Biocompatible coated magnetosome minerals with various organization and cellular interaction properties induce cytotoxicity towards RG-2 and GL-261 glioma cells in the presence of an alternating magnetic field.

PubMed

Hamdous, Yasmina; Chebbi, Imène; Mandawala, Chalani; Le Fèvre, Raphael; Guyot, François; Seksek, Olivier; Alphandéry, Edouard

2017-10-17

Biologics magnetics nanoparticles, magnetosomes, attract attention because of their magnetic characteristics and potential applications. The aim of the present study was to develop and characterize novel magnetosomes, which were extracted from magnetotactic bacteria, purified to produce apyrogen magnetosome minerals, and then coated with Chitosan, Neridronate, or Polyethyleneimine. It yielded stable magnetosomes designated as M-Chi, M-Neri, and M-PEI, respectively. Nanoparticle biocompatibility was evaluated on mouse fibroblast cells (3T3), mouse glioblastoma cells (GL-261) and rat glioblastoma cells (RG-2). We also tested these nanoparticles for magnetic hyperthermia treatment of tumor in vitro on two tumor cell lines GL-261 and RG-2 under the application of an alternating magnetic field. Heating, efficacy and internalization properties were then evaluated. Nanoparticles coated with chitosan, polyethyleneimine and neridronate are apyrogen, biocompatible and stable in aqueous suspension. The presence of a thin coating in M-Chi and M-PEI favors an arrangement in chains of the magnetosomes, similar to that observed in magnetosomes directly extracted from magnetotactic bacteria, while the thick matrix embedding M-Neri leads to structures with an average thickness of 3.5 µm 2 per magnetosome mineral. In the presence of GL-261 cells and upon the application of an alternating magnetic field, M-PEI and M-Chi lead to the highest specific absorption rates of 120-125 W/g Fe . Furthermore, while M-Chi lead to rather low rates of cellular internalization, M-PEI strongly associate to cells, a property modulated by the application of an alternating magnetic field. Coating of purified magnetosome minerals can therefore be chosen to control the interactions of nanoparticles with cells, organization of the minerals, as well as heating and cytotoxicity properties, which are important parameters to be considered in the design of a magnetic hyperthermia treatment of tumor.

Investigation of welded interconnection of large area wraparound contacted silicon solar cells

NASA Technical Reports Server (NTRS)

Lott, D. R.

1984-01-01

An investigation was conducted to evaluate the welding and temperature cycle testing of large area 5.9 x 5.9 wraparound silicon solar cells utilizing printed circuit substrates with SSC-155 interconnect copper metals and the LMSC Infrared Controlled weld station. An initial group of 5 welded modules containing Phase 2 developmental 5.9 x 5.9 cm cells were subjected to cyclical temperatures of + or 80 C at a rate of 120 cycles per day. Anomalies were noted in the adhesion of the cell contact metallization; therefore, 5 additional modules were fabricated and tested using available Phase I cells with demonstrated contact integrity. Cycling of the later module type through 12,000 cycles indicated the viability of this type of lightweight flexible array concept. This project demonstrated acceptable use of an alternate interconnect copper in combination with large area wraparound cells and emphasized the necessity to implement weld pull as opposed to solder pull procedures at the cell vendors for cells that will be interconnected by welding.

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.

DNA Duplex-Based Photodynamic Molecular Beacon for Targeted Killing of Retinoblastoma Cell.

PubMed

Wei, Yanchun; Lu, Cuixia; Chen, Qun; Xing, Da

2016-11-01

Retinoblastoma (RB) is the most common primary intraocular malignancy of infancy. An alternative RB treatment protocol is proposed and tested. It is based on a photodynamic therapy (PDT) with a designed molecular beacon that specifically targets the murine double minute x (MDMX) high-expressed RB cells. A MDMX mRNA triggered photodynamic molecular beacon is designed by binding a photosensitizer molecule (pyropheophorbide-a, or PPa) and a black hole quencher-3 (BHQ3) through a complementary oligonucleotide sequence. Cells with and without MDMX high-expression are incubated with the beacon and then irradiated with a laser. The fluorescence and reactive oxygen species are detected in solution to verify the specific activation of PPa by the perfectly matched DNA targets. The cell viabilities are evaluated with CCK-8 and flow cytometry assay. The fluorescence and photo-cytoxicity of PPa is recovered and significantly higher in the MDMX high-expressed Y79 and WERI-Rb1 cells, compared to that with the MDMX low-expressed cells. The synthesized beacon exhibits high PDT efficiency toward MDMX high-expressed RB cells. The data suggest that the designed beacon may provide a potential alternative for RB therapy and secures the ground for future investigation.

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.

Comprehensive analysis of alternative splicing and functionality in neuronal differentiation of P19 cells.

PubMed

Suzuki, Hitoshi; Osaki, Ken; Sano, Kaori; Alam, A H M Khurshid; Nakamura, Yuichiro; Ishigaki, Yasuhito; Kawahara, Kozo; Tsukahara, Toshifumi

2011-02-18

Alternative splicing, which produces multiple mRNAs from a single gene, occurs in most human genes and contributes to protein diversity. Many alternative isoforms are expressed in a spatio-temporal manner, and function in diverse processes, including in the neural system. The purpose of the present study was to comprehensively investigate neural-splicing using P19 cells. GeneChip Exon Array analysis was performed using total RNAs purified from cells during neuronal cell differentiation. To efficiently and readily extract the alternative exon candidates, 9 filtering conditions were prepared, yielding 262 candidate exons (236 genes). Semiquantitative RT-PCR results in 30 randomly selected candidates suggested that 87% of the candidates were differentially alternatively spliced in neuronal cells compared to undifferentiated cells. Gene ontology and pathway analyses suggested that many of the candidate genes were associated with neural events. Together with 66 genes whose functions in neural cells or organs were reported previously, 47 candidate genes were found to be linked to 189 events in the gene-level profile of neural differentiation. By text-mining for the alternative isoform, distinct functions of the isoforms of 9 candidate genes indicated by the result of Exon Array were confirmed. Alternative exons were successfully extracted. Results from the informatics analyses suggested that neural events were primarily governed by genes whose expression was increased and whose transcripts were differentially alternatively spliced in the neuronal cells. In addition to known functions in neural cells or organs, the uninvestigated alternative splicing events of 11 genes among 47 candidate genes suggested that cell cycle events are also potentially important. These genes may help researchers to differentiate the roles of alternative splicing in cell differentiation and cell proliferation.

Low-cost, disposable microfluidics device for blood plasma extraction using continuously alternating paramagnetic and diamagnetic capture modes

PubMed Central

Kim, Pilkee; Ong, Eng Hui; Yoon, Yong-Jin; Ng, Sum Huan Gary; Puttachat, Khuntontong

2016-01-01

Blood plasma contains biomarkers and substances that indicate the physiological state of an organism, and it can be used to diagnose various diseases or body condition. To improve the accuracy of diagnostic test, it is required to obtain the high purity of blood plasma. This paper presents a low-cost, disposable microfluidics device for blood plasma extraction using magnetophoretic behaviors of blood cells. This device uses alternating magnetophoretic capture modes to trap and separate paramagnetic and diamagnetic cells away from blood plasma. The device system is composed of two parts, a disposable microfluidics chip and a non-disposable (reusable) magnetic field source. Such modularized device helps the structure of the disposable part dramatically simplified, which is beneficial for low-cost mass production. A series of numerical simulation and parametric study have been performed to describe the mechanism of blood cell separation in the microchannel, and the results are discussed. Furthermore, experimental feasibility test has been carried out in order to demonstrate the blood plasma extraction process of the proposed device. In this experiment, pure blood plasma has been successfully extracted with yield of 21.933% from 75 μl 1:10 dilution of deoxygenated blood. PMID:27042252

Good cell culture practices &in vitro toxicology.

PubMed

Eskes, Chantra; Boström, Ann-Charlotte; Bowe, Gerhard; Coecke, Sandra; Hartung, Thomas; Hendriks, Giel; Pamies, David; Piton, Alain; Rovida, Costanza

2017-12-01

Good Cell Culture Practices (GCCP) is of high relevance to in vitro toxicology. The European Society of Toxicology In Vitro (ESTIV), the Center for Alternatives for Animal Testing (CAAT) and the In Vitro Toxicology Industrial Platform (IVTIP) joined forces to address by means of an ESTIV 2016 pre-congress session the different aspects and applications of GCCP. The covered aspects comprised the current status of the OECD guidance document on Good In Vitro Method Practices, the importance of quality assurance for new technological advances in in vitro toxicology including stem cells, and the optimized implementation of Good Manufacturing Practices and Good Laboratory Practices for regulatory testing purposes. General discussions raised the duality related to the difficulties in implementing GCCP in an academic innovative research framework on one hand, and on the other hand, the need for such GCCP principles in order to ensure reproducibility and robustness of in vitro test methods for toxicity testing. Indeed, if good cell culture principles are critical to take into consideration for all uses of in vitro test methods for toxicity testing, the level of application of such principles may depend on the stage of development of the test method as well as on the applications of the test methods, i.e., academic innovative research vs. regulatory standardized test method. Copyright © 2017 Elsevier Ltd. All rights reserved.

Comparison of primary human fibroblasts and keratinocytes with immortalized cell lines regarding their sensitivity to sodium dodecyl sulfate in a neutral red uptake cytotoxicity assay.

PubMed

Olschläger, Veronika; Schrader, Andreas; Hockertz, Stefan

2009-01-01

Cell lines present a valuable tool for in vitro assessment of skin damage caused by application of cosmeticals or pharmaceuticals. They form a reproducible test system under controllable test conditions and, in many cases, can be used as alternatives to animal testing in order to assess the compatibility of drugs or cosmetics and human skin. Yet, it can not necessarily be assumed that the behavior of cultured cells, when treated with different substances, is exactly consistent with the behavior of cells being part of a live organism. Becoming immortal, cells exhibit changes in genotype and/or phenotype, possibly resulting in modified reactions to external influences. Therefore, to obtain results close to in vivo studies, it seems apparent to use primary cells for testing that have not yet undergone any modifications. To compare the properties of primary fibroblasts (Normal Human Dermal Fibroblasts, NHDF) and primary keratinocytes (Normal Human Epidermal Keratinocytes, NHEK) with those of immortal cell lines (3T3 (ACC 173) Swiss albino mouse fibroblasts and HaCaT (human, adult, low calcium, high temperature, human adult skin keratinocytes) cells), their sensitivities in cytotoxicity assays have been assessed. While both fibroblast cell cultures showed similar sensitivities towards sodium dodecyl sulfate (SDS), primary keratinocytes died at SDS concentrations about three times lower than the immortal HaCaT cells.

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.

Reference compounds for alternative test methods to indicate developmental neurotoxicity (DNT) potential of chemicals: example lists and criteria for their selection and use.

PubMed

Aschner, Michael; Ceccatelli, Sandra; Daneshian, Mardas; Fritsche, Ellen; Hasiwa, Nina; Hartung, Thomas; Hogberg, Helena T; Leist, Marcel; Li, Abby; Mundi, William R; Padilla, Stephanie; Piersma, Aldert H; Bal-Price, Anna; Seiler, Andrea; Westerink, Remco H; Zimmer, Bastian; Lein, Pamela J

2017-01-01

There is a paucity of information concerning the developmental neurotoxicity (DNT) hazard posed by industrial and environmental chemicals. New testing approaches will most likely be based on batteries of alternative and complementary (non-animal) tests. As DNT is assumed to result from the modulation of fundamental neurodevelopmental processes (such as neuronal differentiation, precursor cell migration or neuronal network formation) by chemicals, the first generation of alternative DNT tests target these processes. The advantage of such types of assays is that they capture toxicants with multiple targets and modes-of-action. Moreover, the processes modelled by the assays can be linked to toxicity endophenotypes, i.e., alterations in neural connectivity that form the basis for neurofunctional deficits in man. The authors of this review convened in a workshop to define criteria for the selection of positive/negative controls, to prepare recommendations on their use, and to initiate the setup of a directory of reference chemicals. For initial technical optimization of tests, a set of > 50 endpoint-specific control compounds was identified. For further test development, an additional "test" set of 33 chemicals considered to act directly as bona fide DNT toxicants is proposed, and each chemical is annotated to the extent it fulfills these criteria. A tabular compilation of the original literature used to select the test set chemicals provides information on statistical procedures, and toxic/non-toxic doses (both for pups and dams). Suggestions are provided on how to use the > 100 compounds (including negative controls) compiled here to address specificity, adversity and use of alternative test systems.

Alternating air-medium exposure in rotating bioreactors optimizes cell metabolism in 3D novel tubular scaffold polyurethane foams.

PubMed

Tresoldi, Claudia; Stefani, Ilaria; Ferracci, Gaia; Bertoldi, Serena; Pellegata, Alessandro F; Farè, Silvia; Mantero, Sara

2017-04-26

In vitro dynamic culture conditions play a pivotal role in developing engineered tissue grafts, where the supply of oxygen and nutrients, and waste removal must be permitted within construct thickness. For tubular scaffolds, mass transfer is enhanced by introducing a convective flow through rotating bioreactors with positive effects on cell proliferation, scaffold colonization and extracellular matrix deposition. We characterized a novel polyurethane-based tubular scaffold and investigated the impact of 3 different culture configurations over cell behavior: dynamic (i) single-phase (medium) rotation and (ii) double-phase exposure (medium-air) rotation; static (iii) single-phase static culture as control. A new mixture of polyol was tested to create polyurethane foams (PUFs) as 3D scaffold for tissue engineering. The structure obtained was morphologically and mechanically analyzed tested. Murine fibroblasts were externally seeded on the novel porous PUF scaffold, and cultured under different dynamic conditions. Viability assay, DNA quantification, SEM and histological analyses were performed at different time points. The PUF scaffold presented interesting mechanical properties and morphology adequate to promote cell adhesion, highlighting its potential for tissue engineering purposes. Results showed that constructs under dynamic conditions contain enhanced viability and cell number, exponentially increased for double-phase rotation; under this last configuration, cells uniformly covered both the external surface and the lumen. The developed 3D structure combined with the alternated exposure to air and medium provided the optimal in vitro biochemical conditioning with adequate nutrient supply for cells. The results highlight a valuable combination of material and dynamic culture for tissue engineering applications.

ECVAM and new technologies for toxicity testing.

PubMed

Bouvier d'Yvoire, Michel; Bremer, Susanne; Casati, Silvia; Ceridono, Mara; Coecke, Sandra; Corvi, Raffaella; Eskes, Chantra; Gribaldo, Laura; Griesinger, Claudius; Knaut, Holger; Linge, Jens P; Roi, Annett; Zuang, Valérie

2012-01-01

The development of alternative empirical (testing) and non-empirical (non-testing) methods to traditional toxicological tests for complex human health effects is a tremendous task. Toxicants may potentially interfere with a vast number of physiological mechanisms thereby causing disturbances on various levels of complexity of human physiology. Only a limited number of mechanisms relevant for toxicity ('pathways' of toxicity) have been identified with certainty so far and, presumably, many more mechanisms by which toxicants cause adverse effects remain to be identified. Recapitulating in empirical model systems (i.e., in vitro test systems) all those relevant physiological mechanisms prone to be disturbed by toxicants and relevant for causing the toxicity effect in question poses an enormous challenge. First, the mechanism(s) of action of toxicants in relation to the most relevant adverse effects of a specific human health endpoint need to be identified. Subsequently, these mechanisms need to be modeled in reductionist test systems that allow assessing whether an unknown substance may operate via a specific (array of) mechanism(s). Ideally, such test systems should be relevant for the species of interest, i.e., based on human cells or modeling mechanisms present in humans. Since much of our understanding about toxicity mechanisms is based on studies using animal model systems (i.e., experimental animals or animal-derived cells), designing test systems that model mechanisms relevant for the human situation may be limited by the lack of relevant information from basic research. New technologies from molecular biology and cell biology, as well as progress in tissue engineering, imaging techniques and automated testing platforms hold the promise to alleviate some of the traditional difficulties associated with improving toxicity testing for complex endpoints. Such new technologies are expected (1) to accelerate the identification of toxicity pathways with human relevance that need to be modeled in test methods for toxicity testing (2) to enable the reconstruction of reductionist test systems modeling at a reduced level of complexity the target system/organ of interest (e.g., through tissue engineering, use of human-derived cell lines and stem cells etc.), (3) to allow the measurement of specific mechanisms relevant for a given health endpoint in such test methods (e.g., through gene and protein expression, changes in metabolites, receptor activation, changes in neural activity etc.), (4) to allow to measure toxicity mechanisms at higher throughput rates through the use of automated testing. In this chapter, we discuss the potential impact of new technologies on the development, optimization and use of empirical testing methods, grouped according to important toxicological endpoints. We highlight, from an ECVAM perspective, the areas of topical toxicity, skin absorption, reproductive and developmental toxicity, carcinogenicity/genotoxicity, sensitization, hematopoeisis and toxicokinetics and discuss strategic developments including ECVAM's database service on alternative methods. Neither the areas of toxicity discussed nor the highlighted new technologies represent comprehensive listings which would be an impossible endeavor in the context of a book chapter. However, we feel that these areas are of utmost importance and we predict that new technologies are likely to contribute significantly to test development in these fields. We summarize which new technologies are expected to contribute to the development of new alternative testing methods over the next few years and point out current and planned ECVAM projects for each of these areas.

Validation of analytical methods in GMP: the disposable Fast Read 102® device, an alternative practical approach for cell counting

PubMed Central

2012-01-01

Background The quality and safety of advanced therapy products must be maintained throughout their production and quality control cycle to ensure their final use in patients. We validated the cell count method according to the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use and European Pharmacopoeia, considering the tests’ accuracy, precision, repeatability, linearity and range. Methods As the cell count is a potency test, we checked accuracy, precision, and linearity, according to ICH Q2. Briefly our experimental approach was first to evaluate the accuracy of Fast Read 102® compared to the Bürker chamber. Once the accuracy of the alternative method was demonstrated, we checked the precision and linearity test only using Fast Read 102®. The data were statistically analyzed by average, standard deviation and coefficient of variation percentages inter and intra operator. Results All the tests performed met the established acceptance criteria of a coefficient of variation of less than ten percent. For the cell count, the precision reached by each operator had a coefficient of variation of less than ten percent (total cells) and under five percent (viable cells). The best range of dilution, to obtain a slope line value very similar to 1, was between 1:8 and 1:128. Conclusions Our data demonstrated that the Fast Read 102® count method is accurate, precise and ensures the linearity of the results obtained in a range of cell dilution. Under our standard method procedures, this assay may thus be considered a good quality control method for the cell count as a batch release quality control test. Moreover, the Fast Read 102® chamber is a plastic, disposable device that allows a number of samples to be counted in the same chamber. Last but not least, it overcomes the problem of chamber washing after use and so allows a cell count in a clean environment such as that in a Cell Factory. In a good manufacturing practice setting the disposable cell counting devices will allow a single use of the count chamber they can then be thrown away, thus avoiding the waste disposal of vital dye (e.g. Trypan Blue) or lysing solution (e.g. Tuerk solution). PMID:22650233

Cross-reactivity and tolerability of aztreonam and cephalosporins in subjects with a T cell-mediated hypersensitivity to penicillins.

PubMed

Romano, Antonino; Gaeta, Francesco; Valluzzi, Rocco Luigi; Maggioletti, Michela; Caruso, Cristiano; Quaratino, Donato

2016-07-01

The few studies performed in adults with T cell-mediated hypersensitivity to penicillins have found a rate of cross-reactivity with cephalosporins ranging from 2.8% to 31.2% and an absence of cross-reactivity with aztreonam. We sought to evaluate the possibility of using cephalosporins and aztreonam in subjects with documented delayed hypersensitivity to penicillins who especially require them. We conducted a prospective study of 214 consecutive subjects who had 307 nonimmediate reactions to penicillins (almost exclusively aminopenicillins) and had positive patch test and/or delayed-reading skin test responses to at least 1 penicillin reagent. To assess cross-reactivity with cephalosporins and aztreonam and the tolerability of such alternative β-lactams, all subjects underwent skin tests with cephalexin, cefaclor, cefadroxil, cefuroxime, ceftriaxone, and aztreonam. Subjects with negative responses were challenged with the alternative β-lactams concerned. All subjects had negative skin test results to cefuroxime, ceftriaxone, and aztreonam and tolerated challenges. Forty (18.7%) of the 214 subjects had positive skin test responses to at least 1 aminocephalosporin. Of the 174 subjects with negative responses, 170 underwent challenges; 1 reacted to cefaclor. These data demonstrate a rate of cross-reactivity between aminopenicillins and aminocephalosporins (ie, cephalexin, cefaclor, and cefadroxil) of around 20%, as well as the absence of cross-reactivity between penicillins and cefuroxime, ceftriaxone, and aztreonam in all subjects with T cell-mediated hypersensitivity to penicillins, almost exclusively aminopenicillins. Therefore these subjects could be treated with cefuroxime, ceftriaxone, and aztreonam. In those who especially require cephalosporin or aztreonam treatment, however, we recommend pretreatment skin tests because negative responses indicate tolerability. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

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

Therapeutic Targeting of Alternative Translation Initiation in Breast Cancer

DTIC Science & Technology

2009-04-01

investigation within the next 6 months. Cell type specific cancer cell killing of the prototype oncolytic poliovirus , PVS-RIPO, depends on selective...demanded by FDA. 15. SUBJECT TERMS Translation, eIF4E, eIF4G, IRES, Cancer, Poliovirus 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18...genetically recombinant poliovirus . Moreover, my work has laid the groundwork for correlative testing and efficacy studies of a vast array of protein kinase

76 FR 1598 - Submission for OMB Review; Comment Request

Federal Register 2010, 2011, 2012, 2013, 2014

2011-01-11

... purpose of the Cell Phone and Debit Card Test is to research alternative survey designs that could....S. Census Bureau. Title: National Survey of Fishing, Hunting, and Wildlife-Associated Recreation... clearance number 1018-0088) the 2011 National Survey of Fishing, Hunting, and Wildlife-Associated Recreation...

Transportation-Related Consumer Preference Data | Transportation Research |

Science.gov Websites

NREL Transportation-Related Consumer Preference Data Transportation-Related Consumer Preference Data Photo of reporters test driving Toyota prototype electric and fuel cell vehicles. Consumer consumer preference data related to alternative fuel and advanced vehicles and their effects on energy

Differential diagnosis of lung carcinoma with three-dimensional quantitative molecular vibrational imaging

NASA Astrophysics Data System (ADS)

Gao, Liang; Hammoudi, Ahmad A.; Li, Fuhai; Thrall, Michael J.; Cagle, Philip T.; Chen, Yuanxin; Yang, Jian; Xia, Xiaofeng; Fan, Yubo; Massoud, Yehia; Wang, Zhiyong; Wong, Stephen T. C.

2012-06-01

The advent of molecularly targeted therapies requires effective identification of the various cell types of non-small cell lung carcinomas (NSCLC). Currently, cell type diagnosis is performed using small biopsies or cytology specimens that are often insufficient for molecular testing after morphologic analysis. Thus, the ability to rapidly recognize different cancer cell types, with minimal tissue consumption, would accelerate diagnosis and preserve tissue samples for subsequent molecular testing in targeted therapy. We report a label-free molecular vibrational imaging framework enabling three-dimensional (3-D) image acquisition and quantitative analysis of cellular structures for identification of NSCLC cell types. This diagnostic imaging system employs superpixel-based 3-D nuclear segmentation for extracting such disease-related features as nuclear shape, volume, and cell-cell distance. These features are used to characterize cancer cell types using machine learning. Using fresh unstained tissue samples derived from cell lines grown in a mouse model, the platform showed greater than 97% accuracy for diagnosis of NSCLC cell types within a few minutes. As an adjunct to subsequent histology tests, our novel system would allow fast delineation of cancer cell types with minimum tissue consumption, potentially facilitating on-the-spot diagnosis, while preserving specimens for additional tests. Furthermore, 3-D measurements of cellular structure permit evaluation closer to the native state of cells, creating an alternative to traditional 2-D histology specimen evaluation, potentially increasing accuracy in diagnosing cell type of lung carcinomas.

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

Alternative cell lines to improve the rescue of infectious human astrovirus from a cDNA clone.

PubMed

Velázquez-Moctezuma, Rodrigo; Baños-Lara, Ma del Rocío; Acevedo, Yunuén; Méndez, Ernesto

2012-02-01

A reverse genetics system for human astrovirus (HAstV) was established previously; however, it has not been exploited mainly because cells used for virus packaging are not permissive, requiring several rounds of replication to obtain acceptable infectious virus. In this work, in the search for alternative permissive cell lines to be used as packaging cells, Hek-293 and Huh7.5.1 were tested. Given that HAstV infection in Hek-293 showed differences with that in Caco-2, the gold standard for HAstV growth but scarcely transfectable, and it was more similar to that observed in the hepatoma Huh7.5.1 cell line, these last cells were further used to transfect viral RNA. Virus titers near to 10(8) infectious particles per ml (ffu/ml) were obtained at 16-20 h after transfection with RNA from infected cells. However, virus titers close to 10(6) ffu/ml were obtained by using in vitro transcribed RNA from a cDNA HAstV-1 clone. In contrast, virus recovery in BHK-21, reported previously as the packaging cells, from this RNA was of about 10(4) ffu/ml, two logarithms less than in Huh7.5.1. Apparently, the 5'-end modification of the viral RNA determined its specific infectivity, since virus recovery was abolished when the total RNA was treated with proteinase-K, probably by removing a protein-linked genome protein, but it increased when capping of the in vitro transcribed RNA was more efficient. Thus, an alternative and more efficient reverse genetics system for HAstV was established by using Huh7.5.1 cells. Copyright © 2011. Published by Elsevier B.V.

An approach for development of alternative test methods based on mechanisms of skin irritation.

PubMed

Osborne, R; Perkins, M A

1994-02-01

Recent advances in techniques for culture of human skin cells have led to their potential for use as in vitro models for skin irritation testing to augment or replace existing rabbit skin patch tests. Our work is directed towards the development of cultured human skin cells, together with endpoints that can be linked to in vivo mechanisms of skin irritation, as in vitro models for prediction of human skin irritation, and for study of mechanisms of contact irritant dermatitis. Three types of commercial human skin cell cultures have been evaluated, epidermal keratinocytes and partially or fully cornified keratinocyte-dermal fibroblast co-cultures. Human epidermal keratinocyte cultures (Clonetics) were treated with product ingredients and formulations, and the extent of cell damage was assessed by incorporation of the vital dye neutral red. Cell damage correlated with human skin patch data for ingredient chemicals with the exception of acids and alkalis, but did not correlate with skin irritation to surfactant-containing product formulations. Cultures of human skin equivalents were evaluated as potential models for measurement of responses to test materials that could not be measured in the keratinocyte/neutral red assay. We developed a battery of in vitro endpoints to measure responses to prototype ingredients and formulations in human epidermal keratinocyte-dermal fibroblast co-cultures grown on a nylon mesh ('Skin2' from Advanced Tissue Sciences) or on a collagen gel ('Testskin' from Organogenesis). The endpoints measure cytotoxicity (neutral red and MTT vital dye staining, lactate dehydrogenase and N-acetyl glucosaminidase release, glucose utilization) and inflammatory mediator (prostaglandin E2) release. Initial experiments indicate a promising correlation between responses of the Skin2 model to prototype surfactants and in vivo human skin irritation. The responses of Testskin cultures to acids and alkalis help to prove the concept that a topical application model can measure responses to these materials. These results suggest that human skin cell models can provide useful systems for preclinical skin irritation assessments, as alternatives to rabbits, for at least certain classes of test substances.

The graphic cell method: a new look at digitizing geologic maps

USGS Publications Warehouse

Hanley, J.T.

1982-01-01

The graphic cell method is an alternative method of digitizing areal geologic information. It involves a discrete-point sampling scheme in which the computer establishes a matrix of cells over the map. Each cell and the whole cell is assigned the identity or value of the geologic information that is recognized at its center. Cell size may be changed to suit the needs of the user. The computer program resolves the matrix and identifies potential errors such as multiple assignments. Input includes the digitized boundaries of each geologic formation. This method should eliminate a primary bottleneck in the creation and testing of geomathematical models in such disciplines as resource appraisal. ?? 1982.

Gene expression profile during testicular development in patients with SRY-negative 46,XX testicular disorder of sex development.

PubMed

Mizuno, Kentaro; Kojima, Yoshiyuki; Kamisawa, Hideyuki; Moritoki, Yoshinobu; Nishio, Hidenori; Kohri, Kenjiro; Hayashi, Yutaro

2013-12-01

To elucidate alternative pathways in testicular development, we attempted to clarify the genetic characteristics of SRY-negative XX testes. We previously reported 5 cases of SRY-negative 46,XX testicular disorders of sex development and demonstrated that coordinated expression of genes such as SOX9, SOX3, and DAX1 was associated with testicular development. We performed a case-control study between the aforementioned boy with 46,XX testicular disorders of sex development and an age-matched patient with hydrocele testis (46,XY). During their consecutive surgeries, testicular biopsy specimens were obtained. Genes with differential expression compared with XY testis were identified using polymerase chain reaction (PCR)-based subtractive hybridization and sequencing. For validation of differential gene expression, real-time RT-PCR was performed using gene-specific primers. The distribution of candidate proteins in the testicular tissue was clarified by immunohistochemistry in human and rodent specimens. Moreover, in vitro inhibitory assays were performed. We identified 13 upregulated and 7 downregulated genes in XX testis. Among the candidate genes, we focused on ROCK1 (Rho-associated, coiled-coil protein kinase 1) in the upregulated gene group, because high expression in XX testis was validated by real-time RT-PCR. ROCK1 protein was detected in germ cells, Leydig cells, and Sertoli cells by immunohistochemistry. Moreover, the addition of specific ROCK1 inhibitor to Sertoli cells decreased SOX9 gene expression. On the basis of in vitro inhibitory assay, it is suggested that ROCK1 phosphorylates and activates SOX9 in Sertoli cells. Testes formation might be initiated by an alternative signaling pathway attributed to ROCK1, not SRY, activation in XX testes. Copyright © 2013 Elsevier Inc. All rights reserved.

Tympanic membrane organ culture using cell culture well inserts engrafted with tympanic membrane tissue explants.

PubMed

Liew, Lawrence J; Day, Richard M; Dilley, Rodney J

2017-03-01

Tissue engineering approaches using growth factors and various materials for repairing chronic perforations of the tympanic membrane are being developed, but there are surprisingly few relevant tissue culture models available to test new treatments. Here, we present a simple three-dimensional model system based on micro-dissecting the rat tympanic membrane umbo and grafting it into the membrane of a cell culture well insert. Cell outgrowth from the graft produced sufficient cells to populate a membrane of similar surface area to the human tympanic membrane within 2 weeks. Tissue grafts from the annulus region also showed cell outgrowth but were not as productive. The umbo organoid supported substantial cell proliferation and migration under the influence of keratinocyte growth medium. Cells from umbo grafts were enzymatically harvested from the polyethylene terephthalate (PET) membrane for expansion in routine culture and cells could be harvested consecutively from the same graft over multiple cycles. We used harvested cells to test cell migration properties and to engraft a porous silk scaffold material as proof-of-principle for tissue engineering applications. This model is simple enough to be widely adopted for tympanic membrane regeneration studies and has promise as a tissue-equivalent model alternative to animal testing.

Evaluation of p-phenylenediamine, o-phenylphenol sodium salt, and 2,4-diaminotoluene in the rat comet assay as part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiated international validation study of in vivo rat alkaline comet assay.

PubMed

De Boeck, Marlies; van der Leede, Bas-jan; De Vlieger, Kathleen; Geys, Helena; Vynckier, An; Van Gompel, Jacky

2015-07-01

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiated international validation study of in vivo rat alkaline comet assay (comet assay), p-phenylenediamine dihydrochloride (PPD), o-phenylphenol sodium salt (OPP), and 2,4-diaminotoluene (2,4-DAT), were analyzed in this laboratory as coded test chemicals. Male Sprague-Dawley rats (7-9 weeks of age) were given three oral doses of the test compounds, 24 and 21 h apart and liver and stomach were sampled 3h after the final dose administration. Under the conditions of the test, no increases in DNA damage were observed in liver and stomach with PPD and OPP up to 100 and 1000 mg/kg/day, respectively. 2,4-DAT, a known genotoxic carcinogen, induced a weak but reproducible, dose-related and statistically significant increase in DNA damage in liver cells while no increases were observed in stomach cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Space power demonstrator engine, phase 1

NASA Technical Reports Server (NTRS)

1987-01-01

The design, analysis, and preliminary test results for a 25 kWe Free-Piston Stirling engine with integral linear alternators are described. The project is conducted by Mechanical Technology under the direction of LeRC as part of the SP-100 Nuclear Space Power Systems Program. The engine/alternator system is designed to demonstrate the following performance: (1) 25 kWe output at a specific weight less than 8 kg/kW; (2) 25 percent efficiency at a temperature ratio of 2.0; (3) low vibration (amplitude less than .003 in); (4) internal gas bearings (no wear, no external pump); and (5) heater temperature/cooler temperature from 630 to 315 K. The design approach to minimize vibration is a two-module engine (12.5 kWe per module) in a linearly-opposed configuration with a common expansion space. The low specific weight is obtained at high helium pressure (150 bar) and high frequency (105 Hz) and by using high magnetic strength (samarium cobalt) alternator magnets. Engine tests began in June 1985; 16 months following initiation of engine and test cell design. Hydrotest and consequent engine testing to date has been intentionally limited to half pressure, and electrical power output is within 15 to 20 percent of design predictions.

In vitro cell transformation assays for an integrated, alternative assessment of carcinogenicity: a data-based analysis.

PubMed

Benigni, Romualdo; Bossa, Cecilia; Tcheremenskaia, Olga

2013-01-01

The study of the chemical carcinogenesis mechanisms and the design of efficient prevention strategies and measures are of crucial importance to protect human health. The long-term carcinogenesis bioassays have played a central role in protecting human health, but for ethical and practical reasons their use is dramatically diminishing, and the genotoxicity short-term tests have taken the pivotal role in the pre-screening of carcinogenicity. However, there is evidence that this strategy is not sensitive enough to detect all genotoxic carcinogens and it cannot detect nongenotoxic carcinogens. In a previous article, we have shown that an integrated strategy consisting of the in vitro Ames and Syrian Hamster Embryo cells transformation assays, combined with structure-activity relationships, is a valid alternative to the present pre-screening strategies. Here, we expand the previous investigation by (i) including results of cell transformation assays on inorganics, together with an additional assay (Bhas 42), and (ii) considering new structural alerts for nongenotoxic carcinogenicity. We also present a new analysis on global relationships between toxicological endpoints. The new results confirm that the previously proposed integrated, alternative strategy is an efficient tool to identify both genotoxic and nongenotoxic carcinogens, with an estimated 90-95% sensitivity.

Reference compounds for alternative test methods to indicate developmental neurotoxicity (DNT) potential of chemicals: example lists and criteria for their selection and use

PubMed Central

Aschner, Michael; Ceccatelli, Sandra; Daneshian, Mardas; Fritsche, Ellen; Hasiwa, Nina; Hartung, Thomas; Hogberg, Helena T.; Leist, Marcel; Li, Abby; Mundy, William R.; Padilla, Stephanie; Piersma, Aldert H.; Bal-Price, Anna; Seiler, Andrea; Westerink, Remco H.; Zimmer, Bastian; Lein, Pamela J.

2016-01-01

Summary There is a paucity of information concerning the developmental neurotoxicity (DNT) hazard posed by industrial and environmental chemicals. New testing approaches will most likely be based on batteries of alternative and complementary (non-animal) tests. As DNT is assumed to result from the modulation of fundamental neurodevelopmental processes (such as neuronal differentiation, precursor cell migration or neuronal network formation) by chemicals, the first generation of alternative DNT tests target these processes. The advantage of such types of assays is that they capture toxicants with multiple targets and modes-of-action. Moreover, the processes modelled by the assays can be linked to toxicity endophenotypes, i.e. alterations in neural connectivity that form the basis for neurofunctional deficits in man. The authors of this review convened in a workshop to define criteria for the selection of positive/negative controls, to prepare recommendations on their use, and to initiate the setup of a directory of reference chemicals. For initial technical optimization of tests, a set of >50 endpoint-specific control compounds was identified. For further test development, an additional “test” set of 33 chemicals considered to act directly as bona fide DNT toxicants is proposed, and each chemical is annotated to the extent it fulfills these criteria. A tabular compilation of the original literature used to select the test set chemicals provides information on statistical procedures, and toxic/non-toxic doses (both for pups and dams). Suggestions are provided on how to use the >100 compounds (including negative controls) compiled here to address specificity, adversity and use of alternative test systems. PMID:27452664

Physical impaction injury effects on bacterial cells during spread plating influenced by cell characteristics of the organisms.

PubMed

Thomas, P; Mujawar, M M; Sekhar, A C; Upreti, R

2014-04-01

To understand the factors that contribute to the variations in colony-forming units (CFU) in different bacteria during spread plating. Employing a mix culture of vegetative cells of ten organisms varying in cell characteristics (Gram reaction, cell shape and cell size), spread plating to the extent of just drying the agar surface (50-60 s) was tested in comparison with the alternate spotting-and-tilt-spreading (SATS) approach where 100 μl inoculum was distributed by mere tilting of plate after spotting as 20-25 microdrops. The former imparted a significant reduction in CFU by 20% over the spreader-independent SATS approach. Extending the testing to single organisms, Gram-negative proteobacteria with relatively larger cells (Escherichia, Enterobacter, Agrobacterium, Ralstonia, Pantoea, Pseudomonas and Sphingomonas spp.) showed significant CFU reduction with spread plating except for slow-growing Methylobacterium sp., while those with small rods (Xenophilus sp.) and cocci (Acinetobacter sp.) were less affected. Among Gram-positive nonspore formers, Staphylococcus epidermidis showed significant CFU reduction while Staphylococcus haemolyticus and actinobacteria (Microbacterium, Cellulosimicrobium and Brachybacterium spp.) with small rods/cocci were unaffected. Vegetative cells of Bacillus pumilus and B. subtilis were generally unaffected while others with larger rods (B. thuringiensis, Brevibacillus, Lysinibacillus and Paenibacillus spp.) were significantly affected. A simulated plating study coupled with live-dead bacterial staining endorsed the chances of cell disruption with spreader impaction in afflicted organisms. Significant reduction in CFU could occur during spread plating due to physical impaction injury to bacterial cells depending on the spreader usage and the variable effects on different organisms are determined by Gram reaction, cell size and cell shape. The inoculum spreader could impart physical disruption of vegetative cells against a hard surface. Possibility of CFU reduction in sensitive organisms and the skewed selection of hardier organisms during spread plating, and the recommendation of SATS as an easier and safer alternative for CFU enumerations. © 2013 The Society for Applied Microbiology.

TCR-Vß8 as alternative to animal testing for quantifying active SEE

USDA-ARS?s Scientific Manuscript database

Staphylococcal food poisoning is a result of ingestion of Staphylococcal enterotoxins (SEs) produced by the bacterium Staphylococcus aureus. SEs cause gastroenteritis and also cause activation of T cells and massive cytokine release. A current method for the detection of active SEs relies on its eme...

Comparative sensitivity of human and rat neural cultures to chemical-induced inhibition of neurite outgrowth

EPA Science Inventory

There is a need for rapid, efficient and cost effective alternatives to traditional in vivo developmental neurotoxicity testing. In vitro cell culture models can recapitulate many of the key cellular processes of nervous system development, including neurite outgrowth, and may be...

Targeting BCR-ABL-Independent TKI Resistance in Chronic Myeloid Leukemia by mTOR and Autophagy Inhibition.

PubMed

Mitchell, Rebecca; Hopcroft, Lisa E M; Baquero, Pablo; Allan, Elaine K; Hewit, Kay; James, Daniel; Hamilton, Graham; Mukhopadhyay, Arunima; O'Prey, Jim; Hair, Alan; Melo, Junia V; Chan, Edmond; Ryan, Kevin M; Maguer-Satta, Véronique; Druker, Brian J; Clark, Richard E; Mitra, Subir; Herzyk, Pawel; Nicolini, Franck E; Salomoni, Paolo; Shanks, Emma; Calabretta, Bruno; Holyoake, Tessa L; Helgason, G Vignir

2018-05-01

Imatinib and second-generation tyrosine kinase inhibitors (TKIs) nilotinib and dasatinib have statistically significantly improved the life expectancy of chronic myeloid leukemia (CML) patients; however, resistance to TKIs remains a major clinical challenge. Although ponatinib, a third-generation TKI, improves outcomes for patients with BCR-ABL-dependent mechanisms of resistance, including the T315I mutation, a proportion of patients may have or develop BCR-ABL-independent resistance and fail ponatinib treatment. By modeling ponatinib resistance and testing samples from these CML patients, it is hoped that an alternative drug target can be identified and inhibited with a novel compound. Two CML cell lines with acquired BCR-ABL-independent resistance were generated following culture in ponatinib. RNA sequencing and gene ontology (GO) enrichment were used to detect aberrant transcriptional response in ponatinib-resistant cells. A validated oncogene drug library was used to identify US Food and Drug Administration-approved drugs with activity against TKI-resistant cells. Validation was performed using bone marrow (BM)-derived cells from TKI-resistant patients (n = 4) and a human xenograft mouse model (n = 4-6 mice per group). All statistical tests were two-sided. We show that ponatinib-resistant CML cells can acquire BCR-ABL-independent resistance mediated through alternative activation of mTOR. Following transcriptomic analysis and drug screening, we highlight mTOR inhibition as an alternative therapeutic approach in TKI-resistant CML cells. Additionally, we show that catalytic mTOR inhibitors induce autophagy and demonstrate that genetic or pharmacological inhibition of autophagy sensitizes ponatinib-resistant CML cells to death induced by mTOR inhibition in vitro (% number of colonies of control[SD], NVP-BEZ235 vs NVP-BEZ235+HCQ: 45.0[17.9]% vs 24.0[8.4]%, P = .002) and in vivo (median survival of NVP-BEZ235- vs NVP-BEZ235+HCQ-treated mice: 38.5 days vs 47.0 days, P = .04). Combined mTOR and autophagy inhibition may provide an attractive approach to target BCR-ABL-independent mechanism of resistance.

Alternative diagnosis in the putative ventilator-associated pneumonia patient not meeting lavage-based diagnostic criteria.

PubMed

Schoemakers, Rik J; Schnabel, Ronny; Oudhuis, Guy J; Linssen, Catharina F M; van Mook, Walther N K A; Verbon, Annelies; Bergmans, Dennis C J J

2014-12-01

The clinical picture of ventilator-associated pneumonia (VAP) can be mimicked by other infectious and non-infectious diseases. The aim of this study was to determine the alternative diagnoses and to develop a diagnostic flow chart for patients suspected of having VAP not meeting the diagnostic broncho-alveolar lavage (BAL) criteria. Adult intensive care patients with a clinical suspicion of VAP and negative BAL results were included. The clinical suspicion of VAP was based on the combination of clinical, radiological, and microbiological criteria. BAL was considered positive if cell differentiation revealed ≥ 2% cells with intracellular organisms and/or quantitative culture results of ≥ 10(4) cfu/ml. The most likely alternative diagnosis of fever and pulmonary densities was retrospectively determined by two authors independently. In all, 110 of 207 patients with suspected VAP did not meet the diagnostic BAL criteria and required further diagnostic evaluation. In 67 patients an alternative diagnosis for fever could be found. In 51 patients an alternative diagnosis of both fever and pulmonary densities could be established. In almost 40% of patients no alternative diagnosis could be provided. Non-bacterial pneumonia was diagnosed in 10 patients with Herpes simplex virus 1 (HSV-1) as the most common pathogen. In eight patients non-infectious pneumonitis was diagnosed. Due to the wide range of alternative diagnoses and applied tests the diagnostic work-up proved to be necessarily individualized and guided by repeated clinical assessment. The most frequently found alternative diagnoses were viral pneumonia and non-infectious pneumonitis.

