliensis (Polychaeta: Nereidae)

NASA Astrophysics Data System (ADS)

Lücht, Joachim; Pfannenstiel, Hans-Dieter

1989-03-01

Stage 1 of spermatogenesis in the protandrous polychaete Platynereis massiliensis is represented by clusters of about 60 spermatogonia which appear in the coelomic cavity. There are no testes in P. massiliensis. The origin of the spermatogonial clusters is not known. Subclusters of approximately 20 primary spermatocytes each represent stage 2. The appearance of synaptonemal figures in the spermatocyte nuclei marks the beginning of stage 3. Cells tend to lose their tight packing during stage 3 but interdigitate with cellular processes. Then very small subclusters of 4 to 8 spermatocytes appear. Meiosis is completed during stage 4, giving rise to secondary spermatocytes and then to spermatid tetrads. Spermatogonia and primary spermatocytes are interconnected by structurally specialized fusomes while secondary spermatocytes and spermatids, which are also in cytoplasmic continuity, show rather simple cell bridges. Synthesis of acrosomal material starts during stage 2. During spermiogenesis the proacrosomal vesicles of Golgi origin travel from the posterior part of the cell to its anterior part to form the acrosome proper. Acrosome formation, nuclear condensation, shaping of the long and slender sperm nucleus, and development of the sperm tail are the main events during spermiogenesis. Sperm morphology is briefly discussed wity respect to its phylogenetic bearings.

Immunohistochemical localization of GLUT3, MCT1, and MCT2 in the testes of mice and rats: the use of different energy sources in spermatogenesis.

PubMed

Kishimoto, Ayuko; Ishiguro-Oonuma, Toshina; Takahashi, Ritei; Maekawa, Mamiko; Toshimori, Kiyotaka; Watanabe, Masahiko; Iwanaga, Toshihiko

2015-01-01

Lactate represents a preferential energy substrate of germ cells rather than glucose. Testicular Sertoli cells are believed to produce lactate and pyruvate and to supply these to germ cells, particularly spermatocytes and spermatids. Monocarboxylate transporter (MCT), responsible for the transport of lactate and other monocarboxylates via the cell membrane, is abundant in the testes and sperm (MCT1, MCT2, and MCT4). For the uptake of glucose, germ cells within the seminiferous tubules and sperm have been known to intensely express GLUT3. The present study investigated expression profiles of MCTs and GLUTs and revealed their cellular and subcellular localization in the mouse and rat testis. An in situ hybridization analysis showed significant expressions of MCT1, MCT2, and GLUT3 mRNA in the testis. Immunohistochemically, spermatogonia, spermatocytes, and spermatids expressed MCT1 on their cell surfaces in a stage-dependent manner: in some seminiferous tubules, an intense expression of MCT1 was unique to the spermatogonia. MCT2 was restricted to the tails of elongated spermatids and sperm. An intense immunoreactivity for GLUT3 was shared by spermatocytes, spermatids, and sperm. Sertoli cells were devoid of any immunoreactivities for MCT1, MCT2, and GLUT3. The predominant energy source of germ cells may be lactate and other monocarboxylates--especially for spermatogonia, but glucose and other hexoses may be responsible for an energy supply to spermatocytes and spermatids.

Three-dimensional Cell Culture Devices for Cancer Migration and Drug Testing

NASA Astrophysics Data System (ADS)

Ma, Liang

Porous polymeric materials are widely used to mimic the extracellular matrix (ECM) environment for applications such as 3D cell culturing and tissue engineering. A series of comparative experiments on 3D cell cultures both in PLA porous scaffolds and alginate gels were conducted to create an in vitro tumor model. A novel 3D cell culture device based on porous polymeric material was developed to study cancer migration. Significant cell migration was observed through the porous channel within 1--2 weeks induced by 20% fetal bovine serum (FBS). A three-dimensional micro-scale perfusion-based two-chamber (3D-muPTC) tissue model system was developed to test the cytotoxicity of anticancer drugs by emulating liver metabolism effects in vitro. Hepatoma cells and glioblastoma multiforme (GBM) cancer cells were cultured in porous polymeric scaffolds in two separate chambers, representing the liver and tumor, respectively. The cytotoxic effect of temozolomide (TMZ) was first tested using this system. It was found that the GBM cells showed a much higher viability under the TMZ treatment with liver cells in the system, suggesting that the drug metabolism in liver is affecting the efficacy of the drug. The favorable metabolism effect of cytochrome P450 (CYP) was tested using a prodrug ifosfamide (IFO). Without the liver cells, IFO showed only slight toxicity to GBM cells. Moreover, it was shown that different expression levels of CYP 3A4, a major drug metabolizing enzyme, in liver cells caused significantly different levels of GBM cell viability. Simulation of the flow characteristics in the 3D-muPTC system was conducted using the finite-element analysis approach. The shear stress was predicted in the porous scaffolds under different flow rate conditions. The predicted shear stress effects agreed well with an experimental cell viability study. A low cost organic solvent free approach to fabricating tissue engineering scaffolds was developed by combining the twin-screw extrusion and particulate leaching. High porosity and interconnected porous PLA scaffolds with the pore size 50 to 200μm were fabricated with this immiscible polymer blending method. This combined extrusion and particulate leaching method provides a new technique to fabricate tissue engineering scaffolds that can be used in the 3D-muPTC device.

Reliability of plant root comet assay in comparison with human leukocyte comet assay for assessment environmental genotoxic agents.

PubMed

Reis, Gabriela Barreto Dos; Andrade-Vieira, Larissa Fonseca; Moraes, Isabella de Campos; César, Pedro Henrique Souza; Marcussi, Silvana; Davide, Lisete Chamma

2017-08-01

Comet assay is an efficient test to detect genotoxic compounds based on observation of DNA damage. The aim of this work was to compare the results obtained from the comet assay in two different type of cells extracted from the root tips from Lactuca sativa L. and human blood. For this, Spent Pot Liner (SPL), and its components (aluminum and fluoride) were applied as toxic agents. SPL is a solid waste generated in industry from the aluminum mining and processing with known toxicity. Three concentrations of all tested solutions were applied and the damages observed were compared to negative and positive controls. It was observed an increase in the frequency of DNA damage for human leukocytes and plant cells, in all treatments. On human leukocytes, SPL induced the highest percentage of damage, with an average of 87.68%. For root tips cells of L. sativa the highest percentage of damage was detected for aluminum (93.89%). Considering the arbitrary units (AU), the average of nuclei with high levels of DNA fragmentation was significant for both cells type evaluated. The tested cells demonstrated equal effectiveness for detection of the genotoxicity induced by the SPL and its chemical components, aluminum and fluoride. Further, using a unique method, the comet assay, we proved that cells from root tips of Lactuca sativa represent a reliable model to detect DNA damage induced by genotoxic pollutants is in agreement of those observed in human leukocytes as model. So far, plant cells may be suggested as important system to assess the toxicological risk of environmental agents. Copyright © 2017 Elsevier Inc. All rights reserved.

A Novel Bioreactor System for the Assessment of Endothelialization on Deformable Surfaces

PubMed Central

Bachmann, Björn J.; Bernardi, Laura; Loosli, Christian; Marschewski, Julian; Perrini, Michela; Ehrbar, Martin; Ermanni, Paolo; Poulikakos, Dimos; Ferrari, Aldo; Mazza, Edoardo

2016-01-01

The generation of a living protective layer at the luminal surface of cardiovascular devices, composed of an autologous functional endothelium, represents the ideal solution to life-threatening, implant-related complications in cardiovascular patients. The initial evaluation of engineering strategies fostering endothelial cell adhesion and proliferation as well as the long-term tissue homeostasis requires in vitro testing in environmental model systems able to recapitulate the hemodynamic conditions experienced at the blood-to-device interface of implants as well as the substrate deformation. Here, we introduce the design and validation of a novel bioreactor system which enables the long-term conditioning of human endothelial cells interacting with artificial materials under dynamic combinations of flow-generated wall shear stress and wall deformation. The wall shear stress and wall deformation values obtained encompass both the physiological and supraphysiological range. They are determined through separate actuation systems which are controlled based on validated computational models. In addition, we demonstrate the good optical conductivity of the system permitting online monitoring of cell activities through live-cell imaging as well as standard biochemical post-processing. Altogether, the bioreactor system defines an unprecedented testing hub for potential strategies toward the endothelialization or re-endothelialization of target substrates. PMID:27941901

A Palladium-Catalyzed Carbonylation Approach to Eight-Membered Lactam Derivatives with Antitumor Activity.

PubMed

Mancuso, Raffaella; Raut, Dnyaneshwar S; Marino, Nadia; De Luca, Giorgio; Giordano, Cinzia; Catalano, Stefania; Barone, Ines; Andò, Sebastiano; Gabriele, Bartolo

2016-02-24

The reactivity of 2-(2-alkynylphenoxy)anilines under PdI2 /KI-catalyzed oxidative carbonylation conditions has been studied. Although a different reaction pathway could have been operating, N-palladation followed by CO insertion was the favored pathway with all substrates tested, including those containing an internal or terminal triple bond. This led to the formation of a carbamoylpalladium species, the fate of which, as predicted by theoretical calculations, strongly depended on the nature of the substituent on the triple bond. In particular, 8-endo-dig cyclization preferentially occurred when the triple bond was terminal, leading to the formation of carbonylated ζ-lactam derivatives, the structures of which have been confirmed by XRD analysis. These novel medium-sized heterocyclic compounds showed antitumor activity against both estrogen receptor-positive (MCF-7) and triple negative (MDA-MB-231) breast cancer cell lines. In particular, ζ-lactam 3 j' may represent a novel and promising antitumor agent because biological tests clearly demonstrate that this compound significantly reduces cell viability and motility in both MCF-7 and MDA-MB-231 breast cancer cell lines, without affecting normal breast epithelial cell viability. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

RANdom SAmple Consensus (RANSAC) algorithm for material-informatics: application to photovoltaic solar cells.

PubMed

Kaspi, Omer; Yosipof, Abraham; Senderowitz, Hanoch

2017-06-06

An important aspect of chemoinformatics and material-informatics is the usage of machine learning algorithms to build Quantitative Structure Activity Relationship (QSAR) models. The RANdom SAmple Consensus (RANSAC) algorithm is a predictive modeling tool widely used in the image processing field for cleaning datasets from noise. RANSAC could be used as a "one stop shop" algorithm for developing and validating QSAR models, performing outlier removal, descriptors selection, model development and predictions for test set samples using applicability domain. For "future" predictions (i.e., for samples not included in the original test set) RANSAC provides a statistical estimate for the probability of obtaining reliable predictions, i.e., predictions within a pre-defined number of standard deviations from the true values. In this work we describe the first application of RNASAC in material informatics, focusing on the analysis of solar cells. We demonstrate that for three datasets representing different metal oxide (MO) based solar cell libraries RANSAC-derived models select descriptors previously shown to correlate with key photovoltaic properties and lead to good predictive statistics for these properties. These models were subsequently used to predict the properties of virtual solar cells libraries highlighting interesting dependencies of PV properties on MO compositions.

New fixed-point mini-cell to investigate thermocouple drift in a high-temperature environment under neutron irradiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Laurie, M.; Vlahovic, L.; Rondinella, V.V.

Temperature measurements in the nuclear field require a high degree of reliability and accuracy. Despite their sheathed form, thermocouples subjected to nuclear radiations undergo changes due to radiation damage and transmutation that lead to significant EMF drift during long-term fuel irradiation experiment. For the purpose of a High Temperature Reactor fuel irradiation to take place in the High Flux Reactor Petten, a dedicated fixed-point cell was jointly developed by LNE-Cnam and JRC-IET. The developed cell to be housed in the irradiation rig was tailor made to quantify the thermocouple drift during the irradiation (about two year duration) and withstand highmore » temperature (in the range 950 deg. C - 1100 deg. C) in the presence of contaminated helium in a graphite environment. Considering the different levels of temperature achieved in the irradiation facility and the large palette of thermocouple types aimed at surveying the HTR fuel pebble during the qualification test both copper (1084.62 deg. C) and gold (1064.18 deg. C) fixed-point materials were considered. The aim of this paper is to first describe the fixed-point mini-cell designed to be embedded in the reactor rig and to discuss the preliminary results achieved during some out of pile tests as much as some robustness tests representative of the reactor scram scenarios. (authors)« less

Cyclohexa-2,5-diene-1,4-dione-based antiproliferative agents: design, synthesis, and cytotoxic evaluation.

PubMed

Petronzi, Carmen; Festa, Michela; Peduto, Antonella; Castellano, Maria; Marinello, Jessica; Massa, Antonio; Capasso, Anna; Capranico, Giovanni; La Gatta, Annalisa; De Rosa, Mario; Caraglia, Michele; Filosa, Rosanna

2013-04-30

Tumors are diseases characterized by uncontrolled cell growth and, in spite of the progress of medicine over the years, continue to represent a major threat to the health, requiring new therapies. Several synthetic compounds, such as those derived from natural sources, have been identified as anticancer drugs; among these compounds quinone represent the second largest class of anticancer agents in use. Several studies have shown that these act on tumor cells through several mechanisms. An important objective of this work is to develop quinoidscompounds showing antitumor activity, but with fewer side effects. The parachinone cannabinol HU-331, is a small molecule that with its core 4-hydroxy-1,4-benzoquinone, exhibits a potent and selective cytotoxic activity on different tumor cell lines. A series of derivatives 3-hydroxy-1,4-benzochinoni were thus developed through HU-331 chemical modifications. The purpose of the work is to test the ability of the compounds to induce proliferative inhibition and study the mechanisms of cell death. The antitumor activities were evaluated in vitro by examining their cytotoxic effects against different human cancer cell lines. All cell lines tested were plated in 96-multiwell and treated with HU-100-V at different concentrations and cell viability was evaluated byMTT assay. Subsequently via flow cytometry (FACS) it was possible to assess apoptosis by the system of double labeling with PI and Annexin-V, and the effect of the compounds on ROS formation by measuring the dichlorofluorescein fluorescence. The substitution by n-hexyl chain considerably enhanced the bioactivity of the compounds. In details, 2-hexyl-5-hydroxycyclohexa-2,5-diene-1,4-dione (V), 2,5-Dimethoxy-3-hexyl-2,5-cyclohexadiene-1,4-dione (XII) and 2-hydroxy-5-methoxy-3-hexyl-cyclohexa-2,5-diene-1,4-dione (XIII) showed most prominent cytotoxicity against almost human tumour cell lines. Compound V was further subjected to downstream apoptotic analysis, demostrating a time-dependent pro-apoptotic activity on human melanoma M14 cell line mediated by caspases activation and poly-(ADP-ribose)-polymerase (PARP) protein cleavage. These findings indicate that 2-hexyl-5-idrossicicloesa-2,5-diene-1,4-dione can be a promising compound for the design of a new class of antineoplastic derivatives.Carmen Petronzi, Michela Festa, Antonella Peduto and Maria Castellano: equally contributed equally to this work.

Forward Technology Solar Cell Experiment (FTSCE) for MISSE-5 Verified and Readied for Flight on STS-114

NASA Technical Reports Server (NTRS)

Jenkins, Phillip P.; Krasowski, Michael J.; Greer, Lawrence C.; Flatico, Joseph M.

2005-01-01

The Forward Technology Solar Cell Experiment (FTSCE) is a space solar cell experiment built as part of the Fifth Materials on the International Space Station Experiment (MISSE-5): Data Acquisition and Control Hardware and Software. It represents a collaborative effort between the NASA Glenn Research Center, the Naval Research Laboratory, and the U.S. Naval Academy. The purpose of this experiment is to place current and future solar cell technologies on orbit where they will be characterized and validated. This is in response to recent on-orbit and ground test results that raised concerns about the in-space survivability of new solar cell technologies and about current ground test methodology. The various components of the FTSCE are assembled into a passive experiment container--a 2- by 2- by 4-in. folding metal container that will be attached by an astronaut to the outer structure of the International Space Station. Data collected by the FTSCE will be relayed to the ground through a transmitter assembled by the U.S. Naval Academy. Data-acquisition electronics and software were designed to be tolerant of the thermal and radiation effects expected on orbit. The experiment has been verified and readied for flight on STS-114.

Remaining useful life assessment of lithium-ion batteries in implantable medical devices

NASA Astrophysics Data System (ADS)

Hu, Chao; Ye, Hui; Jain, Gaurav; Schmidt, Craig

2018-01-01

This paper presents a prognostic study on lithium-ion batteries in implantable medical devices, in which a hybrid data-driven/model-based method is employed for remaining useful life assessment. The method is developed on and evaluated against data from two sets of lithium-ion prismatic cells used in implantable applications exhibiting distinct fade performance: 1) eight cells from Medtronic, PLC whose rates of capacity fade appear to be stable and gradually decrease over a 10-year test duration; and 2) eight cells from Manufacturer X whose rates appear to be greater and show sharp increase after some period over a 1.8-year test duration. The hybrid method enables online prediction of remaining useful life for predictive maintenance/control. It consists of two modules: 1) a sparse Bayesian learning module (data-driven) for inferring capacity from charge-related features; and 2) a recursive Bayesian filtering module (model-based) for updating empirical capacity fade models and predicting remaining useful life. A generic particle filter is adopted to implement recursive Bayesian filtering for the cells from the first set, whose capacity fade behavior can be represented by a single fade model; a multiple model particle filter with fixed-lag smoothing is proposed for the cells from the second data set, whose capacity fade behavior switches between multiple fade models.

Treatment of internal sources in the finite-volume ELLAM

USGS Publications Warehouse

Healy, R.W.; ,; ,; ,; ,; ,

2000-01-01

The finite-volume Eulerian-Lagrangian localized adjoint method (FVELLAM) is a mass-conservative approach for solving the advection-dispersion equation. The method has been shown to be accurate and efficient for solving advection-dominated problems of solute transport in ground water in 1, 2, and 3 dimensions. Previous implementations of FVELLAM have had difficulty in representing internal sources because the standard assumption of lowest order Raviart-Thomas velocity field does not hold for source cells. Therefore, tracking of particles within source cells is problematic. A new approach has been developed to account for internal sources in FVELLAM. It is assumed that the source is uniformly distributed across a grid cell and that instantaneous mixing takes place within the cell, such that concentration is uniform across the cell at any time. Sub-time steps are used in the time-integration scheme to track mass outflow from the edges of the source cell. This avoids the need for tracking within the source cell. We describe the new method and compare results for a test problem with a wide range of cell Peclet numbers.

A Micromechanics Finite Element Model for Studying the Mechanical Behavior of Spray-On Foam Insulation (SOFI)

NASA Technical Reports Server (NTRS)

Ghosn, Louis J.; Sullivan, Roy M.; Lerch, Bradley A.

2006-01-01

A micromechanics model has been constructed to study the mechanical behavior of spray-on foam insulation (SOFI) for the external tank. The model was constructed using finite elements representing the fundamental repeating unit of the SOFI microstructure. The details of the micromechanics model were based on cell observations and measured average cell dimensions discerned from photomicrographs. The unit cell model is an elongated Kelvin model (fourteen-sided polyhedron with 8 hexagonal and six quadrilateral faces), which will pack to a 100% density. The cell faces and cell edges are modeled using three-dimensional 20-node brick elements. Only one-eighth of the cell is modeled due to symmetry. By exercising the model and correlating the results with the macro-mechanical foam behavior obtained through material characterization testing, the intrinsic stiffness and Poisson s Ratio of the polymeric cell walls and edges are determined as a function of temperature. The model is then exercised to study the unique and complex temperature-dependent mechanical behavior as well as the fracture initiation and propagation at the microscopic unit cell level.

Vessel-associated stem cells from skeletal muscle: From biology to future uses in cell therapy

PubMed Central

Sancricca, Cristina; Mirabella, Massimiliano; Gliubizzi, Carla; Broccolini, Aldobrando; Gidaro, Teresa; Morosetti, Roberta

2010-01-01

Over the last years, the existence of different stem cells with myogenic potential has been widely investigated. Besides the classical skeletal muscle progenitors represented by satellite cells, numerous multipotent and embryologically unrelated progenitors with a potential role in muscle differentiation and repair have been identified. In order to conceive a therapeutic approach for degenerative muscle disorders, it is of primary importance to identify an ideal stem cell endowed with all the features for a possible use in vivo. Among all emerging populations, vessel-associated stem cells are a novel and promising class of multipotent progenitors of mesodermal origin and with high myogenic potential which seem to best fit all the requirements for a possible cell therapy. In vitro and in vivo studies have already tested the effectiveness and safety of vessel-associated stem cells in animal models. This leads to the concrete possibility in the future to start pilot human clinical trials, hopefully opening the way to a turning point in the treatment of genetic and acquired muscle disorders. PMID:21607121

Ulex europaeus agglutinin-I binds to developing gastrin cells.

PubMed

Ge, Z H; Blom, J; Larsson, L I

1998-03-01

We have previously reported that antropyloric gastrin (G) and somatostatin (D) cells derive from precursor (G/D) cells that coexpress both hormones. We have now analyzed this endocrine cell pedigree for binding of Ulex europaeus agglutinin-I (UEA-I), which previously has been reported to represent a useful marker for cell differentiation. Subpopulations of G/D, D, and G cells were all found to express UEA-I binding. Labelling with bromodeoxyuridine showed that UEA-I positive G cells possessed a higher labelling index than UEA-I negative G cells. These data suggest that the UEA-I positive G cells represent maturing cells still involved in DNA synthesis and cell division. Electron microscopically, specific UEA-I binding sites were localized to the secretory granules and the apical cell membrane of G cells. We conclude that UEA-I represents a differentiation marker for G cells. Moreover, the presence of UEA-I binding sites in these cells may be relevant for Helicobacter pylori-mediated disturbances of gastric acid secretion and gastrin hypersecretion.

(Eco)toxicity and biodegradability of selected protic and aprotic ionic liquids.

PubMed

Peric, Brezana; Sierra, Jordi; Martí, Esther; Cruañas, Robert; Garau, Maria Antonia; Arning, Jürgen; Bottin-Weber, Ulrike; Stolte, Stefan

2013-10-15

Ionic liquids (ILs) are a promising group of compounds with a large variety of possible structures and uses. They are considered as a potential "green" replacement for traditional volatile organic solvents, but their impact on the environment is often neglected or not studied enough. In the present study, selected representatives of two ILs groups were analyzed: a new family of protic ILs (derived from aliphatic amines and organic acids) and some frequently used aprotic ILs (substituted imidazolium and piridinium chlorides). The aquatic toxicity (test organisms Vibrio fischeri, Pseudokirchneriella subcapitata and Lemna minor) and biodegradability tests were carried out. The additional tests with enzyme (acetylcholinesterase) and leukemia rat cells (IPC-81) provided more in-depth evaluation of toxicity. In our comparative hazard assessment protic ILs have EC50 values >100 mg L(-1) in all of the tests performed, except in the case of three representatives toward Lemna minor. They also show good biodegradability rates. The EC50 values for aprotic ILs are various orders of magnitude lower than the ones for protic ILs in most of the tests and they show a lower biodegradability potential. These findings indicate that protic ILs can be considered as environmentally safer alternatives for more toxic ILs and organic solvents. Copyright © 2013 Elsevier B.V. All rights reserved.

Cytotoxic cassaine diterpenoid-diterpenoid amide dimers and diterpenoid amides from the leaves of Erythrophleum fordii.

PubMed

Du, Dan; Qu, Jing; Wang, Jia-Ming; Yu, Shi-Shan; Chen, Xiao-Guang; Xu, Song; Ma, Shuang-Gang; Li, Yong; Ding, Guang-Zhi; Fang, Lei

2010-10-01

Detailed phytochemical investigation from the leaves of Erythrophleum fordii resulted in the isolation of 13 compounds, including three cassaine diterpenoid-diterpenoid amide dimers (1, 3 and 5), and seven cassaine diterpenoid amides (6 and 8-13), together with three previously reported ones, erythrophlesins D (2), C (4) and 3beta-hydroxynorerythrosuamide (7). Compounds 1, 3 and 5 are further additions to the small group of cassaine diterpenoid dimers represented by erythrophlesins A-D. Their structures were determined by analysis of extensive one- and two-dimensional NMR experiments and ESIMS methods. Cytotoxic activities of the isolated compounds were tested against HCT-8, Bel-7402, BGC-823, A549 and A2780 human cancer cell lines in the MTT test. Results showed that compounds 1 and 3-5 exhibited significantly selective cytotoxic activities (IC(50)<10 microM) against these cells, respectively. Copyright © 2010 Elsevier Ltd. All rights reserved.

Successful treatment of toxoplasmic encephalitis diagnosed early by polymerase chain reaction after allogeneic hematopoietic stem cell transplantation: two case reports and review of the literature.

PubMed

Miyagi, T; Itonaga, H; Aosai, F; Taguchi, J; Norose, K; Mochizuki, K; Fujii, H; Furumoto, A; Ohama, M; Karimata, K; Yamanoha, A; Taniguchi, H; Sato, S; Taira, N; Moriuchi, Y; Fukushima, T; Masuzaki, H; Miyazaki, Y

2015-08-01

Toxoplasmic encephalitis represents a rare, but often fatal infection after allogeneic hematopoietic stem cell transplantation. Polymerase chain reaction (PCR)-based preemptive therapy is considered promising for this disease, but is not routinely applied, especially in low seroprevalence countries including Japan. We encountered 2 cases of toxoplasmic encephalitis after transplantation that were successfully treated. The diagnosis of toxoplasmic encephalitis in these cases was confirmed by PCR testing when neurological symptoms were observed. Both patients received pyrimethamine and sulfadiazine treatments within 2 weeks of the development of neurological symptoms, and remained free of recurrence for 32 and 12 months. These results emphasized the importance of the PCR test and immediate treatment after diagnosis for the management of toxoplasmic encephalitis. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Latest developments in the Advanced Photovoltaic Solar Array Program

NASA Technical Reports Server (NTRS)

Stella, Paul M.; Kurland, Richard M.

1990-01-01

In 1985, the Advanced Photovoltaic Solar Array (APSA) Program was established to demonstrate a producible array system with a specific power greater than 130 W/kg at a 10-kW (BOL) power level. The latest program phase completed fabrication and initial functional testing of a prototype wing representative of a full-scale 5-kW (BOL) wing (except truncated in length to about 1 kW), with weight characteristics that could meet the 130-W/kg (BOL) specific power goal using thin silicon solar cell modules and weight-efficient structural components. The wing configuration and key design details are reviewed, along with results from key component-level and wing-level tests. Projections for future enhancements that may be expected through the use of advanced solar cells and structural components are shown. Performance estimates are given for solar electric propulsion orbital transfer missions through the Van Allen radiation belts. The latest APSA program plans are presented.

New stent surface materials: the impact of polymer-dependent interactions of human endothelial cells, smooth muscle cells, and platelets.

PubMed

Busch, Raila; Strohbach, Anne; Rethfeldt, Stefanie; Walz, Simon; Busch, Mathias; Petersen, Svea; Felix, Stephan; Sternberg, Katrin

2014-02-01

Despite the development of new coronary stent technologies, in-stent restenosis and stent thrombosis are still clinically relevant. Interactions of blood and tissue cells with the implanted material may represent an important cause of these side effects. We hypothesize material-dependent interaction of blood and tissue cells. The aim of this study is accordingly to investigate the impact of vascular endothelial cells, smooth muscle cells and platelets with various biodegradable polymers to identify a stent coating or platform material that demonstrates excellent endothelial-cell-supportive and non-thrombogenic properties. Human umbilical venous endothelial cells, human coronary arterial endothelial cells and human coronary arterial smooth muscle cells were cultivated on the surfaces of two established biostable polymers used for drug-eluting stents, namely poly(ethylene-co-vinylacetate) (PEVA) and poly(butyl methacrylate) (PBMA). We compared these polymers to new biodegradable polyesters poly(l-lactide) (PLLA), poly(3-hydroxybutyrate) (P(3HB)), poly(4-hydroxybutyrate) (P(4HB)) and a polymeric blend of PLLA/P(4HB) in a ratio of 78/22% (w/w). Biocompatibility tests were performed under static and dynamic conditions. Measurement of cell proliferation, viability, glycocalix width, eNOS and PECAM-1 mRNA expression revealed strong material dependency among the six polymer samples investigated. Only the polymeric blend of PLLA/P(4HB) achieved excellent endothelial markers of biocompatibility. Data show that PLLA and P(4HB) tend to a more thrombotic response, whereas the polymer blend is characterized by a lower thrombotic potential. These data demonstrate material-dependent endothelialization, smooth muscle cell growth and thrombogenicity. Although polymers such as PEVA and PBMA are already commonly used for vascular implants, they did not sufficiently meet the criteria for biocompatibility. The investigated biodegradable polymeric blend PLLA/P(4HB) evidently represents a promising material for vascular stents and stent coatings. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

A Lactose-Binding Lectin from the Marine Sponge Cinachyrella Apion (Cal) Induces Cell Death in Human Cervical Adenocarcinoma Cells

PubMed Central

Rabelo, Luciana; Monteiro, Norberto; Serquiz, Raphael; Santos, Paula; Oliveira, Ruth; Oliveira, Adeliana; Rocha, Hugo; Morais, Ana Heloneida; Uchoa, Adriana; Santos, Elizeu

2012-01-01

Cancer represents a set of more than 100 diseases, including malignant tumors from different locations. Strategies inducing differentiation have had limited success in the treatment of established cancers. Marine sponges are a biological reservoir of bioactive molecules, especially lectins. Several animal and plant lectins were purified with antitumor activity, mitogenic, anti-inflammatory and antiviral, but there are few reports in the literature describing the mechanism of action of lectins purified from marine sponges to induce apoptosis in human tumor cells. In this work, a lectin purified from the marine sponge Cinachyrella apion (CaL) was evaluated with respect to its hemolytic, cytotoxic and antiproliferative properties, besides the ability to induce cell death in tumor cells. The antiproliferative activity of CaL was tested against HeLa, PC3 and 3T3 cell lines, with highest growth inhibition for HeLa, reducing cell growth at a dose dependent manner (0.5–10 µg/mL). Hemolytic activity and toxicity against peripheral blood cells were tested using the concentration of IC50 (10 µg/mL) for both trials and twice the IC50 for analysis in flow cytometry, indicating that CaL is not toxic to these cells. To assess the mechanism of cell death caused by CaL in HeLa cells, we performed flow cytometry and western blotting. Results showed that lectin probably induces cell death by apoptosis activation by pro-apoptotic protein Bax, promoting mitochondrial membrane permeabilization, cell cycle arrest in S phase and acting as both dependent and/or independent of caspases pathway. These results indicate the potential of CaL in studies of medicine for treating cancer. PMID:22690140

Standardized cell samples for midIR technology development

NASA Astrophysics Data System (ADS)

Kastl, Lena; Rommel, Christina E.; Kemper, Björn; Schnekenburger, Jürgen

2015-03-01

The application of midIR spectroscopy towards human cell and tissue samples is impaired by the need for technical solutions and lacking sample standards for technology development. We here present the standardization of stable test samples for the continuous development and testing of novel optical system components. We have selected cell lines representing the major cellular skin constituents keratinocytes and fibroblasts (NIH-3T3, HaCaT). In addition, two skin cancer cell types (A-375 and SK-MEL-28 cells) were analyzed. Cells were seeded on CaF2 substrates and measured dried and under aqueous medium as well as fixated and unfixated. Several independent cell preparations were analyzed with an FTIR spectrometer in the wave number range from 1000 - 4000 cm-1. The obtained data demonstrate that fixed and dehydrated cells on CaF2 can be stored in pure ethanol for several weeks without significant losses in quality of the spectral properties. The established protocol of cell seeding on CaF2 substrates, chemical fixation, dehydration, storage under ethanol and air-drying is suitable for the production of reliable midIR standards. The retrieved spectra demonstrate that fixed cells on CaF2 can be prepared reproducibly; with stable midIR spectral properties over several weeks and properties mimicking reliable unfixed cells. Moreover, the fixated samples on CaF2 show clear differences in the cell type specific spectra that can be identified by principle component analysis. In summary, the standardized cell culture samples on CaF2 substrates are suitable for the development of a midIR device and the optimization of the specific midIR spectra.

Preparation of PEMFC Electrodes from Milligram-Amounts of Catalyst Powder

DOE PAGES

Yarlagadda, Venkata; McKinney, Samuel E.; Keary, Cristin L.; ...

2017-06-03

Development of electrocatalysts with higher activity and stability is one of the highest priorities in enabling cost-competitive hydrogen-air fuel cells. Although the rotating disk electrode (RDE) technique is widely used to study new catalyst materials, it has been often shown to be an unreliable predictor of catalyst performance in actual fuel cell operation. Fabrication of membrane electrode assemblies (MEA) for evaluation which are more representative of actual fuel cells generally requires relatively large amounts (>1 g) of catalyst material which are often not readily available in early stages of development. In this study, we present two MEA preparation techniques usingmore » as little as 30 mg of catalyst material, providing methods to conduct more meaningful MEA-based tests using research-level catalysts amounts.« less

Separation of granulocytes from whole blood by leukoadhesion, phase 1

NASA Technical Reports Server (NTRS)

1976-01-01

Capillary glass tubes are investigated for the separation and retrieval of large quantities of viable granulocytes and monocytes from whole blood on a continuous basis from a single donor. This effort represented the feasibility demonstration of a three phase program for development of a capillary tube cell separation device. The activity included the analysis and parametric laboratory testing with subscale models required to design a prototype device. Capillary tubes 40 cm long with a nominal 0.030 cm internal diameter yielded the highest total process efficiency. Recovery efficiencies as high as 89% of the adhering cell population were obtained. Granulocyte phagocytosis of latex particles indicated approximately 90% viability. Monocytes recovered from the separation column retained their capability to stimulate human bone marrow colony growth, as demonstrated in an in vitro cell culture assay.

The 7.5 kW solar array simulator

NASA Technical Reports Server (NTRS)

Robson, R. R.

1975-01-01

A high power solar array simulator capable of providing the input power to simultaneously operate two 30 cm diameter ion thruster power processors was designed, fabricated, and tested. The maximum power point is set to between 150 and 7500 watts representing an open circuit voltage from 50 to 300 volts and a short circuit current from 4 to 36 amps. Illuminated solar cells are used as the control element to provide a true solar cell characteristic and permit the option of simulating changes in this characteristic due to variations in solar intensity and/or temperature of the solar array. This is accomplished by changing the illumination and/or temperature of the control cells. The response of the output to a step change in load closely approximates that of an actual solar array.

Tamoxifen synergizes with 4-(E)-{(4-hydroxyphenylimino)-methylbenzene, 1,2-diol} and 4-(E)-{(p-tolylimino)-methylbenzene-1,2-diol}, novel azaresveratrol analogs, in inhibiting the proliferation of breast cancer cells

PubMed Central

Ronghe, Amruta; Chatterjee, Anwesha; Bhat, Nimee K.; Padhye, Subhash; Bhat, Hari K.

2016-01-01

We have recently shown that 4-(E)-{(4-hydroxyphenylimino)-methylbenzene, 1,2-diol} (HPIMBD) and 4-(E)-{(p-tolylimino)-methylbenzene-1,2-diol} (TIMBD), novel analogs of resveratrol (Res), selectively inhibited the proliferation of breast cancer cells. In the current study, we tested HPIMBD and TIMBD individually in combination with tamoxifen (Tam) for inhibition of growth of breast cancer cells. Tamoxifen was first tested on non-neoplastic breast epithelial cell lines and its dose that does not inhibit their growth was determined. A combination of this low dose of Tam with either of the Res analogs HPIMBD or TIMBD, resulted in synergistic inhibition of proliferation of breast cancer cells. Both estrogen receptor (ER)-positive and negative breast cancer cell lines responded to the combination. The combination resulted in a substantial decrease in IC50 values of Res analogs in all breast cancer cell lines tested. Mechanistic studies showed a synergistic increase in apoptosis and autophagy genes (beclin-1 and LC3BII/I) with the combination in ER-negative MDA-MB-231 cells. In ER-positive MCF-7 and T47D cells, the mechanism of synergy was found to be inhibition of expression of ERα and oncogene c-Myc. The combination treatment had a synergistic effect in inhibiting the colony forming and spheroid forming ability of cancer cells. Taken together, our findings indicate that a combination of Tam and Res analogs HPIMBD or TIMBD represents a novel approach to enhancing the use of Tam in therapy for breast cancers. Considering the urgent need for novel therapeutic strategies to treat ER-negative breast cancers and overcoming resistance in ER-positive cancers, this combinatorial approach is worthy of continued investigation. PMID:27351134

Flow cytometric assay for analysis of cytotoxic effects of potential drugs on human peripheral blood leukocytes

NASA Astrophysics Data System (ADS)

Nieschke, Kathleen; Mittag, Anja; Golab, Karolina; Bocsi, Jozsef; Pierzchalski, Arkadiusz; Kamysz, Wojciech; Tarnok, Attila

2014-03-01

Toxicity test of new chemicals belongs to the first steps in the drug screening, using different cultured cell lines. However, primary human cells represent the human organism better than cultured tumor derived cell lines. We developed a very gentle toxicity assay for isolation and incubation of human peripheral blood leukocytes (PBL) and tested it using different bioactive oligopeptides (OP). Effects of different PBL isolation methods (red blood cell lysis; Histopaque isolation among others), different incubation tubes (e.g. FACS tubes), anticoagulants and blood sources on PBL viability were tested using propidium iodide-exclusion as viability measure (incubation time: 60 min, 36°C) and flow cytometry. Toxicity concentration and time-depended effects (10-60 min, 36 °C, 0-100 μg /ml of OP) on human PBL were analyzed. Erythrocyte lysis by hypotonic shock (dH2O) was the fastest PBL isolation method with highest viability (>85%) compared to NH4Cl-Lysis (49%). Density gradient centrifugation led to neutrophil granulocyte cell loss. Heparin anticoagulation resulted in higher viability than EDTA. Conical 1.5 mL and 2 mL micro-reaction tubes (both polypropylene (PP)) had the highest viability (99% and 97%) compared to other tubes, i.e. three types of 5.0 mL round-bottom tubes PP (opaque-60%), PP (blue-62%), Polystyrene (PS-64%). Viability of PBL did not differ between venous and capillary blood. A gentle reproducible preparation and analytical toxicity-assay for human PBL was developed and evaluated. Using our assay toxicity, time-course, dose-dependence and aggregate formation by OP could be clearly differentiated and quantified. This novel assay enables for rapid and cost effective multiparametric toxicological screening and pharmacological testing on primary human PBL and can be adapted to high-throughput-screening.°z

Tofacitinib induces G1 cell-cycle arrest and inhibits tumor growth in Epstein-Barr virus-associated T and natural killer cell lymphoma cells.

PubMed

Ando, Shotaro; Kawada, Jun-Ichi; Watanabe, Takahiro; Suzuki, Michio; Sato, Yoshitaka; Torii, Yuka; Asai, Masato; Goshima, Fumi; Murata, Takayuki; Shimizu, Norio; Ito, Yoshinori; Kimura, Hiroshi

2016-11-22

Epstein-Barr virus (EBV) infects not only B cells, but also T cells and natural killer (NK) cells, and is associated with T or NK cell lymphoma. These lymphoid malignancies are refractory to conventional chemotherapy. We examined the activation of the JAK3/STAT5 pathway in EBV-positive and -negative B, T and NK cell lines and in cell samples from patients with EBV-associated T cell lymphoma. We then evaluated the antitumor effects of the selective JAK3 inhibitor, tofacitinib, against these cell lines in vitro and in a murine xenograft model. We found that all EBV-positive T and NK cell lines and patient samples tested displayed activation of the JAK3/STAT5 pathway. Treatment of these cell lines with tofacitinib reduced the levels of phospho-STAT5, suppressed proliferation, induced G1 cell-cycle arrest and decreased EBV LMP1 and EBNA1 expression. An EBV-negative NK cell line was also sensitive to tofacitinib, whereas an EBV-infected NK cell line was more sensitive to tofacitinib than its parental line. Tofacitinib significantly inhibited the growth of established tumors in NOG mice. These findings suggest that tofacitinib may represent a useful therapeutic agent for patients with EBV-associated T and NK cell lymphoma.

Field Test of a Hybrid Finite-Difference and Analytic Element Regional Model.

PubMed

Abrams, D B; Haitjema, H M; Feinstein, D T; Hunt, R J

2016-01-01

Regional finite-difference models often have cell sizes that are too large to sufficiently model well-stream interactions. Here, a steady-state hybrid model is applied whereby the upper layer or layers of a coarse MODFLOW model are replaced by the analytic element model GFLOW, which represents surface waters and wells as line and point sinks. The two models are coupled by transferring cell-by-cell leakage obtained from the original MODFLOW model to the bottom of the GFLOW model. A real-world test of the hybrid model approach is applied on a subdomain of an existing model of the Lake Michigan Basin. The original (coarse) MODFLOW model consists of six layers, the top four of which are aggregated into GFLOW as a single layer, while the bottom two layers remain part of MODFLOW in the hybrid model. The hybrid model and a refined "benchmark" MODFLOW model simulate similar baseflows. The hybrid and benchmark models also simulate similar baseflow reductions due to nearby pumping when the well is located within the layers represented by GFLOW. However, the benchmark model requires refinement of the model grid in the local area of interest, while the hybrid approach uses a gridless top layer and is thus unaffected by grid discretization errors. The hybrid approach is well suited to facilitate cost-effective retrofitting of existing coarse grid MODFLOW models commonly used for regional studies because it leverages the strengths of both finite-difference and analytic element methods for predictions in mildly heterogeneous systems that can be simulated with steady-state conditions. © 2015, National Ground Water Association.

Synthesis and biological evaluation of benzo[d]isothiazole, benzothiazole and thiazole Schiff bases.