On-Site Fuel Cell Energy Systems: The U.S. Air Force Field Test Demonstration Plan.

DTIC Science & Technology

1980-12-01

Continue on reverse -, de if necessary and identify by block number) Fuel cells Cogererati on Energy conversion ABSTRACT (Continue an reverse ide If...fuel electrode, water at the oxygen electrode, and to act as a mechanical barrier between the two gases to prevent mixing and direct combustion . When the...possibility of more effective utilization of hydrocarbon fuels, especially when compared with the alternative heat engine combustion technologies. Figure 12

A simple and novel modification of comet assay for determination of bacteriophage mediated bacterial cell lysis.

PubMed

Khairnar, Krishna; Sanmukh, Swapnil; Chandekar, Rajshree; Paunikar, Waman

2014-07-01

The comet assay is the widely used method for in vitro toxicity testing which is also an alternative to the use of animal models for in vivo testing. Since, its inception in 1984 by Ostling and Johansson, it is being modified frequently for a wide range of application. In spite of its wide applicability, unfortunately there is no report of its application in bacteriophages research. In this study, a novel application of comet assay for the detection of bacteriophage mediated bacterial cell lysis was described. The conventional methods in bacteriophage research for studying bacterial lysis by bacteriophages are plaque assay method. It is time consuming, laborious and costly. The lytic activity of bacteriophage devours the bacterial cell which results in the release of bacterial genomic material that gets detected by ethidium bromide staining method by the comet assay protocol. The objective of this study was to compare efficacy of comet assay with different assay used to study phage mediated bacterial lysis. The assay was performed on culture isolates (N=80 studies), modified comet assay appear to have relatively higher sensitivity and specificity than other assay. The results of the study showed that the application of comet assay can be an economical, time saving and less laborious alternative to conventional plaque assay for the detection of bacteriophage mediated bacterial cell lysis. Copyright © 2014 Elsevier B.V. All rights reserved.

Genome-wide profiling of chemoradiation‑induced changes in alternative splicing in colon cancer cells.

PubMed

Xiong, Wei; Gao, Depei; Li, Yunfeng; Liu, Xin; Dai, Peiling; Qin, Jiyong; Wang, Guanshun; Li, Kangming; Bai, Han; Li, Wenhui

2016-10-01

Alternative splicing is a key mechanism that regulates protein diversity and has been found to be associated with colon cancer progression and metastasis. However, the function of alternative splicing in chemoradiation‑resistant colon cancer remains elusive. In this study, we constructed a chemoradiation‑resistant colon cancer cell line. Through RNA-sequencing of normal and chemoradiation‑resistant colon cancer cells (HCT116), we found 818 genes that were highly expressed in the normal HCT116 cells, whereas 285 genes were highly expressed in the chemoradiation-resistant HCT116 (RCR-HCT116) cells. Gene ontology (GO) analysis showed that genes that were highly expressed in the HCT116 cells were enriched in GO categories related to cell cycle and cell division, whereas genes that were highly expressed in the RCR-HCT116 cells were associated with regulation of system processes and response to wounding. Analysis of alternative splicing events revealed that exon skipping was significantly increased in the chemoradiation‑resistant colon cancer cells. Moreover, we identified 323 alternative splicing events in 293 genes that were significantly different between the two different HCT116 cell types. These alternative splicing‑related genes were clustered functionally into several groups related with DNA replication, such as deoxyribonucleotide metabolic/catabolic processes, response to DNA damage stimulus and helicase activity. These findings enriched our knowledge by elucidating the function of alternative splicing in chemoradiation-resistant colon cancer.

Screening of toxic potential of graphene family nanomaterials using in vitro and alternative in vivo toxicity testing systems.

PubMed

Chatterjee, Nivedita; Yang, Ji Su; Park, Kwangsik; Oh, Seung Min; Park, Jeonggue; Choi, Jinhee

2015-01-01

The widely promising applications of graphene nanomaterials raise considerable concerns regarding their environmental and human health risk assessment. The aim of the current study was to evaluate the toxicity profiling of graphene family nananomaterials (GFNs) in alternative in vitro and in vivo toxicity testing models. The GFNs used in this study are graphene nanoplatelets ([GNPs]-pristine, carboxylate [COOH] and amide [NH2]) and graphene oxides (single layer [SLGO] and few layers [FLGO]). The human bronchial epithelial cells (Beas2B cells) as in vitro system and the nematode Caenorhabditis elegans as in vivo system were used to profile the toxicity response of GFNs. Cytotoxicity assays, colony formation assay for cellular toxicity and reproduction potentiality in C. elegans were used as end points to evaluate the GFNs' toxicity. In general, GNPs exhibited higher toxicity than GOs in Beas2B cells, and among the GNPs the order of toxicity was pristine>NH2>COOH. Although the order of toxicity of the GNPs was maintained in C. elegans reproductive toxicity, but GOs were found to be more toxic in the worms than GNPs. In both systems, SLGO exhibited profoundly greater dose dependency than FLGO. The possible reason of their differential toxicity lay in their distinctive physicochemical characteristics and agglomeration behavior in the exposure media. The present study revealed that the toxicity of GFNs is dependent on the graphene nanomaterial's physical forms, surface functionalizations, number of layers, dose, time of exposure and obviously, on the alternative model systems used for toxicity assessment.

Developmental toxicity testing in the 21st century: the sword of Damocles shattered by embryonic stem cell assays?

PubMed

Seiler, Andrea; Oelgeschläger, Michael; Liebsch, Manfred; Pirow, Ralph; Riebeling, Christian; Tralau, Tewes; Luch, Andreas

2011-11-01

Modern society faces an inherent dilemma. In our globalized society, we are spoilt for choice by an ever-increasing number of products, many of which are made of new materials and compound mixtures. At the same time, as consumers we got accustomed to the idea of a life minimized for risk, including our own exposure to chemicals from the environment or to compounds present in and released from everyday products. Chemical safety testing bridges these obviously diverging interests, and the corresponding legislation has hence been tremendously extended (e.g., introduction of the European legislation REACH in 2007). However, the underlying regulatory toxicology still relies mainly on animal testing, which is relatively slow, expensive, and ethically arguable. Meanwhile, recent years have seen a surge in efforts to develop alternative testing systems and strategies. Expectations are particularly high for the applicability of stem cells as test systems especially for developmental toxicity testing in vitro. For the first time in history, test systems can be based on differentiating cells and tissue progenitors in culture, thus bringing the 'vision of toxicity testing in the 21st century' a step closer.

Affimer proteins for F-actin: novel affinity reagents that label F-actin in live and fixed cells.

PubMed

Lopata, Anna; Hughes, Ruth; Tiede, Christian; Heissler, Sarah M; Sellers, James R; Knight, Peter J; Tomlinson, Darren; Peckham, Michelle

2018-04-26

Imaging the actin cytoskeleton in cells uses a wide range of approaches. Typically, a fluorescent derivative of the small cyclic peptide phalloidin is used to image F-actin in fixed cells. Lifeact and F-tractin are popular for imaging the cytoskeleton in live cells. Here we characterised novel affinity reagents called Affimers that specifically bind to F-actin in vitro to determine if they are suitable alternatives as eGFP-fusion proteins, to label actin in live cells, or for labeling F-actin in fixed cells. In vitro experiments showed that 3 out of the 4 Affimers (Affimers 6, 14 and 24) tested bind tightly to purified F-actin, and appear to have overlapping binding sites. As eGFP-fusion proteins, the same 3 Affimers label F-actin in live cells. FRAP experiments suggest that eGFP-Affimer 6 behaves most similarly to F-tractin and Lifeact. However, it does not colocalise with mCherry-actin in dynamic ruffles, and may preferentially bind stable actin filaments. All 4 Affimers label F-actin in methanol fixed cells, while only Affimer 14 labels F-actin after paraformaldehyde fixation. eGFP-Affimer 6 has potential for use in selectively imaging the stable actin cytoskeleton in live cells, while all 4 Affimers are strong alternatives to phalloidin for labelling F-actin in fixed cells.

Investigating dye-sensitised solar cells

NASA Astrophysics Data System (ADS)

Tobin, Laura L.; O'Reilly, Thomas; Zerulla, Dominic; Sheridan, John T.

2010-05-01

At present there is considerable global concern in relation to environmental issues and future energy supplies, for instance climate change (global warming) and the rapid depletion of fossil fuel resources. This trepidation has initiated a more critical investigation into alternative and renewable sources of power such as geothermal, biomass, hydropower, wind and solar energy. The immense dependence on electrical power in today's society has prompted the manufacturing of devices such as photovoltaic (PV) cells to help alleviate and replace current electrical demands of the power grid. The most popular and commercially available PV cells are silicon solar cells which have to date the greatest efficiencies for PV cells. The drawback however is that the manufacturing of these cells is complex and costly due to the expense and difficulty of producing and processing pure silicon. One relatively inexpensive alternative to silicon PV cells that we are currently studying are dye-sensitised solar cells (DSSC or Grätzel Cells). DSSC are biomimetic solar cells which are based on the process of photosynthesis. The SFI Strategic Research Centre for Solar Energy Conversion is a research cluster based in Ireland formed with the express intention of bringing together industry and academia to produce renewable energy solutions. Our specific research area is in DSSC and their electrical properties. We are currently developing testing equipment for arrays of DSSC and developing optoelectronic models which todescribe the performance and behaviour of DSSCs.

40 CFR 86.162-00 - Approval of alternative air conditioning test simulations and descriptions of AC1 and AC2.

Code of Federal Regulations, 2012 CFR

2012-07-01

... power requirements to roadload dynamometer requirements. AC2 simulates, in standard test cell ambient...)(i) (A) and (B) are replaced with 76 °F and 50 grains of water/pound of dry air and the solar heat...)(5)(i) (A) and (B) are replaced with 76 °F and 50 grains of water/pound of dry air and the solar heat...

40 CFR 86.162-00 - Approval of alternative air conditioning test simulations and descriptions of AC1 and AC2.

Code of Federal Regulations, 2013 CFR

2013-07-01

... power requirements to roadload dynamometer requirements. AC2 simulates, in standard test cell ambient...)(i) (A) and (B) are replaced with 76 °F and 50 grains of water/pound of dry air and the solar heat...)(5)(i) (A) and (B) are replaced with 76 °F and 50 grains of water/pound of dry air and the solar heat...

Beam dynamics validation of the Halbach Technology FFAG Cell for Cornell-BNL Energy Recovery Linac

NASA Astrophysics Data System (ADS)

Méot, F.; Tsoupas, N.; Brooks, S.; Trbojevic, D.

2018-07-01

The Cornell-BNL Electron Test Accelerator (CBETA), a 150 MeV energy recovery linac (ERL) now in construction at Cornell, employs a fixed-field alternating gradient optics return loop: a single beam line comprised of FFAG cells, which accepts four recirculated energies. CBETA FFAG cell uses Halbach permanent magnet technology, its design studies have covered an extended period of time supported by extensive particle dynamics simulations using computed 3-D field map models. This approach is discussed, and illustrated here, based on the final stage in these beam dynamics studies, namely the validation of a ultimate, optimized design of the Halbach cell.

The consensus sequence of FAMLF alternative splice variants is overexpressed in undifferentiated hematopoietic cells.

PubMed

Chen, W L; Luo, D F; Gao, C; Ding, Y; Wang, S Y

2015-07-01

The familial acute myeloid leukemia related factor gene (FAMLF) was previously identified from a familial AML subtractive cDNA library and shown to undergo alternative splicing. This study used real-time quantitative PCR to investigate the expression of the FAMLF alternative-splicing transcript consensus sequence (FAMLF-CS) in peripheral blood mononuclear cells (PBMCs) from 119 patients with de novo acute leukemia (AL) and 104 healthy controls, as well as in CD34+ cells from 12 AL patients and 10 healthy donors. A 429-bp fragment from a novel splicing variant of FAMLF was obtained, and a 363-bp consensus sequence was targeted to quantify total FAMLF expression. Kruskal-Wallis, Nemenyi, Spearman's correlation, and Mann-Whitney U-tests were used to analyze the data. FAMLF-CS expression in PBMCs from AL patients and CD34+ cells from AL patients and controls was significantly higher than in control PBMCs (P < 0.0001). Moreover, FAMLF-CS expression in PBMCs from the AML group was positively correlated with red blood cell count (rs =0.317, P=0.006), hemoglobin levels (rs = 0.210, P = 0.049), and percentage of peripheral blood blasts (rs = 0.256, P = 0.027), but inversely correlated with hemoglobin levels in the control group (rs = -0.391, P < 0.0001). AML patients with high CD34+ expression showed significantly higher FAMLF-CS expression than those with low CD34+ expression (P = 0.041). Our results showed that FAMLF is highly expressed in both normal and malignant immature hematopoietic cells, but that expression is lower in normal mature PBMCs.

High-level production of recombinant trypsin in transgenic rice cell culture through utilization of an alternative carbon source and recycling system.

PubMed

Kim, Nan-Sun; Yu, Hwa-Young; Chung, Nguyen-Duc; Kwon, Tae-Ho; Yang, Moon-Sik

2014-09-01

Productivity of recombinant bovine trypsin using a rice amylase 3D promoter has been studied in transgenic rice suspension culture. Alternative carbon sources were added to rice cell suspension cultures in order to improve the production of recombinant bovine trypsin. It was demonstrated that addition of alternative carbon sources such as succinic acid, fumaric acid and malic acid in the culture medium could increase the productivity of recombinant bovine trypsin 3.8-4.3-fold compared to those in the control medium without carbon sources. The highest accumulated trypsin reached 68.2 mg/L on day 5 in the culture medium with 40 mM fumaric acid. The feasibility of repeated use of the cells for recombinant trypsin production was tested in transgenic rice cell suspension culture with the culture medium containing the combination of variable sucrose concentration and 40 mM fumaric acid. Among the used combinations, the combination of 1% sucrose and 40 mM fumaric acid resulted in a yield of up to 53 mg/L five days after incubation. It also increased 31% (W/W) of dry cell weight and improved 43% of cell viability compared to that in control medium without sucrose. Based on these data, recycling of the trypsin production process with repeated 1% sucrose and 40 mM fumaric acid supplying-harvesting cycles was developed in flask scale culture. Recombinant bovine trypsin could be stably produced with a yield of up to 53-39 mg/L per cycle during five recycling cycles. Copyright © 2014 Elsevier Inc. All rights reserved.

Alternative Fuels Data Center: Test Your Alternative Fuel IQ

Science.gov Websites

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Virus-like particle (VLP)-based vaccines for pandemic influenza

PubMed Central

López-Macías, Constantino

2012-01-01

The influenza pandemic of 2009 demonstrated the inability of the established global capacity for egg-based vaccine production technology to provide sufficient vaccine for the population in a timely fashion. Several alternative technologies for developing influenza vaccines have been proposed, among which non-replicating virus-like particles (VLPs) represent an attractive option because of their safety and immunogenic characteristics. VLP vaccines against pandemic influenza have been developed in tobacco plant cells and in Sf9 insect cells infected with baculovirus that expresses protein genes from pandemic influenza strains. These technologies allow rapid and large-scale production of vaccines (3–12 weeks). The 2009 influenza outbreak provided an opportunity for clinical testing of a pandemic influenza VLP vaccine in the midst of the outbreak at its epicenter in Mexico. An influenza A(H1N1)2009 VLP pandemic vaccine (produced in insect cells) was tested in a phase II clinical trial involving 4,563 healthy adults. Results showed that the vaccine is safe and immunogenic despite high preexisting anti-A(H1N1)2009 antibody titers present in the population. The safety and immunogenicity profile presented by this pandemic VLP vaccine during the outbreak in Mexico suggests that VLP technology is a suitable alternative to current influenza vaccine technologies for producing pandemic and seasonal vaccines. PMID:22330956

Nonimmediate hypersensitivity reactions to iodinated contrast media.

PubMed

Gómez, Enrique; Ariza, Adriana; Blanca-López, Natalia; Torres, Maria J

2013-08-01

To provide a detailed analysis of the latest findings on the mechanisms underlying the nonimmediate reactions to iodinated contrast media and comment on the recent advances in diagnosis, focusing on the roles of the skin test, drug provocation test (DPT), and lymphocyte transformation test (LTT). Several studies have reported new findings supporting an important role for T-lymphocytes in the nonimmediate reactions to iodinated contrast media. The LTT has been used as an in-vitro tool for diagnosis, but with variable results. However, the inclusion of autologous monocyte-derived dendritic cells as professional antigen-presenting cells has improved the sensitivity of this test. Regarding in-vivo diagnosis, although skin testing has been routine, it has now been shown that its sensitivity and negative predictive value are low. Recent studies have demonstrated that the DPT is a well tolerated and useful procedure that is necessary to confirm the diagnosis of nonimmediate hypersensitivity reactions to iodinated contrast media. Nonimmediate reactions to contrast media are usually T-cell mediated. Diagnosis is based on skin testing, although its sensitivity and negative predictive value are not optimal. Consequently, drug provocation testing is often needed to confirm the diagnosis and also to seek alternative contrast media that can be tolerated.

Metabolomics in transfusion medicine.

PubMed

Nemkov, Travis; Hansen, Kirk C; Dumont, Larry J; D'Alessandro, Angelo

2016-04-01

Biochemical investigations on the regulatory mechanisms of red blood cell (RBC) and platelet (PLT) metabolism have fostered a century of advances in the field of transfusion medicine. Owing to these advances, storage of RBCs and PLT concentrates has become a lifesaving practice in clinical and military settings. There, however, remains room for improvement, especially with regard to the introduction of novel storage and/or rejuvenation solutions, alternative cell processing strategies (e.g., pathogen inactivation technologies), and quality testing (e.g., evaluation of novel containers with alternative plasticizers). Recent advancements in mass spectrometry-based metabolomics and systems biology, the bioinformatics integration of omics data, promise to speed up the design and testing of innovative storage strategies developed to improve the quality, safety, and effectiveness of blood products. Here we review the currently available metabolomics technologies and briefly describe the routine workflow for transfusion medicine-relevant studies. The goal is to provide transfusion medicine experts with adequate tools to navigate through the otherwise overwhelming amount of metabolomics data burgeoning in the field during the past few years. Descriptive metabolomics data have represented the first step omics researchers have taken into the field of transfusion medicine. However, to up the ante, clinical and omics experts will need to merge their expertise to investigate correlative and mechanistic relationships among metabolic variables and transfusion-relevant variables, such as 24-hour in vivo recovery for transfused RBCs. Integration with systems biology models will potentially allow for in silico prediction of metabolic phenotypes, thus streamlining the design and testing of alternative storage strategies and/or solutions. © 2015 AABB.

Alternate cathodes for sodium-metal chloride batteries

NASA Technical Reports Server (NTRS)

Ratnakumar, B. V.; Attia, A. I.; Halpert, G.

1991-01-01

Various metal chlorides were tested as possible cathode materials for sodium-metal batteries (in addition to Fe and Ni chlorides, which have been already developed to a stage of commercialization), using an electrochemical cell consisting of a pyrex tube, heated to 250 C, with the metal wire as working electrode, concentric Ni foil as counterelectrode, and high-purity Al as reference electrode. In particular, the aim of this study was to identify metal chlorides insoluble even in neutral melts, possible at the interface during overcharge, in order to eliminate the failure mode of the cell through a cationic exchange of the dissolved metal ions with sodium beta-double-prime alumina solid electrolyte. Results indicate that Mo and Co are likely alternatives to FeCl2 and NiCl2 cathodes in sodium batteries. The open circuit voltages of Na/CoCl(x) and Na/MoCl(x) cells at 250 C would be 2.55 V and 2.64 V, respectively.

Fluorescent carbon quantum dots synthesized by chemical vapor deposition: An alternative candidate for electron acceptor in polymer solar cells

NASA Astrophysics Data System (ADS)

Cui, Bo; Yan, Lingpeng; Gu, Huimin; Yang, Yongzhen; Liu, Xuguang; Ma, Chang-Qi; Chen, Yongkang; Jia, Husheng

2018-01-01

Excitation-wavelength-dependent blue-greenish fluorescent carbon quantum dots (CQDs) with graphite structure were synthesized by chemical vapor deposition (CVD) method. In comparison with those synthesized by hydrothermal method (named H-CQDs), C-CQDs have less hydrophilic terminal groups, showing good solubility in common organic solvents. Furthermore, these synthesized C-CQDs show a low LUMO energy level (LUMO = -3.84 eV), which is close to that of phenyl-C61-butyric acid methyl ester (PC61BM, LUMO = -4.01 eV), the most widely used electron acceptor in polymer solar cells. Photoluminescence quenching of the poly(3-hexylthiophene-2,5-diyl):C-CQDs blended film (P3HT:C-CQDs) indicated that a photo-induced charge transfer between P3HT and C-CQDs occurs in such a composite film. Bulk heterojunction solar cells using C-CQDs as electron acceptors or doping materials were fabricated and tested. High fill factors were achieved for these C-CQDs based polymer solar cells, demonstrating that CQDs synthesized by CVD could be alternative to the fullerene derivatives for applying in polymer solar cells.

Intron-mediated alternative splicing of WOOD-ASSOCIATED NAC TRANSCRIPTION FACTOR1B regulates cell wall thickening during fiber development in Populus species.

PubMed

Zhao, Yunjun; Sun, Jiayan; Xu, Peng; Zhang, Rui; Li, Laigeng

2014-02-01

Alternative splicing is an important mechanism involved in regulating the development of multicellular organisms. Although many genes in plants undergo alternative splicing, little is understood of its significance in regulating plant growth and development. In this study, alternative splicing of black cottonwood (Populus trichocarpa) wood-associated NAC domain transcription factor (PtrWNDs), PtrWND1B, is shown to occur exclusively in secondary xylem fiber cells. PtrWND1B is expressed with a normal short-transcript PtrWND1B-s as well as its alternative long-transcript PtrWND1B-l. The intron 2 structure of the PtrWND1B gene was identified as a critical sequence that causes PtrWND1B alternative splicing. Suppression of PtrWND1B expression specifically inhibited fiber cell wall thickening. The two PtrWND1B isoforms play antagonistic roles in regulating cell wall thickening during fiber cell differentiation in Populus spp. PtrWND1B-s overexpression enhanced fiber cell wall thickening, while overexpression of PtrWND1B-l repressed fiber cell wall thickening. Alternative splicing may enable more specific regulation of processes such as fiber cell wall thickening during wood formation.

Intron-Mediated Alternative Splicing of WOOD-ASSOCIATED NAC TRANSCRIPTION FACTOR1B Regulates Cell Wall Thickening during Fiber Development in Populus Species1[W

PubMed Central

Zhao, Yunjun; Sun, Jiayan; Xu, Peng; Zhang, Rui; Li, Laigeng

2014-01-01

Alternative splicing is an important mechanism involved in regulating the development of multicellular organisms. Although many genes in plants undergo alternative splicing, little is understood of its significance in regulating plant growth and development. In this study, alternative splicing of black cottonwood (Populus trichocarpa) wood-associated NAC domain transcription factor (PtrWNDs), PtrWND1B, is shown to occur exclusively in secondary xylem fiber cells. PtrWND1B is expressed with a normal short-transcript PtrWND1B-s as well as its alternative long-transcript PtrWND1B-l. The intron 2 structure of the PtrWND1B gene was identified as a critical sequence that causes PtrWND1B alternative splicing. Suppression of PtrWND1B expression specifically inhibited fiber cell wall thickening. The two PtrWND1B isoforms play antagonistic roles in regulating cell wall thickening during fiber cell differentiation in Populus spp. PtrWND1B-s overexpression enhanced fiber cell wall thickening, while overexpression of PtrWND1B-l repressed fiber cell wall thickening. Alternative splicing may enable more specific regulation of processes such as fiber cell wall thickening during wood formation. PMID:24394777

Computerized In Vitro Test for Chemical Toxicity Based on Tetrahymena Swimming Patterns

NASA Technical Reports Server (NTRS)

Noever, David A.; Matsos, Helen C.; Cronise, Raymond J.; Looger, Loren L.; Relwani, Rachna A.; Johnson, Jacqueline U.

1994-01-01

An apparatus and a method for rapidly determining chemical toxicity have been evaluated as an alternative to the rabbit eye initancy test (Draize). The toxicity monitor includes an automated scoring of how motile biological cells (Tetrahymena pyriformis) slow down or otherwise change their swimming patterns in a hostile chemical environment. The method, called the motility assay (MA), is tested for 30 s to determine the chemical toxicity in 20 aqueous samples containing trace organics and salts. With equal or better detection limits, results compare favorably to in vivo animal tests of eye irritancy.

Industrial Technology Modernization Program. Project 80. Increase Efficiency of Card Test/Device Test Areas by the Usage of Improved Material Handling Systems. Revision 1. Phase 2

DTIC Science & Technology

1988-03-01

INDUSTRY ANALYSIS/FINDINGS 132 12 EQUIPMENT/MACHINERY ALTERNATIVES 134 13 MIS REQUIREMENTS/IMPROVEMENTS 135 14 COST BENEFIT ANALYSIS AND PROCEDURE 137 15...SOFTWARE DIAGRAM 14.0-1 COST BENEFIT ANALYSIS METHODOLOGY 138 14.3-1 PROJECT 80 EXPENDITURE SCHEDULE 141 14.4-1 PROJECT 80 CASH FLOWS 142 15.1-1 PROJECT 80...testing, streamlining work flow, and installation of ergonomically designed work cell/work centers. The benefits associated with the implementation of ITM

Utility of human embryonic kidney cell line HEK-293 for rapid isolation of fixed and street rabies viruses: comparison with Neuro-2a and BHK-21 cell lines.

PubMed

Madhusudana, Shampur Narayan; Sundaramoorthy, Subha; Ullas, Padinjaremattatthil Thankappan

2010-12-01

A confirmatory rabies diagnosis can be achieved by rapid virus isolation in cell culture using brain tissue from the suspect animal. Several cell lines have been used for this purpose and the murine neuroblastoma cell line Neuro-2a has been found to be the most sensitive. The human embryonic kidney cell line HEK-293 is known to express several neuronal proteins and is believed to be of neuronal origin. We hypothesized that this cell line could be susceptible to rabies virus, which is highly neurotropic. First we tested the sensitivity of HEK-293 cells to the laboratory strain, challenge virus standard (CVS). We then tested 120 brain samples from different animals and humans suspected to have died of rabies by fluorescent antibody test (FAT). Both FAT-positive and FAT-negative brains were tested for virus isolation using Neuro-2a, BHK-21, and HEK-293 cell lines and also by mouse inoculation. There was 100% correlation between FAT, virus isolation in Neuro-2a and HEK-293 cells, and mouse inoculation. However, the rate of virus isolation in the BHK-21 cell line was only 28% when compared to the other cell lines. The sensitivity of HEK-293 to CVS strain of virus was similar to that of Neuro-2a. We conclude that the HEK-293 cell line is as sensitive as the Neuro-2a cell line for the rapid isolation of rabies virus and may serve as an alternative cell line for rabies diagnosis and future research. Copyright © 2010 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Engineering cell-fluorescent ion track hybrid detectors.

PubMed

Niklas, Martin; Greilich, Steffen; Melzig, Claudius; Akselrod, Mark S; Debus, Jürgen; Jäkel, Oliver; Abdollahi, Amir

2013-06-11

The lack of sensitive biocompatible particle track detectors has so far limited parallel detection of physical energy deposition and biological response. Fluorescent nuclear track detectors (FNTDs) based on Al₂O₃:C,Mg single crystals combined with confocal laser scanning microscopy (CLSM) provide 3D information on ion tracks with a resolution limited by light diffraction. Here we report the development of next generation cell-fluorescent ion track hybrid detectors (Cell-Fit-HD). The biocompatibility of FNTDs was tested using six different cell lines, i.e. human non-small cell lung carcinoma (A549), glioblastoma (U87), androgen independent prostate cancer (PC3), epidermoid cancer (A431) and murine (VmDk) glioma SMA-560. To evaluate cell adherence, viability and conformal coverage of the crystals different seeding densities and alternative coating with extracellular matrix (fibronectin) was tested. Carbon irradiation was performed in Bragg peak (initial 270.55 MeV u⁻¹). A series of cell compartment specific fluorescence stains including nuclear (HOECHST), membrane (Glut-1), cytoplasm (Calcein AM, CM-DiI) were tested on Cell-Fit-HDs and a single CLSM was employed to co-detect the physical (crystal) as well as the biological (cell layer) information. The FNTD provides a biocompatible surface. Among the cells tested, A549 cells formed the most uniform, viable, tightly packed epithelial like monolayer. The ion track information was not compromised in Cell-Fit-HD as compared to the FNTD alone. Neither cell coating and culturing, nor additional staining procedures affected the properties of the FNTD surface to detect ion tracks. Standard immunofluorescence and live staining procedures could be employed to co-register cell biology and ion track information. The Cell-Fit-Hybrid Detector system is a promising platform for a multitude of studies linking biological response to energy deposition at high level of optical microscopy resolution.

Assuring safety without animal testing: the case for the human testis in vitro.

PubMed

Chapin, Robert E; Boekelheide, Kim; Cortvrindt, Rita; van Duursen, Majorie B M; Gant, Tim; Jegou, Bernard; Marczylo, Emma; van Pelt, Ans M M; Post, Janine N; Roelofs, Maarke J E; Schlatt, Stefan; Teerds, Katja J; Toppari, Jorma; Piersma, Aldert H

2013-08-01

From 15 to 17 June 2011, a dedicated workshop was held on the subject of in vitro models for mammalian spermatogenesis and their applications in toxicological hazard and risk assessment. The workshop was sponsored by the Dutch ASAT initiative (Assuring Safety without Animal Testing), which aims at promoting innovative approaches toward toxicological hazard and risk assessment on the basis of human and in vitro data, and replacement of animal studies. Participants addressed the state of the art regarding human and animal evidence for compound mediated testicular toxicity, reviewed existing alternative assay models, and brainstormed about future approaches, specifically considering tissue engineering. The workshop recognized the specific complexity of testicular function exemplified by dedicated cell types with distinct functionalities, as well as different cell compartments in terms of microenvironment and extracellular matrix components. This complexity hampers quick results in the realm of alternative models. Nevertheless, progress has been achieved in recent years, and innovative approaches in tissue engineering may open new avenues for mimicking testicular function in vitro. Although feasible, significant investment is deemed essential to be able to bring new ideas into practice in the laboratory. For the advancement of in vitro testicular toxicity testing, one of the most sensitive end points in regulatory reproductive toxicity testing, such an investment is highly desirable. Copyright © 2013. Published by Elsevier Inc. All rights reserved.

A study on the electrolysis of sulfur dioxide and water for the sulfur cycle hydrogen production process

NASA Technical Reports Server (NTRS)

1980-01-01

Experimental electrolysis cells using various platinum catalyzed carbon electrodes were tested. When operated at 200 mA/sq cm current density using 50 w/o acid at 50 C and 1 atm, a reference cell required 1.22 volts and degraded rapidly. After several improvements were incorporated into electrodes and the test cell configuration, a later cell required only 0.77 volts under identical operating conditions. At a lower current density, 100 mA/sq cm, the cell required only 0.63 volts. Kinetic studies on metal electrodes, measurements of temperature effects on electrode kinetics, investigations of electrocatalytic activities of metal electrodes over a wide range of acid concentrations, cyclic voltametric studies and evaluation of alternate catalysts were also conducted. From diffusivity experiments, a cation exchange membrane material, P-4010, exhibited an excellent diffusion coefficient, more than two orders of magnitude lower than that of rubber. Ionic resistivity measurements of eight materials showed that microporous rubber had the lowest resistivity.

In vitro approaches to evaluate toxicity induced by organotin compounds tributyltin (TBT), dibutyltin (DBT), and monobutyltin (MBT) in neuroblastoma cells.

PubMed

Ferreira, Martiña; Blanco, Lucía; Garrido, Alejandro; Vieites, Juan M; Cabado, Ana G

2013-05-01

The toxic effects of the organotin compounds (OTCs) monobutyltin (MBT), dibutyltin (DBT), and tributyltin (TBT) were evaluated in vitro in a neuroblastoma human cell line. Mechanisms of cell death, apoptosis versus necrosis, were studied by using several markers: inhibition of cell viability and proliferation, F-actin, and mitochondrial membrane potential changes as well as reactive oxygen species (ROS) production and DNA fragmentation. The most toxic effects were detected with DBT and TBT even at very low concentrations (0.1-1 μM). In contrast, MBT induced lighter cytotoxic changes at the higher doses tested. None of the studied compounds stimulated propidium iodide uptake, although the most toxic chemical, TBT, caused lactate dehydrogenase release at the higher concentrations tested. These findings suggest that in neuroblastoma, OTC-induced cytotoxicity involves different pathways depending on the compound, concentration, and incubation time. A screening method for DBT and TBT quantification based on cell viability loss was developed, allowing a fast detection alternative to complex methodology.

Regulation of alternative splicing at the single-cell level.

PubMed

Faigenbloom, Lior; Rubinstein, Nimrod D; Kloog, Yoel; Mayrose, Itay; Pupko, Tal; Stein, Reuven

2015-12-28

Alternative splicing is a key cellular mechanism for generating distinct isoforms, whose relative abundances regulate critical cellular processes. It is therefore essential that inclusion levels of alternative exons be tightly regulated. However, how the precision of inclusion levels among individual cells is governed is poorly understood. Using single-cell gene expression, we show that the precision of inclusion levels of alternative exons is determined by the degree of evolutionary conservation at their flanking intronic regions. Moreover, the inclusion levels of alternative exons, as well as the expression levels of the transcripts harboring them, also contribute to this precision. We further show that alternative exons whose inclusion levels are considerably changed during stem cell differentiation are also subject to this regulation. Our results imply that alternative splicing is coordinately regulated to achieve accuracy in relative isoform abundances and that such accuracy may be important in determining cell fate. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

Cloning from stem cells: different lineages, different species, same story.

PubMed

Oback, Björn

2009-01-01

Following nuclear transfer (NT), the most stringent measure of extensive donor cell reprogramming is development into viable offspring. This is referred to as cloning efficiency and quantified as the proportion of cloned embryos transferred into surrogate mothers that survive into adulthood. Cloning efficiency depends on the ability of the enucleated recipient cell to carry out the reprogramming reactions ('reprogramming ability') and the ability of the nuclear donor cell to be reprogrammed ('reprogrammability'). It has been postulated that reprogrammability of the somatic donor cell epigenome is inversely proportional to its differentiation status. In order to test this hypothesis, reprogrammability was compared between undifferentiated stem cells and their differentiated isogenic progeny. In the mouse, cells of divergent differentiation status from the neuronal, haematopoietic and skin epithelial lineage were tested. In cattle and deer, skeletal muscle and antler cells, respectively, were used as donors. No conclusive correlation between differentiation status and cloning efficiency was found, indicating that somatic donor cell type may not be the limiting factor for cloning success. This may reflect technical limitations of the NT-induced reprogramming assay. Alternatively, differentiation status and reprogrammability may be unrelated, making all cells equally difficult to reprogramme once they have left the ground state of pluripotency.

Lower growth temperature increases alternative pathway capacity and alternative oxidase protein in tobacco.

PubMed

Vanlerberghe, G C; McIntosh, L

1992-09-01

Suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow) have been used to study the effect of growth temperature on the CN-resistant, salicylhydroxamic acid-sensitive alternative pathway of respiration. Mitochondria isolated from cells maintained at 30 degrees C had a low capacity to oxidize succinate via the alternative pathway, whereas mitochondria isolated from cells 24 h after transfer to 18 degrees C displayed, on average, a 5-fold increase in this capacity (from 7 to 32 nanoatoms oxygen per milligram protein per minute). This represented an increase in alternative pathway capacity from 18 to 45% of the total capacity of electron transport. This increased capacity was lost upon transfer of cells back to 30 degrees C. A monoclonal antibody to the terminal oxidase of the alternative pathway (the alternative oxidase) from Sauromatum guttatum (T.E. Elthon, R.L. Nickels, L. McIntosh [1989] Plant Physiology 89: 1311-1317) recognized a 35-kilodalton mitochondrial protein in tobacco. There was an excellent correlation between the capacity of the alternative path in isolated tobacco mitochondria and the levels of this 35-kilodalton alternative oxidase protein. Cycloheximide could inhibit both the increased level of the 35-kilodalton alternative oxidase protein and the increased alternative pathway capacity normally seen upon transfer to 18 degrees C. We conclude that transfer of tobacco cells to the lower temperature increases the capacity of the alternative pathway due, at least in part, to de novo synthesis of the 35-kilodalton alternative oxidase protein.

Corrective Action Investigation Plan for Corrective Action Unit 528: Polychlorinated Biphenyls Contamination, Nevada Test Site, Nevada, Rev. 0

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

U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office

This Corrective Action Investigation Plan contains the U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office's approach to collect the data necessary to evaluate corrective action alternatives appropriate for the closure of Corrective Action Unit (CAU) 528, Polychlorinated Biphenyls Contamination (PCBs), Nevada Test Site (NTS), Nevada, under the Federal Facility Agreement and Consent Order. Located in the southwestern portion of Area 25 on the NTS in Jackass Flats (adjacent to Test Cell C [TCC]), CAU 528 consists of Corrective Action Site 25-27-03, Polychlorinated Biphenyls Surface Contamination. Test Cell C was built to support the Nuclear Rocket Development Stationmore » (operational between 1959 and 1973) activities including conducting ground tests and static firings of nuclear engine reactors. Although CAU 528 was not considered as a direct potential source of PCBs and petroleum contamination, two potential sources of contamination have nevertheless been identified from an unknown source in concentrations that could potentially pose an unacceptable risk to human health and/or the environment. This CAU's close proximity to TCC prompted Shaw to collect surface soil samples, which have indicated the presence of PCBs extending throughout the area to the north, east, south, and even to the edge of the western boundary. Based on this information, more extensive field investigation activities are being planned, the results of which are to be used to support a defensible evaluation of corrective action alternatives in the corrective action decision document.« less

Strategy for selecting disposable bags for cell culture media applications based on a root-cause investigation.

PubMed

Wood, Joseph; Mahajan, Ekta; Shiratori, Masaru

2013-01-01

The use of disposable bags for cell culture media storage has grown significantly in the past decade. Some of the key advantages of using disposable bags relative to non-disposable containers include increased product throughput, decreased cleaning validation costs, reduced risk of cross contamination and lower facility costs. As the scope of use of disposable bags for cell culture applications increases, problematic bags and scenarios should be identified and addressed to continue improving disposables technologies and meet the biotech industry's needs. In this article, we examine a cell culture application wherein media stored in disposable bags is warmed at 37°C before use for cell culture operations. A problematic bag film was identified through a prospective and retrospective cell culture investigation. The investigation provided information on the scope and variation of the issue with respect to different Chinese hamster ovary (CHO) cell lines, cell culture media, and application-specific parameters. It also led to the development of application-specific test methods and enabled a strategy for disposable bag film testing. The strategy was implemented for qualifying an alternative bag film for use in our processes. In this test strategy, multiple lots of 13 bag film types, encompassing eight vendors were evaluated using a three round, cell culture-based test strategy. The test strategy resulted in the determination of four viable bag film options based on the technical data. The results of this evaluation were used to conclude that a volatile or air-quenched compound, likely generated by gamma irradiation of the problematic bag film, negatively impacted cell culture performance. © 2013 American Institute of Chemical Engineers.

Epigenetic changes and alternate promoter usage by human colon cancers for expressing DCLK1-isoforms: Clinical Implications

PubMed Central

O’Connell, Malaney R.; Sarkar, Shubhashish; Luthra, Gurinder K.; Okugawa, Yoshinaga; Toiyama, Yuji; Gajjar, Aakash H.; Qiu, Suimin; Goel, Ajay; Singh, Pomila

2015-01-01

DCLK1 specifically marks colon/pancreatic cancers in mice, and is expressed by human colon adenocarcinomas (hCRCs). Down-regulation of DCLK1 results in loss of cancer-stem-cells (CSCs), and inhibits spheroidal/xenograft growths from hCRC-cells. The 5′-promoter of DCLK1-gene is reportedly hypermethylated in hCRCs, resulting in loss of expression of DCLK1-transcripts, originating from 5′(α)-promoter (termed DCLK1-L, in here). However, in mouse colon-tumors, 5′-promoter of DCLK1-gene remains unchanged, and DCLK1-L, originating from 5′(α)-promoter, is expressed. We hypothesized that elevated levels of DCLK1-protein in hCRC-cells, may be transcribed/translated from an alternate-promoter. Several in silico and molecular biology approaches were used to test our hypothesis. We report for the first time that majority of hCRCs express short-transcripts of DCLK1 (termed DCLK1-S, in here) from an alternate β-promoter in IntronV of the gene, while normal-colons mainly express DCLK1-L from 5′(α)-promoter. We additionally report an important role of β-catenin and TCF4/LEF binding-sites for activating (α)-promoter, while activated NF-κBp65 (bound to NF-κB-cis-element), activates (β)-promoter in cancer-cells. DCLK1-S expression was examined in a cohort of 92 CRC patients; high-expressors had significantly worse overall-survival compared to low-expressors. Our novel findings’ regarding usage of alternate (β)-promoter by hCRCs, suggests that DCLK1-S may represent an important target for preventing/inhibiting colon-cancers, and for eliminating colon-CSCs. PMID:26447334

Differences of Cytotoxicity of Orthodontic Bands Assessed by Survival Tests in Saccharomyces cerevisiae

PubMed Central

Gonçalves, Tatiana Siqueira; de Menezes, Luciane Macedo; Ribeiro, Luciele Gonzaga; Lindholz, Catieli Gobetti; Medina-Silva, Renata

2014-01-01

The aim of this study was to evaluate the cytotoxicity induced by orthodontic bands through survival tests on Saccharomyces cerevisiae, a microorganism that presents several genetic and biochemical characteristics similar to human cells. Three groups of bands were evaluated: silver soldered (SSB), laser soldered (LSB), and bands without any solder (WSB). Yeast cells were directly exposed to the bands and indirectly, when a previous elution of the metals in artificial saliva was performed. The negative control was composed of yeast cells or artificial saliva not exposed to any kind of metal. In the direct exposure experiments, all tested groups of bands induced a slight reduction in yeast viability compared to the control. This effect was more intense for the SSB, although not statistically significant. For the indirect exposure experiments, the SSB induced a statistically significant decrease in cell viability compared to the LSB. There were no significant differences between the survival rates of the negative control and the LSB group in both direct and saliva tests. SSBs were cytotoxic, whilst LSBs were not, confirming that laser soldering may be a more biocompatible alternative for use in connecting wires to orthodontic appliances. PMID:24511527

PAI-1 (Plasminogen Activator Inhibitor-1) Expression Renders Alternatively Activated Human Macrophages Proteolytically Quiescent

PubMed Central

Hohensinner, Philipp J.; Baumgartner, Johanna; Kral-Pointner, Julia B.; Uhrin, Pavel; Ebenbauer, Benjamin; Thaler, Barbara; Doberer, Konstantin; Stojkovic, Stefan; Demyanets, Svitlana; Fischer, Michael B.; Huber, Kurt; Schabbauer, Gernot; Speidl, Walter S.

2017-01-01

Objective— Macrophages are versatile immune cells capable of polarizing into functional subsets depending on environmental stimulation. In atherosclerotic lesions, proinflammatory polarized macrophages are associated with symptomatic plaques, whereas Th2 (T-helper cell type 2) cytokine–polarized macrophages are inversely related with disease progression. To establish a functional cause for these observations, we analyzed extracellular matrix degradation phenotypes in polarized macrophages. Approach and Results— We provide evidence that proinflammatory polarized macrophages rely on membrane-bound proteases including MMP-14 (matrix metalloproteinase-14) and the serine protease uPA (urokinase plasminogen activator) together with its receptor uPAR for extracellular matrix degradation. In contrast, Th2 cytokine alternatively primed macrophages do not show different proteolytic activity in comparison to unpolarized macrophages and lack increased localization of MMP-14 and uPA receptor to the cell membrane. Nonetheless, they express the highest amount of the serine protease uPA. However, uPA activity is blocked by similarly increased expression of its inhibitor PAI-1 (plasminogen activator inhibitor 1). When inhibiting PAI-1 or when analyzing macrophages deficient in PAI-1, Th2 cytokine–polarized macrophages display the same matrix degradation capability as proinflammatory-primed macrophages. Within atherosclerotic lesions, macrophages positive for the alternative activation marker CD206 express high levels of PAI-1. In addition, to test changed tissue remodeling capacities of alternatively activated macrophages, we used a bleomycin lung injury model in mice reconstituted with PAI-1−/− bone marrow. These results supported an enhanced remodeling phenotype displayed by increased fibrosis and elevated MMP activity in the lung after PAI-1 loss. Conclusions— We were able to demonstrate matrix degradation dependent on membrane-bound proteases in proinflammatory stimulated macrophages and a forced proteolytical quiescence in alternatively polarized macrophages by the expression of PAI-1. PMID:28818858

Low Intensity Low Temperature (LILT) measurements and coefficients on new photovoltaic structures

NASA Technical Reports Server (NTRS)

Schelman, David A.; Jenkins, Philip 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 (RTG's). 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 photovoltaic (PV) and thermophotovoltaic (TPV). Both of these alternate energy sources convert light/thermal energy directly into electricity. In order to create a viable PV and TPV data base for planetary mission planners and cell designers, we have compiled low temperature low intensity (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 dI(sub SC)/dT, dV(sub OC)/dT, dFF/dT, and also as a function of intensity, an accurate prediction of cell performance under the AMO 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/Ge, InP, InGaAs/InP, InP/InGaAs/InP, and GainP. Temperatures range as low as -175 C and intensities range from 1 sun to .02 suns.

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.

Human platelet lysate in mesenchymal stromal cell expansion according to a GMP grade protocol: a cell factory experience.

PubMed

Becherucci, Valentina; Piccini, Luisa; Casamassima, Serena; Bisin, Silvia; Gori, Valentina; Gentile, Francesca; Ceccantini, Riccardo; De Rienzo, Elena; Bindi, Barbara; Pavan, Paola; Cunial, Vanessa; Allegro, Elisa; Ermini, Stefano; Brugnolo, Francesca; Astori, Giuseppe; Bambi, Franco

2018-05-02

The use of platelet lysate (PL) for the ex-vivo expansion of mesenchymal stromal/stem cells (MSCs) was initially proposed by Doucet et al. in 2005, as an alternative to animal serum. Moreover, regulatory authorities discourage the use of fetal bovine serum (FBS) or other animal derivatives, to avoid risk of zoonoses and xenogeneic immune reactions. Even if many studies investigated PL composition, there still are some open issues related to its use in ex-vivo MSC expansion, especially according to good manufacturing practice (GMP) grade protocols. As an authorized cell factory, we report our experience using standardized PL produced by Azienda Ospedaliero Universitaria Meyer Transfusion Service for MSC expansion according to a GMP grade clinical protocol. As suggested by other authors, we performed an in-vitro test on MSCs versus MSCs cultured with FBS that still represents the best way to test PL batches. We compared 12 MSC batches cultured with DMEM 5% PL with similar batches cultured with DMEM 10% FBS, focusing on the MSC proliferation rate, MSC surface marker expression, MSC immunomodulatory and differentiation potential, and finally MSC relative telomere length. Results confirmed the literature data as PL increases cell proliferation without affecting the MSC immunophenotype, immunomodulatory potential, differentiation potential and relative telomere length. PL can be considered a safe alternative to FBS for ex-vivo expansion of MSC according to a GMP grade protocol. Our experience confirms the literature data: a large number of MSCs for clinical applications can be obtained by expansion with PL, without affecting the MSC main features. Our experience underlines the benefits of a close collaboration between the PL producers (transfusion service) and the end users (cell factory) in a synergy of skills and experiences that can lead to standardized PL production.

Economic analysis of ALK testing and crizotinib therapy for advanced non-small-cell lung cancer.

PubMed

Lu, Shun; Zhang, Jie; Ye, Ming; Wang, Baoai; Wu, Bin

2016-06-01

The economic outcome of crizotinib in advanced non-small-cell lung cancer harboring anaplastic lymphoma kinase rearrangement would be investigated. Based on a mathematical model, the economic outcome of three techniques for testing ALK gene rearrangement combing with crizotinib would be evaluated and compared with traditional regimen. The impact of the crizotinib patient assistance program (PAP) was assessed. Ventana immunohistochemistry, quantitative real-time reverse transcription-polymerase chain reaction and IHC testing plus fluorescent in situ hybridization confirmation for anaplastic lymphoma kinase testing following crizotinib treatment leaded to the incremental cost-effectiveness ratios of US$16,820 and US$223,242, US$24,424 and US$223,271, and US$16,850 and US$254,668 per quality-adjusted life-year gained with and without PAP, respectively. Gene-guided crizotinib therapy might be a cost-effective alternative comparing with the traditional regimen in the PAP setting.

A novel approach on accelerated ageing towards reliability optimization of high concentration photovoltaic cells

NASA Astrophysics Data System (ADS)

Tsanakas, John A.; Jaffre, Damien; Sicre, Mathieu; Elouamari, Rachid; Vossier, Alexis; de Salins, Jean-Edouard; Bechou, Laurent; Levrier, Bruno; Perona, Arnaud; Dollet, Alain

2014-09-01

This paper presents a preliminary study upon a novel approach proposed for highly accelerated ageing and reliability optimization of high concentrating photovoltaic (HCPV) cells and assemblies. The intended approach aims to overcome several limitations of some current accelerated ageing tests (AAT) adopted up today, proposing the use of an alternative experimental set-up for performing faster and more realistic thermal cycles, under real sun, without the involvement of environmental chamber. The study also includes specific characterization techniques, before and after each AAT sequence, which respectively provide the initial and final diagnosis on the condition of the tested sample. The acquired data from these diagnostic/characterization methods are then used as indices to determine both quantitatively and qualitatively the severity of degradation and, thus, the ageing level for each tested HCPV assembly or cell sample. Ultimate goal of such "initial diagnosis - AAT - final diagnosis" sequences is to provide the basis for a future work on the reliability analysis of the main degradation mechanisms and confident prediction of failure propagation in HCPV cells, by means of acceleration factor (AF) and mean-time-to-failure (MTTF) estimations.

Inactivation of CUG-BP1/CELF1 causes growth, viability, and spermatogenesis defects in mice.

PubMed

Kress, Chantal; Gautier-Courteille, Carole; Osborne, H Beverley; Babinet, Charles; Paillard, Luc

2007-02-01

CUG-BP1/CELF1 is a multifunctional RNA-binding protein involved in the regulation of alternative splicing and translation. To elucidate its role in mammalian development, we produced mice in which the Cugbp1 gene was inactivated by homologous recombination. These Cugbp1(-/-) mice were viable, although a significant portion of them did not survive after the first few days of life. They displayed growth retardation, and most Cugbp1(-/-) males and females exhibited impaired fertility. Male infertility was more thoroughly investigated. Histological examination of testes from Cugbp1(-/-) males showed an arrest of spermatogenesis that occurred at step 7 of spermiogenesis, before spermatid elongation begins, and an increased apoptosis. A quantitative reverse transcriptase PCR analysis showed a decrease of all the germ cell markers tested but not of Sertoli and Leydig markers, suggesting a general decrease in germ cell number. In wild-type testes, CUG-BP1 is expressed in germ cells from spermatogonia to round spermatids and also in Sertoli and Leydig cells. These findings demonstrate that CUG-BP1 is required for completion of spermatogenesis.

Beam dynamics validation of the Halbach Technology FFAG Cell for Cornell-BNL Energy Recovery Linac

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

Meot, Francois; Tsoupas, N.; Brooks, S.

The Cornell-BNL Electron Test Accelerator (CBETA), a 150 MeV energy recovery linac (ERL) now in construction at Cornell, employs a fixed-field alternating gradient optics return loop: a single beam line comprised of FFAG cells, which accepts four recirculated energies. CBETA FFAG cell uses Halbach permanent magnet technology, its design studies have covered an extended period of time supported by extensive particle dynamics simulations using computed 3-D field map models. As a result, this approach is discussed, and illustrated here, based on the final stage in these beam dynamics studies, namely the validation of a ultimate, optimized design of the Halbachmore » cell.« less

Beam dynamics validation of the Halbach Technology FFAG Cell for Cornell-BNL Energy Recovery Linac

DOE PAGES

Meot, Francois; Tsoupas, N.; Brooks, S.; ...

2018-04-16

The Cornell-BNL Electron Test Accelerator (CBETA), a 150 MeV energy recovery linac (ERL) now in construction at Cornell, employs a fixed-field alternating gradient optics return loop: a single beam line comprised of FFAG cells, which accepts four recirculated energies. CBETA FFAG cell uses Halbach permanent magnet technology, its design studies have covered an extended period of time supported by extensive particle dynamics simulations using computed 3-D field map models. As a result, this approach is discussed, and illustrated here, based on the final stage in these beam dynamics studies, namely the validation of a ultimate, optimized design of the Halbachmore » cell.« less

The current status of alternatives to animal testing and predictive toxicology methods using liver microfluidic biochips.

PubMed

Prot, Jean Matthieu; Leclerc, Eric

2012-06-01

In this paper, we will consider new in vitro cell culture platforms and the progress made, based on the microfluidic liver biochips dedicated to pharmacological and toxicological studies. Particular emphasis will be given to recent developments in the microfluidic tools dedicated to cell culture (more particularly liver cell culture), in silico opportunities for Physiologically Based PharmacoKinetic (PBPK) modelling, the challenge of the mechanistic interpretations offered by the approaches resulting from "multi-omics" data (transcriptomics, proteomics, metabolomics, cytomics) and imaging microfluidic platforms. Finally, we will discuss the critical features regarding microfabrication, design and materials, and cell functionality as the key points for the future development of new microfluidic liver biochips.

Instrumentation for accelerated life tests of concentrator solar cells.

PubMed

Núñez, N; Vázquez, M; González, J R; Jiménez, F J; Bautista, J

2011-02-01

Concentrator photovoltaic is an emergent technology that may be a good economical and efficient alternative for the generation of electricity at a competitive cost. However, the reliability of these new solar cells and systems is still an open issue due to the high-irradiation level they are subjected to as well as the electrical and thermal stresses that they are expected to endure. To evaluate the reliability in a short period of time, accelerated aging tests are essential. Thermal aging tests for concentrator photovoltaic solar cells and systems under illumination are not available because no technical solution to the problem of reaching the working concentration inside a climatic chamber has been available. This work presents an automatic instrumentation system that overcomes the aforementioned limitation. Working conditions have been simulated by forward biasing the solar cells to the current they would handle at the working concentration (in this case, 700 and 1050 times the irradiance at one standard sun). The instrumentation system has been deployed for more than 10 000 h in a thermal aging test for III-V concentrator solar cells, in which the generated power evolution at different temperatures has been monitored. As a result of this test, the acceleration factor has been calculated, thus allowing for the degradation evolution at any temperature in addition to normal working conditions to be obtained.

Cytotoxic effects of octenidine mouth rinse on human fibroblasts and epithelial cells - an in vitro study.

PubMed

Schmidt, J; Zyba, V; Jung, K; Rinke, S; Haak, R; Mausberg, R F; Ziebolz, D

2016-01-01

This study compared the cytotoxicity of a new octenidine mouth rinse (MR) against gingival fibroblasts and epithelial cells with different established MRs. The following MRs were used: Octenidol (OCT), Chlorhexidine 0.2% (CHX), Listerine (LIS), Meridol (MER), Betaisodona (BET); and control (medium only). Human primary gingiva fibroblasts and human primary nasal epithelial cells were cultivated in cell-specific media (2 × 10(5) cells/ml) and treated with MR for 1, 5, and 15 min. Each test was performed 12 times. Metabolism activity was measured using a cytotoxicity assay. A cellometer analyzed cell viability, cell number, and cell diameter. The data were analyzed by two-way analysis of variance with subsequent Dunnett's test and additional t-tests. The cytotoxic effects of all MRs on fibroblasts and epithelial cells compared to the control depended on the contact time (p < 0.001). OCT and BET showed less influence on cell metabolism in fibroblasts than other MRs. OCT also demonstrated comparable but not significant results in epithelial cells (p > 0.005). Cell numbers of both cell types at all contact times revealed that OCT showed a less negative effect (p > 0.005), especially for epithelial cells compared to CHX after 15 min (p < 0.005). OCT and BET showed the best results for viability in fibroblasts (p > 0.005), but MER showed less influence than OCT in epithelial cells (p < 0.005). OCT is a potential alternative to CHX regarding cytotoxicity because of its lower cell-toxic effect against fibroblasts and epithelial cells.

The evolution of bacterial cell size: the internal diffusion-constraint hypothesis.

PubMed

Gallet, Romain; Violle, Cyrille; Fromin, Nathalie; Jabbour-Zahab, Roula; Enquist, Brian J; Lenormand, Thomas

2017-07-01

Size is one of the most important biological traits influencing organismal ecology and evolution. However, we know little about the drivers of body size evolution in unicellulars. A long-term evolution experiment (Lenski's LTEE) in which Escherichia coli adapts to a simple glucose medium has shown that not only the growth rate and the fitness of the bacterium increase over time but also its cell size. This increase in size contradicts prominent 'external diffusion' theory (EDC) predicting that cell size should have evolved toward smaller cells. Among several scenarios, we propose and test an alternative 'internal diffusion-constraint' (IDC) hypothesis for cell size evolution. A change in cell volume affects metabolite concentrations in the cytoplasm. The IDC states that a higher metabolism can be achieved by a reduction in the molecular traffic time inside of the cell, by increasing its volume. To test this hypothesis, we studied a population from the LTEE. We show that bigger cells with greater growth and CO 2 production rates and lower mass-to-volume ratio were selected over time in the LTEE. These results are consistent with the IDC hypothesis. This novel hypothesis offers a promising approach for understanding the evolutionary constraints on cell size.

Fluorescent Protein-Based Quantification of Alternative Splicing of a Target Cassette Exon in Mammalian Cells.

PubMed

Gurskaya, N G; Staroverov, D B; Lukyanov, K A

2016-01-01

Alternative splicing is an important mechanism of regulation of gene expression and expansion of proteome complexity. Recently we developed a new fluorescence reporter for quantitative analysis of alternative splicing of a target cassette exon in live cells (Gurskaya et al., 2012). It consists of a specially designed minigene encoding red and green fluorescent proteins (Katushka and TagGFP2) and a fragment of the target gene between them. Skipping or inclusion of the alternative exon induces a frameshift; ie, alternative exon length must not be a multiple of 3. Finally, red and green fluorescence intensities of cells expressing this reporter are used to estimate the percentage of alternative (exon-skipped) and normal (exon-retained) transcripts. Here, we provide a detailed description of design and application of the fluorescence reporter of a target alternative exon splicing in mammalian cell lines. © 2016 Elsevier Inc. All rights reserved.

Reflex high-risk human papilloma virus DNA test is useful in the triage of women with atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion.

PubMed

Wu, Howard Her-Juing; Allen, Susan L; Kirkpatrick, Joseph L; Elsheikh, Tarik M

2006-10-01

This study is aimed to investigate the role of reflex high-risk human papilloma virus (HPV) DNA testing as an alternative triage method to colposcopy for women with atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion (ASC-H) on Papanicolaou (Pap) tests. Reflex HPV DNA testing using Hybrid Capture II method was carried out on 88 women with ASC-H diagnosed by Thin Prep Pap test. Correlation with follow-up biopsies was available on 42 of these patients. The reflex HPV DNA test showed an overall positive rate of 67% and negative rate of 33% in 88 patients with ASC-H. Using age 30 as the cut off point, the positive rate had increased to 83.3% (35/42) in patients 30 yr or younger, while the positive rate for patients older than 30 yr had decreased to 52.2% (24/46). Follow-up colposcopic biopsy results were available in 35 of 59 HPV-positive women, which revealed 15 (43%) high-grade squamous intraepithelial lesions (HSIL), 12 low-grade squamous intraepithelial lesions (LSIL), and 8 negative for dysplasia. In 7 HPV-negative patients, the follow-up biopsies showed no evidence of HSIL or LSIL. Correlation between clinical risk factors and the HPV results demonstrated no significant differences in HPV positivity between the high-risk and low-risk patients. The high sensitivity (100%) and negative predictive rate (100%) in detecting HSIL in our study provide strong evidence that, instead of automatic referral to colposcopy, reflex HPV DNA testing may be used as an alternative triage method for women diagnosed with ASC-H on Thin Prep Pap test, especially for women older than 30 yr of age.

Application of succulent plant leaves for Agrobacterium infiltration-mediated protein production.

PubMed

Jones, Richard W

2016-01-01

When expressing plant cell wall degrading enzymes in the widely used tobacco (Nicotiana benthamiana) after Agrobacterium infiltration, difficulties arise due to the thin leaf structure. Thick leaved succulents, Kalanchoe blossfeldiana and Hylotelephium telephium, were tested as alternatives. A xyloglucanase, as well as a xyloglucanase inhibitor protein was successfully produced. Published by Elsevier B.V.

Catabolite-mediated mutations in alternate toluene degradative pathways in Pseudomonas putida.

PubMed Central

Leddy, M B; Phipps, D W; Ridgway, H F

1995-01-01

Pseudomonas putida 54g grew on mineral salts with toluene and exhibited catechol-2,3-dioxygenase (C23O) activity, indicating a meta pathway. After 10 to 15 days on toluene, nondegrading (Tol-) variants approached nearly 10% of total CFU. Auxotrophs were not detected among variants, suggesting selective loss of catabolic function(s). Variant formation was substrate dependent, since Tol- cells were observed on neither ethylbenzene, glucose, nor peptone-based media nor when toluene catabolism was suppressed by glucose. Unlike wild-type cells, variants did not grow on gasoline, toluene, benzene, ethylbenzene, benzoate, or catechol, suggesting loss of meta pathway function. Catabolic and C23O activities were restored to variants via transfer of a 78-mDa TOL-like plasmid from a wild-type Tol+ donor. Tests for reversion of variants to Tol+ were uniformly negative, suggesting possible delection or excision of catabolic genes. Deletions were confirmed in some variants by failure to hybridize with a DNA probe specific for the xylE gene encoding C23O. Cells grown on benzoate remained Tol+ but were C23O- and contained a plasmid of reduced size or were plasmid free, suggesting an alternate chromosomal catabolic pathway, also defective in variants. Cells exposed to benzyl alcohol, the initial oxidation product of toluene, accumulated > 13% variants in 5 days, even when cell division was repressed by nitrogen deprivation to abrogate selection processes. No variants formed in identical ethylbenzene-exposed controls. The results suggest that benzyl alcohol mediates irreversible defects in both a plasmid-associated meta pathway and an alternate chromosomal pathway. PMID:7642499

DNA Damage Potential of Engine Emissions Measured In Vitro by Micronucleus Test in Human Bronchial Epithelial Cells.

PubMed

Cervena, Tereza; Rossnerova, Andrea; Sikorova, Jitka; Beranek, Vit; Vojtisek-Lom, Michal; Ciganek, Miroslav; Topinka, Jan; Rossner, Pavel

2017-09-01

Internal combustion engine emissions belong among the major anthropogenic sources of air pollution in urban areas. According to the International Agency for Research on Cancer, there is sufficient evidence of the carcinogenicity of diesel exhaust in human beings. Although alternative fuels, mainly biodiesel, have recently become popular, little is still known about the genotoxicity of emissions from these fuels. We analysed DNA damage expressed as the frequency of micronuclei (MN) in human bronchial epithelial cells (BEAS-2B), induced by extractable organic matter (EOM; tested concentrations: 1, 10 and 25 μg/ml) obtained from particle emissions from various blends of biodiesel with diesel fuels (including neat diesel fuel (B0), a blend of 70% B0 and 30% biodiesel (B30) and neat biodiesel (B100)). We also tested the effect of selected diesel exhaust organic/genotoxic components [benzo[a]pyrene (B[a]P) concentrations: 25, 100 and 200 μM; 1-nitropyrene (1-NP) concentrations: 1, 5 and 10 μM; 3-nitrobenzanthrone (3-NBA) concentrations: 1, 5 and 50 μM]. The cells were treated with the compounds for 28 and 48 hr. Our results showed that most of the tested compounds (except for the 25 μM B[a]P, 28-hr treatment) significantly increased MN frequency. The genotoxicity of EOMs from the engine emissions of diesel and biodiesel engines was comparable. Both nitro-PAH compounds demonstrated higher genotoxic potential in comparison with B[a]P. Considering our results and due to increasing popularity of alternative fuels, it is prudent that the potential genotoxic effects of various fuels are investigated across engine technologies and operating conditions in a relevant model system. © 2016 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

The IKK Inhibitor Bay 11-7082 Induces Cell Death Independent from Inhibition of Activation of NFκB Transcription Factors

PubMed Central

Rauert-Wunderlich, Hilka; Siegmund, Daniela; Maier, Eduard; Giner, Tina; Bargou, Ralf C.; Wajant, Harald; Stühmer, Thorsten

2013-01-01

Multiple myeloma (MM) displays an NFκB activity-related gene expression signature and about 20% of primary MM samples harbor genetic alterations conducive to intrinsic NFκB signaling activation. The relevance of blocking the classical versus the alternative NFκB signaling pathway and the molecular execution mechanisms involved, however, are still poorly understood. Here, we comparatively tested NFκB activity abrogation through TPCA-1 (an IKK2 inhibitor), BAY 11-7082 (an IKK inhibitor poorly selective for IKK1 and IKK2), and MLN4924 (an NEDD8 activating enzyme (NAE)-inhibitor), and analyzed their anti-MM activity. Whereas TPCA-1 interfered selectively with activation of the classical NFκB pathway, the other two compounds inhibited classical and alternative NFκB signaling without significant discrimination. Noteworthy, whereas TPCA-1 and MLN4924 elicited rather mild anti-MM effects with slight to moderate cell death induction after 1 day BAY 11-7082 was uniformly highly toxic to MM cell lines and primary MM cells. Treatment with BAY 11-7082 induced rapid cell swelling and its initial effects were blocked by necrostatin-1 or the ROS scavenger BHA, but a lasting protective effect was not achieved even with additional blockade of caspases. Because MLN4924 inhibits the alternative NFκB pathway downstream of IKK1 at the level of p100 processing, the quite discordant effects between MLN4924 and BAY 11-7082 must thus be due to blockade of IKK1-mediated NFκB-independent necrosis-inhibitory functions or represent an off-target effect of BAY 11-7082. In accordance with the latter, we further observed that concomitant knockdown of IKK1 and IKK2 did not have any major short-term adverse effect on the viability of MM cells. PMID:23527154

Engineering behavior of small-scale foundation piers constructed from alternative materials

NASA Astrophysics Data System (ADS)

Prokudin, Maxim Mikhaylovich

Testing small-scale prototype pier foundations to evaluate engineering behavior is an alternative to full-scale testing that facilitates testing of several piers and pier groups at relatively low cost. In this study, various pier systems and pier groups at one tenth scale were subjected to static vertical loading under controlled conditions to evaluate stiffness, bearing capacity, and group efficiency. Pier length, material properties and methods of installation were evaluated. Pier length to diameter ratios varied between four and eight. A unique soil pit with dimensions of 2.1 m in width, 1.5 m in length and 2.0 m in depth was designed to carry out this research. The test pit was filled with moisture conditioned and compacted Western Iowa loess. A special load test frame was designed and fabricated to provide up to 25,000 kg vertical reaction force for load testing. A load cell and displacement instrumentation was setup to capture the load test data. Alternative materials to conventional cement concrete were studied. The pier materials evaluated in this study included compacted aggregate, cement stabilized silt, cementitious grouts, and fiber reinforced silt. Key findings from this study demonstrated that (1) the construction method influences the behavior of aggregate piers, (2) the composition of the pier has a significant impact on the stiffness, (3) group efficiencies were found to be a function of pier length and pier material, (4) in comparison to full-scale testing the scaled piers were found to produce a stiffer response with load-settlement and bearing capacities to be similar. Further, although full-scale test results were not available for all pier materials, the small-scale testing provided a means for comparing results between pier systems. Finally, duplicate pier tests for a given length and material were found to be repeatable.

IARC classes 1 and 2 carcinogens are successfully identified by an alternative strategy that detects DNA-reactivity and cell transformation ability of chemicals.

PubMed

Benigni, Romualdo; Bossa, Cecilia; Battistelli, Chiara Laura; Tcheremenskaia, Olga

2013-12-12

For decades, traditional toxicology has been the ultimate source of information on the carcinogenic potential of chemicals; however with increasing demand on regulation of chemicals and decreasing resources for testing, opportunities to accept "alternative" approaches have dramatically expanded. The need for tools able to identify carcinogens in shorter times and at a lower cost in terms of animal lives and money is still an open issue, and the present strategies and regulations for carcinogenicity pre-screening do not adequately protect human health. In previous papers, we have proposed an integrated in vitro/in silico strategy that detects DNA-reactivity and tissue disorganization/disruption by chemicals, and we have shown that the combination of Salmonella and Structural Alerts for the DNA-reactive carcinogens, and in vitro cell transformation assays for nongenotoxic carcinogens permits the identification of a very large proportion (up to 95%) of rodent carcinogens, while having a considerable specificity with the rodent noncarcinogens. In the present paper we expand the previous investigation and show that this alternative strategy identifies correctly IARC Classes 1 and 2 carcinogens. If implemented, this alternative strategy can contribute to improve the protection of human health while decreasing the use of animals. Copyright © 2013 Elsevier B.V. All rights reserved.

Lower Growth Temperature Increases Alternative Pathway Capacity and Alternative Oxidase Protein in Tobacco 1

PubMed Central

Vanlerberghe, Greg C.; McIntosh, Lee

1992-01-01

Suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow) have been used to study the effect of growth temperature on the CN-resistant, salicylhydroxamic acid-sensitive alternative pathway of respiration. Mitochondria isolated from cells maintained at 30°C had a low capacity to oxidize succinate via the alternative pathway, whereas mitochondria isolated from cells 24 h after transfer to 18°C displayed, on average, a 5-fold increase in this capacity (from 7 to 32 nanoatoms oxygen per milligram protein per minute). This represented an increase in alternative pathway capacity from 18 to 45% of the total capacity of electron transport. This increased capacity was lost upon transfer of cells back to 30°C. A monoclonal antibody to the terminal oxidase of the alternative pathway (the alternative oxidase) from Sauromatum guttatum (T.E. Elthon, R.L. Nickels, L. McIntosh [1989] Plant Physiology 89: 1311-1317) recognized a 35-kilodalton mitochondrial protein in tobacco. There was an excellent correlation between the capacity of the alternative path in isolated tobacco mitochondria and the levels of this 35-kilodalton alternative oxidase protein. Cycloheximide could inhibit both the increased level of the 35-kilodalton alternative oxidase protein and the increased alternative pathway capacity normally seen upon transfer to 18°C. We conclude that transfer of tobacco cells to the lower temperature increases the capacity of the alternative pathway due, at least in part, to de novo synthesis of the 35-kilodalton alternative oxidase protein. Images Figure 3 Figure 4 PMID:16652932

Vehicle Integrated Photovoltaics for Compression Ignition Vehicles: An Experimental Investigation of Solar Alkaline Water Electrolysis for Improving Diesel Combustion and a Solar Charging System for Reducing Auxiliary Engine Loads

NASA Astrophysics Data System (ADS)

Negroni, Garry Inocentes

Vehicle-integrated photovoltaic electricity can be applied towards aspiration of hydrogen-oxygen-steam gas produced through alkaline electrolysis and reductions in auxiliary alternator load for reducing hydrocarbon emissions in low nitrogen oxide indirect-injection compression-ignition engines. Aspiration of 0.516 ± 0.007 liters-per-minute of gas produced through alkaline electrolysis of potassium-hydroxide 2wt.% improves full-load performance; however, part-load performance decreases due to auto-ignition of aspirated gas prior to top-dead center. Alternator load reductions offer improved part-load and full-load performance with practical limitations resulting from accessory electrical loads. In an additive approach, solar electrolysis can electrochemically convert solar photovoltaic electricity into a gas comprised of stoichiometric hydrogen and oxygen gas. Aspiration of this hydrogen-oxygen gas enhances combustion properties decreasing emissions and increased combustion efficiency in light-duty diesel vehicles. The 316L stainless steel (SS) electrolyser plates are arranged with two anodes and three cathodes space with four bipolar plates delineating four stacks in parallel with five cells per stack. The electrolyser was tested using potassium hydroxide 2 wt.% and hydronium 3wt.% at measured voltage and current inputs. The flow rate output from the reservoir cell was measured in parallel with the V and I inputs producing a regression model correlating current input to flow rate. KOH 2 wt.% produced 0.005 LPM/W, while H9O44 3 wt.% produced less at 0.00126 LPM/W. In a subtractive approach, solar energy can be used to charge a larger energy storage device, as is with plug-in electric vehicles, in order to alleviate the engine of the mechanical load placed upon it by the vehicles electrical accessories through the alternator. Solar electrolysis can improve part-load emissions and full-load performance. The average solar-to-battery efficiency based on the OEM rated efficiency was 11.4%. The average voltage efficiency of the electrolyser during dynamometer testing was 69.16%, producing a solar-to-electrolysis efficiency of 7.88%. At varying engine speeds, HC emissions decreased an average of 54.4% at multiple engine speeds at part-load, while CO2 increased by 2.54% due to oxygen enrichment of intake air. However, the auto-ignition of a small amount of hydrogen (0.0035% of diesel fuel energy) had a negative impact on part-load power (-3.671%) and torque (-3.296%). Full-load sweep testing showed an increase in peak power (1.562%) and peak torque (2.608%). Solar electrolysis gas aspiration reduced soot opacity by 31.5%. The alternator-less part-load step tests show average HC and CO2 emissions decrease on average 25.05% and 1.14% respectively. The test also indicates an increase in average part-load power (1.57%) and torque (2.12%). Alternator-less operation can reduce soot opacity by 56.76%. Full-load testing of the vehicle with alternator unplugged indicates that alternator load upon an engine increase with engine ne speed even with no load and no pilot excitation. Alternator load elimination's performance and emissions improvements should be considered, however, practical limitations exist in winter-night, summer-midday scenarios and for longer duration of operation.

COMMUNICATION: Alternative splicing and genomic stability

NASA Astrophysics Data System (ADS)

Cahill, Kevin

2004-06-01

Alternative splicing allows an organism to make different proteins in different cells at different times, all from the same gene. In a cell that uses alternative splicing, the total length of all the exons is much shorter than in a cell that encodes the same set of proteins without alternative splicing. This economical use of exons makes genes more stable during reproduction and development because a genome with a shorter exon length is more resistant to harmful mutations. Genomic stability may be the reason why higher vertebrates splice alternatively. For a broad class of alternatively spliced genes, a formula is given for the increase in their stability.

An alternative anionic bio-sustainable anti-fungal agent: Investigation of its mode of action on the fungal cell membrane.

PubMed

Stenbæk, Jonas; Löf, David; Falkman, Peter; Jensen, Bo; Cárdenas, Marité

2017-07-01

The potential of a lactylate (the sodium caproyl lactylate or C10 lactylate), a typical food grade emulsifier, as an anionic environmental friendly anti-fungal additive was tested in growth medium and formulated in a protective coating for exterior wood. Different laboratory growth tests on the blue stain fungus Aureobasidium pullulans were performed and its interactions on a model fungal cell membrane were studied. Promising short term anti-fungal effects in growth tests were observed, although significant but less dramatic effects took place in coating test on wood panels. Scanning electron microscope analysis shows clear differences in the amount of fungal slime on the mycelium of Aureobasidium pullulans when the fungus was exposed of C10 lactylate. This could indicate an effect on the pullulan and melanin production by the fungus. Moreover, the interaction studies on model fungal cell membranes show that C10 lactylate affects the phospholipid bilayer in a similar manner to other negative charged detergents. Copyright © 2017 Elsevier Inc. All rights reserved.

Ensuring the Quality of Stem Cell-Derived In Vitro Models for Toxicity Testing.

PubMed

Stacey, Glyn N; Coecke, Sandra; Price, Anna-Bal; Healy, Lyn; Jennings, Paul; Wilmes, Anja; Pinset, Christian; Ingelman-Sundberg, Magnus; Louisse, Jochem; Haupt, Simone; Kidd, Darren; Robitski, Andrea; Jahnke, Heinz-Georg; Lemaitre, Gilles; Myatt, Glenn

Quality control of cell cultures used in new in vitro toxicology assays is crucial to the provision of reliable, reproducible and accurate toxicity data on new drugs or constituents of new consumer products. This chapter explores the key scientific and ethical criteria that must be addressed at the earliest stages of developing toxicology assays based on human pluripotent stem cell (hPSC) lines. It also identifies key considerations for such assays to be acceptable for regulatory, laboratory safety and commercial purposes. Also addressed is the development of hPSC-based assays for the tissue and cell types of greatest interest in drug toxicology. The chapter draws on a range of expert opinion within the European Commission/Cosmetics Europe-funded alternative testing cluster SEURAT-1 and consensus from international groups delivering this guidance such as the International Stem Cell Banking Initiative. Accordingly, the chapter summarizes the most up-date best practices in the use and quality control of human Pluripotent Stem Cell lines in the development of in vitro toxicity assays from leading experts in the field.

Enumeration of residual white blood cells in leukoreduced blood products: Comparing flow cytometry with a portable microscopic cell counter.

PubMed

Castegnaro, Silvia; Dragone, Patrizia; Chieregato, Katia; Alghisi, Alberta; Rodeghiero, Francesco; Astori, Giuseppe

2016-04-01

Transfusion of blood components is potentially associated to the risk of cell-mediated adverse events and current guidelines require a reduction of residual white blood cells (rWBC) below 1 × 10(6) WBC/unit. The reference method to enumerate rare events is the flow cytometry (FCM). The ADAM-rWBC microscopic cell counter has been proposed as an alternative: it measures leukocytes after their staining with propidium iodide. We have tested the Adam-rWBC for the ability to enumerate rWBC in red blood cells and concentrates. We have validated the flow cytometry (FCM) for linearity, precision accuracy and robustness and then the ADAM-rWBC results have been compared with the FCM. Our data confirm the linearity, accuracy, precision and robustness of the FCM. The ADAM-rWBC has revealed an adequate precision and accuracy. Even if the Bland-Altman analysis of the paired data has indicated that the two systems are comparable, it should be noted that the rWBC values obtained by the ADAM-rWBC were significantly higher compared to FCM. In conclusion, the Adam-rWBC cell counter could represent an alternative where FCM technology expertise is not available, even if the risk that borderline products could be misclassified exists. Copyright © 2015 Elsevier Ltd. All rights reserved.

Bioengineering of Artificial Antigen Presenting Cells and Lymphoid Organs

PubMed Central

Wang, Chao; Sun, Wujin; Ye, Yanqi; Bomba, Hunter N.; Gu, Zhen

2017-01-01

The immune system protects the body against a wide range of infectious diseases and cancer by leveraging the efficiency of immune cells and lymphoid organs. Over the past decade, immune cell/organ therapies based on the manipulation, infusion, and implantation of autologous or allogeneic immune cells/organs into patients have been widely tested and have made great progress in clinical applications. Despite these advances, therapy with natural immune cells or lymphoid organs is relatively expensive and time-consuming. Alternatively, biomimetic materials and strategies have been applied to develop artificial immune cells and lymphoid organs, which have attracted considerable attentions. In this review, we survey the latest studies on engineering biomimetic materials for immunotherapy, focusing on the perspectives of bioengineering artificial antigen presenting cells and lymphoid organs. The opportunities and challenges of this field are also discussed. PMID:28912891

Bioengineering of Artificial Antigen Presenting Cells and Lymphoid Organs.

PubMed

Wang, Chao; Sun, Wujin; Ye, Yanqi; Bomba, Hunter N; Gu, Zhen

2017-01-01

The immune system protects the body against a wide range of infectious diseases and cancer by leveraging the efficiency of immune cells and lymphoid organs. Over the past decade, immune cell/organ therapies based on the manipulation, infusion, and implantation of autologous or allogeneic immune cells/organs into patients have been widely tested and have made great progress in clinical applications. Despite these advances, therapy with natural immune cells or lymphoid organs is relatively expensive and time-consuming. Alternatively, biomimetic materials and strategies have been applied to develop artificial immune cells and lymphoid organs, which have attracted considerable attentions. In this review, we survey the latest studies on engineering biomimetic materials for immunotherapy, focusing on the perspectives of bioengineering artificial antigen presenting cells and lymphoid organs. The opportunities and challenges of this field are also discussed.

Small capillary pumped AMTEC systems

NASA Astrophysics Data System (ADS)

Hunt, Thomas K.; Sievers, Robert K.; Butkiewicz, David A.; Pantolin, Jan E.; Ivanenok, Joseph F.

1993-01-01

Alkali Metal Thermoelectric Converter (AMTEC) systems offer significant potential advantages for space power. Recent experiments have shown that electromagnetic pumps can operate with a negative priming head and so may be suitable for space applications in microgravity (Hunt et al. 1992). Capillary pumped cells offer an alternative approach to microgravity compatibility. We have designed, built, and operated capillary pumped AMTEC cells in various orientations with respect to gravity in order to provide a presumptive demonstration of zero-G capability (Sievers et al. 1992). We report lifetime and performance data for these capillary pumped AMTEC cells. Progress on other issues relating to space flight testing of AMTEC systems is also discussed.

A quantitative risk assessment of exposure to adventitious agents in a cell culture-derived subunit influenza vaccine.

PubMed

Gregersen, Jens-Peter

2008-06-19

A risk-assessment model has demonstrated the ability of a new cell culture-based vaccine manufacturing process to reduce the level of any adventitious agent to a million-fold below infectious levels. The cell culture-derived subunit influenza vaccine (OPTAFLU), Novartis Vaccines and Diagnostics) is produced using Madin-Darby canine kidney (MDCK) cells to propagate seasonal viral strains, as an alternative to embryonated chicken-eggs. As only a limited range of mammalian viruses can grow in MDCK cells, similar to embryonated eggs, MDCK cells can act as an effective filter for a wide range of adventitious agents that might be introduced during vaccine production. However, the introduction of an alternative cell substrate (for example, MDCK cells) into a vaccine manufacturing process requires thorough investigations to assess the potential for adventitious agent risk in the final product, in the unlikely event that contamination should occur. The risk assessment takes into account the entire manufacturing process, from initial influenza virus isolation, through to blending of the trivalent subunit vaccine and worst-case residual titres for the final vaccine formulation have been calculated for >20 viruses or virus families. Maximum residual titres for all viruses tested were in the range of 10(-6) to 10(-16) infectious units per vaccine dose. Thus, the new cell culture-based vaccine manufacturing process can reduce any adventitious agent to a level that is unable to cause infection.

Possible alternative to European Pharmacopoeia's method of analysis Test for Fc Function of Immunoglobulin (2.7.9) by using tetanus toxoid as antigen.

PubMed

Perez-del-Pulgar, S; Lopez, M; Gensana, M; Jorquera, J I

2006-08-01

Preparations of intravenous immunoglobulins must keep functional integrity throughout the purification process. In order to assess Fc fragment functionality, the European Pharmacopoeia proposes the Test for Fc function of immunoglobulin (2.7.9), which is based on a rubella antigen of high titre. Sometimes, such antigen is difficult to obtain. In the present study, we develop the same assay using tetanus toxoid instead of rubella antigen, adapting the procedure for the use of tetanus toxoid. The comparison between rubella-based and tetanus-based assays showed that the slopes of the haemolysis curves were higher if red blood cells had been sensitised with the rubella antigen than with tetanus toxoid. Nonetheless, the tetanus-based assay gave satisfactory results and it could be a good alternative antigen target.

A fuel cell energy storage system concept for the Space Station Freedom Extravehicular Mobility Unit

NASA Technical Reports Server (NTRS)

Adlhart, Otto J.; Rosso, Matthew J., Jr.; Marmolejo, Jose

1989-01-01

An update is given on work to design and build a Fuel Cell Energy Storage System (FCESS) bench-tested unit for the Space Station Freedom Extravehicular Mobility Unit (EMU). Fueled by oxygen and hydride-stored hydrogen, the FCESS is being considered as an alternative to the EMU zinc-silver oxide battery. Superior cycle life and quick recharge are the main attributes of FCESS. The design and performance of a nonventing, 28 V, 34 Ahr system with 7 amp rating are discussed.

Crab Cavity and Cryomodule Prototype Development for the Advanced Photon Source

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

Wang, H; Ciovati, G; Clemens, W A

2011-03-01

We review the single-cell, superconducting crab cavity designs for the short-pulse x-ray (SPX) project at the Advanced Photon Source (APS). The 'on-cell' waveguide scheme is expected to have a more margin for the impedance budget of the APS storage ring, as well as offering a more compact design compared with the original design consisting of a low order mode damping waveguide on the beam pipe. We will report recent fabrication progress, cavity test performance on original and alternate prototypes, and concept designs and analysis for various cryomodule components.

A fuel cell energy storage system concept for the Space Station Freedom Extravehicular Mobility Unit

NASA Astrophysics Data System (ADS)

Adlhart, Otto J.; Rosso, Matthew J., Jr.; Marmolejo, Jose

1989-03-01

An update is given on work to design and build a Fuel Cell Energy Storage System (FCESS) bench-tested unit for the Space Station Freedom Extravehicular Mobility Unit (EMU). Fueled by oxygen and hydride-stored hydrogen, the FCESS is being considered as an alternative to the EMU zinc-silver oxide battery. Superior cycle life and quick recharge are the main attributes of FCESS. The design and performance of a nonventing, 28 V, 34 Ahr system with 7 amp rating are discussed.

Operations Analysis for Lunar Surface Construction: Results of Two Office of Exploration Case Studies

DTIC Science & Technology

1991-08-01

photovoltaic array (PVA) and regenerative fuel cell (RFC) is a critical construction activity during the first manned visit to the lunar surface ( February...An alternative design would be to have a standoff structure, possibly integrated with the photovoltaic material, in rigid panels. The difference in...8 Unload Fuel Cell Power (1CI) Cart 5 1 1 Ig item 0.2 Test FCP Can 1 1 4 systems 4 10 Unload Thermal Control (TC) Cans 5 1 1 Ig item U -.- 1 II lrest

The relationship between CD86/CD54 expression and THP-1 cell viability in an in vitro skin sensitization test--human cell line activation test (h-CLAT).