PubMed

Vicini, Paola; Geronikaki, Athina; Incerti, Matteo; Busonera, Bernadetta; Poni, Graziella; Cabras, Carla Alba; La Colla, Paolo

2003-11-03

Three new series of benzo[d]isothiazole, benzothiazole and thiazole Schiff bases were synthesized and tested in vitro with the aim of identifying novel lead compounds active against emergent and re-emergent human and cattle infectious diseases (AIDS, hepatitis B and C, tuberculosis, bovine viral diarrhoea) or against drug-resistant cancers (leukaemia, carcinoma, melanoma, MDR tumors) for which no definitive cure or efficacious vaccine is available at present. In particular, these compounds were evaluated in vitro against representatives of different virus classes, such as a HIV-1 (Retrovirus), a HBV (Hepadnavirus) and the single-stranded RNA(+) viruses Yellow fever virus (YFV) and Bovine viral diarrhoea virus (BVDV), both belonging to Flaviviridae. Title compounds were also tested against representatives of Gram-positive and Gram-negative bacteria (Staphylococcus aureus, Salmonella spp.), various atypic mycobacterial strains (Mycobacterium fortuitum and Mycobacterium smegmatis), yeast (Candida albicans) and mould (Aspergillus fumigatus). None of the compounds showed antiviral or antimicrobial activity. The benzo[d]isothiazole compounds showed a marked cytotoxicity (CC(50)=4-9 microM) against the human CD4(+) lymphocytes (MT-4) that were used to support HIV-1 growth. For this reason, the most cytotoxic compounds of this series were evaluated for their antiproliferative activity against a panel of human cell lines derived from haematological and solid tumors. The results highlighted that all the benzo[d]isothiazole derivatives inhibited the growth of leukaemia cell lines, whereas only one of the above mentioned compounds (1e) showed antiproliferative activity against two solid tumor-derived cell lines.

Reverse Osmosis Water Purification Unit: Efficacy of Cartridge Filters for Removal of Bacteria and Protozoan Cysts when Ro Elements are Bypassed

DTIC Science & Technology

1993-04-01

were Klebsiella terrigena, Cryptosporidium parvum oocysts, Rhodotorula rubra, and 3.7 pm latex beads. Challenge waters were dechlorinated tap water and...The morphological and size characteristics of Rhodotorula rubra (ATCC 36053) made the yeast suitable as a protozoan cyst simulant. The yeast cells...representative enteric bacterium), Cryptosporidium parvum (an enteric protozoan pathogen) oocysts, Rhodotorula rubra (a yeast, used to test prefilters only

Cleaner mites: sanitary mutualism in the miniature ecosystem of neotropical bee nests.

PubMed

Biani, Natalia B; Mueller, Ulrich G; Wcislo, William T

2009-06-01

Cleaning symbioses represent classic models of mutualism, and some bee mites are thought to perform cleaning services for their hosts in exchange for suitable environments for reproduction and dispersal. These mutual benefits, however, have not been rigorously demonstrated. We tested the sanitary role of bee mites by correlating mite loads with fungal contamination in natural nests of Megalopta genalis and Megalopta ecuadoria and by experimentally manipulating mite loads in artificial cells with developing brood. Field observations revealed significant correlations between the presence of mites and the absence of fungi inside the brood cells, as well as between the absence of mites and increased bee mortality. Likewise, experimental brood cells with mites have fewer fungal colonies than do cells without mites. Field observations and experimental manipulations, therefore, provide clear evidence of the sanitary effect of mites in nests of Megalopta bees. This bee-mite association constitutes one of the few examples of terrestrial cleaning mutualisms.

Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy.

PubMed

Gualda, Emilio J; Simão, Daniel; Pinto, Catarina; Alves, Paula M; Brito, Catarina

2014-01-01

The development of three dimensional (3D) cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex 3D matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy (LSFM) is becoming an excellent tool for fast imaging of such 3D biological structures. We demonstrate the potential of this technique for the imaging of human differentiated 3D neural aggregates in fixed and live samples, namely calcium imaging and cell death processes, showing the power of imaging modality compared with traditional microscopy. The combination of light sheet microscopy and 3D neural cultures will open the door to more challenging experiments involving drug testing at large scale as well as a better understanding of relevant biological processes in a more realistic environment.

Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy

PubMed Central

Gualda, Emilio J.; Simão, Daniel; Pinto, Catarina; Alves, Paula M.; Brito, Catarina

2014-01-01

The development of three dimensional (3D) cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex 3D matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy (LSFM) is becoming an excellent tool for fast imaging of such 3D biological structures. We demonstrate the potential of this technique for the imaging of human differentiated 3D neural aggregates in fixed and live samples, namely calcium imaging and cell death processes, showing the power of imaging modality compared with traditional microscopy. The combination of light sheet microscopy and 3D neural cultures will open the door to more challenging experiments involving drug testing at large scale as well as a better understanding of relevant biological processes in a more realistic environment. PMID:25161607

Selective bactericidal activity of nanopatterned superhydrophobic cicada Psaltoda claripennis wing surfaces.

PubMed

Hasan, Jafar; Webb, Hayden K; Truong, Vi Khanh; Pogodin, Sergey; Baulin, Vladimir A; Watson, Gregory S; Watson, Jolanta A; Crawford, Russell J; Ivanova, Elena P

2013-10-01

The nanopattern on the surface of Clanger cicada (Psaltoda claripennis) wings represents the first example of a new class of biomaterials that can kill bacteria on contact based solely on its physical surface structure. As such, they provide a model for the development of novel functional surfaces that possess an increased resistance to bacterial contamination and infection. Their effectiveness against a wide spectrum of bacteria, however, is yet to be established. Here, the bactericidal properties of the wings were tested against several bacterial species, possessing a range of combinations of morphology and cell wall type. The tested species were primarily pathogens, and included Bacillus subtilis, Branhamella catarrhalis, Escherichia coli, Planococcus maritimus, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Staphylococcus aureus. The wings were found to consistently kill Gram-negative cells (i.e., B. catarrhalis, E. coli, P. aeruginosa, and P. fluorescens), while Gram-positive cells (B. subtilis, P. maritimus, and S. aureus) remained resistant. The morphology of the cells did not appear to play any role in determining cell susceptibility. The bactericidal activity of the wing was also found to be quite efficient; 6.1 ± 1.5 × 10(6) P. aeruginosa cells in suspension were inactivated per square centimeter of wing surface after 30-min incubation. These findings demonstrate the potential for the development of selective bactericidal surfaces incorporating cicada wing nanopatterns into the design.

Primary cutaneous perivascular epithelioid cell tumor (PEComa): Five new cases and review of the literature.

PubMed

Stuart, Lauren N; Tipton, Russell G; DeWall, Michael R; Parker, Douglas C; Stelton, Christina D; Morrison, Annie O; Coleman, Landon W; Fosko, Scott W; Vidal, Claudia I; Yadira Hurley, Maria; Deeken, Amy H; Gardner, Jerad M

2017-08-01

PEComas represent a family of uncommon mesenchymal tumors composed of "perivascular epithelioid cells" with a distinct immunophenotype that typically shows both myogenic and melanocytic differentiation. The PEComa family includes angiomyolipoma (AML), clear cell "sugar" tumor of the lung and extra pulmonary sites, lymphangioleiomyomatosis and clear cell myomelanocytic tumor of the falciform ligament/ligamentum teres. Very rarely, PEComas may arise in the skin. Primary cutaneous PEComas typically display a dermal proliferation of epithelioid cells with pale, clear, or granular pink cytoplasm arranged in nests and trabecula with an intervening arborizing network of delicate capillaries. Primary cutaneous PEComas have a lower frequency of myogenic marker expression than their deep soft tissue and visceral counterparts. They also often express strong diffuse CD10, leading to potential confusion with metastatic renal cell carcinoma. Most cases behave indolently. We report 5 additional cases of this rare entity. All showed classic histologic features and expression of either HMB-45 and/or Melan-A/MART-1. Four cases were tested for myogenic markers (2 were positive & 2 were negative). Three cases were tested for CD10 (all 3 were positive). All of our cases with clinical follow-up behaved indolently. Table 1 provides a summary of findings for all 5 cases in our series. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Investigation of representing hysteresis in macroscopic models of two-phase flow in porous media using intermediate scale experimental data

NASA Astrophysics Data System (ADS)

Cihan, Abdullah; Birkholzer, Jens; Trevisan, Luca; Gonzalez-Nicolas, Ana; Illangasekare, Tissa

2017-01-01

Incorporating hysteresis into models is important to accurately capture the two phase flow behavior when porous media systems undergo cycles of drainage and imbibition such as in the cases of injection and post-injection redistribution of CO2 during geological CO2 storage (GCS). In the traditional model of two-phase flow, existing constitutive models that parameterize the hysteresis associated with these processes are generally based on the empirical relationships. This manuscript presents development and testing of mathematical hysteretic capillary pressure—saturation—relative permeability models with the objective of more accurately representing the redistribution of the fluids after injection. The constitutive models are developed by relating macroscopic variables to basic physics of two-phase capillary displacements at pore-scale and void space distribution properties. The modeling approach with the developed constitutive models with and without hysteresis as input is tested against some intermediate-scale flow cell experiments to test the ability of the models to represent movement and capillary trapping of immiscible fluids under macroscopically homogeneous and heterogeneous conditions. The hysteretic two-phase flow model predicted the overall plume migration and distribution during and post injection reasonably well and represented the postinjection behavior of the plume more accurately than the nonhysteretic models. Based on the results in this study, neglecting hysteresis in the constitutive models of the traditional two-phase flow theory can seriously overpredict or underpredict the injected fluid distribution during post-injection under both homogeneous and heterogeneous conditions, depending on the selected value of the residual saturation in the nonhysteretic models.

Clonal Populations of Amniotic Cells by Dilution and Direct Plating: Evidence for Hidden Diversity

PubMed Central

Wilson, Patricia G.; Devkota, Lorna; Payne, Tiffany; Crisp, Laddie; Winter, Allison; Wang, Zhan

2012-01-01

Fetal cells are widely considered a superior cell source for regenerative medicine; fetal cells show higher proliferative capacity and have undergone fewer replicative cycles that could generate spontaneous mutations. Fetal cells in amniotic fluid were among the first normal primary cells to be cultured ex vivo, but the undefined composition of amniotic fluid has hindered advance for regenerative applications. We first developed a highly efficient method to generate clonal populations by dilution of amniocentesis samples in media and direct plating without intervening refrigeration, centrifugation, or exposure of cells to the paracrine effects in mixed cell cultures. More than 40 clonal populations were recovered from 4 amniocentesis samples and representative clones were characterized by flow cytometry, conventional assays for differentiation potential, immunofluorescence imaging, and transcript analysis. The results revealed previously unreported diversity among stromal and epithelial cell types and identified unique cell types that could be lost or undetected in mixed cell populations. The differentiation potential of amniotic cells proved to be uncoupled from expression of definitive cell surface or cytoplasmic markers for stromal and epithelial cells. Evidence for diversity among stromal and epithelial cells in amniotic fluid bears on interpretations applied to molecular and functional tests of amniotic cell populations. PMID:23024659

O2-Dependent Efficacy of Novel Piperidine- and Piperazine-Based Chalcones against the Human Parasite Giardia intestinalis

PubMed Central

Bahadur, Vijay; Mastronicola, Daniela; Tiwari, Hemandra Kumar; Kumar, Yogesh; Falabella, Micol; Pucillo, Leopoldo Paolo; Sarti, Paolo

2014-01-01

Giardia intestinalis is the most frequent protozoan agent of intestinal diseases worldwide. Though commonly regarded as an anaerobic pathogen, it preferentially colonizes the fairly oxygen-rich mucosa of the proximal small intestine. Therefore, when testing new potential antigiardial drugs, O2 should be taken into account, since it also reduces the efficacy of metronidazole, the gold standard drug against giardiasis. In this study, 46 novel chalcones were synthesized by microwave-assisted Claisen-Schmidt condensation, purified, characterized by high-resolution mass spectrometry, 1H and 13C nuclear magnetic resonance, and infrared spectroscopy, and tested for their toxicity against G. intestinalis under standard anaerobic conditions. As a novel approach, compounds showing antigiardial activity under anaerobiosis were also assayed under microaerobic conditions, and their selectivity against parasitic cells was assessed in a counterscreen on human epithelial colorectal adenocarcinoma cells. Among the tested compounds, three [30(a), 31(e), and 33] were more effective in the presence of O2 than under anaerobic conditions and killed the parasite 2 to 4 times more efficiently than metronidazole under anaerobiosis. Two of them [30(a) and 31(e)] proved to be selective against parasitic cells, thus representing potential candidates for the design of novel antigiardial drugs. This study highlights the importance of testing new potential antigiardial agents not only under anaerobic conditions but also at low, more physiological O2 concentrations. PMID:24217695

The Culture-Repopulating Ability Assays and Incubation in Low Oxygen: A Simple Way to Test Drugs on Leukaemia Stem or Progenitor Cells

PubMed Central

Cipolleschi, Maria Grazia; Rovida, Elisabetta; Sbarba, Persio Dello

2013-01-01

The Culture-Repopulating Ability (CRA) assays is a method to measure in vitro the bone marrow-repopulating potential of haematopoietic cells. The method was developed in our laboratory in the course of studies based on the use of growth factor-supplemented liquid cultures to study haematopoietic stem/progenitor cell resistance to, and selection at, low oxygen tensions in the incubation atmosphere. These studies led us to put forward the first hypothesis of the existence in vivo of haematopoietic stem cell niches where oxygen tension is physiologically lower than in other bone marrow areas. The CRA assays and incubation in low oxygen were later adapted to the study of leukaemias. Stabilized leukaemia cell lines, ensuring genetically homogeneous cells and enhancing repeatability of results, were found nevertheless phenotypically heterogeneous, comprising cell subsets exhibiting functional phenotypes of stem or progenitor cells. These subsets can be assayed separately, provided an experimental system capable to select one from another (such as different criteria for incubation in low oxygen) is established. On this basis, a two-step procedure was designed, including a primary culture of leukaemia cells in low oxygen for different times, where drug treatment is applied, followed by the transfer of residual cell population (CRA assay) to a drug-free secondary culture incubated at standard oxygen tension, where the expansion of population is allowed. The CRA assays, applied to cell lines first and then to primary cells, represent a simple and relatively rapid, yet accurate and reliable, method for the pre-screening of drugs potentially active on leukaemias which in our opinion could be adopted systematically before they are tested in vivo. PMID:23394087

Protective Effect of Boric Acid on Oxidative DNA Damage In Chinese Hamster Lung Fibroblast V79 Cell Lines

PubMed Central

Yılmaz, Sezen; Ustundag, Aylin; Cemiloglu Ulker, Ozge; Duydu, Yalcın

2016-01-01

Objective Many studies have been published on the antioxidative effects of boric acid (BA) and sodium borates in in vitro studies. However, the boron (B) concentrations tested in these in vitro studies have not been selected by taking into account the realistic blood B concentrations in humans due to the lack of comprehensive epidemiological studies. The recently published epidemiological studies on B exposure conducted in China and Turkey provided blood B concentrations for both humans in daily life and workers under extreme exposure conditions in occupational setting. The results of these studies have made it possible to test antioxidative effects of BA in in vitro studies within the concentra- tion range relevant to humans. The aim of this study was to investigate the protective ef- fects of BA against oxidative DNA damage in V79 (Chinese hamster lung fibroblast) cells. The concentrations of BA tested for its protective effect was selected by taking the blood B concentrations into account reported in previously published epidemiological studies. Therefore, the concentrations of BA tested in this study represent the exposure levels for humans in both daily life and occupational settings. Materials and Methods In this experimental study, comet assay and neutral red uptake (NRU) assay methods were used to determinacy to toxicity and genotoxicity of BA and hydrogen peroxide (H2O2). Results The results of the NRU assay showed that BA was not cytotoxic within the tested concentrations (3, 10, 30, 100 and 200 µM). These non-cytotoxic concentrations were used for comet assay. BA pre-treatment significantly reduced (P<0.05, one-way ANOVA) the DNA damaging capacity of H2O2 at each tested BA concentrations in V79 cells. Conclusion Consequently, pre-incubation of V79 cells with BA has significantly reduced the H2O2-induced oxidative DNA damage in V79 cells. The protective effect of BA against oxidative DNA damage in V79 cells at 5, 10, 50, 100 and 200 μM (54, 108, 540, 1080, and 2161 ng/ml B equivalents) concentrations was proved in this in vitro study. PMID:26862534

Protective Effect of Boric Acid on Oxidative DNA Damage In Chinese Hamster Lung Fibroblast V79 Cell Lines.

PubMed

Yılmaz, Sezen; Ustundag, Aylin; Cemiloglu Ulker, Ozge; Duydu, Yalcın

2016-01-01

Many studies have been published on the antioxidative effects of boric acid (BA) and sodium borates in in vitro studies. However, the boron (B) concentrations tested in these in vitro studies have not been selected by taking into account the realistic blood B concentrations in humans due to the lack of comprehensive epidemiological studies. The recently published epidemiological studies on B exposure conducted in China and Turkey provided blood B concentrations for both humans in daily life and workers under extreme exposure conditions in occupational setting. The results of these studies have made it possible to test antioxidative effects of BA in in vitro studies within the concentra- tion range relevant to humans. The aim of this study was to investigate the protective ef- fects of BA against oxidative DNA damage in V79 (Chinese hamster lung fibroblast) cells. The concentrations of BA tested for its protective effect was selected by taking the blood B concentrations into account reported in previously published epidemiological studies. Therefore, the concentrations of BA tested in this study represent the exposure levels for humans in both daily life and occupational settings. In this experimental study, comet assay and neutral red uptake (NRU) assay methods were used to determinacy to toxicity and genotoxicity of BA and hydrogen peroxide (H2O2). The results of the NRU assay showed that BA was not cytotoxic within the tested concentrations (3, 10, 30, 100 and 200 µM). These non-cytotoxic concentrations were used for comet assay. BA pre-treatment significantly reduced (P<0.05, one-way ANOVA) the DNA damaging capacity of H2O2 at each tested BA concentrations in V79 cells. Consequently, pre-incubation of V79 cells with BA has significantly reduced the H2O2-induced oxidative DNA damage in V79 cells. The protective effect of BA against oxidative DNA damage in V79 cells at 5, 10, 50, 100 and 200 μM (54, 108, 540, 1080, and 2161 ng/ml B equivalents) concentrations was proved in this in vitro study.

Short-term spheroid culture of primary colorectal cancer cells as an in vitro model for personalizing cancer medicine

PubMed Central

Jeppesen, Maria; Hagel, Grith; Glenthoj, Anders; Vainer, Ben; Ibsen, Per; Harling, Henrik; Thastrup, Ole; Jørgensen, Lars N.

2017-01-01

Chemotherapy treatment of cancer remains a challenge due to the molecular and functional heterogeneity displayed by tumours originating from the same cell type. The pronounced heterogeneity makes it difficult for oncologists to devise an effective therapeutic strategy for the patient. One approach for increasing treatment efficacy is to test the chemosensitivity of cancer cells obtained from the patient’s tumour. 3D culture represents a promising method for modelling patient tumours in vitro. The aim of this study was therefore to evaluate how closely short-term spheroid cultures of primary colorectal cancer cells resemble the original tumour. Colorectal cancer cells were isolated from human tumour tissue and cultured as spheroids. Spheroid cultures were established with a high success rate and remained viable for at least 10 days. The spheroids exhibited significant growth over a period of 7 days and no difference in growth rate was observed for spheroids of different sizes. Comparison of spheroids with the original tumour revealed that spheroid culture generally preserved adenocarcinoma histology and expression patterns of cytokeratin 20 and carcinoembryonic antigen. Interestingly, spheroids had a tendency to resemble tumour protein expression more closely after 10 days of culture compared to 3 days. Chemosensitivity screening using spheroids from five patients demonstrated individual response profiles. This indicates that the spheroids maintained patient-to-patient differences in sensitivity towards the drugs and combinations most commonly used for treatment of colorectal cancer. In summary, short-term spheroid culture of primary colorectal adenocarcinoma cells represents a promising in vitro model for use in personalized medicine. PMID:28877221

Alginate and DNA Gels Are Suitable Delivery Systems for Diabetic Wound Healing.

PubMed

Tellechea, Ana; Silva, Eduardo A; Min, Jianghong; Leal, Ermelindo C; Auster, Michael E; Pradhan-Nabzdyk, Leena; Shih, William; Mooney, David J; Veves, Aristidis

2015-06-01

Diabetic foot ulcers (DFU) represent a severe health problem and an unmet clinical challenge. In this study, we tested the efficacy of novel biomaterials in improving wound healing in mouse models of diabetes mellitus (DM). The biomaterials are composed of alginate- and deoxyribonucleic acid (DNA)-based gels that allow incorporation of effector cells, such as outgrowth endothelial cells (OEC), and provide sustained release of bioactive factors, such as neuropeptides and growth factors, which have been previously validated in experimental models of DM wound healing or hind limb ischemia. We tested these biomaterials in mice and demonstrate that they are biocompatible and can be injected into the wound margins without major adverse effects. In addition, we show that the combination of OEC and the neuropeptide Substance P has a better healing outcome than the delivery of OEC alone, while subtherapeutic doses of vascular endothelial growth factor (VEGF) are required for the transplanted cells to exert their beneficial effects in wound healing. In summary, alginate and DNA scaffolds could serve as potential delivery systems for the next-generation DFU therapies. © The Author(s) 2015.

Characterization of instrumented sites for the onsite fuel-cell field-test project. Volume 4. Topical report, 1983-1985

DOE Office of Scientific and Technical Information (OSTI.GOV)

Racine, W.C.; Campillo, C.J.

During the site-selection phase of the Onsite Fuel-Cell Field Test, nearly one hundred sites throughout the U.S. were each instrumented with a standard data-acquisition system (DAS) to collect hourly electrical and thermal data for one year. Seventy of those sites are included in the report. Each site's electrical and thermal systems were instrumented including ambient temperature, electrical demands, building gas usage, and other parameters necessary to calculate building thermal loads. Multifamily residential, commercial, and light industrial sites were instrumented. Approximately twenty market sectors were represented including restaurants, hospitals, hotels, apartments, health clubs, nursing homes, and food-processing plants. The primary usemore » of the data was to determine site compatibility for the installation of 40-kW fuel-cell power plants. However, the collected energy data and site-specific information summarized in this comprehensive report may also be useful for other applications such as market characterization and simulation of new or improved energy-utilization equipment in actual sites. This volume covers metal-plating facilities, nurseries, nursing homes, office buildings and other industrial applications.« less

Advances in 3D cell culture technologies enabling tissue-like structures to be created in vitro.

PubMed

Knight, Eleanor; Przyborski, Stefan

2015-12-01

Research in mammalian cell biology often relies on developing in vitro models to enable the growth of cells in the laboratory to investigate a specific biological mechanism or process under different test conditions. The quality of such models and how they represent the behavior of cells in real tissues plays a critical role in the value of the data produced and how it is used. It is particularly important to recognize how the structure of a cell influences its function and how co-culture models can be used to more closely represent the structure of real tissue. In recent years, technologies have been developed to enhance the way in which researchers can grow cells and more readily create tissue-like structures. Here we identify the limitations of culturing mammalian cells by conventional methods on two-dimensional (2D) substrates and review the popular approaches currently available that enable the development of three-dimensional (3D) tissue models in vitro. There are now many ways in which the growth environment for cultured cells can be altered to encourage 3D cell growth. Approaches to 3D culture can be broadly categorized into scaffold-free or scaffold-based culture systems, with scaffolds made from either natural or synthetic materials. There is no one particular solution that currently satisfies all requirements and researchers must select the appropriate method in line with their needs. Using such technology in conjunction with other modern resources in cell biology (e.g. human stem cells) will provide new opportunities to create robust human tissue mimetics for use in basic research and drug discovery. Application of such models will contribute to advancing basic research, increasing the predictive accuracy of compounds, and reducing animal usage in biomedical science. © 2014 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.

Inhibition of Fatty Acid Metabolism Reduces Human Myeloma Cells Proliferation

PubMed Central

Tirado-Vélez, José Manuel; Joumady, Insaf; Sáez-Benito, Ana; Cózar-Castellano, Irene; Perdomo, Germán

2012-01-01

Multiple myeloma is a haematological malignancy characterized by the clonal proliferation of plasma cells. It has been proposed that targeting cancer cell metabolism would provide a new selective anticancer therapeutic strategy. In this work, we tested the hypothesis that inhibition of β-oxidation and de novo fatty acid synthesis would reduce cell proliferation in human myeloma cells. We evaluated the effect of etomoxir and orlistat on fatty acid metabolism, glucose metabolism, cell cycle distribution, proliferation, cell death and expression of G1/S phase regulatory proteins in myeloma cells. Etomoxir and orlistat inhibited β-oxidation and de novo fatty acid synthesis respectively in myeloma cells, without altering significantly glucose metabolism. These effects were associated with reduced cell viability and cell cycle arrest in G0/G1. Specifically, etomoxir and orlistat reduced by 40–70% myeloma cells proliferation. The combination of etomoxir and orlistat resulted in an additive inhibitory effect on cell proliferation. Orlistat induced apoptosis and sensitized RPMI-8226 cells to apoptosis induction by bortezomib, whereas apoptosis was not altered by etomoxir. Finally, the inhibitory effect of both drugs on cell proliferation was associated with reduced p21 protein levels and phosphorylation levels of retinoblastoma protein. In conclusion, inhibition of fatty acid metabolism represents a potential therapeutic approach to treat human multiple myeloma. PMID:23029529

Characterization of multi-drug tolerant persister cells in Streptococcus suis.

PubMed

Willenborg, Jörg; Willms, Daniela; Bertram, Ralph; Goethe, Ralph; Valentin-Weigand, Peter

2014-05-12

Persister cells constitute a subpopulation of dormant cells within a microbial population which are genetically identical but phenotypically different to regular cells. Notably, persister cells show an elevated tolerance to antimicrobial agents. Thus, they are considered to represent a microbial 'bet-hedging' strategy and are of particular importance in pathogenic bacteria. We studied the ability of the zoonotic pathogen Streptococcus (S.) suis to form multi-drug tolerant variants and identified persister cells dependent on the initial bacterial growth phase. We observed lower numbers of persisters in exponential phase cultures than in stationary growth phase populations. S. suis persister cells showed a high tolerance to a variety of antibiotics, and the phenotype was not inherited as tested with four passages of S. suis populations. Furthermore, we provide evidence that the persister phenotype is related to expression of genes involved in general metabolic pathways since we found higher numbers of persister cells in a mutant strain defective in the catabolic arginine deiminase system as compared to its parental wild type strain. Finally, we observed persister cell formation also in other S. suis strains and pathogenic streptococcal species. Taken together, this is the first study that reports multi-drug tolerant persister cells in the zoonotic pathogen S. suis.

[Stem cells therapy in amyotrophic lateral sclerosis treatment. A critical view].

PubMed

Soler, Bernardita; Fadic, Ricardo; von Bernhardi, Rommy

2011-04-01

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease. At present, there are not curative therapies for ALS. Pathogenic and progression mechanisms suggest the existence of oxidative stress, abnormal intracellular protein aggregation, mitochondrial dysfunction, axonal transport impairment, impairment of trophic support, altered glial cell function, and glutamate excitoxicity. To evaluate therapeutic results with adult stem cell for ALS treatment. Stem cells represent a potential therapeutic strategy, because their biological mechanisms could act on several of the pathogenic mechanisms proposed for ALS. Bone marrow mesenchymal stem cells are especially interesting among adult stem cells. Mesenchymal stem cells can differentiate in all central nervous system cells and potentially replace them. Furthermore, they have immunomodulatory effects, secreting, especially in neuroinflammatory environments, neurotrophic and antiinflammatory factors. Studies in murine models of ALS show decrease of inflammation and disease progression, and increase on animal highly heterogeneous, suggest that mesenchymal stem cells transplant in ALS appears to be safe. However, they fail showing clinical improvement of patients. Additional preclinical studies are necessary to refine this therapeutic approach, to assess long term survival and differentiation of mesenchymal stem cells, dosing, biological activity and safety should be conducted before any planning further human testing occurs.

Multi-100 kW: Planar low cost solar array development

NASA Technical Reports Server (NTRS)

1982-01-01

The applicability of selected low cost options to solar array blanket design was studied by fabricating representative modules and submitting them to thermal cycle environment. Large area (5.9 x 5.9 cm) solar cells of 3 varieties were purchased: (1) Standard wraparound, (2) Copper contacts substituted for the conventional Titanium-Palladium-Silver, and (3) Standard wraparound except with gridded back contact instead of continuous metallization. The baseline cell was purchased to compare fabrication cost and to serve as a control cell during test evaluation of the other two cells. All cells were assembled into either substrate modules where the cell is individually filtered and welded to an integrated Kapton-copper circuit or into a superstrate configuration with 4 cells jointly adhered to a single sheet of microsheet and then welded to the integrated Kapton-copper circuit. Cell quality, particularly in the metallization of contacts, was less than desired. Problems were encountered with copper metallization in laying down a barrier metal which would ohmically bond to the silicon. The cells received were shunted (sintered) or with low contact pull strength (non-sintered), thus leading to the decision to solder rather than weld the copper cells to the Kapton substrate.

Toxicological and efficacy assessment of post-transition metal (Indium) phthalocyanine for photodynamic therapy in neuroblastoma

PubMed Central

Neagu, Monica; Constantin, Carolina; Tampa, Mircea; Matei, Clara; Lupu, Andreea; Manole, Emilia; Ion, Rodica-Mariana; Fenga, Concettina; Tsatsakis, Aristidis M.

2016-01-01

Metallo-phthalocyanines due to their photophysical characteristics as high yield of triplet state and long lifetimes, appear to be good candidates for photodynamic therapy (PDT). Complexes with diamagnetic metals such as Zn2+, Al3+ Ga3+ and In3+meet such requirements and are recognized as potential PDT agents. Clinically, Photofrin® PDT in neuroblastoma therapy proved in pediatric subjects diagnosed with progressive/recurrent malignant brain tumors increased progression free survival and overall survival outcome. Our study focuses on the dark toxicity testing of a Chloro-Indium-phthalocyanine photosensitizer (In-Pc) upon SH-SY5Y neuroblastoma cell line and its experimental in vitro PDT. Upon testing, In-Pc has shown a relatively high singlet oxygen quantum yield within the cells subjected to PDT (0.553), and 50 μg/mL IC50. Classical toxicological and efficacy assessment were completed with dynamic cellular impedance measurement methodology. Using this technology we have shown that long time incubation of neuroblastoma cell lines in In-Pc (over 5 days) does not significantly hinder cell proliferation when concentration are ≤ 10 μg/mL. When irradiating neuroblastoma cells loaded with non-toxic concentration of In-Pc, 50% of cells entered apoptosis. Transmission electron microscopy has confirmed apoptotic characteristics of cells. Investigating the proliferative capacity of the in vitro treated cells we have shown that cells that “escape” the irradiation protocol, present a reduced proliferative capacity. In conclusion, In-Pc represents another photosensitizer that can display sound PDT properties enhancing neuroblastoma therapy armentarium. PMID:27626486

Toxicological and efficacy assessment of post-transition metal (Indium) phthalocyanine for photodynamic therapy in neuroblastoma.

PubMed

Neagu, Monica; Constantin, Carolina; Tampa, Mircea; Matei, Clara; Lupu, Andreea; Manole, Emilia; Ion, Rodica-Mariana; Fenga, Concettina; Tsatsakis, Aristidis M

2016-10-25

Metallo-phthalocyanines due to their photophysical characteristics as high yield of triplet state and long lifetimes, appear to be good candidates for photodynamic therapy (PDT). Complexes with diamagnetic metals such as Zn2+, Al3+ Ga3+ and In3+meet such requirements and are recognized as potential PDT agents. Clinically, Photofrin® PDT in neuroblastoma therapy proved in pediatric subjects diagnosed with progressive/recurrent malignant brain tumors increased progression free survival and overall survival outcome. Our study focuses on the dark toxicity testing of a Chloro-Indium-phthalocyanine photosensitizer (In-Pc) upon SH-SY5Y neuroblastoma cell line and its experimental in vitro PDT. Upon testing, In-Pc has shown a relatively high singlet oxygen quantum yield within the cells subjected to PDT (0.553), and 50 μg/mL IC50. Classical toxicological and efficacy assessment were completed with dynamic cellular impedance measurement methodology. Using this technology we have shown that long time incubation of neuroblastoma cell lines in In-Pc (over 5 days) does not significantly hinder cell proliferation when concentration are ≤ 10 μg/mL. When irradiating neuroblastoma cells loaded with non-toxic concentration of In-Pc, 50% of cells entered apoptosis. Transmission electron microscopy has confirmed apoptotic characteristics of cells. Investigating the proliferative capacity of the in vitro treated cells we have shown that cells that "escape" the irradiation protocol, present a reduced proliferative capacity. In conclusion, In-Pc represents another photosensitizer that can display sound PDT properties enhancing neuroblastoma therapy armentarium.

The impact of in-vehicle cell-phone use on accidents or near-accidents among college students.

PubMed

Seo, Dong-Chul; Torabi, Mohammad R

2004-01-01

With in-vehicle use of cell phones rapidly increasing, the safety of young drivers, who represent 14% of licensed drivers but 26% of drivers involved in fatal crashes, may be disproportionately threatened. The authors used a questionnaire to examine the association between in-vehicle cell-phone use and accidents or near-accidents among 1,291 conveniently recruited college students in 4 states. Of the 1,185 respondents who were drivers, 87% had a cell phone, and 86% of the cell-phone owners reported talking while driving at least occasionally. Of the 762 reported accidents or near-accidents, 21% (n = 159) involved at least 1 of the drivers talking while driving. Chi-square tests and logistic regression analyses showed that the frequency, not the duration, of drivers talking while driving was related to experiencing accidents or near-accidents. Differences between drivers who used cell phones and nonusers in unsafe driving behaviors and attitudes were also examined, and target groups for intervention efforts against talking on a cell phone while driving are suggested.

Vertex Models of Epithelial Morphogenesis

PubMed Central

Fletcher, Alexander G.; Osterfield, Miriam; Baker, Ruth E.; Shvartsman, Stanislav Y.

2014-01-01

The dynamic behavior of epithelial cell sheets plays a central role during numerous developmental processes. Genetic and imaging studies of epithelial morphogenesis in a wide range of organisms have led to increasingly detailed mechanisms of cell sheet dynamics. Computational models offer a useful means by which to investigate and test these mechanisms, and have played a key role in the study of cell-cell interactions. A variety of modeling approaches can be used to simulate the balance of forces within an epithelial sheet. Vertex models are a class of such models that consider cells as individual objects, approximated by two-dimensional polygons representing cellular interfaces, in which each vertex moves in response to forces due to growth, interfacial tension, and pressure within each cell. Vertex models are used to study cellular processes within epithelia, including cell motility, adhesion, mitosis, and delamination. This review summarizes how vertex models have been used to provide insight into developmental processes and highlights current challenges in this area, including progressing these models from two to three dimensions and developing new tools for model validation. PMID:24896108

Newborn Screening for Severe Combined Immunodeficiency in 11 Screening Programs in the United States

PubMed Central

Kwan, Antonia; Abraham, Roshini S.; Currier, Robert; Brower, Amy; Andruszewski, Karen; Abbott, Jordan K.; Baker, Mei; Ballow, Mark; Bartoshesky, Louis E.; Bonagura, Vincent R.; Bonilla, Francisco A.; Brokopp, Charles; Brooks, Edward; Caggana, Michele; Celestin, Jocelyn; Church, Joseph A.; Comeau, Anne Marie; Connelly, James A.; Cowan, Morton J.; Cunningham-Rundles, Charlotte; Dasu, Trivikram; Dave, Nina; De La Morena, Maria T.; Duffner, Ulrich; Fong, Chin-To; Forbes, Lisa; Freedenberg, Debra; Gelfand, Erwin W.; Hale, Jaime E.; Celine Hanson, I.; Hay, Beverly N.; Hu, Diana; Infante, Anthony; Johnson, Daisy; Kapoor, Neena; Kay, Denise M.; Kohn, Donald B.; Lee, Rachel; Lehman, Heather; Lin, Zhili; Lorey, Fred; Abdel-Mageed, Aly; Manning, Adrienne; McGhee, Sean; Moore, Theodore B.; Naides, Stanley J.; Notarangelo, Luigi D.; Orange, Jordan S.; Pai, Sung-Yun; Porteus, Matthew; Rodriguez, Ray; Romberg, Neil; Routes, John; Ruehle, Mary; Rubenstein, Arye; Saavedra-Matiz, Carlos A.; Scott, Ginger; Scott, Patricia M.; Secord, Elizabeth; Seroogy, Christine; Shearer, William T.; Siegel, Subhadra; Silvers, Stacy K.; Stiehm, E. Richard; Sugerman, Robert W.; Sullivan, John L.; Tanksley, Susan; Tierce, Millard L.; Verbsky, James; Vogel, Beth; Walker, Rosalyn; Walkovich, Kelly; Walter, Jolan E.; Wasserman, Richard L.; Watson, Michael S.; Weinberg, Geoffrey A.; Weiner, Leonard B.; Wood, Heather; Yates, Anne B.; Puck, Jennifer M.

2015-01-01

IMPORTANCE Newborn screening for severe combined immunodeficiency (SCID) using assays to detect T-cell receptor excision circles (TRECs) began in Wisconsin in 2008, and SCID was added to the national recommended uniform panel for newborn screened disorders in 2010. Currently 23 states, the District of Columbia, and the Navajo Nation conduct population-wide newborn screening for SCID. The incidence of SCID is estimated at 1 in 100 000 births. OBJECTIVES To present data from a spectrum of SCID newborn screening programs, establish population-based incidence for SCID and other conditions with T-cell lymphopenia, and document early institution of effective treatments. DESIGN Epidemiological and retrospective observational study. SETTING Representatives in states conducting SCID newborn screening were invited to submit their SCID screening algorithms, test performance data, and deidentified clinical and laboratory information regarding infants screened and cases with nonnormal results. Infants born from the start of each participating program from January 2008 through the most recent evaluable date prior to July 2013 were included. Representatives from 10 states plus the Navajo Area Indian Health Service contributed data from 3 030 083 newborns screened with a TREC test. MAIN OUTCOMES AND MEASURES Infants with SCID and other diagnoses of T-cell lymphopenia were classified. Incidence and, where possible, etiologies were determined. Interventions and survival were tracked. RESULTS Screening detected 52 cases of typical SCID, leaky SCID, and Omenn syndrome, affecting 1 in 58 000 infants (95%CI, 1/46 000-1/80 000). Survival of SCID-affected infants through their diagnosis and immune reconstitution was 87%(45/52), 92%(45/49) for infants who received transplantation, enzyme replacement, and/or gene therapy. Additional interventions for SCID and non-SCID T-cell lymphopenia included immunoglobulin infusions, preventive antibiotics, and avoidance of live vaccines. Variations in definitions and follow-up practices influenced the rates of detection of non-SCID T-cell lymphopenia. CONCLUSIONS AND RELEVANCE Newborn screening in 11 programs in the United States identified SCID in 1 in 58 000 infants, with high survival. The usefulness of detection of non-SCID T-cell lymphopenias by the same screening remains to be determined. PMID:25138334

Growth Patterns Inferred from Anatomical Records 1

PubMed Central

Silk, Wendy Kuhn; Lord, Elizabeth M.; Eckard, Kathleen J.

1989-01-01

Our objective was to test whether accurate growth analyses can be obtained from anatomical records and some mathematical formulas. Roots of Zea mays L. were grown at one of two temperatures (19°C or 29°C) and were prepared with standard techniques for light microscopy. Positions of cell walls were digitized from micrographs. The digitized data were averaged and smoothed and used in formulas to estimate growth trajectories, Z(t), velocities, v(z), and strain rates, r(z), where Z(t) is the location occupied by the cellular particle at time t; and v(z) and r(z) are, respectively, the fields of growth velocity and strain rate. The relationships tested are: for Z(t), t = n * c; v(z) = l(z) * f; and r(z) = f * (∂/∂z (l(z))). In the formulas, n represents the number of cells between the origin and the position Z(t); l(z) is local cell length; the constant c, named the `cellochron,' denotes the time for successive cells to pass a spatial point distal to the meristem; l(z) is local cell length, and f is cell flux. Growth trajectories and velocity fields from the anatomical method are in good agreement with earlier analyses based on marking experiments at the two different temperatures. Growth strain rate fields show an unexpected oscillation which may be due to numerical artifacts or to a real oscillation in cell production rate. Images Figure 2 PMID:16666832

Novel scalable 3D cell based model for in vitro neurotoxicity testing: Combining human differentiated neurospheres with gene expression and functional endpoints.

PubMed

Terrasso, Ana Paula; Pinto, Catarina; Serra, Margarida; Filipe, Augusto; Almeida, Susana; Ferreira, Ana Lúcia; Pedroso, Pedro; Brito, Catarina; Alves, Paula Marques

2015-07-10

There is an urgent need for new in vitro strategies to identify neurotoxic agents with speed, reliability and respect for animal welfare. Cell models should include distinct brain cell types and represent brain microenvironment to attain higher relevance. The main goal of this study was to develop and validate a human 3D neural model containing both neurons and glial cells, applicable for toxicity testing in high-throughput platforms. To achieve this, a scalable bioprocess for neural differentiation of human NTera2/cl.D1 cells in stirred culture systems was developed. Endpoints based on neuronal- and astrocytic-specific gene expression and functionality in 3D were implemented in multi-well format and used for toxicity assessment. The prototypical neurotoxicant acrylamide affected primarily neurons, impairing synaptic function; our results suggest that gene expression of the presynaptic marker synaptophysin can be used as sensitive endpoint. Chloramphenicol, described as neurotoxicant affected both cell types, with cytoskeleton markers' expression significantly reduced, particularly in astrocytes. In conclusion, a scalable and reproducible process for production of differentiated neurospheres enriched in mature neurons and functional astrocytes was obtained. This 3D approach allowed efficient production of large numbers of human differentiated neurospheres, which in combination with gene expression and functional endpoints are a powerful cell model to evaluate human neuronal and astrocytic toxicity. Copyright © 2014 Elsevier B.V. All rights reserved.

Mycoplasma testing of cell substrates and biologics: Review of alternative non-microbiological techniques.

PubMed

Volokhov, Dmitriy V; Graham, Laurie J; Brorson, Kurt A; Chizhikov, Vladimir E

2011-01-01

Mycoplasmas, particularly species of the genera Mycoplasma and Acholeplasma, are known to be occasional microbial contaminants of cell cultures that produce biologics. This presents a serious concern regarding the risk of mycoplasma contamination for research laboratories and commercial facilities developing and manufacturing cell-derived biological and biopharmaceutical products for therapeutic use. Potential undetected contamination of these products or process intermediates with mycoplasmas represents a potential safety risk for patients and a business risk for producers of biopharmaceuticals. To minimize these risks, monitoring for adventitious agents, such as viruses and mycoplasmas, is performed during the manufacture of biologics produced in cell culture substrates. The "gold standard" microbiological assay, currently recommended by the USP, EP, JP and the US FDA, for the mycoplasma testing of biologics, involves the culture of viable mycoplasmas in broth, agar plates and indicator cells. Although the procedure enables highly efficient mycoplasma detection in cell substrates and cell-derived products, the overall testing strategy is time consuming (a minimum of 28 days) and requires skilled interpretation of the results. The long time period required for these conventional assays does not permit their use for products with short shelf-lives or for timely 'go/no-go' decisions during routine in-process testing. PCR methodology has existed for decades, however PCR based and other alternative methods for mycoplasma detection have only recently been considered for application to biologics manufacture. The application of alternative nucleic acid-based, enzyme-based and/or recombinant cell-culture methods, particularly in combination with efficient sample preparation procedures, could provide advantages over conventional microbiological methods in terms of analytical throughput, simplicity, and turnaround time. However, a challenge to the application of alternative methods for detection of mycoplasmas remains whether these alternative methods can provide a limit of detection comparable or superior to those of the culture methods. An additional challenge is that nucleic acid amplification technique (NAT) methods do not allow for accurate discrimination between viable and non-viable mycoplasma contaminants, which might lead to false-positive results (e.g. from inactivated raw materials, etc.). Our review provides an overview of these alternative methods and discusses the pros and cons of their application for the testing of mycoplasmas in biologics and cell substrates. Published by Elsevier Ltd.

Procedure for developing experimental designs for accelerated tests for service-life prediction. [for solar cell modules

NASA Technical Reports Server (NTRS)

Thomas, R. E.; Gaines, G. B.

1978-01-01

Recommended design procedures to reduce the complete factorial design by retaining information on anticipated important interaction effects, and by generally giving up information on unconditional main effects are discussed. A hypothetical photovoltaic module used in the test design is presented. Judgments were made of the relative importance of various environmental stresses such as UV radiation, abrasion, chemical attack, temperature, mechanical stress, relative humidity and voltage. Consideration is given to a complete factorial design and its graphical representation, elimination of selected test conditions, examination and improvement of an engineering design, and parametric study. The resulting design consists of a mix of conditional main effects and conditional interactions and represents a compromise between engineering and statistical requirements.

Stability, antimicrobial activity, and effect of nisin on the physico-chemical properties of fruit juices.

PubMed

de Oliveira Junior, Adelson Alves; de Araújo Couto, Hyrla Grazielle Silva; Barbosa, Ana Andréa Teixeira; Carnelossi, Marcelo Augusto Guitierrez; de Moura, Tatiana Rodrigues

2015-10-15

Heat processing is the most commonly used hurdle for inactivating microorganisms in fruit juices. However, this preservation method could interfere with the organoleptic characteristics of the product. Alternative methods have been proposed and bacteriocins such as nisin are potential candidates. However, the approval of bacteriocins as food additives is limited, especially in foods from vegetal origin. We aimed to verify the stability, the effect on physico-chemical properties, and the antimicrobial activity of nisin in different fruit juices. Nisin remained stable in fruit juices (cashew, soursop, peach, mango, passion fruit, orange, guava, and cupuassu) for at least 30 days at room or refrigerated temperature and did not cause any significant alterations in the physico-chemical characteristics of the juices. Besides, nisin favored the preservation of vitamin C content in juices. The antimicrobial activity of nisin was tested against Alicyclobacillus acidoterrestris, Bacillus cereus, Staphylococcus aureus and Listeria monocytogenes in cashew, soursop, peach, and mango juices. Nisin caused a 4-log reduction in viable cells of A. acidoterrestris in soursop, peach, and mango juices after 8h of incubation, and no viable cells were detected in cashew juices. After 24h of incubation in the presence of nisin, no viable cells were detected, independently of the juices. To S. aureus, at 24h of incubation in the presence of nisin, viable cells were only detected in mango juices, representing a 4-log decrease as compared with the control treatment. The number of viable cells of B. cereus at 24h of incubation in the presence of nisin represented at least a 4-log decrease compared to the control treatment. When the antimicrobial activity of nisin was tested against L. monocytogenes in cashew and soursop juices, no reduction in the viable cell number was observed compared to the control treatment after 24h of incubation. Viable cells were four and six times less than in the control treatment, in peach and mango juices respectively. The most sensitive microorganism to nisin was A. acidoterrestris and the least sensitive was L. monocytogenes. Still, a reduction of up to 90% of viable cells was observed in peach and mango juices inoculated with L. monocytogenes. These results indicate that the use of nisin could be an alternative in fruit juice processing. Copyright © 2015 Elsevier B.V. All rights reserved.

Acoustical sensing of cardiomyocyte cluster beating

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tymchenko, Nina; Kunze, Angelika; Dahlenborg, Kerstin

2013-06-14

Highlights: •An example of the application of QCM-D to live cell studies. •Detection of human pluripotent stem cell-derived cardiomyocyte cluster beating. •Clusters were studied in a thin liquid film and in a large liquid volume. •The QCM-D beating profile provides an individual fingerprint of the hPS-CMCs. -- Abstract: Spontaneously beating human pluripotent stem cell-derived cardiomyocytes clusters (CMCs) represent an excellent in vitro tool for studies of human cardiomyocyte function and for pharmacological cardiac safety assessment. Such testing typically requires highly trained operators, precision plating, or large cell quantities, and there is a demand for real-time, label-free monitoring of small cellmore » quantities, especially rare cells and tissue-like structures. Array formats based on sensing of electrical or optical properties of cells are being developed and in use by the pharmaceutical industry. A potential alternative to these techniques is represented by the quartz crystal microbalance with dissipation monitoring (QCM-D) technique, which is an acoustic surface sensitive technique that measures changes in mass and viscoelastic properties close to the sensor surface (from nm to μm). There is an increasing number of studies where QCM-D has successfully been applied to monitor properties of cells and cellular processes. In the present study, we show that spontaneous beating of CMCs on QCM-D sensors can be clearly detected, both in the frequency and the dissipation signals. Beating rates in the range of 66–168 bpm for CMCs were detected and confirmed by simultaneous light microscopy. The QCM-D beating profile was found to provide individual fingerprints of the hPS-CMCs. The presented results point towards acoustical assays for evaluation cardiotoxicity.« less

Functional integrative levels in the human interactome recapitulate organ organization.

PubMed

Souiai, Ouissem; Becker, Emmanuelle; Prieto, Carlos; Benkahla, Alia; De las Rivas, Javier; Brun, Christine

2011-01-01

Interactome networks represent sets of possible physical interactions between proteins. They lack spatio-temporal information by construction. However, the specialized functions of the differentiated cell types which are assembled into tissues or organs depend on the combinatorial arrangements of proteins and their physical interactions. Is tissue-specificity, therefore, encoded within the interactome? In order to address this question, we combined protein-protein interactions, expression data, functional annotations and interactome topology. We first identified a subnetwork formed exclusively of proteins whose interactions were observed in all tested tissues. These are mainly involved in housekeeping functions and are located at the topological center of the interactome. This 'Largest Common Interactome Network' represents a 'functional interactome core'. Interestingly, two types of tissue-specific interactions are distinguished when considering function and network topology: tissue-specific interactions involved in regulatory and developmental functions are central whereas tissue-specific interactions involved in organ physiological functions are peripheral. Overall, the functional organization of the human interactome reflects several integrative levels of functions with housekeeping and regulatory tissue-specific functions at the center and physiological tissue-specific functions at the periphery. This gradient of functions recapitulates the organization of organs, from cells to organs. Given that several gradients have already been identified across interactomes, we propose that gradients may represent a general principle of protein-protein interaction network organization.

Systematic Identification of Combinatorial Drivers and Targets in Cancer Cell Lines

PubMed Central

Tabchy, Adel; Eltonsy, Nevine; Housman, David E.; Mills, Gordon B.

2013-01-01

There is an urgent need to elicit and validate highly efficacious targets for combinatorial intervention from large scale ongoing molecular characterization efforts of tumors. We established an in silico bioinformatic platform in concert with a high throughput screening platform evaluating 37 novel targeted agents in 669 extensively characterized cancer cell lines reflecting the genomic and tissue-type diversity of human cancers, to systematically identify combinatorial biomarkers of response and co-actionable targets in cancer. Genomic biomarkers discovered in a 141 cell line training set were validated in an independent 359 cell line test set. We identified co-occurring and mutually exclusive genomic events that represent potential drivers and combinatorial targets in cancer. We demonstrate multiple cooperating genomic events that predict sensitivity to drug intervention independent of tumor lineage. The coupling of scalable in silico and biologic high throughput cancer cell line platforms for the identification of co-events in cancer delivers rational combinatorial targets for synthetic lethal approaches with a high potential to pre-empt the emergence of resistance. PMID:23577104

Systematic identification of combinatorial drivers and targets in cancer cell lines.

PubMed

Tabchy, Adel; Eltonsy, Nevine; Housman, David E; Mills, Gordon B

2013-01-01

There is an urgent need to elicit and validate highly efficacious targets for combinatorial intervention from large scale ongoing molecular characterization efforts of tumors. We established an in silico bioinformatic platform in concert with a high throughput screening platform evaluating 37 novel targeted agents in 669 extensively characterized cancer cell lines reflecting the genomic and tissue-type diversity of human cancers, to systematically identify combinatorial biomarkers of response and co-actionable targets in cancer. Genomic biomarkers discovered in a 141 cell line training set were validated in an independent 359 cell line test set. We identified co-occurring and mutually exclusive genomic events that represent potential drivers and combinatorial targets in cancer. We demonstrate multiple cooperating genomic events that predict sensitivity to drug intervention independent of tumor lineage. The coupling of scalable in silico and biologic high throughput cancer cell line platforms for the identification of co-events in cancer delivers rational combinatorial targets for synthetic lethal approaches with a high potential to pre-empt the emergence of resistance.

Micromechanics Fatigue Damage Analysis Modeling for Fabric Reinforced Ceramic Matrix Composites

NASA Technical Reports Server (NTRS)

Min, J. B.; Xue, D.; Shi, Y.

2013-01-01

A micromechanics analysis modeling method was developed to analyze the damage progression and fatigue failure of fabric reinforced composite structures, especially for the brittle ceramic matrix material composites. A repeating unit cell concept of fabric reinforced composites was used to represent the global composite structure. The thermal and mechanical properties of the repeating unit cell were considered as the same as those of the global composite structure. The three-phase micromechanics, the shear-lag, and the continuum fracture mechanics models were integrated with a statistical model in the repeating unit cell to predict the progressive damages and fatigue life of the composite structures. The global structure failure was defined as the loss of loading capability of the repeating unit cell, which depends on the stiffness reduction due to material slice failures and nonlinear material properties in the repeating unit cell. The present methodology is demonstrated with the analysis results evaluated through the experimental test performed with carbon fiber reinforced silicon carbide matrix plain weave composite specimens.

Cytosine-based nucleoside analogs are selectively lethal to DNA mismatch repair-deficient tumour cells by enhancing levels of intracellular oxidative stress

PubMed Central

Hewish, M; Martin, S A; Elliott, R; Cunningham, D; Lord, C J; Ashworth, A

2013-01-01

Background: DNA mismatch repair deficiency is present in a significant proportion of a number of solid tumours and is associated with distinct clinical behaviour. Methods: To identify the therapeutic agents that might show selectivity for mismatch repair-deficient tumour cells, we screened a pair of isogenic MLH1-deficient and MLH1-proficient tumour cell lines with a library of clinically used drugs. To test the generality of hits in the screen, selective agents were retested in cells deficient in the MSH2 mismatch repair gene. Results: We identified cytarabine and other related cytosine-based nucleoside analogues as being selectively toxic to MLH1 and MSH2-deficient tumour cells. The selective cytotoxicity we observed was likely caused by increased levels of cellular oxidative stress, as it could be abrogated by antioxidants. Conclusion: We propose that cytarabine-based chemotherapy regimens may represent a tumour-selective treatment strategy for mismatch repair-deficient cancers. PMID:23361057

2-D Model for Normal and Sickle Cell Blood Microcirculation

NASA Astrophysics Data System (ADS)

Tekleab, Yonatan; Harris, Wesley

2011-11-01

Sickle cell disease (SCD) is a genetic disorder that alters the red blood cell (RBC) structure and function such that hemoglobin (Hb) cannot effectively bind and release oxygen. Previous computational models have been designed to study the microcirculation for insight into blood disorders such as SCD. Our novel 2-D computational model represents a fast, time efficient method developed to analyze flow dynamics, O2 diffusion, and cell deformation in the microcirculation. The model uses a finite difference, Crank-Nicholson scheme to compute the flow and O2 concentration, and the level set computational method to advect the RBC membrane on a staggered grid. Several sets of initial and boundary conditions were tested. Simulation data indicate a few parameters to be significant in the perturbation of the blood flow and O2 concentration profiles. Specifically, the Hill coefficient, arterial O2 partial pressure, O2 partial pressure at 50% Hb saturation, and cell membrane stiffness are significant factors. Results were found to be consistent with those of Le Floch [2010] and Secomb [2006].

Simulated Tip Rub Testing of Low-Density Metal Foam

NASA Technical Reports Server (NTRS)

Bowman, Cheryl L.; Jones, Michael G.

2009-01-01

Preliminary acoustic studies have indicated that low-density, open-cell, metal foams may be suitable acoustic liner material for noise suppression in high by-pass engines. Metal foam response under simulated tip rub conditions was studied to assess whether its durability would be sufficient for the foam to serve both as a rub strip above the rotor as well as an acoustic treatment. Samples represented four metal alloys, nominal cell dimensions ranging from 60 to 120 cells per inch (cpi), and relative densities ranging from 3.4 to 10 percent. The resulting rubbed surfaces were relatively smooth and the open cell structure of the foam was not adversely affected. Sample relative density appeared to have significant influence on the forces induced by the rub event. Acoustic responses of various surface preparations were measured using a normal incidence tube. The results of this study indicate that the foam s open-cell structure was retained after rubbing and that the acoustic absorption spectra variation was minimal.

Dual-beam optical trapping of cells in an optofluidic device fabricated by femtosecond lasers

NASA Astrophysics Data System (ADS)

Bellini, N.; Bragheri, F.; Vishnubhatla, K. C.; Ferrara, L.; Minzioni, P.; Cerullo, G.; Ramponi, R.; Cristiani, I.; Osellame, R.

2010-02-01

We present design and optimization of an optofluidic monolithic chip, able to provide optical trapping and controlled stretching of single cells. The chip is fabricated in a fused silica glass substrate by femtosecond laser micromachining, which can produce both optical waveguides and microfluidic channels with great accuracy. Versatility and three-dimensional capabilities of this fabrication technology provide the possibility to fabricate circular cross-section channels with enlarged access holes for an easy connection with an external fluidic circuit. Moreover, a new fabrication procedure adopted allows the demonstration of microchannels with a square cross-section, thus guaranteeing an improved quality of the trapped cell images. Optical trapping and stretching of single red blood cells are demonstrated, thus proving the effectiveness of the proposed device as a monolithic optical stretcher. We believe that femtosecond laser micromachining represents a promising technique for the development of multifunctional integrated biophotonic devices that can be easily coupled to a microscope platform, thus enabling a complete characterization of the cells under test.

Selective arylsulfonamide inhibitors of ADAM-17: hit optimization and activity in ovarian cancer cell models.

PubMed

Nuti, Elisa; Casalini, Francesca; Santamaria, Salvatore; Fabbi, Marina; Carbotti, Grazia; Ferrini, Silvano; Marinelli, Luciana; La Pietra, Valeria; Novellino, Ettore; Camodeca, Caterina; Orlandini, Elisabetta; Nencetti, Susanna; Rossello, Armando

2013-10-24

Activated leukocyte cell adhesion molecule (ALCAM) is expressed at the surface of epithelial ovarian cancer (EOC) cells and is released in a soluble form (sALCAM) by ADAM-17-mediated shedding. This process is relevant to EOC cell motility and invasiveness, which is reduced by inhibitors of ADAM-17. In addition, ADAM-17 plays a key role in EGFR signaling and thus may represent a useful target in anticancer therapy. Herein we report our hit optimization effort to identify potent and selective ADAM-17 inhibitors, starting with previously identified inhibitor 1. A new series of secondary sulfonamido-based hydroxamates was designed and synthesized. The biological activity of the newly synthesized compounds was tested in vitro on isolated enzymes and human EOC cell lines. The optimization process led to compound 21, which showed an IC50 of 1.9 nM on ADAM-17 with greatly increased selectivity. This compound maintained good inhibitory properties on sALCAM shedding in several in vitro assays.

Chronic active EBV infection: the experience of the Samsung Medical Center in South Korea.

PubMed

Lee, Tae-Hee; Ko, Young-Hyeh

Chronic active EBV infection (CAEBV) of T-cell or NK-cell type is an EBV+ polyclonal, oligoclonal or often monoclonal lymphoproliferative disorder (LPD) recognized as representing the spectrum of EBV-associated T-cell and NK-cell LPD with different clinical presentations; one systemic and two cutaneous disorders including hydroa vacciniforme-like T-cell LPD and mosquito bite hypersensitivity. The systemic form of the disease is characterized by fever, persistent hepatitis, hepatosplenomegaly and lymphadenopathy, which shows varying degrees of clinical severity depending on the immune response of the host and the EBV viral load. We described the clinicopathological findings of two children with CAEBV with a brief review of the literature. Recognition of the disease is important for adequate management of the patient. EBV analysis should be included in the principal diagnostic tests for febrile children. Copyright © 2015 Hospital Infantil de México Federico Gómez. Publicado por Masson Doyma México S.A. All rights reserved.

Models of collective cell spreading with variable cell aspect ratio: a motivation for degenerate diffusion models.

PubMed

Simpson, Matthew J; Baker, Ruth E; McCue, Scott W

2011-02-01

Continuum diffusion models are often used to represent the collective motion of cell populations. Most previous studies have simply used linear diffusion to represent collective cell spreading, while others found that degenerate nonlinear diffusion provides a better match to experimental cell density profiles. In the cell modeling literature there is no guidance available with regard to which approach is more appropriate for representing the spreading of cell populations. Furthermore, there is no knowledge of particular experimental measurements that can be made to distinguish between situations where these two models are appropriate. Here we provide a link between individual-based and continuum models using a multiscale approach in which we analyze the collective motion of a population of interacting agents in a generalized lattice-based exclusion process. For round agents that occupy a single lattice site, we find that the relevant continuum description of the system is a linear diffusion equation, whereas for elongated rod-shaped agents that occupy L adjacent lattice sites we find that the relevant continuum description is connected to the porous media equation (PME). The exponent in the nonlinear diffusivity function is related to the aspect ratio of the agents. Our work provides a physical connection between modeling collective cell spreading and the use of either the linear diffusion equation or the PME to represent cell density profiles. Results suggest that when using continuum models to represent cell population spreading, we should take care to account for variations in the cell aspect ratio because different aspect ratios lead to different continuum models.

A Novel Three-Dimensional Immune Oncology Model for High-Throughput Testing of Tumoricidal Activity.

PubMed

Sherman, Hilary; Gitschier, Hannah J; Rossi, Ann E

2018-01-01

The latest advancements in oncology research are focused on autologous immune cell therapy. However, the effectiveness of this type of immunotherapy for cancer remediation is not equivalent for all patients or cancer types. This suggests the need for better preclinical screening models that more closely recapitulate in vivo tumor biology. The established method for investigating tumoricidal activity of immunotherapies has been study of two-dimensional (2D) monolayer cultures of immortalized cancer cell lines or primary tumor cells in standard tissue culture vessels. Indeed, a proven means to examine immune cell migration and invasion are 2D chemotaxis assays in permeabilized supports or Boyden chambers. Nevertheless, the more in vivo -like three-dimensional (3D) multicellular tumor spheroids are quickly becoming the favored model to examine immune cell invasion and tumor cell cytotoxicity. Accordingly, we have developed a 3D immune oncology model by combining 96-well permeable support systems and 96-well low-attachment microplates. The use of the permeable support system enables assessment of immune cell migration, which was tested in this study as chemotactic response of natural killer NK-92MI cells to human stromal-cell derived factor-1 (SDF-1α). Immune invasion was assessed by measuring NK-92MI infiltration into lung carcinoma A549 cell spheroids that were formed in low-attachment microplates. The novel pairing of the permeable support system with low-attachment microplates permitted simultaneous investigation of immune cell homing, immune invasion of tumor spheroids, and spheroid cytotoxicity. In effect, the system represents a more comprehensive and in vivo -like immune oncology model that can be utilized for high-throughput study of tumoricidal activity.

Inadequate Reference Datasets Biased toward Short Non-epitopes Confound B-cell Epitope Prediction*

PubMed Central

Rahman, Kh. Shamsur; Chowdhury, Erfan Ullah; Sachse, Konrad; Kaltenboeck, Bernhard

2016-01-01

X-ray crystallography has shown that an antibody paratope typically binds 15–22 amino acids (aa) of an epitope, of which 2–5 randomly distributed amino acids contribute most of the binding energy. In contrast, researchers typically choose for B-cell epitope mapping short peptide antigens in antibody binding assays. Furthermore, short 6–11-aa epitopes, and in particular non-epitopes, are over-represented in published B-cell epitope datasets that are commonly used for development of B-cell epitope prediction approaches from protein antigen sequences. We hypothesized that such suboptimal length peptides result in weak antibody binding and cause false-negative results. We tested the influence of peptide antigen length on antibody binding by analyzing data on more than 900 peptides used for B-cell epitope mapping of immunodominant proteins of Chlamydia spp. We demonstrate that short 7–12-aa peptides of B-cell epitopes bind antibodies poorly; thus, epitope mapping with short peptide antigens falsely classifies many B-cell epitopes as non-epitopes. We also show in published datasets of confirmed epitopes and non-epitopes a direct correlation between length of peptide antigens and antibody binding. Elimination of short, ≤11-aa epitope/non-epitope sequences improved datasets for evaluation of in silico B-cell epitope prediction. Achieving up to 86% accuracy, protein disorder tendency is the best indicator of B-cell epitope regions for chlamydial and published datasets. For B-cell epitope prediction, the most effective approach is plotting disorder of protein sequences with the IUPred-L scale, followed by antibody reactivity testing of 16–30-aa peptides from peak regions. This strategy overcomes the well known inaccuracy of in silico B-cell epitope prediction from primary protein sequences. PMID:27189949

Quality by control: Towards model predictive control of mammalian cell culture bioprocesses.

PubMed

Sommeregger, Wolfgang; Sissolak, Bernhard; Kandra, Kulwant; von Stosch, Moritz; Mayer, Martin; Striedner, Gerald

2017-07-01

The industrial production of complex biopharmaceuticals using recombinant mammalian cell lines is still mainly built on a quality by testing approach, which is represented by fixed process conditions and extensive testing of the end-product. In 2004 the FDA launched the process analytical technology initiative, aiming to guide the industry towards advanced process monitoring and better understanding of how critical process parameters affect the critical quality attributes. Implementation of process analytical technology into the bio-production process enables moving from the quality by testing to a more flexible quality by design approach. The application of advanced sensor systems in combination with mathematical modelling techniques offers enhanced process understanding, allows on-line prediction of critical quality attributes and subsequently real-time product quality control. In this review opportunities and unsolved issues on the road to a successful quality by design and dynamic control implementation are discussed. A major focus is directed on the preconditions for the application of model predictive control for mammalian cell culture bioprocesses. Design of experiments providing information about the process dynamics upon parameter change, dynamic process models, on-line process state predictions and powerful software environments seem to be a prerequisite for quality by control realization. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Weissella ghanensis sp. nov., isolated from a Ghanaian cocoa fermentation.

PubMed

De Bruyne, Katrien; Camu, Nicholas; Lefebvre, Karen; De Vuyst, Luc; Vandamme, Peter

2008-12-01

During a study on lactic acid bacteria (and their species diversity) in spontaneous heap fermentations of Ghanaian cocoa beans, two strains, designated 215(T) and 194B, were isolated. A phylogenetic analysis based on 16S rRNA gene sequences demonstrated that these strains represented a distinct lineage close to the genus Weissella and showing only 92.1 % 16S rRNA gene sequence similarity with respect to their closest neighbour, Weissella soli LMG 20113(T). Whole-cell protein electrophoresis, fluorescent amplified fragment length polymorphism fingerprinting of whole genomes and physiological and biochemical tests confirmed the unique taxonomic position of the two novel isolates. On the basis of the results of the morphological and biochemical tests and 16S rRNA gene sequence analysis, strains 215(T) and 194B represent the most peripheral lineage of the genus Weissella, for which we propose the name Weissella ghanensis sp. nov. The type strain is 215(T) (=LMG 24286(T)=DSM 19935(T)).

Ultrafiltration and endotoxin removal from dialysis fluids.

PubMed

Di Felice, A; Cappelli, G; Facchini, F; Tetta, C; Cornia, F; Aimo, G; Lusvarghi, E

1993-06-01

Biocompatibility in hemodialysis is now regarded as a multifactorial problem and dialysate represents a main risk. Pyrogenic fractions mostly coming from gram-negative bacteria easily pass through dialysis membrane, either by backdiffusion or by backfiltration, and induce blood cell activation. To demonstrate the long-term efficiency of a 2 m2 polyamide ultrafilter in producing a pyrogen free solution, we used an experimental circuit ultrafiltering for 240 hours (500 ml/min) a bicarbonate dialysate contaminated (5 to 48 EU/ml) by a Pseudomonas aeruginosa filtrate. The efficiency was monitored by LAL-test and IL-1 PBMC so to detect not only lipid A containing endotoxins but also other cytokines inducing bacterial fractions. At the post-ultrafilter sampling port the LAL-test was < 0.005 to 0.034 EU/ml; IL-1 PBMC was below the detection limit (20 pg/ml) being 27 to 63 pg/ml at the pre-ultrafilter level. Polyamide ultrafiltration represents an efficient system to obtain an endotoxin-free dialysate and a single filter works up to 240 hours.

Medial Orbitofrontal Neurons Preferentially Signal Cues Predicting Changes in Reward during Unblocking

PubMed Central

Lopatina, Nina; McDannald, Michael A.; Styer, Clay V.; Peterson, Jacob F.; Sadacca, Brian F.; Cheer, Joseph F.

2016-01-01

The orbitofrontal cortex (OFC) has been broadly implicated in the ability to use the current value of expected outcomes to guide behavior. Although value correlates have been prominently reported in lateral OFC, they are more often associated with more medial areas. Further, recent studies in primates have suggested a dissociation in which the lateral OFC is involved in credit assignment and representation of reward identity and more medial areas are critical to representing value. Previously, we used unblocking to test more specifically what information about outcomes is represented by OFC neurons in rats; consistent with the proposed dichotomy between the lateral and medial OFC, we found relatively little linear value coding in the lateral OFC (Lopatina et al., 2015). Here we have repeated this experiment, recording in the medial OFC, to test whether such value signals might be found there. Neurons were recorded in an unblocking task as rats learned about cues that signaled either more, less, or the same amount of reward. We found that medial OFC neurons acquired responses to these cues; however, these responses did not signal different reward values across cues. Surprisingly, we found that cells developed responses to cues predicting a change, particularly a decrease, in reward value. This is consistent with a special role for medial OFC in representing current value to support devaluation/revaluation sensitive changes in behavior. SIGNIFICANCE STATEMENT This study uniquely examines encoding in rodent mOFC at the single-unit level in response to cues that predict more, less, or no change in reward in rats during training in a Pavlovian unblocking task, finding more cells responding to change-predictive cues and stronger activity in response to cues predictive of less reward. PMID:27511013

On the corrosion behavior and biocompatibility of palladium-based dental alloys

NASA Astrophysics Data System (ADS)

Sun, Desheng

Palladium-based alloys have been used as dental restorative materials for about two decades with good clinical history. But there have been clinical case reports showing possible allergy effects from these alloys. The aim of this study was to characterize the corrosion behavior and mechanisms of several palladium-based dental alloys by potentiodynamic polarization methods, electrochemical impedance spectroscopy (EIS), and scanning Kelvin probe force microscopy/atomic force microscopy (SKPFM/AFM), and to evaluate their biocompatibility by a cell culture technique and an animal model. Using SKPFM/AFM and scanning electron microscopy, the Ru-enriched phase from the use of ruthenium as a grain-refining element was identified as being slightly more noble than the palladium solid solution matrix in a high-palladium alloy. Other secondary precipitates that exist in the microstructures of these high-palladium alloys have minimal differences in Volta potential compared to the matrix. For high-palladium alloys, corrosion is generally uniform due to the predominant palladium content in the different phases. Potentiodynamic polarization and EIS have shown that representative palladium-silver alloys have low corrosion tendency and high corrosion resistance, which are equivalent to a well-known high-noble gold-palladium alloy in simulated body fluid and oral environments. The palladium-silver alloys tested are resistant to chloride ion corrosion. Passivation and dealloying have been identified for all of the tested palladium-silver alloys. The great similarity in corrosion behavior among the palladium-silver alloys is attributed to their similar chemical compositions. The variation in microstructures of palladium-silver alloys tested does not cause significant difference in corrosion behavior. The corrosion resistance of these palladium-silver alloys at elevated potentials relevant to oral environment is still satisfactory. The release of elements from representative dental palladium alloys into cell culture media did not significantly affect the proliferation and viability of human fibroblast cells. Subcutaneous implantation of samples of one high-palladium alloy, one palladium-silver alloy and a gold alloy into mice did not cause any significant histological change in their skin and spleen. The presence of an oxide layer from dental laboratory processing of these alloys did not cause any adverse reactions from the cells or animals. The biocompatibility of the dental palladium-based alloys evaluated by the cell culture and animal models is satisfactory, suggesting that these alloys are safe for clinical usage.

Evaluation of cytotoxicity, genotoxicity, and apoptosis of wastewater before and after disinfection with performic acid.

PubMed

Ragazzo, Patrizia; Feretti, Donatella; Monarca, Silvano; Dominici, Luca; Ceretti, Elisabetta; Viola, Gaia; Piccolo, Valentina; Chiucchini, Nicoletta; Villarini, Milena

2017-06-01

Disinfection with performic acid (PFA) represents an emerging technology in wastewater treatment. Many recent studies indicate its effectiveness and suitability as a disinfectant for different applications; several have demonstrated its reliability as an alternative to chlorine for disinfecting secondary effluents from urban wastewater treatment plants (WWTPs). Some disinfection technologies, in relation to their oxidative power, lead to the formation of disinfection by-products (DBPs), some of which are of concern for their toxic and carcinogenic potential. The aim of this study was to investigate potential genotoxic, cytotoxic, and mutagenic effects of this disinfection agent on treated secondary effluent coming from a municipal WWTP. A strategy with multiple short-term tests and different target cells (bacterial, plant, and mammalian) was adopted to explore a relatively wide range of potential genotoxic events. The Ames test (point mutation in Salmonella), the micronucleus (chromosomal damage) and Comet tests (primary DNA damage) on human hepatic cells (HepG2) were conducted to detect mutagenicity and chromosomal DNA alterations. DNA fragmentation and mitochondrial potential assays were conducted to evaluate apoptosis in the same kinds of cells. Mutagenic and clastogenic effect potentials were evaluated by examining micronucleus formation in Allium cepa root cells. In all the in vitro tests, carried out on both disinfected and non-disinfected effluents, negative results were always obtained for mutagenic and genotoxic effects. In the Allium cepa tests, however, some non-concentrated wastewater samples after PFA treatment induced a slight increase in micronucleus frequencies in root cells, but not in a dose-related manner. In conclusion, PFA applied for disinfection to a secondary effluent from a municipal wastewater treatment plant did not contribute to the release of genotoxic or mutagenic compounds. Further studies are required to establish to which extent these findings can be generalized to support PFA for other disinfection applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mycobacterium avium subspecies paratuberculosis isolates from sheep and goats show reduced persistence in bovine macrophages than cattle, bison, deer and wild boar strains regardless of genotype.

PubMed

Abendaño, Naiara; Sevilla, Iker A; Prieto, José Miguel; Garrido, Joseba M; Juste, Ramon A; Alonso-Hearn, Marta

2013-05-03

Assessment of the virulence of isolates of Mycobacterium avium subsp. paratuberculosis (Map) exhibiting distinct genotypes and isolated from different hosts may help to clarify the degree to which clinical manifestations of the disease in cattle can be attributed to bacterial or to host factors. The objective of this study was to test the ability of 10 isolates of Map representing distinct genotypes and isolated from domestic (cattle, sheep, and goat), and wildlife animal species (fallow deer, deer, wild boar, and bison) to enter and grow in bovine macrophages. The isolates were previously typed using IS1311 PCR followed by restriction endonuclease analysis into types C, S or B. Intracellular growth of the isolates in a bovine macrophage-like cell line (BoMac) and in primary bovine monocyte-derived macrophages (MDM) was evaluated by quantification of CFU numbers in the initial inoculum and inside of the host cells at 2h and 7 d p.i. using an automatic liquid culture system (Bactec MGIT 960). Individual data illustrated that growth was less variable in BoMac than in MDM cells. All the isolates from goat and sheep hosts persisted within BoMac cells in lower CFU numbers than the other tested isolates after 7 days of infection regardless of genotype. In addition, BoMac cells exhibited differential inflammatory, apoptotic and destructive responses when infected with a bovine or an ovine isolate; which correlated with the differential survival of these strains within BoMac cells. Our results indicated that the survival of the tested Map isolates within bovine macrophages is associated with the specific host from which the isolates were initially isolated. Copyright © 2013 Elsevier B.V. All rights reserved.

Estimating milk yield and value losses from increased somatic cell count on US dairy farms.

PubMed

Hadrich, J C; Wolf, C A; Lombard, J; Dolak, T M

2018-04-01

Milk loss due to increased somatic cell counts (SCC) results in economic losses for dairy producers. This research uses 10 mo of consecutive dairy herd improvement data from 2013 and 2014 to estimate milk yield loss using SCC as a proxy for clinical and subclinical mastitis. A fixed effects regression was used to examine factors that affected milk yield while controlling for herd-level management. Breed, milking frequency, days in milk, seasonality, SCC, cumulative months with SCC greater than 100,000 cells/mL, lactation, and herd size were variables included in the regression analysis. The cumulative months with SCC above a threshold was included as a proxy for chronic mastitis. Milk yield loss increased as the number of test days with SCC ≥100,000 cells/mL increased. Results from the regression were used to estimate a monetary value of milk loss related to SCC as a function of cow and operation related explanatory variables for a representative dairy cow. The largest losses occurred from increased cumulative test days with a SCC ≥100,000 cells/mL, with daily losses of $1.20/cow per day in the first month to $2.06/cow per day in mo 10. Results demonstrate the importance of including the duration of months above a threshold SCC when estimating milk yield losses. Cows with chronic mastitis, measured by increased consecutive test days with SCC ≥100,000 cells/mL, resulted in higher milk losses than cows with a new infection. This provides farm managers with a method to evaluate the trade-off between treatment and culling decisions as it relates to mastitis control and early detection. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Monoclonal antibodies against LipL32, the major outer membrane protein of pathogenic Leptospira: production, characterization, and testing in diagnostic applications.

PubMed

Fernandes, Cláudia P H; Seixas, Fabiana K; Coutinho, Mariana L; Vasconcellos, Flávia A; Seyffert, Núbia; Croda, Julio; McBride, Alan J; Ko, Albert I; Dellagostin, Odir A; Aleixo, José A G

2007-02-01

Pathogenic serovars of Leptospira have a wide antigenic diversity attributed mainly to the lipopolysaccharide present in the outer membrane. In contrast, antigens conserved among pathogenic serovars are mainly represented by outer membrane proteins. Surface exposure of a major and highly conserved outer membrane lipoprotein (LipL32) was recently demonstrated on pathogenic Leptospira. LipL32 in its recombinant form (rLipL32) was used to immunize BALB/c mice to develop murine monoclonal antibodies (MAbs). Three MAbs against rLipL32 were produced, isotyped, and evaluated for further use in diagnostic tests of leptospirosis using different approaches. MAbs were conjugated to peroxidase and evaluated in a native protein enzyme-linked immunosorbent assay (ELISA) with intact and heat-treated leptospiral cells, conjugated to fluorescein isothiocyanate (FITC) for indirect immunofluorescence with intact and methanol fixed cells and were used for LipL32 immunoprecipitation from leptospiral cells. rLipL32 MAbs conjugated to peroxidase or used as primary antibody bound to intact and heat-treated cells in ELISA, proving that they could be used in enzyme immunoassays for detection of the native protein. In immunofluorescence assay, MAbs labeled bacterial cells either intact or methanol fixed. Two MAbs were able to immunoprecipitate the native protein from live and motile leptospiral cells and, adsorbed onto magnetic beads, captured intact bacteria from artificially contaminated human sera for detection by polymerase chain reaction (PCR) amplification. Results of this study suggest that the MAbs produced can be useful for the development of diagnostic tests based on detection of LipL32 leptospiral antigen in biological fluids.

Excess Circulating Alternatively Activated Myeloid (M2) Cells Accelerate ALS Progression While Inhibiting Experimental Autoimmune Encephalomyelitis

PubMed Central

Miller, Omer; Butovsky, Oleg; Bukshpan, Shay; Beers, David R.; Henkel, Jenny S.; Yoles, Eti; Appel, Stanley H.; Schwartz, Michal

2011-01-01

Background Circulating immune cells including autoreactive T cells and monocytes have been documented as key players in maintaining, protecting and repairing the central nervous system (CNS) in health and disease. Here, we hypothesized that neurodegenerative diseases might be associated, similarly to tumors, with increased levels of circulating peripheral myeloid derived suppressor cells (MDSCs), representing a subset of suppressor cells that often expand under pathological conditions and inhibit possible recruitment of helper T cells needed for fighting off the disease. Methods and Findings We tested this working hypothesis in amyotrophic lateral sclerosis (ALS) and its mouse model, which are characterized by a rapid progression once clinical symptoms are evident. Adaptive transfer of alternatively activated myeloid (M2) cells, which homed to the spleen and exhibited immune suppressive activity in G93A mutant superoxide dismutase-1 (mSOD1) mice at a stage before emergence of disease symptoms, resulted in earlier appearance of disease symptoms and shorter life expectancy. The same protocol mitigated the inflammation-induced disease model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), which requires circulating T cells for disease induction. Analysis of whole peripheral blood samples obtained from 28 patients suffering from sporadic ALS (sALS), revealed a two-fold increase in the percentage of circulating MDSCs (LIN−/LowHLA-DR−CD33+) compared to controls. Conclusions Taken together, these results emphasize the distinct requirements for fighting the inflammatory neurodegenerative disease, multiple sclerosis, and the neurodegenerative disease, ALS, though both share a local inflammatory component. Moreover, the increased levels of circulating MDSCs in ALS patients indicates the operation of systemic mechanisms that might lead to an impairment of T cell reactivity needed to overcome the disease conditions within the CNS. This high level of suppressive immune cells might represent a risk factor and a novel target for therapeutic intervention in ALS at least at the early stage. PMID:22073221

Three-dimensional bioprinting of stem-cell derived tissues for human regenerative medicine.

PubMed

Skeldon, Gregor; Lucendo-Villarin, Baltasar; Shu, Wenmiao

2018-07-05

Stem cell technology in regenerative medicine has the potential to provide an unlimited supply of cells for drug testing, medical transplantation and academic research. In order to engineer a realistic tissue model using stem cells as an alternative to human tissue, it is essential to create artificial stem cell microenvironment or niches. Three-dimensional (3D) bioprinting is a promising tissue engineering field that offers new opportunities to precisely place stem cells within their niches layer-by-layer. This review covers bioprinting technologies, the current development of 'bio-inks' and how bioprinting has already been applied to stem-cell culture, as well as their applications for human regenerative medicine. The key considerations for bioink properties such as stiffness, stability and biodegradation, biocompatibility and printability are highlighted. Bioprinting of both adult and pluriopotent stem cells for various types of artificial tissues from liver to brain has been reviewed. 3D bioprinting of stem-cell derived tissues for human regenerative medicine is an exciting emerging area that represents opportunities for new research, industries and products as well as future challenges in clinical translation.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'. © 2018 The Author(s).

Towards a Biohybrid Lung: Endothelial Cells Promote Oxygen Transfer through Gas Permeable Membranes.

PubMed

Menzel, Sarah; Finocchiaro, Nicole; Donay, Christine; Thiebes, Anja Lena; Hesselmann, Felix; Arens, Jutta; Djeljadini, Suzana; Wessling, Matthias; Schmitz-Rode, Thomas; Jockenhoevel, Stefan; Cornelissen, Christian Gabriel

2017-01-01

In patients with respiratory failure, extracorporeal lung support can ensure the vital gas exchange via gas permeable membranes but its application is restricted by limited long-term stability and hemocompatibility of the gas permeable membranes, which are in contact with the blood. Endothelial cells lining these membranes promise physiological hemocompatibility and should enable prolonged application. However, the endothelial cells increase the diffusion barrier of the blood-gas interface and thus affect gas transfer. In this study, we evaluated how the endothelial cells affect the gas exchange to optimize performance while maintaining an integral cell layer. Human umbilical vein endothelial cells were seeded on gas permeable cell culture membranes and cultivated in a custom-made bioreactor. Oxygen transfer rates of blank and endothelialized membranes in endothelial culture medium were determined. Cell morphology was assessed by microscopy and immunohistochemistry. Both setups provided oxygenation of the test fluid featuring small standard deviations of the measurements. Throughout the measuring range, the endothelial cells seem to promote gas transfer to a certain extent exceeding the blank membranes gas transfer performance by up to 120%. Although the underlying principles hereof still need to be clarified, the results represent a significant step towards the development of a biohybrid lung.

Cytotoxic activity against human neuroblastoma and melanoma cells mediated by IgM antibodies derived from peripheral blood of healthy donors.