PubMed

Sakaguchi, Hitoshi; Ashikaga, Takao; Miyazawa, Masaaki; Kosaka, Nanae; Ito, Yuichi; Yoneyama, Katsurako; Sono, Sakiko; Itagaki, Hiroshi; Toyoda, Hidekazu; Suzuki, Hiroyuki

2009-04-01

Recent regulations for cosmetics in Europe prohibit animal testing for evaluating the sensitization potential of chemicals to improve animal welfare. Yet, there is not an acceptable Organization for Economic Co-operation and Development non-animal skin sensitization test method. Several in vitro skin sensitization methods that focus on the activation of Langerhans cells, including human cell lines, are being evaluated as possible alternatives. In our previous study, we optimized our human cell line activation test (h-CLAT) using THP-1 cells (monocytic leukemia cell line) and conducted an inter-laboratory study. We found that measuring CD86/CD54 expression may be useful for predicting skin sensitization. The aim of this study was to confirm the relationship between CD86/CD54 expression and THP-1 cell viability in the h-CLAT. In this study, 21 allergens (e.g., dinitrochlorobenzene, p-phenylenediamine, Ni) and 8 non-allergens (e.g., SLS, lactic acid) were evaluated. For each chemical, more than 10 concentrations that gave a predicted cell viability range of 20-95% were used. The data showed that expression patterns of CD86/CD54 differed depending on chemical. For most allergens, cytotoxicity (65-90% cell viability) was needed for enhancement of CD86/CD54 expression. The criteria of "CD86 > or = 150 or CD54 > or = 200" resulted in an accuracy of 93%, which confirms appropriate cut-off criteria for h-CLAT. Furthermore, a good correlation was observed between EC3 of local lymph node assay and EC150(CD86) or EC200(CD54) of h-CLAT (12 or 16 chemicals, respectively), which would provide a useful estimate of allergic potency. These findings suggest that h-CLAT would be a good robust in vitro skin sensitization test.

Pathway Based Toxicology and Fit-for-Purpose Assays.

PubMed

Clewell, Rebecca A; McMullen, Patrick D; Adeleye, Yeyejide; Carmichael, Paul L; Andersen, Melvin E

The field of toxicity testing for non-pharmaceutical chemicals is in flux with multiple initiatives in North America and the EU to move away from animal testing to mode-of-action based in vitro assays. In this arena, there are still obstacles to overcome, such as developing appropriate cellular assays, creating pathway-based dose-response models and refining in vitro-in vivo extrapolation (IVIVE) tools. Overall, it is necessary to provide assurances that these new approaches are adequately protective of human and ecological health. Another major challenge for individual scientists and regulatory agencies is developing a cultural willingness to shed old biases developed around animal tests and become more comfortable with mode-of-action based assays in human cells. At present, most initiatives focus on developing in vitro alternatives and assessing how well these alternative methods reproduce past results related to predicting organism level toxicity in intact animals. The path forward requires looking beyond benchmarking against high dose animal studies. We need to develop targeted cellular assays, new cell biology-based extrapolation models for assessing regions of safety for chemical exposures in human populations, and mode-of-action-based approaches which are constructed on an understanding of human biology. Furthermore, it is essential that assay developers have the flexibility to 'validate' against the most appropriate mode-of-action data rather than against apical endpoints in high dose animal studies. This chapter demonstrates the principles of fit-for-purpose assay development using pathway-targeted case studies. The projects include p53-mdm2-mediated DNA-repair, estrogen receptor-mediated cell proliferation and PPARα receptor-mediated liver responses.

Atypical Squamous Cells: Cytopathological Findings and Correlation with HPV Genotype and Histopathology.

PubMed

Oliveira, Geilson Gomes de; Oliveira, Judite Maria da Silva Costa de; Eleutério, Renata Mirian Nunes; Barbosa, Rita de Cássia Carvalho; Almeida, Paulo Roberto Carvalho de; Eleutério, José

2018-06-13

We aimed to assess potential associations between atypical squamous cell (ASC) subgroups: ASC-US (undetermined significance) and ASC-H (cannot exclude high-grade squamous intraepithelial lesion), regarding cytomorphological features, high-risk (HR) human papillomavirus (HPV) testing, and histological outcomes in a sample of Brazilian women. Cross-sectional study which evaluated 1,346 liquid-based cytologies between January 2010 and July 2016 with ASC results. ASC-US and ASC-H were analyzed for frequency, diagnostic criteria, and cytological findings and compared with HR-HPV tests and histological outcomes. Enlarged nucleus was the most frequent ASC-US criterion, but alternative criteria were present in 20% of the total cases. No ASC-US criteria were associated with histological outcomes or HR-HPV positivity. Parakeratosis, corneal pearl, giant cells, and binucleation were strongly associated with ASC-US while hyperkeratosis was associated with high-grade squamous intraepithelial lesions (HSIL) or a superior outcome. HR-HPV was positive in 64.39% of ASC-US and 65.38% of ASC-H. HSIL or superior outcomes also occurred in 13.33% of ASC-US and 64.71% of ASC-H cases. Alternative criteria for ASC-US were relatively frequent. Reactive cellular changes suggestive of atypias were more abundant in ASC-US. Although ASC-H is associated with worse histological outcomes, no differences in HPV positivity were found in comparison to ASC-US. © 2018 S. Karger AG, Basel.

More human, more humane: a new approach for testing airborne pollutants.

PubMed

Potera, Carol

2007-03-01

People not only inhale airborne contaminants but also absorb them through the skin. Both routes can set off localized toxic reactions or damage internal organs such as the liver, kidney, and brain. Conventional tests of the toxicity of gases and vapors, in which laboratory animals are exposed to lethal or sub-lethal doses of chemicals, have been criticized as expensive, unethical, inhumane, and time-consuming. Now researchers at the University of New South Wales (UNSW) in Sydney, Australia, have developed an animal-free alternative that uses human cells to test the effects of exposure to airborne toxicants.

A roadmap for the development of alternative (non-animal) methods for systemic toxicity testing - t4 report*.

PubMed

Basketter, David A; Clewell, Harvey; Kimber, Ian; Rossi, Annamaria; Blaauboer, Bas; Burrier, Robert; Daneshian, Mardas; Eskes, Chantra; Goldberg, Alan; Hasiwa, Nina; Hoffmann, Sebastian; Jaworska, Joanna; Knudsen, Thomas B; Landsiedel, Robert; Leist, Marcel; Locke, Paul; Maxwell, Gavin; McKim, James; McVey, Emily A; Ouédraogo, Gladys; Patlewicz, Grace; Pelkonen, Olavi; Roggen, Erwin; Rovida, Costanza; Ruhdel, Irmela; Schwarz, Michael; Schepky, Andreas; Schoeters, Greet; Skinner, Nigel; Trentz, Kerstin; Turner, Marian; Vanparys, Philippe; Yager, James; Zurlo, Joanne; Hartung, Thomas

2012-01-01

Systemic toxicity testing forms the cornerstone for the safety evaluation of substances. Pressures to move from traditional animal models to novel technologies arise from various concerns, including: the need to evaluate large numbers of previously untested chemicals and new products (such as nanoparticles or cell therapies), the limited predictivity of traditional tests for human health effects, duration and costs of current approaches, and animal welfare considerations. The latter holds especially true in the context of the scheduled 2013 marketing ban on cosmetic ingredients tested for systemic toxicity. Based on a major analysis of the status of alternative methods (Adler et al., 2011) and its independent review (Hartung et al., 2011), the present report proposes a roadmap for how to overcome the acknowledged scientific gaps for the full replacement of systemic toxicity testing using animals. Five whitepapers were commissioned addressing toxicokinetics, skin sensitization, repeated-dose toxicity, carcinogenicity, and reproductive toxicity testing. An expert workshop of 35 participants from Europe and the US discussed and refined these whitepapers, which were subsequently compiled to form the present report. By prioritizing the many options to move the field forward, the expert group hopes to advance regulatory science.

The journey of islet cell transplantation and future development.

PubMed

Gamble, Anissa; Pepper, Andrew R; Bruni, Antonio; Shapiro, A M James

2018-03-04

Intraportal islet transplantation has proven to be efficacious in preventing severe hypoglycemia and restoring insulin independence in selected patients with type 1 diabetes. Multiple islet infusions are often required to achieve and maintain insulin independence. Many challenges remain in clinical islet transplantation, including substantial islet cell loss early and late after islet infusion. Contributions to graft loss include the instant blood-mediated inflammatory reaction, potent host auto- and alloimmune responses, and beta cell toxicity from immunosuppressive agents. Protective strategies are being tested to circumvent several of these events including exploration of alternative transplantation sites, stem cell-derived insulin producing cell therapies, co-transplantation with mesenchymal stem cells or exploration of novel immune protective agents. Herein, we provide a brief introduction and history of islet cell transplantation, limitations associated with this procedure and methods to alleviate islet cell loss as a means to improve engraftment outcomes.

Tandem concentrator photovoltaic array applied to Space Station Freedom evolutionary power requirements

NASA Technical Reports Server (NTRS)

Fisher, Edward M., Jr.

1991-01-01

Additional power is required to support Space Station Freedom (SSF) evolution. Boeing Defense and Space Group, LeRC, and Entech Corporation have participated in the development of efficiency gallium arsenide and gallium antimonide solar cells make up the solar array tandem cell stacks. Entech's Mini-Dome Fresnel Lens Concentrators focus solar energy onto the active area of the solar cells at 50 times one solar energy flux. Development testing for a flight array, to be launched in Nov. 1992 is under way with support from LeRC. The tandem cells, interconnect wiring, concentrator lenses, and structure were integrated into arrays subjected to environmental testing. A tandem concentrator array can provide high mass and area specific power and can provide equal power with significantly less array area and weight than the baseline array design. Alternatively, for SSF growth, an array of twice the baseline power can be designed which still has a smaller drag area than the baseline.

Microengineered physiological biomimicry: organs-on-chips.

PubMed

Huh, Dongeun; Torisawa, Yu-suke; Hamilton, Geraldine A; Kim, Hyun Jung; Ingber, Donald E

2012-06-21

Microscale engineering technologies provide unprecedented opportunities to create cell culture microenvironments that go beyond current three-dimensional in vitro models by recapitulating the critical tissue-tissue interfaces, spatiotemporal chemical gradients, and dynamic mechanical microenvironments of living organs. Here we review recent advances in this field made over the past two years that are focused on the development of 'Organs-on-Chips' in which living cells are cultured within microfluidic devices that have been microengineered to reconstitute tissue arrangements observed in living organs in order to study physiology in an organ-specific context and to develop specialized in vitro disease models. We discuss the potential of organs-on-chips as alternatives to conventional cell culture models and animal testing for pharmaceutical and toxicology applications. We also explore challenges that lie ahead if this field is to fulfil its promise to transform the future of drug development and chemical safety testing.

Oxygen separation from air using zirconia solid electrolyte membranes

NASA Technical Reports Server (NTRS)

Suitor, J. W.; Marner, W. J.; Schroeder, J. E.; Losey, R. W.; Ferrall, J. F.

1988-01-01

Air separation using a zirconia solid electrolyte membrane is a possible alternative source of oxygen. The process of zirconia oxygen separation is reviewed, and an oxygen plant concept using such separation is described. Potential cell designs, stack designs, and testing procedures are examined. Fabrication of the materials used in a zirconia module as well as distribution plate design and fabrication are examined.

Diverse alternative back-splicing and alternative splicing landscape of circular RNAs

PubMed Central

Zhang, Xiao-Ou; Dong, Rui; Zhang, Yang; Zhang, Jia-Lin; Luo, Zheng; Zhang, Jun; Chen, Ling-Ling; Yang, Li

2016-01-01

Circular RNAs (circRNAs) derived from back-spliced exons have been widely identified as being co-expressed with their linear counterparts. A single gene locus can produce multiple circRNAs through alternative back-splice site selection and/or alternative splice site selection; however, a detailed map of alternative back-splicing/splicing in circRNAs is lacking. Here, with the upgraded CIRCexplorer2 pipeline, we systematically annotated different types of alternative back-splicing and alternative splicing events in circRNAs from various cell lines. Compared with their linear cognate RNAs, circRNAs exhibited distinct patterns of alternative back-splicing and alternative splicing. Alternative back-splice site selection was correlated with the competition of putative RNA pairs across introns that bracket alternative back-splice sites. In addition, all four basic types of alternative splicing that have been identified in the (linear) mRNA process were found within circRNAs, and many exons were predominantly spliced in circRNAs. Unexpectedly, thousands of previously unannotated exons were detected in circRNAs from the examined cell lines. Although these novel exons had similar splice site strength, they were much less conserved than known exons in sequences. Finally, both alternative back-splicing and circRNA-predominant alternative splicing were highly diverse among the examined cell lines. All of the identified alternative back-splicing and alternative splicing in circRNAs are available in the CIRCpedia database (http://www.picb.ac.cn/rnomics/circpedia). Collectively, the annotation of alternative back-splicing and alternative splicing in circRNAs provides a valuable resource for depicting the complexity of circRNA biogenesis and for studying the potential functions of circRNAs in different cells. PMID:27365365

Running wheel training does not change neurogenesis levels or alter working memory tasks in adult rats

PubMed Central

Rojas, Manuel J.; Cardenas P., Fernando

2017-01-01

Background Exercise can change cellular structure and connectivity (neurogenesis or synaptogenesis), causing alterations in both behavior and working memory. The aim of this study was to evaluate the effect of exercise on working memory and hippocampal neurogenesis in adult male Wistar rats using a T-maze test. Methods An experimental design with two groups was developed: the experimental group (n = 12) was subject to a forced exercise program for five days, whereas the control group (n = 9) stayed in the home cage. Six to eight weeks after training, the rats’ working memory was evaluated in a T-maze test and four choice days were analyzed, taking into account alternation as a working memory indicator. Hippocampal neurogenesis was evaluated by means of immunohistochemistry of BrdU positive cells. Results No differences between groups were found in the behavioral variables (alternation, preference index, time of response, time of trial or feeding), or in the levels of BrdU positive cells. Discussion Results suggest that although exercise may have effects on brain structure, a construct such as working memory may require more complex changes in networks or connections to demonstrate a change at behavioral level. PMID:28503368

Development of the EpiOcular(TM) eye irritation test for hazard identification and labelling of eye irritating chemicals in response to the requirements of the EU cosmetics directive and REACH legislation.

PubMed

Kaluzhny, Yulia; Kandárová, Helena; Hayden, Patrick; Kubilus, Joseph; d'Argembeau-Thornton, Laurence; Klausner, Mitchell

2011-09-01

The recently implemented 7th Amendment to the EU Cosmetics Directive and the EU REACH legislation have heightened the need for in vitro ocular test methods. To address this need, the EpiOcular(TM) eye irritation test (EpiOcular-EIT), which utilises the normal (non-transformed) human cell-based EpiOcular tissue model, has been developed. The EpiOcular-EIT prediction model is based on an initial training set of 39 liquid and 21 solid test substances and uses a single exposure period and a single cut-off in tissue viability, as determined by the MTT assay. A chemical is classified as an irritant (GHS Category 1 or 2), if the tissue viability is ≤ 60%, and as a non-irritant (GHS unclassified), if the viability is > 60%. EpiOcular-EIT results for the training set, along with results for an additional 52 substances, which included a range of alcohols, hydrocarbons, amines, esters, and ketones, discriminated between ocular irritants and non-irritants with 98.1% sensitivity, 72.9% specificity, and 84.8% accuracy. To ensure the long-term commercial viability of the assay, EpiOcular tissues produced by using three alternative cell culture inserts were evaluated in the EpiOcular-EIT with 94 chemicals. The assay results obtained with the initial insert and the three alternative inserts were very similar, as judged by correlation coefficients (r²) that ranged from 0.82 to 0.96. The EpiOcular-EIT was pre-validated in 2007/2008, and is currently involved in a formal, multi-laboratory validation study sponsored by the European Cosmetics Association (COLIPA) under the auspices of the European Centre for the Validation of Alternative Methods (ECVAM). The EpiOcular-EIT, together with EpiOcular's long history of reproducibility and proven utility for ultra-mildness testing, make EpiOcular a useful model for addressing current legislation related to animal use in the testing of potential ocular irritants. 2011 FRAME.

Osmotic modulation of chromatin impacts on efficiency and kinetics of cell fate modulation.

PubMed

Lima, A F; May, G; Colunga, J; Pedreiro, S; Paiva, A; Ferreira, L; Enver, T; Iborra, F J; Pires das Neves, R

2018-05-08

Chromatin structure is a major regulator of transcription and gene expression. Herein we explore the use of osmotic modulation to modify the chromatin structure and reprogram gene expression. In this study we use the extracellular osmotic pressure as a chromatin structure and transcriptional modulator. Hyposmotic modulation promotes chromatin loosening and induces changes in RNA polymerase II (Pol II) activity. The chromatin decondensation opens space for higher amounts of DNA engaged RNA Pol II. Hyposmotic modulation constitutes an alternative route to manipulate cell fate decisions. This technology was tested in model protocols of induced pluripotency and transdifferentiation in cells growing in suspension and adherent to substrates, CD34 + umbilical-cord-blood (UCB), fibroblasts and B-cells. The efficiency and kinetics of these cell fate modulation processes were improved by transient hyposmotic modulation of the cell environment.

Topography and refractometry of sperm cells using spatial light interference microscopy

NASA Astrophysics Data System (ADS)

Liu, Lina; Kandel, Mikhail E.; Rubessa, Marcello; Schreiber, Sierra; Wheeler, Mathew B.; Popescu, Gabriel

2018-02-01

Characterization of spermatozoon viability is a common test in treating infertility. Recently, it has been shown that label-free, phase-sensitive imaging can provide a valuable alternative for this type of assay. We employ spatial light interference microscopy (SLIM) to perform high-accuracy single-cell phase imaging and decouple the average thickness and refractive index information for the population. This procedure was enabled by quantitative-phase imaging cells on media of two different refractive indices and using a numerical tool to remove the curvature from the cell tails. This way, we achieved ensemble averaging of topography and refractometry of 100 cells in each of the two groups. The results show that the thickness profile of the cell tail goes down to 150 nm and the refractive index can reach values of 1.6 close to the head.

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: phase II enzymes.

PubMed

Götz, Christine; Pfeiffer, Roland; Tigges, Julia; Ruwiedel, Karsten; Hübenthal, Ulrike; Merk, Hans F; Krutmann, Jean; Edwards, Robert J; Abel, Josef; Pease, Camilla; Goebel, Carsten; Hewitt, Nicola; Fritsche, Ellen

2012-05-01

The 7th Amendment to the EU Cosmetics Directive prohibits the use of animals in cosmetic testing for certain endpoints, such as genotoxicity. Therefore, skin in vitro models have to replace chemical testing in vivo. However, the metabolic competence neither of human skin nor of alternative in vitro models has so far been fully characterized, although skin is the first-pass organ for accidentally or purposely (cosmetics and pharmaceuticals) applied chemicals. Thus, there is an urgent need to understand the xenobiotic-metabolizing capacities of human skin and to compare these activities to models developed to replace animal testing. We have measured the activity of the phase II enzymes glutathione S-transferase, UDP-glucuronosyltransferase and N-acetyltransferase in ex vivo human skin, the 3D epidermal model EpiDerm 200 (EPI-200), immortalized keratinocyte-based cell lines (HaCaT and NCTC 2544) and primary normal human epidermal keratinocytes. We show that all three phase II enzymes are present and highly active in skin as compared to phase I. Human skin, therefore, represents a more detoxifying than activating organ. This work systematically compares the activities of three important phase II enzymes in four different in vitro models directly to human skin. We conclude from our studies that 3D epidermal models, like the EPI-200 employed here, are superior over monolayer cultures in mimicking human skin xenobiotic metabolism and thus better suited for dermatotoxicity testing. © 2012 John Wiley & Sons A/S.

Changes in cell wall polysaccharide composition, gene transcription and alternative splicing in germinating barley embryos.

PubMed

Zhang, Qisen; Zhang, Xiaoqi; Pettolino, Filomena; Zhou, Gaofeng; Li, Chengdao

2016-02-01

Barley (Hordeum vulgare L.) seed germination initiates many important biological processes such as DNA, membrane and mitochondrial repairs. However, little is known on cell wall modifications in germinating embryos. We have investigated cell wall polysaccharide composition change, gene transcription and alternative splicing events in four barley varieties at 24h and 48 h germination. Cell wall components in germinating barley embryos changed rapidly, with increases in cellulose and (1,3)(1,4)-β-D-glucan (20-100%) within 24h, but decreases in heteroxylan and arabinan (3-50%). There were also significant changes in the levels of type I arabinogalactans and heteromannans. Alternative splicing played very important roles in cell wall modifications. At least 22 cell wall transcripts were detected to undergo either alternative 3' splicing, alternative 5' splicing or intron retention type of alternative splicing. These genes coded enzymes catalyzing synthesis and degradation of cellulose, heteroxylan, (1,3)(1,4)-β-D-glucan and other cell wall polymers. Furthermore, transcriptional regulation also played very important roles in cell wall modifications. Transcript levels of primary wall cellulase synthase, heteroxylan synthesizing and nucleotide sugar inter-conversion genes were very high in germinating embryos. At least 50 cell wall genes changed transcript levels significantly. Expression patterns of many cell wall genes coincided with changes in polysaccharide composition. Our data showed that cell wall polysaccharide metabolism was very active in germinating barley embryos, which was regulated at both transcriptional and post-transcriptional levels. Copyright © 2015 Elsevier GmbH. All rights reserved.

Evaluation of royal jelly as an alternative to fetal bovine serum in cell culture using cell proliferation assays and live cell imaging.

PubMed

Musa, Marahaini; Nasir, Nurul Fatihah Mohamad; Thirumulu, Kannan Ponnuraj

2014-01-01

Royal jelly is a nutritious substance produced by the young nurse bees and contains significant amounts of proteins which are important for cell growth and proliferation. The aim of this study was to evaluate the effect of royal jelly as an alternative to fetal bovine serum (FBS) in cell culture using cell proliferation assays and live cell imaging. MRC-5 cells were treated with various concentrations of royal jelly extract in MTT assay. The control groups were comprised of Alpha-Minimal Essential Medium (α-MEM) alone and α-MEM with 10% FBS. Subsequently, the cell proliferation was studied for 10 days using Alamar Blue assay and live cell imaging from 48 to 72 h. The population doubling time (PDT) was determined using trypan blue assay after live cell imaging. In MTT assay, 0.156 and 0.078 mg/ml of royal jelly produced higher cell viability compared to positive control group but were not significantly different (P > 0.05). In the Alamar Blue assay, 0.156 and 0.078 mg/ml of royal jelly produced greater percentage of reduction at day 3 even though no significant difference was found (P > 0.05). Based on live cell imaging, the PDT for positive, negative, 0.156 and 0.078 mg/ml of royal jelly groups were 29.09, 62.50, 41.67 and 41.67 h respectively. No significant difference was found in the PDT between all the groups (P > 0.05). Royal jelly does not exhibit similar ability like FBS to facilitate cell growth under the present test conditions.

Peroxisome Proliferator-Activated Receptor γ Deficiency in T Cells Accelerates Chronic Rejection by Influencing the Differentiation of CD4+ T Cells and Alternatively Activated Macrophages

PubMed Central

Ye, Ping; Cheng, Chao; Wu, Jie; Wang, Sihua; Sun, Yuan; Liu, Zheng; Xie, Aini; Xia, Jiahong

2014-01-01

Background In a previous study, activation of the peroxisome proliferator–activated receptor γ (PPARγ) inhibited chronic cardiac rejection. However, because of the complexity of chronic rejection and the fact that PPARγ is widely expressed in immune cells, the mechanism of the PPARγ - induced protective effect was unclear. Materials and Methods A chronic rejection model was established using B6.C-H-2bm12KhEg (H-2bm12) mice as donors, and MHC II-mismatched T-cell-specific PPARγ knockout mice or wild type (WT) littermates as recipients. The allograft lesion was assessed by histology and immunohistochemistry. T cells infiltrates in the allograft were isolated, and cytokines and subpopulations were detected using cytokine arrays and flow cytometry. Transcription levels in the allograft were measured by RT-PCR. In vitro, the T cell subset differentiation was investigated after culture in various polarizing conditions. PPARγ-deficient regularory T cells (Treg) were cocultured with monocytes to test their ability to induce alternatively activated macrophages (AAM). Results T cell-specific PPARγ knockout recipients displayed reduced cardiac allograft survival and an increased degree of pathology compared with WT littermates. T cell-specific PPARγ knockout resulted in more CD4+ T cells infiltrating into the allograft and altered the Th1/Th2 and Th17/Treg ratios. The polarization of AAM was also reduced by PPARγ deficiency in T cells through the action of Th2 and Treg. PPARγ-deficient T cells eliminated the pioglitazone-induced polarization of AAM and reduced allograft survival. Conclusions PPARγ-deficient T cells influenced the T cell subset and AAM polarization in chronic allograft rejection. The mechanism of PPARγ activation in transplantation tolerance could yield a novel treatment without side effects. PMID:25383620

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.

Iron deficiency anaemia in chronic kidney disease.

PubMed

Wittwer, Iain

2013-09-01

Iron Deficiency Anaemia (IDA) has been shown to be the most common cause of anaemia worldwide. It is accepted that people with chronic kidney disease (CKD) develop anaemia as their kidney function declines. To better understand IDA in CKD, it is necessary to appreciate the normal iron metabolism and utilisation of iron and how these processes can be disordered in patients with CKD. The problems related to infection / inflammation and oxidative stress are examined. Whilst National and international guidelines recommend specific tests for IDA, these and alternative tests are reviewed. Whilst iron supplementation is necessary for CKD patients with IDA, iron metabolism and utilisation can be affected by factors such as infection or inflammation. Iron is essential element for all life, it can be toxic to cells through the process of oxidative stress. The recommended tests for IDA may be affected by factors such as infection and inflammation. Alternative tests are available, which may be a more accurate indicator of IDA as they are not affected by external factors. © 2013 European Dialysis and Transplant Nurses Association/European Renal Care Association.

Function of alternative splicing

PubMed Central

Kelemen, Olga; Convertini, Paolo; Zhang, Zhaiyi; Wen, Yuan; Shen, Manli; Falaleeva, Marina; Stamm, Stefan

2017-01-01

Almost all polymerase II transcripts undergo alternative pre-mRNA splicing. Here, we review the functions of alternative splicing events that have been experimentally determined. The overall function of alternative splicing is to increase the diversity of mRNAs expressed from the genome. Alternative splicing changes proteins encoded by mRNAs, which has profound functional effects. Experimental analysis of these protein isoforms showed that alternative splicing regulates binding between proteins, between proteins and nucleic acids as well as between proteins and membranes. Alternative splicing regulates the localization of proteins, their enzymatic properties and their interaction with ligands. In most cases, changes caused by individual splicing isoforms are small. However, cells typically coordinate numerous changes in ‘splicing programs’, which can have strong effects on cell proliferation, cell survival and properties of the nervous system. Due to its widespread usage and molecular versatility, alternative splicing emerges as a central element in gene regulation that interferes with almost every biological function analyzed. PMID:22909801

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.

The development of magnetic degradable DP-Bioglass for hyperthermia cancer therapy.

PubMed

Wang, Tzu-Wei; Wu, Hsi-Chin; Wang, Wei-Ren; Lin, Feng-Huei; Lou, Pei-Jen; Shieh, Ming-Jium; Young, Tai-Horng

2007-12-01

In this study, a novel magnetic degradable material was developed by adding Fe ions into DP-Bioglass (Na(2)O-CaO-P(2)O(5)-SiO(2)) as thermoseed for hyperthermia cancer therapy under an alternating magnetic field. We have investigated the properties of developed magnetic DP-Bioglass including morphology, chemical composition, and magnetism. The degradability was conducted by measuring the released concentrations of Na, Ca, Si, P, and Fe ions. The biocompatibility was analyzed by biological assays, and the functional hyperthermia effect to cancer cells was evaluated by in vitro cell culture test. In the results, the morphology of synthesized magnetic DP-Bioglass was revealed in sphere and rod shape with particle size around 50-100 nm. From the hysteresis loop analysis, it showed that the group of Fe/Bioglass = 0.2 possessed the maximum magnetization property. When cultured with fibroblasts, the magnetic DP-Bioglass had no significant influence on cell viability and mediated low cytotoxicity. The thermal-induced property demonstrated that after exposure to an alternating magnetic field, the cell number of human Caucasian lung carcinoma cells (A549) was significantly decreased when temperature was increasing to 45 degrees C. In brief, successfully incorporated with Fe ions by sol-gel method, this magnetic degradable DP-Bioglass possessed the potential and properties of hyperthermia effect to lung carcinoma cells. Copyright 2007 Wiley Periodicals, Inc.

Biological activity of Ganoderma lucidum basidiocarps cultivated on alternative and commercial substrate.

PubMed

Cilerdžić, Jasmina; Vukojević, Jelena; Stajić, Mirjana; Stanojković, Tatjana; Glamočlija, Jasmina

2014-08-08

Ganoderma lucidum (W. Curt.:Fr.) P. Karst. has been used for several thousand years in traditional medicine of the Far East for the treatment of a various diseases including: cancer, immunological disorders, hepatitis, arthritis, nephritis, bronchitis, asthma etc. Ganoderma species are natural sources of various antimicrobial, antioxidant and antitumor active compounds which can be alternative to commercial antibiotics, antimycotics, antioxidants and cytostatics. As the amount of wild Ganoderma lucidum basidiocarps is not sufficient, the aim of this study was to compare the bioactivity of basidiaocarps obtained on alternative substrate (wheat straw) with commercially cultivated ones. Antimicrobial potential of the tested Ganoderma lucidum extracts was studied by disc-diffusion and microdilution method. Cytotoxic effect of tested extracts on two cancer cell lines was determined by microculture tetrazolium test. DPPH scavenging ability, total phenols and flavonoids content were measured in order to determine the antioxidative potential of Ganoderma lucidum extracts. Ganoderma lucidum BEOFB 434 extract was the most effective antibacterial agent, while commercial strain was the weakest. DPPH· scavenging activity of extracts of basidiocarps from alternative substrate was also significantly higher than that of commercially obtained basidiocarps. The extracts of Ganoderma lucidum BEOFB 431 exhibited the strongest cytotoxic activity significantly higher than those in commercial strain. The study showed that fruiting bodies produced on one of the most accessible and cheapest crop residue, wheat straw, are better antimicrobial, antioxidant and cytotoxic agents than those obtained on conventional substrate, oak sawdust. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Noninvasive inductive stent heating: alternative approach to prevent instent restenosis?

PubMed

Floren, Michael G; Günther, Rolf W; Schmitz-Rode, Thomas

2004-05-01

To test noninvasive inductive heating of implanted vascular stents as an alternative approach for reduction or prevention of neointimal hyperplasia. Calorimetric pretests were performed to get an orientation on the different parameters of influence for inductive heating of stents. The field strength was set to a maximum of 90 kA/m within a frequency range from 80 kHz to 320 kHz. The electromagnetic field was emitted by a custom-made water-cooled copper winding antenna. A flow model for stent heating was set up to assess the increase in temperature of an expanded 316L stainless steel stent with typical coronary stent dimensions of 3.5 mm diameter and 14.5 mm in length, and in a second setup with 4.5 mm diameter and 13 mm in length, respectively. The stent was located in a bioartificial artery, simulated by a fibrinogen matrix with a defined number of vital cells. The system was exposed to a pulsating perfusion and to an electromagnetic field of 200 kHz over a period of 20 minutes and in a second setup to an electromagnetic field of 300 kHz and increasing intensity up to maximum power-output. Afterward, the artificial vessel was sliced and examined by fluorescence microscopy to evaluate the number and location of damaged cells. The calorimetric tests show an exponential correlation of energy uptake in the stent with an increase in frequency and a constant generator output. At a frequency of 80 kHz, the power uptake accounts for 0.1 W (250 kHz 1.0 W; 320 kHz 1.9 W, respectively). The flow tests confirmed feasibility to elevate the stent temperature from 37 degrees C body temperature to 44 degrees C at 200 kHz within 55 seconds. The temperature increase of the fluid passing the heated vessel region was only marginal (maximum of 0.5 degrees C). Cell necrosis after 20 minutes of treatment was not observed. In a second set-up with 4.5 mm stent diameter, a frequency of 300 kHz and with maximum power output, the stent temperature was increased to 80 degrees C and there was extensive necrosis area around the stent. Treatment time and stent temperature were optimized in further tests. Selective noninvasive energy transfer to coronary stainless steel stents by inductive heating is possible within a wide range of power. By thermal conduction, vital cells close to the stent struts can be affected. The frequency of 200 kHz turned out to be favorable. There is still room for further optimization of energy dosage with regard to material and stent design, to induce controlled cell death. The method has potential to serve as an alternative approach for prevention of instent restenosis.

Hepatocyte transplantation and advancements in alternative cell sources for liver-based regenerative medicine.

PubMed

Lee, Charlotte A; Sinha, Siddharth; Fitzpatrick, Emer; Dhawan, Anil

2018-06-01

Human hepatocyte transplantation has been actively perused as an alternative to liver replacement for acute liver failure and liver-based metabolic defects. Current challenges in this field include a limited cell source, reduced cell viability following cryopreservation and poor engraftment of cells into the recipient liver with consequent limited life span. As a result, alternative stem cell sources such as pluripotent stem cells, fibroblasts, hepatic progenitor cells, amniotic epithelial cells and mesenchymal stem/stromal cells (MSCs) can be used to generate induced hepatocyte like cells (HLC) with each technique exhibiting advantages and disadvantages. HLCs may have comparable function to primary human hepatocytes and could offer patient-specific treatment. However, long-term functionality of transplanted HLCs and the potential oncogenic risks of using stem cells have yet to be established. The immunomodulatory effects of MSCs are promising, and multiple clinical trials are investigating their effect in cirrhosis and acute liver failure. Here, we review the current status of hepatocyte transplantation, alternative cell sources to primary human hepatocytes and their potential in liver regeneration. We also describe recent clinical trials using hepatocytes derived from stem cells and their role in improving the phenotype of several liver diseases.

High efficiency solar cells for concentrator systems: silicon or multi-junction?

NASA Astrophysics Data System (ADS)

Slade, Alexander; Stone, Kenneth W.; Gordon, Robert; Garboushian, Vahan

2005-08-01

Amonix has become the first company to begin production of high concentration silicon solar cells where volumes are over 10 MW/year. Higher volumes are available due to the method of manufacture; Amonix solely uses semiconductor foundries for solar cell production. In the previous years of system and cell field testing, this method of manufacturing enabled Amonix to maintain a very low overhead while incurring a high cost for the solar cell. However, recent simplifications to the solar cell processing sequence resulted in cost reduction and increased yield. This new process has been tested by producing small qualities in very short time periods, enabling a simulation of high volume production. Results have included over 90% wafer yield, up to 100% die yield and world record performance (η =27.3%). This reduction in silicon solar cell cost has increased the required efficiency for multi-junction concentrator solar cells to be competitive / advantageous. Concentrator systems are emerging as a low-cost, high volume option for solar-generated electricity due to the very high utilization of the solar cell, leading to a much lower $/Watt cost of a photovoltaic system. Parallel to this is the onset of alternative solar cell technologies, such as the very high efficiency multi-junction solar cells developed at NREL over the last two decades. The relatively high cost of these type of solar cells has relegated their use to non-terrestrial applications. However, recent advancements in both multi-junction concentrator cell efficiency and their stability under high flux densities has made their large-scale terrestrial deployment significantly more viable. This paper presents Amonix's experience and testing results of both high-efficiency silicon rear-junction solar cells and multi-junction solar cells made for concentrated light operation.

Engineering cell-fluorescent ion track hybrid detectors

PubMed Central

2013-01-01

Background The lack of sensitive biocompatible particle track detectors has so far limited parallel detection of physical energy deposition and biological response. Fluorescent nuclear track detectors (FNTDs) based on Al2O3:C,Mg single crystals combined with confocal laser scanning microscopy (CLSM) provide 3D information on ion tracks with a resolution limited by light diffraction. Here we report the development of next generation cell-fluorescent ion track hybrid detectors (Cell-Fit-HD). Methods The biocompatibility of FNTDs was tested using six different cell lines, i.e. human non-small cell lung carcinoma (A549), glioblastoma (U87), androgen independent prostate cancer (PC3), epidermoid cancer (A431) and murine (VmDk) glioma SMA-560. To evaluate cell adherence, viability and conformal coverage of the crystals different seeding densities and alternative coating with extracellular matrix (fibronectin) was tested. Carbon irradiation was performed in Bragg peak (initial 270.55 MeV u−1). A series of cell compartment specific fluorescence stains including nuclear (HOECHST), membrane (Glut-1), cytoplasm (Calcein AM, CM-DiI) were tested on Cell-Fit-HDs and a single CLSM was employed to co-detect the physical (crystal) as well as the biological (cell layer) information. Results The FNTD provides a biocompatible surface. Among the cells tested, A549 cells formed the most uniform, viable, tightly packed epithelial like monolayer. The ion track information was not compromised in Cell-Fit-HD as compared to the FNTD alone. Neither cell coating and culturing, nor additional staining procedures affected the properties of the FNTD surface to detect ion tracks. Standard immunofluorescence and live staining procedures could be employed to co-register cell biology and ion track information. Conclusions The Cell-Fit-Hybrid Detector system is a promising platform for a multitude of studies linking biological response to energy deposition at high level of optical microscopy resolution. PMID:23758749

Development of a Test Method for the Evaluation of DNA Damage in Mouse Spermatogonial Stem Cells

PubMed Central

Jeon, Hye Lyun; Yi, Jung-Sun; Kim, Tae Sung; Oh, Youkyung; Lee, Hye Jeong; Lee, Minseong; Bang, Jin Seok; Ko, Kinarm; Ahn, Il Young; Ko, Kyungyuk; Kim, Joohwan; Park, Hye-Kyung; Lee, Jong Kwon; Sohn, Soo Jung

2017-01-01

Although alternative test methods based on the 3Rs (Replacement, Reduction, Refinement) are being developed to replace animal testing in reproductive and developmental toxicology, they are still in an early stage. Consequently, we aimed to develop alternative test methods in male animals using mouse spermatogonial stem cells (mSSCs). Here, we modified the OECD TG 489 and optimized the in vitro comet assay in our previous study. This study aimed to verify the validity of in vitro tests involving mSSCs by comparing their results with those of in vivo tests using C57BL/6 mice by gavage. We selected hydroxyurea (HU), which is known to chemically induce male reproductive toxicity. The 50% inhibitory concentration (IC50) value of HU was 0.9 mM, as determined by the MTT assay. In the in vitro comet assay, % tail DNA and Olive tail moment (OTM) after HU administration increased significantly, compared to the control. Annexin V, PI staining and TUNEL assays showed that HU caused apoptosis in mSSCs. In order to compare in vitro tests with in vivo tests, the same substances were administered to male C57BL/6 mice. Reproductive toxicity was observed at 25, 50, 100, and 200 mg/kg/day as measured by clinical measures of reduction in sperm motility and testicular weight. The comet assay, DCFH-DA assay, H&E staining, and TUNEL assay were also performed. The results of the test with C57BL/6 mice were similar to those with mSSCs for HU treatment. Finally, linear regression analysis showed a strong positive correlation between results of in vitro tests and those of in vivo. In conclusion, the present study is the first to demonstrate the effect of HU-induced DNA damage, ROS formation, and apoptosis in mSSCs. Further, the results of the current study suggest that mSSCs could be a useful model to predict male reproductive toxicity. PMID:28443181

Preliminary evaluation of glass resin materials for solar cell cover use. [on spacecraft

NASA Technical Reports Server (NTRS)

Marsik, S. J.; Swartz, C. K.; Baraona, C. R.

1978-01-01

Silicon solar cells and silicon wafers coated with a heat-curable resin consisting of alternating Si-O atoms were subjected to three tests to evaluate the potential utility of this coating in space environments. These included UV irradiation in vacuum at an intensity of 10 air mass zero UV energy-equivalent solar constants for 728 hours followed by a long thermal cycle; 15 thermal shock cycles between 100 C and minus 196 C; and high temperature and humidity (65 C at 90% relative humidity). The UV tests resulted in a 8 to 24% loss in short-circuit current and darkening of the covers. Modification of the resin to provide a better match between the coefficients of expansion of the resin and silicon improved resistance to thermal shock, but also increased the darkening effect under UV irradiation. Silicon wafers coated with the resin were not adversely affected by the temperature/humidity test.

Suitability of Daphnia similis as an alternative organism in ecotoxicological tests: implications for metal toxicity.

PubMed

Rodgher, Suzelei; Espíndola, Evaldo Luiz Gaeta; Lombardi, Ana Teresa

2010-08-01

The acute toxicity of metals to Daphnia similis was determined and compared to other daphnid species to evaluate the suitability of this organism in ecotoxicology bioassays. To verify the performance D. similis in toxicity tests, we also investigated the effect of Pseudokirchneriella subcapitata at 1 x 10(5) and 1 x 10(6) cells ml(-1) on Cd and Cr acute toxicity to the cladoceran. Daphnid neonates were exposed to a range of chromium and cadmium concentrations in the absence and presence of the algal cells. Metal speciation calculations using MINEQL(+) showed that total dissolved metal concentrations in zooplankton culture corresponded to 96.2% free Cd and 100% free Cr concentrations. Initial total dissolved metal concentrations were used for 48 h-LC(50) determination. LC(50) for D. similis was 5.15 x 10(-7) mol l(-1) dissolved Cd without algal cells, whereas with 1 x 10(5) cells ml(-1), it was significantly higher (7.15 x 10(-7) mol l(-1) dissolved Cd). For Cr, the 48 h-LC(50) value of 9.17 x 10(-7) mol l(-1) obtained for the cladoceran in tests with 1 x 10(6) cells ml(-1) of P. subcapitata was also significantly higher than that obtained in tests without algal cells (5.28 x 10(-7) mol l(-1) dissolved Cr). The presence of algal cells reduced the toxicity of metals to D. similis, as observed in other studies that investigated the effects of food on metal toxicity to standard cladocerans. Comparing our results to those of literature, we observed that D. similis is as sensitive to metals as other standardized Daphnia species and may serve as a potential test species in ecotoxicological evaluations.

Inactivation of CUG-BP1/CELF1 Causes Growth, Viability, and Spermatogenesis Defects in Mice▿

PubMed Central

Kress, Chantal; Gautier-Courteille, Carole; Osborne, H. Beverley; Babinet, Charles; Paillard, Luc

2007-01-01

CUG-BP1/CELF1 is a multifunctional RNA-binding protein involved in the regulation of alternative splicing and translation. To elucidate its role in mammalian development, we produced mice in which the Cugbp1 gene was inactivated by homologous recombination. These Cugbp1−/− mice were viable, although a significant portion of them did not survive after the first few days of life. They displayed growth retardation, and most Cugbp1−/− males and females exhibited impaired fertility. Male infertility was more thoroughly investigated. Histological examination of testes from Cugbp1−/− males showed an arrest of spermatogenesis that occurred at step 7 of spermiogenesis, before spermatid elongation begins, and an increased apoptosis. A quantitative reverse transcriptase PCR analysis showed a decrease of all the germ cell markers tested but not of Sertoli and Leydig markers, suggesting a general decrease in germ cell number. In wild-type testes, CUG-BP1 is expressed in germ cells from spermatogonia to round spermatids and also in Sertoli and Leydig cells. These findings demonstrate that CUG-BP1 is required for completion of spermatogenesis. PMID:17130239

Stem Cells for Skeletal Muscle Tissue Engineering.

PubMed

Pantelic, Molly N; Larkin, Lisa M

2018-04-19

Volumetric muscle loss (VML) is a debilitating condition wherein muscle loss overwhelms the body's normal physiological repair mechanism. VML is particularly common among military service members who have sustained war injuries. Because of the high social and medical cost associated with VML and suboptimal current surgical treatments, there is great interest in developing better VML therapies. Skeletal muscle tissue engineering (SMTE) is a promising alternative to traditional VML surgical treatments that use autogenic tissue grafts, and rather uses isolated stem cells with myogenic potential to generate de novo skeletal muscle tissues to treat VML. Satellite cells are the native precursors to skeletal muscle tissue, and are thus the most commonly studied starting source for SMTE. However, satellite cells are difficult to isolate and purify, and it is presently unknown whether they would be a practical source in clinical SMTE applications. Alternative myogenic stem cells, including adipose-derived stem cells, bone marrow-derived mesenchymal stem cells, perivascular stem cells, umbilical cord mesenchymal stem cells, induced pluripotent stem cells, and embryonic stem cells, each have myogenic potential and have been identified as possible starting sources for SMTE, although they have yet to be studied in detail for this purpose. These alternative stem cell varieties offer unique advantages and disadvantages that are worth exploring further to advance the SMTE field toward highly functional, safe, and practical VML treatments. The following review summarizes the current state of satellite cell-based SMTE, details the properties and practical advantages of alternative myogenic stem cells, and offers guidance to tissue engineers on how alternative myogenic stem cells can be incorporated into SMTE research.

Noninvasive prenatal diagnosis for single gene disorders.

PubMed

Allen, Stephanie; Young, Elizabeth; Bowns, Benjamin

2017-04-01

Noninvasive prenatal diagnosis for single gene disorders is coming to fruition in its clinical utility. The presence of cell-free DNA in maternal plasma has been recognized for many years, and a number of applications have developed from this. Noninvasive prenatal diagnosis for single gene disorders has lagged behind due to complexities of technology development, lack of investment and the need for validation samples for rare disorders. Publications are emerging demonstrating a variety of technical approaches and feasibility of clinical application. Techniques for analysis of cell-free DNA including digital PCR, next-generation sequencing and relative haplotype dosage have been used most often for assay development. Analysis of circulating fetal cells in the maternal blood is still being investigated as a viable alternative and more recently transcervical trophoblast cells. Studies exploring ethical and social issues are generally positive but raise concerns around the routinization of prenatal testing. Further work is necessary to make testing available to all patients with a pregnancy at risk of a single gene disorder, and it remains to be seen if the development of more powerful technologies such as isolation and analysis of single cells will shift the emphasis of noninvasive prenatal diagnosis. As testing becomes possible for a wider range of conditions, more ethical questions will become relevant.

Generation of Murine Monoclonal Antibodies by Hybridoma Technology.

PubMed

Holzlöhner, Pamela; Hanack, Katja

2017-01-02

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

DNA Methylation Pyrosequencing Assay Is Applicable for the Assessment of Epigenetic Active Environmental or Clinical Relevant Chemicals

PubMed Central

Florea, Ana-Maria

2013-01-01

Exposure of cells and organisms to stressors might result in epigenetic changes. Here it is shown that investigation of DNA methylation using pyrosequencing is an alternative for in vitro and in vivo toxicological testing of epigenetic effects induced by chemicals and drugs. An in vitro evaluation of global and CpG site specific DNA methylation upon treatment of cells with chemicals/drugs is shown. Bisulfite genomic sequencing of methylation controls showed high methylation of LINE1 in methylation positive control and low methylation in the negative controls. The CpG sites within the LINE1 element are methylated at different levels. In vitro cell cultures show a methylation level ranging from 56% to 49%. Cultures of drug resistant tumor cells show significant hypomethylation as compared with the originating nonresistant tumor cells. The in vitro testing of epigenetically active chemicals (5-methyl-2'-deoxycytidine and trichostatin A) revealed a significant change of LINE1 methylation status upon treatment, while specific CpG sites were more prone to demethylation than others (focal methylation). In conclusion, DNA methylation using pyrosequencing might be used not only for testing epigenetic toxins/drugs but also in risk assessment of drugs, food, and environmental relevant pollutants. PMID:24093099

Effect of convection on osteoblastic cell growth and function in biodegradable polymer foam scaffolds

NASA Technical Reports Server (NTRS)

Goldstein, A. S.; Juarez, T. M.; Helmke, C. D.; Gustin, M. C.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

2001-01-01

Culture of seeded osteoblastic cells in three-dimensional osteoconductive scaffolds in vitro is a promising approach to produce an osteoinductive material for repair of bone defects. However, culture of cells in scaffolds sufficiently large to bridge critical-sized defects is a challenge for tissue engineers. Diffusion may not be sufficient to supply nutrients into large scaffolds and consequently cells may grow preferentially at the periphery under static culture conditions. Three alternative culturing schemes that convect media were considered: a spinner flask, a rotary vessel, and a perfusion flow system. Poly(DL-lactic-co-glycolic acid) (PLGA) foam discs (12.7 mm diameter, 6.0 mm thick, 78.8% porous) were seeded with osteoblastic marrow stromal cells and cultured in the presence of dexamethasone and L-ascorbic acid for 7 and 14 days. Cell numbers per foam were found to be similar with all culturing schemes indicating that cell growth could not be enhanced by convection, but histological analysis indicated that the rotary vessel and flow system produced a more uniform distribution of cells throughout the foams. Alkaline phosphatase (ALP) activity per cell was higher with culture in the flow system and spinner flask after 7 days, while no differences in osteocalcin (OC) activity per cell were observed among culturing methods after 14 days in culture. Based on the higher ALP activity and better cell uniformity throughout the cultured foams, the flow system appears to be the superior culturing method, although equally important is the fact that in none of the tests did any of the alternative culturing techniques underperform the static controls. Thus, this study demonstrates that culturing techniques that utilize fluid flow, and in particular the flow perfusion system, improve the properties of the seeded cells over those maintained in static culture.

False Operation of Static Random Access Memory Cells under Alternating Current Power Supply Voltage Variation

NASA Astrophysics Data System (ADS)

Sawada, Takuya; Takata, Hidehiro; Nii, Koji; Nagata, Makoto

2013-04-01

Static random access memory (SRAM) cores exhibit susceptibility against power supply voltage variation. False operation is investigated among SRAM cells under sinusoidal voltage variation on power lines introduced by direct RF power injection. A standard SRAM core of 16 kbyte in a 90 nm 1.5 V technology is diagnosed with built-in self test and on-die noise monitor techniques. The sensitivity of bit error rate is shown to be high against the frequency of injected voltage variation, while it is not greatly influenced by the difference in frequency and phase against SRAM clocking. It is also observed that the distribution of false bits is substantially random in a cell array.

Granulocyte-colony stimulating factor (G-CSF)-primed, delayed marrow harvests as a source of hematopoietic stem and progenitor cells for allogeneic transplantation.

PubMed

Phillips, G L; Davey, D D; Hale, G A; Marshall, K W; Munn, R K; Nath, R; Reece, D E; Van Zant, G

1999-10-01

We evaluated the ability of G-CSF to increase the number of hematopoietic stem cells obtained by "delayed" BM harvest for allogeneic transplantation. Five normal donors received G-CSF @ 10 mcg/kg/day x 5 followed by repeat PB and BM assays at day 6 and 16, and BM harvest at day 16. Stem cells were not increased in the BM at day 16. Five patients underwent BMT and engrafted at +10 to +19 days. While the tested strategy offers no intrinsic advantages, its potential cannot be evaluated fully without alternative timing and/or additional, "early acting" growth factors.

What Undergraduates Misunderstand about Stem Cell Research

NASA Astrophysics Data System (ADS)

Halverson, Kristy Lynn; Freyermuth, Sharyn K.; Siegel, Marcelle A.; Clark, Catharine G.

2010-11-01

As biotechnology-related scientific advances, such as stem cell research (SCR), are increasingly permeating the popular media, it has become ever more important to understand students' ideas about this issue. Very few studies have investigated learners' ideas about biotechnology. Our study was designed to understand the types of alternative conceptions students hold concerning SCR. The qualitative research design allowed us to examine college students' understandings about stem cells and SCR. More specifically, we addressed the following questions: How can alternative conceptions about stem cell topics be categorized? What types of alternative conceptions are most common? Participants included 132 students enrolled in a biotechnology course that focused on the scientific background of biotechnology applications relevant to citizens. In this study, we used an inductive approach to develop a taxonomy of alternative ideas about SCR by analyzing student responses to multiple open-ended data sources. We identified five categories of conceptions: alternative conceptions about what, alternative conceptions about how, alternative conceptions about medical potential, terminology confusion, and political and legal alternative conceptions. In order to improve instruction, it is important to understand students' ideas when entering the classroom. Our findings highlight a need to teach how science can be applied to societal issues and improve science literacy and citizenship.

Evaluation of the major royal jelly proteins as an alternative to fetal bovine serum in culturing human cell lines.

PubMed

Chen, Di; Xin, Xiao-Xuan; Qian, Hao-Cheng; Yu, Zhang-Yin; Shen, Li-Rong

2016-06-01

Royal jelly (RJ) is a well-known bioactive substance. It contains large amounts of major royal jelly proteins (MRJPs), which express growth-factor-like activity in several animal and human cell lines. However, the question on whether MRJPs possess growth-factor-like activity on all types of cell cultures remains. In order to determine whether MRJPs can be used as an alternative to fetal bovine serum (FBS) in different types of human cell culture, the proliferation of the complex serum with different ratios of MRJPs/FBS (M/F) was evaluated on five cell lines: 293T, HFL-I, 231, HCT116, and Changliver using MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay. The proliferation activity of the combination of the complex M/F serum with cytokines on the test cell lines was also measured. The results demonstrated that the complex serum with M/F 6/4 possessed the highest proliferation activity similar to or in excess of FBS. However, no activity of complex medium with M/F 6/4 was observed in 231 cells, indicating a selectivity of MRJPs on cell types. Compared with the complex medium with M/F 6/4, the complex medium with M/F 6/4 together with two cytokines, epidermal growth factor (EGF) and insulin-transferrin-selenium (ITS), promoted proliferations of Changliver, 293T, HCT116, and HFL-I by 18.73%‒56.19% (P<0.01). Our findings demonstrate that MRJPs could partially replace FBS in culturing many human cell lines.

Development and evaluation of a self-regulating alternating pressure air cushion.

PubMed

Nakagami, Gojiro; Sanada, Hiromi; Sugama, Junko

2015-03-01

To investigate the effect of alternating air cells of a newly developed dynamic cushion on interface pressure and tissue oxygenation levels. This cross-over experimental study included 19 healthy volunteers. The dynamic cushion used has an automatic self-regulating alternating pressure air-cell system with 35 small and four large air cells for maintaining posture while seated. This cushion also has 17 bottoming-out detectors that automatically inflate the air cells to release a high interface pressure. To assess the effect of this alternating system, participants sat on the new cushion with an alternating system or static system for 30 min and then performed push-ups. The interface pressure was monitored by pressure-sensitive and conductive ink film sensors and tissue oxygenation levels were monitored by near-infrared spectroscopy. A reactive hyperaemia indicator was calculated using tissue oxygenation levels as an outcome measure. The peak interface pressure was not significantly different between the groups. The reactive hyperaemia indicator was significantly higher in the static group than in the alternating group. An alternating system has beneficial effects on blood oxygenation levels without increasing interface pressure. Therefore, our new cushion is promising for preventing pressure ulcers with patients with limited ability to perform push-ups. Implications for Rehabilitation A dynamic cushion was developed, which consists of a uniquely-designed air-cell layout, detectors for bottoming out, and an alternating system with multiple air-cell lines. The alternating system did not increase interface pressure and it significantly reduced reactive hyperaemia after 30 min of sitting in healthy volunteers. This cushion is a new option for individuals who require stable posture but have limitations in performing scheduled push-ups for prevention of pressure ulcers.

The Asymmetric Cell Division Regulators Par3, Scribble and Pins/Gpsm2 Are Not Essential for Erythroid Development or Enucleation

PubMed Central

Wölwer, Christina B.; Gödde, Nathan; Pase, Luke B.; Elsum, Imogen A.; Lim, Krystle Y. B.; Sacirbegovic, Faruk; Walkley, Carl R.; Ellis, Sarah; Ohno, Shigeo; Matsuzaki, Fumio; Russell, Sarah M.; Humbert, Patrick O.

2017-01-01

Erythroid enucleation is the process by which the future red blood cell disposes of its nucleus prior to entering the blood stream. This key event during red blood cell development has been likened to an asymmetric cell division (ACD), by which the enucleating erythroblast divides into two very different daughter cells of alternate molecular composition, a nucleated cell that will be removed by associated macrophages, and the reticulocyte that will mature to the definitive erythrocyte. Here we investigated gene expression of members of the Par, Scribble and Pins/Gpsm2 asymmetric cell division complexes in erythroid cells, and functionally tested their role in erythroid enucleation in vivo and ex vivo. Despite their roles in regulating ACD in other contexts, we found that these polarity regulators are not essential for erythroid enucleation, nor for erythroid development in vivo. Together our results put into question a role for cell polarity and asymmetric cell division in erythroid enucleation. PMID:28095473

Evidence that "brain-specific" FOX-1, FOX-2, and nPTB alternatively spliced isoforms are produced in the lens.

PubMed

Bitel, Claudine L; Nathan, Rachel; Wong, Patrick; Kuppasani, Sunil; Matsushita, Masafumi; Kanazawa, Hrioshi; Frederikse, Peter H

2011-04-01

Alternative RNA splicing is essential in development and more rapid physiological processes that include disease mechanisms. Studies over the last 20 years demonstrated that RNA binding protein families, which mediate the alternative splicing of a large percentage of genes in mammals, contain isoforms with mutually exclusive expression in non-neural and neural progenitor cells vs. post-mitotic neurons, and regulate the comprehensive reprogramming of alternative splicing during neurogenesis. Polypyrimidine tract binding (PTB) proteins and Fox-1 proteins also undergo mutually exclusive alternative splicing in neural and non-neural cells that regulates their tissue-specific expression and splicing activities. Over the past 50 years, striking morphological similarities noted between lens fiber cells and neurons suggested that cell biology processes and gene expression profiles may be shared as well. Here, we examined mouse and rat lenses to determine if alternative splicing of neuronal nPTB and Fox-1/Fox-2 isoforms also occurs in lenses. Immunoblot, immunofluorescence, and RT-PCR were used to examine expression and alternative splicing of transcripts in lens and brain. We demonstrated that exon 10 is predominantly included in nPTB transcripts consistent with nPTB protein in lenses, and that alternatively spliced Fox-1/-2 lens transcripts contain exons that have been considered neuron-specific. We identified a 3' alternative Fox-1 exon in lenses that encodes a nuclear localization signal consistent with its protein distribution detected in fiber cells. Neuronal alternative splicing of kinesin KIF1Bβ2 has been associated with PTB/nPTB and Fox-2, and we found that two 'neuron-specific' exons are also included in lenses. The present study provides evidence that alternative neuronal nPTB and Fox-1/Fox-2 isoforms are also produced in lenses. These findings raise questions regarding the extent these factors contribute to a similar reprogramming of alternative splicing during lens differentiation, and the degree that alternative gene transcripts produced during neurogenesis are also expressed in the lens.

In vitro pituitary and thyroid cell proliferation assays and their relevance as alternatives to animal testing.

PubMed

Jomaa, Barae; Aarts, Jac M M J G; de Haan, Laura H J; Peijnenburg, Ad A C M; Bovee, Toine F H; Murk, Albertinka J; Rietjens, Ivonne M C M

2013-01-01

This study investigates the in vitro effect of eleven thyroid-active compounds known to affect pituitary and/or thyroid weights in vivo, using the proliferation of GH3 rat pituitary cells in the so-called "T-screen," and of FRTL-5 rat thyroid cells in a newly developed test denoted "TSH-screen" to gain insight into the relative value of these in vitro proliferation tests for an integrated testing strategy (ITS) for thyroid activity. Pituitary cell proliferation in the T-screen was stimulated by three out of eleven tested compounds, namely thyrotropin releasing hormone (TRH), triiodothyronine (T3) and thyroxine (T4). Of these three compounds, only T4 causes an increase in relative pituitary weight, and thus T4 was the only compound for which the effect in the in vitro assay correlated with a reported in vivo effect. As to the newly developed TSH-screen, two compounds had an effect, namely, thyroid-stimulating hormone (TSH) induced and T4 antagonized FRTL-5 cell proliferation. These effects correlated with in vivo changes induced by these compounds on thyroid weight. Altogether, the results indicate that most of the selected compounds affect pituitary and thyroid weights by modes of action different from a direct thyroid hormone receptor (THR) or TSH receptor (TSHR)-mediated effect, and point to the need for additional in vitro tests for an ITS. Additional analysis of the T-screen revealed a positive correlation between the THR-mediated effects of the tested compounds in vitro and their effects on relative heart weight in vivo, suggesting that the T-screen may directly predict this THR-mediated in vivo adverse effect.

Solar cell system having alternating current output

NASA Technical Reports Server (NTRS)

Evans, J. C., Jr. (Inventor)

1980-01-01

A monolithic multijunction solar cell was modified by fabricating an integrated circuit inverter on the back of the cell to produce a device capable of generating an alternating current output. In another embodiment, integrated curcuit power conditioning electronics was incorporated in a module containing a solar cell power supply.

Comparison of human platelet lysate alternatives using expired and freshly isolated platelet concentrates for adipose-derived stromal cell expansion.

PubMed

Dessels, Carla; Durandt, Chrisna; Pepper, Michael S

2018-03-19

Pooled human platelet lysate (pHPL) has been used to expand adipose-derived stromal cells (ASCs) and can be formulated using fresh or expired buffy coats (BCs) which are then resuspended in either plasma or an additive solution. Not much is known about the effects that expired products and additive solutions have on ASC expansion, and the need for quality control and release criteria has been expressed. This pilot study compared proliferation, cell size, morphology and immunophenotype of ASCs expanded in the different pHPL alternatives versus foetal bovine serum (FBS). Quality control criteria were assessed prior to and during the manufacture of the pHPL alternatives. ASCs were then expanded in 1%, 2.5%, 5% or 10% of the different pHPL alternatives or in 10% FBS. Cell size, morphology, cell number and immunophenotype were measured using microscopy and flow cytometry. The majority of the pHPL alternatives were within the recommended ranges for the quality control criteria. ASCs expanded in the pHPL alternatives were smaller in size, displayed a tighter spindle-shaped morphology, increased cell growth and had a similar immunophenotype (with the exception of CD34 and CD36) when compared to ASCs expanded in FBS. Here we report on the effects that expired BC products and additive solutions have on ASC expansion. When taken together, our findings indicate that all of the pHPL alternatives can be considered to be suitable replacements for FBS for ASC expansion, and that expired BC products can be used as an alternative to fresh BC products.

Evaluation of mechanical properties of human mesenchymal stem cells during differentiation to smooth muscle cells.

PubMed

Khani, Mohammad-Mehdi; Tafazzoli-Shadpour, Mohammad; Rostami, Mostafa; Peirovi, Habibollah; Janmaleki, Mohsen

2014-07-01

Human mesenchymal stem cells (hMSCs) are multipotent cells appropriate for a variety of tissue engineering and cell therapy applications. Mechanical properties of hMSCs during differentiation are associated with their particular metabolic activity and regulate cell function due to alternations in cytoskeleton and structural elements. The objective of this study is to evaluate elastic and viscoelastic properties of hMSCs during long term cultivation in control and transforming growth factor-β1 treatment groups using micropipette aspiration technique. The mean Young's modulus (E) of the control samples remained nearly unchanged during 6 days of cultivation, but that of the test samples showed an initial reduction compared to its relevant control sample after 2 days of treatment by biological growth factor, followed by a significant rise after 4 and 6 days. The viscoelastic creep tests showed that both instantaneous and equilibrium moduli significantly increased with the treatment time and reached to maximum values of 622.9 ± 114.2 and 144.3 ± 11.6 Pa at the sixth day, respectively, while increase in apparent viscosity was not statistically significant. Such change of mechanical properties of hMSCs during specific lineage commitment contributes to regenerative medicine as well as stem-cell-based therapy in which biophysical signals regulate stem cell fate.

Alternative fuel car

NASA Image and Video Library

2011-02-27

John C. Stennis Space Center, America's largest rocket engine test complex, and one of the country's leading consumers of liquid hydrogen, was the location Feb. 27 for a fuel stop of three Mercedes B-Class F-CELL vehicles. The B-Class F-CELL is an electric vehicle, which is powered by electricity produced on board the vehicle from hydrogen gas. The only emission by this unique vehicle is pure water vapor. Due to the limited number of existing hydrogen locations, Stennis Space Center provided a logical choice for a refueling location as the vehicle made its way across the United States as part of a worldwide tour.

Topography and refractometry of sperm cells using spatial light interference microscopy.

PubMed

Liu, Lina; Kandel, Mikhail E; Rubessa, Marcello; Schreiber, Sierra; Wheeler, Mathew B; Popescu, Gabriel

2018-02-01

Characterization of spermatozoon viability is a common test in treating infertility. Recently, it has been shown that label-free, phase-sensitive imaging can provide a valuable alternative for this type of assay. We employ spatial light interference microscopy (SLIM) to perform high-accuracy single-cell phase imaging and decouple the average thickness and refractive index information for the population. This procedure was enabled by quantitative-phase imaging cells on media of two different refractive indices and using a numerical tool to remove the curvature from the cell tails. This way, we achieved ensemble averaging of topography and refractometry of 100 cells in each of the two groups. The results show that the thickness profile of the cell tail goes down to 150 nm and the refractive index can reach values of 1.6 close to the head. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

In Vivo Imaging and Monitoring of Transplanted Stem Cells: Clinical Applications

PubMed Central

Rodriguez-Porcel, Martin

2010-01-01

Regenerative medicine using stem cells has appeared as a potential therapeutic alternative for coronary artery disease, and stem cell clinical studies are currently on their way. However, initial results of these studies have provided mixed information, in part because of the inability to correlate organ functional information with the presence/absence of transplanted stem cells. Recent advances in molecular biology and imaging have allowed the successful noninvasive monitoring of transplanted stem cells in the living subject. In this article, different imaging strategies (direct labeling, indirect labeling with reporter genes) to study the viability and biology of stem cells are discussed. In addition, the limitations of each approach and imaging modality (eg, single photon emission computed tomography, positron emission tomography, and MRI) and their requirements for clinical use are addressed. Use of these strategies will be critical as the different regenerative therapies are being tested for clinical use. PMID:20425184

76 FR 9777 - Recent Postings of Broadly Applicable Alternative Test Methods

Federal Register 2010, 2011, 2012, 2013, 2014

2011-02-22

... Applicable Alternative Test Methods AGENCY: Environmental Protection Agency (EPA). ACTION: Notice of availability. SUMMARY: This notice announces the broadly applicable alternative test method approval decisions... technical questions about individual alternative test method decisions, refer to the contact person...

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

Bio-inactivation of human malignant cells through highly responsive diluted colloidal suspension of functionalized magnetic iron oxide nanoparticles

NASA Astrophysics Data System (ADS)

Ferreira, Roberta V.; Silva-Caldeira, Priscila P.; Pereira-Maia, Elene C.; Fabris, José D.; Cavalcante, Luis Carlos D.; Ardisson, José D.; Domingues, Rosana Z.

2016-04-01

Magnetic fluids, more specifically aqueous colloidal suspensions containing certain magnetic nanoparticles (MNPs), have recently been gaining special interest due to their potential use in clinical treatments of cancerous formations in mammalians. The technological application arises mainly from their hyperthermic behavior, which means that the nanoparticles dissipate heat upon being exposed to an alternating magnetic field (AMF). If the temperature is raised to slightly above 43 °C, cancer cells are functionally inactivated or killed; however, normal cells tend to survive under those same conditions, entirely maintaining their bioactivity. Recent in vitro studies have revealed that under simultaneous exposure to an AMF and magnetic nanoparticles, certain lines of cancer cells are bio-inactivated even without experiencing a significant temperature increase. This non-thermal effect is cell specific, indicating that MNPs, under alternating magnetic fields, may effectively kill cancer cells under conditions that were previously thought to be implausible, considering that the temperature does not increase more than 5 °C, which is also true in cases for which the concentration of MNPs is too low. To experimentally test for this effect, this study focused on the feasibility of inducing K562 cell death using an AMF and aqueous suspensions containing very low concentrations of MNPs. The assay was designed for a ferrofluid containing magnetite nanoparticles, which were obtained through the co-precipitation method and were functionalized with citric acid; the particles had an average diameter of 10 ± 2 nm and a mean hydrodynamic diameter of approximately 40 nm. Experiments were first performed to test for the ability of the ferrofluid to release heat under an AMF. The results show that for concentrations ranging from 2.5 to 1.0 × 103 mg L-1, the maximum temperature increase was actually less than 2 °C. However, the in vitro test results from K562 cells and suspensions containing these MNPs at concentrations varying within a narrower range from 2.5 to 10 mg L-1, typically under an AMF of 15 kA m-1 at 356 kHz, indicate efficient cytotoxic activity against malignant cells and inhibition of cell growth, even at very low hyperthermally induced temperature increases. The IC50 value varied with time, reaching 3.5 mg L-1 after 10 min under the AMF. Our results effectively demonstrate new prospective uses for such nanoparticles in advanced medical practices in oncology.

Pig StAR: mRNA expression and alternative splicing in testis and Leydig cells, and association analyses with testicular morphology traits.

PubMed

Zhang, Yanghai; Cui, Yang; Zhang, Xuelian; Wang, Yimin; Gao, Jiayang; Yu, Ting; Lv, Xiaoyan; Pan, Chuanying

2018-05-31

Steroidogenic acute regulatory protein (StAR), primarily expressed in Leydig cells (LCs) in the mammalian testes, is essential for testosterone biosynthesis and male fertility. However, no previous reports have explored the expression profiles, alternative splicing and genetic variations of StAR gene in pig. The aim of current study was to explore the expression profiles in different tissues and different types of testicular cells (LCs; spermatogonial stem cells, SSCs; Sertoli cells, SCs), to identify different splice variants and their expression levels, as well as to detect the indel polymorphism in pig StAR gene. Expression analysis results revealed that StAR was widely expressed in all tested tissues and the expression level in testis was significantly higher than that in other tissues (P < 0.01); among different types of testicular cells, the StAR mRNA expression level was significantly higher in LCs than others (P < 0.05). Furthermore, three splice variants, StAR-a, StAR-b and StAR-c, were first found in pig. Further study showed StAR-a was highly expressed in both testis and LCs when compared with other variants (P < 0.01), suggesting StAR-a was the primary variant at StAR gene post-transcription and may facilitate the combination and transportation of cholesterol with StAR. In addition, a 5-bp duplicated deletion (NC_010457.5:g.5524-5528 delACTTG) was verified in the porcine StAR gene, which was closely related to male testicular morphology traits (P < 0.05), and we speculated that the allele "D" of StAR gene might be a positive allele. Briefly, the current findings suggest that StAR and StAR-a play imperative roles in male fertility and the 5-bp indel can be a potential DNA marker for the marker-assisted selection in boar. Copyright © 2018 Elsevier Inc. All rights reserved.

In-depth analysis of switchable glycerol based polymeric coatings for cell sheet engineering.

PubMed

Becherer, Tobias; Heinen, Silke; Wei, Qiang; Haag, Rainer; Weinhart, Marie

2015-10-01

Scaffold-free cell sheet engineering using thermoresponsive substrates provides a promising alternative to conventional tissue engineering which in general employs biodegradable scaffold materials. We have previously developed a thermoresponsive coating with glycerol based linear copolymers that enables gentle harvesting of entire cell sheets. In this article we present an in-depth analysis of these thermoresponsive linear polyglycidyl ethers and their performance as coating for substrates in cell culture in comparison with commercially available poly(N-isopropylacrylamide) (PNIPAM) coated culture dishes. A series of copolymers of glycidyl methyl ether (GME) and glycidyl ethyl ether (EGE) was prepared in order to study their thermoresponsive properties in solution and on the surface with respect to the comonomer ratio. In both cases, when grafted to planar surfaces or spherical nanoparticles, the applied thermoresponsive polyglycerol coatings render the respective surfaces switchable. Protein adsorption experiments on copolymer coated planar surfaces with surface plasmon resonance (SPR) spectroscopy reveal the ability of the tested thermoresponsive coatings to be switched between highly protein resistant and adsorptive states. Cell culture experiments demonstrate that these thermoresponsive coatings allow for adhesion and proliferation of NIH 3T3 fibroblasts comparable to TCPS and faster than on PNIPAM substrates. Temperature triggered detachment of complete cell sheets from copolymer coated substrates was accomplished within minutes while maintaining high viability of the harvested cells. Thus such glycerol based copolymers present a promising alternative to PNIPAM as a thermoresponsive coating of cell culture substrates. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Empirical correlations of the performance of vapor-anode PX-series AMTEC cells

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

Huang, L.; Merrill, J.M.; Mayberry, C.

Power systems based on AMTEC technology will be used for future NASA missions, including a Pluto-Express (PX) or Europa mission planned for approximately year 2004. AMTEC technology may also be used as an alternative to photovoltaic based power systems for future Air Force missions. An extensive development program of Alkali-Metal Thermal-to-Electric Conversion (AMTEC) technology has been underway at the Vehicle Technologies Branch of the Air Force Research Laboratory (AFRL) in Albuquerque, New Mexico since 1992. Under this program, numerical modeling and experimental investigations of the performance of the various multi-BASE tube, vapor-anode AMTEC cells have been and are being performed.more » Vacuum testing of AMTEC cells at AFRL determines the effects of changing the hot and cold end temperatures, T{sub hot} and T{sub cold}, and applied external load, R{sub ext}, on the cell electric power output, current-voltage characteristics, and conversion efficiency. Test results have traditionally been used to provide feedback to cell designers, and to validate numerical models. The current work utilizes the test data to develop empirical correlations for cell output performance under various working conditions. Because the empirical correlations are developed directly from the experimental data, uncertainties arising from material properties that must be used in numerical modeling can be avoided. Empirical correlations of recent vapor-anode PX-series AMTEC cells have been developed. Based on AMTEC theory and the experimental data, the cell output power (as well as voltage and current) was correlated as a function of three parameters (T{sub hot}, T{sub cold}, and R{sub ext}) for a given cell. Correlations were developed for different cells (PX-3C, PX-3A, PX-G3, and PX-5A), and were in good agreement with experimental data for these cells. Use of these correlations can greatly reduce the testing required to determine electrical performance of a given type of AMTEC cell over a wide range of operating conditions.« less

Transfection using DEAE-dextran.

PubMed

Selden, R F

2001-05-01

Two protocols for DEAE-dextran transfection of cells are provided in this unit. The Basic Protocol describes a procedure used to transfect adherent cells and the first Alternate Protocol presents a method used to transfect suspension cells. If an increase in transfection efficiency is needed, cells can be treated with chloroquine as described in the second Alternate Protocol.

The Dr-nanos gene is essential for germ cell specification in the planarian Dugesia ryukyuensis.

PubMed

Nakagawa, Haruka; Ishizu, Hirotsugu; Chinone, Ayako; Kobayashi, Kazuya; Matsumoto, Midori

2012-01-01

Homologs of nanos are required for the formation and maintenance of germline stem cell (GSC) systems and for gametogenesis in many metazoans. Planarians can change their reproductive mode seasonally, alternating between asexual and sexual reproduction; they develop and maintain their somatic stem cells (SSCs) and GCSs from pluripotent stem cells known as neoblasts. We isolated a nanos homolog, Dr-nanos, from the expressed sequence tags (ESTs) of the sexualized form of Dugesia ryukyuensis. We examined the expression of Dr-nanos in asexual and sexualized planarians by in situ hybridization and analyzed its function using RNA interference (RNAi) together with a planarian sexualization assay. A nanos homolog, Dr-nanos, was identified in the planarian D. ryukyuensis. Dr-nanos expression was observed in the ovarian primordial cells of the asexual worms. This expression increased in proportion to sexualization and was localized in the early germline cells of the ovaries and testes. In X-ray-irradiated worms, the expression of Dr-nanos decreased to a large extent, indicating that Dr-nanos is expressed in some subpopulations of stem cells, especially in GSCs. During the sexualization process, worms in which Dr-nanos was knocked down by RNAi exhibited decreased numbers of oogonia in the ovaries and failed to develop testes, whereas the somatic sexual organs were not affected. We conclude that Dr-nanos is essential for the development of germ cells in the ovaries and testes and may have a function in the early stages of germ cell specification, but not in the development of somatic sexual organs.

Impact of plant extracts tested in attention-deficit/hyperactivity disorder treatment on cell survival and energy metabolism in human neuroblastoma SH-SY5Y cells.

PubMed

Schmidt, Andreas Johannes; Krieg, Jürgen-Christian; Hemmeter, Ulrich Michael; Kircher, Tilo; Schulz, Eberhard; Clement, Hans-Willi; Heiser, Philip

2010-10-01

Plant extracts such as Hypericum perforatum and Pycnogenol have been tested as alternatives to the classical ADHD drugs. It has been possible to describe neuroprotective effects of such plant extracts. A reduction of ADHD symptoms could be shown in clinical studies after the application of Pycnogenol, which is a pine bark extract. The impacts of the standardized herbal extracts Hypericum perforatum, Pycnogenol and Enzogenol up to a concentration of 5000 ng/mL on cell survival and energy metabolism in human SH-SY5Y neuroblastoma cells has been investigated in the present examination. Hypericum perforatum significantly decreased the survival of cells after treatment with a concentration of 5000 ng/mL, whereas lower concentrations exerted no significant effects. Pycnogenol( induced a significant increase of cell survival after incubation with a concentration of 32.25 ng/mL and a concentration of 250 ng/mL. Other applied concentrations of Pycnogenol failed to exert significant effects. Treatment with Enzogenol did not lead to significant changes in cell survival.Concerning energy metabolism, the treatment of cells with a concentration of 5000 ng/mL Hypericum perforatum led to a significant increase of ATP levels, whereas treatment with a concentration of 500 ng/mL had no significant effect. Incubation of cells with Pycnogenol and Enzogenol exerted no significant effects.None of the tested substances caused any cytotoxic effect when used in therapeutically relevant concentrations. Copyright © 2010 John Wiley & Sons, Ltd.

Extracts of Edible and Medicinal Plants Damage Membranes of Vibrio cholerae▿

PubMed Central

Sánchez, Eduardo; García, Santos; Heredia, Norma

2010-01-01

The use of natural compounds from plants can provide an alternative approach against food-borne pathogens. The mechanisms of action of most plant extracts with antimicrobial activity have been poorly studied. In this work, changes in membrane integrity, membrane potential, internal pH (pHin), and ATP synthesis were measured in Vibrio cholerae cells after exposure to extracts of edible and medicinal plants. A preliminary screen of methanolic, ethanolic, and aqueous extracts of medicinal and edible plants was performed. Minimal bactericidal concentrations (MBCs) were measured for extracts showing high antimicrobial activity. Our results indicate that methanolic extracts of basil (Ocimum basilicum L.), nopal cactus (Opuntia ficus-indica var. Villanueva L.), sweet acacia (Acacia farnesiana L.), and white sagebrush (Artemisia ludoviciana Nutt.) are the most active against V. cholera, with MBCs ranging from 0.5 to 3.0 mg/ml. Using four fluorogenic techniques, we studied the membrane integrity of V. cholerae cells after exposure to these four extracts. Extracts from these plants were able to disrupt the cell membranes of V. cholerae cells, causing increased membrane permeability, a clear decrease in cytoplasmic pH, cell membrane hyperpolarization, and a decrease in cellular ATP concentration in all strains tested. These four plant extracts could be studied as future alternatives to control V. cholerae contamination in foods and the diseases associated with this microorganism. PMID:20802077

Extracts of edible and medicinal plants damage membranes of Vibrio cholerae.

PubMed

Sánchez, Eduardo; García, Santos; Heredia, Norma

2010-10-01

The use of natural compounds from plants can provide an alternative approach against food-borne pathogens. The mechanisms of action of most plant extracts with antimicrobial activity have been poorly studied. In this work, changes in membrane integrity, membrane potential, internal pH (pH(in)), and ATP synthesis were measured in Vibrio cholerae cells after exposure to extracts of edible and medicinal plants. A preliminary screen of methanolic, ethanolic, and aqueous extracts of medicinal and edible plants was performed. Minimal bactericidal concentrations (MBCs) were measured for extracts showing high antimicrobial activity. Our results indicate that methanolic extracts of basil (Ocimum basilicum L.), nopal cactus (Opuntia ficus-indica var. Villanueva L.), sweet acacia (Acacia farnesiana L.), and white sagebrush (Artemisia ludoviciana Nutt.) are the most active against V. cholera, with MBCs ranging from 0.5 to 3.0 mg/ml. Using four fluorogenic techniques, we studied the membrane integrity of V. cholerae cells after exposure to these four extracts. Extracts from these plants were able to disrupt the cell membranes of V. cholerae cells, causing increased membrane permeability, a clear decrease in cytoplasmic pH, cell membrane hyperpolarization, and a decrease in cellular ATP concentration in all strains tested. These four plant extracts could be studied as future alternatives to control V. cholerae contamination in foods and the diseases associated with this microorganism.

Oral mucosa: an alternative epidermic cell source to develop autologous dermal-epidermal substitutes from diabetic subjects.

PubMed

Guzmán-Uribe, Daniela; Alvarado-Estrada, Keila Neri; Pierdant-Pérez, Mauricio; Torres-Álvarez, Bertha; Sánchez-Aguilar, Jesus Martin; Rosales-Ibáñez, Raúl

2017-01-01

The aim of this study was to obtain autologous dermal-epidermal skin substitutes from oral mucosa from diabetic subjects as a first step towards a possible clinical application for cases of diabetic foot. Oral mucosa was obtained from diabetic and healthy subjects (n=20 per group). Epidermal cells were isolated and cultured using autologous fibrin to develop dermal-epidermal in vitro substitutes by the air-liquid technique with autologous human serum as a supplement media. Substitutes were immunocharacterized with collagen IV and cytokeratin 5-14 as specific markers. A Student´s t- test was performed to assess the differences between both groups. It was possible to isolate epidermal cells from the oral mucosa of diabetic and healthy subjects and develop autologous dermal-epidermal skin substitutes using autologous serum as a supplement. Differences in the expression of specific markers were observed and the cytokeratin 5-14 expression was lower in the diabetic substitutes, and the collagen IV expression was higher in the diabetic substitutes when compared with the healthy group, showing a significant difference. Cells from oral mucosa could be an alternative and less invasive source for skin substitutes and wound healing. A difference in collagen production of diabetic cells suggests diabetic substitutes could improve diabetic wound healing. More research is needed to determine the crosstalk between components of these skin substitutes and damaged tissues.

77 FR 8865 - Recent Postings of Broadly Applicable Alternative Test Methods

Federal Register 2010, 2011, 2012, 2013, 2014

2012-02-15

... Applicable Alternative Test Methods AGENCY: Environmental Protection Agency (EPA). ACTION: Notice of availability. SUMMARY: This notice announces the broadly applicable alternative test method approval decisions... INFORMATION CONTACT: An electronic copy of each alternative test method approval document is available on the...

75 FR 7593 - Recent Postings of Broadly Applicable Alternative Test Methods

Federal Register 2010, 2011, 2012, 2013, 2014

2010-02-22

... Applicable Alternative Test Methods AGENCY: Environmental Protection Agency (EPA). ACTION: Notice of availability. SUMMARY: This notice announces the broadly applicable alternative test method approval decisions... electronic copy of each alternative test method approval document is available on EPA's Web site at http...

78 FR 11174 - Recent Postings of Broadly Applicable Alternative Test Methods

Federal Register 2010, 2011, 2012, 2013, 2014

2013-02-15

... Applicable Alternative Test Methods AGENCY: Environmental Protection Agency (EPA). ACTION: Notice of availability. SUMMARY: This notice announces the broadly applicable alternative test method approval decisions... INFORMATION CONTACT: An electronic copy of each alternative test method approval document is available on the...

40 CFR 136.5 - Approval of alternate test procedures.

Code of Federal Regulations, 2011 CFR

2011-07-01

... and to the Alternate Test Procedure Program Coordinator, Office of Science and Technology (4303... to the Alternate Test Procedure Program Coordinator, Office of Science and Technology (4303), Office... will be forwarded to the Alternate Test Procedure Program Coordinator, Office of Science and Technology...

40 CFR 136.5 - Approval of alternate test procedures.

Code of Federal Regulations, 2010 CFR

2010-07-01

... and to the Alternate Test Procedure Program Coordinator, Office of Science and Technology (4303... to the Alternate Test Procedure Program Coordinator, Office of Science and Technology (4303), Office... will be forwarded to the Alternate Test Procedure Program Coordinator, Office of Science and Technology...

Cultured meat from stem cells: challenges and prospects.

PubMed

Post, Mark J

2012-11-01

As one of the alternatives for livestock meat production, in vitro culturing of meat is currently studied. The generation of bio-artificial muscles from satellite cells has been ongoing for about 15 years, but has never been used for generation of meat, while it already is a great source of animal protein. In order to serve as a credible alternative to livestock meat, lab or factory grown meat should be efficiently produced and should mimic meat in all of its physical sensations, such as visual appearance, smell, texture and of course, taste. This is a formidable challenge even though all the technologies to create skeletal muscle and fat tissue have been developed and tested. The efficient culture of meat will primarily depend on culture conditions such as the source of medium and its composition. Protein synthesis by cultured skeletal muscle cells should further be maximized by finding the optimal combination of biochemical and physical conditions for the cells. Many of these variables are known, but their interactions are numerous and need to be mapped. This involves a systematic, if not systems, approach. Given the urgency of the problems that the meat industry is facing, this endeavor is worth undertaking. As an additional benefit, culturing meat may provide opportunities for production of novel and healthier products. Copyright © 2012 Elsevier Ltd. All rights reserved.

Economic Analysis of Alternative Strategies for Detection of ALK Rearrangements in Non Small Cell Lung Cancer.

PubMed

Doshi, Shivang; Ray, David; Stein, Karen; Zhang, Jie; Koduru, Prasad; Fogt, Franz; Wellman, Axel; Wat, Ricky; Mathews, Charles

2016-01-06

Identification of alterations in ALK gene and development of ALK-directed therapies have increased the need for accurate and efficient detection methodologies. To date, research has focused on the concordance between the two most commonly used technologies, fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC). However, inter-test concordance reflects only one, albeit important, aspect of the diagnostic process; laboratories, hospitals, and payors must understand the cost and workflow of ALK rearrangement detection strategies. Through literature review combined with interviews of pathologists and laboratory directors in the U.S. and Europe, a cost-impact model was developed that compared four alternative testing strategies-IHC only, FISH only, IHC pre-screen followed by FISH confirmation, and parallel testing by both IHC and FISH. Interviews were focused on costs of reagents, consumables, equipment, and personnel. The resulting model showed that testing by IHC alone cost less ($90.07 in the U.S., $68.69 in Europe) than either independent or parallel testing by both FISH and IHC ($441.85 in the U.S. and $279.46 in Europe). The strategies differed in cost of execution, turnaround time, reimbursement, and number of positive results detected, suggesting that laboratories must weigh the costs and the clinical benefit of available ALK testing strategies.

Economic Analysis of Alternative Strategies for Detection of ALK Rearrangements in Non Small Cell Lung Cancer

PubMed Central

Doshi, Shivang; Ray, David; Stein, Karen; Zhang, Jie; Koduru, Prasad; Fogt, Franz; Wellman, Axel; Wat, Ricky; Mathews, Charles

2016-01-01

Identification of alterations in ALK gene and development of ALK-directed therapies have increased the need for accurate and efficient detection methodologies. To date, research has focused on the concordance between the two most commonly used technologies, fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC). However, inter-test concordance reflects only one, albeit important, aspect of the diagnostic process; laboratories, hospitals, and payors must understand the cost and workflow of ALK rearrangement detection strategies. Through literature review combined with interviews of pathologists and laboratory directors in the U.S. and Europe, a cost-impact model was developed that compared four alternative testing strategies—IHC only, FISH only, IHC pre-screen followed by FISH confirmation, and parallel testing by both IHC and FISH. Interviews were focused on costs of reagents, consumables, equipment, and personnel. The resulting model showed that testing by IHC alone cost less ($90.07 in the U.S., $68.69 in Europe) than either independent or parallel testing by both FISH and IHC ($441.85 in the U.S. and $279.46 in Europe). The strategies differed in cost of execution, turnaround time, reimbursement, and number of positive results detected, suggesting that laboratories must weigh the costs and the clinical benefit of available ALK testing strategies. PMID:26838801

Compound-specific effects of diverse neurodevelopmental toxicants on global gene expression in the neural embryonic stem cell test (ESTn)

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

Theunissen, P.T., E-mail: Peter.Theunissen@rivm.nl; Department of Toxicogenomics, Maastricht University, Maastricht; Robinson, J.F.