PubMed

Devarapu, Satish Kumar; Mamidi, Srinivas; Plöger, Frank; Dill, Othmar; Blixt, Ola; Kirschfink, Michael; Schwartz-Albiez, Reinhard

2016-06-15

A small percentage of healthy donors identified in the Western population carry antibodies in their peripheral blood which convey cytotoxic activity against certain human melanoma and neuroblastoma cell lines. We measured the cytotoxic activity of sera and plasmas from healthy donors on the human neuroblastoma cell line Kelly and various melanoma cell lines. Antibodies of IgM isotype, presumably belonging to the class of naturally occurring antibodies, exerted cytotoxic activity in a complement-dependent fashion. Apart from complement-dependent tumor cell lysis, we observed C3 opsonization in all tumor cell lines upon treatment with cytotoxic plasmas. Cell lines tested primarily expressed membrane complement regulatory proteins (mCRP) CD46, CD55 and CD59 to various extents. Blocking of mCRPs by monoclonal antibodies enhanced cell lysis and opsonization, though some melanoma cells remained resistant to complement attack. Epitopes recognized by cytotoxic antibodies were represented by gangliosides such as GD2 and GD3, as evidenced by cellular sialidase pretreatment and enhanced expression of distinct gangliosides. It remains to be clarified why only a small fraction of healthy persons carry these antitumor cytotoxic antibodies. © 2016 UICC.

Stem cell-based therapies in Parkinson's disease: future hope or current treatment option?

PubMed

Loewenbrück, Kai; Storch, Alexander

2011-05-01

Parkinson's disease (PD) is one of the most frequent neurodegenerative diseases and represents a major therapeutic challenge because of the so far missing therapeutic means to influence the ongoing loss of dopaminergic innervation to the striatum. Cell replacement has raised hope to offer the first restorative treatment option. Clinical trials have provided "proof of principle" that transplantation of dopamine-producing neurons into the striatum of PD patients can achieve symptomatic relief given that the striatum is sufficiently re-innervated. Various cell sources have been tested, including fetal ventral midbrain tissue, embryonic stem cells, fetal and adult neural stem cells and, after a ground-breaking discovery, induced pluripotent stem cells. Although embryonic and induced pluripotent stem cells have emerged as the most promising candidates to overcome most of the obstacles to clinical successful cell replacement, each cell source has its unique drawbacks. This review does not only provide a comprehensive overview of the different cellular candidates, including their assets and drawbacks, but also of the various additional issues that need to be addressed in order to convert cellular replacement therapies from an experimental to a clinically relevant therapeutic alternative.

Expression of surface markers on the human monocytic leukaemia cell line, THP-1, as indicators for the sensitizing potential of chemicals.

PubMed

An, Susun; Kim, Seoyoung; Huh, Yong; Lee, Tae Ryong; Kim, Han-Kon; Park, Kui-Lea; Eun, Hee Chul

2009-04-01

Evaluation of skin sensitization potential is an important part of the safety assessment of cosmetic ingredients and topical drugs. Recently, evaluation of changes in surface marker expression induced in dendritic cells (DC) or DC surrogate cell lines following exposure to chemicals represents one approach for in vitro test methods. The study aimed to test the change of expression patterns of surface markers on THP-1 cells by chemicals as a predictive in vitro method for contact sensitization. We investigated the expression of CD54, CD86, CD83, CD80, and CD40 after a 1-day exposure to sensitizers (1-chloro-2,4-dinitrobenzene; 2,4-dinitrofluorobenzene; benzocaine; 5-chloro-2-methyl-4-isothiazolin-3-one; hexyl cinnamic aldehyde; eugenol; nickel sulfate hexahydrate; potassium dichromate; cobalt sulfate; 2-mercaptobenzothiazole; and ammonium tetrachloroplatinate) and non-sensitizers (sodium lauryl sulfate, benzalkonium chloride, lactic acid, salicylic acid, isopropanol, and dimethyl sulphoxide). The test concentrations were 0.1x, 0.5x, and 1x of the 50% inhibitory concentration, and the relative fluorescence intensity was used as an expression indicator. By evaluating the expression patterns of CD54, CD86, and CD40, we could classify the chemicals as sensitizers or non-sensitizers, but CD80 and CD83 showed non-specific patterns of expression. These data suggest that the THP-1 cells are good model for screening contact sensitizers and CD40 could be a useful marker complementary to CD54 and CD86.

Three-dimensional HepaRG model as an attractive tool for toxicity testing.

PubMed

Leite, Sofia B; Wilk-Zasadna, Iwona; Zaldivar, Jose M; Airola, Elodie; Reis-Fernandes, Marcos A; Mennecozzi, Milena; Guguen-Guillouzo, Christiane; Chesne, Christopher; Guillou, Claude; Alves, Paula M; Coecke, Sandra

2012-11-01

The culture of HepaRG cells as three dimensional (3D) structures in the spinner-bioreactor may represent added value as a hepatic system for toxicological purposes. The use of a cost-effective commercially available bioreactor, which is compatible with high-throughput cell analysis, constitutes an attractive approach for routine use in the drug testing industry. In order to assess specific aspects of the biotransformation capacity of the bioreactor-based HepaRG system, the induction of CYP450 enzymes (i.e., CYP1A2, 2B6, 2C9, and 3A4) and the activity of the phase II enzyme, uridine diphosphate glucuronoltransferase (UGT), were tested. The long-term functionality of the system was demonstrated by 7-week stable profiles of albumin secretion, CYP3A4 induction, and UGT activities. Immunofluorescence-based staining showed formation of tissue-like arrangements including bile canaliculi-like structures and polar distribution of transporters. The use of in silico models to analyze the in vitro data related to hepatotoxic activity of acetaminophen (APAP) demonstrated the advantage of the integration of kinetic and dynamic aspects for a better understanding of the in vitro cell behavior. The bioactivation of APAP and its related cytotoxicity was assessed in a system compatible to high-throughput screening. The approach also proved to be a good strategy to reduce the time necessary to obtain fully differentiated cell cultures. In conclusion, HepaRG cells cultured in 3D spinner-bioreactors are an attractive tool for toxicological studies, showing a liver-like performance and demonstrating a practical applicability for toxicodynamic approaches.

Comparison of local grid refinement methods for MODFLOW

USGS Publications Warehouse

Mehl, S.; Hill, M.C.; Leake, S.A.

2006-01-01

Many ground water modeling efforts use a finite-difference method to solve the ground water flow equation, and many of these models require a relatively fine-grid discretization to accurately represent the selected process in limited areas of interest. Use of a fine grid over the entire domain can be computationally prohibitive; using a variably spaced grid can lead to cells with a large aspect ratio and refinement in areas where detail is not needed. One solution is to use local-grid refinement (LGR) whereby the grid is only refined in the area of interest. This work reviews some LGR methods and identifies advantages and drawbacks in test cases using MODFLOW-2000. The first test case is two dimensional and heterogeneous; the second is three dimensional and includes interaction with a meandering river. Results include simulations using a uniform fine grid, a variably spaced grid, a traditional method of LGR without feedback, and a new shared node method with feedback. Discrepancies from the solution obtained with the uniform fine grid are investigated. For the models tested, the traditional one-way coupled approaches produced discrepancies in head up to 6.8% and discrepancies in cell-to-cell fluxes up to 7.1%, while the new method has head and cell-to-cell flux discrepancies of 0.089% and 0.14%, respectively. Additional results highlight the accuracy, flexibility, and CPU time trade-off of these methods and demonstrate how the new method can be successfully implemented to model surface water-ground water interactions. Copyright ?? 2006 The Author(s).

Curvatures Estimation in Orientation Selection

DTIC Science & Technology

1991-01-31

processes are run at the same scale ). Not only is the L/L edge operator as accurate, it makes explicit a great deal of information which is either...Figure 11: An artificial image used to test the image operators. This is an anti-alia sed grey- scale image of lines and curves, which represent the...MacKay, "Influence of luminance gra- dient reversal on simple cells in feline striate cortex," J. Physiology (London), vol. 337, pp. 69--87, 1983

Complement and Immunoregulation in Tissue Injury

DTIC Science & Technology

2014-10-01

and neutrophil infiltration represent the initiating events in this process (9). Recently, the role of different T cell subsets ( Tregs , Th17) and B...first tested the hypothesis that ILCs , which rapidly release IL-17A in response to epithelial injury, are the relevant source of IL- 17A in small...intestinal IRI. To address this question we took advantage of the Rag2-/-γc-/- mouse, which lacks ILCs because it is deficient in IL-7 signaling, which

Use of the gamma distribution to represent monthly rainfall in Africa for drought monitoring applications

USGS Publications Warehouse

Husak, Gregory J.; Michaelsen, Joel C.; Funk, Christopher C.

2007-01-01

Evaluating a range of scenarios that accurately reflect precipitation variability is critical for water resource applications. Inputs to these applications can be provided using location- and interval-specific probability distributions. These distributions make it possible to estimate the likelihood of rainfall being within a specified range. In this paper, we demonstrate the feasibility of fitting cell-by-cell probability distributions to grids of monthly interpolated, continent-wide data. Future work will then detail applications of these grids to improved satellite-remote sensing of drought and interpretations of probabilistic climate outlook forum forecasts. The gamma distribution is well suited to these applications because it is fairly familiar to African scientists, and capable of representing a variety of distribution shapes. This study tests the goodness-of-fit using the Kolmogorov–Smirnov (KS) test, and compares these results against another distribution commonly used in rainfall events, the Weibull. The gamma distribution is suitable for roughly 98% of the locations over all months. The techniques and results presented in this study provide a foundation for use of the gamma distribution to generate drivers for various rain-related models. These models are used as decision support tools for the management of water and agricultural resources as well as food reserves by providing decision makers with ways to evaluate the likelihood of various rainfall accumulations and assess different scenarios in Africa. 

Development of a tester for evaluation of prototype thermal cells and batteries

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guidotti, R.A.

1994-10-01

A tester was developed to evaluate prototype thermal cells and batteries--especially high-voltage units--under a wide range of constant-current and constant-resistance discharge conditions. Programming of the steady-state and pulsing conditions was by software control or by hardware control via an external pulse generator. The tester was assembled from primarily Hewlett-Packard (H-P) instrumentation and was operated under H-P`s Rocky Mountain Basic (RMB). Constant-current electronic loads rated up to 4 kW (400 V at up to 100 A) were successfully used with the setup. For testing under constant-resistance conditions, power metal-oxide field-effect transistors (MOSFETs) controlled by a programmable pulse generator were used tomore » switch between steady-state and pulse loads. The pulses were digitized at up to a 50 kHz rate (20 {mu} s/pt) using high-speed DVMs; steady-state voltages were monitored with standard DVMs. This paper describes several of the test configurations used and discusses the limitations of each. Representative data are presented for a number of the test conditions.« less

Fusion proteins of flagellin and the major birch pollen allergen Bet v 1 show enhanced immunogenicity, reduced allergenicity, and intrinsic adjuvanticity.

PubMed

Kitzmüller, Claudia; Kalser, Julia; Mutschlechner, Sonja; Hauser, Michael; Zlabinger, Gerhard J; Ferreira, Fatima; Bohle, Barbara

2018-01-01

Recombinant fusion proteins of flagellin and antigens have been demonstrated to induce strong innate and adaptive immune responses. Such fusion proteins can enhance the efficacy of allergen-specific immunotherapy. We sought to characterize different fusion proteins of flagellin and the major birch pollen allergen Bet v 1 for suitability as allergy vaccines. A truncated version of flagellin (NtCFlg) was genetically fused to the N- or C-terminus of Bet v 1. Toll-like receptor (TLR) 5 binding was assessed with HEK293 cells expressing TLR5. Upregulation of CD40, CD80, CD83, and CD86 on monocyte-derived dendritic cells from allergic patients was analyzed by using flow cytometry. The T cell-stimulatory capacity of the fusion proteins was assessed with naive and Bet v 1-specific T cells. IgE binding was tested in inhibition ELISAs and basophil activation tests. Mice were immunized with the fusion proteins in the absence and presence of aluminum hydroxide. Cellular and antibody responses were monitored. Murine antibodies were tested for blocking capacity in basophil activation tests. Both fusion proteins matured monocyte-derived dendritic cells through TLR5. Compared with Bet v 1, the fusion proteins showed stronger T cell-stimulatory and reduced IgE-binding capacity and induced murine Bet v 1-specific antibodies in the absence of aluminum hydroxide. However, only antibodies induced by means of immunization with NtCFlg fused to the C-terminus of Bet v 1 inhibited binding of patients' IgE antibodies to Bet v 1. Bet v 1-flagellin fusion proteins show enhanced immunogenicity, reduced allergenicity, and intrinsic adjuvanticity and thus represent promising vaccines for birch pollen allergen-specific immunotherapy. However, the sequential order of allergen and adjuvant within a fusion protein determines its immunologic characteristics. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Testing the biocompatibility of a glutathione-containing intra-ocular irrigation solution by using an isolated perfused bovine retina organ culture model - an alternative to animal testing.

PubMed

Januschowski, Kai; Zhour, Ahmad; Lee, Albert; Maddani, Ramin; Mueller, Sebastien; Spitzer, Martin S; Schnichels, Sven; Schultheiss, Maximilian; Doycheva, Deshka; Bartz-Schmidt, Karl-Ulrich; Szurman, Peter

2012-03-01

The effects of a glutathione-containing intra-ocular irrigation solution, BSS Plus©, on retinal function and on the survival of ganglion cells in whole-mount retinal explants were studied. Evidence is provided that the perfused ex vivo bovine retina can serve as an alternative to in vivo animal testing. Isolated bovine retinas were prepared and perfused with an oxygen-saturated standard irrigation solution, and an electroretinogram was recorded to assess retinal function. After stable b-waves were detected, the isolated retinas were perfused with BSS Plus for 45 minutes. To investigate the effects of BSS Plus on photoreceptor function, 1mM aspartate was added to the irrigation solution in order to obtain a-waves, and the ERG trace was monitored for 75 minutes. For histological analysis, isolated whole retinal mounts were stored for 24 hours at 4°C, in the dark. The percentages of cell death in the retinal ganglion cell layer and in the outer and inner nuclear layers were estimated by using an ethidium homodimer-1 stain and the TUNEL assay. General swelling of the retina was examined with high-resolution optical coherence tomography. During perfusion with BSS Plus, no significant changes in a-wave and b-wave amplitudes were recorded. Retinas stored for 24 hours in BSS Plus showed a statistically significant smaller percentage (52.6%, standard deviation [SD] = 16.1%) of cell death in the retinal ganglion cell layer compared to the control group (69.6%, SD = 3.9, p = 0.0031). BSS Plus did not seem to affect short-term retinal function, and had a beneficial effect on the survival of retinal ganglion cells. This method for analysing the isolated perfused retina represents a valuable alternative for testing substances for their retinal biocompatibility and toxicity. 2012 FRAME.

Pancreatic cancer cell detection by targeted lipid microbubbles and multiphoton imaging

NASA Astrophysics Data System (ADS)

Cromey, Benjamin; McDaniel, Ashley; Matsunaga, Terry; Vagner, Josef; Kieu, Khanh Quoc; Banerjee, Bhaskar

2018-04-01

Surgical resection of pancreatic cancer represents the only chance of cure and long-term survival in this common disease. Unfortunately, determination of a cancer-free margin at surgery is based on one or two tiny frozen section biopsies, which is far from ideal. Not surprisingly, cancer is usually left behind and is responsible for metastatic disease. We demonstrate a method of receptor-targeted imaging using peptide ligands, lipid microbubbles, and multiphoton microscopy that could lead to a fast and accurate way of examining the entire cut surface during surgery. Using a plectin-targeted microbubble, we performed a blinded in-vitro study to demonstrate avid binding of targeted microbubbles to pancreatic cancer cells but not noncancerous cell lines. Further work should lead to a much-needed point-of-care diagnostic test for determining clean margins in oncologic surgery.

Solar array module plasma interactions experiment (SAMPIE) - Science and technology objectives

NASA Technical Reports Server (NTRS)

Hillard, G. B.; Ferguson, Dale C.

1993-01-01

The solar array module plasma interactions experiment (SAMPIE) is an approved NASA flight experiment manifested for Shuttle deployment in early 1994. The SAMPIE experiment is designed to investigate the interaction of high voltage space power systems with ionospheric plasma. To study the behavior of solar cells, a number of solar cell coupons (representing design technologies of current interest) will be biased to high voltages to measure both arcing and current collection. Various theories of arc suppression will be tested by including several specially modified cell coupons. Finally, SAMPIE will include experiments to study the basic nature of arcing and current collection. This paper describes the rationale for a space flight experiment, the measurements to be made, and the significance of the expected results. A future paper will present a detailed discussion of the engineering design.

Comparison of ThinPrep and conventional preparations on fine needle aspiration cytology material.

PubMed

Dey, P; Luthra, U K; George, J; Zuhairy, F; George, S S; Haji, B I

2000-01-01

To compare the various cytologic features on ThinPrep 2000 (TP) (Cytyc Corporation, Marlborough, Massachusetts, U.S.A.) and conventional preparation (CP) specimens from fine needle aspiration cytology (FNAC) material by a semiquantitative scoring system. In this prospective study a total of 71 consecutive cases were included. In each case, two passes were performed. The first pass was used for conventional preparations, with direct smears made and fixed immediately in 95% alcohol for Papanicolaou stain. For TP preparation a second pass produced material for processing in the ThinPrep 2000. The TP and CP slides were studied independently by two observers and representative slides of CP and TP compared for cellularity, background blood and necrotic cell debris, cell architecture, informative background, presence of monolayer cells, and nuclear and cytoplasmic details by a semiquantitative scoring system. Statistical analysis was performed by Wilcoxon's signed rank test on an SPSS program (Chicago, Illinois, U.S.A.). TP preparations contained adequate diagnostic cells in all cases and were tangibly superior to CP preparations concerning monolayer cells, absence of blood and necrosis, and preservation of nuclear and cytoplasmic detail (statistically significant, Wilcoxon's signed rank test, P < .000). TP preparations are superior to conventional preparations with regard to clear background, monolayer cell preparation and cell preservation. It is easier and less time consuming to screen and interpret TP preparations because the cells are limited to smaller areas on clear backgrounds, with excellent cellular preservation. However, TP preparations are more expensive than CP and require some experience for interpretation.

Metal Free Graphene Oxide (GO) Nanosheets and Pristine-Single Wall Carbon Nanotubes (p-SWCNTs) Biocompatibility Investigation: A Comparative Study in Different Human Cell Lines.

PubMed

Valentini, Federica; Mari, Emanuela; Zicari, Alessandra; Calcaterra, Andrea; Talamo, Maurizio; Scioli, Maria Giovanna; Orlandi, Augusto; Mardente, Stefania

2018-04-28

The in vitro biocompatibility of Graphene Oxide (GO) nanosheets, which were obtained by the electrochemical exfoliation of graphite electrodes in an electrolytic bath containing salts, was compared with the pristine Single Wall Carbon Nanotubes (p-SWCNTs) under the same experimental conditions in different human cell lines. The cells were treated with different concentrations of GO and SWCNTs for up to 48 h. GO did not induce any significant morphological or functional modifications (demonstrating a high biocompatibility), while SWNCTs were toxic at any concentration used after a few hours of treatment. The cell viability or cytotoxicity were detected by the trypan blue assay and the lactate dehydrogenase LDH quantitative enzymatic test. The Confocal Laser Scanning Microscopy (CLSM) and transmission electron microscopy (TEM) analysis demonstrated the uptake and internalization of GO sheets into cells, which was localized mainly in the cytoplasm. Different results were observed in the same cell lines treated with p-SWCNTs. TEM and CLSM (Confocal Laser Scanning Microscopy) showed that the p-SWCNTs induced vacuolization in the cytoplasm, disruption of cellular architecture and damage to the nuclei. The most important result of this study is our finding of a higher GO biocompatibility compared to the p-SWCNTs in the same cell lines. This means that GO nanosheets, which are obtained by the electrochemical exfoliation of a graphite-based electrode (carried out in saline solutions or other physiological working media) could represent an eligible nanocarrier for drug delivery, gene transfection and molecular cell imaging tests.

Bioluminescence Truth Data Measurement and Signature Detection

DTIC Science & Technology

2007-09-30

cell phone based communications module attached to the top of the piling. A cell phone tower represents communication of data to shore. Also shown...representing each Kilroy installation are located based on GPS coordinates telemetered by the cell phone module. Icons point in direction of most recently

Application of in vitro cell transformation assays in regulatory toxicology for pharmaceuticals, chemicals, food products and cosmetics.

PubMed

Vanparys, Philippe; Corvi, Raffaella; Aardema, Marilyn J; Gribaldo, Laura; Hayashi, Makoto; Hoffmann, Sebastian; Schechtman, Leonard

2012-04-11

Two year rodent bioassays play a key role in the assessment of carcinogenic potential of chemicals to humans. The seventh amendment to the European Cosmetics Directive will ban in 2013 the marketing of cosmetic and personal care products that contain ingredients that have been tested in animal models. Thus 2-year rodent bioassays will not be available for cosmetics/personal care products. Furthermore, for large testing programs like REACH, in vivo carcinogenicity testing is impractical. Alternative ways to carcinogenicity assessment are urgently required. In terms of standardization and validation, the most advanced in vitro tests for carcinogenicity are the cell transformation assays (CTAs). Although CTAs do not mimic the whole carcinogenesis process in vivo, they represent a valuable support in identifying transforming potential of chemicals. CTAs have been shown to detect genotoxic as well as non-genotoxic carcinogens and are helpful in the determination of thresholds for genotoxic and non-genotoxic carcinogens. The extensive review on CTAs by the OECD (OECD (2007) Environmental Health and Safety Publications, Series on Testing and Assessment, No. 31) and the proven within- and between-laboratories reproducibility of the SHE CTAs justifies broader use of these methods to assess carcinogenic potential of chemicals. Copyright © 2012 Elsevier B.V. All rights reserved.

Automated point-of-care testing for ABO agglutination test: proof of concept and validation.

PubMed

El Kenz, H; Corazza, F

2015-07-01

ABO-incompatible red blood cell transfusions still represent an important hazard in transfusion medicine. Therefore, some countries have introduced a systematic bedside ABO agglutination test checking that the right blood is given to the right patient. However, this strategy requires an extremely time-consuming learning programme and relies on a subjective interpretation of ABO test cards agglutination. We developed a prototype of a fully automated device performing the bedside agglutination test that could be completed by reading of a barcoded wristband. This POCT checks the ABO compatibility between the patient and the blood bag. Proof of concept and analytical validation of the prototype has been completed on 451 blood samples: 238 donor packed red blood cells, 137 consecutive unselected patients for whom a blood group determination had been ordered and on 76 patient samples selected with pathology that could possibly interfere with or impair performances of the assay. We observed 100% concordance for ABO blood groups between the POCT and the laboratory instrument. These preliminary results demonstrate the feasibility of ABO determination with a simple POCT device eliminating manipulation and subjective interpretation responsible for transfusion errors. This device should be linked to the blood bank system allowing all cross-check of the results. © 2015 International Society of Blood Transfusion.

Simulation of solute transport across low-permeability barrier walls

USGS Publications Warehouse

Harte, P.T.; Konikow, Leonard F.; Hornberger, G.Z.

2006-01-01

Low-permeability, non-reactive barrier walls are often used to contain contaminants in an aquifer. Rates of solute transport through such barriers are typically many orders of magnitude slower than rates through the aquifer. Nevertheless, the success of remedial actions may be sensitive to these low rates of transport. Two numerical simulation methods for representing low-permeability barriers in a finite-difference groundwater-flow and transport model were tested. In the first method, the hydraulic properties of the barrier were represented directly on grid cells and in the second method, the intercell hydraulic-conductance values were adjusted to approximate the reduction in horizontal flow, allowing use of a coarser and computationally efficient grid. The alternative methods were tested and evaluated on the basis of hypothetical test problems and a field case involving tetrachloroethylene (PCE) contamination at a Superfund site in New Hampshire. For all cases, advective transport across the barrier was negligible, but preexisting numerical approaches to calculate dispersion yielded dispersive fluxes that were greater than expected. A transport model (MODFLOW-GWT) was modified to (1) allow different dispersive and diffusive properties to be assigned to the barrier than the adjacent aquifer and (2) more accurately calculate dispersion from concentration gradients and solute fluxes near barriers. The new approach yields reasonable and accurate concentrations for the test cases. ?? 2006.

A Comprehensive Panel of Three-Dimensional Models for Studies of Prostate Cancer Growth, Invasion and Drug Responses

PubMed Central

Härmä, Ville; Virtanen, Johannes; Mäkelä, Rami; Happonen, Antti; Mpindi, John-Patrick; Knuuttila, Matias; Kohonen, Pekka; Lötjönen, Jyrki; Kallioniemi, Olli; Nees, Matthias

2010-01-01

Prostate epithelial cells from both normal and cancer tissues, grown in three-dimensional (3D) culture as spheroids, represent promising in vitro models for the study of normal and cancer-relevant patterns of epithelial differentiation. We have developed the most comprehensive panel of miniaturized prostate cell culture models in 3D to date (n = 29), including many non-transformed and most currently available classic prostate cancer (PrCa) cell lines. The purpose of this study was to analyze morphogenetic properties of PrCa models in 3D, to compare phenotypes, gene expression and metabolism between 2D and 3D cultures, and to evaluate their relevance for pre-clinical drug discovery, disease modeling and basic research. Primary and non-transformed prostate epithelial cells, but also several PrCa lines, formed well-differentiated round spheroids. These showed strong cell-cell contacts, epithelial polarization, a hollow lumen and were covered by a complete basal lamina (BL). Most PrCa lines, however, formed large, poorly differentiated spheroids, or aggressively invading structures. In PC-3 and PC-3M cells, well-differentiated spheroids formed, which were then spontaneously transformed into highly invasive cells. These cell lines may have previously undergone an epithelial-to-mesenchymal transition (EMT), which is temporarily suppressed in favor of epithelial maturation by signals from the extracellular matrix (ECM). The induction of lipid and steroid metabolism, epigenetic reprogramming, and ECM remodeling represents a general adaptation to 3D culture, regardless of transformation and phenotype. In contrast, PI3-Kinase, AKT, STAT/interferon and integrin signaling pathways were particularly activated in invasive cells. Specific small molecule inhibitors targeted against PI3-Kinase blocked invasive cell growth more effectively in 3D than in 2D monolayer culture, or the growth of normal cells. Our panel of cell models, spanning a wide spectrum of phenotypic plasticity, supports the investigation of different modes of cell migration and tumor morphologies, and will be useful for predictive testing of anti-cancer and anti-metastatic compounds. PMID:20454659

Pectin-coated boron nitride nanotubes: In vitro cyto-/immune-compatibility on RAW 264.7 macrophages.

PubMed

Rocca, Antonella; Marino, Attilio; Del Turco, Serena; Cappello, Valentina; Parlanti, Paola; Pellegrino, Mario; Golberg, Dmitri; Mattoli, Virgilio; Ciofani, Gianni

2016-04-01

Boron nitride nanotubes (BNNTs) represent a new opportunity for drug delivery and clinical therapy. The present work has the objective to investigate pectin-coated BNNTs (P-BNNTs) for their biocompatibility on macrophage cultures, since these cells are among the first components of the immune system to interact with administered nanoparticles. As first step, the potential toxicity of P-BNNTs is verified in terms of proliferation, oxidative stress induction and apoptosis/necrosis phenomena. Thereafter, the modulation of immune cell response following P-BNNT exposure is evaluated at gene and protein level, in particular focusing on cytokine release. Finally, P-BNNT internalization is assessed through transmission electron microscopy and confocal microscopy. The results proved that P-BNNTs are not toxic for macrophages up to 50 μg/ml after 24 h of incubation. The cytokine expression is not affected by P-BNNT administration both at gene and protein level. Moreover, P-BNNTs are internalized by macrophages without impairments of the cell structures. Collected data suggest that P-BNNTs cause neither adverse effects nor inflammation processes in macrophages. These findings represent the first and fundamental step in immune compatibility evaluation of BNNTs, mandatory before any further pre-clinical testing. Copyright © 2016 Elsevier B.V. All rights reserved.

A multi-directional and multi-scale roughness filter to detect lineament segments on digital elevation models - analyzing spatial objects in R

NASA Astrophysics Data System (ADS)

Baumann, Sebastian; Robl, Jörg; Wendt, Lorenz; Willingshofer, Ernst; Hilberg, Sylke

2016-04-01

Automated lineament analysis on remotely sensed data requires two general process steps: The identification of neighboring pixels showing high contrast and the conversion of these domains into lines. The target output is the lineaments' position, extent and orientation. We developed a lineament extraction tool programmed in R using digital elevation models as input data to generate morphological lineaments defined as follows: A morphological lineament represents a zone of high relief roughness, whose length significantly exceeds the width. As relief roughness any deviation from a flat plane, defined by a roughness threshold, is considered. In our novel approach a multi-directional and multi-scale roughness filter uses moving windows of different neighborhood sizes to identify threshold limited rough domains on digital elevation models. Surface roughness is calculated as the vertical elevation difference between the center cell and the different orientated straight lines connecting two edge cells of a neighborhood, divided by the horizontal distance of the edge cells. Thus multiple roughness values depending on the neighborhood sizes and orientations of the edge connecting lines are generated for each cell and their maximum and minimum values are extracted. Thereby negative signs of the roughness parameter represent concave relief structures as valleys, positive signs convex relief structures as ridges. A threshold defines domains of high relief roughness. These domains are thinned to a representative point pattern by a 3x3 neighborhood filter, highlighting maximum and minimum roughness peaks, and representing the center points of lineament segments. The orientation and extent of the lineament segments are calculated within the roughness domains, generating a straight line segment in the direction of least roughness differences. We tested our algorithm on digital elevation models of multiple sources and scales and compared the results visually with shaded relief map of these digital elevation models. The lineament segments trace the relief structure to a great extent and the calculated roughness parameter represents the physical geometry of the digital elevation model. Modifying the threshold for the surface roughness value highlights different distinct relief structures. Also the neighborhood size at which lineament segments are detected correspond with the width of the surface structure and may be a useful additional parameter for further analysis. The discrimination of concave and convex relief structures perfectly matches with valleys and ridges of the surface.

Modelling human disease with pluripotent stem cells.

PubMed

Siller, Richard; Greenhough, Sebastian; Park, In-Hyun; Sullivan, Gareth J

2013-04-01

Recent progress in the field of cellular reprogramming has opened up the doors to a new era of disease modelling, as pluripotent stem cells representing a myriad of genetic diseases can now be produced from patient tissue. These cells can be expanded and differentiated to produce a potentially limitless supply of the affected cell type, which can then be used as a tool to improve understanding of disease mechanisms and test therapeutic interventions. This process requires high levels of scrutiny and validation at every stage, but international standards for the characterisation of pluripotent cells and their progeny have yet to be established. Here we discuss the current state of the art with regard to modelling diseases affecting the ectodermal, mesodermal and endodermal lineages, focussing on studies which have demonstrated a disease phenotype in the tissue of interest. We also discuss the utility of pluripotent cell technology for the modelling of cancer and infectious disease. Finally, we spell out the technical and scientific challenges which must be addressed if the field is to deliver on its potential and produce improved patient outcomes in the clinic.

Cytotoxicity screening of 23 engineered nanomaterials using a test matrix of ten cell lines and three different assays

PubMed Central

2011-01-01

Background Engineered nanomaterials display unique properties that may have impact on human health, and thus require a reliable evaluation of their potential toxicity. Here, we performed a standardized in vitro screening of 23 engineered nanomaterials. We thoroughly characterized the physicochemical properties of the nanomaterials and adapted three classical in vitro toxicity assays to eliminate nanomaterial interference. Nanomaterial toxicity was assessed in ten representative cell lines. Results Six nanomaterials induced oxidative cell stress while only a single nanomaterial reduced cellular metabolic activity and none of the particles affected cell viability. Results from heterogeneous and chemically identical particles suggested that surface chemistry, surface coating and chemical composition are likely determinants of nanomaterial toxicity. Individual cell lines differed significantly in their response, dependent on the particle type and the toxicity endpoint measured. Conclusion In vitro toxicity of the analyzed engineered nanomaterials cannot be attributed to a defined physicochemical property. Therefore, the accurate identification of nanomaterial cytotoxicity requires a matrix based on a set of sensitive cell lines and in vitro assays measuring different cytotoxicity endpoints. PMID:21345205

Tofacitinib induces G1 cell-cycle arrest and inhibits tumor growth in Epstein-Barr virus-associated T and natural killer cell lymphoma cells

PubMed Central

Ando, Shotaro; Kawada, Jun-ichi; Watanabe, Takahiro; Suzuki, Michio; Sato, Yoshitaka; Torii, Yuka; Asai, Masato; Goshima, Fumi; Murata, Takayuki; Shimizu, Norio; Ito, Yoshinori; Kimura, Hiroshi

2016-01-01

Epstein-Barr virus (EBV) infects not only B cells, but also T cells and natural killer (NK) cells, and is associated with T or NK cell lymphoma. These lymphoid malignancies are refractory to conventional chemotherapy. We examined the activation of the JAK3/STAT5 pathway in EBV-positive and -negative B, T and NK cell lines and in cell samples from patients with EBV-associated T cell lymphoma. We then evaluated the antitumor effects of the selective JAK3 inhibitor, tofacitinib, against these cell lines in vitro and in a murine xenograft model. We found that all EBV-positive T and NK cell lines and patient samples tested displayed activation of the JAK3/STAT5 pathway. Treatment of these cell lines with tofacitinib reduced the levels of phospho-STAT5, suppressed proliferation, induced G1 cell-cycle arrest and decreased EBV LMP1 and EBNA1 expression. An EBV-negative NK cell line was also sensitive to tofacitinib, whereas an EBV-infected NK cell line was more sensitive to tofacitinib than its parental line. Tofacitinib significantly inhibited the growth of established tumors in NOG mice. These findings suggest that tofacitinib may represent a useful therapeutic agent for patients with EBV-associated T and NK cell lymphoma. PMID:27732937

Strigolactone analogues induce apoptosis through activation of p38 and the stress response pathway in cancer cell lines and in conditionally reprogrammed primary prostate cancer cells.

PubMed

Pollock, Claire B; McDonough, Sara; Wang, Victor S; Lee, Hyojung; Ringer, Lymor; Li, Xin; Prandi, Cristina; Lee, Richard J; Feldman, Adam S; Koltai, Hinanit; Kapulnik, Yoram; Rodriguez, Olga C; Schlegel, Richard; Albanese, Christopher; Yarden, Ronit I

2014-03-30

Strigolactones are a novel class of plant hormones produced in roots and regulate shoot and root development. We have previously shown that synthetic strigolactone analogues potently inhibit growth of breast cancer cells and breast cancer stem cells. Here we show that strigolactone analogues inhibit the growth and survival of an array of cancer-derived cell lines representing solid and non-solid cancer cells including: prostate, colon, lung, melanoma, osteosarcoma and leukemic cell lines, while normal cells were minimally affected. Treatment of cancer cells with strigolactone analogues was hallmarked by activation of the stress-related MAPKs: p38 and JNK and induction of stress-related genes; cell cycle arrest and apoptosis evident by increased percentages of cells in the sub-G1 fraction and Annexin V staining. In addition, we tested the response of patient-matched conditionally reprogrammed primary prostate normal and cancer cells. The tumor cells exhibited significantly higher sensitivity to the two most potent SL analogues with increased apoptosis confirmed by PARP1 cleavage compared to their normal counterpart cells. Thus, Strigolactone analogues are promising candidates for anticancer therapy by their ability to specifically induce cell cycle arrest, cellular stress and apoptosis in tumor cells with minimal effects on growth and survival of normal cells.

Fluorescence Microspectroscopy for Testing the Dimerization Hypothesis of BACE1 Protein in Cultured HEK293 Cells

NASA Astrophysics Data System (ADS)

Gardeen, Spencer; Johnson, Joseph L.; Heikal, Ahmed A.

2016-06-01

Alzheimer's Disease (AD) is a neurodegenerative disorder that results from the formation of beta-amyloid plaques in the brain that trigger the known symptoms of memory loss in AD patients. The beta-amyloid plaques are formed by the proteolytic cleavage of the amyloid precursor protein (APP) by the proteases BACE1 and gamma-secretase. These enzyme-facilitated cleavages lead to the production of beta-amyloid fragments that aggregate to form plaques, which ultimately lead to neuronal cell death. Recent detergent protein extraction studies suggest that BACE1 protein forms a dimer that has significantly higher catalytic activity than its monomeric counterpart. In this contribution, we examine the dimerization hypothesis of BACE1 in cultured HEK293 cells using complementary fluorescence spectroscopy and microscopy methods. Cells were transfected with a BACE1-EGFP fusion protein construct and imaged using confocal, and differential interference contrast to monitor the localization and distribution of intracellular BACE1. Complementary fluorescence lifetime and anisotropy measurements enabled us to examine the conformational and environmental changes of BACE1 as a function of substrate binding. Using fluorescence correlation spectroscopy, we also quantified the diffusion coefficient of BACE1-EGFP on the plasma membrane as a means to test the dimerization hypothesis as a fucntion of substrate-analog inhibitition. Our results represent an important first towards examining the substrate-mediated dimerization hypothesis of BACE1 in live cells.

Seasonal spatial patterns in seabird and marine mammal distribution in the eastern Chukchi and western Beaufort seas: Identifying biologically important pelagic areas

NASA Astrophysics Data System (ADS)

Kuletz, Kathy J.; Ferguson, Megan C.; Hurley, Brendan; Gall, Adrian E.; Labunski, Elizabeth A.; Morgan, Tawna C.

2015-08-01

The Chukchi and Beaufort seas are undergoing rapid climate change and increased human activity. Conservation efforts for upper trophic level predators such as seabirds and marine mammals require information on species' distributions and identification of important marine areas. Here we describe broad-scale distributions of seabirds and marine mammals. We examined spatial patterns of relative abundance of seabirds and marine mammals in the eastern Chukchi and western Beaufort seas during summer (15 June-31 August) and fall (1 September-20 November) from 2007 to 2012. We summarized 49,206 km of shipboard surveys for seabirds and 183,157 km of aerial surveys for marine mammals into a grid of 40-km × 40-km cells. We used Getis-Ord Gi∗ hotspot analysis to test for cells with higher relative abundance than expected when compared to all cells within the study area. We identified cells representing single species and taxonomic group hotspots, cells representing hotspots for multiple species, and cells representing hotspots for both seabirds and marine mammals. The locations of hotspots varied among species but often were located near underwater canyons or over continental shelf features and slopes. Hotspots for seabirds, walrus, and gray whales occurred primarily in the Chukchi Sea. Hotspots for bowhead whales and other pinnipeds (i.e., seals) occurred near Barrow Canyon and along the Beaufort Sea shelf and slope. Hotspots for belugas occurred in both the Chukchi and Beaufort seas. There were three hotspots shared by both seabirds and marine mammals in summer: off Wainwright in the eastern Chukchi Sea, south of Hanna Shoal, and at the mouth of Barrow Canyon. In fall, the only identified shared hotspot occurred at the mouth of Barrow Canyon. Shared hotspots are characterized by strong fronts caused by upwelling and currents, and these areas can have high densities of euphausiids in summer and fall. Due to the high relative abundance of animals and diversity of taxa, these sites are clearly important areas of congregation for seabirds and marine mammals that should be prioritized in the development of management and conservation plans.

Heart grafts tolerized through third-party multipotent adult progenitor cells can be retransplanted to secondary hosts with no immunosuppression.

PubMed

Eggenhofer, Elke; Popp, Felix C; Mendicino, Michael; Silber, Paula; Van't Hof, Wouter; Renner, Philipp; Hoogduijn, Martin J; Pinxteren, Jef; van Rooijen, Nico; Geissler, Edward K; Deans, Robert; Schlitt, Hans J; Dahlke, Marc H

2013-08-01

Multipotent adult progenitor cells (MAPCs) are an adherent stem cell population that belongs to the mesenchymal-type progenitor cell family. Although MAPCs are emerging as candidate agents for immunomodulation after solid organ transplantation, their value requires further validation in a clinically relevant cell therapy model using an organ donor- and organ recipient-independent, third-party cell product. We report that stable allograft survival can be achieved following third-party MAPC infusion in a rat model of fully allogeneic, heterotopic heart transplantation. Furthermore, long-term accepted heart grafts recovered from MAPC-treated animals can be successfully retransplanted to naïve animals without additional immunosuppression. This prolongation of MAPC-mediated allograft acceptance depends upon a myeloid cell population since depletion of macrophages by clodronate abrogates the tolerogenic MAPC effect. We also show that MAPC-mediated allograft acceptance differs mechanistically from drug-induced tolerance regarding marker gene expression, T regulatory cell induction, retransplantability, and macrophage dependence. MAPC-based immunomodulation represents a promising pathway for clinical immunotherapy that has led us to initiate a phase I clinical trial for testing safety and feasibility of third-party MAPC therapy after liver transplantation.

A combined cell based approach to identify P-glycoprotein substrates and inhibitors in a single assay.

PubMed

Balimane, Praveen V; Chong, Saeho

2005-09-14

The objective of this project was to develop a cell based in vitro experimental procedure that can differentiate P-glycoprotein (P-gp) substrates from inhibitors in a single assay. Caco-2 cells grown to confluency on 12-well Transwell were used for this study. The efflux permeability (B to A) of P-gp specific probe (viz., digoxin) in the presence of test compounds (e.g. substrates, inhibitors and non-substrates of P-gp) was monitored, and the influx permeability (A to B) of test compounds was evaluated after complete P-gp blockade. Radiolabelled digoxin was added on the basolateral side with buffer on the apical side. The digoxin concentration appearing on the apical side represents digoxin efflux permeability during the control phase (0-1 h period). After 1 h, a test compound (10 microM) was added on the apical side. The reduced efflux permeability of digoxin suggests that the added test compound is an inhibitor. The influx permeability of test compound is also determined during the 1-2 h study period by measuring the concentration of the test compound in the basolateral side. At the end of 2 h, a potent P-gp inhibitor (GF120918) was added. The increased influx permeability of test compound during the 2-3 h incubation period indicates that the added test compound is a substrate. Samples were taken from both sides at the end of 1-3 h and the concentrations of the test compounds and digoxin were quantitated. Digoxin efflux permeability remained unchanged when incubated with P-gp substrates (e.g., etoposide, rhodamine123, taxol). However, when a P-gp inhibitor was added to the apical side, the digoxin efflux (B to A permeability) was significantly reduced (ketoconazole=51% reduction) as expected. The influx permeability of substrates increased significantly (rhodamine123=70%, taxol=220%, digoxin=290%) after the P-gp inhibitor (GF120918) was introduced, whereas the influx permeability of P-gp inhibitor and non-substrates was not affected by GF120918. Thus, this combined assay provides an efficient cell based in vitro screening tool to simultaneously distinguish compounds that are P-gp substrates from P-gp inhibitors.

Regulatory aspects on the use of fish embryos in environmental toxicology.