Alternative assays for developmental toxicity testing are needed to reduce animal use in regulatory toxicology. The in vitro murine neural embryonic stem cell test (ESTn) was designed as an alternative for neurodevelopmental toxicity testing. The integration of toxicogenomic-based approaches may further increase predictivity as well as provide insight into underlying mechanisms of developmental toxicity. In the present study, we investigated concentration-dependent effects of six mechanistically diverse compounds, acetaldehyde (ACE), carbamazepine (CBZ), flusilazole (FLU), monoethylhexyl phthalate (MEHP), penicillin G (PENG) and phenytoin (PHE), on the transcriptome and neural differentiation in the ESTn. All compounds with the exception of PENG altered ESTnmore » morphology (cytotoxicity and neural differentiation) in a concentration-dependent manner. Compound induced gene expression changes and corresponding enriched gene ontology biological processes (GO–BP) were identified after 24 h exposure at equipotent differentiation-inhibiting concentrations of the compounds. Both compound-specific and common gene expression changes were observed between subsets of tested compounds, in terms of significance, magnitude of regulation and functionality. For example, ACE, CBZ and FLU induced robust changes in number of significantly altered genes (≥ 687 genes) as well as a variety of GO–BP, as compared to MEHP, PHE and PENG (≤ 55 genes with no significant changes in GO–BP observed). Genes associated with developmentally related processes (embryonic morphogenesis, neuron differentiation, and Wnt signaling) showed diverse regulation after exposure to ACE, CBZ and FLU. In addition, gene expression and GO–BP enrichment showed concentration dependence, allowing discrimination of non-toxic versus toxic concentrations on the basis of transcriptomics. This information may be used to define adaptive versus toxic responses at the transcriptome level.« less

Trichostatin A Enhances the Apoptotic Potential of Palladium Nanoparticles in Human Cervical Cancer Cells.

PubMed

Zhang, Xi-Feng; Yan, Qi; Shen, Wei; Gurunathan, Sangiliyandi

2016-08-19

Cervical cancer ranks seventh overall among all types of cancer in women. Although several treatments, including radiation, surgery and chemotherapy, are available to eradicate or reduce the size of cancer, many cancers eventually relapse. Thus, it is essential to identify possible alternative therapeutic approaches for cancer. We sought to identify alternative and effective therapeutic approaches, by first synthesizing palladium nanoparticles (PdNPs), using a novel biomolecule called saponin. The synthesized PdNPs were characterized by several analytical techniques. They were significantly spherical in shape, with an average size of 5 nm. Recently, PdNPs gained much interest in various therapies of cancer cells. Similarly, histone deacetylase inhibitors are known to play a vital role in anti-proliferative activity, gene expression, cell cycle arrest, differentiation and apoptosis in various cancer cells. Therefore, we selected trichostatin A (TSA) and PdNPs and studied their combined effect on apoptosis in cervical cancer cells. Cells treated with either TSA or PdNPs showed a dose-dependent effect on cell viability. The combinatorial effect, tested with 50 nM TSA and 50 nMPdNPs, had a more dramatic inhibitory effect on cell viability, than either TSA or PdNPs alone. The combination of TSA and PdNPs had a more pronounced effect on cytotoxicity, oxidative stress, mitochondrial membrane potential (MMP), caspase-3/9 activity and expression of pro- and anti-apoptotic genes. Our data show a strong synergistic interaction between TSA and PdNPs in cervical cancer cells. The combinatorial treatment increased the therapeutic potential and demonstrated relevant targeted therapy for cervical cancer. Furthermore, we provide the first evidence for the combinatory effect and cytotoxicity mechanism of TSA and PdNPs in cervical cancer cells.

Trichostatin A Enhances the Apoptotic Potential of Palladium Nanoparticles in Human Cervical Cancer Cells

PubMed Central

Zhang, Xi-Feng; Yan, Qi; Shen, Wei; Gurunathan, Sangiliyandi

2016-01-01

Cervical cancer ranks seventh overall among all types of cancer in women. Although several treatments, including radiation, surgery and chemotherapy, are available to eradicate or reduce the size of cancer, many cancers eventually relapse. Thus, it is essential to identify possible alternative therapeutic approaches for cancer. We sought to identify alternative and effective therapeutic approaches, by first synthesizing palladium nanoparticles (PdNPs), using a novel biomolecule called saponin. The synthesized PdNPs were characterized by several analytical techniques. They were significantly spherical in shape, with an average size of 5 nm. Recently, PdNPs gained much interest in various therapies of cancer cells. Similarly, histone deacetylase inhibitors are known to play a vital role in anti-proliferative activity, gene expression, cell cycle arrest, differentiation and apoptosis in various cancer cells. Therefore, we selected trichostatin A (TSA) and PdNPs and studied their combined effect on apoptosis in cervical cancer cells. Cells treated with either TSA or PdNPs showed a dose-dependent effect on cell viability. The combinatorial effect, tested with 50 nM TSA and 50 nMPdNPs, had a more dramatic inhibitory effect on cell viability, than either TSA or PdNPs alone. The combination of TSA and PdNPs had a more pronounced effect on cytotoxicity, oxidative stress, mitochondrial membrane potential (MMP), caspase-3/9 activity and expression of pro- and anti-apoptotic genes. Our data show a strong synergistic interaction between TSA and PdNPs in cervical cancer cells. The combinatorial treatment increased the therapeutic potential and demonstrated relevant targeted therapy for cervical cancer. Furthermore, we provide the first evidence for the combinatory effect and cytotoxicity mechanism of TSA and PdNPs in cervical cancer cells. PMID:27548148

Alternative cells for regeneration.

PubMed

Slack, Jonathan M W

2012-04-17

Normally, in fish fin regeneration, bone regenerates from bone. But what happens when there is no bone? Singh et al. (2012) show in this issue of Developmental Cell that the bony rays still regenerate from an alternative cell source. Copyright © 2012 Elsevier Inc. All rights reserved.

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.

Fermentation of lignocellulosic hydrolysate by the alternative industrial ethanol yeast Dekkera bruxellensis.

PubMed

Blomqvist, J; South, E; Tiukova, I; Tiukova, L; Momeni, M H; Hansson, H; Ståhlberg, J; Horn, S J; Schnürer, J; Passoth, V

2011-07-01

Testing the ability of the alternative ethanol production yeast Dekkera bruxellensis to produce ethanol from lignocellulose hydrolysate and comparing it to Saccharomyces cerevisiae. Industrial isolates of D. bruxellensis and S. cerevisiae were cultivated in small-scale batch fermentations of enzymatically hydrolysed steam exploded aspen sawdust. Different dilutions of hydrolysate were tested. None of the yeasts grew in undiluted or 1:2 diluted hydrolysate [final glucose concentration always adjusted to 40 g l⁻¹ (0.22 mol l⁻¹)]. This was most likely due to the presence of inhibitors such as acetate or furfural. In 1:5 hydrolysate, S. cerevisiae grew, but not D. bruxellensis, and in 1:10 hydrolysate, both yeasts grew. An external vitamin source (e.g. yeast extract) was essential for growth of D. bruxellensis in this lignocellulosic hydrolysate and strongly stimulated S. cerevisiae growth and ethanol production. Ethanol yields of 0.42 ± 0.01 g ethanol (g glucose)⁻¹ were observed for both yeasts in 1:10 hydrolysate. In small-scale continuous cultures with cell recirculation, with a gradual increase in the hydrolysate concentration, D. bruxellensis was able to grow in 1:5 hydrolysate. In bioreactor experiments with cell recirculation, hydrolysate contents were increased up to 1:2 hydrolysate, without significant losses in ethanol yields for both yeasts and only slight differences in viable cell counts, indicating an ability of both yeasts to adapt to toxic compounds in the hydrolysate. Dekkera bruxellensis and S. cerevisiae have a similar potential to ferment lignocellulose hydrolysate to ethanol and to adapt to fermentation inhibitors in the hydrolysate. This is the first study investigating the potential of D. bruxellensis to ferment lignocellulosic hydrolysate. Its high competitiveness in industrial fermentations makes D. bruxellensis an interesting alternative for ethanol production from those substrates. © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.

Lactoferrin Adsorbed onto Biomimetic Hydroxyapatite Nanocrystals Controlling - In Vivo - the Helicobacter pylori Infection

PubMed Central

Fulgione, Andrea; Nocerino, Nunzia; Iannaccone, Marco; Roperto, Sante; Capuano, Federico; Roveri, Norberto; Lelli, Marco; Crasto, Antonio; Calogero, Armando; Pilloni, Argenia Paola; Capparelli, Rosanna

2016-01-01

Background The resistance of Helicobacter pylori to the antibiotic therapy poses the problem to discover new therapeutic approaches. Recently it has been stated that antibacterial, immunomodulatory, and antioxidant properties of lactoferrin are increased when this protein is surface-linked to biomimetic hydroxyapatite nanocrystals. Objective Based on these knowledge, the aim of the study was to investigate the efficacy of lactoferrin delivered by biomimetic hydroxyapatite nanoparticles with cell free supernatant from probiotic Lactobacillus paracasei as an alternative therapy against Helicobacter pylori infection. Methods Antibacterial and antinflammatory properties, humoral antibody induction, histopathological analysis and absence of side effects were evaluated in both in vitro and in vivo studies. Results The tests carried out have been demonstrated better performance of lactoferrin delivered by biomimetic hydroxyapatite nanoparticles combined with cell free supernatant from probiotic Lactobacillus paracasei compared to both lactoferrin and probiotic alone or pooled. Conclusion These findings indicate the effectiveness and safety of our proposed therapy as alternative treatment for Helicobacter pylori infection. PMID:27384186

Alternate NASDA Payload Specialists in the Huntsville Operations Support Center (HOSC) Spacelab

NASA Technical Reports Server (NTRS)

1992-01-01

The science laboratory, Spacelab-J (SL-J), flown aboard the STS-47 flight was a joint venture between NASA and the National Space Development Agency of Japan (NASDA) utilizing a manned Spacelab module. The mission conducted 24 materials science and 20 life science experiments, of which 35 were sponsored by NASDA, 7 by NASA, and two collaborative efforts. Materials science investigations covered such fields as biotechnology, electronic materials, fluid dynamics and transport phenomena, glasses and ceramics, metals and alloys, and acceleration measurements. Life sciences included experiments on human health, cell separation and biology, developmental biology, animal and human physiology and behavior, space radiation, and biological rhythms. Test subjects included the crew, Japanese koi fish (carp), cultured animal and plant cells, chicken embryos, fruit flies, fungi and plant seeds, and frogs and frog eggs. Pictured along with George Norris in the Huntsville Operations Support Center (HOSC) Spacelab Payload Operations Control Center (SL POCC) at Marshall Space Flight Center (MSFC) are NASDA alternate payload specialists Dr. Doi and Dr. Mukai.

Alternate NASDA Payload Specialists in the Huntsville Operations Support Center (HOSC) Spacelab

NASA Technical Reports Server (NTRS)

1992-01-01

The science laboratory, Spacelab-J (SL-J), flown aboard the STS-47 flight was a joint venture between NASA and the National Space Development Agency of Japan (NASDA) utilizing a manned Spacelab module. The mission conducted 24 materials science and 20 life science experiments, of which 35 were sponsored by NASDA, 7 by NASA, and two collaborative efforts. Materials science investigations covered such fields as biotechnology, electronic materials, fluid dynamics and transport phenomena, glasses and ceramics, metals and alloys, and acceleration measurements. Life sciences included experiments on human health, cell separation and biology, developmental biology, animal and human physiology and behavior, space radiation, and biological rhythms. Test subjects included the crew, Japanese koi fish (carp), cultured animal and plant cells, chicken embryos, fruit flies, fungi and plant seeds, and frogs and frog eggs. Pictured in the Huntsville Operations Support Center (HOSC) Spacelab Payload Operations Control Center (SL POCC) of Marshall Space Flight Center (MSFC) are NASDA alternate payload specialists Dr. Doi and Dr. Mukai.

Transient acquisition of pluripotency during somatic cell transdifferentiation with iPSC reprogramming factors.

PubMed

Maza, Itay; Caspi, Inbal; Zviran, Asaf; Chomsky, Elad; Rais, Yoach; Viukov, Sergey; Geula, Shay; Buenrostro, Jason D; Weinberger, Leehee; Krupalnik, Vladislav; Hanna, Suhair; Zerbib, Mirie; Dutton, James R; Greenleaf, William J; Massarwa, Rada; Novershtern, Noa; Hanna, Jacob H

2015-07-01

Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced 'transdifferentiation' pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by various methods.

Transient Acquisition of Pluripotency During Somatic Cell Transdifferentiation with iPSC Reprogramming Factors

PubMed Central

Maza, Itay; Caspi, Inbal; Zviran, Asaf; Chomsky, Elad; Rais, Yoach; Viukov, Sergey; Geula, Shay; Buenrostro, Jason D.; Weinberger, Leehee; Krupalnik, Vladislav; Hanna, Suhair; Zerbib, Mirie; Dutton, James R.; Greenleaf, William J.; Massarwa, Rada; Novershtern, Noa; Hanna, Jacob H.

2015-01-01

Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors1,2. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation3–6. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced transdifferentiation pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by different methods. PMID:26098448

The RNA-binding landscape of RBM10 and its role in alternative splicing regulation in models of mouse early development.

PubMed

Rodor, Julie; FitzPatrick, David R; Eyras, Eduardo; Cáceres, Javier F

2017-01-02

Mutations in the RNA-binding protein, RBM10, result in a human syndromic form of cleft palate, termed TARP syndrome. A role for RBM10 in alternative splicing regulation has been previously demonstrated in human cell lines. To uncover the cellular functions of RBM10 in a cell line that is relevant to the phenotype observed in TARP syndrome, we used iCLIP to identify its endogenous RNA targets in a mouse embryonic mandibular cell line. We observed that RBM10 binds to pre-mRNAs with significant enrichment in intronic regions, in agreement with a role for this protein in pre-mRNA splicing. In addition to protein-coding transcripts, RBM10 also binds to a variety of cellular RNAs, including non-coding RNAs, such as spliceosomal small nuclear RNAs, U2 and U12. RNA-seq was used to investigate changes in gene expression and alternative splicing in RBM10 KO mouse mandibular cells and also in mouse ES cells. We uncovered a role for RBM10 in the regulation of alternative splicing of common transcripts in both cell lines but also identified cell-type specific events. Importantly, those pre-mRNAs that display changes in alternative splicing also contain RBM10 iCLIP tags, suggesting a direct role of RBM10 in these events. Finally, we show that depletion of RBM10 in mouse ES cells leads to proliferation defects and to gross alterations in their differentiation potential. These results demonstrate a role for RBM10 in the regulation of alternative splicing in two cell models of mouse early development and suggests that mutations in RBM10 could lead to splicing changes that affect normal palate development and cause human disease.

The determinants of alternative RNA splicing in human cells.

PubMed

Ramanouskaya, Tatsiana V; Grinev, Vasily V

2017-12-01

Alternative splicing represents an important level of the regulation of gene function in eukaryotic organisms. It plays a critical role in virtually every biological process within an organism, including regulation of cell division and cell death, differentiation of tissues in the embryo and the adult organism, as well as in cellular response to diverse environmental factors. In turn, studies of the last decade have shown that alternative splicing itself is controlled by different mechanisms. Unfortunately, there is no clear understanding of how these diverse mechanisms, or determinants, regulate and constrain the set of alternative RNA species produced from any particular gene in every cell of the human body. Here, we provide a consolidated overview of alternative splicing determinants including RNA-protein interactions, epigenetic regulation via chromatin remodeling, coupling of transcription-to-alternative splicing, effect of secondary structures in pre-RNA, and function of the RNA quality control systems. We also extensively and critically discuss some mechanistic insights on coordinated inclusion/exclusion of exons during the formation of mature RNA molecules. We conclude that the final structure of RNA is pre-determined by a complex interplay between cis- and trans-acting factors. Altogether, currently available empirical data significantly expand our understanding of the functioning of the alternative splicing machinery of cells in normal and pathological conditions. On the other hand, there are still many blind spots that require further deep investigations.

Catalyst Stability Benchmarking for the Oxygen Evolution Reaction: The Importance of Backing Electrode Material and Dissolution in Accelerated Aging Studies.

PubMed

Geiger, Simon; Kasian, Olga; Mingers, Andrea M; Nicley, Shannon S; Haenen, Ken; Mayrhofer, Karl J J; Cherevko, Serhiy

2017-09-18

In searching for alternative oxygen evolution reaction (OER) catalysts for acidic water splitting, fast screening of the material intrinsic activity and stability in half-cell tests is of vital importance. The screening process significantly accelerates the discovery of new promising materials without the need of time-consuming real-cell analysis. In commonly employed tests, a conclusion on the catalyst stability is drawn solely on the basis of electrochemical data, for example, by evaluating potential-versus-time profiles. Herein important limitations of such approaches, which are related to the degradation of the backing electrode material, are demonstrated. State-of-the-art Ir-black powder is investigated for OER activity and for dissolution as a function of the backing electrode material. Even at very short time intervals materials like glassy carbon passivate, increasing the contact resistance and concealing the degradation phenomena of the electrocatalyst itself. Alternative backing electrodes like gold and boron-doped diamond show better stability and are thus recommended for short accelerated aging investigations. Moreover, parallel quantification of dissolution products in the electrolyte is shown to be of great importance for comparing OER catalyst feasibility. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Anatomy of Caprine Female Urethra and Characteristics of Muscle and Bone Marrow Derived Caprine Cells for Autologous Cell Therapy Testing.

PubMed

Burdzinska, Anna; Dybowski, Bartosz; Zarychta-Wisniewska, Weronika; Kulesza, Agnieszka; Zagozdzon, Radoslaw; Gajewski, Zdzislaw; Paczek, Leszek

2017-03-01

Cell therapy is emerging as an alternative treatment of stress urinary incontinence. However, many aspects of the procedure require further optimization. A large animal model is needed to reliably test cell delivery methods. In this study, we aim to determine suitability of the goat as an experimental animal for testing intraurethral autologous cell transplantation in terms of urethral anatomy and cell culture parameters. The experiments were performed in 12 mature/aged female goats. Isolated caprine muscle derived cells (MDC) were myogenic in vitro and mesenchymal stem cells (MSC) population was able to differentiate into adipo-, osteo- and chondrogenic lineages. The median yield of cells after 3 weeks of culture amounted 47 × 10(6) for MDC and 37 × 10(6) for MSC. Urethral pressure profile measurements revealed the mean functional urethral length of 3.75 ± 0.7 cm. The mean maximal urethral closure pressure amounted 63.5 ± 5.9 cmH 2 O and the mean functional area was 123.3 ± 19.4 cm*cmH 2 O. The omega- shaped striated urethral sphincter was well developed in the middle and distal third of the urethra and its mean thickness on cross section was 2.3 mm. In the proximal part of the urethra only loosely arranged smooth muscle fibers were identified. To conclude, presented data demonstrate that caprine MDC and MSC can be expanded in vitro in a repeatable manner even when mature or aged animals are cell donors. Results suggest that female caprine urethra has similar parameters to those reported in human and therefore the goat can be an appropriate experimental animal for testing intraurethral cell transplantation. Anat Rec, 00:000-000, 2016. © 2016 Wiley Periodicals, Inc. Anat Rec, 300:577-588, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

ToxCast Profiling in a Human Stem Cell Assay for ...

EPA Pesticide Factsheets

Standard practice for assessing disruptions in embryogenesis involves testing pregnant animals of two species, typically rats and rabbits, exposed during major organogenesis and evaluated just prior to term. Under this design the major manifestations of developmental toxicity are observed as one or more apical endpoints including intrauterine death, fetal growth retardation, structural malformations and variations. Alternative approaches to traditional developmental toxicity testing have been proposed in the form of in vitro data (e.g., embryonic stem cells, zebrafish embryos, HTS assays) and in silico models (e.g., computational toxicology). To increase the diversity of assays used to assess developmental toxicity in EPA’s ToxCast program, we tested the chemicals in Stemina’s metabolomics-based platform that utilizes the commecrially available H9 human embryonic stem cell line. The devTOXqP dataset for ToxCast of high-quality based on replicate samples and model performance (82% balanced accuracy, 0.71 sensitivity and 1.00 specificity). To date, 136 ToxCast chemicals (12.8% of 1065 tested) were positive in this platform; 48 triggered the biomarker signal without any change in hESC viability and 88 triggered activity concurrent with effects on cell viability. Work is in progress to complete the STM dataset entry into the TCPL, compare data with results from zFish and mESC platforms, profile bioactivity (ToxCastDB), endpoints (ToxRefDB), chemotypes (DSSTox)

Establishment of an immortal chicken embryo liver-derived cell line.

PubMed

Lee, Jeongyoon; Foster, Douglas N; Bottje, Walter G; Jang, Hyeon-Min; Chandra, Yohanna G; Gentles, Lauren E; Kong, Byung-Whi

2013-06-01

A continuously growing immortal cell substrate can be used for virus propagation, diagnostic purposes, and vaccine production. The aim of this study was to develop an immortal chicken cell line for efficient propagation of avian infectious viruses. From the various chicken embryo cells that were tested for life span extension, an immortalized chicken embryo liver (CEL) cell line, named CEL-im, was derived spontaneously without either oncogenic viruses or carcinogenic chemical treatment. Currently, CEL-im cells are growing 0.8 to 1.1 population doublings per day and have reached 120 passages. The CEL-im cell line is permissive for poultry infectious viruses, including avian metapneumovirus (AMPV), Marek's disease virus serotype 1 (MDV-1), and infectious laryngotracheitis virus. The CEL-im cells produced high AMPV titer (>10(5) pfu/mL), whereas very low titers (~10 pfu/mL) for MDV-1 and infectious laryngotracheitis virus were produced. To identify genetic alterations in the immortal CEL-im cell line, telomerase activity and mRNA expression for major cell cycle regulatory genes were determined during the immortalizing process. The CEL-im cell line has negative telomerase activity, and when compared with the primary passage 2 CEL cell counterpart, mRNA expression of tumor suppressor protein p53, mouse double minute 2 (Mdm2), cyclin dependent kinase (CDK) inhibitor p21 (p21(WAF)), and CDK inhibitor p16 (p16(INK4)) were downregulated in the CEL-im cell line, whereas retinoblastoma (Rb), transcription factor E2F, member 1 (E2F-1), and alternative reading frame of p16(INK4) (ARF) were upregulated. These results are similar to genetic alterations found previously in immortal chicken embryo fibroblast (CEF) cell lines that showed efficient propagation of MDV-1. Therefore, this newly established CEL-im cell line can serve as an alternative cell substrate for the propagation of poultry viruses, such as AMPV.

Alternator insulation evaluation tests

NASA Technical Reports Server (NTRS)

Penn, W. B.; Schaefer, R. F.; Balke, R. L.

1972-01-01

Tests were conducted to predict the remaining electrical insulation life of a 60 KW homopolar inductor alternator following completion of NASA turbo-alternator endurance tests for SNAP-8 space electrical power systems application. The insulation quality was established for two alternators following completion of these tests. A step-temperature aging test procedure was developed for insulation life prediction and applied to one of the two alternators. Armature winding insulation life of over 80,000 hours for an average winding temperature of 248 degrees C was predicted using the developed procedure.

Identification of Alternative Splice Variants Using Unique Tryptic Peptide Sequences for Database Searches.

PubMed

Tran, Trung T; Bollineni, Ravi C; Strozynski, Margarita; Koehler, Christian J; Thiede, Bernd

2017-07-07

Alternative splicing is a mechanism in eukaryotes by which different forms of mRNAs are generated from the same gene. Identification of alternative splice variants requires the identification of peptides specific for alternative splice forms. For this purpose, we generated a human database that contains only unique tryptic peptides specific for alternative splice forms from Swiss-Prot entries. Using this database allows an easy access to splice variant-specific peptide sequences that match to MS data. Furthermore, we combined this database without alternative splice variant-1-specific peptides with human Swiss-Prot. This combined database can be used as a general database for searching of LC-MS data. LC-MS data derived from in-solution digests of two different cell lines (LNCaP, HeLa) and phosphoproteomics studies were analyzed using these two databases. Several nonalternative splice variant-1-specific peptides were found in both cell lines, and some of them seemed to be cell-line-specific. Control and apoptotic phosphoproteomes from Jurkat T cells revealed several nonalternative splice variant-1-specific peptides, and some of them showed clear quantitative differences between the two states.

Potential feasibility of dental stem cells for regenerative therapies: stem cell transplantation and whole-tooth engineering.

PubMed

Nakahara, Taka

2011-07-01

Multipotent mesenchymal stem cells from bone marrow are expected to be a somatic stem cell source for the development of new cell-based therapy in regenerative medicine. However, dental clinicians are unlikely to carry out autologous cell/tissue collection from patients (i.e., marrow aspiration) as a routine procedure in their clinics; hence, the utilization of bone marrow stem cells seems impractical in the dental field. Dental tissues harvested from extracted human teeth are well known to contain highly proliferative and multipotent stem cell compartments and are considered to be an alternative autologous cell source in cell-based medicine. This article provides a short overview of the ongoing studies for the potential application of dental stem cells and suggests the utilization of 2 concepts in future regenerative medicine: (1) dental stem cell-based therapy for hepatic and other systemic diseases and (2) tooth replacement therapy using the bioengineered human whole tooth, called the "test-tube dental implant." Regenerative therapies will bring new insights and benefits to the fields of clinical medicine and dentistry.

Green Toxicology-Know Early About and Avoid Toxic Product Liabilities.

PubMed

Maertens, Alexandra; Hartung, Thomas

2018-02-01

Toxicology uniquely among the life sciences relies largely on methods which are more than 40-years old. Over the last 3 decades with more or less success some additions to and few replacements in this toolbox took place, mainly as alternatives to animal testing. The acceptance of such new approaches faces the needs of formal validation and the conservative attitude toward change in safety assessments. Only recently, there is growing awareness that the same alternative methods, especially in silico and in vitro tools can also much earlier and before validation inform decision-taking in the product life cycle. As similar thoughts developed in the context of Green Chemistry, the term of Green Toxicology was coined to describe this change in approach. Here, the current developments in the alternative field, especially computational and more organo-typic cell cultures are reviewed, as they lend themselves to front-loaded chemical safety assessments. The initiatives of the Center for Alternatives to Animal Testing Green Toxicology Collaboration are presented. They aim first of all for forming a community to promote this concept and then for a cultural change in companies with the necessary training of chemists, product stewards and later regulators. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Tuberculin purified protein derivative (PPD) immunoassay as an in vitro alternative assay for identity and confirmation of potency.

PubMed

Ho, Mei M; Kairo, Satnam K; Corbel, Michael J

2006-01-01

Tuberculin purified protein derivative (PPD) currently can only be standardised by delayed hypersensitivity skin reactions in sensitised guinea pigs. An in vitro dot blot immunoassay was developed for both identity and confirmation of potency estimation of PPD. Polyclonal antibodies (mainly IgG) were generated and immunoreacted with human, bovine and, to lesser extent, avian PPD preparations. Combining size exclusion chromatography (FPLC-SEC) and dot blot immunoassay, the results showed that PPD preparations were mixtures of very heterogeneous tuberculoproteins ranging in size from very large aggregates to very small degraded molecules. All individual fractions of PPD separated by size were immunoreactive, although those of the largest molecular sizes appeared the most immunoreactive in this in vitro dot blot immunoassay. This method is very sensitive and specific to tuberculoproteins and can be an in vitro alternative for the in vivo intradermal skin assay which uses guinea pigs for identity of PPD preparations. Although the capacity of PPD to elicit cell-mediated immune responses on intradermal testing has to be confirmed by in vivo assay, the dot blot immunoassay offers a rapid, sensitive and animal-free alternative to in vivo testing for confirming the identity of PPD preparations with appropriate potencies. This alternative assay would be particularly useful for national regulatory laboratories for confirming the data of manufacturers and thus reducing the use of animals.

Prolonged viability of human organotypic skin explant in culture method (hOSEC)*

PubMed Central

Frade, Marco Andrey Cipriani; de Andrade, Thiago Antônio Moretti; Aguiar, Andréia Fernanda Carvalho Leone; Guedes, Flávia Araújo; Leite, Marcel Nani; Passos, Williane Rodrigues; Coelho, Eduardo Barbosa; Das, Pranab Kummar

2015-01-01

BACKGROUND: Currently, the cosmetic industry is overwhelmed in keeping up with the safety assessment of the increasing number of new products entering the market. To meet such demand, research centers have explored alternative methods to animal testing and also the large number of volunteers necessary for preclinical and clinical tests. OBJECTIVES: This work describes the human skin ex-vivo model (hOSEC: Human Organotypic Skin Explant Culture) as an alternative to test the effectiveness of cosmetics and demonstrate its viability through cutaneous keratinocytes' proliferative capacity up to 75 days in culture. METHODS: The skin explants obtained from surgeries were cultured in CO2-humid incubator. After 1, 7, 30 and 75 days in culture, skin fragments were harvested for analysis with histomorphological exam (HE staining) on all days of follow-up and immunohistochemistry for Ck5/6, Ck10 and Ki-67 only on the 75th day. RESULTS: On the 7th day, the epidermis was perfect in the dermoepidermal junction, showing the viability of the model. On the 30th day, the epidermis was thicker, with fewer layers on the stratum corneum, although the cutaneous structure was unaltered. On the 75th day, the skin became thinner but the dermoepidermal junctions were preserved and epidermal proliferation was maintained. After the 75th day on culture, the skin was similar to normal skin, expressing keratinocytes with Ck5/6 on supra-basal layers; Ck10 on differentiated layers; and viability could be assessed by the positivity of basal cells by Ki-67. CONCLUSION: The hOSEC model seems a good alternative to animal testing; it can be used as a preclinical test analogous to clinical human skin test with similar effectiveness and viability proven by immunohistological analyses. PMID:26131864

Prolonged viability of human organotypic skin explant in culture method (hOSEC).

PubMed

Frade, Marco Andrey Cipriani; Andrade, Thiago Antônio Moretti de; Aguiar, Andréia Fernanda Carvalho Leone; Guedes, Flávia Araújo; Leite, Marcel Nani; Passos, Williane Rodrigues; Coelho, Eduardo Barbosa; Das, Pranab Kummar

2015-01-01

Currently, the cosmetic industry is overwhelmed in keeping up with the safety assessment of the increasing number of new products entering the market. To meet such demand, research centers have explored alternative methods to animal testing and also the large number of volunteers necessary for preclinical and clinical tests. This work describes the human skin ex-vivo model (hOSEC: Human Organotypic Skin Explant Culture) as an alternative to test the effectiveness of cosmetics and demonstrate its viability through cutaneous keratinocytes' proliferative capacity up to 75 days in culture. The skin explants obtained from surgeries were cultured in CO2-humid incubator. After 1, 7, 30 and 75 days in culture, skin fragments were harvested for analysis with histomorphological exam (HE staining) on all days of follow-up and immunohistochemistry for Ck5/6, Ck10 and Ki-67 only on the 75th day. On the 7th day, the epidermis was perfect in the dermoepidermal junction, showing the viability of the model. On the 30th day, the epidermis was thicker, with fewer layers on the stratum corneum, although the cutaneous structure was unaltered. On the 75th day, the skin became thinner but the dermoepidermal junctions were preserved and epidermal proliferation was maintained. After the 75th day on culture, the skin was similar to normal skin, expressing keratinocytes with Ck5/6 on supra-basal layers; Ck10 on differentiated layers; and viability could be assessed by the positivity of basal cells by Ki-67. The hOSEC model seems a good alternative to animal testing; it can be used as a preclinical test analogous to clinical human skin test with similar effectiveness and viability proven by immunohistological analyses.

Evaluation of cooling concepts and specimen geometries for high heat flux tests on neutron irradiated divertor elements

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

Linke, J.; Bolt. H.; Breitbach, G.

1994-12-31

To assess the lifetime and the long term heat removal capabilities of plasma facing components in future thermonuclear fusion reactors such as ITER, neutron irradiation and subsequent high heat flux tests will be most essential. The effect of neutron damage will be simulated in material test reactors (such as the HFR-Petten) in a fission neutron environment. To investigate the heat loads during normal and off-normal operation scenarios a 60 kW electron beam test stand (Juelich Divertor Test Facility in Hot Cells, JUDITH) has been installed in a hot cell which can be operated by remote handling techniques. In this facilitymore » inertially cooled test coupons can be handled as well as small actively cooled divertor mock-ups. A special clamping mechanism for small test coupons (25 mm x 25 mm x 35 mm) with an integrated coolant channel within a copper or TZM heat sink has been developed and tested in an electron beam test bed. This method is an attractive alternative to costly large scale tests on complete divertor modules. The temperature and stress fields in individual CFC or beryllium tiles brazed to metallic heat sink (e.g. copper or TZM) can be investigated before and after neutron irradiation with moderate efforts.« less

Improved Electrodes for High Temperature Proton Exchange Membrane Fuel Cells using Carbon Nanospheres.

PubMed

Zamora, Héctor; Plaza, Jorge; Cañizares, Pablo; Lobato, Justo; Rodrigo, Manuel A

2016-05-23

This work evaluates the use of carbon nanospheres (CNS) in microporous layers (MPL) of high temperature proton exchange membrane fuel cell (HT-PEMFC) electrodes and compares the characteristics and performance with those obtained using conventional MPL based on carbon black. XRD, hydrophobicity, Brunauer-Emmett-Teller theory, and gas permeability of MPL prepared with CNS were the parameters evaluated. In addition, a short life test in a fuel cell was carried out to evaluate performance under accelerated stress conditions. The results demonstrate that CNS is a promising alternative to traditional carbonaceous materials because of its high electrochemical stability and good electrical conductivity, suitable to be used in this technology. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cross-species malaria immunity induced by chemically attenuated parasites

PubMed Central

Good, Michael F.; Reiman, Jennifer M.; Rodriguez, I. Bibiana; Ito, Koichi; Yanow, Stephanie K.; El-Deeb, Ibrahim M.; Batzloff, Michael R.; Stanisic, Danielle I.; Engwerda, Christian; Spithill, Terry; Hoffman, Stephen L.; Lee, Moses; McPhun, Virginia

2013-01-01

Vaccine development for the blood stages of malaria has focused on the induction of antibodies to parasite surface antigens, most of which are highly polymorphic. An alternate strategy has evolved from observations that low-density infections can induce antibody-independent immunity to different strains. To test this strategy, we treated parasitized red blood cells from the rodent parasite Plasmodium chabaudi with seco-cyclopropyl pyrrolo indole analogs. These drugs irreversibly alkylate parasite DNA, blocking their ability to replicate. After administration in mice, DNA from the vaccine could be detected in the blood for over 110 days and a single vaccination induced profound immunity to different malaria parasite species. Immunity was mediated by CD4+ T cells and was dependent on the red blood cell membrane remaining intact. The human parasite, Plasmodium falciparum, could also be attenuated by treatment with seco-cyclopropyl pyrrolo indole analogs. These data demonstrate that vaccination with chemically attenuated parasites induces protective immunity and provide a compelling rationale for testing a blood-stage parasite-based vaccine targeting human Plasmodium species. PMID:23863622

Coordinate regulation of cytochrome and alternative pathway respiration in tobacco.

PubMed

Vanlerberghe, G C; McIntosh, L

1992-12-01

In suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow), inhibition of the cytochrome pathway of respiration with antimycin A induced a large increase in the capacity of the alternative pathway over a period of approximately 12 h, as confirmed in both whole cells and isolated mitochondria. The increase in alternative pathway capacity required de novo RNA and protein synthesis and correlated closely with the increase of a 35-kD alternative oxidase protein. When the cytochrome pathway of intact cells was inhibited by antimycin A, respiration proceeded exclusively through the alternative pathway, reached rates significantly higher than before antimycin A addition, and was not stimulated by p-trifluoromethoxycarbonylcyanide (FCCP). When inhibition of the cytochrome pathway was relieved, alternative pathway capacity and the level of the 35-kD alternative oxidase protein declined. Respiration rate also declined and could once again be stimulated by FCCP. These observations show that the capacities of the mitochondrial electron transport pathways can be regulated in a coordinate fashion.

Novel application of human periodontal ligament stem cells and water-soluble chitin for collagen tissue regeneration: in vitro and in vivo investigations.

PubMed

Jung, Im Hee; Park, Jung Chul; Kim, Jane C; Jeon, Dong Won; Choi, Seong Ho; Cho, Kyoo Sung; Im, Gun Il; Kim, Byung Soo; Kim, Chang Sung

2012-03-01

Human periodontal ligament stem cells (hPDLSCs) have been proposed as an alternative to conventional cosmetic fillers because they display an innate ability to synthesize collagen. The aims of this study were to determine the effects of water-soluble chitin (WSC) on the proliferation and migration of hPDLSCs, and to quantify collagen synthesis in vitro and in vivo compared with human adipose-derived stem cell (hADSC)s. hPDLSCs were isolated from healthy extracted teeth, and the cell proliferation and cell migration capacities of untreated hPDLSCs (control group) and WSC-treated hPDLSCs (test group) were compared. Insoluble/soluble collagen synthesis were also assessed, and collagen related markers were evaluated including lysyl oxidase (LOX), lysyl oxidase like (LOXL)1, LOXL2, and hydroxyproline. In vivo collagen formation was examined by transplanting hyaluronic acid as a cell carrier into the subcutaneous pockets of immunocompromised mice in the control and test groups; histology and immunohistochemistry analyses were performed 4 (n=4) and 8 (n=4) weeks later. There was a dose-dependent enhancement of hPDLSCs proliferation in the test group, and a concomitant reduction in cell migration. The amount of insoluble collagen formed was greater in the test group than in the control group (p<0.05), whereas soluble collagen formation was significantly reduced in the test group (p<0.05). The histology and immunohistochemistry results revealed that the amount of collagen formed in vivo was greater in WSC-treated hPDLSCs than in the control cells at 4 and 8 weeks (p<0.05), and histometric analysis at 8 weeks revealed that enhancement of collagen formation by hPDLSCs was greater than by hADSCs. These results indicate that WSC modulates the properties of hPDLSCs, rendering them more suitable for cosmetic soft-tissue augmentation.

Inflammatory Effects of the Plant Protection Product Stifenia (FEN560) on Vertebrates.

PubMed

Teyssier, Lény; Colussi, Julie; Delemasure, Stéphanie; Chluba, Johanna; Wendehenne, David; Lamotte, Olivier; Connat, Jean-Louis

2017-01-01

Plant defense stimulators (PDSs) rely on the activation of plant innate immunity in order to protect crops against various pests. These molecules are thought to be a safer alternative to classical plant protection products. Given that innate immune systems share common features in plants and vertebrates, PDS can potentially cross-react with innate immunity of non-target organisms. To test this hypothesis, we studied effects of the commercial PDS Stifenia (FEN560), which is composed of crushed fenugreek seeds. We tested various concentrations of Stifenia (0.03-1 mg mL -1 ) on human peripheral blood mononuclear cells and checked, 20 h later, cell metabolic activity (MA) using XTT assay, cell death by flow cytometry analysis, and IL-1β inflammatory cytokine released in the culture medium using ELISA. Stifenia induced a general decrease of the cell MA, which was concomitant with a dose-dependent release of IL-1β. Our results highlight the activation of human immune cells. The inflammatory effect of Stifenia was partially inhibited by pan-caspase inhibitor. Accordingly, Stifenia induced the release of p20 caspase-1 fragment into the culture medium suggesting the involvement of the NLRP3 inflammasome. Furthermore, we observed that Stifenia can induce cell death. We also tested the effect of Stifenia on Zebrafish larvae. After 24 h of exposure, Stifenia induced a dose-dependent IL-1β and TNFα gene expression. The human-cell-based approach developed in this work revealed a high sensitivity concerning inflammatory properties of a plant protection product. These tests could be routinely used to screen the potential adverse effects of this type of compounds. Finally, our results suggest a potential danger of using extensively certain PDS for crop protection.

Inflammatory Effects of the Plant Protection Product Stifenia (FEN560) on Vertebrates

PubMed Central

Teyssier, Lény; Colussi, Julie; Delemasure, Stéphanie; Chluba, Johanna; Wendehenne, David; Lamotte, Olivier; Connat, Jean-Louis

2017-01-01

Plant defense stimulators (PDSs) rely on the activation of plant innate immunity in order to protect crops against various pests. These molecules are thought to be a safer alternative to classical plant protection products. Given that innate immune systems share common features in plants and vertebrates, PDS can potentially cross-react with innate immunity of non-target organisms. To test this hypothesis, we studied effects of the commercial PDS Stifenia (FEN560), which is composed of crushed fenugreek seeds. We tested various concentrations of Stifenia (0.03–1 mg mL−1) on human peripheral blood mononuclear cells and checked, 20 h later, cell metabolic activity (MA) using XTT assay, cell death by flow cytometry analysis, and IL-1β inflammatory cytokine released in the culture medium using ELISA. Stifenia induced a general decrease of the cell MA, which was concomitant with a dose-dependent release of IL-1β. Our results highlight the activation of human immune cells. The inflammatory effect of Stifenia was partially inhibited by pan-caspase inhibitor. Accordingly, Stifenia induced the release of p20 caspase-1 fragment into the culture medium suggesting the involvement of the NLRP3 inflammasome. Furthermore, we observed that Stifenia can induce cell death. We also tested the effect of Stifenia on Zebrafish larvae. After 24 h of exposure, Stifenia induced a dose-dependent IL-1β and TNFα gene expression. The human-cell-based approach developed in this work revealed a high sensitivity concerning inflammatory properties of a plant protection product. These tests could be routinely used to screen the potential adverse effects of this type of compounds. Finally, our results suggest a potential danger of using extensively certain PDS for crop protection. PMID:28484691

Study of the dopamine effect into cell solutions by impedance analysis

NASA Astrophysics Data System (ADS)

Paivana, G.; Apostolou, T.; Kaltsas, G.; Kintzios, S.

2017-11-01

Electrochemical Impedance Spectroscopy (EIS) has become a technique that is frequently used for biological assays. Impedance is defined as a complex - valued generalization of resistance and varies depending on its use per application field. In health sciences, bioimpedance is widely used as non-invasive and low cost alternative in many medical areas that provides valuable information about health status. This work focuses on assessing the effects of a bioactive substance applied to immobilized cells. Dopamine was used as a stimulant in order to implement impedance analysis with a specific type of cells. Dopamine constitutes one of the most important catecholamine neurotransmitters in both the mammalian central and peripheral nervous systems. The main purpose is to extract calibration curves at different frequencies with known dopamine concentrations in order to describe the behavior of cells applied to dopamine using an impedance measurement device. For comparison purposes, non-immobilized cells were tested for the same dopamine concentrations.

Hyaluronic acid as a potential boron carrier for BNCT: Preliminary evaluation.