PubMed

Halder, Marlies; Léonard, Marc; Iguchi, Taisen; Oris, James T; Ryder, Kathy; Belanger, Scott E; Braunbeck, Thomas A; Embry, Michelle R; Whale, Graham; Norberg-King, Teresa; Lillicrap, Adam

2010-07-01

Animal alternative tests are gaining serious consideration in an array of environmental sciences, particularly as they relate to sound management of chemicals and wastewater discharges. The ILSI Health and Environmental Sciences Institute and the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) held an International Workshop on the Application of the Fish Embryo Test in March, 2008. This relatively young discipline is following advances in animal alternatives for human safety sciences, and it is advisable to develop a broad comparison of how animal alternative tests involving fish are viewed in a regulatory context over a wide array of authorities or advising bodies. These include OECD, Western Europe, North America, and Japan. This paper summarizes representative practices from these regions. Presently, the global regulatory environment has varying stances regarding the protection of fish for use as an experimental animal. Such differences have a long-term potential to lead to a lack of harmony in approaches to fish toxicity testing, especially for chemicals in commerce across multiple geographic regions. Implementation of alternative methods and approaches will be most successful if accepted globally, including methods of fish toxicity testing. An important area for harmonization would be in the interpretation of protected and nonprotected life stages of fish. Use of fish embryos represent a promising alternative and allow bridging to more technically challenging alternatives with longer prospective timelines, including cell-based assays, ecotoxicogenomics, and QSARs. (c) 2010 SETAC.

Optimization and experimental validation of a thermal cycle that maximizes entropy coefficient fisher identifiability for lithium iron phosphate cells

NASA Astrophysics Data System (ADS)

Mendoza, Sergio; Rothenberger, Michael; Hake, Alison; Fathy, Hosam

2016-03-01

This article presents a framework for optimizing the thermal cycle to estimate a battery cell's entropy coefficient at 20% state of charge (SOC). Our goal is to maximize Fisher identifiability: a measure of the accuracy with which a parameter can be estimated. Existing protocols in the literature for estimating entropy coefficients demand excessive laboratory time. Identifiability optimization makes it possible to achieve comparable accuracy levels in a fraction of the time. This article demonstrates this result for a set of lithium iron phosphate (LFP) cells. We conduct a 24-h experiment to obtain benchmark measurements of their entropy coefficients. We optimize a thermal cycle to maximize parameter identifiability for these cells. This optimization proceeds with respect to the coefficients of a Fourier discretization of this thermal cycle. Finally, we compare the estimated parameters using (i) the benchmark test, (ii) the optimized protocol, and (iii) a 15-h test from the literature (by Forgez et al.). The results are encouraging for two reasons. First, they confirm the simulation-based prediction that the optimized experiment can produce accurate parameter estimates in 2 h, compared to 15-24. Second, the optimized experiment also estimates a thermal time constant representing the effects of thermal capacitance and convection heat transfer.

Calibrations between the variables of microbial TTI response and ground pork qualities.

PubMed

Kim, Eunji; Choi, Dong Yeol; Kim, Hyun Chul; Kim, Keehyuk; Lee, Seung Ju

2013-10-01

A time-temperature indicator (TTI) based on a lactic acid bacterium, Weissella cibaria CIFP009, was applied to ground pork packaging. Calibration curves between TTI response and pork qualities were obtained from storage tests at 2°C, 10°C, and 13°C. The curves of the TTI vs. total cell number at different temperatures coincided to the greatest extent, indicating the highest representativeness of calibration, by showing the least coefficient of variance (CV=11%) of the quality variables at a given TTI response (titratable acidity) on the curves, followed by pH (23%), volatile basic nitrogen (VBN) (25%), and thiobarbituric acid-reactive substances (TBARS) (47%). Similarity of Arrhenius activation energy (Ea) could also reflect the representativeness of calibration. The total cell number (104.9 kJ/mol) was found to be the most similar to that of the TTI response (106.2 kJ/mol), followed by pH (113.6 kJ/mol), VBN (77.4 kJ/mol), and TBARS (55.0 kJ/mol). Copyright © 2013 Elsevier Ltd. All rights reserved.

Targeted cellular ablation based on the morphology of malignant cells

NASA Astrophysics Data System (ADS)

Ivey, Jill W.; Latouche, Eduardo L.; Sano, Michael B.; Rossmeisl, John H.; Davalos, Rafael V.; Verbridge, Scott S.

2015-11-01

Treatment of glioblastoma multiforme (GBM) is especially challenging due to a shortage of methods to preferentially target diffuse infiltrative cells, and therapy-resistant glioma stem cell populations. Here we report a physical treatment method based on electrical disruption of cells, whose action depends strongly on cellular morphology. Interestingly, numerical modeling suggests that while outer lipid bilayer disruption induced by long pulses (~100 μs) is enhanced for larger cells, short pulses (~1 μs) preferentially result in high fields within the cell interior, which scale in magnitude with nucleus size. Because enlarged nuclei represent a reliable indicator of malignancy, this suggested a means of preferentially targeting malignant cells. While we demonstrate killing of both normal and malignant cells using pulsed electric fields (PEFs) to treat spontaneous canine GBM, we proposed that properly tuned PEFs might provide targeted ablation based on nuclear size. Using 3D hydrogel models of normal and malignant brain tissues, which permit high-resolution interrogation during treatment testing, we confirmed that PEFs could be tuned to preferentially kill cancerous cells. Finally, we estimated the nuclear envelope electric potential disruption needed for cell death from PEFs. Our results may be useful in safely targeting the therapy-resistant cell niches that cause recurrence of GBM tumors.

The costs of using unauthenticated, over-passaged cell lines: how much more data do we need?

PubMed

Hughes, Peyton; Marshall, Damian; Reid, Yvonne; Parkes, Helen; Gelber, Cohava

2007-11-01

Increasing data demonstrate that cellular cross-contamination, misidentified cell lines, and the use of cultures at high-passage levels contribute to the generation of erroneous and misleading results as well as wasted research funds. Contamination of cell lines by other lines has been recognized and documented back to the 1950s. Based on submissions to major cell repositories in the last decade, it is estimated that between 18% and 36% of cell lines may be contaminated or misidentified. More recently, problems surrounding practices of over-subculturing cells are being identified. As a result of selective pressures and genetic drift, cell lines, when kept in culture too long, exhibit reduced or altered key functions and often no longer represent reliable models of their original source material. A review of papers showing significant experimental variances between low- and high-passage cell culture numbers, as well as contaminated lines, makes a strong case for using verified, tested cell lines at low- or defined passage numbers. In the absence of cell culture guidelines, mandates from the National Institutes of Health (NIH) and other funding agencies or journal requirements, it becomes the responsibility of the scientific community to perform due diligence to ensure the integrity of cell cultures used in research.

Targeted cellular ablation based on the morphology of malignant cells

PubMed Central

Ivey, Jill W.; Latouche, Eduardo L.; Sano, Michael B.; Rossmeisl, John H.; Davalos, Rafael V.; Verbridge, Scott S.

2015-01-01

Treatment of glioblastoma multiforme (GBM) is especially challenging due to a shortage of methods to preferentially target diffuse infiltrative cells, and therapy-resistant glioma stem cell populations. Here we report a physical treatment method based on electrical disruption of cells, whose action depends strongly on cellular morphology. Interestingly, numerical modeling suggests that while outer lipid bilayer disruption induced by long pulses (~100 μs) is enhanced for larger cells, short pulses (~1 μs) preferentially result in high fields within the cell interior, which scale in magnitude with nucleus size. Because enlarged nuclei represent a reliable indicator of malignancy, this suggested a means of preferentially targeting malignant cells. While we demonstrate killing of both normal and malignant cells using pulsed electric fields (PEFs) to treat spontaneous canine GBM, we proposed that properly tuned PEFs might provide targeted ablation based on nuclear size. Using 3D hydrogel models of normal and malignant brain tissues, which permit high-resolution interrogation during treatment testing, we confirmed that PEFs could be tuned to preferentially kill cancerous cells. Finally, we estimated the nuclear envelope electric potential disruption needed for cell death from PEFs. Our results may be useful in safely targeting the therapy-resistant cell niches that cause recurrence of GBM tumors. PMID:26596248

Matrix diffusion coefficients in volcanic rocks at the Nevada test site: influence of matrix porosity, matrix permeability, and fracture coating minerals.

PubMed

Reimus, Paul W; Callahan, Timothy J; Ware, S Doug; Haga, Marc J; Counce, Dale A

2007-08-15

Diffusion cell experiments were conducted to measure nonsorbing solute matrix diffusion coefficients in forty-seven different volcanic rock matrix samples from eight different locations (with multiple depth intervals represented at several locations) at the Nevada Test Site. The solutes used in the experiments included bromide, iodide, pentafluorobenzoate (PFBA), and tritiated water ((3)HHO). The porosity and saturated permeability of most of the diffusion cell samples were measured to evaluate the correlation of these two variables with tracer matrix diffusion coefficients divided by the free-water diffusion coefficient (D(m)/D*). To investigate the influence of fracture coating minerals on matrix diffusion, ten of the diffusion cells represented paired samples from the same depth interval in which one sample contained a fracture surface with mineral coatings and the other sample consisted of only pure matrix. The log of (D(m)/D*) was found to be positively correlated with both the matrix porosity and the log of matrix permeability. A multiple linear regression analysis indicated that both parameters contributed significantly to the regression at the 95% confidence level. However, the log of the matrix diffusion coefficient was more highly-correlated with the log of matrix permeability than with matrix porosity, which suggests that matrix diffusion coefficients, like matrix permeabilities, have a greater dependence on the interconnectedness of matrix porosity than on the matrix porosity itself. The regression equation for the volcanic rocks was found to provide satisfactory predictions of log(D(m)/D*) for other types of rocks with similar ranges of matrix porosity and permeability as the volcanic rocks, but it did a poorer job predicting log(D(m)/D*) for rocks with lower porosities and/or permeabilities. The presence of mineral coatings on fracture walls did not appear to have a significant effect on matrix diffusion in the ten paired diffusion cell experiments.

Evaluation of the osteoinductive potential of a bio-inspired scaffold mimicking the osteogenic niche for bone augmentation.

PubMed

Minardi, Silvia; Corradetti, Bruna; Taraballi, Francesca; Sandri, Monica; Van Eps, Jeffrey; Cabrera, Fernando J; Weiner, Bradley K; Tampieri, Anna; Tasciotti, Ennio

2015-09-01

Augmentation of regenerative osteogenesis represents a premier clinical need, as hundreds of thousands of patients are left with insufficient healing of bony defects related to a host of insults ranging from congenital abnormalities to traumatic injury to surgically-induced deficits. A synthetic material that closely mimics the composition and structure of the human osteogenic niche represents great potential to successfully address this high demand. In this study, a magnesium-doped hydroxyapatite/type I collagen scaffold was fabricated through a biologically-inspired mineralization process and designed to mimic human trabecular bone. The composition of the scaffold was fully characterized by XRD, FTIR, ICP and TGA, and compared to human bone. Also, the scaffold microstructure was evaluated by SEM, while its nano-structure and nano-mechanical properties were evaluated by AFM. Human bone marrow-derived mesenchymal stem cells were used to test the in vitro capability of the scaffold to promote osteogenic differentiation. The cell/scaffold constructs were cultured up to 7 days and the adhesion, organization and proliferation of the cells were evaluated. The ability of the scaffold to induce osteogenic differentiation of the cells was assessed over 3 weeks and the correlate gene expression for classic genes of osteogenesis was assessed. Finally, when tested in an ectopic model in rabbit, the scaffold produced a large volume of trabecular bone in only two weeks, that subsequently underwent maturation over time as expected, with increased mature cortical bone formation, supporting its ability to promote bone regeneration in clinically-relevant scenarios. Altogether, these results confirm a high level of structural mimicry by the scaffold to the composition and structure of human osteogenic niche that translated to faster and more efficient osteoinduction in vivo--features that suggest such a biomaterial may have great utility in future clinical applications where bone regeneration is required. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Matrix diffusion coefficients in volcanic rocks at the Nevada test site: Influence of matrix porosity, matrix permeability, and fracture coating minerals

NASA Astrophysics Data System (ADS)

Reimus, Paul W.; Callahan, Timothy J.; Ware, S. Doug; Haga, Marc J.; Counce, Dale A.

2007-08-01

Diffusion cell experiments were conducted to measure nonsorbing solute matrix diffusion coefficients in forty-seven different volcanic rock matrix samples from eight different locations (with multiple depth intervals represented at several locations) at the Nevada Test Site. The solutes used in the experiments included bromide, iodide, pentafluorobenzoate (PFBA), and tritiated water ( 3HHO). The porosity and saturated permeability of most of the diffusion cell samples were measured to evaluate the correlation of these two variables with tracer matrix diffusion coefficients divided by the free-water diffusion coefficient ( Dm/ D*). To investigate the influence of fracture coating minerals on matrix diffusion, ten of the diffusion cells represented paired samples from the same depth interval in which one sample contained a fracture surface with mineral coatings and the other sample consisted of only pure matrix. The log of ( Dm/ D*) was found to be positively correlated with both the matrix porosity and the log of matrix permeability. A multiple linear regression analysis indicated that both parameters contributed significantly to the regression at the 95% confidence level. However, the log of the matrix diffusion coefficient was more highly-correlated with the log of matrix permeability than with matrix porosity, which suggests that matrix diffusion coefficients, like matrix permeabilities, have a greater dependence on the interconnectedness of matrix porosity than on the matrix porosity itself. The regression equation for the volcanic rocks was found to provide satisfactory predictions of log( Dm/ D*) for other types of rocks with similar ranges of matrix porosity and permeability as the volcanic rocks, but it did a poorer job predicting log( Dm/ D*) for rocks with lower porosities and/or permeabilities. The presence of mineral coatings on fracture walls did not appear to have a significant effect on matrix diffusion in the ten paired diffusion cell experiments.

Induction of Multifunctional Broadly Reactive T Cell Responses by a Plasmodium vivax Circumsporozoite Protein Recombinant Chimera.

PubMed

Cabrera-Mora, Monica; Fonseca, Jairo Andres; Singh, Balwan; Oliveira-Ferreira, Joseli; Lima-Junior, Josué da Costa; Calvo-Calle, J Mauricio; Moreno, Alberto

2015-09-01

Plasmodium vivax is the most widespread species of Plasmodium, causing up to 50% of the malaria cases occurring outside sub-Saharan Africa. An effective vaccine is essential for successful control and potential eradication. A well-characterized vaccine candidate is the circumsporozoite protein (CSP). Preclinical and clinical trials have shown that both antibodies and cellular immune responses have been correlated with protection induced by immunization with CSP. On the basis of our reported approach of developing chimeric Plasmodium yoelii proteins to enhance protective efficacy, we designed PvRMC-CSP, a recombinant chimeric protein based on the P. vivax CSP (PvCSP). In this engineered protein, regions of the PvCSP predicted to contain human T cell epitopes were genetically fused to an immunodominant B cell epitope derived from the N-terminal region I and to repeat sequences representing the two types of PvCSP repeats. The chimeric protein was expressed in soluble form with high yield. As the immune response to PvCSP has been reported to be genetically restricted in the murine model, we tested the immunogenicity of PvRMC-CSP in groups of six inbred strains of mice. PvRMC-CSP was able to induce robust antibody responses in all the mouse strains tested. Synthetic peptides representing the allelic forms of the P. vivax CSP were also recognized to a similar extent regardless of the mouse strain. Furthermore, the immunization regimen induced high frequencies of multifunctional CD4(+) and CD8(+) PvRMC-CSP-specific T cells. The depth and breadth of the immune responses elicited suggest that immunization with PvRMC-CSP can circumvent the genetic restriction of the immune response to P. vivax CSP. Interestingly, PvRMC-CSP was also recognized by naturally acquired antibodies from individuals living in areas where malaria is endemic. These features make PvRMC-CSP a promising vaccine candidate for further development. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Induction of Multifunctional Broadly Reactive T Cell Responses by a Plasmodium vivax Circumsporozoite Protein Recombinant Chimera

PubMed Central

Cabrera-Mora, Monica; Fonseca, Jairo Andres; Singh, Balwan; Oliveira-Ferreira, Joseli; Lima-Junior, Josué da Costa; Calvo-Calle, J. Mauricio

2015-01-01

Plasmodium vivax is the most widespread species of Plasmodium, causing up to 50% of the malaria cases occurring outside sub-Saharan Africa. An effective vaccine is essential for successful control and potential eradication. A well-characterized vaccine candidate is the circumsporozoite protein (CSP). Preclinical and clinical trials have shown that both antibodies and cellular immune responses have been correlated with protection induced by immunization with CSP. On the basis of our reported approach of developing chimeric Plasmodium yoelii proteins to enhance protective efficacy, we designed PvRMC-CSP, a recombinant chimeric protein based on the P. vivax CSP (PvCSP). In this engineered protein, regions of the PvCSP predicted to contain human T cell epitopes were genetically fused to an immunodominant B cell epitope derived from the N-terminal region I and to repeat sequences representing the two types of PvCSP repeats. The chimeric protein was expressed in soluble form with high yield. As the immune response to PvCSP has been reported to be genetically restricted in the murine model, we tested the immunogenicity of PvRMC-CSP in groups of six inbred strains of mice. PvRMC-CSP was able to induce robust antibody responses in all the mouse strains tested. Synthetic peptides representing the allelic forms of the P. vivax CSP were also recognized to a similar extent regardless of the mouse strain. Furthermore, the immunization regimen induced high frequencies of multifunctional CD4+ and CD8+ PvRMC-CSP-specific T cells. The depth and breadth of the immune responses elicited suggest that immunization with PvRMC-CSP can circumvent the genetic restriction of the immune response to P. vivax CSP. Interestingly, PvRMC-CSP was also recognized by naturally acquired antibodies from individuals living in areas where malaria is endemic. These features make PvRMC-CSP a promising vaccine candidate for further development. PMID:26169267

Development of Novel Patient-Derived Xenografts from Breast Cancer Brain Metastases

PubMed Central

Contreras-Zárate, María J.; Ormond, D. Ryan; Gillen, Austin E.; Hanna, Colton; Day, Nicole L.; Serkova, Natalie J.; Jacobsen, Britta M.; Edgerton, Susan M.; Thor, Ann D.; Borges, Virginia F.; Lillehei, Kevin O.; Graner, Michael W.; Kabos, Peter; Cittelly, Diana M.

2017-01-01

Brain metastases are an increasing burden among breast cancer patients, particularly for those with HER2+ and triple negative (TN) subtypes. Mechanistic insight into the pathophysiology of brain metastases and preclinical validation of therapies has relied almost exclusively on intracardiac injection of brain-homing cells derived from highly aggressive TN MDA-MB-231 and HER2+ BT474 breast cancer cell lines. Yet, these well characterized models are far from representing the tumor heterogeneity observed clinically and, due to their fast progression in vivo, their suitability to validate therapies for established brain metastasis remains limited. The goal of this study was to develop and characterize novel human brain metastasis breast cancer patient-derived xenografts (BM-PDXs) to study the biology of brain metastasis and to serve as tools for testing novel therapeutic approaches. We obtained freshly resected brain metastases from consenting donors with breast cancer. Tissue was immediately implanted in the mammary fat pad of female immunocompromised mice and expanded as BM-PDXs. Brain metastases from 3/4 (75%) TN, 1/1 (100%) estrogen receptor positive (ER+), and 5/9 (55.5%) HER2+ clinical subtypes were established as transplantable BM-PDXs. To facilitate tracking of metastatic dissemination using BM-PDXs, we labeled PDX-dissociated cells with EGFP-luciferase followed by reimplantation in mice, and generated a BM-derived cell line (F2-7). Immunohistologic analyses demonstrated that parental and labeled BM-PDXs retained expression of critical clinical markers such as ER, progesterone receptor, epidermal growth factor receptor, HER2, and the basal cell marker cytokeratin 5. Similarly, RNA sequencing analysis showed clustering of parental, labeled BM-PDXs and their corresponding cell line derivative. Intracardiac injection of dissociated cells from BM-E22-1, resulted in magnetic resonance imaging-detectable macrometastases in 4/8 (50%) and micrometastases (8/8) (100%) mice, suggesting that BM-PDXs remain capable of colonizing the brain at high frequencies. Brain metastases developed 8–12 weeks after ic injection, located to the brain parenchyma, grew around blood vessels, and elicited astroglia activation characteristic of breast cancer brain metastasis. These novel BM-PDXs represent heterogeneous and clinically relevant models to study mechanisms of brain metastatic colonization, with the added benefit of a slower progression rate that makes them suitable for preclinical testing of drugs in therapeutic settings. PMID:29164052

Physical models of collective cell motility: from cell to tissue

NASA Astrophysics Data System (ADS)

Camley, B. A.; Rappel, W.-J.

2017-03-01

In this article, we review physics-based models of collective cell motility. We discuss a range of techniques at different scales, ranging from models that represent cells as simple self-propelled particles to phase field models that can represent a cell’s shape and dynamics in great detail. We also extensively review the ways in which cells within a tissue choose their direction, the statistics of cell motion, and some simple examples of how cell-cell signaling can interact with collective cell motility. This review also covers in more detail selected recent works on collective cell motion of small numbers of cells on micropatterns, in wound healing, and the chemotaxis of clusters of cells.

The Quest for Targets Executing MYC-Dependent Cell Transformation.

PubMed

Hartl, Markus

2016-01-01

MYC represents a transcription factor with oncogenic potential converting multiple cellular signals into a broad transcriptional response, thereby controlling the expression of numerous protein-coding and non-coding RNAs important for cell proliferation, metabolism, differentiation, and apoptosis. Constitutive activation of MYC leads to neoplastic cell transformation, and deregulated MYC alleles are frequently observed in many human cancer cell types. Multiple approaches have been performed to isolate genes differentially expressed in cells containing aberrantly activated MYC proteins leading to the identification of thousands of putative targets. Functional analyses of genes differentially expressed in MYC-transformed cells had revealed that so far more than 40 upregulated or downregulated MYC targets are actively involved in cell transformation or tumorigenesis. However, further systematic and selective approaches are required for determination of the known or yet unidentified targets responsible for processing the oncogenic MYC program. The search for critical targets in MYC-dependent tumor cells is exacerbated by the fact that during tumor development, cancer cells progressively evolve in a multistep process, thereby acquiring their characteristic features in an additive manner. Functional expression cloning, combinatorial gene expression, and appropriate in vivo tests could represent adequate tools for dissecting the complex scenario of MYC-specified cell transformation. In this context, the central goal is to identify a minimal set of targets that suffices to phenocopy oncogenic MYC. Recently developed genomic editing tools could be employed to confirm the requirement of crucial transformation-associated targets. Knowledge about essential MYC-regulated genes is beneficial to expedite the development of specific inhibitors to interfere with growth and viability of human tumor cells in which MYC is aberrantly activated. Approaches based on the principle of synthetic lethality using MYC-overexpressing cancer cells and chemical or RNAi libraries have been employed to search for novel anticancer drugs, also leading to the identification of several druggable targets. Targeting oncogenic MYC effector genes instead of MYC may lead to compounds with higher specificities and less side effects. This class of drugs could also display a wider pharmaceutical window because physiological functions of MYC, which are important for normal cell growth, proliferation, and differentiation would be less impaired.

The Quest for Targets Executing MYC-Dependent Cell Transformation

PubMed Central

Hartl, Markus

2016-01-01

MYC represents a transcription factor with oncogenic potential converting multiple cellular signals into a broad transcriptional response, thereby controlling the expression of numerous protein-coding and non-coding RNAs important for cell proliferation, metabolism, differentiation, and apoptosis. Constitutive activation of MYC leads to neoplastic cell transformation, and deregulated MYC alleles are frequently observed in many human cancer cell types. Multiple approaches have been performed to isolate genes differentially expressed in cells containing aberrantly activated MYC proteins leading to the identification of thousands of putative targets. Functional analyses of genes differentially expressed in MYC-transformed cells had revealed that so far more than 40 upregulated or downregulated MYC targets are actively involved in cell transformation or tumorigenesis. However, further systematic and selective approaches are required for determination of the known or yet unidentified targets responsible for processing the oncogenic MYC program. The search for critical targets in MYC-dependent tumor cells is exacerbated by the fact that during tumor development, cancer cells progressively evolve in a multistep process, thereby acquiring their characteristic features in an additive manner. Functional expression cloning, combinatorial gene expression, and appropriate in vivo tests could represent adequate tools for dissecting the complex scenario of MYC-specified cell transformation. In this context, the central goal is to identify a minimal set of targets that suffices to phenocopy oncogenic MYC. Recently developed genomic editing tools could be employed to confirm the requirement of crucial transformation-associated targets. Knowledge about essential MYC-regulated genes is beneficial to expedite the development of specific inhibitors to interfere with growth and viability of human tumor cells in which MYC is aberrantly activated. Approaches based on the principle of synthetic lethality using MYC-overexpressing cancer cells and chemical or RNAi libraries have been employed to search for novel anticancer drugs, also leading to the identification of several druggable targets. Targeting oncogenic MYC effector genes instead of MYC may lead to compounds with higher specificities and less side effects. This class of drugs could also display a wider pharmaceutical window because physiological functions of MYC, which are important for normal cell growth, proliferation, and differentiation would be less impaired. PMID:27313991

A grid-doubling finite-element technique for calculating dynamic three-dimensional spontaneous rupture on an earthquake fault

USGS Publications Warehouse

Barall, Michael

2009-01-01

We present a new finite-element technique for calculating dynamic 3-D spontaneous rupture on an earthquake fault, which can reduce the required computational resources by a factor of six or more, without loss of accuracy. The grid-doubling technique employs small cells in a thin layer surrounding the fault. The remainder of the modelling volume is filled with larger cells, typically two or four times as large as the small cells. In the resulting non-conforming mesh, an interpolation method is used to join the thin layer of smaller cells to the volume of larger cells. Grid-doubling is effective because spontaneous rupture calculations typically require higher spatial resolution on and near the fault than elsewhere in the model volume. The technique can be applied to non-planar faults by morphing, or smoothly distorting, the entire mesh to produce the desired 3-D fault geometry. Using our FaultMod finite-element software, we have tested grid-doubling with both slip-weakening and rate-and-state friction laws, by running the SCEC/USGS 3-D dynamic rupture benchmark problems. We have also applied it to a model of the Hayward fault, Northern California, which uses realistic fault geometry and rock properties. FaultMod implements fault slip using common nodes, which represent motion common to both sides of the fault, and differential nodes, which represent motion of one side of the fault relative to the other side. We describe how to modify the traction-at-split-nodes method to work with common and differential nodes, using an implicit time stepping algorithm.

Integrative analysis for identification of shared markers from various functional cells/tissues for rheumatoid arthritis.

PubMed

Xia, Wei; Wu, Jian; Deng, Fei-Yan; Wu, Long-Fei; Zhang, Yong-Hong; Guo, Yu-Fan; Lei, Shu-Feng

2017-02-01

Rheumatoid arthritis (RA) is a systemic autoimmune disease. So far, it is unclear whether there exist common RA-related genes shared in different tissues/cells. In this study, we conducted an integrative analysis on multiple datasets to identify potential shared genes that are significant in multiple tissues/cells for RA. Seven microarray gene expression datasets representing various RA-related tissues/cells were downloaded from the Gene Expression Omnibus (GEO). Statistical analyses, testing both marginal and joint effects, were conducted to identify significant genes shared in various samples. Followed-up analyses were conducted on functional annotation clustering analysis, protein-protein interaction (PPI) analysis, gene-based association analysis, and ELISA validation analysis in in-house samples. We identified 18 shared significant genes, which were mainly involved in the immune response and chemokine signaling pathway. Among the 18 genes, eight genes (PPBP, PF4, HLA-F, S100A8, RNASEH2A, P2RY6, JAG2, and PCBP1) interact with known RA genes. Two genes (HLA-F and PCBP1) are significant in gene-based association analysis (P = 1.03E-31, P = 1.30E-2, respectively). Additionally, PCBP1 also showed differential protein expression levels in in-house case-control plasma samples (P = 2.60E-2). This study represented the first effort to identify shared RA markers from different functional cells or tissues. The results suggested that one of the shared genes, i.e., PCBP1, is a promising biomarker for RA.

Comparative anatomy, morphology, and molecular phylogenetics of the African genus Satanocrater (Acanthaceae).

PubMed

Tripp, Erin A; Fatimah, Siti

2012-06-01

Anatomical and morphological features of Satanocrater were studied to test hypotheses of xeric adaptations in the genus, which is endemic to arid tropical Africa. These features, together with molecular data, were used to test the phylogenetic placement of Satanocrater within the large plant family Acanthaceae. We undertook a comparative study of four species of Satanocrater. Carbon isotope ratios were generated to test a hypothesis of C(4) photosynthesis. Molecular data from chloroplast (trnG-trnS, trnG-trnR, psbA-trnH) and nuclear (Eif3E) loci were used to test the placement of Satanocrater within Acanthaceae. Anatomical features reflecting xeric adaptations of species of Satanocrater included a thick-walled epidermis, thick cuticle, abundant trichomes and glandular scales, stomata overarched by subsidiary cells, tightly packed mesophyll cells, and well-developed palisade parenchyma on both leaf surfaces. Although two species had enlarged bundle sheath cells, a feature often implicated in C(4) photosynthesis, isotope ratios indicated all species of Satanocrater use the C(3) pathway. Molecular data resolved Satanocrater within tribe Ruellieae with strong support. Within Ruellieae, our data suggest that pollen morphology of Satanocrater may represent an intermediate stage in a transition series. Anatomical and morphological features of Satanocrater reflect adaptation to xeric environments and add new information about the biology of xerophytes. Morphological and molecular data place Satanocrater in the tribe Ruellieae with confidence. This study adds to our capacity to test hypotheses of broad evolutionary and ecological interest in a diverse and important family of flowering plants.

Responses of human birch pollen allergen-reactive T cells to chemically modified allergens (allergoids).

PubMed

Dormann, D; Ebner, C; Jarman, E R; Montermann, E; Kraft, D; Reske-Kunz, A B

1998-11-01

Allergoids are widely used in specific immunotherapy for the treatment of IgE-mediated allergic diseases. The aim of this study was to analyse whether a modification of birch pollen allergens with formaldehyde affects the availability of T-cell epitopes. Efficient modification of the allergens was verified by determining IgE and IgG binding activity using ELISA inhibition tests. T-cell responses to birch pollen allergoids were analysed in polyclonal systems, using peripheral blood mononuclear cells (PBMC) of five birch pollen-allergic individuals, as well as birch pollen extract-reactive T-cell lines (TCL), established from the peripheral blood of 14 birch pollen-allergic donors. To determine whether the modification of natural (n)Bet v 1 with formaldehyde or maleic anhydride results in epitope-specific changes in T-cell reactivities, 22 Bet v 1-specific T-cell clones (TCC), established from nine additional birch pollen-allergic individuals, were tested for their reactivity with these products. The majority of PBMC and TCL showed a reduced response to the birch pollen extract allergoid. Bet v 1-specific TCC could be divided into allergoid-reactive and -non-reactive TCC. No simple correlation between possible modification sites of formaldehyde in the respective T-cell epitopes and the stimulatory potential of the allergoid was observed. Mechanisms of suppression or of anergy induction were excluded as an explanation for the non-reactivity of representative TCC. All TCC could be stimulated by maleylated and unmodified nBet v 1 to a similar extent. These results demonstrate differences in the availability of T-cell epitopes between allergoids and unmodified allergens, which are most likely due to structural changes within the allergen molecule.

Correlation of visual in vitro cytotoxicity ratings of biomaterials with quantitative in vitro cell viability measurements.

PubMed

Bhatia, Sujata K; Yetter, Ann B

2008-08-01

Medical devices and implanted biomaterials are often assessed for biological reactivity using visual scores of cell-material interactions. In such testing, biomaterials are assigned cytotoxicity ratings based on visual evidence of morphological cellular changes, including cell lysis, rounding, spreading, and proliferation. For example, ISO 10993 cytotoxicity testing of medical devices allows the use of a visual grading scale. The present study compared visual in vitro cytotoxicity ratings to quantitative in vitro cytotoxicity measurements for biomaterials to determine the level of correlation between visual scoring and a quantitative cell viability assay. Biomaterials representing a spectrum of biological reactivity levels were evaluated, including organo-tin polyvinylchloride (PVC; a known cytotoxic material), ultra-high molecular weight polyethylene (a known non-cytotoxic material), and implantable tissue adhesives. Each material was incubated in direct contact with mouse 3T3 fibroblast cell cultures for 24 h. Visual scores were assigned to the materials using a 5-point rating scale; the scorer was blinded to the material identities. Quantitative measurements of cell viability were performed using a 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay; again, the assay operator was blinded to material identities. The investigation revealed a high degree of correlation between visual cytotoxicity ratings and quantitative cell viability measurements; a Pearson's correlation gave a correlation coefficient of 0.90 between the visual cytotoxicity score and the percent viable cells. An equation relating the visual cytotoxicity score and the percent viable cells was derived. The results of this study are significant for the design and interpretation of in vitro cytotoxicity studies of novel biomaterials.

In vitro cytotoxicity of maxillofacial silicone elastomers: effect of accelerated aging.

PubMed

Bal, Bilge Turhan; Yilmaz, Handan; Aydin, Cemal; Karakoca, Seçil; Yilmaz, Sükran

2009-04-01

The purpose of this in vitro study was to evaluate the cytotoxicity of three maxillofacial silicone elastomers at 24, 48, and 72 h on L-929 cells and to determine the effect of accelerated aging on the cytotoxicity of these silicone elastomers. Disc-shaped test samples of maxillofacial silicone elastomers (Cosmesil, Episil, Multisil) were fabricated according to manufacturers' instructions under aseptic conditions. Samples were then divided into three groups: (1) not aged; (2) aged for 150 h with an accelerated weathering tester; and (3) aged for 300 h. Then the samples were placed in Dulbecco's Modified Eagle Medium/Ham's F12 (DMEM/F12) for 24, 48, and 72 h. After the incubation periods, cytotoxicity of the extracts to cultured fibroblasts (L-929) was measured by MTT assay. The degree of cytotoxicity of each sample was determined according to the reference value represented by the cells with a control (culture without sample). Statistical significance was determined by repeated measurement ANOVA (p < 0.01) followed by Duncan's test (p < 0.05). All test materials in each group demonstrated high survival rates in MTT assay (Episil; 93.84%, Multisil; 88.30%, Cosmesil; 87.50%, respectively); however, in all groups, Episil material demonstrated significantly higher cell survival rate after each of the experimental incubation periods (p < 0.05). Accelerated aging for 150 and 300 h had no significant effect on the biocompatibility of maxillofacial silicone elastomers tested (p > 0.05).

Engineering biological systems using automated biofoundries

PubMed Central

Chao, Ran; Mishra, Shekhar; Si, Tong; Zhao, Huimin

2017-01-01

Engineered biological systems such as genetic circuits and microbial cell factories have promised to solve many challenges in the modern society. However, the artisanal processes of research and development are slow, expensive, and inconsistent, representing a major obstacle in biotechnology and bioengineering. In recent years, biological foundries or biofoundries have been developed to automate design-build-test engineering cycles in an effort to accelerate these processes. This review summarizes the enabling technologies for such biofoundries as well as their early successes and remaining challenges. PMID:28602523

NANOS2 acts downstream of glial cell line-derived neurotrophic factor signaling to suppress differentiation of spermatogonial stem cells.

PubMed

Sada, Aiko; Hasegawa, Kazuteru; Pin, Pui Han; Saga, Yumiko

2012-02-01

Stem cells are maintained by both stem cell-extrinsic niche signals and stem cell-intrinsic factors. During murine spermatogenesis, glial cell line-derived neurotrophic factor (GDNF) signal emanated from Sertoli cells and germ cell-intrinsic factor NANOS2 represent key regulators for the maintenance of spermatogonial stem cells. However, it remains unclear how these factors intersect in stem cells to control their cellular state. Here, we show that GDNF signaling is essential to maintain NANOS2 expression, and overexpression of Nanos2 can alleviate the stem cell loss phenotype caused by the depletion of Gfra1, a receptor for GDNF. By using an inducible Cre-loxP system, we show that NANOS2 expression is downregulated upon the conditional knockout (cKO) of Gfra1, while ectopic expression of Nanos2 in GFRA1-negative spermatogonia does not induce de novo GFRA1 expression. Furthermore, overexpression of Nanos2 in the Gfra1-cKO testes prevents precocious differentiation of the Gfra1-knockout stem cells and partially rescues the stem cell loss phenotypes of Gfra1-deficient mice, indicating that the stem cell differentiation can be suppressed by NANOS2 even in the absence of GDNF signaling. Taken together, we suggest that NANOS2 acts downstream of GDNF signaling to maintain undifferentiated state of spermatogonial stem cells. Copyright © 2011 AlphaMed Press.

Decreasing Enterobacter sakazakii (Cronobacter spp.) food contamination level with bacteriophages: prospects and problems

PubMed Central

Zuber, Sophie; Boissin‐Delaporte, Catherine; Michot, Lise; Iversen, Carol; Diep, Benjamin; Brüssow, Harald; Breeuwer, Pieter

2008-01-01

Summary Enterobacter sakazakii (Cronobacter spp.) is an opportunistic pathogen, which can cause rare, but life‐threatening infections in neonates and infants through feeding of a contaminated milk formula. We isolated 67 phages from environmental samples and tested their lytic host range on a representative collection of 40 E. sakazakii strains. A cocktail of five phages prevented the outgrowth of 35 out of 40 test strains in artificially contaminated infant formula. Two E. sakazakii phages represented prolate head Myoviridae. Molecular tests identified them as close relatives of Escherichia coli phage T4. The remaining three phages represented isometric head Myoviridae with large genome size of 140 and 200 kb, respectively, which belonged to two different DNA hybridization groups. A high dose of 108 pfu ml−1 of phage could effectively sterilize a broth contaminated with both high and low pathogen counts (106 and 102 cfu ml−1). In contrast, broth inoculated with 104 phage and 102 bacteria per ml first showed normal bacterial growth until reaching a cell titre of 105 cfu ml−1. Only when crossing this threshold, phage replication started, but it could not reduce the contamination level below 100 cfu ml−1. Phages could be produced with titres of 1010 pfu ml−1 in broth culture, but they were not stable upon freeze‐drying. Addition of trehalose or milk formula stabilized the phage preparation, which then showed excellent storage stability even at elevated temperature. PMID:21261874

The Development and Validation of an In Vitro Airway Model to Assess Realistic Airway Deposition and Drug Permeation Behavior of Orally Inhaled Products Across Synthetic Membranes.

PubMed

Huynh, Bao K; Traini, Daniela; Farkas, Dale R; Longest, P Worth; Hindle, Michael; Young, Paul M

2018-04-01

Current in vitro approaches to assess lung deposition, dissolution, and cellular transport behavior of orally inhaled products (OIPs) have relied on compendial impactors to collect drug particles that are likely to deposit in the airway; however, the main drawback with this approach is that these impactors do not reflect the airway and may not necessarily represent drug deposition behavior in vivo. The aim of this article is to describe the development and method validation of a novel hybrid in vitro approach to assess drug deposition and permeation behavior in a more representative airway model. The medium-sized Virginia Commonwealth University (VCU) mouth-throat (MT) and tracheal-bronchial (TB) realistic upper airway models were used in this study as representative models of the upper airway. The TB model was modified to accommodate two Snapwell ® inserts above the first TB airway bifurcation region to collect deposited nebulized ciprofloxacin-hydrochloride (CIP-HCL) droplets as a model drug aerosol system. Permeation characteristics of deposited nebulized CIP-HCL droplets were assessed across different synthetic membranes using the Snapwell test system. The Snapwell test system demonstrated reproducible and discriminatory drug permeation profiles for already dissolved and nebulized CIP-HCL droplets through a range of synthetic permeable membranes under different test conditions. The rate and extent of drug permeation depended on the permeable membrane material used, presence of a stirrer in the receptor compartment, and, most importantly, the drug collection method. This novel hybrid in vitro approach, which incorporates a modified version of a realistic upper airway model, coupled with the Snapwell test system holds great potential to evaluate postairway deposition characteristics, such as drug permeation and particle dissolution behavior of OIPs. Future studies will expand this approach using a cell culture-based setup instead of synthetic membranes, within a humidified chamber, to assess airway epithelia transport behavior in a more representative manner.

Evaluation of Radioresponse and Radiosensitizers in Glioblastoma Organotypic Cultures.

PubMed

Bayin, N Sumru; Ma, Lin; Placantonakis, Dimitris G; Barcellos-Hoff, Mary Helen

2018-01-01

Glioblastoma (GBM), a deadly primary brain malignancy, manifests pronounced radioresistance. Identifying agents that improve the sensitivity of tumor tissue to radiotherapy is critical for improving patient outcomes. The response to ionizing radiation is regulated by both cell-intrinsic and -extrinsic mechanisms. In particular, the tumor microenvironment is known to promote radioresistance in GBM. Therefore, model systems used to test radiosensitizing agents need to take into account the tumor microenvironment. We recently showed that GBM explant cultures represent an adaptable ex vivo platform for rapid and personalized testing of radiosensitizers. These explants preserve the cellular composition and tissue architecture of parental patient tumors and therefore capture the microenvironmental context that critically determines the response to radiotherapy. This chapter focuses on the detailed protocol for testing candidate radiosensitizing agents in GBM explants.

High-resolution imaging of basal cell carcinoma: a comparison between multiphoton microscopy with fluorescence lifetime imaging and reflectance confocal microscopy.

PubMed

Manfredini, Marco; Arginelli, Federica; Dunsby, Christopher; French, Paul; Talbot, Clifford; König, Karsten; Pellacani, Giovanni; Ponti, Giovanni; Seidenari, Stefania

2013-02-01

The aim of this study was to compare morphological aspects of basal cell carcinoma (BCC) as assessed by two different imaging methods: in vivo reflectance confocal microscopy (RCM) and multiphoton tomography with fluorescence lifetime imaging implementation (MPT-FLIM). The study comprised 16 BCCs for which a complete set of RCM and MPT-FLIM images were available. The presence of seven MPT-FLIM descriptors was evaluated. The presence of seven RCM equivalent parameters was scored in accordance to their extension. Chi-squared test with Fisher's exact test and Spearman's rank correlation coefficient were determined between MPT-FLIM scores and adjusted-RCM scores. MPT-FLIM and RCM descriptors of BCC were coupled to match the descriptors that define the same pathological structures. The comparison included: Streaming and Aligned elongated cells, Streaming with multiple directions and Double alignment, Palisading (RCM) and Palisading (MPT-FLIM), Typical tumor islands, and Cell islands surrounded by fibers, Dark silhouettes and Phantom islands, Plump bright cells and Melanophages, Vessels (RCM), and Vessels (MPT-FLIM). The parameters that were significantly correlated were Melanophages/Plump Bright Cells, Aligned elongated cells/Streaming, Double alignment/Streaming with multiple directions, and Palisading (MPT-FLIM)/Palisading (RCM). According to our data, both methods are suitable to image BCC's features. The concordance between MPT-FLIM and RCM is high, with some limitations due to the technical differences between the two devices. The hardest difficulty when comparing the images generated by the two imaging modalities is represented by their different field of view. © 2012 John Wiley & Sons A/S.

Davallialactone reduces inflammation and repairs dentinogenesis on glucose oxidase-induced stress in dental pulp cells.

PubMed

Lee, Young-Hee; Kim, Go-Eun; Song, Yong-Beom; Paudel, Usha; Lee, Nan-Hee; Yun, Bong-Sik; Yu, Mi-Kyung; Yi, Ho-Keun

2013-11-01

The chronic nature of diabetes mellitus (DM) raises the risk of oral complication diseases. In general, DM causes oxidative stress to organs. This study aimed to evaluate the cellular change of dental pulp cells against glucose oxidative stress by glucose oxidase with a high glucose state. The purpose of this study was to test the antioxidant character of davallialactone and to reduce the pathogenesis of dental pulp cells against glucose oxidative stress. The glucose oxidase with a high glucose concentration was tested for hydroxy peroxide (H2O2) production, cellular toxicity, reactive oxygen species (ROS) formation, induction of inflammatory molecules and disturbance of dentin mineralization in human dental pulp cells. The anti-oxidant effect of Davallilactone was investigated to restore dental pulp cells' vitality and dentin mineralization via reduction of H2O2 production, cellular toxicity, ROS formation and inflammatory molecules. The treatment of glucose oxidase with a high glucose concentration increased H2O2 production, cellular toxicity, and inflammatory molecules and disturbed dentin mineralization by reducing pulp cell activity. However, davallialactone reduced H2O2 production, cellular toxicity, ROS formation, inflammatory molecules, and dentin mineralization disturbances even with a long-term glucose oxidative stress state. The results of this study imply that the development of oral complications is related to the irreversible damage of dental pulp cells by DM-induced oxidative stress. Davallialactone, a natural antioxidant, may be useful to treat complicated oral disease, representing an improvement for pulp vital therapy. Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Implementing vertex dynamics models of cell populations in biology within a consistent computational framework.

PubMed

Fletcher, Alexander G; Osborne, James M; Maini, Philip K; Gavaghan, David J

2013-11-01

The dynamic behaviour of epithelial cell sheets plays a central role during development, growth, disease and wound healing. These processes occur as a result of cell adhesion, migration, division, differentiation and death, and involve multiple processes acting at the cellular and molecular level. Computational models offer a useful means by which to investigate and test hypotheses about these processes, and have played a key role in the study of cell-cell interactions. However, the necessarily complex nature of such models means that it is difficult to make accurate comparison between different models, since it is often impossible to distinguish between differences in behaviour that are due to the underlying model assumptions, and those due to differences in the in silico implementation of the model. In this work, an approach is described for the implementation of vertex dynamics models, a discrete approach that represents each cell by a polygon (or polyhedron) whose vertices may move in response to forces. The implementation is undertaken in a consistent manner within a single open source computational framework, Chaste, which comprises fully tested, industrial-grade software that has been developed using an agile approach. This framework allows one to easily change assumptions regarding force generation and cell rearrangement processes within these models. The versatility and generality of this framework is illustrated using a number of biological examples. In each case we provide full details of all technical aspects of our model implementations, and in some cases provide extensions to make the models more generally applicable. Copyright © 2013 Elsevier Ltd. All rights reserved.

Centroacinar Cells Are Progenitors That Contribute to Endocrine Pancreas Regeneration

PubMed Central

Delaspre, Fabien; Beer, Rebecca L.; Rovira, Meritxell; Huang, Wei; Wang, Guangliang; Gee, Stephen; Vitery, Maria del Carmen; Wheelan, Sarah J.

2015-01-01

Diabetes is associated with a paucity of insulin-producing β-cells. With the goal of finding therapeutic routes to treat diabetes, we aim to find molecular and cellular mechanisms involved in β-cell neogenesis and regeneration. To facilitate discovery of such mechanisms, we use a vertebrate organism where pancreatic cells readily regenerate. The larval zebrafish pancreas contains Notch-responsive progenitors that during development give rise to adult ductal, endocrine, and centroacinar cells (CACs). Adult CACs are also Notch responsive and are morphologically similar to their larval predecessors. To test our hypothesis that adult CACs are also progenitors, we took two complementary approaches: 1) We established the transcriptome for adult CACs. Using gene ontology, transgenic lines, and in situ hybridization, we found that the CAC transcriptome is enriched for progenitor markers. 2) Using lineage tracing, we demonstrated that CACs do form new endocrine cells after β-cell ablation or partial pancreatectomy. We concluded that CACs and their larval predecessors are the same cell type and represent an opportune model to study both β-cell neogenesis and β-cell regeneration. Furthermore, we show that in cftr loss-of-function mutants, there is a deficiency of larval CACs, providing a possible explanation for pancreatic complications associated with cystic fibrosis. PMID:26153247

Small Molecule Inhibition of cAMP Response Element Binding Protein in Human Acute Myeloid Leukemia Cells

PubMed Central

Mitton, Bryan; Chae, Hee-Don; Hsu, Katie; Dutta, Ritika; Aldana-Masangkay, Grace; Ferrari, Roberto; Davis, Kara; Tiu, Bruce C.; Kaul, Arya; Lacayo, Norman; Dahl, Gary; Xie, Fuchun; Li, Bingbing X.; Breese, Marcus R.; Landaw, Elliot M.; Nolan, Garry; Pellegrini, Matteo; Romanov, Sergei; Xiao, Xiangshu; Sakamoto, Kathleen M.

2016-01-01

The transcription factor CREB (cAMP Response Element Binding Protein) is overexpressed in the majority of acute myeloid leukemia (AML) patients, and this is associated with a worse prognosis. Previous work revealed that CREB overexpression augmented AML cell growth, while CREB knockdown disrupted key AML cell functions in vitro. In contrast, CREB knockdown had no effect on long-term hematopoietic stem cell activity in mouse transduction/transplantation assays. Together, these studies position CREB as a promising drug target for AML. To test this concept, a small molecule inhibitor of CREB, XX-650-23, was developed. This molecule blocks a critical interaction between CREB and its required co-activator CBP (CREB Binding Protein), leading to disruption of CREB-driven gene expression. Inhibition of CBP-CREB interaction induced apoptosis and cell cycle arrest in AML cells, and prolonged survival in vivo in mice injected with human AML cells. XX-650-23 had little toxicity on normal human hematopoietic cells and tissues in mice. To understand the mechanism of XX-650-23, we performed RNA-seq, ChIP-seq and Cytometry Time of Flight with human AML cells. Our results demonstrate that small molecule inhibition of CBP-CREB interaction mostly affects apoptotic, cell cycle, and survival pathways, which may represent a novel approach for AML therapy. PMID:27211267

Characterization of multi-drug tolerant persister cells in Streptococcus suis

PubMed Central

2014-01-01

Background Persister cells constitute a subpopulation of dormant cells within a microbial population which are genetically identical but phenotypically different to regular cells. Notably, persister cells show an elevated tolerance to antimicrobial agents. Thus, they are considered to represent a microbial ‘bet-hedging’ strategy and are of particular importance in pathogenic bacteria. Results We studied the ability of the zoonotic pathogen Streptococcus (S.) suis to form multi-drug tolerant variants and identified persister cells dependent on the initial bacterial growth phase. We observed lower numbers of persisters in exponential phase cultures than in stationary growth phase populations. S. suis persister cells showed a high tolerance to a variety of antibiotics, and the phenotype was not inherited as tested with four passages of S. suis populations. Furthermore, we provide evidence that the persister phenotype is related to expression of genes involved in general metabolic pathways since we found higher numbers of persister cells in a mutant strain defective in the catabolic arginine deiminase system as compared to its parental wild type strain. Finally, we observed persister cell formation also in other S. suis strains and pathogenic streptococcal species. Conclusions Taken together, this is the first study that reports multi-drug tolerant persister cells in the zoonotic pathogen S. suis. PMID:24885389

T cell virological synapses and HIV-1 pathogenesis.

PubMed

Chen, Benjamin K

2012-12-01

Human immunodeficiency virus type 1 is the cause of a modern global pandemic associated with progressive acquired immune deficiency. The infection is characterized by the loss of the primary target of viral infection, the CD4+ T cell. The measurement of plasma viremia in patients can predict the rate of CD4+ cell decline; however, it is not clear whether this cell-free plasma virus represents the engine that drives viral spread. Active viral replication is mainly observed within lymphoid tissues that are hotbeds of cell-cell interactions that initiate and organize immune responses. It is well established that cell-cell interactions enhance viral spread in vitro. Dendritic cell-T cell interactions, which lie at the heart of adaptive immune responses, enhance viral infection in vitro. Interactions between infected and uninfected CD4+ T cells are a dominant route of viral spread in vitro and are likely to play a central role in viral dissemination in vivo. Future studies will test existing paradigms of HIV-1 dissemination to determine whether virus-transmitting contacts between infected and uninfected T cells called virological synapses are the dominant mode of viral spread in vivo. Here, we review the status of our understanding of this mode of infection with a focus on T cell-T cell interactions and examine how it may explain resistance to neutralizing antibodies and or the generation of genetic diversity of HIV.

The Cell Cycle: An Activity Using Paper Plates to Represent Time Spent in Phases of the Cell Cycle

ERIC Educational Resources Information Center

Scherer, Yvette D.

2014-01-01

In this activity, students are given the opportunity to combine skills in math and geometry for a biology lesson in the cell cycle. Students utilize the data they collect and analyze from an online onion-root-tip activity to create a paper-plate time clock representing a 24-hour cell cycle. By dividing the paper plate into appropriate phases of…

Exceptionally well-preserved Cretaceous microfossils reveal new biomineralization styles.

PubMed

Wendler, Jens E; Bown, Paul

2013-01-01

Calcareous microplankton shells form the dominant components of ancient and modern pelagic sea-floor carbonates and are widely used in palaeoenvironmental reconstructions. The efficacy of these applications, however, is dependent upon minimal geochemical alteration during diagenesis, but these modifying processes are poorly understood. Here we report on new biomineralization architectures of previously unsuspected complexity in calcareous cell-wall coverings of extinct dinoflagellates (pithonellids) from a Tanzanian microfossil-lagerstätte. These Cretaceous 'calcispheres' have previously been considered biomineralogically unremarkable but our new observations show that the true nature of these tests has been masked by recrystallization. The pristine Tanzanian fossils are formed from fibre-like crystallites and show archeopyles and exquisitely constructed opercula, demonstrating the dinoflagellate affinity of pithonellids, which has long been uncertain. The interwoven fibre-like structures provide strength and flexibility enhancing the protective function of these tests. The low-density wall fabrics may represent specific adaptation for oceanic encystment life cycles, preventing the cells from rapid sinking.

Modeling and visualizing cell type switching.

PubMed

Ghaffarizadeh, Ahmadreza; Podgorski, Gregory J; Flann, Nicholas S

2014-01-01

Understanding cellular differentiation is critical in explaining development and for taming diseases such as cancer. Differentiation is conventionally represented using bifurcating lineage trees. However, these lineage trees cannot readily capture or quantify all the types of transitions now known to occur between cell types, including transdifferentiation or differentiation off standard paths. This work introduces a new analysis and visualization technique that is capable of representing all possible transitions between cell states compactly, quantitatively, and intuitively. This method considers the regulatory network of transcription factors that control cell type determination and then performs an analysis of network dynamics to identify stable expression profiles and the potential cell types that they represent. A visualization tool called CellDiff3D creates an intuitive three-dimensional graph that shows the overall direction and probability of transitions between all pairs of cell types within a lineage. In this study, the influence of gene expression noise and mutational changes during myeloid cell differentiation are presented as a demonstration of the CellDiff3D technique, a new approach to quantify and envision all possible cell state transitions in any lineage network.

Performance comparison of tin oxide anodes to commercially available dimensionally stable anodes.

PubMed

Watts, Richard J; Finn, Dennis D; Wyeth, Megan S; Teel, Amy L

2008-06-01

Dimensionally stable anodes (DSAs) demonstrate potential for the electrochemical treatment of industrial waste streams and disinfection of effluent. Oxidation by laboratory-prepared tin oxide DSAs was compared with that of commercially available ruthenium oxide, iridium oxide, and mixed metal oxide DSAs, using hexanol as a probe molecule. The performance of the four anodes was similar in two-chamber reactors, in which the anode cell was separated from the cathode cell by a Nafion membrane, which allows transmission of current between the chambers, but not passage of chemical constituents. The anodes were then evaluated in single-cell reactors, which are more representative of potential treatment and disinfection applications. However, in the single-cell reactors, the tin oxide anodes were significantly more effective at oxidation and generated higher quality cyclic voltammograms than the other DSAs. These results suggest that tin oxide anodes have greater potential than the three commercially available DSAs tested for industrial waste stream treatment and effluent disinfection.

STUDIES ON THE PATHOGENESIS OF FEVER

PubMed Central

Kaiser, Hans Klaus; Wood, W. Barry

1962-01-01

Determination of the dose-response curve for rabbit leucocytic pyrogen reveals a hyperthermic "ceiling" at which there is a marked insensitivity to dosage. This finding has important implications in relation to the quantitative assay of leucocytic pyrogen. Polymorphonuclear leucocytes separated from normal rabbit blood possess the capacity to produce less than 5 per cent of the pyrogen generated by the same number of rabbit granulocytes collected from acute peritoneal exudates. Blood granulocytes, separated in the cold from the buffy coat, contain no detectable preformed pyrogen. The amount of preformed pyrogen within exudate granulocytes represents but a small fraction of the pyrogen which the cells are capable of generating when incubated in normal saline at 37°C. It is suggested that the active pyrogen is formed from an inactive precursor within the cells. Under the conditions tested, cell fragments of rabbit granulocytes fail to produce endogenous pyrogen. The fact that the production of pyrogen is blocked at 4°C is in keeping with the hypothesis that it involves metabolic reactions within the cell. PMID:14453159

Analysis of 12 AH aerospace nickel-cadmium cells from the design variable program

NASA Technical Reports Server (NTRS)

Vasanth, Kunigahalli L.; Morrow, George

1987-01-01

The Design Variable Program of NASA/GSFC provided a systematic approach to evaluate the performance of 12 Ampere-Hour Nickel-Cadmium cells of different designs. Design Variables tested in this program included teflonated negative plates, silver treated negative plates, lightly loaded negative plates, positive plates with no cadmium treatment, plate design of 1968 utilizing old and new processing techniques and electrochemically impregnated positive plates. These cells were life cycled in a Low-Earth Orbit (LEO) regime for 3 to 4 years. Representative cells taken from the Design Variable Program were examined via chemical, electrochemical and surface analyses. The results indicate the following: (1) positive swelling and carbonate content in the electrolyte increase as a function of number of cycles; (2) electrolyte distribution follows a general order NEG greater than POS greater than SEP; (3) control and No PQ groups outperformed the rest of the groups; and (4) the polyproylene group exhibited heavy cadmium migration and poor performance.

Can the Stark-Einstein law resolve the measurement problem from an animate perspective?

PubMed

Thaheld, Fred H

2015-09-01

Analysis of the Stark-Einstein law as it applies to the retinal molecule, which is part of the rhodopsin molecule within the rod cells of the retina, reveals that it may provide the solution to the measurement problem from an animate perspective. That it represents a natural boundary where the Schrödinger equation or wave function automatically goes from linear to nonlinear while remaining in a deterministic state. It will be possible in the near future to subject this theory to empirical tests as has been previously proposed. This analysis provides a contrast to the many decades well studied and debated inanimate measurement problem and would represent an addition to the Stark-Einstein law involving information carried by the photon. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Cell-Based Sensor System Using L6 Cells for Broad Band Continuous Pollutant Monitoring in Aquatic Environments

PubMed Central

Kubisch, Rebekka; Bohrn, Ulrich; Fleischer, Maximilian; Stütz, Evamaria

2012-01-01

Pollution of drinking water sources represents a continuously emerging problem in global environmental protection. Novel techniques for real-time monitoring of water quality, capable of the detection of unanticipated toxic and bioactive substances, are urgently needed. In this study, the applicability of a cell-based sensor system using selected eukaryotic cell lines for the detection of aquatic pollutants is shown. Readout parameters of the cells were the acidification (metabolism), oxygen consumption (respiration) and impedance (morphology) of the cells. A variety of potential cytotoxic classes of substances (heavy metals, pharmaceuticals, neurotoxins, waste water) was tested with monolayers of L6 cells (rat myoblasts). The cytotoxicity or cellular effects induced by inorganic ions (Ni2+ and Cu2+) can be detected with the metabolic parameters acidification and respiration down to 0.5 mg/L, whereas the detection limit for other substances like nicotine and acetaminophen are rather high, in the range of 0.1 mg/L and 100 mg/L. In a close to application model a real waste water sample shows detectable signals, indicating the existence of cytotoxic substances. The results support the paradigm change from single substance detection to the monitoring of overall toxicity. PMID:22737014

Cell-based sensor system using L6 cells for broad band continuous pollutant monitoring in aquatic environments.

PubMed

Kubisch, Rebekka; Bohrn, Ulrich; Fleischer, Maximilian; Stütz, Evamaria

2012-01-01

Pollution of drinking water sources represents a continuously emerging problem in global environmental protection. Novel techniques for real-time monitoring of water quality, capable of the detection of unanticipated toxic and bioactive substances, are urgently needed. In this study, the applicability of a cell-based sensor system using selected eukaryotic cell lines for the detection of aquatic pollutants is shown. Readout parameters of the cells were the acidification (metabolism), oxygen consumption (respiration) and impedance (morphology) of the cells. A variety of potential cytotoxic classes of substances (heavy metals, pharmaceuticals, neurotoxins, waste water) was tested with monolayers of L6 cells (rat myoblasts). The cytotoxicity or cellular effects induced by inorganic ions (Ni(2+) and Cu(2+)) can be detected with the metabolic parameters acidification and respiration down to 0.5 mg/L, whereas the detection limit for other substances like nicotine and acetaminophen are rather high, in the range of 0.1 mg/L and 100 mg/L. In a close to application model a real waste water sample shows detectable signals, indicating the existence of cytotoxic substances. The results support the paradigm change from single substance detection to the monitoring of overall toxicity.

Advances in cell-free protein array methods.

PubMed

Yu, Xiaobo; Petritis, Brianne; Duan, Hu; Xu, Danke; LaBaer, Joshua

2018-01-01

Cell-free protein microarrays represent a special form of protein microarray which display proteins made fresh at the time of the experiment, avoiding storage and denaturation. They have been used increasingly in basic and translational research over the past decade to study protein-protein interactions, the pathogen-host relationship, post-translational modifications, and antibody biomarkers of different human diseases. Their role in the first blood-based diagnostic test for early stage breast cancer highlights their value in managing human health. Cell-free protein microarrays will continue to evolve to become widespread tools for research and clinical management. Areas covered: We review the advantages and disadvantages of different cell-free protein arrays, with an emphasis on the methods that have been studied in the last five years. We also discuss the applications of each microarray method. Expert commentary: Given the growing roles and impact of cell-free protein microarrays in research and medicine, we discuss: 1) the current technical and practical limitations of cell-free protein microarrays; 2) the biomarker discovery and verification pipeline using protein microarrays; and 3) how cell-free protein microarrays will advance over the next five years, both in their technology and applications.

In vitro membrane protein synthesis inside Sec translocon-reconstituted cell-sized liposomes

PubMed Central

Ohta, Naoki; Kato, Yasuhiko; Watanabe, Hajime; Mori, Hirotada; Matsuura, Tomoaki

2016-01-01

Protein synthesis using an in vitro transcription-translation system (IVTT) inside cell-sized liposomes has become a valuable tool to study the properties of biological systems under cell-mimicking conditions. However, previous liposome systems lacked the machinery for membrane protein translocation. Here, we reconstituted the translocon consisting of SecYEG from Escherichia coli inside cell-sized liposomes. The cell-sized liposomes also carry the reconstituted IVTT, thereby providing a cell-mimicking environment for membrane protein synthesis. By using EmrE, a multidrug transporter from E. coli, as a model membrane protein, we found that both the amount and activity of EmrE synthesized inside the liposome is increased approximately three-fold by incorporating the Sec translocon. The topological change of EmrE induced by the translocon was also identified. The membrane integration of 6 out of 9 E. coli inner membrane proteins that was tested was increased by incorporation of the translocon. By introducing the Sec translocon, the membrane integration efficiency of the membrane protein of interest was increased, and enabled the integration of membrane proteins that otherwise cannot be inserted. In addition, this work represents an essential step toward the construction of an artificial cell through a bottom-up approach. PMID:27808179

Morphological description of limbal epithelium: searching for stem cells crypts in the dog, cat, pig, cow, sheep and horse.

PubMed

Patruno, M; Perazzi, A; Martinello, T; Blaseotto, A; Di Iorio, E; Iacopetti, I

2017-06-01

The cornea provides protection and transparency to the eye, allowing an optimal sharpness view. In some pathological conditions the cornea is able to regenerate thanks to the presence of a stem cells reservoir present at the level of the transition area between cornea and sclera (limbus). Corneal cell therapies in Veterinary Medicine are really limited due to the lacking of knowledge about the anatomy of the limbal area, the putative presence of stem cells and their identification in domestic species. The aim of this study was to provide an overview of the main distinctive structural features of the sclero-corneal junction and conjunctival-corneal junction areas in some species of veterinary importance, using optic microscope observations of histological sections. The resulting data were compared with cornea from humans adapting protocols already used to identify stem cells by means of a specific cellular marker. We tested the expression of ΔNp63α isoform in the cornea basal cells, trying to correlate the distribution profile with areas of highly proliferative turnover. The results obtained from this study represent a first step towards the identification of a corneal stem cells reservoir in different animals.

High Efficient Differentiation of Functional Hepatocytes from Porcine Induced Pluripotent Stem Cells

PubMed Central

Ao, Ying; Mich-Basso, Jocelyn Danielle; Lin, Bo; Yang, Lei

2014-01-01

Hepatocyte transplantation is considered to be a promising therapy for patients with liver diseases. Induced pluripotent stem cells (iPSCs) provide an unlimited source for the generation of functional hepatocytes. In this study, we generated iPSCs from porcine ear fibroblasts (PEFs) by overexpressing Sox2, Klf4, Oct4, and c-Myc (SKOM), and developed a novel strategy for the efficient differentiation of hepatocyte-like cells from porcine iPSCs by following the processes of early liver development. The differentiated cells displayed the phenotypes of hepatocytes, exhibited classic hepatocyte-associated bio-functions, such as LDL uptake, glycogen storage and urea secretion, as well as possessed the metabolic activities of cytochrome P-450 (CYP) 3A and 2C. Furthermore, we compared the hepatocyte differentiation efficacy of our protocol with another published method, and the results demonstrated that our differentiation strategy could significantly improve the generation of morphological and functional hepatocyte-like cells from porcine iPSCs. In conclusion, this study establishes an efficient method for in vitro generation of functional hepatocytes from porcine iPSCs, which could represent a promising cell source for preclinical testing of cell-based therapeutics for liver failure and for pharmacological applications. PMID:24949734

Cardiomyocyte H9c2 cells present a valuable alternative to fish lethal testing for azoxystrobin.

PubMed

Rodrigues, Elsa T; Pardal, Miguel Â; Laizé, Vincent; Cancela, M Leonor; Oliveira, Paulo J; Serafim, Teresa L

2015-11-01

The present study aims at identifying, among six mammalian and fish cell lines, a sensitive cell line whose in vitro median inhibitory concentration (IC50) better matches the in vivo short-term Sparus aurata median lethal concentration (LC50). IC50s and LC50 were assessed after exposure to the widely used fungicide azoxystrobin (AZX). Statistical results were relevant for most cell lines after 48 h of AZX exposure, being H9c2 the most sensitive cells, as well as the ones which provided the best prediction of fish toxicity, with a LC50,96h/IC50,48h = 0.581. H9c2 cell proliferation upon 72 h of AZX exposure revealed a LC50,96h/IC50,72h = 0.998. Therefore, identical absolute sensitivities were attained for both in vitro and in vivo assays. To conclude, the H9c2 cell-based assay is reliable and represents a suitable ethical alternative to conventional fish assays for AZX, and could be used to get valuable insights into the toxic effects of other pesticides. Copyright © 2015 Elsevier Ltd. All rights reserved.

STUDIES ON THE IMMUNE RESPONSE TO A CHARACTERIZED ANTIGENIC DETERMINANT OF THE TOBACCO MOSAIC VIRUS PROTEIN

PubMed Central

Spitler, Lynn; Benjamini, E.; Young, Janis D.; Kaplan, Harvey; Fudenberg, H. H.

1970-01-01

The following peptides have previously been shown to bind specifically with antibodies to TMVP: (a) An eicosapeptide representing residues 93–112 of TMVP and having the sequence Ileu-Ileu-Glu-Val-Glu-AspNH2-GluNH2-Ala-AspNH2-Pro-Thr-Thr-Ala-Glu-Thr-Leu-Asp-Ala-Thr-Arg. (b) Its C-terminal decapeptide. (c) Its C-terminal pentapeptide. (d) N-octanoyl-C-terminal-tripeptide. (e) (Lys)4-C-terminal-pentapeptide. (f) (Lys)7 C-terminal-pentapeptide. The present communication deals with the investigation of several parameters of the immunological activity of the peptides. The results show that none of the peptides tested were immunogenic in guinea pigs, nor did they stimulate the incorporation of 14C-thymidine by spleen cells derived from TMVP-primed animals. Results also showed that all of the peptides tested could elicit specific delayed and immediate skin reactions in TMVP-sensitized guinea pigs, and furthermore, that the peptides could specifically inhibit the migration of peritoneal exudate cells derived from these animals. The elicitation of delayed skin reactions and the ability to inhibit migration of peritoneal exudate cells were independent of carrier specificity. PMID:5409944

Characterization of Time-Dependent Behavior of Ramming Paste Used in an Aluminum Electrolysis Cell

NASA Astrophysics Data System (ADS)

Orangi, Sakineh; Picard, Donald; Alamdari, Houshang; Ziegler, Donald; Fafard, Mario

2015-12-01

A new methodology was proposed for the characterization of time-dependent behavior of materials in order to develop a constitutive model. The material used for the characterization was ramming paste, a porous material used in an aluminum electrolysis cell, which is baked in place under varying loads induced by the thermal expansion of other components of the cell. In order to develop a constitutive model representing the paste mechanical behavior, it was necessary to get some insight into its behavior using samples which had been baked at different temperatures ranging from 200 to 1000 °C. Creep stages, effect of testing temperature on the creep, creep-recovery, as well as nonlinear creep were observed for designing a constitutive law. Uniaxial creep-recovery tests were carried out at two temperatures on the baked paste: ambient and higher. Results showed that the shape of creep curves was similar to a typical creep; recovery happened and the creep was shown to be nonlinear. Those experimental observations and the identification of nonlinear parameters of developed constitutive model demonstrated that the baked paste experiences nonlinear viscoelastic-viscoplastic behavior at different temperatures.

Microinjection of cytoplasm as a test of complementation in Paramecium

PubMed Central

1982-01-01

Mutants in Paramecium tetraurelia, unable to generate action potentials, have been isolated as cells which show no backward swimming in response to ionic stimulation. These "pawn" mutants belong to at least three complementation groups designated pwA, pwB, and pwC. We have found that microinjection of cytoplasm from a wild-type donor into a pawn recipient of any of the three complementation groups restores the ability of the pawn to generate action potentials and hence swim backward. In addition, the cytoplasm from a pawn cannot restore a recipient of the same complementation group, but that from a pawn of a different group can. Electrophysiological analysis had demonstrated that the restoration of backward swimming is not due to a simple addition of ions but represents a profound change in the excitable membrane of the recipient pawn cells. Using known pawn mutants and those which had previously been unclassified, we have been able to establish a perfect concordance of genetic complementation and complementation by cytoplasmic transfer through microinjection. This method has been used to classify pawn mutants that are sterile or hard- to-mate and to examine the ability of cytoplasms from different species of ciliated protozoa to restore the ability to swim backward in the pawn mutants of P. tetraurelia. A cell homogenate has also been fractionated by centrifugation to further purify the active components. These results demonstrate that transfer of cytoplasm between cells by microinjection can be a valid and systematic method to classify mutants. This test is simpler to perform than the genetic complementation test and can be used under favorable conditions in mutants that are sterile and in cells of different species. PMID:7061597

Enhancement of HIV-1 VLP production using gene inhibition strategies.

PubMed

Fuenmayor, Javier; Cervera, Laura; Rigau, Cristina; Gòdia, Francesc

2018-05-01

Gag polyprotein from HIV-1 is able to generate virus-like particles (VLPs) when recombinantly expressed in animal cell platforms. HIV-1 VLP production in HEK293 cells can be improved by the use of different strategies for increasing product titers. One of them is the so-called extended gene expression (EGE), based on repeated medium exchanges and retransfections of the cell culture to prolong the production phase. Another approach is the media supplementation with gene expression enhancers such as valproic acid and caffeine, despite their detrimental effect on cell viability. Valproic acid is a histone deacetylase inhibitor while caffeine has a phosphodiesterase inhibition effect. Here, the combination of the EGE protocol with additive supplementation to maximize VLP production is first tested. As an alternative to the direct additive supplementation, the replacement of these chemical additives by iRNA for obtaining the same inhibition action is also tested. The combination of the EGE protocol with caffeine and valproic acid supplementation resulted in a 1.5-fold improvement in HIV-1 VLP production compared with the EGE protocol alone, representing an overall 18-fold improvement over conventional batch cultivation. shRNAs encoded in the expression vector were tested to substitute valproic acid and caffeine. This novel strategy enhanced VLP production by 2.3 fold without any detrimental effect on cell viability (91.7%) compared with the batch cultivation (92.0%). Finally, the combination of shRNA with EGE resulted in more than 15.6-fold improvement compared with the batch standard protocol traditionally used. The methodology developed enables the production of high titers of HIV-1 VLPs avoiding the toxic effects of additives.

Chemical composition and in vitro cytotoxic, genotoxic effects of essential oil from Urtica dioica L.

PubMed

Gül, Süleyman; Demirci, Betül; Başer, Kemal Hüsnü Can; Akpulat, H Aşkin; Aksu, Pinar

2012-05-01

The aim of this study was to determine the chemical composition of Urtica dioica essential oil, and to evaluate its cytotoxic and genotoxic effects, using cytogenetic tests such as the cytokinesis-block micronucleus assay and chromosomal aberration analysis in human lymphocyte cultures in vitro. GC-MS analysis of U. dioica essential oil identified 43 compounds, representing 95.8% of the oil. GC and GC-MS analysis of the essential oil of U. dioica revealed that carvacrol (38.2%), carvone (9.0%), naphthalene (8.9%), (E)-anethol (4.7%), hexahydrofarnesyl acetone (3.0%), (E)-geranyl acetone (2.9%), (E)-β-ionone (2.8%) and phytol (2.7%) are the main components, comprising 72.2% of the oil. A significant correlation was found between the concentration of essential oil and the following: chromosomal aberrations, micronuclei frequency, apoptotic cells, necrotic cells, and binucleated cells.

Pancreatic cancer cell detection by targeted lipid microbubbles and multiphoton imaging.

PubMed

Cromey, Benjamin; McDaniel, Ashley; Matsunaga, Terry; Vagner, Josef; Kieu, Khanh Quoc; Banerjee, Bhaskar

2018-04-01

Surgical resection of pancreatic cancer represents the only chance of cure and long-term survival in this common disease. Unfortunately, determination of a cancer-free margin at surgery is based on one or two tiny frozen section biopsies, which is far from ideal. Not surprisingly, cancer is usually left behind and is responsible for metastatic disease. We demonstrate a method of receptor-targeted imaging using peptide ligands, lipid microbubbles, and multiphoton microscopy that could lead to a fast and accurate way of examining the entire cut surface during surgery. Using a plectin-targeted microbubble, we performed a blinded in-vitro study to demonstrate avid binding of targeted microbubbles to pancreatic cancer cells but not noncancerous cell lines. Further work should lead to a much-needed point-of-care diagnostic test for determining clean margins in oncologic surgery. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

Structural and functional identification of vasculogenic mimicry in vitro.

PubMed

Racordon, Dusan; Valdivia, Andrés; Mingo, Gabriel; Erices, Rafaela; Aravena, Raúl; Santoro, Felice; Bravo, Maria Loreto; Ramirez, Carolina; Gonzalez, Pamela; Sandoval, Alejandra; González, Alfonso; Retamal, Claudio; Kogan, Marcelo J; Kato, Sumie; Cuello, Mauricio A; Osorio, German; Nualart, Francisco; Alvares, Pedro; Gago-Arias, Araceli; Fabri, Daniella; Espinoza, Ignacio; Sanchez, Beatriz; Corvalán, Alejandro H; Pinto, Mauricio P; Owen, Gareth I

2017-08-01

Vasculogenic mimicry (VM) describes a process by which cancer cells establish an alternative perfusion pathway in an endothelial cell-free manner. Despite its strong correlation with reduced patient survival, controversy still surrounds the existence of an in vitro model of VM. Furthermore, many studies that claim to demonstrate VM fail to provide solid evidence of true hollow channels, raising concerns as to whether actual VM is actually being examined. Herein, we provide a standardized in vitro assay that recreates the formation of functional hollow channels using ovarian cancer cell lines, cancer spheres and primary cultures derived from ovarian cancer ascites. X-ray microtomography 3D-reconstruction, fluorescence confocal microscopy and dye microinjection conclusively confirm the existence of functional glycoprotein-rich lined tubular structures in vitro and demonstrate that many of structures reported in the literature may not represent VM. This assay may be useful to design and test future VM-blocking anticancer therapies.

Physiological significance of polyploidization in mammalian cells.

PubMed

Pandit, Shusil K; Westendorp, Bart; de Bruin, Alain

2013-11-01

Programmed polyploidization occurs in all mammalian species during development and aging in selected tissues, but the biological properties of polyploid cells remain obscure. Spontaneous polyploidization arises during stress and has been observed in a variety of pathological conditions, such as cancer and degenerative diseases. A major challenge in the field is to test the predicted functions of polyploidization in vivo. However, recent genetic mouse models with diminished polyploidization phenotypes represent novel, powerful tools to unravel the biological function of polyploidization. Contrary to a longstanding hypothesis, polyploidization appears to not be required for differentiation and has no obvious impact on proliferation. Instead, polyploidization leads to increased cell size and genetic diversity, which could promote better adaptation to chronic injury or stress. We discuss here the consequences of reducing polyploidization in mice and review which stress responses and molecular signals trigger polyploidization during development and disease. Copyright © 2013 Elsevier Ltd. All rights reserved.

Polymer substrates for flexible photovoltaic cells application in personal electronic system

NASA Astrophysics Data System (ADS)

Znajdek, K.; Sibiński, M.; Strąkowska, A.; Lisik, Z.

2016-01-01

The article presents an overview of polymeric materials for flexible substrates in photovoltaic (PV) structures that could be used as power supply in the personal electronic systems. Four types of polymers have been elected for testing. The first two are the most specialized and heat resistant polyimide films. The third material is transparent polyethylene terephthalate film from the group of polyesters which was proposed as a cheap and commercially available substrate for the technology of photovoltaic cells in a superstrate configuration. The last selected polymeric material is a polysiloxane, which meets the criteria of high elasticity, is temperature resistant and it is also characterized by relatively high transparency in the visible light range. For the most promising of these materials additional studies were performed in order to select those of them which represent the best optical, mechanical and temperature parameters according to their usage for flexible substrates in solar cells.

Functional Integrative Levels in the Human Interactome Recapitulate Organ Organization

PubMed Central

Prieto, Carlos; Benkahla, Alia; De Las Rivas, Javier; Brun, Christine

2011-01-01

Interactome networks represent sets of possible physical interactions between proteins. They lack spatio-temporal information by construction. However, the specialized functions of the differentiated cell types which are assembled into tissues or organs depend on the combinatorial arrangements of proteins and their physical interactions. Is tissue-specificity, therefore, encoded within the interactome? In order to address this question, we combined protein-protein interactions, expression data, functional annotations and interactome topology. We first identified a subnetwork formed exclusively of proteins whose interactions were observed in all tested tissues. These are mainly involved in housekeeping functions and are located at the topological center of the interactome. This ‘Largest Common Interactome Network’ represents a ‘functional interactome core’. Interestingly, two types of tissue-specific interactions are distinguished when considering function and network topology: tissue-specific interactions involved in regulatory and developmental functions are central whereas tissue-specific interactions involved in organ physiological functions are peripheral. Overall, the functional organization of the human interactome reflects several integrative levels of functions with housekeeping and regulatory tissue-specific functions at the center and physiological tissue-specific functions at the periphery. This gradient of functions recapitulates the organization of organs, from cells to organs. Given that several gradients have already been identified across interactomes, we propose that gradients may represent a general principle of protein-protein interaction network organization. PMID:21799769

Novel antiproliferative flavonoids induce cell cycle arrest in human prostate cancer cell lines.

PubMed

Haddad, A Q; Venkateswaran, V; Viswanathan, L; Teahan, S J; Fleshner, N E; Klotz, L H

2006-01-01

Epidemiologic studies have demonstrated an inverse association between flavonoid intake and prostate cancer (PCa) risk. The East Asian diet is very high in flavonoids and, correspondingly, men in China and Japan have the lowest incidence of PCa worldwide. There are thousands of different naturally occurring and synthetic flavonoids. However, only a few have been studied in PCa. Our aim was to identify novel flavonoids with antiproliferative effect in PCa cell lines, as well as determine their effects on cell cycle. We have screened a representative subgroup of 26 flavonoids for antiproliferative effect on the human PCa (LNCaP and PC3), breast cancer (MCF-7), and normal prostate stromal cell lines (PrSC). Using a fluorescence-based cell proliferation assay (Cyquant), we have identified five flavonoids, including the novel compounds 2,2'-dihydroxychalcone and fisetin, with antiproliferative and cell cycle arresting properties in human PCa in vitro. Most of the flavonoids tested exerted antiproliferative effect at lower doses in the PCa cell lines compared to the non-PCa cells. Flow cytometry was used as a means to determine the effects on cell cycle. PC3 cells were arrested in G2/M phase by flavonoids. LNCaP cells demonstrated different cell cycle profiles. Further studies are warranted to determine the molecular mechanism of action of 2,2'-DHC and fisetin in PCa, and to establish their effectiveness in vivo.

Microsatellites within the feline androgen receptor are suitable for X chromosome-linked clonality testing in archival material.

PubMed

Farwick, Nadine M; Klopfleisch, Robert; Gruber, Achim D; Weiss, Alexander Th A

2017-04-01

Objectives A hallmark of neoplasms is their origin from a single cell; that is, clonality. Many techniques have been developed in human medicine to utilise this feature of tumours for diagnostic purposes. One approach is X chromosome-linked clonality testing using polymorphisms of genes encoded by genes on the X chromosome. The aim of this study was to determine if the feline androgen receptor gene was suitable for X chromosome-linked clonality testing. Methods The feline androgen receptor gene was characterised and used to test clonality of feline lymphomas by PCR and polyacrylamide gel electrophoresis, using archival formalin-fixed, paraffin-embedded material. Results Clonality of the feline lymphomas under study was confirmed and the gene locus was shown to represent a suitable target in clonality testing. Conclusions and relevance Because there are some pitfalls of using X chromosome-linked clonality testing, further studies are necessary to establish this technique in the cat.

The Embryonic Stem Cell Test as Tool to Assess Structure-Dependent Teratogenicity: The Case of Valproic Acid

PubMed Central

Riebeling, Christian; Pirow, Ralph; Becker, Klaus; Buesen, Roland; Eikel, Daniel; Kaltenhäuser, Johanna; Meyer, Frauke; Nau, Heinz; Slawik, Birgitta; Visan, Anke; Volland, Jutta; Spielmann, Horst; Luch, Andreas; Seiler, Andrea

2011-01-01

Teratogenicity can be predicted in vitro using the embryonic stem cell test (EST). The EST, which is based on the morphometric measurement of cardiomyocyte differentiation and cytotoxicity parameters, represents a scientifically validated method for the detection and classification of chemicals according to their teratogenic potency. Furthermore, an abbreviated protocol applying flow cytometry of intracellular marker proteins to determine differentiation into the cardiomyocyte lineage is available. Although valproic acid (VPA) is in worldwide clinical use as antiepileptic drug, it exhibits two severe side effects, i.e., teratogenicity and hepatotoxicity. These limitations have led to extensive research into derivatives of VPA. Here we chose VPA as model compound to test the applicability domain and to further evaluate the reliability of the EST. To this end, we study six closely related congeners of VPA and demonstrate that both the standard and the molecular flow cytometry-based EST are well suited to indicate differences in the teratogenic potency among VPA analogs that differ only in chirality or side chain length. Our data show that identical results can be obtained by using the standard EST or a shortened protocol based on flow cytometry of intracellular marker proteins. Both in vitro protocols enable to reliably determine differentiation of murine stem cells toward the cardiomyocyte lineage and to assess its chemical-mediated inhibition. PMID:21227905

[Antimicrobial effect on some zoonotic bacteria, of the cell-free fermentation fluid and purified peptide fraction of the entomopathogenic bacterium, Xenorhabdus budapestensis].