PubMed

Zaboronok, A; Yamamoto, T; Nakai, K; Yoshida, F; Uspenskii, S; Selyanin, M; Zelenetskii, A; Matsumura, Akira

2015-12-01

Hyaluronic acid (HA), a nonimmunogenic, biocompatible polymer found in different biological tissues, has the potential to attach to CD44 receptors on the surface of certain cancer cells, where the receptor is overexpressed compared with normal cells. Boron-hyaluronic acid (BHA) was tested for its feasibility as a potential agent for BNCT. BHA with low-viscosity 30 kDa HA could be administered by intravenous injection. The compound showed a certain degree of cytotoxicity and accumulation in C6 rat glioma cells in vitro. Instability of the chelate bonds between boron and HA and/or insufficient specificity of CD44 receptors on C6 cells to BHA could account for the insufficient in vitro accumulation. To ensure the future eligibility of BHA for BNCT experiments, using alternative tumor cell lines and chemically securing the chelate bonds or synthesizing BHA with boron covalently attached to HA might be required. Copyright © 2015 Elsevier Ltd. All rights reserved.

Preparation of a Three-Dimensional Full Thickness Skin Equivalent.

PubMed

Reuter, Christian; Walles, Heike; Groeber, Florian

2017-01-01

In vitro test systems are a promising alternative to animal models. Due to the use of human cells in a three-dimensional arrangement that allows cell-cell or cell-matrix interactions these models may be more predictive for the human situation compared to animal models or two-dimensional cell culture systems. Especially for dermatological research, skin models such as epidermal or full-thickness skin equivalents (FTSE) are used for different applications. Although epidermal models provide highly standardized conditions for risk assessment, FTSE facilitate a cellular crosstalk between the dermal and epidermal layer and thus can be used as more complex models for the investigation of processes such as wound healing, skin development, or infectious diseases. In this chapter, we describe the generation and culture of an FTSE, based on a collagen type I matrix and provide troubleshooting tips for commonly encountered technical problems.

Alternative Splicing in Neurogenesis and Brain Development.

PubMed

Su, Chun-Hao; D, Dhananjaya; Tarn, Woan-Yuh

2018-01-01

Alternative splicing of precursor mRNA is an important mechanism that increases transcriptomic and proteomic diversity and also post-transcriptionally regulates mRNA levels. Alternative splicing occurs at high frequency in brain tissues and contributes to every step of nervous system development, including cell-fate decisions, neuronal migration, axon guidance, and synaptogenesis. Genetic manipulation and RNA sequencing have provided insights into the molecular mechanisms underlying the effects of alternative splicing in stem cell self-renewal and neuronal fate specification. Timely expression and perhaps post-translational modification of neuron-specific splicing regulators play important roles in neuronal development. Alternative splicing of many key transcription regulators or epigenetic factors reprograms the transcriptome and hence contributes to stem cell fate determination. During neuronal differentiation, alternative splicing also modulates signaling activity, centriolar dynamics, and metabolic pathways. Moreover, alternative splicing impacts cortical lamination and neuronal development and function. In this review, we focus on recent progress toward understanding the contributions of alternative splicing to neurogenesis and brain development, which has shed light on how splicing defects may cause brain disorders and diseases.

Effect of 3-Bromopyruvate and Atovaquone on Infection during In Vitro Interaction of Toxoplasma gondii and LLC-MK2 Cells

PubMed Central

de Lima, Loyze Paola O.; Seabra, Sergio H.; Carneiro, Henrique

2015-01-01

Toxoplasma gondii infection can be severe during pregnancy and in immunocompromised patients. Current therapies for toxoplasmosis are restricted to tachyzoites and have little or no effect on bradyzoites, which are maintained in tissue cysts. Consequently, new therapeutic alternatives have been proposed as the use of atovaquone has demonstrated partial efficacy against tachyzoites and bradyzoites. This work studies the effect of 3-bromopyruvate (3-BrPA), a compound that is being tested against cancer cells, on the infection of LLC-MK2 cells with T. gondii tachyzoites, RH strain. No effect of 3-BrPA on host cell proliferation or viability was observed, but it inhibited the proliferation of T. gondii. The incubation of cultures with lectin Dolichos biflorus agglutinin (DBA) showed the development of cystogenesis, and an ultrastructural analysis of parasite intracellular development confirmed morphological characteristics commonly found in tissue cysts. Moreover, the presence of degraded parasites and the influence of 3-BrPA on endodyogeny were observed. Infected cultures were alternatively treated with a combination of this compound plus atovaquone. This resulted in a 73% reduction in intracellular parasites after 24 h of treatment and a 71% reduction after 48 h; cyst wall formation did not occur in these cultures. Therefore, we conclude that the use of 3-BrPA may serve as an important tool for the study of (i) in vitro cystogenesis; (ii) parasite metabolism, requiring a deeper understanding of the target of action of this compound on T. gondii; (iii) the alternative parasite metabolic pathways; and (iv) the molecular/cellular mechanisms that trigger parasite death. PMID:26077255

Effect of 3-bromopyruvate and atovaquone on infection during in vitro interaction of Toxoplasma gondii and LLC-MK2 cells.

PubMed

de Lima, Loyze Paola O; Seabra, Sergio H; Carneiro, Henrique; Barbosa, Helene S

2015-09-01

Toxoplasma gondii infection can be severe during pregnancy and in immunocompromised patients. Current therapies for toxoplasmosis are restricted to tachyzoites and have little or no effect on bradyzoites, which are maintained in tissue cysts. Consequently, new therapeutic alternatives have been proposed as the use of atovaquone has demonstrated partial efficacy against tachyzoites and bradyzoites. This work studies the effect of 3-bromopyruvate (3-BrPA), a compound that is being tested against cancer cells, on the infection of LLC-MK2 cells with T. gondii tachyzoites, RH strain. No effect of 3-BrPA on host cell proliferation or viability was observed, but it inhibited the proliferation of T. gondii. The incubation of cultures with lectin Dolichos biflorus agglutinin (DBA) showed the development of cystogenesis, and an ultrastructural analysis of parasite intracellular development confirmed morphological characteristics commonly found in tissue cysts. Moreover, the presence of degraded parasites and the influence of 3-BrPA on endodyogeny were observed. Infected cultures were alternatively treated with a combination of this compound plus atovaquone. This resulted in a 73% reduction in intracellular parasites after 24 h of treatment and a 71% reduction after 48 h; cyst wall formation did not occur in these cultures. Therefore, we conclude that the use of 3-BrPA may serve as an important tool for the study of (i) in vitro cystogenesis; (ii) parasite metabolism, requiring a deeper understanding of the target of action of this compound on T. gondii; (iii) the alternative parasite metabolic pathways; and (iv) the molecular/cellular mechanisms that trigger parasite death. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

RNA-Seq of Arabidopsis Pollen Uncovers Novel Transcription and Alternative Splicing1[C][W][OA

PubMed Central

Loraine, Ann E.; McCormick, Sheila; Estrada, April; Patel, Ketan; Qin, Peng

2013-01-01

Pollen grains of Arabidopsis (Arabidopsis thaliana) contain two haploid sperm cells enclosed in a haploid vegetative cell. Upon germination, the vegetative cell extrudes a pollen tube that carries the sperm to an ovule for fertilization. Knowing the identity, relative abundance, and splicing patterns of pollen transcripts will improve our understanding of pollen and allow investigation of tissue-specific splicing in plants. Most Arabidopsis pollen transcriptome studies have used the ATH1 microarray, which does not assay splice variants and lacks specific probe sets for many genes. To investigate the pollen transcriptome, we performed high-throughput sequencing (RNA-Seq) of Arabidopsis pollen and seedlings for comparison. Gene expression was more diverse in seedling, and genes involved in cell wall biogenesis were highly expressed in pollen. RNA-Seq detected at least 4,172 protein-coding genes expressed in pollen, including 289 assayed only by nonspecific probe sets. Additional exons and previously unannotated 5′ and 3′ untranslated regions for pollen-expressed genes were revealed. We detected regions in the genome not previously annotated as expressed; 14 were tested and 12 were confirmed by polymerase chain reaction. Gapped read alignments revealed 1,908 high-confidence new splicing events supported by 10 or more spliced read alignments. Alternative splicing patterns in pollen and seedling were highly correlated. For most alternatively spliced genes, the ratio of variants in pollen and seedling was similar, except for some encoding proteins involved in RNA splicing. This study highlights the robustness of splicing patterns in plants and the importance of ongoing annotation and visualization of RNA-Seq data using interactive tools such as Integrated Genome Browser. PMID:23590974

Prognostic significance of equivocal human epidermal growth factor receptor 2 results and clinical utility of alternative chromosome 17 genes in patients with invasive breast cancer: A cohort study.

PubMed

Sneige, Nour; Hess, Kenneth R; Multani, Asha S; Gong, Yun; Ibrahim, Nuhad K

2017-04-01

The 2013 testing guidelines for determining the human epidermal growth factor receptor 2 (HER2) status include new cutoff points for the HER2/chromosome enumeration probe 17 (CEP17) ratio and the average HER2 copy number per cell, and they recommend using a reflex test with alternative chromosome 17 probes (Ch17Ps) to resolve equivocal HER2 results. This study sought to determine the clinical utility of alternative Ch17Ps in equivocal cases and the effects of equivocal results and/or a change in the HER2 status on patients' outcomes. The University of Texas MD Anderson Cancer Center database of HER2 dual-probe fluorescence in situ hybridization results from 2000 to 2010 was searched for cases of invasive breast cancer with HER2/CEP17 ratios < 2 and average HER2 copy numbers < 6 per cell. Cases with HER2 copy numbers of 4 to < 6 (the definition of equivocal HER2 results) were analyzed with alternative Ch17Ps for Smith-Magenis syndrome and retinoic acid receptor α genes. Disease-free survival (DFS) and overall survival (OS) were evaluated with respect to the HER2 copy number with multivariate Cox proportional hazards regression. Among the 3630 patients meeting the inclusion criteria, 137 (4%) had equivocal HER2 results. With alternative Ch17Ps, 35 of 57 equivocal HER2 cases (61%) were upgraded to a positive HER2 status, and 22 cases (39%) remained unchanged. The 5-year DFS and OS adjusted hazard ratios (HRs) for copy numbers of 4 to < 6 versus < 4 were 0.6 (95% confidence interval [CI], 0.3-1.2) and 0.5 (95% CI, 0.2-1.0) with P values of .16 and .66, respectively. In comparison with HER2-negative cases, these CIs indicated that equivocal HER2 results were associated with either a protective effect (HR, < 0.5) or no effect (HR, 1.0). These findings rule out a significant deleterious effect of equivocal HER2 results. Alternative Ch17Ps may erroneously upgrade the HER2 status; therefore, they cannot be considered reliable in clinical practice. Cancer 2017;123:1115-1123. © 2016 American Cancer Society. © 2016 American Cancer Society.

Molecular Viability Testing of UV-Inactivated Bacteria.

PubMed

Weigel, Kris M; Nguyen, Felicia K; Kearney, Moira R; Meschke, John S; Cangelosi, Gerard A

2017-05-15

PCR is effective in detecting bacterial DNA in samples, but it is unable to differentiate viable bacteria from inactivated cells or free DNA fragments. New PCR-based analytical strategies have been developed to address this limitation. Molecular viability testing (MVT) correlates bacterial viability with the ability to rapidly synthesize species-specific rRNA precursors (pre-rRNA) in response to brief nutritional stimulation. Previous studies demonstrated that MVT can assess bacterial inactivation by chlorine, serum, and low-temperature pasteurization. Here, we demonstrate that MVT can detect inactivation of Escherichia coli , Aeromonas hydrophila , and Enterococcus faecalis cells by UV irradiation. Some UV-inactivated E. coli cells transiently retained the ability to synthesize pre-rRNA postirradiation (generating false-positive MVT results), but this activity ceased within 1 h following UV exposure. Viable but transiently undetectable (by culture) E. coli cells were consistently detected by MVT. An alternative viability testing method, viability PCR (vPCR), correlates viability with cell envelope integrity. This method did not distinguish viable bacteria from UV-inactivated bacteria under some conditions, indicating that the inactivated cells retained intact cell envelopes. MVT holds promise as a means to rapidly assess microbial inactivation by UV treatment. IMPORTANCE UV irradiation is increasingly being used to disinfect water, food, and other materials for human use. Confirming the effectiveness of UV disinfection remains a challenging task. In particular, microbiological methods that rely on rapid detection of microbial DNA can yield misleading results, due to the detection of remnant DNA associated with dead microbial cells. This report describes a novel method that rapidly distinguishes living microbial cells from dead microbial cells after UV disinfection. Copyright © 2017 American Society for Microbiology.

Optimizing multiple-choice tests as tools for learning.

PubMed

Little, Jeri L; Bjork, Elizabeth Ligon

2015-01-01

Answering multiple-choice questions with competitive alternatives can enhance performance on a later test, not only on questions about the information previously tested, but also on questions about related information not previously tested-in particular, on questions about information pertaining to the previously incorrect alternatives. In the present research, we assessed a possible explanation for this pattern: When multiple-choice questions contain competitive incorrect alternatives, test-takers are led to retrieve previously studied information pertaining to all of the alternatives in order to discriminate among them and select an answer, with such processing strengthening later access to information associated with both the correct and incorrect alternatives. Supporting this hypothesis, we found enhanced performance on a later cued-recall test for previously nontested questions when their answers had previously appeared as competitive incorrect alternatives in the initial multiple-choice test, but not when they had previously appeared as noncompetitive alternatives. Importantly, however, competitive alternatives were not more likely than noncompetitive alternatives to be intruded as incorrect responses, indicating that a general increased accessibility for previously presented incorrect alternatives could not be the explanation for these results. The present findings, replicated across two experiments (one in which corrective feedback was provided during the initial multiple-choice testing, and one in which it was not), thus strongly suggest that competitive multiple-choice questions can trigger beneficial retrieval processes for both tested and related information, and the results have implications for the effective use of multiple-choice tests as tools for learning.

In vitro cell culture, platelet adhesion tests and in vivo implant tests of plasma-polymerized para-xylene films

NASA Astrophysics Data System (ADS)

Chou, Chia-Man; Yeh, Chou-Ming; Chung, Chi-Jen; He, Ju-Liang

2013-09-01

Plasma-polymerized para-xylene (PPX) was developed in a previous study by adjusting the process parameters: pulse frequency of the power supply (ωp) and para-xylene monomer flow rate (fp). All the obtained PPX films exhibit an amorphous structure and present hydrophobicity (water contact angle ranging from 98.5° to 121.1°), higher film growth rate and good fibroblast cell proliferation. In this study, in vitro tests (fibroblast cell compatibility and platelet adhesion) and an in vivo animal study were performed by using PPX deposited industrial-grade silicone sheets (IGS) and compared with medical-grade silicone ones (MS), which were commonly manufactured into catheters or drainage tubes in clinical use. The results reveal that PPX deposited at high ωp or high fp, in comparison with MS, exhibit better cell proliferation and clearly shows less cell adhesion regardless of ωp and fp. PPX also exhibit a comparatively lower level of platelet adhesion than MS. In the animal study, PPX-coated IGS result in similar local tissue responses at 3, 7 and 28 days (short-term) and 84 days (long-term) after subcutaneous implantation the abdominal wall of rodents compared with respective responses to MS. These results suggest that PPX-coated industrial-grade silicone is one alternative to high cost medical-grade silicone.

Toxicity study in blood and tumor cells of laser produced medicines for application in fabrics.

PubMed

Morán, M Carmen; Tozar, Tatiana; Simon, Agota; Dinache, Andra; Smarandache, Adriana; Andrei, Ionut Relu; Boni, Mihai; Pascu, Mihail Lucian; Cirisano, Francesca; Ferrari, Michele

2016-01-01

Phenothiazine derivatives are non-antibiotics with antimicrobial, fungistatic and fungicidal effects. We exposed to a high energy UV laser beam phenothiazines solutions in water at 20mg/mL concentration to increase antibacterial activity of resulting mixtures. Compared to previous results obtained on bacteria, more research is needed about UV laser irradiated phenothiazines applications on cancer cell cultures to evidence possible anticancerous properties. Evaluation of the safety of the newly obtained photoproducts in view of use on humans is also needed. Due to expensive animal testing in toxicology and pressure from general public and governments to develop alternatives to in vivo testing, in vitro cell-based models are attractive for preliminary testing of new materials. Cytotoxicity screening reported here shows that laser irradiated (4h exposure time length) chlorpromazine and promazine are more efficient against some cell cultures. Interaction of laser irradiated phenothiazines with fabrics show that promethazine and chlorpromazine have improved wetting properties. Correlation of these two groups of properties shows that chlorpromazine appears to be more recommended for applications on tissues using fabrics as transport vectors. The reported results concern stability study of phenothiazines water solutions to know the time limits within which they are stable and may be used. Copyright © 2015 Elsevier B.V. All rights reserved.

Experience with local lymph node assay performance standards using standard radioactivity and nonradioactive cell count measurements.

PubMed

Basketter, David; Kolle, Susanne N; Schrage, Arnhild; Honarvar, Naveed; Gamer, Armin O; van Ravenzwaay, Bennard; Landsiedel, Robert

2012-08-01

The local lymph node assay (LLNA) is the preferred test for identification of skin-sensitizing substances by measuring radioactive thymidine incorporation into the lymph node. To facilitate acceptance of nonradioactive variants, validation authorities have published harmonized minimum performance standards (PS) that the alternative endpoint assay must meet. In the present work, these standards were applied to a variant of the LLNA based on lymph node cell counts (LNCC) run in parallel as a control with the standard LLNA with radioactivity measurements, with threshold concentrations (EC3) being determined for the sensitizers. Of the 22 PS chemicals tested in this study, 21 yielded the same results from standard radioactivity and cell count measurements; only 2-mercaptobenzothiazole was positive by LLNA but negative by LNCC. Of the 16 PS positives, 15 were positive by LLNA and 14 by LNCC; methylmethacrylate was not identified as sensitizer by either of the measurements. Two of the six PS negatives tested negative in our study by both LLNA and LNCC. Of the four PS negatives which were positive in our study, chlorobenzene and methyl salicylate were tested at higher concentrations than the published PS, whereas the corresponding concentrations resulted in consistent negative results. Methylmethacrylate and nickel chloride tested positive within the concentration range used for the published PS. The results indicate cell counts and radioactive measurements are in good accordance within the same LLNA using the 22 PS test substances. Comparisons with the published PS results may, however, require balanced analysis rather than a simple checklist approach. Copyright © 2011 John Wiley & Sons, Ltd.

Modifications to the algal growth inhibition test for use as a regulatory assay

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

Geis, S.W.; Fleming, K.L.; Korthals, E.T.

2000-01-01

Biological assays using aquatic invertebrates and fish do not necessarily predict protection levels for primary producers such as algae and aquatic macrophytes. State regulatory programs may not be protecting the environment from many phytotoxic compounds. Recent modifications of the US Environmental Protection Agency's algal test were evaluated for their potential use as a regulatory assay. Primary goals of this investigation were to downsize the algal assay and to evaluate various methods of automation. Disposable microplates with 2-ml sample wells were evaluated as an alternative testing chamber for the 96-h growth inhibition test with Raphidocelis subcapitata (formerly known as Selenastrum capricornutum).more » The authors compared the standardized Erlenmeyer {reg_sign} flask test to the microplate test using CuCl{sub 2}, NaCl, phenol, ZnCl{sub 2}, and a surfactant. They noted improved control performance with the microplate test, whereas median inhibitory concentration values were similar for both methods. Other procedures they addressed included the use of EDTA, filtration of samples, and the effect of colored samples on algal growth. They also evaluated growth estimates by comparing manual cell counting to more automated growth estimates using fluorescence and absorbance endpoints. The use of fluorescence and absorbance measurements demonstrated reductions in replicate variability over manual counting and may offer time-saving alternatives for laboratory analysts.« less

Predictive performance and inter-laboratory reproducibility in assessing eye irritation potential of water- and oil-soluble mixtures using the Short Time Exposure test method.

PubMed

Abo, Takayuki; Hilberer, Allison; Behle-Wagner, Christine; Watanabe, Mika; Cameron, David; Kirst, Annette; Nukada, Yuko; Yuki, Takuo; Araki, Daisuke; Sakaguchi, Hitoshi; Itagaki, Hiroshi

2018-04-01

The Short Time Exposure (STE) test method is an alternative method for assessing eye irritation potential using Statens Seruminstitut Rabbit Cornea cells and has been adopted as test guideline 491 by the Organisation for Economic Co-operation and Development. Its good predictive performance in identifying the Globally Harmonized System (GHS) No Category (NC) or Irritant Category has been demonstrated in evaluations of water-soluble substances, oil-soluble substances, and water-soluble mixtures. However, the predictive performance for oil-soluble mixtures was not evaluated. Twenty-four oil-soluble mixtures were evaluated using the STE test method. The GHS NC or Irritant Category of 22 oil-soluble mixtures were consistent with that of a Reconstructed human Cornea-like Epithelium (RhCE) test method. Inter-laboratory reproducibility was then confirmed using 20 water- and oil-soluble mixtures blind-coded. The concordance in GHS NC or Irritant Category among four laboratories was 90%-100%. In conclusion, the concordance in comparison with the results of RhCE test method using 24 oil-soluble mixtures and inter-laboratory reproducibility using 20 water- and oil-soluble mixtures blind-coded were good, indicating that the STE test method is a suitable alternative for predicting the eye irritation potential of both substances and mixtures. Copyright © 2018 Elsevier Ltd. All rights reserved.

Immunohistochemistry and alternative FISH testing in breast cancer with HER2 equivocal amplification.

PubMed

Agersborg, Sally; Mixon, Christopher; Nguyen, Thanh; Aithal, Sramila; Sudarsanam, Sucha; Blocker, Forrest; Weiss, Lawrence; Gasparini, Robert; Jiang, Shiping; Chen, Wayne; Hess, Gregory; Albitar, Maher

2018-03-22

While HER2 testing is well established in directing appropriate treatment for breast cancer, a small percentage of cases show equivocal results by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Alternative probes may be used in equivocal cases. We present a single community-based institution's experience in further evaluating these cases. Between 2014 and 2016, 4255 samples were submitted for HER2 amplification testing by alternative probes, TP53, RAI1, and RARA. Of the patients tested by FISH, 505/3908 (12.9%) also had IHC data. Most (73.9%) FISH equivocal cases remained equivocal after IHC testing. However, 50.5% of equivocal cases were classified as HER2 amplified by alternative probes. Most cases were positive by more than one probe: 78% of positive cases by RAI1 and 73.9% by TP53. There was a significant difference between IHC and FISH alternative testing (p < 0.0001) among the equivocal cases by conventional FISH testing, 44% of IHC negative cases became positive while 36% of the positive IHC cases became negative by alternative FISH testing. Available data showed that 41% of patients were treated with palbociclib and were positive by alternative FISH. The prevalence of double HER2 equivocal cases and the discrepancy between IHC and alternative FISH testing suggest that FISH alternative testing using both RAI1 and TP53 probes is necessary for conclusive classification. Because almost half of FISH equivocal cases converted to HER2 amplified upon alternative testing, clinical studies to determine the benefit of anti-HER2 therapy in these patients are urgently needed.

Classical and alternative activation of rat hepatic sinusoidal endothelial cells by inflammatory stimuli.

PubMed

Liu, Yinglin; Gardner, Carol R; Laskin, Jeffrey D; Laskin, Debra L

2013-02-01

The ability of rat hepatic sinusoidal endothelial cells (HSEC) to become activated in response to diverse inflammatory stimuli was analyzed. Whereas the classical macrophage activators, IFNγ and/or LPS upregulated expression of iNOS in HSEC, the alternative macrophage activators, IL-10 or IL-4+IL-13 upregulated arginase-1 and mannose receptor. Similar upregulation of iNOS and arginase-1 was observed in classically and alternatively activated Kupffer cells, respectively. Removal of inducing stimuli from the cells had no effect on expression of these markers, demonstrating that activation is persistent. Washing and incubation of IFNγ treated cells with IL-4+IL-13 resulted in decreased iNOS and increased arginase-1 expression, while washing and incubation of IL-4+IL-13 treated cells with IFNγ resulted in decreased arginase-1 and increased iNOS, indicating that classical and alternative activation of the cells is reversible. HSEC were more sensitive to phenotypic switching than Kupffer cells, suggesting greater functional plasticity. Hepatocyte viability and expression of PCNA, β-catenin and MMP-9 increased in the presence of alternatively activated HSEC. In contrast, the viability of hepatocytes pretreated for 2 h with 5 mM acetaminophen decreased in the presence of classically activated HSEC. These data demonstrate that activated HSEC can modulate hepatocyte responses following injury. The ability of hepatocytes to activate HSEC was also investigated. Co-culture of HSEC with acetaminophen-injured hepatocytes, but not control hepatocytes, increased the sensitivity of HSEC to classical and alternative activating stimuli. The capacity of HSEC to respond to phenotypic activators may represent an important mechanism by which they participate in inflammatory responses associated with hepatotoxicity. Copyright © 2012 Elsevier Inc. All rights reserved.

Genome-wide analysis of alternative splicing during dendritic cell response to a bacterial challenge.

PubMed

Rodrigues, Raquel; Grosso, Ana Rita; Moita, Luís

2013-01-01

The immune system relies on the plasticity of its components to produce appropriate responses to frequent environmental challenges. Dendritic cells (DCs) are critical initiators of innate immunity and orchestrate the later and more specific adaptive immunity. The generation of diversity in transcriptional programs is central for effective immune responses. Alternative splicing is widely considered a key generator of transcriptional and proteomic complexity, but its role has been rarely addressed systematically in immune cells. Here we used splicing-sensitive arrays to assess genome-wide gene- and exon-level expression profiles in human DCs in response to a bacterial challenge. We find widespread alternative splicing events and splicing factor transcriptional signatures induced by an E. coli challenge to human DCs. Alternative splicing acts in concert with transcriptional modulation, but these two mechanisms of gene regulation affect primarily distinct functional gene groups. Alternative splicing is likely to have an important role in DC immunobiology because it affects genes known to be involved in DC development, endocytosis, antigen presentation and cell cycle arrest.

Engineering an in vitro air-blood barrier by 3D bioprinting

PubMed Central

Horváth, Lenke; Umehara, Yuki; Jud, Corinne; Blank, Fabian; Petri-Fink, Alke; Rothen-Rutishauser, Barbara

2015-01-01

Intensive efforts in recent years to develop and commercialize in vitro alternatives in the field of risk assessment have yielded new promising two- and three dimensional (3D) cell culture models. Nevertheless, a realistic 3D in vitro alveolar model is not available yet. Here we report on the biofabrication of the human air-blood tissue barrier analogue composed of an endothelial cell, basement membrane and epithelial cell layer by using a bioprinting technology. In contrary to the manual method, we demonstrate that this technique enables automatized and reproducible creation of thinner and more homogeneous cell layers, which is required for an optimal air-blood tissue barrier. This bioprinting platform will offer an excellent tool to engineer an advanced 3D lung model for high-throughput screening for safety assessment and drug efficacy testing. PMID:25609567

Direct and Dry Deposited Single-Walled Carbon Nanotube Films Doped with MoO(x) as Electron-Blocking Transparent Electrodes for Flexible Organic Solar Cells.

PubMed

Jeon, Il; Cui, Kehang; Chiba, Takaaki; Anisimov, Anton; Nasibulin, Albert G; Kauppinen, Esko I; Maruyama, Shigeo; Matsuo, Yutaka

2015-07-01

Organic solar cells have been regarded as a promising electrical energy source. Transparent and conductive carbon nanotube film offers an alternative to commonly used ITO in photovoltaics with superior flexibility. This communication reports carbon nanotube-based indium-free organic solar cells and their flexible application. Direct and dry deposited carbon nanotube film doped with MoO(x) functions as an electron-blocking transparent electrode, and its performance is enhanced further by overcoating with PSS. The single-walled carbon nanotube organic solar cell in this work shows a power conversion efficiency of 6.04%. This value is 83% of the leading ITO-based device performance (7.48%). Flexible application shows 3.91% efficiency and is capable of withstanding a severe cyclic flex test.

Radiation-induced alternative transcripts as detected in total and polysome-bound mRNA.

PubMed

Wahba, Amy; Ryan, Michael C; Shankavaram, Uma T; Camphausen, Kevin; Tofilon, Philip J

2018-01-02

Alternative splicing is a critical event in the posttranscriptional regulation of gene expression. To investigate whether this process influences radiation-induced gene expression we defined the effects of ionizing radiation on the generation of alternative transcripts in total cellular mRNA (the transcriptome) and polysome-bound mRNA (the translatome) of the human glioblastoma stem-like cell line NSC11. For these studies, RNA-Seq profiles from control and irradiated cells were compared using the program SpliceSeq to identify transcripts and splice variations induced by radiation. As compared to the transcriptome (total RNA) of untreated cells, the radiation-induced transcriptome contained 92 splice events suggesting that radiation induced alternative splicing. As compared to the translatome (polysome-bound RNA) of untreated cells, the radiation-induced translatome contained 280 splice events of which only 24 were overlapping with the radiation-induced transcriptome. These results suggest that radiation not only modifies alternative splicing of precursor mRNA, but also results in the selective association of existing mRNA isoforms with polysomes. Comparison of radiation-induced alternative transcripts to radiation-induced gene expression in total RNA revealed little overlap (about 3%). In contrast, in the radiation-induced translatome, about 38% of the induced alternative transcripts corresponded to genes whose expression level was affected in the translatome. This study suggests that whereas radiation induces alternate splicing, the alternative transcripts present at the time of irradiation may play a role in the radiation-induced translational control of gene expression and thus cellular radioresponse.

Bringing fuel cell vehicles to market : scenarios and challenges with fuel alternatives : consultant study report [version 1.1

DOT National Transportation Integrated Search

2001-10-01

The California Fuel Cell Partnership (CaFCP) commissioned a study on bringing fuel cell vehicles to market and the scenarios and challenges with fuel alternatives. The study's overall goal was to identify challenges and solutions for four different f...

Upgrading cytochrome P450 activity in HepG2 cells co-transfected with adenoviral vectors for drug hepatotoxicity assessment.

PubMed

Tolosa, Laia; Donato, M Teresa; Pérez-Cataldo, Gabriela; Castell, José Vicente; Gómez-Lechón, M José

2012-12-01

In a number of adverse drug reactions leading to hepatotoxicity, drug metabolism is thought to be involved by the generation of reactive metabolites from non-toxic drugs. The use of hepatoma cell lines, such as HepG2 cell line, for the evaluation of drug-induced hepatotoxicity is hampered by their low cytochrome P450 expression which makes impossible the study of the toxicity produced by bioactivable compounds. Genetically manipulated cells constitute promising tools for hepatotoxicity applications. HepG2 cells were simultaneously transfected with recombinant adenoviruses encoding CYP1A2, CYP2C9 and CYP3A4 to confer them drug-metabolic competence. Upgraded cells (Adv-HepG2) were highly able to metabolize the toxin studied in contrast to the reduced metabolic capacity of HepG2 cells. Aflatoxin B1-induced hepatotoxicity was studied as a proof of concept in metabolically competent and non-competent HepG2 cells by using high content screening technology. Significant differences in mitochondrial membrane potential, intracellular calcium concentration, nuclear morphology and cell viability after treatment with aflatoxin B1 were observed in Adv-HepG2 when compared to HepG2 cells. Rotenone (non bioactivable) and citrate (non hepatotoxic) were analysed as negative controls. This cell model showed to be a suitable hepatic model to test hepatotoxicity of bioactivable drugs and constitutes a valuable alternative for hepatotoxicity testing. Copyright © 2011 Elsevier Ltd. All rights reserved.

Pulsed magnetic field enhances therapeutic efficiency of mesenchymal stem cells in chronic neuropathic pain model.

PubMed

Mert, Tufan; Kurt, Akif Hakan; Altun, İdiris; Celik, Ahmet; Baran, Furkan; Gunay, Ismail

2017-05-01

Cell-based or magnetic field therapies as alternative approaches to pain management have been tested in several experimental pain models. The aim of this study therefore was to investigate the actions of the cell-based therapy (adipose tissue derived mesenchymal stem cells; ADMSC) or pulsed magnetic field (PMF) therapy and magneto-cell therapy (combination of ADMSC and PMF) in chronic constriction nerve injury model (CCI). The actions of individual ADMSC (route dependent [systemic or local], time-dependent [a day or a week after surgery]), or PMF and their combination (magneto-cell) therapies on hyperalgesia and allodynia were investigated by using thermal plantar test and a dynamic plantar aesthesiometer, respectively. In addition, various cytokine levels (IL-1β, IL-6, and IL-10) of rat sciatic nerve after CCI were analyzed. Following the CCI, both latency and threshold significantly decreased. ADMSC or PMF significantly increased latencies and thresholds. The combination of ADMSC with PMF even more significantly increased latency and threshold when compared with ADMSC alone. However, ADMSC-induced decrease in pro-inflammatory or increase in anti-inflammatory cytokines levels were partially prevented by PMF treatments. Present findings may suggest that both cell-based and magnetic therapies can effectively attenuate chronic neuropathic pain symptoms. Combined magneto-cell therapy may also efficiently reverse neuropathic signs. Bioelectromagnetics. 38:255-264, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

PSMA-targeted bispecific Fab conjugates that engage T cells.

PubMed

Patterson, James T; Isaacson, Jason; Kerwin, Lisa; Atassi, Ghazi; Duggal, Rohit; Bresson, Damien; Zhu, Tong; Zhou, Heyue; Fu, Yanwen; Kaufmann, Gunnar F

2017-12-15

Bioconjugate formats provide alternative strategies for antigen targeting with bispecific antibodies. Here, PSMA-targeted Fab conjugates were generated using different bispecific formats. Interchain disulfide bridging of an αCD3 Fab enabled installation of either the PSMA-targeting small molecule DUPA (SynFab) or the attachment of an αPSMA Fab (BisFab) by covalent linkage. Optimization of the reducing conditions was critical for selective interchain disulfide reduction and good bioconjugate yield. Activity of αPSMA/CD3 Fab conjugates was tested by in vitro cytotoxicity assays using prostate cancer cell lines. Both bispecific formats demonstrated excellent potency and antigen selectivity. Copyright © 2017. Published by Elsevier Ltd.

Improved multistage wide band laser frequency stabilization

NASA Astrophysics Data System (ADS)

Kawamura, Seiji; Abramovici, Alex; Zucker, Michael E.

1997-01-01

Suppression of laser frequency fluctuations is an essential technology for planned interferometric detectors for astrophysical gravitational waves. Because of the low degree of residual frequency noise which is ultimately required, control topologies comprising two or more cascaded loops are favored. One such topology, used in the Laser Interferometer Gravitational-Wave Observatory 40 m interferometer, relied on electro-optic Pockels cell phase correction as a fast actuator for the final stage. This actuation method proved susceptible to spurious amplitude modulation effects, which provided an unintended parasitic feedback path. An alternate arrangement, which achieves comparably effective frequency stabilization without using a phase correcting Pockels cell, was introduced and successfully tested.

Use of solar cell in electrokinetic remediation of cadmium-contaminated soil.

PubMed

Yuan, Songhu; Zheng, Zhonghua; Chen, Jing; Lu, Xiaohua

2009-03-15

This preliminary study used a solar cell, instead of direct current (DC) power supply, to generate electric field for electrokinetic (EK) remediation of cadmium-contaminated soil. Three EK tests were conducted and compared; one was conducted on a cloudy and rainy day with solar cell, one was conducted on a sunny day with solar cell and another was conducted periodically with DC power supply. It was found that the output potential of solar cell depended on daytime and was influenced by weather conditions; the applied potential in soil was affected by the output potential and weather conditions, and the current achieved by solar cell was comparable with that achieved by DC power supply. Solar cell could be used to drive the electromigration of cadmium in contaminated soil, and removal efficiency achieved by solar cell was comparable with that achieved by DC power supply. Compared with traditional DC power supply, using solar cell as power supply for EK remediation can greatly reduce energy expenditure. This study provided an alternative to improve the EK soil remediation and expanded the use of solar cell in environmental remediation.

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.

The initiation of lateral roots in the primary roots of maize (Zea mays L.) implies a reactivation of cell proliferation in a group of founder pericycle cells.

PubMed

Alarcón, M Victoria; Lloret, Pedro G; Martín-Partido, Gervasio; Salguero, Julio

2016-03-15

The initiation of lateral roots (LRs) has generally been viewed as a reactivation of proliferative activity in pericycle cells that are committed to initiate primordia. However, it is also possible that pericycle founder cells that initiate LRs never cease proliferative activity but rather are displaced to the most distal root zones while undertaking successive stages of LR initiation. In this study, we tested these two alternative hypotheses by examining the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into the DNA of meristematic root cells of Zea mays. According to the values for the length of the cell cycle and values for cell displacement along the maize root, our results strongly suggest that pericycle cells that initiate LR primordia ceased proliferative activity upon exiting the meristematic zone. This finding is supported by the existence of a root zone between 4 and 20mm from the root cap junction, in which neither mitotic cells nor labelled nuclei were observed in phloem pericycle cells. Copyright © 2016 Elsevier GmbH. All rights reserved.

Personalized medicine and pharmacogenetic biomarkers: progress in molecular oncology testing

PubMed Central

Ong, Frank S; Das, Kingshuk; Wang, Jay; Vakil, Hana; Kuo, Jane Z; Blackwell, Wendell-Lamar B; Lim, Stephen W; Goodarzi, Mark O; Bernstein, Kenneth E; Rotter, Jerome I; Grody, Wayne W

2012-01-01

In the field of oncology, clinical molecular diagnostics and biomarker discoveries are constantly advancing as the intricate molecular mechanisms that transform a normal cell into an aberrant state in concert with the dysregulation of alternative complementary pathways are increasingly understood. Progress in biomarker technology, coupled with the companion clinical diagnostic laboratory tests, continue to advance this field, where individualized and customized treatment appropriate for each individual patient define the standard of care. Here, we discuss the current commonly used predictive pharmacogenetic biomarkers in clinical oncology molecular testing: BRAF V600E for vemurafenib in melanoma; EML4–ALK for crizotinib and EGFR for erlotinib and gefitinib in non-small-cell lung cancer; KRAS against the use of cetuximab and panitumumab in colorectal cancer; ERBB2 (HER2/neu) for trastuzumab in breast cancer; BCR–ABL for tyrosine kinase inhibitors in chronic myeloid leukemia; and PML/RARα for all-trans-retinoic acid and arsenic trioxide treatment for acute promyelocytic leukemia. PMID:22845480

The role of in vitro methods as alternatives to animals in toxicity testing.

PubMed

Anadón, Arturo; Martínez, María Aranzazu; Castellano, Victor; Martínez-Larrañaga, María Rosa

2014-01-01

It is accepted that animal testing should be reduced, refined or replaced as far as it is practicably possible. There are also a wide variety of in vitro models, which are used as screening studies and mechanistic investigations. The ability of an in vitro assay to be reliable, biomedically, is essential in pharmaceutical development. Furthermore, it is necessary that cells used in in vitro testing mimic the phenotype of cells within the human target tissue. The focus of this review article is to identify the key points of in vitro assays. In doing so, the authors take into account the chemical agents that are assessed and the integrated in vitro testing strategies. There is a transfer of toxicological data from primary in vivo animal studies to in vitro assays. The key element for designing an integrated in vitro testing strategy is summarized as follows: exposure modeling of chemical agents for in vitro testing; data gathering, sharing and read-across for testing a class of chemical; a battery of tests to assemble a broad spectrum of data on different mechanisms of action to predict toxic effects; and applicability of the test and the integrated in vitro testing strategies and flexibility to adjust the integrated in vitro testing strategies to test substance. While these methods will be invaluable if effective, more studies must be done to ensure reliability and suitability of these tests for humans.

Evaluation of Two Statistical Methods Provides Insights into the Complex Patterns of Alternative Polyadenylation Site Switching

PubMed Central

Li, Jie; Li, Rui; You, Leiming; Xu, Anlong; Fu, Yonggui; Huang, Shengfeng

2015-01-01

Switching between different alternative polyadenylation (APA) sites plays an important role in the fine tuning of gene expression. New technologies for the execution of 3’-end enriched RNA-seq allow genome-wide detection of the genes that exhibit significant APA site switching between different samples. Here, we show that the independence test gives better results than the linear trend test in detecting APA site-switching events. Further examination suggests that the discrepancy between these two statistical methods arises from complex APA site-switching events that cannot be represented by a simple change of average 3’-UTR length. In theory, the linear trend test is only effective in detecting these simple changes. We classify the switching events into four switching patterns: two simple patterns (3’-UTR shortening and lengthening) and two complex patterns. By comparing the results of the two statistical methods, we show that complex patterns account for 1/4 of all observed switching events that happen between normal and cancerous human breast cell lines. Because simple and complex switching patterns may convey different biological meanings, they merit separate study. We therefore propose to combine both the independence test and the linear trend test in practice. First, the independence test should be used to detect APA site switching; second, the linear trend test should be invoked to identify simple switching events; and third, those complex switching events that pass independence testing but fail linear trend testing can be identified. PMID:25875641

Essiac? and Flor-Essence? herbal tonics stimulate the in vitro growth of human breast cancer cells

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

Kulp, K S; Montgomery, J L; McLimans, B

People diagnosed with cancer often self-administer complementary and alternative medicines (CAMs) to supplement their conventional treatments, improve health, or prevent recurrence. Flor-Essence{reg_sign} and Essiac{reg_sign} Herbal Tonics are commercially available complex mixtures of herbal extracts sold as dietary supplements and used by cancer patients based on anecdotal evidence that they can treat or prevent disease. In this study, we evaluated Flor-Essence{reg_sign} and Essiac{reg_sign} for their effects on the growth of human tumor cells in culture. The effect of Flor-Essence{reg_sign} and Essiac{reg_sign} herbal tonics on cell proliferation was tested in MCF-7, MDA-MB-436, MDA-MB-231, and T47D cancer cells isolated from human breast tumors.more » Estrogen receptor (ER) dependent activation of a luciferase reporter construct was tested in MCF-7 cells. Specific binding to the ER was tested using an ICI 182,780 competition assay. Flor-Essence{reg_sign} and Essiac{reg_sign} herbal tonics at 1%, 2%, 4% and 8% stimulated cell proliferation relative to untreated controls and activated ER dependent luciferase activity in MCF-7 cells. A 10{sup -7} M concentration of ICI 870,780 inhibited the induction of ER dependent luciferase activity by Flor-Essence{reg_sign} and Essiac{reg_sign}, but did not affect cell proliferation. Flor-Essence{reg_sign} and Essiac{reg_sign} Herbal Tonics can stimulate the growth of human breast cancer cells through ER mediated as well as ER independent mechanisms of action. Cancer patients and health care providers can use this information to make informed decisions about the use of these CAMs.« less

Alternative plasticizer, 4-cyclohexene-1,2-dicarboxylic acid dinonyl ester, for blood containers with protective effects on red blood cells and improved cold resistance.