PubMed

Burgettiné Böszörményi, Erzsébet; Barcs, István; Domján, Gyula; Bélafiné Bakó, Katalin; Fodor, András; Makrai, László; Vozik, Dávid

2015-11-01

Many multi-resistant patogens appear continuously resulting in a permanent need for the development of novel antibiotics. A large number of antibiotics introduced in clinical and veterinary practices are not effective. Antibacterial peptides with unusual mode of action may represent a promising option against multi-resistant pathogens. The entomopathogenic Xenorhabdus budapestensis bacteria produce several different antimicrobial peptides compounds such as bicornutin-A and fabclavin. The aim of the authors was to evaluate the in vitro antibacterial effect of Xenorhabdus budapestensis using zoonotic patogen bacteria. Cell-free conditioned media and purified peptide fractions of Xenorhabdus budapestensis were tested on Gram-positive (Rhodococcus equi, Erysipelothrix rhusiopathia, Staphylococcus aureus, Streptococcus equi, Corynebacterium pseudotuberculosis, Listeria monocytagenes) and Gram-negative bacteria (Salmonella gallinarum, Salmonella derbi, Bordatella bronchoseptica, Escherichia coli, Pasteurella multocida, Aeromonas hydrophila) using agar diffusion test on blood agar plates. It was found that Xenorhabdus budapestensis bacteria produced compounds with strong and dose-dependent effects on the tested organisms. Purified peptid fraction exerted a more marked effect than cell free conditioned media. Gram-positive bacteria were more sensitive to this antibacterial effect than Gram-negative bacteria. Antibacterial peptide compound from Xenorhabdus budapestensis exert marked antibacterial effect on zoonotic patogen bacteria and they should be further evaluated in future for their potential use in the control or prevention of zoonoses.

Reconstruction of Orion Engineering Development Unit (EDU) Parachute Inflation Loads

NASA Technical Reports Server (NTRS)

Ray, Eric S.

2013-01-01

The process of reconstructing inflation loads of Capsule Parachute Assembly System (CPAS) has been updated as the program transitioned to testing Engineering Development Unit (EDU) hardware. The equations used to reduce the test data have been re-derived based on the same physical assumptions made by simulations. Due to instrumentation challenges, individual parachute loads are determined from complementary accelerometer and load cell measurements. Cluster inflations are now simulated by modeling each parachute individually to better represent different inflation times and non-synchronous disreefing. The reconstruction procedure is tailored to either infinite mass or finite mass events based on measurable characteristics from the test data. Inflation parameters are determined from an automated optimization routine to reduce subjectivity. Infinite mass inflation parameters have been re-defined to avoid unrealistic interactions in Monte Carlo simulations. Sample cases demonstrate how best-fit inflation parameters are used to generate simulated drag areas and loads which favorably agree with test data.

Collagen gel droplet-embedded culture drug sensitivity testing in squamous cell carcinoma cell lines derived from human oral cancers: Optimal contact concentrations of cisplatin and fluorouracil.

PubMed

Sakuma, Kaname; Tanaka, Akira; Mataga, Izumi

2016-12-01

The collagen gel droplet-embedded culture drug sensitivity test (CD-DST) is an anticancer drug sensitivity test that uses a method of three-dimensional culture of extremely small samples, and it is suited to primary cultures of human cancer cells. It is a useful method for oral squamous cell carcinoma (OSCC), in which the cancer tissues available for testing are limited. However, since the optimal contact concentrations of anticancer drugs have yet to be established in OSCC, CD-DST for detecting drug sensitivities of OSCC is currently performed by applying the optimal contact concentrations for stomach cancer. In the present study, squamous carcinoma cell lines from human oral cancer were used to investigate the optimal contact concentrations of cisplatin (CDDP) and fluorouracil (5-FU) during CD-DST for OSCC. CD-DST was performed in 7 squamous cell carcinoma cell lines derived from human oral cancers (Ca9-22, HSC-3, HSC-4, HO-1-N-1, KON, OSC-19 and SAS) using CDDP (0.15, 0.3, 1.25, 2.5, 5.0 and 10.0 µg/ml) and 5-FU (0.4, 0.9, 1.8, 3.8, 7.5, 15.0 and 30.0 µg/ml), and the optimal contact concentrations were calculated from the clinical response rate of OSCC to single-drug treatment and the in vitro efficacy rate curve. The optimal concentrations were 0.5 µg/ml for CDDP and 0.7 µg/ml for 5-FU. The antitumor efficacy of CDDP at this optimal contact concentration in CD-DST was compared to the antitumor efficacy in the nude mouse method. The T/C values, which were calculated as the ratio of the colony volume of the treatment group and the colony volume of the control group, at the optimal contact concentration of CDDP and of the nude mouse method were almost in agreement (P<0.05) and predicted clinical efficacy, indicating that the calculated optimal contact concentration is valid. Therefore, chemotherapy for OSCC based on anticancer drug sensitivity tests offers patients a greater freedom of choice and is likely to assume a greater importance in the selection of treatment from the perspectives of function preservation and quality of life, as well as representing a treatment option for unresectable, intractable or recurrent cases.

CD13 as target for tissue factor induced tumor vascular infarction in small cell lung cancer.

PubMed

Schmidt, Lars Henning; Stucke-Ring, Janine; Brand, Caroline; Schliemann, Christoph; Harrach, Saliha; Muley, Thomas; Herpel, Esther; Kessler, Torsten; Mohr, Michael; Görlich, Dennis; Kreuter, Michael; Lenz, Georg; Wardelmann, Eva; Thomas, Michael; Berdel, Wolfgang E; Schwöppe, Christian; Hartmann, Wolfgang

2017-11-01

Zinc-binding protease aminopeptidase N (CD13) is expressed on tumor vascular cells and tumor cells. It represents a potential candidate for molecular targeted therapy, e.g. employing truncated tissue factor (tTF)-NGR, which can bind CD13 and thereby induce tumor vascular infarction. We performed a comprehensive analysis of CD13 expression in a clinically well characterized cohort of patients with small cell lung cancer (SCLC) to evaluate its potential use for targeted therapies in this disease. CD13 expression was analyzed immunohistochemically in 27 SCLC patients and correlated with clinical course and outcome. In CD-1 nude mice bearing human HTB119 SCLC xenotransplants, the systemic effects of the CD13-targeting fusion protein tTF-NGR on tumor growth were tested. In 52% of the investigated SCLC tissue samples, CD13 was expressed in tumor stroma cells, while the tumor cells were negative for CD13. No prognostic effect was found in the investigated SCLC study collective with regard to overall survival (p>0.05). In CD-1 nude mice, xenografts of CD13 negative HTB119 SCLC cells showed CD13 expression in the intratumoral vascular and perivascular cells, and the systemic application of CD13-targeted tissue factor tTF-NGR led to a significant reduction of tumor growth. We here present first data on the expression of CD13 in SCLC tumor samples. Our results strongly recommend the further investigation of tTF-NGR and other molecules targeted by NGR-peptides in SCLC patients. Considering the differential expression of CD13 in SCLC samples pre-therapeutic CD13 analysis is proposed for testing as investigational predictive biomarker for patient selection. Copyright © 2017 Elsevier B.V. All rights reserved.

Regulation of Chromatin Assembly and Cell Transformation by Formaldehyde Exposure in Human Cells

PubMed Central

Chen, Danqi; Fang, Lei; Mei, Shenglin; Li, Hongjie; Xu, Xia; Des Marais, Thomas L.; Lu, Kun; Liu, X. Shirley

2017-01-01

Background: Formaldehyde (FA) is an environmental and occupational chemical carcinogen. Recent studies have shown that exogenous FA causes only a modest increase in DNA adduct formation compared with the amount of adducts formed by endogenous FA, raising the possibility that epigenetic mechanisms may contribute to FA-mediated carcinogenicity. Objectives: We investigated the effects of FA exposure on histone modifications and chromatin assembly. We also examined the role of defective chromatin assembly in FA-mediated transcription and cell transformation. Methods: Cellular fractionation and Western blot analysis were used to measure the levels of histone modifications in human bronchial epithelial BEAS-2B cells and human nasal RPMI2650 cells in the presence of FA. Chromatin immunoprecipitation (ChIP) and micrococcal nuclease (MNase) digest assays were performed to examine the changes in chromatin assembly and accessibility after FA exposure. RNA sequencing (RNA-seq) and real-time polymerase chain reaction (PCR) were used to examine transcriptional dysregulation. Finally, anchorage-independent cell growth ability was tested by soft agar assay following FA exposure. Results: Exposure to FA dramatically decreased the acetylation of the N-terminal tails of cytosolic histones. These modifications are important for histone nuclear import and subsequent chromatin assembly. Histone proteins were depleted in both the chromatin fraction and at most of the genomic loci tested following FA exposure, suggesting that FA compromises chromatin assembly. Moreover, FA increased chromatin accessibility and altered the expression of hundreds of cancer-related genes. Knockdown of the histone H3.3 gene (an H3 variant), which mimics inhibition of chromatin assembly, facilitated FA-mediated anchorage-independent cell growth. Conclusions: We propose that the inhibition of chromatin assembly represents a novel mechanism of cell transformation induced by the environmental and occupational chemical carcinogen FA. https://doi.org/10.1289/EHP1275 PMID:28937961

Venetoclax Synergizes with Radiotherapy for Treatment of B-cell Lymphomas.

PubMed

O'Steen, Shyril; Green, Damian J; Gopal, Ajay K; Orozco, Johnnie J; Kenoyer, Aimee L; Lin, Yukang; Wilbur, D Scott; Hamlin, Donald K; Fisher, Darrell R; Hylarides, Mark D; Gooley, Theodore A; Waltman, Amelia; Till, Brian G; Press, Oliver W

2017-07-15

Constitutive B-cell receptor signaling leads to overexpression of the antiapoptotic BCL-2 protein and is implicated in the pathogenesis of many types of B-cell non-Hodgkin lymphoma (B-NHL). The BCL-2 small-molecule inhibitor venetoclax shows promising clinical response rates in several lymphomas, but is not curative as monotherapy. Radiotherapy is a rational candidate for combining with BCL-2 inhibition, as DNA damage caused by radiotherapy increases the activity of pro-apoptotic BCL-2 pathway proteins, and lymphomas are exquisitely sensitive to radiation. We tested B-NHL responses to venetoclax combined with either external beam radiotherapy or radioimmunotherapy (RIT), which joins the selectivity of antibody targeting with the effectiveness of irradiation. We first tested cytotoxicity of cesium-137 irradiation plus venetoclax in 14 B-NHL cell lines representing five lymphoma subtypes. Combination treatment synergistically increased cell death in 10 of 14 lines. Lack of synergy was predicted by resistance to single-agent venetoclax and high BCL-XL expression. We then assessed the efficacy of external beam radiotherapy plus venetoclax in murine xenograft models of mantle cell (MCL), germinal-center diffuse large B-cell (GCB-DLBCL), and activated B-cell (ABC-DLBCL) lymphomas. In each model, external beam radiotherapy plus venetoclax synergistically increased mouse survival time, curing up to 10%. We finally combined venetoclax treatment of MCL and ABC-DLBCL xenografts with a pretargeted RIT (PRIT) system directed against the CD20 antigen. Optimal dosing of PRIT plus venetoclax cured 100% of mice with no detectable toxicity. Venetoclax combined with radiotherapy may be a promising treatment for a wide range of lymphomas Cancer Res; 77(14); 3885-93. ©2017 AACR . ©2017 American Association for Cancer Research.

Direct transplantation of native pericytes from adipose tissue: A new perspective to stimulate healing in critical size bone defects.

PubMed

König, Matthias A; Canepa, Daisy D; Cadosch, Dieter; Casanova, Elisa; Heinzelmann, Michael; Rittirsch, Daniel; Plecko, Michael; Hemmi, Sonja; Simmen, Hans-Peter; Cinelli, Paolo; Wanner, Guido A

2016-01-01

Fractures with a critical size bone defect (e.g., open fracture with segmental bone loss) are associated with high rates of delayed union and non-union. The prevention and treatment of these complications remain a serious issue in trauma and orthopaedic surgery. Autologous cancellous bone grafting is a well-established and widely used technique. However, it has drawbacks related to availability, increased morbidity and insufficient efficacy. Mesenchymal stromal cells can potentially be used to improve fracture healing. In particular, human fat tissue has been identified as a good source of multilineage adipose-derived stem cells, which can be differentiated into osteoblasts. The main issue is that mesenchymal stromal cells are a heterogeneous population of progenitors and lineage-committed cells harboring a broad range of regenerative properties. This heterogeneity is also mirrored in the differentiation potential of these cells. In the present study, we sought to test the possibility to enrich defined subpopulations of stem/progenitor cells for direct therapeutic application without requiring an in vitro expansion. We enriched a CD146+NG2+CD45- population of pericytes from freshly isolated stromal vascular fraction from mouse fat tissue and tested their osteogenic differentiation capacity in vitro and in vivo in a mouse model for critical size bone injury. Our results confirm the ability of enriched CD146+NG2+CD45- cells to efficiently generate osteoblasts in vitro, to colonize cancellous bone scaffolds and to successfully contribute to regeneration of large bone defects in vivo. This study represents proof of principle for the direct use of enriched populations of cells with stem/progenitor identity for therapeutic applications. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Combined use of the ASK and SHK-1 cell lines to enhance the detection of infectious salmon anemia virus

USGS Publications Warehouse

Rolland, J.B.; Bouchard, D.; Coll, J.; Winton, J.R.

2005-01-01

Infectious salmon anemia (ISA) is a severe disease primarily affecting commercially farmed Atlantic salmon (Salmo salar) in seawater. The disease has been reported in portions of Canada, the United Kingdom, the Faroe Islands, and the United States. Infectious salmon anemia virus (ISAV), the causative agent of ISA, has also been isolated from several asymptomatic marine and salmonid fish species. Diagnostic assays for the detection of ISAV include virus isolation in cell culture, a reverse transcriptase-PCR, an enzyme-linked immunosorbent assay, and an indirect fluorescent antibody test. Virus isolation is considered the gold standard, and 5 salmonid cell lines are known to support growth of ISAV. In this study, the relative performance of the salmon head kidney 1 (SHK-1), Atlantic salmon kidney (ASK), and CHSE-214 cell lines in detecting ISAV was evaluated using samples from both experimentally and naturally infected Atlantic salmon. Interlaboratory comparisons were conducted using a quality control-quality assurance ring test. Both the ASK and SHK-1 cell lines performed well in detecting ISAV, although the SHK-1 line was more variable in its sensitivity to infection and somewhat slower in the appearance of cytopathic effect. Relative to the SHK-1 and ASK lines, the CHSE-214 cell line performed poorly. Although the ASK line appeared to represent a good alternative to the more commonly used SHK-1 line, use of a single cell line for diagnostic assays may increase the potential for false-negative results. Thus, the SHK-1 and ASK cell lines can be used in combination to provide enhanced ability to detect ISAV.

Circulating tumour cells from patients with colorectal cancer have cancer stem cell hallmarks in ex vivo culture.

PubMed

Grillet, Fanny; Bayet, Elsa; Villeronce, Olivia; Zappia, Luke; Lagerqvist, Ebba Louise; Lunke, Sebastian; Charafe-Jauffret, Emmanuelle; Pham, Kym; Molck, Christina; Rolland, Nathalie; Bourgaux, Jean François; Prudhomme, Michel; Philippe, Claire; Bravo, Sophie; Boyer, Jean Christophe; Canterel-Thouennon, Lucile; Taylor, Graham Roy; Hsu, Arthur; Pascussi, Jean Marc; Hollande, Frédéric; Pannequin, Julie

2017-10-01

Although counting of circulating tumour cells (CTC) has attracted a broad interest as potential markers of tumour progression and treatment response, the lack of functional characterisation of these cells had become a bottleneck in taking these observations to the clinic. Our objective was to culture these cells in order to understand them and exploit their therapeutic potential to the full. Here, hypothesising that some CTC potentially have cancer stem cell (CSC) phenotype, we generated several CTC lines from the blood of patients with advanced metastatic colorectal cancer (CRC) based on their self-renewal abilities. Multiple standard tests were then employed to characterise these cells. Our CTC lines self-renew, express CSC markers and have multilineage differentiation ability, both in vitro and in vivo . Patient-derived CTC lines are tumorigenic in subcutaneous xenografts and are also able to colonise the liver after intrasplenic injection. RNA sequencing analyses strikingly demonstrate that drug metabolising pathways represent the most upregulated feature among CTC lines in comparison with primary CRC cells grown under similar conditions. This result is corroborated by the high resistance of the CTC lines to conventional cytotoxic compounds. Taken together, our results directly demonstrate the existence of patient-derived colorectal CTCs that bear all the functional attributes of CSCs. The CTC culture model described here is simple and takes <1 month from blood collection to drug testing, therefore, routine clinical application could facilitate access to personalised medicine. ClinicalTrial.gov NCT01577511. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Genotoxicity of 11 heavy metals detected as food contaminants in two human cell lines.

PubMed

Kopp, B; Zalko, D; Audebert, M

2018-04-01

Heavy metals, such as arsenic (As), antimony (Sb), barium (Ba), cadmium (Cd), cobalt (Co), germanium (Ge), lead (Pb), nickel (Ni), tellurium (Te), and vanadium (V) are widely distributed in the environment and in the food chain. Human exposure to heavy metals through water and food has been reported by different international agencies. Although some of these heavy metals are essential elements for human growth and development, they may also be toxic at low concentrations due to indirect mechanisms. In this study, the genotoxic and cytotoxic properties of 15 different oxidation statuses of 11 different heavy metals were investigated using high-throughput screening (γH2AX assay) in two human cell lines (HepG2 and LS-174T) representative of target organs (liver and colon) for food contaminants. Base on their lowest observed adverse effect concentration, the genotoxic potency of each heavy metal in each cell line was ranked in decreasing order, NaAsO 2  > CdCl 2  > PbCl 2 (only in LS-174T cells) > As 2 O 5  > SbCl 3  > K 2 TeO 3  > As 2 O 3 . No significant genotoxicity was observed with the other heavy metals tested. Cell viability data indicate that several heavy metals (As, Cd, Co, Ni, Sb, and Te) induce cytotoxicity at high concentrations, whereas an increase in the number of cells was observed for lead concentrations >100 µM in both cell lines tested, suggesting that lead stimulates cell growth. All these results highlight the possible human health hazards associated with the presence of heavy metals present in food. Environ. Mol. Mutagen. 59:202-210, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Percutaneous fiber-optic sensor for the detection of chemotherapy-induced apoptosis in vivo

NASA Astrophysics Data System (ADS)

O'Kelly, James; Liao, Kuo-Chih; Clifton, William; Lu, Daning; Koeffler, Phillip; Loeb, Gerald

2010-02-01

Early imaging of tumor response to chemotherapy has the potential for significant clinical benefits. We are developing a family of fiber-optic sensors called SencilsTM (sensory cilia), which are disposable, minimally invasive, and can provide in vivo monitoring of various analytes for several weeks. The objective of this study was to develop and test our sensor to image the labeling of phosphatidylserine by apoptotic cells in response to chemotherapeutic drugs. FM1-43 was a better fluorescent marker for detecting phosphatidylserine expression than Annexin V-FITC; both the proportion of labeled cells (Annexin V, 15%; FM1-43, 58%) and the relative fluorescent increase (Annexin V-FITC, 1.5-fold; FM1-43, 4.5-fold) was greater when FM1-43 was used to detect apoptosis. Initial testing of the optical sensing technology using Taxol-treated MCF-7 cells demonstrated that injection of FM1-43 resulted in a rapid, transient increase in fluorescence that was greater in apoptotic cells compared to control cells (apoptotic cells, 4-fold increase; control cells, 2-fold increase). Using an established animal model, mice were injected with cyclophosphamide and hepatic apoptosis was assessed by imaging of PS expression. Both the amplitude of fluorescence increase and the time taken for the amplitude to decay to half of its peak were increased in livers from animals treated with cyclophosphamide. Our optical sensing technology can be used to detect the early apoptotic response of cells to chemotherapeutic drugs both in vitro and in vivo. This novel technology represents a unique option for the imaging of tumor responses in vivo, and provides an inexpensive, specific system for the detection of early-stage apoptosis.

Photosensitizer adhered to cell culture microplates induces phototoxicity in carcinoma cells.

PubMed

Ziegler, Verena; Kiesslich, Tobias; Krammer, Barbara; Plaetzer, Kristjan

2013-01-01

In vitro experiments in plastic receptacles are the basis of characterization of new photosensitizers (PSs) for the photodynamic therapy. We recently reported that lipophilic PSs adhere to cell culture microplates in a kinetic-like manner (Engelhardt et al., 2011). In the current study, we examined the interaction and phototoxic effects of the microplate-adhered PS in cancer cells. Therefore, we preloaded microplates with hypericin, Foscan, PVP-hypericin, or aluminum (III) phthalocyanine tetrasulfonate chloride (AlPCS4) for 24 hours and measured the PS distribution after addition of A431 human carcinoma cells: following another 24 hours up to 68% of hypericin were detected in the cell fraction. The hydrophilic PVP-hypericin and AlPCS4 also diffused into the cells, but the quantities of PS adherence were considerably lower. Microplate-adhered Foscan appeared not to be redistributed. In contrast to the hydrophilic PSs, the cellular phototoxicity of microplate-adhered lipophilic PS was high, independent of whether the PS (i) was pre-loaded onto microplates or (ii) added simultaneously with the cells or (iii) one day after cell seeding. Based on these results, we suggest testing lipophilic PS dyes for their adherence to microplates. Furthermore, the ability of plastic materials to (reversibly) store PSs might represent a new approach for the PS delivery or the development of antimicrobial coatings.

Photosensitizer Adhered to Cell Culture Microplates Induces Phototoxicity in Carcinoma Cells

PubMed Central

Ziegler, Verena; Kiesslich, Tobias; Krammer, Barbara; Plaetzer, Kristjan

2013-01-01

In vitro experiments in plastic receptacles are the basis of characterization of new photosensitizers (PSs) for the photodynamic therapy. We recently reported that lipophilic PSs adhere to cell culture microplates in a kinetic-like manner (Engelhardt et al., 2011). In the current study, we examined the interaction and phototoxic effects of the microplate-adhered PS in cancer cells. Therefore, we preloaded microplates with hypericin, Foscan, PVP-hypericin, or aluminum (III) phthalocyanine tetrasulfonate chloride (AlPCS4) for 24 hours and measured the PS distribution after addition of A431 human carcinoma cells: following another 24 hours up to 68% of hypericin were detected in the cell fraction. The hydrophilic PVP-hypericin and AlPCS4 also diffused into the cells, but the quantities of PS adherence were considerably lower. Microplate-adhered Foscan appeared not to be redistributed. In contrast to the hydrophilic PSs, the cellular phototoxicity of microplate-adhered lipophilic PS was high, independent of whether the PS (i) was pre-loaded onto microplates or (ii) added simultaneously with the cells or (iii) one day after cell seeding. Based on these results, we suggest testing lipophilic PS dyes for their adherence to microplates. Furthermore, the ability of plastic materials to (reversibly) store PSs might represent a new approach for the PS delivery or the development of antimicrobial coatings. PMID:23509741

The novel anticancer agent JNJ-26854165 is active in chronic myeloid leukemic cells with unmutated BCR/ABL and T315I mutant BCR/ABL through promoting proteosomal degradation of BCR/ABL proteins.

PubMed

You, Liangshun; Liu, Hui; Huang, Jian; Xie, Wanzhuo; Wei, Jueying; Ye, Xiujin; Qian, Wenbin

2017-01-31

Chronic myeloid leukemia (CML) is a clonal malignant disease caused by the expression of BCR/ABL. MDM2 (human homolog of the murine double minute-2) inhibitors such as Nutlin-3 have been shown to induce apoptosis in a p53-dependent manner in CML cells and sensitize cells to Imatinib. Here, we demonstrate that JNJ-26854165, an inhibitor of MDM2, inhibits proliferation and triggers cell death in a p53-independent manner in various BCR/ABL-expressing cells, which include primary leukemic cells from patients with CML blast crisis and cells expressing the Imatinib-resistant T315I BCR/ABL mutant. The response to JNJ-26854165 is associated with the downregulation of BCR/ABL dependently of proteosome activation. Moreover, in all tested CML cells, with the exception of T315I mutation cells, combining JNJ-26854165 and tyrosine kinase inhibitor (TKI) Imatinib or PD180970 leads to a synergistic effect. In conclusion, our results suggest that JNJ-26854165, used either alone or in combination with TKIs, represents a promising novel targeted approach to overcome TKI resistance and improve patient outcome in CML.

Inverse associations between obesity indicators and thymic T-cell production levels in aging atomic-bomb survivors.

PubMed

Yoshida, Kengo; Nakashima, Eiji; Kubo, Yoshiko; Yamaoka, Mika; Kajimura, Junko; Kyoizumi, Seishi; Hayashi, Tomonori; Ohishi, Waka; Kusunoki, Yoichiro

2014-01-01

Reduction of the naive T-cell population represents a deteriorating state in the immune system that occurs with advancing age. In animal model studies, obesity compromises the T-cell immune system as a result of enhanced adipogenesis in primary lymphoid organs and systemic inflammation. In this study, to test the hypothesis that obesity may contribute to the aging of human T-cell immunity, a thousand atomic-bomb survivors were examined for obesity status and ability to produce naive T cells, i.e., T-cell receptor excision circle (TREC) numbers in CD4 and CD8 T cells. The number of TRECs showed a strong positive correlation with naive T cell numbers, and lower TREC numbers were associated with higher age. We found that the TREC number was inversely associated with levels of obesity indicators (BMI, hemoglobin A1c) and serum CRP levels. Development of type-2 diabetes and fatty liver was also associated with lower TREC numbers. This population study suggests that obesity with enhanced inflammation is involved in aging of the human T-cell immune system. Given the fact that obesity increases the risk of numerous age-related diseases, attenuated immune competence is a possible mechanistic link between obesity and disease development among the elderly.

Inverse Associations between Obesity Indicators and Thymic T-Cell Production Levels in Aging Atomic-Bomb Survivors

PubMed Central

Yoshida, Kengo; Nakashima, Eiji; Kubo, Yoshiko; Yamaoka, Mika; Kajimura, Junko; Kyoizumi, Seishi; Hayashi, Tomonori; Ohishi, Waka; Kusunoki, Yoichiro

2014-01-01

Reduction of the naive T-cell population represents a deteriorating state in the immune system that occurs with advancing age. In animal model studies, obesity compromises the T-cell immune system as a result of enhanced adipogenesis in primary lymphoid organs and systemic inflammation. In this study, to test the hypothesis that obesity may contribute to the aging of human T-cell immunity, a thousand atomic-bomb survivors were examined for obesity status and ability to produce naive T cells, i.e., T-cell receptor excision circle (TREC) numbers in CD4 and CD8 T cells. The number of TRECs showed a strong positive correlation with naive T cell numbers, and lower TREC numbers were associated with higher age. We found that the TREC number was inversely associated with levels of obesity indicators (BMI, hemoglobin A1c) and serum CRP levels. Development of type-2 diabetes and fatty liver was also associated with lower TREC numbers. This population study suggests that obesity with enhanced inflammation is involved in aging of the human T-cell immune system. Given the fact that obesity increases the risk of numerous age-related diseases, attenuated immune competence is a possible mechanistic link between obesity and disease development among the elderly. PMID:24651652

Cell-wall recovery after irreversible deformation of wood

NASA Astrophysics Data System (ADS)

Keckes, Jozef; Burgert, Ingo; Frühmann, Klaus; Müller, Martin; Kölln, Klaas; Hamilton, Myles; Burghammer, Manfred; Roth, Stephan V.; Stanzl-Tschegg, Stefanie; Fratzl, Peter

2003-12-01

The remarkable mechanical properties of biological materials reside in their complex hierarchical architecture and in specific molecular mechanistic phenomena. The fundamental importance of molecular interactions and bond recovery has been suggested by studies on deformation and fracture of bone and nacre. Like these mineral-based materials, wood also represents a complex nanocomposite with excellent mechanical performance, despite the fact that it is mainly based on polymers. In wood, however, the mechanistic contribution of processes in the cell wall is not fully understood. Here we have combined tensile tests on individual wood cells and on wood foils with simultaneous synchrotron X-ray diffraction analysis in order to separate deformation mechanisms inside the cell wall from those mediated by cell-cell interactions. We show that tensile deformation beyond the yield point does not deteriorate the stiffness of either individual cells or foils. This indicates that there is a dominant recovery mechanism that re-forms the amorphous matrix between the cellulose microfibrils within the cell wall, maintaining its mechanical properties. This stick-slip mechanism, rather like Velcro operating at the nanometre level, provides a 'plastic response' similar to that effected by moving dislocations in metals. We suggest that the molecular recovery mechanism in the cell matrix is a universal phenomenon dominating the tensile deformation of different wood tissue types.

Establishment and characterization of a differentiated epithelial cell culture model derived from the porcine cervix uteri

PubMed Central

2012-01-01

Background Cervical uterine epithelial cells maintain a physiological and pathogen-free milieu in the female mammalian reproductive tract and are involved in sperm-epithelium interaction. Easily accessible, differentiated model systems of the cervical epithelium are not yet available to elucidate the underlying molecular mechanisms within these highly specialized cells. Therefore, the aim of the study was to establish a cell culture of the porcine cervical epithelium representing in vivo-like properties of the tissue. Results We tested different isolation methods and culture conditions and validated purity of the cultured cells by immunohistochemistry against keratins. We could reproducibly culture pure epithelial cells from cervical tissue explants. Based on a morphology score and the WST-1 Proliferation Assay, we optimized the growth medium composition. Primary porcine cervical cells performed best in conditioned Ham's F-12, containing 10% FCS, EGF and insulin. After cultivation in an air-liquid interface for three weeks, the cells showed a discontinuously multilayered phenotype. Finally, differentiation was validated via immunohistochemistry against beta catenin. Mucopolysaccharide production could be shown via alcian blue staining. Conclusions We provide the first suitable protocol to establish a differentiated porcine epithelial model of the cervix uteri, based on easily accessible cells using slaughterhouse material. PMID:22429795

Assessment of Safety and Functional Efficacy of Stem Cell-Based Therapeutic Approaches Using Retinal Degenerative Animal Models

PubMed Central

Lin, Tai-Chi; Zhu, Danhong; Hinton, David R.; Clegg, Dennis O.; Humayun, Mark S.

2017-01-01

Dysfunction and death of retinal pigment epithelium (RPE) and or photoreceptors can lead to irreversible vision loss. The eye represents an ideal microenvironment for stem cell-based therapy. It is considered an “immune privileged” site, and the number of cells needed for therapy is relatively low for the area of focused vision (macula). Further, surgical placement of stem cell-derived grafts (RPE, retinal progenitors, and photoreceptor precursors) into the vitreous cavity or subretinal space has been well established. For preclinical tests, assessments of stem cell-derived graft survival and functionality are conducted in animal models by various noninvasive approaches and imaging modalities. In vivo experiments conducted in animal models based on replacing photoreceptors and/or RPE cells have shown survival and functionality of the transplanted cells, rescue of the host retina, and improvement of visual function. Based on the positive results obtained from these animal experiments, human clinical trials are being initiated. Despite such progress in stem cell research, ethical, regulatory, safety, and technical difficulties still remain a challenge for the transformation of this technique into a standard clinical approach. In this review, the current status of preclinical safety and efficacy studies for retinal cell replacement therapies conducted in animal models will be discussed. PMID:28928775

Applying standardized uptake values in gallium-67-citrate single-photon emission computed tomography/computed tomography studies and their correlation with blood test results in representative organs.

PubMed

Toriihara, Akira; Daisaki, Hiromitsu; Yamaguchi, Akihiro; Yoshida, Katsuya; Isogai, Jun; Tateishi, Ukihide

2018-05-21

Recently, semiquantitative analysis using standardized uptake value (SUV) has been introduced in bone single-photon emission computed tomography/computed tomography (SPECT/CT). Our purposes were to apply SUV-based semiquantitative analytic method for gallium-67 (Ga)-citrate SPECT/CT and to evaluate correlation between SUV of physiological uptake and blood test results in representative organs. The accuracy of semiquantitative method was validated using an National Electrical Manufacturers Association body phantom study (radioactivity ratio of sphere : background=4 : 1). Thereafter, 59 patients (34 male and 25 female; mean age, 66.9 years) who had undergone Ga-citrate SPECT/CT were retrospectively enrolled in the study. A mean SUV of physiological uptake was calculated for the following organs: the lungs, right atrium, liver, kidneys, spleen, gluteal muscles, and bone marrow. The correlation between physiological uptakes and blood test results was evaluated using Pearson's correlation coefficient. The phantom study revealed only 1% error between theoretical and actual SUVs in the background, suggesting the sufficient accuracy of scatter and attenuation corrections. However, a partial volume effect could not be overlooked, particularly in small spheres with a diameter of less than 28 mm. The highest mean SUV was observed in the liver (range: 0.44-4.64), followed by bone marrow (range: 0.33-3.60), spleen (range: 0.52-2.12), and kidneys (range: 0.42-1.45). There was no significant correlation between hepatic uptake and liver function, renal uptake and renal function, or bone marrow uptake and blood cell count (P>0.05). The physiological uptake in Ga-citrate SPECT/CT can be represented as SUVs, which are not significantly correlated with corresponding blood test results.

Comparative proteome analysis of monolayer and spheroid culture of canine osteosarcoma cells.

PubMed

Gebhard, Christiane; Miller, Ingrid; Hummel, Karin; Neschi Née Ondrovics, Martina; Schlosser, Sarah; Walter, Ingrid

2018-04-15

Osteosarcoma is an aggressive bone tumor with high metastasis rate in the lungs and affects both humans and dogs in a similar way. Three-dimensional tumor cell cultures mimic the in vivo situation of micro-tumors and metastases and are therefore better experimental in vitro models than the often applied two-dimensional monolayer cultures. The aim of the present study was to perform comparative proteomics of standard monolayer cultures of canine osteosarcoma cells (D17) and three-dimensional spheroid cultures, to better characterize the 3D model before starting with experiments like migration assays. Using DIGE in combination with MALDI-TOF/TOF we found 27 unique canine proteins differently represented between these two culture systems, most of them being part of a functional network including mainly chaperones, structural proteins, stress-related proteins, proteins of the glycolysis/gluconeogenesis pathway and oxidoreductases. In monolayer cells, a noticeable shift to more acidic pI values was noticed for several proteins of medium to high abundance; two proteins (protein disulfide isomerase A3, stress-induced-phosphoprotein 1) showed an increase of phosphorylated protein species. Protein distribution within the cells, as detected by immunohistochemistry, displayed a switch of stress-induced-phosphoprotein 1 from the cytoplasm (in monolayer cultures) to the nucleus (in spheroid cultures). Additionally, Western blot testing revealed upregulated concentrations of metastasin (S100A4), triosephosphate isomerase 1 and septin 2 in spheroid cultures, in contrast to decreased concentrations of CCT2, a subunit of the T-complex. Results indicate regulation of stress proteins in the process of three-dimensional organization characterized by a hypoxic and nutrient-deficient environment comparable to tumor micro-metastases. Osteosarcoma is an aggressive bone tumor that early spreads to the lungs. Three-dimensional tumor cell cultures represent the avascular stage of micro-tumors and metastases, and should therefore represent a better experimental in vitro model compared to two-dimensional monolayer cultures. Significant differences have been reported in response to drug and radiation treatment between these two culture systems. A gel-based proteomic investigation was performed to compare protein patterns of a canine osteosarcoma cell line cultivated under those two conditions, to learn more about altered cell composition and its impact on cell behaviour. Due to the fact that the canine osteosarcoma is an accepted model for the human disease, results will be relevant for the human species as well. Copyright © 2018 Elsevier B.V. All rights reserved.

First steps towards the successful surface-based cultivation of human embryonic stem cells in hanging drop systems.

PubMed

Schulz, Julia C; Stumpf, Patrick S; Katsen-Globa, Alisa; Sachinidis, Agapios; Hescheler, Jürgen; Zimmermann, Heiko

2012-11-01

Miniaturization and parallelization of cell culture procedures are in focus of research in order to develop test platforms with low material consumption and increased standardization for toxicity and drug screenings. The cultivation in hanging drops (HDs) is a convenient and versatile tool for biological applications and represents an interesting model system for the screening applications due to its uniform shape, the advantageous gas supply, and the small volume. However, its application has so far been limited to non-adherent and aggregate forming cells. Here, we describe for the first time the proof-of-principle regarding the adherent cultivation of human embryonic stem cells in HD. For this microcarriers were added to the droplet as dynamic cultivation surfaces resulting in a maintained pluripotency and proliferation capacity for 10 days. This enables the HD technique to be extended to the cultivation of adherence-dependent stem cells. Also, the possible automation of this method by implementation of liquid handling systems opens new possibilities for miniaturized screenings, the improvement of cultivation and differentiation conditions, and toxicity and drug development.

First steps towards the successful surface-based cultivation of human embryonic stem cells in hanging drop systems

PubMed Central

Schulz, Julia C; Stumpf, Patrick S; Katsen-Globa, Alisa; Sachinidis, Agapios; Hescheler, Jürgen; Zimmermann, Heiko

2012-01-01

Miniaturization and parallelization of cell culture procedures are in focus of research in order to develop test platforms with low material consumption and increased standardization for toxicity and drug screenings. The cultivation in hanging drops (HDs) is a convenient and versatile tool for biological applications and represents an interesting model system for the screening applications due to its uniform shape, the advantageous gas supply, and the small volume. However, its application has so far been limited to non‐adherent and aggregate forming cells. Here, we describe for the first time the proof-of-principle regarding the adherent cultivation of human embryonic stem cells in HD. For this microcarriers were added to the droplet as dynamic cultivation surfaces resulting in a maintained pluripotency and proliferation capacity for 10 days. This enables the HD technique to be extended to the cultivation of adherence-dependent stem cells. Also, the possible automation of this method by implementation of liquid handling systems opens new possibilities for miniaturized screenings, the improvement of cultivation and differentiation conditions, and toxicity and drug development. PMID:23486530

Apoptotic effect of chalcone derivatives of 2-acetylthiophene in human breast cancer cells.

PubMed

Fogaça, Tatiana B; Martins, Rosiane M; Begnini, Karine R; Carapina, Caroline; Ritter, Marina; de Pereira, Claudio M P; Seixas, Fabiana K; Collares, Tiago

2017-02-01

A variety of chalcones have demonstrated cytotoxic activity toward several cancer cell lines. This study aimed to investigate the cytotoxicity of four chalcones derivatives of 2-acetylthiophene in human breast cancer cell lines. MCF-7 and MDA-MB-231 cells were treated with synthesized chalcones and the cytotoxicity was evaluated by tetrazolium dye (MTT), live/dead, and DAPI assays. Chalcones significantly decreased MCF-7 and MDA-MB-231 cells viability in vitro in a dose dependent manner. After 48h treatment, the IC 50 values ranging from 5.52 to 34.23μM. Chalcone 3c displayed the highest cytotoxic activity from all the tested compounds. Cytotoxic effects of compounds were confirmed in the live/dead assay. In addition, DAPI staining revealed that these compounds induce death by apoptosis. The data speculate that chalcone derivatives of 2-acetylthiophene may represent a source of therapeutic agents for human breast cancer. Copyright © 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

[Embryonic stem cells. Future perspectives].

PubMed

Groebner, M; David, R; Franz, W M

2006-05-01

Embryonic stem cells (ES cells) are able to differentiate into any cell type, and therefore represent an excellent source for cellular replacement therapies in the case of widespread diseases, for example heart failure, diabetes, Parkinson's disease and spinal cord injury. A major prerequisite for their efficient and safe clinical application is the availability of pure populations for direct cell transplantation or tissue engineering as well as the immunological compatibility of the transplanted cells. The expression of human surface markers under the control of cell type specific promoters represents a promising approach for the selection of cardiomyocytes and other cell types for therapeutic applications. The first human clinical trial using ES cells will start in the United States this year.

Bortezomib sensitises TRAIL-resistant HPV-positive head and neck cancer cells to TRAIL through a caspase-dependent, E6-independent mechanism.

PubMed

Bullenkamp, J; Raulf, N; Ayaz, B; Walczak, H; Kulms, D; Odell, E; Thavaraj, S; Tavassoli, M

2014-10-23

Human papillomavirus (HPV) is causative for a new and increasing form of head and neck squamous cell carcinomas (HNSCCs). Although localised HPV-positive cancers have a favourable response to radio-chemotherapy (RT/CT), the impact of HPV in advanced or metastatic HNSCC remains to be defined and targeted therapeutics need to be tested for cancers resistant to RT/CT. To this end, we investigated the sensitivity of HPV-positive and -negative HNSCC cell lines to TRAIL (tumour necrosis factor-related apoptosis-inducing ligand), which induces tumour cell-specific apoptosis in various cancer types. A clear correlation was observed between HPV positivity and resistance to TRAIL compared with HPV-negative head and neck cancer cell lines. All TRAIL-resistant HPV-positive cell lines tested were sensitised to TRAIL-induced cell death by treatment with bortezomib, a clinically approved proteasome inhibitor. Bortezomib-mediated sensitisation to TRAIL was associated with enhanced activation of caspase-8, -9 and -3, elevated membrane expression levels of TRAIL-R2, cytochrome c release and G2/M arrest. Knockdown of caspase-8 significantly blocked cell death induced by the combination therapy, whereas the BH3-only protein Bid was not required for induction of apoptosis. XIAP depletion increased the sensitivity of both HPV-positive and -negative cells to TRAIL alone or in combination with bortezomib. In contrast, restoration of p53 following E6 knockdown in HPV-positive cells had no effect on their sensitivity to either single or combination therapy, suggesting a p53-independent pathway for the observed response. In summary, bortezomib-mediated proteasome inhibition sensitises previously resistant HPV-positive HNSCC cells to TRAIL-induced cell death through a mechanism involving both the extrinsic and intrinsic pathways of apoptosis. The cooperative effect of these two targeted anticancer agents therefore represents a promising treatment strategy for RT/CT-resistant HPV-associated head and neck cancers.

Whole-Body Single-Cell Sequencing Reveals Transcriptional Domains in the Annelid Larval Body

PubMed Central

Achim, Kaia; Eling, Nils; Vergara, Hernando Martinez; Bertucci, Paola Yanina; Musser, Jacob; Vopalensky, Pavel; Brunet, Thibaut; Collier, Paul; Benes, Vladimir; Marioni, John C; Arendt, Detlev

2018-01-01

Abstract Animal bodies comprise diverse arrays of cells. To characterize cellular identities across an entire body, we have compared the transcriptomes of single cells randomly picked from dissociated whole larvae of the marine annelid Platynereis dumerilii. We identify five transcriptionally distinct groups of differentiated cells, each expressing a unique set of transcription factors and effector genes that implement cellular phenotypes. Spatial mapping of cells into a cellular expression atlas, and wholemount in situ hybridization of group-specific genes reveals spatially coherent transcriptional domains in the larval body, comprising, for example, apical sensory-neurosecretory cells versus neural/epidermal surface cells. These domains represent new, basic subdivisions of the annelid body based entirely on differential gene expression, and are composed of multiple, transcriptionally similar cell types. They do not represent clonal domains, as revealed by developmental lineage analysis. We propose that the transcriptional domains that subdivide the annelid larval body represent families of related cell types that have arisen by evolutionary diversification. Their possible evolutionary conservation makes them a promising tool for evo–devo research. PMID:29373712

Sensitization prevalence, antibody cross-reactivity and immunogenic peptide profile of Api g 2, the non-specific lipid transfer protein 1 of celery.

PubMed

Gadermaier, Gabriele; Hauser, Michael; Egger, Matthias; Ferrara, Rosetta; Briza, Peter; Santos, Keity Souza; Zennaro, Danila; Girbl, Tamara; Zuidmeer-Jongejan, Laurian; Mari, Adriano; Ferreira, Fatima

2011-01-01

Celery (Apium graveolens) represents a relevant allergen source that can elicit severe reactions in the adult population. To investigate the sensitization prevalence and cross-reactivity of Api g 2 from celery stalks in a Mediterranean population and in a mouse model. 786 non-randomized subjects from Italy were screened for IgE reactivity to rApi g 2, rArt v 3 (mugwort pollen LTP) and nPru p 3 (peach LTP) using an allergen microarray. Clinical data of 32 selected patients with reactivity to LTP under investigation were evaluated. Specific IgE titers and cross-inhibitions were performed in ELISA and allergen microarray. Balb/c mice were immunized with purified LTPs; IgG titers were determined in ELISA and mediator release was examined using RBL-2H3 cells. Simulated endolysosomal digestion was performed using microsomes obtained from human DCs. IgE testing showed a sensitization prevalence of 25.6% to Api g 2, 18.6% to Art v 3, and 28.6% to Pru p 3 and frequent co-sensitization and correlating IgE-reactivity was observed. 10/32 patients suffering from LTP-related allergy reported symptoms upon consumption of celery stalks which mainly presented as OAS. Considerable IgE cross-reactivity was observed between Api g 2, Art v 3, and Pru p 3 with varying inhibition degrees of individual patients' sera. Simulating LTP mono-sensitization in a mouse model showed development of more congruent antibody specificities between Api g 2 and Art v 3. Notably, biologically relevant murine IgE cross-reactivity was restricted to the latter and diverse from Pru p 3 epitopes. Endolysosomal processing of LTP showed generation of similar clusters, which presumably represent T-cell peptides. Api g 2 represents a relevant celery stalk allergen in the LTP-sensitized population. The molecule displays common B cell epitopes and endolysosomal peptides that encompass T cell epitopes with pollen and plant-food derived LTP.

Magnetic resonance imaging with superparamagnetic iron oxide fails to track the long-term fate of mesenchymal stem cells transplanted into heart.

PubMed

Ma, Ning; Cheng, Huaibing; Lu, Minjie; Liu, Qiong; Chen, Xiuyu; Yin, Gang; Zhu, Hao; Zhang, Lianfeng; Meng, Xianmin; Tang, Yue; Zhao, Shihua

2015-03-12

MRI for in vivo stem cell tracking remains controversial. Here we tested the hypothesis that MRI can track the long-term fate of the superparamagnetic iron oxide (SPIO) nanoparticles labelled mesenchymal stem cells (MSCs) following intramyocardially injection in AMI rats. MSCs (1 × 10(6)) from male rats doubly labeled with SPIO and DAPI were injected 2 weeks after myocardial infarction. The control group received cell-free media injection. In vivo serial MRI was performed at 24 hours before cell delivery (baseline), 3 days, 1, 2, and 4 weeks after cell delivery, respectively. Serial follow-up MRI demonstrated large persistent intramyocardial signal-voids representing SPIO during the follow-up of 4 weeks, and MSCs did not moderate the left ventricular dysfunction. The TUNEL analysis confirmed that MSCs engrafted underwent apoptosis. The histopathological studies revealed that the site of cell injection was infiltrated by inflammatory cells progressively and the iron-positive cells were macrophages identified by CD68 staining, but very few or no DAPI-positive stem cells at 4 weeks after cells transplantation. The presence of engrafted cells was confirmed by real-time PCR, which showed that the amount of Y-chromosome-specific SRY gene was consistent with the results. MRI may not reliably track the long-term fate of SPIO-labeled MSCs engraftment in heart.

The use of a computerized algorithm to determine single cardiac cell volumes.

PubMed

Marino, T A; Cook, L; Cook, P N; Dwyer, S J

1981-04-01

Single cardiac muscles cell volume data have been difficult to obtain, especially because the shape of a cell is quite complex. With the aid of a surface reconstruction method, a cell volume estimation algorithm has been developed that can be used on serial of cells. The cell surface is reconstructed by means of triangular tiles so that the cell is represented as a polyhedron. When this algorithm was tested on computer generated surfaces of a known volume, the difference was less than 1.6%. Serial sections of two phantoms of a known volume were also reconstructed and a comparison of the mathematically derived volumes and the computed volume estimations gave a per cent difference of between 2.8% and 4.1%. Finally cell volumes derived using conventional methods and volumes calculated using the algorithm were compared. The mean atrial muscle cell volume derived using conventional methods was 7752.7 +/- 644.7 micrometers3, while the mean computerized algorithm estimated atrial muscle cell volume was 7110.6 +/- 625.5 micrometers3. For AV bundle cells the mean cell volume obtained by conventional methods was 484.4 +/- 88.8 micrometers3 and the volume derived from the computer algorithm was 506.0 +/- 78.5 micrometers3. The differences between the volumes calculated using conventional methods and the algorithm were not significantly different.

iAB-RBC-283: A proteomically derived knowledge-base of erythrocyte metabolism that can be used to simulate its physiological and patho-physiological states.

PubMed

Bordbar, Aarash; Jamshidi, Neema; Palsson, Bernhard O

2011-07-12

The development of high-throughput technologies capable of whole cell measurements of genes, proteins, and metabolites has led to the emergence of systems biology. Integrated analysis of the resulting omic data sets has proved to be hard to achieve. Metabolic network reconstructions enable complex relationships amongst molecular components to be represented formally in a biologically relevant manner while respecting physical constraints. In silico models derived from such reconstructions can then be queried or interrogated through mathematical simulations. Proteomic profiling studies of the mature human erythrocyte have shown more proteins present related to metabolic function than previously thought; however the significance and the causal consequences of these findings have not been explored. Erythrocyte proteomic data was used to reconstruct the most expansive description of erythrocyte metabolism to date, following extensive manual curation, assessment of the literature, and functional testing. The reconstruction contains 281 enzymes representing functions from glycolysis to cofactor and amino acid metabolism. Such a comprehensive view of erythrocyte metabolism implicates the erythrocyte as a potential biomarker for different diseases as well as a 'cell-based' drug-screening tool. The analysis shows that 94 erythrocyte enzymes are implicated in morbid single nucleotide polymorphisms, representing 142 pathologies. In addition, over 230 FDA-approved and experimental pharmaceuticals have enzymatic targets in the erythrocyte. The advancement of proteomic technologies and increased generation of high-throughput proteomic data have created the need for a means to analyze these data in a coherent manner. Network reconstructions provide a systematic means to integrate and analyze proteomic data in a biologically meaning manner. Analysis of the red cell proteome has revealed an unexpected level of complexity in the functional capabilities of human erythrocyte metabolism.

Blastocentesis: a source of DNA for preimplantation genetic testing. Results from a pilot study.

PubMed

Gianaroli, Luca; Magli, M Cristina; Pomante, Alessandra; Crivello, Anna M; Cafueri, Giulia; Valerio, Marzia; Ferraretti, Anna P

2014-12-01

To investigate the presence of DNA in blastocyst fluids (BFs) and to estimate whether the chromosomal status predicted by its analysis corresponds with the ploidy condition in trophectoderm (TE) cells, the whole embryo, and that predicted by polar bodies (PBs) or blastomeres. Prospective study. In vitro fertilization unit. Seventeen couples undergoing preimplantation genetic screening with the use of array comparative genomic hybridization on PBs (n = 12) or blastomeres (n = 5). BFs and TE cells were retrieved from 51 blastocysts for separate chromosomal analysis. Presence of DNA in BFs and assessment of the corresponding chromosome condition; correlation with the results in TE cells and those predicted by the analysis done at earlier stages. DNA was detected in 39 BFs (76.5%). In 38 of 39 cases (97.4%) the ploidy condition of BFs was confirmed in TE cells, and the rate of concordance per single chromosome was 96.6% (904/936). In relation to the whole embryo, the ploidy condition corresponded in all cases with a per-chromosome concordance of 98.1%. The testing of PBs and blastomeres had 93.3% and 100% prediction of BF ploidy condition with a concordance per chromosome of 93.5% and 94%, respectively. Blastocentesis could represent an alternative source of material for chromosomal testing, because the BF is highly predictive of the embryo ploidy condition and chromosome content. Our data confirm the relevance of the oocyte and of the early-cleavage embryo in determining the ploidy condition of the resulting blastocyst. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

[Diagnostic contribution of abnormal delayed-type hypersensitivity to Candida albicans. Characterization test by activation of cells sensitized to successive dilutions of Candida].

PubMed

Brunet, J L; Cozon, G; Sainte-Laudy, J; Boissel, J P; Delair, S; Peyramond, D

1997-10-01

By measuring the activation of different cell models (lymphocytes and lymphocytic subsets) in the presence of Candida albicans with flow cytometry reading, it is possible to show that successive dilutions of Candida albicans can lead to lymphocyte activation in abnormally-sensitized subjects. In a first trial, 10 subjects were tested in duplicate. The decrease of activity of the dilutions does not appear to be regular in relation to the progression of the dilutions. The activity of the dilutions wanes relatively rapidly with the first dilutions, then recurs later very distinctly, at the 6th dilution, then ebbs, then reappears in similar manner at the 9th, the 14th, and finally, the 19th dilution. Cell reactivity appears to differ depending on the subject. It can be represented through the calculated slope of the regression line, for each series of data. It therefore appears feasible to determine a threshold of reactivity and a scale of sensitivity, to make it possible to specify the degree of abnormal reactivity existing at a given time for a given subject. The constancy of the activity of the different dilutions tested, on 10 cultures of a single cell suspension, is especially well demonstrated in the second trial, showing unusually small standard deviations. Thus, the question arises as to the exact nature of the observed phenomenon and of its analysis from a physical-chemical point of view, with regard to the pharmacological effect of successive dilutions of Candida albicans.

Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer

PubMed Central

Wolff, Antonio C.; Hammond, M. Elizabeth H.; Hicks, David G.; Dowsett, Mitch; McShane, Lisa M.; Allison, Kimberly H.; Allred, Donald C.; Bartlett, John M.S.; Bilous, Michael; Fitzgibbons, Patrick; Hanna, Wedad; Jenkins, Robert B.; Mangu, Pamela B.; Paik, Soonmyung; Perez, Edith A.; Press, Michael F.; Spears, Patricia A.; Vance, Gail H.; Viale, Giuseppe; Hayes, Daniel F.

2014-01-01

Purpose To update the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guideline recommendations for human epidermal growth factor receptor 2 (HER2) testing in breast cancer to improve the accuracy of HER2 testing and its utility as a predictive marker in invasive breast cancer. Methods ASCO/CAP convened an Update Committee that included coauthors of the 2007 guideline to conduct a systematic literature review and update recommendations for optimal HER2 testing. Results The Update Committee identified criteria and areas requiring clarification to improve the accuracy of HER2 testing by immunohistochemistry (IHC) or in situ hybridization (ISH). The guideline was reviewed and approved by both organizations. Recommendations The Update Committee recommends that HER2 status (HER2 negative or positive) be determined in all patients with invasive (early stage or recurrence) breast cancer on the basis of one or more HER2 test results (negative, equivocal, or positive). Testing criteria define HER2-positive status when (on observing within an area of tumor that amounts to >10% of contiguous and homogeneous tumor cells) there is evidence of protein overexpression (IHC) or gene amplification (HER2 copy number or HER2/CEP17 ratio by ISH based on counting at least 20 cells within the area). If results are equivocal (revised criteria), reflex testing should be performed using an alternative assay (IHC or ISH). Repeat testing should be considered if results seem discordant with other histopathologic findings. Laboratories should demonstrate high concordance with a validated HER2 test on a sufficiently large and representative set of specimens. Testing must be performed in a laboratory accredited by CAP or another accrediting entity. The Update Committee urges providers and health systems to cooperate to ensure the highest quality testing. PMID:24099077

A GRAPH PARTITIONING APPROACH TO PREDICTING PATTERNS IN LATERAL INHIBITION SYSTEMS

PubMed Central

RUFINO FERREIRA, ANA S.; ARCAK, MURAT

2017-01-01

We analyze spatial patterns on networks of cells where adjacent cells inhibit each other through contact signaling. We represent the network as a graph where each vertex represents the dynamics of identical individual cells and where graph edges represent cell-to-cell signaling. To predict steady-state patterns we find equitable partitions of the graph vertices and assign them into disjoint classes. We then use results from monotone systems theory to prove the existence of patterns that are structured in such a way that all the cells in the same class have the same final fate. To study the stability properties of these patterns, we rely on the graph partition to perform a block decomposition of the system. Then, to guarantee stability, we provide a small-gain type criterion that depends on the input-output properties of each cell in the reduced system. Finally, we discuss pattern formation in stochastic models. With the help of a modal decomposition we show that noise can enhance the parameter region where patterning occurs. PMID:29225552

Real-time garbage collection for list processing

NASA Technical Reports Server (NTRS)

Shuler, R. L., Jr. (Inventor)

1986-01-01

In a list processing system, small reference counters are maintained in conjunction with memory cells for the purpose of identifying memory cells that become available for re-use. The counters are updated as references to the cells are created and destroyed, and when a counter of a cell is decremented to logical zero the cell is immediately returned to a list of free cells. In those cases where a counter must be incremented beyond the maximum value that can be represented in a small counter, the cell is restructured so that the additional reference count can be represented. The restructuring involves allocating an additional cell, distributing counter, tag, and pointer information among the two cells, and linking both cells appropriately into the existing list structure.

A linear programming approach to reconstructing subcellular structures from confocal images for automated generation of representative 3D cellular models.

PubMed

Wood, Scott T; Dean, Brian C; Dean, Delphine

2013-04-01

This paper presents a novel computer vision algorithm to analyze 3D stacks of confocal images of fluorescently stained single cells. The goal of the algorithm is to create representative in silico model structures that can be imported into finite element analysis software for mechanical characterization. Segmentation of cell and nucleus boundaries is accomplished via standard thresholding methods. Using novel linear programming methods, a representative actin stress fiber network is generated by computing a linear superposition of fibers having minimum discrepancy compared with an experimental 3D confocal image. Qualitative validation is performed through analysis of seven 3D confocal image stacks of adherent vascular smooth muscle cells (VSMCs) grown in 2D culture. The presented method is able to automatically generate 3D geometries of the cell's boundary, nucleus, and representative F-actin network based on standard cell microscopy data. These geometries can be used for direct importation and implementation in structural finite element models for analysis of the mechanics of a single cell to potentially speed discoveries in the fields of regenerative medicine, mechanobiology, and drug discovery. Copyright © 2012 Elsevier B.V. All rights reserved.

Estimating the Impact of Earlier ART Initiation and Increased Testing Coverage on HIV Transmission among Men Who Have Sex with Men in Mexico using a Mathematical Model.

PubMed

Caro-Vega, Yanink; del Rio, Carlos; Lima, Viviane Dias; Lopez-Cervantes, Malaquias; Crabtree-Ramirez, Brenda; Bautista-Arredondo, Sergio; Colchero, M Arantxa; Sierra-Madero, Juan

2015-01-01

To estimate the impact of late ART initiation on HIV transmission among men who have sex with men (MSM) in Mexico. An HIV transmission model was built to estimate the number of infections transmitted by HIV-infected men who have sex with men (MSM-HIV+) MSM-HIV+ in the short and long term. Sexual risk behavior data were estimated from a nationwide study of MSM. CD4+ counts at ART initiation from a representative national cohort were used to estimate time since infection. Number of MSM-HIV+ on treatment and suppressed were estimated from surveillance and government reports. Status quo scenario (SQ), and scenarios of early ART initiation and increased HIV testing were modeled. We estimated 14239 new HIV infections per year from MSM-HIV+ in Mexico. In SQ, MSM take an average 7.4 years since infection to initiate treatment with a median CD4+ count of 148 cells/mm3(25th-75th percentiles 52-266). In SQ, 68% of MSM-HIV+ are not aware of their HIV status and transmit 78% of new infections. Increasing the CD4+ count at ART initiation to 350 cells/mm3 shortened the time since infection to 2.8 years. Increasing HIV testing to cover 80% of undiagnosed MSM resulted in a reduction of 70% in new infections in 20 years. Initiating ART at 500 cells/mm3 and increasing HIV testing the reduction would be of 75% in 20 years. A substantial number of new HIV infections in Mexico are transmitted by undiagnosed and untreated MSM-HIV+. An aggressive increase in HIV testing coverage and initiating ART at a CD4 count of 500 cells/mm3 in this population would significantly benefit individuals and decrease the number of new HIV infections in Mexico.

Design and testing of digitally manufactured paraffin Acrylonitrile-butadiene-styrene hybrid rocket motors

NASA Astrophysics Data System (ADS)

McCulley, Jonathan M.

This research investigates the application of additive manufacturing techniques for fabricating hybrid rocket fuel grains composed of porous Acrylonitrile-butadiene-styrene impregnated with paraffin wax. The digitally manufactured ABS substrate provides mechanical support for the paraffin fuel material and serves as an additional fuel component. The embedded paraffin provides an enhanced fuel regression rate while having no detrimental effect on the thermodynamic burn properties of the fuel grain. Multiple fuel grains with various ABS-to-Paraffin mass ratios were fabricated and burned with nitrous oxide. Analytical predictions for end-to-end motor performance and fuel regression are compared against static test results. Baseline fuel grain regression calculations use an enthalpy balance energy analysis with the material and thermodynamic properties based on the mean paraffin/ABS mass fractions within the fuel grain. In support of these analytical comparisons, a novel method for propagating the fuel port burn surface was developed. In this modeling approach the fuel cross section grid is modeled as an image with white pixels representing the fuel and black pixels representing empty or burned grid cells.

Stem cell media culture of melanoma results in the induction of a nonrepresentative neural expression profile.

PubMed

Anaka, Matthew; Freyer, Claudia; Gedye, Craig; Caballero, Otavia; Davis, Ian D; Behren, Andreas; Cebon, Jonathan

2012-02-01

The ability of cell lines to accurately represent cancer is a major concern in preclinical research. Culture of glioma cells as neurospheres in stem cell media (SCM) has been shown to better represent the genotype and phenotype of primary glioblastoma in comparison to serum cell lines. Despite the use of neurosphere-like models of many malignancies, there has been no robust analysis of whether other cancers benefit from a more representative phenotype and genotype when cultured in SCM. We analyzed the growth properties, transcriptional profile, and genotype of melanoma cells grown de novo in SCM, as while melanocytes share a common precursor with neural cells, melanoma frequently demonstrates divergent behavior in cancer stem cell assays. SCM culture of melanoma cells induced a neural lineage gene expression profile that was not representative of matched patient tissue samples and which could be induced in serum cell lines by switching them into SCM. There was no enrichment for expression of putative melanoma stem cell markers, but the SCM expression profile did overlap significantly with that of SCM cultures of glioma, suggesting that the observed phenotype is media-specific rather than melanoma-specific. Xenografts derived from either culture condition provided the best representation of melanoma in situ. Finally, SCM culture of melanoma did not prevent ongoing acquisition of DNA copy number abnormalities. In conclusion, SCM culture of melanoma does not provide a better representation of the phenotype or genotype of metastatic melanoma, and the resulting neural bias could potentially confound therapeutic target identification. Copyright © 2011 AlphaMed Press.

Flexible Lab-Tailored Cut-Offs for Suitability of Formalin-Fixed Tumor Samples for Diagnostic Mutational Analyses

PubMed Central

Mariani, Sara; Tondat, Fabrizio; Pacchioni, Donatella; Molinaro, Luca; Barreca, Antonella; Macrì, Luigia; Chiusa, Luigi; di Celle, Paola Francia; Cassoni, Paola; Sapino, Anna

2015-01-01

The selection of proper tissues from formalin-fixed and paraffin-embedded tumors before diagnostic molecular testing is responsibility of the pathologist and represents a crucial step to produce reliable test results. The international guidelines suggest two cut-offs, one for the percentage and one for the number of tumor cells, in order to enrich the tumor content before DNA extraction. The aim of the present work was two-fold: to evaluate to what extent a low percentage or absolute number of tumor cells can be qualified for somatic mutation testing; and to determine how assay sensitivities can guide pathologists towards a better definition of morphology-based adequacy cut-offs. We tested 1797 tumor specimens from melanomas, colorectal and lung adenocarcinomas. Respectively, their BRAF, K-RAS and EGFR genes were analyzed at specific exons by mutation-enriched PCR, pyrosequencing, direct sequencing and real-time PCR methods. We demonstrate that poorly cellular specimens do not modify the frequency distribution of either mutated or wild-type DNA samples nor that of specific mutations. This observation suggests that currently recommended cut-offs for adequacy of specimens to be processed for molecular assays seem to be too much stringent in a laboratory context that performs highly sensitive routine analytical methods. In conclusion, new cut-offs are needed based on test sensitivities and documented tumor heterogeneity. PMID:25844806

Sertoli-Leydig cell tumors: hormonal profile after dynamic test with GnRH analogue: triptorelin represents a useful tool to evaluate tumoral hyperandrogenism.

PubMed

Turra, J; Granzotto, M; Gallea, M; Faggian, D; Conte, L; Litta, P; Vettor, R; Mioni, R

2015-01-01

We report the case of a 15-year-old woman with signs of hyperandrogenism affected by a Sertoli-Leydig cell tumor (SLCT). In our patient, blood analysis showed a high testosterone (T) level (T: 8.53 nmol/L; nv < 1.87 nmol/L) while the GnRH-analogue test demonstrated an exaggerated secretion of 17-hydroxyprogesterone (OHP), T, and androstenedione (A) by the ovary after stimulation. We compared the GnRH-analogue test of our patient with that obtained in a group of normal and healthy women (no. 8 subjects, 16-26 years old), men (no. 4 subjects, 18-28 years old), and in a group of PCOS patients with age and body weight compared. We found in our patient a value of OHP, 17-beta estradiol (E2) and T, from 2 to 18 times higher than healthy women. When we compared our patient with healthy men, we differently observed a comparable response of T. The response of our patient was also comparable with that observed in the PCOS group for E2. During the post-surgical follow up, the GnRH-analogue test of our patient showed a response of OHP, T, and E2 comparable with that of the PCOS group. The GnRH-analogue test is a useful tool to characterize steroidogenesis in SLCT.

Minimal Residual Disease Evaluation in Childhood Acute Lymphoblastic Leukemia: An Economic Analysis

PubMed Central

Gajic-Veljanoski, O.; Pham, B.; Pechlivanoglou, P.; Krahn, M.; Higgins, Caroline; Bielecki, Joanna

2016-01-01

Background Minimal residual disease (MRD) testing by higher performance techniques such as flow cytometry and polymerase chain reaction (PCR) can be used to detect the proportion of remaining leukemic cells in bone marrow or peripheral blood during and after the first phases of chemotherapy in children with acute lymphoblastic leukemia (ALL). The results of MRD testing are used to reclassify these patients and guide changes in treatment according to their future risk of relapse. We conducted a systematic review of the economic literature, cost-effectiveness analysis, and budget-impact analysis to ascertain the cost-effectiveness and economic impact of MRD testing by flow cytometry for management of childhood precursor B-cell ALL in Ontario. Methods A systematic literature search (1998–2014) identified studies that examined the incremental cost-effectiveness of MRD testing by either flow cytometry or PCR. We developed a lifetime state-transition (Markov) microsimulation model to quantify the cost-effectiveness of MRD testing followed by risk-directed therapy to no MRD testing and to estimate its marginal effect on health outcomes and on costs. Model input parameters were based on the literature, expert opinion, and data from the Pediatric Oncology Group of Ontario Networked Information System. Using predictions from our Markov model, we estimated the 1-year cost burden of MRD testing versus no testing and forecasted its economic impact over 3 and 5 years. Results In a base-case cost-effectiveness analysis, compared with no testing, MRD testing by flow cytometry at the end of induction and consolidation was associated with an increased discounted survival of 0.0958 quality-adjusted life-years (QALYs) and increased discounted costs of $4,180, yielding an incremental cost-effectiveness ratio (ICER) of $43,613/QALY gained. After accounting for parameter uncertainty, incremental cost-effectiveness of MRD testing was associated with an ICER of $50,249/QALY gained. In the budget-impact analysis, the 1-year cost expenditure for MRD testing by flow cytometry in newly diagnosed patients with precursor B-cell ALL was estimated at $340,760. We forecasted that the province would have to pay approximately $1.3 million over 3 years and $2.4 million over 5 years for MRD testing by flow cytometry in this population. Conclusions Compared with no testing, MRD testing by flow cytometry in newly diagnosed patients with precursor B-cell ALL represents good value for money at commonly used willingness-to-pay thresholds of $50,000/QALY and $100,000/QALY. PMID:27099644

Minimal Residual Disease Evaluation in Childhood Acute Lymphoblastic Leukemia: An Economic Analysis.

PubMed

2016-01-01

Minimal residual disease (MRD) testing by higher performance techniques such as flow cytometry and polymerase chain reaction (PCR) can be used to detect the proportion of remaining leukemic cells in bone marrow or peripheral blood during and after the first phases of chemotherapy in children with acute lymphoblastic leukemia (ALL). The results of MRD testing are used to reclassify these patients and guide changes in treatment according to their future risk of relapse. We conducted a systematic review of the economic literature, cost-effectiveness analysis, and budget-impact analysis to ascertain the cost-effectiveness and economic impact of MRD testing by flow cytometry for management of childhood precursor B-cell ALL in Ontario. A systematic literature search (1998-2014) identified studies that examined the incremental cost-effectiveness of MRD testing by either flow cytometry or PCR. We developed a lifetime state-transition (Markov) microsimulation model to quantify the cost-effectiveness of MRD testing followed by risk-directed therapy to no MRD testing and to estimate its marginal effect on health outcomes and on costs. Model input parameters were based on the literature, expert opinion, and data from the Pediatric Oncology Group of Ontario Networked Information System. Using predictions from our Markov model, we estimated the 1-year cost burden of MRD testing versus no testing and forecasted its economic impact over 3 and 5 years. In a base-case cost-effectiveness analysis, compared with no testing, MRD testing by flow cytometry at the end of induction and consolidation was associated with an increased discounted survival of 0.0958 quality-adjusted life-years (QALYs) and increased discounted costs of $4,180, yielding an incremental cost-effectiveness ratio (ICER) of $43,613/QALY gained. After accounting for parameter uncertainty, incremental cost-effectiveness of MRD testing was associated with an ICER of $50,249/QALY gained. In the budget-impact analysis, the 1-year cost expenditure for MRD testing by flow cytometry in newly diagnosed patients with precursor B-cell ALL was estimated at $340,760. We forecasted that the province would have to pay approximately $1.3 million over 3 years and $2.4 million over 5 years for MRD testing by flow cytometry in this population. Compared with no testing, MRD testing by flow cytometry in newly diagnosed patients with precursor B-cell ALL represents good value for money at commonly used willingness-to-pay thresholds of $50,000/QALY and $100,000/QALY.

Methods for Analysis of Nanoparticle Immunosuppressive Properties In Vitro and In Vivo.

PubMed

Potter, Timothy M; Neun, Barry W; Dobrovolskaia, Marina A

2018-01-01

Adverse drug effects on the immune system function represent a significant concern in the pharmaceutical industry, because 10-20% of the drug withdrawal from the market is accounted to immunotoxicity. Immunosuppression is one such adverse effect. The traditional immune function test used to estimate materials' immunosuppression is a T-cell-dependent antibody response (TDAR). This method involves a 28 day in vivo study evaluating the animal's antibody titer to a known antigen (KLH) with and without challenge. Due to the limited quantities of novel drug candidates, an in vitro method called human leukocyte activation (HuLa) assay has been developed to substitute the traditional TDAR assay during early preclinical development. In this test, leukocytes isolated from healthy donors vaccinated with the current year's flu vaccine are incubated with Fluzone in the presence or absence of a test material. The antigen-specific leukocyte proliferation is then measured by ELISA analyzing incorporation of BrdU into DNA of the proliferating cells. Here, we describe the experimental procedures for investigating immunosuppressive properties of nanoparticles by both TDAR and HuLa assays, discuss the in vitro-in vivo correlation of these methods, and show a case study using the iron oxide nanoparticle formulation, Feraheme.

Skin photosensitivity as a model in photodynamic therapy

NASA Astrophysics Data System (ADS)

Richter, Anna M.; Jain, Ashok K.; Canaan, Alice J.; Meadows, Howard; Levy, Julia G.

1996-01-01

Skin photosensitivity is the most common side effect of photodynamic therapy (PDT) and in clinical situations needs to be avoided or at least minimized. However, because of the accessibility of skin tissue, skin photosensitivity represents a useful test system in vivo for evaluation of the pharmacokinetics of photosensitizers and light sources. Pig skin resembles in many aspects human skin and, therefore, is most suitable for these tests. Using pig skin photosensitivity as an end point, we evaluate the effect of cell loading with a photosensitizer, benzoporphyrin derivative (BPD verteporfin) following its intravenous administration either as a rapid bolus or slow infusion. Skin response to light activation indicated a very similar cell content of BPD. These results were in agrement with those obtained in an in vitro model. In addition, in the same pig skin photosensitivity model we compared the efficiency of activation of BPD with either laser (690 plus or minus 3 nm) or light-emitting diode (LED; 690 plus or minus 12 nm) light. Results indicated the equivalency of the two light sources in this test system, with LED light being slightly more efficient, due possibly to a fluence rate lower than laser light.

Photodynamic effects of 31 different phthalocyanines on a human keratinocyte cell line.

PubMed

Jančula, Daniel; Maršálek, Blahoslav; Babica, Pavel

2013-10-01

Phthalocyanines (Pcs, colored macromolecular compounds with the ability to generate singlet oxygen) represent a promising group of photosensitizers due to their intense absorption in the red and UV portion of the spectrum which leads to their excitation. In order to characterize possible toxic effects associated with eventual practical use and application of these chemicals, we employed an in vitro cell culture model to evaluate cytotoxic effects of 31 different phthalocyanines using neutral red uptake assay. An immortalized human keratinocyte cell line HaCaT was exposed to the tested chemicals for 2 or 24h, either with or without illumination in the last 60 min of the exposure period. After 2- or 24-h exposure without illumination, no cytotoxic effects or weak cytotoxic effects were induced by any Pc under the study and EC50 values could not be obtained within the tested concentration ranges (1.25-20 mg L(-1) or 0.625-10 mg L(-1)). On the other hand, exposure to phthalocyanines under illumination induced a significant cytotoxic effect. The most pronounced cytotoxicity was elicited by Pcs previously shown to have high positive charge densities at peripheral parts of substituent groups, which is most likely the factor responsible for the binding of Pc to negatively charged membranes on the cell surface and thus guaranteeing the tight connection necessary for the singlet oxygen attack on the cell surface. Copyright © 2013 Elsevier Ltd. All rights reserved.

A revisionist history of adult marrow stem cell biology or 'they forgot about the discard'.

PubMed

Quesenberry, P; Goldberg, L

2017-08-01

The adult marrow hematopoietic stem cell biology has largely been based on studies of highly purified stem cells. This is unfortunate because during the stem cell purification the great bulk of stem cells are discarded. These cells are actively proliferating. The final purified stem cell is dormant and not representative of the whole stem cell compartment. Thus, a large number of studies on the cellular characteristics, regulators and molecular details of stem cells have been carried on out of non-represented cells. Niche studies have largely pursued using these purified stem cells and these are largely un-interpretable. Other considerations include the distinction between baseline and transplant stem cells and the modulation of stem cell phenotype by extracellular vesicles, to cite a non-inclusive list. Work needs to proceed on characterizing the true stem cell population.

Mitochondrial filaments and clusters as intracellular power-transmitting cables.

PubMed

Skulachev, V P

2001-01-01

Mitochondria exist in two interconverting forms; as small isolated particles, and as extended filaments, networks or clusters connected with intermitochondrial junctions. Extended mitochondria can represent electrically united systems, which can facilitate energy delivery from the cell periphery to the cell core and organize antioxidant defence of the cell interior when O2 is consumed by mitochondrial clusters near the the outer cell membrane, and protonic potential is transmitted to the cell core mitochondria to form ATP. As to small mitochondria, they might represent a transportable form of these organelles.

Atypical Fibroxanthoma Revisited.

PubMed

Mentzel, Thomas; Requena, Luis; Brenn, Thomas

2017-06-01

Atypical fibroxanthoma (AFX) represents a rare mesenchymal neoplasm arising predominantly in the head and neck area of elderly patients. Clinically, the neoplasm is characterized by a rapid and exophytic growth with frequent ulceration of the epidermis. Histopathologically, AFX represents a well-circumscribed, dermal-based neoplasm composed of a variable admixture of large histiocytoid cells, enlarged spindled and epithelioid tumor cells, and multinucleated tumor giant cells with bizarre and pleomorphic nuclei. If strict diagnostic criteria are applied, the clinical behavior of AFX is benign in most cases, and complete excision represents the treatment of choice. Copyright © 2017 Elsevier Inc. All rights reserved.

[Perspectives of cell therapy in sequelae from cerebrovascular accidents].

PubMed

Otero, Laura; Zurita, Mercedes; Bonilla, Celia; Aguayo, Concepción; Rico, Miguel Angel; Vaquero, Jesús

2012-09-01

Spontaneous intracerebral hemorrhage (ICH) is associated with mortality between 40 and 50% of cases. Among the survivors, only 10% are independent after one month, there is no effective treatment of sequelae, except for the limited possibilities providing for rehabilitation. We review the current experience with intracerebral transplantation of mesenchymal stem cells (MSCs) obtained from bone marrow as a potential treatment of neurological sequelae occurring after experimental ICH. We describe the model of ICH by intracerebral administration of collagenaseIV at basal ganglia level in Wistar rats. Neurological deficits caused by ICH can be quantified through a variety of functional assessment test (NMSS, Rota-rod, VTB-test). 5×10allogeneic MSCs in 10μl of saline were administered intracerebrally in 10 animals, 2 months after ICH. In another 10 animals (controls) the same volume of saline was administered. Changes in the functional deficits were assessed during the next 6 months in both experimental groups. The results suggested therapeutic efficacy of MSCs transplantation and showed that transplanted stem cells can survive in the injured brain, transforming into neurons and glial cells. This form of cell therapy induces reactivation of endogenous neurogenesis at the subventricular zone (SVZ) and achieves antiapoptotic protective effect in the injured brain. Cell therapy represents an important field of research with potential clinical application to treatment of neurological sequels, currently considered irreversible. Neurosurgeons should become involved in the development of these new techniques that are likely to shape the future of this specialty. Copyright © 2011 Sociedad Española de Neurocirugía. Published by Elsevier España. All rights reserved.

Energy and Technology Review

NASA Astrophysics Data System (ADS)

Poggio, Andrew J.

1988-10-01

This issue of Energy and Technology Review contains: Neutron Penumbral Imaging of Laser-Fusion Targets--using our new penumbral-imaging diagnostic, we have obtained the first images that can be used to measure directly the deuterium-tritium burn region in laser-driven fusion targets; Computed Tomography for Nondestructive Evaluation--various computed tomography systems and computational techniques are used in nondestructive evaluation; Three-Dimensional Image Analysis for Studying Nuclear Chromatin Structure--we have developed an optic-electronic system for acquiring cross-sectional views of cell nuclei, and computer codes to analyze these images and reconstruct the three-dimensional structures they represent; Imaging in the Nuclear Test Program--advanced techniques produce images of unprecedented detail and resolution from Nevada Test Site data; and Computational X-Ray Holography--visible-light experiments and numerically simulated holograms test our ideas about an X-ray microscope for biological research.

Engraftment and reversal of diabetes after intramuscular transplantation of neonatal porcine islet-like clusters.

PubMed

Wolf-van Buerck, Lelia; Schuster, Marion; Baehr, Andrea; Mayr, Tanja; Guethoff, Sonja; Abicht, Jan; Reichart, Bruno; Nam-Apostolopoulos, Yun-Chung; Klymiuk, Nikolai; Wolf, Eckhard; Seissler, Jochen

2015-01-01

Intraportal infusion is currently the method of choice for clinical islet cell transplantation but suffers from poor efficacy. As the liver may not represent an optimal transplantation site for Langerhans islets, we examined the potential of neonatal porcine islet-like clusters (NPICCs) to engraft in skeletal muscle as an alternative transplantation site. Neonatal porcine islet-like clusters were isolated from 2- to 5-day-old piglets and either transplanted under the kidney capsule (s.k.) or injected into the lower hindlimb muscle (i.m.) of streptozotocin-diabetic NOD-SCID IL2rγ(-/-) (NSG) mice. Survival, vascularization, maturation, and functional activity were analyzed by intraperitoneal glucose tolerance testing and immunohistochemical analyses. Intramuscular transplantation of NPICCs resulted in development of normoglycemia and restored glucose homeostasis. Time to reversal of diabetes and glucose tolerance (AUC glucose and AUC insulin) did not significantly differ as compared to s.k. transplantation. Intramuscular grafts exhibited rapid neovascularization and graft composition with cytokeratin-positive ductal cells and beta cells at post-transplant weeks 2 and 8 and after establishment of normoglycemia was comparable in both groups. Intramuscular injection represents a minimally invasive but efficient alternative for transplantation of NPICCs and, thus, offers an attractive alternative site for xenotransplantation approaches. These findings may have important implications for improving the outcome and the monitoring of pig islet xenotransplantation. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

An anthocyanin-rich strawberry extract protects against oxidative stress damage and improves mitochondrial functionality in human dermal fibroblasts exposed to an oxidizing agent.

PubMed

Giampieri, Francesca; Alvarez-Suarez, José M; Mazzoni, Luca; Forbes-Hernandez, Tamara Y; Gasparrini, Massimiliano; Gonzàlez-Paramàs, Ana M; Santos-Buelga, Celestino; Quiles, Josè L; Bompadre, Stefano; Mezzetti, Bruno; Battino, Maurizio

2014-08-01

This study investigates the protective effect of the Sveva strawberry polyphenol-rich extract on human dermal fibroblasts against AAPH-induced oxidative stress. The HPLC-DAD/ESI-MS analysis was used for evaluating the phenolic composition of the fruits. Sveva strawberry presented a high anthocyanin content (639.79 mg per kg fresh fruit), representing ∼86.08% of the total phenolic content, with Pg-3-glc as the most abundant representative (611.18 mg per kg fresh fruit). Only one ellagitannin (agrimoniin) was identified, while two quercetins, three kaempherol derivates, and three ellagic acid derivatives were detected and quantified. Strawberry pre-treatment (0.5 mg ml(-1)) markedly increased human dermal fibroblast viability, with a significant reduction of apoptotic and dead cells, and suppressed AAPH-induced ROS generation, after only 30 minutes of incubation with the oxidizing agent, and lipid peroxidation, against a range of AAPH concentrations tested. Notably, the strawberry extract also improved the mitochondrial functionality: the basal respiratory performance after treatment was ∼1.59-fold higher compared to control cells, while pre-treatment with strawberry extract before oxidative damage increased ∼2.70-fold compared to stressed cells. Our results confirm that the strawberry possesses antioxidant properties, and may be useful for the prevention of free radical-induced skin damage.

Hematopoietic and mesenchymal stem cells for the treatment of chronic respiratory diseases: role of plasticity and heterogeneity.

PubMed

Conese, Massimo; Piro, Donatella; Carbone, Annalucia; Castellani, Stefano; Di Gioia, Sante

2014-01-01

Chronic lung diseases, such as cystic fibrosis (CF), asthma, and chronic obstructive pulmonary disease (COPD) are incurable and represent a very high social burden. Stem cell-based treatment may represent a hope for the cure of these diseases. In this paper, we revise the overall knowledge about the plasticity and engraftment of exogenous marrow-derived stem cells into the lung, as well as their usefulness in lung repair and therapy of chronic lung diseases. The lung is easily accessible and the pathophysiology of these diseases is characterized by injury, inflammation, and eventually by remodeling of the airways. Bone marrow-derived stem cells, including hematopoietic stem/progenitor cells (HSPCs) and mesenchymal stromal (stem) cells (MSCs), encompass a wide array of cell subsets with different capacities of engraftment and injured tissue regenerating potential. Proof-of-principle that marrow cells administered locally may engraft and give rise to specialized epithelial cells has been given, but the efficiency of this conversion is too limited to give a therapeutic effect. Besides the identification of plasticity mechanisms, the characterization/isolation of the stem cell subpopulations represents a major challenge to improving the efficacy of transplantation protocols used in regenerative medicine for lung diseases.

Detection of viability of micro-algae cells by optofluidic hologram pattern.

PubMed

Wang, Junsheng; Yu, Xiaomei; Wang, Yanjuan; Pan, Xinxiang; Li, Dongqing

2018-03-01

A rapid detection of micro-algae activity is critical for analysis of ship ballast water. A new method for detecting micro-algae activity based on lens-free optofluidic holographic imaging is presented in this paper. A compact lens-free optofluidic holographic imaging device was developed. This device is mainly composed of a light source, a small through-hole, a light propagation module, a microfluidic chip, and an image acquisition and processing module. The excited light from the light source passes through a small hole to reach the surface of the micro-algae cells in the microfluidic chip, and a holographic image is formed by the diffraction light of surface of micro-algae cells. The relation between the characteristics in the hologram pattern and the activity of micro-algae cells was investigated by using this device. The characteristics of the hologram pattern were extracted to represent the activity of micro-algae cells. To demonstrate the accuracy of the presented method and device, four species of micro-algae cells were employed as the test samples and the comparison experiments between the alive and dead cells of four species of micro-algae were conducted. The results show that the developed method and device can determine live/dead microalgae cells accurately.