PubMed

Morishita, Yuki; Nomura, Yusuke; Fukui, Chie; Fujisawa, Ayano; Watanabe, Kayo; Fujimaki, Hideo; Kumada, Hidefumi; Inoue, Kaoru; Morikawa, Tomomi; Takahashi, Miwa; Kawakami, Tsuyoshi; Sakoda, Hideyuki; Mukai, Tomokazu; Yuba, Toshiyasu; Inamura, Ken-Ichi; Tanoue, Akito; Miyazaki, Ken-Ichi; Chung, Ung-Il; Ogawa, Kumiko; Yoshida, Midori; Haishima, Yuji

2018-04-01

Di (2-ethylhexyl) phthalate (DEHP), a typical plasticizer used for polyvinyl chloride (PVC), is eluted from PVC-made blood containers and protects against red blood cell (RBC) hemolysis. However, concerns have arisen regarding the reproductive and developmental risks of DEHP in humans, and the use of alternative plasticizers for medical devices has been recommended worldwide. In this study, we propose that the use of a novel plasticizer, 4-cyclohexene-1,2-dicarboxylic acid dinonyl ester (DL9TH), could help produce more useful and safe blood containers. PVC sheet containing DL9TH and di (2-ethylhexyl) 4-cyclohexene-1,2-dicarboxylate (DOTH) provides comparable or superior protective effects to RBCs relative to PVC sheet containing DEHP or di-isononyl-cyclohexane-1,2-dicarboxylate (DINCH ® , an alternative plasticizer that has been used in PVC sheets for blood containers). The total amount of plasticizer eluted from DOTH/DL9TH-PVC sheets is nearly the same as that eluted from DEHP-PVC sheets. In addition, DOTH/DL9TH-PVC has better cold resistance than DEHP- and DINCH ® -PVC sheets. In vitro and in vivo tests for biological safety based on International Organization for Standardization guidelines (10993 series) suggest that the DOTH/DL9TH-PVC sheet can be used safely. Subchronic toxicity testing of DL9TH in male rats in accordance with the principles of Organisation for Economic Co-operation and Development Test Guideline 408 showed that DL9TH did not induce adverse effects up to the highest dose level tested (717 mg/kg body weight/day). There were no effects on testicular histopathology and sperm counts, and no indications of endocrine effects: testosterone, thyroid-stimulating hormone, follicle-stimulating hormone, and 17β-estradiol were unchanged by the treatment, compared with the control group. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1052-1063, 2018. © 2017 Wiley Periodicals, Inc.

Neural Stem Cells Derived from Human Parthenogenetic Stem Cells Engraft and Promote Recovery in a Nonhuman Primate Model of Parkinson's Disease.

PubMed

Gonzalez, Rodolfo; Garitaonandia, Ibon; Poustovoitov, Maxim; Abramihina, Tatiana; McEntire, Caleb; Culp, Ben; Attwood, Jordan; Noskov, Alexander; Christiansen-Weber, Trudy; Khater, Marwa; Mora-Castilla, Sergio; To, Cuong; Crain, Andrew; Sherman, Glenn; Semechkin, Andrey; Laurent, Louise C; Elsworth, John D; Sladek, John; Snyder, Evan Y; Redmond, D Eugene; Kern, Russell A

2016-11-01

Cell therapy has attracted considerable interest as a promising therapeutic alternative for patients with Parkinson's disease (PD). Clinical studies have shown that grafted fetal neural tissue can achieve considerable biochemical and clinical improvements in PD. However, the source of fetal tissue grafts is limited and ethically controversial. Human parthenogenetic stem cells offer a good alternative because they are derived from unfertilized oocytes without destroying potentially viable human embryos and can be used to generate an unlimited supply of neural cells for transplantation. We have previously reported that human parthenogenetic stem cell-derived neural stem cells (hpNSCs) successfully engraft, survive long term, and increase brain dopamine (DA) levels in rodent and nonhuman primate models of PD. Here we report the results of a 12-month transplantation study of hpNSCs in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned African green monkeys with moderate to severe clinical parkinsonian symptoms. The hpNSCs manufactured under current good manufacturing practice (cGMP) conditions were injected bilaterally into the striatum and substantia nigra of immunosuppressed monkeys. Transplantation of hpNSCs was safe and well tolerated by the animals with no dyskinesia, tumors, ectopic tissue formation, or other test article-related serious adverse events. We observed that hpNSCs promoted behavioral recovery; increased striatal DA concentration, fiber innervation, and number of dopaminergic neurons; and induced the expression of genes and pathways downregulated in PD compared to vehicle control animals. These results provide further evidence for the clinical translation of hpNSCs and support the approval of the world's first pluripotent stem cell-based phase I/IIa study for the treatment of PD (Clinical Trial Identifier NCT02452723).

Alternatives to animal testing: information resources via the Internet and World Wide Web.

PubMed

Hakkinen, P J Bert; Green, Dianne K

2002-04-25

Many countries, including the United States, Canada, European Union member states, and others, require that a comprehensive search for possible alternatives be completed before beginning some or all research involving animals. Completing comprehensive alternatives searches and keeping current with information associated with alternatives to animal testing is a challenge that will be made easier as people throughout the world gain access to the Internet and World Wide Web. Numerous Internet and World Wide Web resources are available to provide guidance and other information on in vitro and other alternatives to animal testing. A comprehensive Web site is Alternatives to Animal Testing on the Web (Altweb), which serves as an online clearinghouse for resources, information, and news about alternatives to animal testing. Examples of other important Web sites include the joint one for the (US) Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) and the Norwegian Reference Centre for Laboratory Animal Science and Alternatives (The NORINA database). Internet mailing lists and online access to bulletin boards, discussion areas, newsletters, and journals are other ways to access and share information to stay current with alternatives to animal testing.

Cost-Benefit Analysis For Alternative Low-Emission Surface Preparation/ Depainting Technologies for Structural Steel

NASA Technical Reports Server (NTRS)

Lewis, Pattie

2007-01-01

Stennis Space Center (SSC), Kennedy Space Center (KSC) and Air Force Space Command (AFSPC) identified particulate emissions and waste generated from the depainting process of steel structures as hazardous materials to be eliminated or reduced. A Potential Alternatives Report, Potential Alternatives Report for Validation of Alternative Low Emission Surface Preparation/Depainting Technologies for Structural Steel, provided a technical analyses of identified alternatives to the current coating removal processes, criteria used to select alternatives for further analysis, and a list of those alternatives recommended for testing. The initial coating removal alternatives list was compiled using literature searches and stakeholder recommendations. The involved project participants initially considered approximately 13 alternatives. In late 2003, core project members selected the following depainting processes to be further evaluated: (1) Plastic Blast Media-Quickstrip(R)-A. (2) Hard Abrasive-Steel-Magic(R). (3) Sponge Blasting-Sponge-Jet(R). (4) Liquid Nitrogen-NItroJet(R). (5) Mechanical Removal with Vacuum Attachment-DESCO and OCM Clean-Air (6) Laser Coating Removal Alternatives were tested in accordance with the Joint Test Protocol for Validation of Alternative Low-Emission Surface Preparation/Depainting Technologies for Structural Steel, and the Field Evaluation Test Plan for Validation of Alternative Low-Emission Surface Preparation/Depainting Technologies for Structural Steel. Results of the testing are documented in the Joint Test Report. This Cost-Benefit Analysis (CBA) focuses on the three alternatives (Quickstrip(R)-A, SteelMagic (R), and Sponge-Jet(R)) that were considered viable alternatives for large area operations based on the results of the field demonstration and lab testing. This CBA was created to help participants determine if implementation of the candidate alternatives is economically justified. Each of the alternatives examined reduced Environmental Activity (EA) Costs-those costs associated with complying with environmental regulations. One alternative, Steel-Magic(R), also showed reduced Direct Costs and reduced total costs.

Context-dependent control of alternative splicing by RNA-binding proteins

PubMed Central

Fu, Xiang-Dong; Ares, Manuel

2015-01-01

Sequence-specific RNA-binding proteins (RBPs) bind to pre-mRNA to control alternative splicing, but it is not yet possible to read the ‘splicing code’ that dictates splicing regulation on the basis of genome sequence. Each alternative splicing event is controlled by multiple RBPs, the combined action of which creates a distribution of alternatively spliced products in a given cell type. As each cell type expresses a distinct array of RBPs, the interpretation of regulatory information on a given RNA target is exceedingly dependent on the cell type. RBPs also control each other’s functions at many levels, including by mutual modulation of their binding activities on specific regulatory RNA elements. In this Review, we describe some of the emerging rules that govern the highly context-dependent and combinatorial nature of alternative splicing regulation. PMID:25112293

Bioengineered humanized livers as better three-dimensional drug testing model system.

PubMed

Vishwakarma, Sandeep Kumar; Bardia, Avinash; Lakkireddy, Chandrakala; Nagarapu, Raju; Habeeb, Md Aejaz; Khan, Aleem Ahmed

2018-01-27

To develop appropriate humanized three-dimensional ex-vivo model system for drug testing. Bioengineered humanized livers were developed in this study using human hepatic stem cells repopulation within the acellularized liver scaffolds which mimics with the natural organ anatomy and physiology. Six cytochrome P-450 probes were used to enable efficient identification of drug metabolism in bioengineered humanized livers. The drug metabolism study in bioengineered livers was evaluated to identify the absorption, distribution, metabolism, excretion and toxicity responses. The bioengineered humanized livers showed cellular and molecular characteristics of human livers. The bioengineered liver showed three-dimensional natural architecture with intact vasculature and extra-cellular matrix. Human hepatic cells were engrafted similar to the human liver. Drug metabolism studies provided a suitable platform alternative to available ex-vivo and in vivo models for identifying cellular and molecular dynamics of pharmacological drugs. The present study paves a way towards the development of suitable humanized preclinical model systems for pharmacological testing. This approach may reduce the cost and time duration of preclinical drug testing and further overcomes on the anatomical and physiological variations in xenogeneic systems.

Kilowatt Isotope Power System: component test report for the ground demonstration system alternator stator. 78-KIPS-17

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

Not Available

1977-12-14

The purpose of this test was to demonstrate that the alternator stator has satisfactorily completed sufficient testing to safisfy the requirements set forth within the Kilowatt Isotope Power System (KIPS) Component Test Specification for the GDS Alternator Stator (TS 2538). The results of the acceptance tests conducted on the alternator stator, S/N 003, are presented, and show that the stator did meet specified requirements.

Characterising dye-sensitized solar cells

NASA Astrophysics Data System (ADS)

Tobin, Laura L.; O'Reilly, Thomas; Zerulla, Dominic; Sheridan, John T.

2009-08-01

With growing energy and environmental concerns due to fossil fuel depletion and global warming there is an increasing attention being attracted by alternative and/or renewable sources of power such as biomass, hydropower, geothermal, wind and solar energy. In today's society there is a vast and in many cases not fully appreciated dependence on electrical power for everyday life and therefore devices such as PV cells are of enormous importance. The more widely used and commercially available silicon (semiconductor) based cells currently have the greatest efficiencies, however the manufacturing of these cells is complex and costly due to the cost and difficulty of producing and processing pure silicon. One new direction being explored is the development of dye-sensitised solar cells (DSSC). The SFI Strategic Research Centre for Solar Energy Conversion is a new research cluster based in Ireland, formed with the express intention of bringing together industry and academia to produce renewable energy solutions. Our specific area of research is in biomimetic dye sensitised solar cells and their electrical properties. We are currently working to develop test equipment, and optoelectronic models describing the performance and behaviors of dye-sensitised solar cells (Grätzel Cells). In this paper we describe some of the background to our work and also some of our initial experimental results. Based on these results we intend to characterise the opto-electrical properties and bulk characteristics of simple dye-sensitised solar cells and then to proceed to test new cell compositions.

Allethrin-Based Mosquito Control Device Causing Knockdown, Morbidity, and Mortality in Four Species of Field-Caught Mosquitoes (Diptera: Culicidae).

PubMed

Bibbs, Christopher S; Fulcher, Ali; Xue, Rui-De

2015-07-01

A mosquito control device marketed for spatial repellency, the ThermaCELL Mosquito Repellent Appliance, was evaluated in semifield trials against multiple field-caught species of mosquito. Using paper and mesh cages, mosquito test groups of at least 30 mosquitoes were suspended in a 2,337 cubic foot outdoor space while two ThermaCELL repellent devices were active. After 30 min of treatment, cages were moved to the laboratory to observe knockdown, morbidity, and mortality for 24 h. Species tested included Aedes atlanticus Dyar and Knab (98% average mortality), Psorophora ferox Humboldt (97% average mortality), Psorophora columbiae Dyar and Knab (96% average mortality), and Aedes taeniorhynchus Wiedemann (84% average mortality). The repellent devices showed effectiveness with high knockdown and mortality across all species tested. Mosquito control devices like the ThermaCELL Mosquito Repellent Appliance may have further practical applications to help combat viral exposures by limiting host mosquitoes. Such devices may provide a functional alternative to DEET dependence in the current state of mosquito management. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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.

Molecular cloning of Brevundimonas diminuta for efficacy assessment of reverse osmosis devices.

PubMed

Donofrio, Robert; Saha, Ratul; Bestervelt, Lori; Bagley, Susan

2012-06-01

Brevundimonas diminuta is the test organism specified in the United States Environmental Protection Agency's (USEPA) reverse osmosis (RO) treatment device verification protocol. As non-selective growth medium is employed, enumeration of B. diminuta may be impaired due to interference by indigenous heterotrophic bacteria. Thus the microbial removal capability of the filtration system may be incorrectly assessed. As these treatment devices are used in emergency situations, the health of the public could be compromised. The objective of this study was to develop selective approaches for enumerating viable B. diminuta in test water. Two molecular approaches were investigated: expression of a kanamycin resistance gene and expression of a fluorescent protein gene. The USEPA protocol specifies a 0.3 μm cell size, so the expression of the selective markers were assessed following growth on media designed to induce this small cell diameter. The kan(R) strain was demonstrated to be equivalent to the wild type in cell dimension and survival following exposure to the test water. The kan(R) strain showed equivalent performance to the wild type in the RO protocol indicating that it is a viable alternative surrogate. By utilizing this strain, a more accurate validation of the RO system can be achieved.

Light Weight Design Nickel-Alkaline Cells Using Fiber Electrodes

NASA Technical Reports Server (NTRS)

Pickett, David F.; Willis, Bob; Britton, Doris; Saelens, Johan

2005-01-01

Using fiber electrode technology, currently produced by Bekaert Corporation (Bekaert), Electro Energy, Inc., (EEI) Mobile Energy Products Group (formerly, Eagle-Picher Technologies, LLC., Power Systems Department) in Colorado Springs, CO has demonstrated that it is feasible to manufacture flight weight nickel-hydrogen cells having about twice the specific energy (80 vs. 40 watt-hr/kg) as state-of-the-art nickel-hydrogen cells that are flown on geosynchronous communications satellites. Although lithium-ion battery technology has made large in-roads to replace the nickel-alkaline technology (nickel-cadmium, nickel-metal hydride), the technology offered here competes with lithium-ion weight and offers alternatives not present in the lithium-ion chemistry such as ability to undergo continuous overcharge, reversal on discharge and sustain rate capability sufficient to start automotive and aircraft engines at subzero temperatures. In development to date seven 50 ampere-hour nickel-hydrogen have been constructed, acceptance tested and briefly tested in a low earth orbit (LEO) cycle regime. The effort was jointly funded by Electro Energy, Inc. and NASA Glenn Research Center, Cleveland, OH. Five of the seven cells have been shipped to NASA GRC for further cycle testing. Two of the cells experienced failure due to internal short circuits during initial cycle testing at EEL Destructive Physical Analysis (DPA) of one of the cells has shown the failure mode to be due to inadequate hydrogen catalyst electrodes that were not capacity balanced with the higher energy density nickel oxide electrodes. In the investigators opinion, rebuild of the cells using proper electrode balance would result in cells that could sustain over 30,000 cycles at moderate depths-of-discharge in a LEO regime or endure over 20 years of geosynchronous orbit (GEO) cycling while realizing a two-fold increase in specific energy for the battery or a 1.1 kg weight savings per 50 ampere-hour cell. Additional information is included in the original extended abstract.

A distinct first replication cycle of DNA introduced in mammalian cells

PubMed Central

Chandok, Gurangad S.; Kapoor, Kalvin K.; Brick, Rachel M.; Sidorova, Julia M.; Krasilnikova, Maria M.

2011-01-01

Many mutation events in microsatellite DNA sequences were traced to the first embryonic divisions. It was not known what makes the first replication cycles of embryonic DNA different from subsequent replication cycles. Here we demonstrate that an unusual replication mode is involved in the first cycle of replication of DNA introduced in mammalian cells. This alternative replication starts at random positions, and occurs before the chromatin is fully assembled. It is detected in various cell lines and primary cells. The presence of single-stranded regions increases the efficiency of this alternative replication mode. The alternative replication cannot progress through the A/T-rich FRA16B fragile site, while the regular replication mode is not affected by it. A/T-rich microsatellites are associated with the majority of chromosomal breakpoints in cancer. We suggest that the alternative replication mode may be initiated at the regions with immature chromatin structure in embryonic and cancer cells resulting in increased genomic instability. This work demonstrates, for the first time, differences in the replication progression during the first and subsequent replication cycles in mammalian cells. PMID:21062817

A light-regulated type I pilus contributes to Acinetobacter baumannii biofilm, motility and virulence functions.

PubMed

Wood, Cecily R; Ohneck, Emily J; Edelmann, Richard E; Actis, Luis A

2018-06-11

Transcriptional analyses of A. baumannii ATCC 17978 showed that the expression of A1S_2091 was enhanced in cells cultured in darkness at 24°C through a process that depended on the BlsA photoreceptor. Disruption of A1S_2091, a component of the A1S_2088-A1S_2091 polycistronic operon predicted to code for a type I chaperone/usher pilus assembly system, abolished surface motility and pellicle formation but significantly enhanced biofilm formation on plastic by bacteria cultured under darkness. Based on these observations, the A1S_2088-A1S_2091 operon was named the p hoto- r egulated p ilus ABCD ( prpABCD ) operon, with A1S_2091 coding for the PrpA pilin subunit. Unexpectedly, the comparative analyses of ATCC 17978 and prpA isogenic mutant cells cultured at 37°C showed the expression of light-regulated biofilm biogenesis and motility functions under a temperature condition that drastically affects BlsA production and its light sensing activity. These assays also suggest that 17978 cells produce alternative light-regulated adhesins and/or pili systems that enhance bacterial adhesion and biofilm formation both at 24°C and 37°C on plastic as well as on the surface of polarized A549 alveolar epithelial cells, where formation of bacterial filaments and cell chains was significantly enhanced. The inactivation of prpA also resulted in a significant reduction in virulence when tested using the Galleria mellonella virulence model. All these observations provide strong evidence showing the capacity of A. baumannii to sense light and interact with biotic and abiotic surfaces using undetermined alternative sensing and regulatory systems as well as alternative adherence and motility cellular functions that allow this pathogen to persist in different ecological niches. Copyright © 2018 American Society for Microbiology.

Alternative splicing of the tyrosinase gene transcript in normal human melanocytes and lymphocytes.

PubMed

Fryer, J P; Oetting, W S; Brott, M J; King, R A

2001-11-01

We have identified and isolated ectopically expressed tyrosinase transcripts in normal human melanocytes and lymphocytes and in a human melanoma (MNT-1) cell line to establish a baseline for the expression pattern of this gene in normal tissue. Tyrosinase mRNA from human lymphoblastoid cell lines was reverse transcribed and amplified using specific "nested" primers. This amplification yielded eight identifiable transcripts; five that resulted from alternative splicing patterns arising from the utilization of normal and alternative splice sequences. Identical splicing patterns were found in transcripts from human primary melanocytes in culture and a melanoma cell line, indicating that lymphoblastoid cell lines provide an accurate reflection of transcript processing in melanocytes. Similar splicing patterns have also been found with murine melanocyte tyrosinase transcripts. Our results demonstrate that alternative splicing of human tyrosinase gene transcript produces a number of predictable and identifiable transcripts, and that human lymphoblastoid cell lines provide a source of ectopically expressed transcripts that can be used to study the biology of tyrosinase gene expression in humans.

Effectiveness and biological compatibility of different generations of dentin adhesives.

PubMed

da Silva, João M F; Rodrigues, José R; Camargo, Carlos H R; Fernandes, Virgilio Vilas Boas; Hiller, Karl-Anton; Schweikl, Helmut; Schmalz, Gottfried

2014-01-01

Besides possessing good mechanical properties, dental materials should present a good biological behavior and should not injure the involved tissues. Bond strength and biocompatibility are both highly significant properties of dentin adhesives. For that matter, these properties of four generations of adhesive systems (Multi-Purpose/Single Bond/SE Plus/Easy Bond) were evaluated. Eighty bovine teeth had their dentin exposed (500- and 200-μm thickness). Adhesive was applied on the dentin layer of each specimen. Following that, the microshearing test was performed for all samples. A dentin barrier test was used for the cytotoxicity evaluation. Cell cultures (SV3NeoB) were collected from testing materials by means of 200- or 500-μm-thick dentin slices and placed in a cell culture perfusion chamber. Cell viability was measured 24 h post-exposition by means of a photometrical test (MTT test). The best bonding performance was shown by the single-step adhesive Easy Bond (21 MPa, 200 μm; 27 MPa, 500 μm) followed by Single Bond (15.6 MPa, 200 μm; 23.4 MPa, 500 μm), SE Plus (18.2 MPa, 200 μm; 20 MPa, 500 μm), and Multi-Purpose (15.2 MPa, 200 μm; 17.9 MPa, 500 μm). Regarding the cytotoxicity, Multi-Purpose slightly reduced the cell viability to 92% (200 μm)/93% (500 μm). Single Bond was reasonably cytotoxic, reducing cell viability to 71% (200 μm)/64% (500 μm). The self-etching adhesive Scotchbond SE decreased cell viability to 85% (200 μm)/71% (500 μm). Conversely, Easy Bond did not reduce cell viability in this test, regardless of the dentin thickness. Results showed that the one-step system had the best bond strength performance and was the least toxic to pulp cells. In multiple-step systems, a correct bonding technique must be done, and a pulp capping strategy is necessary for achieving good performance in both properties. The study showed a promising system (one-step self-etching), referring to it as a good alternative for specific cases, mainly due to its technical simplicity and good biological responses.

The level of PPD-specific IFN-gamma-producing CD4+ T cells in the blood predicts the in vivo response to PPD.

PubMed

Martins, Marcia Valéria B S; Lima, Mônica Cristina B S; Duppre, Nadia C; Matos, Haroldo J; Spencer, John S; Brennan, Patrick J; Sarno, Euzenir N; Fonseca, Leila; Pereira, Geraldo M B; Pessolani, Maria Cristina V

2007-05-01

There are no reliable means for detecting subclinical mycobacterial infections. The recent sequencing of several mycobacterial genomes has now afforded new opportunities for the development of pathogen-specific diagnostic tests, critical in the context of leprosy and tuberculosis control. In the present study, we applied a multi-parametric flow cytometric analysis that allowed the investigation of T-cell functions in order to define immunological markers that measure previous exposure to mycobacteria. We compared the in vivo response to PPD, the gold standard skin test reagent for measuring previous exposure to Mycobacterium tuberculosis, with in vitro parameters of leukocyte activation in five PPD positive and five PPD negative healthy volunteers. PPD-stimulated peripheral leukocytes expressing CD4, CD69, cutaneous lymphocyte-associated antigen (CLA) and intracellular IFN-gamma were enumerated in whole blood and compared with the size of in vivo PPD-induced induration and IFN-gamma production levels as measured by ELISA in supernatants of PPD-stimulated peripheral blood mononuclear cells. The reactivity to the tuberculin skin test (TST) was associated with markedly increased frequencies of PPD-responsive activated (CD69+) and IFN-gamma-producing CD4+T cells. Detection of PPD-specific IFN-gamma producing leukocytes was restricted to CD4+T cells and a subset of these cells was shown to express the skin homing molecule CLA. Multiple linear regression modeling of responses to PPD showed the highest association between skin test indurations and frequencies of PPD-responsive IFN-gamma-producing CD4+CD69+ T cells. Our data show that the in vitro enumeration of antigen-specific IFN-gamma-producing CD4+ T cells can provide an alternative to the in vivo tuberculin test for the detection of latent Mycobacterium tuberculosis infection. Moreover, the measurement of these immunological parameters can be useful for the screening of new specific antigens defined by the genome sequence allowing selection of the best candidates for new diagnostics (including new skin tests), and vaccines for leprosy and tuberculosis.

Medical waste tissues - breathing life back into respiratory research.

PubMed

BéruBé, Kelly A

2013-12-01

With the advent of biobanks to store human lung cells and tissues from patient donations and from the procurement of medical waste tissues, it is now possible to integrate (both spatially and temporally) cells into anatomically-correct and physiologically-functional tissues. Modern inhalation toxicology relies on human data on exposure and adverse effects, to determine the most appropriate risk assessments and mitigations for beneficial respiratory health. A point in case is the recapitulation of airway tissue, such as the bronchial epithelium, to investigate the impact of air pollution on human respiratory health. The bronchi are the first point of contact for inhaled substances that bypass defences in the upper respiratory tract. Animal models have been used to resolve such inhalation toxicology hazards. However, the access to medical waste tissues has enabled the Lung Particle Research Group to tissue-engineer the Micro-Lung (TM) and Metabo-Lung(TM) cell culture models, as alternatives to animals in basic research and in the safety testing of aerosolised consumer goods. The former model favours investigations focused on lung injury and repair mechanisms, and the latter model provides the element of metabolism, through the co-culturing of lung and liver (hepatocyte) cells. These innovations represent examples of the animal-free alternatives advocated by the 21st century toxicology paradigm, whereby human-derived cell/tissue data will lead to more-accurate and more-reliable public health risk assessments and therapeutic mitigations (e.g. exposure to ambient air pollutants and adverse drug reactions) for lung disease. 2013 FRAME.

Enzyme-Linked Immunospot Assay Detection of Mumps-Specific Antibody-Secreting B Cells as an Alternative Method of Laboratory Diagnosis ▿

PubMed Central

Latner, Donald R.; McGrew, Marcia; Williams, Nobia; Lowe, Luis; Werman, Roniel; Warnock, Eli; Gallagher, Kathleen; Doyle, Peter; Smole, Sandra; Lett, Susan; Cocoros, Noelle; DeMaria, Alfred; Konomi, Raimond; Brown, Cedric J.; Rota, Paul A.; Bellini, William J.; Hickman, Carole J.

2011-01-01

Although high measles, mumps, and rubella (MMR) vaccination coverage has been successful in dramatically reducing mumps disease in the United States, mumps (re)infections occasionally occur in individuals who have been either previously vaccinated or naturally infected. Standard diagnostics that detect virus or virus-specific antibody are dependable for confirming primary mumps infection in immunologically naïve persons, but these methods perform inconsistently for individuals with prior immune exposure. We hypothesized that detection of activated mumps-specific antibody-secreting B cells (ASCs) by enzyme-linked immunospot (ELISPOT) assay could be used as a more reliable diagnostic. To test this, a time course of virus-specific ASC responses was measured by ELISPOT assay following MMR vaccination of 16 previously vaccinated or naturally exposed adult volunteers. Mumps-specific ASCs were detectable in 68% of these individuals at some point during the first 3 weeks following revaccination. In addition, mumps-specific ASCs were detected in 7/7 previously vaccinated individuals who recently had been infected as part of a confirmed mumps outbreak. These data suggest that ELISPOT detection of mumps-specific ASCs has the potential for use as an alternative method of diagnosis when suspect cases cannot be confirmed by detection of IgM or virus. In addition, it was determined that mumps-specific memory B cells are detected at a much lower frequency than measles- or rubella-specific cells, suggesting that mumps infection may not generate robust B-cell memory. PMID:21047998

77 FR 41370 - Proposed Information Collection; Comment Request; 2013 Alternative Contact Strategy Test

Federal Register 2010, 2011, 2012, 2013, 2014

2012-07-13

... Alternative Contact Strategy Test AGENCY: U.S. Census Bureau, Commerce. ACTION: Notice. SUMMARY: The...-response. This research will be conducted through a series of projects and tests throughout the decade... 2013 Alternative Contact Strategy Test is the first test to support this research. The Census Bureau...

Sensing Cell-Culture Assays with Low-Cost Circuitry.

PubMed

Pérez, Pablo; Huertas, Gloria; Maldonado-Jacobi, Andrés; Martín, María; Serrano, Juan A; Olmo, Alberto; Daza, Paula; Yúfera, Alberto

2018-06-11

An alternative approach for cell-culture end-point protocols is proposed herein. This new technique is suitable for real-time remote sensing. It is based on Electrical Cell-substrate Impedance Spectroscopy (ECIS) and employs the Oscillation-Based Test (OBT) method. Simple and straightforward circuit blocks form the basis of the proposed measurement system. Oscillation parameters - frequency and amplitude - constitute the outcome, directly correlated with the culture status. A user can remotely track the evolution of cell cultures in real time over the complete experiment through a web tool continuously displaying the acquired data. Experiments carried out with commercial electrodes and a well-established cell line (AA8) are described, obtaining the cell number in real time from growth assays. The electrodes have been electrically characterized along the design flow in order to predict the system performance and the sensitivity curves. Curves for 1-week cell growth are reported. The obtained experimental results validate the proposed OBT for cell-culture characterization. Furthermore, the proposed electrode model provides a good approximation for the cell number and the time evolution of the studied cultures.

High-throughput alternative splicing detection using dually constrained correspondence analysis (DCCA).

PubMed

Baty, Florent; Klingbiel, Dirk; Zappa, Francesco; Brutsche, Martin

2015-12-01

Alternative splicing is an important component of tumorigenesis. Recent advent of exon array technology enables the detection of alternative splicing at a genome-wide scale. The analysis of high-throughput alternative splicing is not yet standard and methodological developments are still needed. We propose a novel statistical approach-Dually Constrained Correspondence Analysis-for the detection of splicing changes in exon array data. Using this methodology, we investigated the genome-wide alteration of alternative splicing in patients with non-small cell lung cancer treated by bevacizumab/erlotinib. Splicing candidates reveal a series of genes related to carcinogenesis (SFTPB), cell adhesion (STAB2, PCDH15, HABP2), tumor aggressiveness (ARNTL2), apoptosis, proliferation and differentiation (PDE4D, FLT3, IL1R2), cell invasion (ETV1), as well as tumor growth (OLFM4, FGF14), tumor necrosis (AFF3) or tumor suppression (TUSC3, CSMD1, RHOBTB2, SERPINB5), with indication of known alternative splicing in a majority of genes. DCCA facilitates the identification of putative biologically relevant alternative splicing events in high-throughput exon array data. Copyright © 2015 Elsevier Inc. All rights reserved.

Immunoconjugates in the management of hairy cell leukemia

PubMed Central

Kreitman, Robert J.; Pastan, Ira

2015-01-01

Hairy cell leukemia (HCL) is an indolent B-cell malignancy effectively treated but not often cured by purine analog therapy; after multiple courses of purine analogs, patients can become purine analog resistant and in need of alternative therapies. Complete remission to single-agent purine analog is often accompanied by minimal residual disease (MRD), residual HCL cells detectable by immunologic methods, considered a risk factor for eventual relapse. Several different non-chemotherapy approaches are being used to target relapsed and refractory HCL, including inhibitors of BRAF, but so far only monoclonal antibody (MAb)-based approaches have been reported to eliminate MRD in a high percentage of patients. One of the MAb-based options for HCL currently under clinical investigation involves recombinant immunotoxins, containing a fragment of a MAb and a bacterial toxin. The bacterial toxin, a highly potent fragment from Pseudomonas exotoxin, catalytically ADP-ribosylates elongation factor 2 (EF2), resulting in protein synthesis inhibition and apoptotic cell death. Recombinant immunotoxins tested in HCL patients include LMB-2, targeting CD25, and BL22, targeting CD22. An affinity matured version of BL22, termed moxetumomab pasudotox (formerly HA22 or CAT-8015) achieved high CR rates in phase I, and is currently undergoing multicenter Phase 3 testing. Phase I testing was without dose-limiting toxicity, although 2 patients had grade 2 hemolytic uremic syndrome (HUS) with transient grade 1 abnormalities in platelets and creatinine. Preclinical work is underway to identify residues on moxetumomab pasudotox leading to immunogenicity. Moxetumomab pasudotox is undergoing pivotal testing for relapsed and refractory HCL. PMID:26614902

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.

Erythroleukemia cells acquire an alternative mitophagy capability.

PubMed

Wang, Jian; Fang, Yixuan; Yan, Lili; Yuan, Na; Zhang, Suping; Xu, Li; Nie, Meilan; Zhang, Xiaoying; Wang, Jianrong

2016-04-19

Leukemia cells are superior to hematopoietic cells with a normal differentiation potential in buffering cellular stresses, but the underlying mechanisms for this leukemic advantage are not fully understood. Using CRISPR/Cas9 deletion of the canonical autophagy-essential gene Atg7, we found that erythroleukemia K562 cells are armed with two sets of autophagic machinery. Alternative mitophagy is functional regardless of whether the canonical autophagic mechanism is intact or disrupted. Although canonical autophagy defects attenuated cell cycling, proliferation and differentiation potential, the leukemia cells retained their abilities for mitochondrial clearance and for maintaining low levels of reactive oxygen species (ROS) and apoptosis. Treatment with a specific inducer of mitophagy revealed that the canonical autophagy-defective erythroleukemia cells preserved a mitophagic response. Selective induction of mitophagy was associated with the upregulation and localization of RAB9A on the mitochondrial membrane in both wild-type and Atg7(-/-) leukemia cells. When the leukemia cells were treated with the alternative autophagy inhibitor brefeldin A or when the RAB9A was knocked down, this mitophagy was prohibited. This was accompanied by elevated ROS levels and apoptosis as well as reduced DNA damage repair. Therefore, the results suggest that erythroleukemia K562 cells possess an ATG7-independent alternative mitophagic mechanism that functions even when the canonical autophagic process is impaired, thereby maintaining the ability to respond to stresses such as excessive ROS and DNA damage.

Measuring bioenergetics in T cells using a Seahorse Extracellular Flux Analyzer

PubMed Central

van der Windt, Gerritje J.W.; Chang, Chih-Hao; Pearce, Erika L.

2016-01-01

This unit contains several protocols to determine the energy utilization of T cells in real-time using a Seahorse Extracellular Flux Analyzer (www.seahorsebio.com). The advantages to using this machine over traditional metabolic assays include the simultaneous measurement of glycolysis and mitochondrial respiration, in real-time, on relatively small numbers of cells, without any radioactivity. The Basic Protocol describes a standard mitochondrial stress test on the XFe96, which yields information about oxidative phosphorylation and glycolysis, two energy-generating pathways. The alternate protocols provide examples of adaptations to the Basic Protocol, including adjustments for the use of the XFe24. A protocol for real-time bioenergetic responses to T cell activation allows for the analysis of immediate metabolic changes after T cell receptor stimulation. Specific substrate utilization can be determined by the use of differential assay media, or the injection of drugs that specifically affect certain metabolic processes. Accurate cell numbers, purity, and viability are critical to obtain reliable results. PMID:27038461

Measuring Bioenergetics in T Cells Using a Seahorse Extracellular Flux Analyzer.

PubMed

van der Windt, Gerritje J W; Chang, Chih-Hao; Pearce, Erika L

2016-04-01

This unit contains several protocols to determine the energy utilization of T cells in real-time using a Seahorse Extracellular Flux Analyzer (http://www.seahorsebio.com). The advantages to using this machine over traditional metabolic assays include the simultaneous measurement of glycolysis and mitochondrial respiration, in real-time, on relatively small numbers of cells, without any radioactivity. The Basic Protocol describes a standard mitochondrial stress test on the XF(e) 96, which yields information about oxidative phosphorylation and glycolysis, two energy-generating pathways. The alternate protocols provide examples of adaptations to the Basic Protocol, including adjustments for the use of the XF(e) 24. A protocol for real-time bioenergetic responses to T cell activation allows for the analysis of immediate metabolic changes after T cell receptor stimulation. Specific substrate utilization can be determined by the use of differential assay media, or the injection of drugs that specifically affect certain metabolic processes. Accurate cell numbers, purity, and viability are critical to obtain reliable results. Copyright © 2016 John Wiley & Sons, Inc.

Mobilization of bone marrow mesenchymal stem cells in vivo augments bone healing in a mouse model of segmental bone defect.

PubMed

Kumar, Sanjay; Ponnazhagan, Selvarangan

2012-04-01

Although the number of mesenchymal stem cells (MSC) in the bone marrow is sufficient to maintain skeletal homeostasis, in osteopenic pathology, aggravated osteoclast activity or insufficient osteoblast numbers ensue, affecting normal bone remodeling. Most of the currently available therapies are anti-resorptive with limited osteogenic potential. Since mobilization of stem/progenitors from the BM is a prerequisite for their participation in tissue repair, amplification of endogenous stem cells may provide an alternative approach in these conditions. The present study determined the potential of MSC mobilization in vivo, using combinations of different growth factors with the CXCR4 antagonist, AMD3100, in a mouse model of segmental bone defect. Results indicated that among several factors tested IGF1 had maximum proliferative ability of MSC in vitro. Results of the in vivo studies indicated that the combination of IGF1 and AMD3100 provided significant augmentation of bone growth as determined by DXA, micro-CT and histomorphometry in mice bearing segmental fractures. Further, characterization of MSC isolated from mice treated with IGF1 and AMD3100 indicated Akt/PI3K, MEK1/2-Erk1/2 and smad2/3 as key signaling pathways mediating this effect. These data indicate the potential of in vivo stem cell mobilization as a novel alternative for bone healing. Copyright © 2012 Elsevier Inc. All rights reserved.

Stem cell therapy and its potential role in pituitary disorders.

PubMed

Lara-Velazquez, Montserrat; Akinduro, Oluwaseun O; Reimer, Ronald; Woodmansee, Whitney W; Quinones-Hinojosa, Alfredo

2017-08-01

The pituitary gland is one of the key components of the endocrine system. Congenital or acquired alterations can mediate destruction of cells in the gland leading to hormonal dysfunction. Even though pharmacological treatment for pituitary disorders is available, exogenous hormone replacement is neither curative nor sustainable. Thus, alternative therapies to optimize management and improve quality of life are desired. An alternative modality to re-establish pituitary function is to promote endocrine cell regeneration through stem cells that can be obtained from the pituitary parenchyma or pluripotent cells. Stem cell therapy has been successfully applied to a plethora of other disorders, and is a promising alternative to hormonal supplementation for resumption of normal hormone homeostasis. In this review, we describe the common causes for pituitary deficiencies and the advances in cellular therapy to restore the physiological pituitary function.

Improving the Efficiency and Durability of Reversible Solid Oxide Cells for Energy Storage

NASA Astrophysics Data System (ADS)

Hughes, Gareth Allen

This thesis presents research on the use of solid oxide cells (SOCs) as energy storage devices, and covers methods to improve their efficiency and durability for this use. It specifically covers two main topics: the durability of the oxygen electrode under forced alternating current, and the effect of pressurization on various oxygen electrode materials. Additionally, research was completed on thermodynamic modeling of a pressurized SOC energy storage system, and a new experimental testing apparatus was constructed to enable investigation of SOC samples operating under pressure. Forced alternating current using a symmetric sample structure was used to simulate the operation of a reversible SOC, effectively isolating the measurement of the performance response of the oxygen electrode. Cells consisting of La 0.8Sr0.2MnO3-delta - 8mol% Y2O 3-stabilized ZrO2 (LSM-YSZ) oxygen electrodes on YSZ electrolytes were tested. Early testing utilizing Ag current collectors showed that forced currents and the elevated operating temperature of SOCs cause silver to vaporize and deposit at the active region of the electrode. To avoid this artifact, a new test setup utilizing LSM current collectors was created. It was found that a shorter current cycling time of 1 hour helps prevent degradation compared to 12 hour cycles. Additionally, both cycling times showed improvement compared cells operated with dc current. Further study showed that operating at current densities of 0.8 A/cm2 and below can prevent degradation entirely. Pressurization of oxygen electrodes showed, as expected, that polarization resistance decreases with increasing oxygen pressure. The materials tested were LSM-YSZ and La0.6Sr0.4Fe0.8Co0.2 O3-d - Ce0.8Gd0.2O1.95 (LSCF-GDC), both in single-phase and composite electrode structures. Additionally, LSM-infiltrated YSZ was tested. The resistance typically decreased following power-law behavior with exponents ranging from -0.17 to -0.30, with similar trends found in all material systems and electrode structures. The electrodes showed resistance decreases of factors between 1.4 and 3.5 on going from 0.1 atm O2 to 10 atm O2. The electrodes containing LSM each showed distinct features in their frequency responses and capacitances, while the two LSCF containing electrode showed very similar features. The resistance decreases were attributed to decreased charge transfer reaction limitations and accelerated adsorption and surface migration of oxygen ions.

Analysis of tumoral spheres growing in a multichamber microfluidic device.

PubMed

Belgorosky, Denise; Fernández-Cabada, Tamara; Peñaherrera-Pazmiño, Ana Belén; Langle, Yanina; Booth, Ross; Bhansali, Shekhar; Pérez, Maximiliano S; Eiján, Ana María; Lerner, Betiana

2018-09-01

Lab on a Chip (LOC) farming systems have emerged as a powerful tool for single cell studies combined with a non-adherent cell culture substrate and single cell capture chips for the study of single cell derived tumor spheres. Cancer is characterized by its cellular heterogeneity where only a small population of cancer stem cells (CSCs) are responsible for tumor metastases and recurrences. Thus, the in vitro strategy to the formation of a single cell-derived sphere is an attractive alternative to identify CSCs. In this study, we test the effectiveness of microdevices for analysis of heterogeneity within CSC populations and its interaction with different components of the extracellular matrix. CSC could be identify using specific markers related to its pluripotency and self-renewal characteristics such as the transcription factor Oct-4 or the surface protein CD44. The results confirm the usefulness of LOC as an effective method for quantification of CSC, through the formation of spheres under conditions of low adhesion or growing on components of the extracellular matrix. The device used is also a good alternative for evaluating the individual growth of each sphere and further identification of these CSC markers by immunofluorescence. In conclusion, LOC devices have not only the already known advantages, but they are also a promising tool since they use small amounts of reagents and are under specific culture parameters. LOC devices could be considered as a novel technology to be used as a complement or replacement of traditional studies on culture plates. © 2018 Wiley Periodicals, Inc.

Alternatively Activated Macrophages Play an Important Role in Vascular Remodeling and Hemorrhaging in Patients with Brain Arteriovenous Malformation.

PubMed

Nakamura, Yukihiko; Sugita, Yasuo; Nakashima, Shinji; Okada, Yousuke; Yoshitomi, Munetake; Kimura, Yoshizou; Miyoshi, Hiroaki; Morioka, Motohiro; Ohshima, Koichi

2016-03-01

Angiogenic and immunoactive lesions in brain arteriovenous malformation (BAVM) contribute to hemorrhagic events and the growth of BAVMs. However, the detailed mechanism is unclear. Our objective is to clarify the relationship between hemorrhagic events of BAVM and alternatively activated macrophages in the perinidal dilated capillary network (PDCN). We examined microsurgical specimens of BVMs (n = 29) and focused on the PDCN area. Ten autopsied brains without intracranial disease were the controls. We performed immunostaining of the inflammatory and endothelial cell markers, macrophage markers (CD163 and CD68), and vascular endothelial growth factor A (VEGF-A). We evaluated each cell's density and the vessel density in the PDCN and analyzed the relationship to hemorrhagic events of BAVM. The PDCN was involved in all the resected arteriovenous malformations, and these vessels showed a high rate of CD105 expression (72.0 ± 10.64%), indicating newly proliferating vessels. Alternatively activated macrophages were found, with a high rate (85.6%) for all macrophages (controls, 56.6%). In the hemorrhagic cases, the cell density was significantly higher than that in the nonhemorrhagic cases and controls (hemorrhagic group, 290 ± 44 cells/mm(2); nonhemorrhagic group, 180 ± 59 cells/mm(2); and control, 19 ± 8 cells/mm(2)). The cell density of alternatively activated macrophages showed a positive correlation with the vessel density of the PDCN. Double immunostaining showed that VEGF-A was secreted by alternatively activated macrophages. Our data suggest that alternatively activated macrophages may have some relationships with angiogenesis of PDCN and hemorrhagic event of BAVM. Copyright © 2016 National Stroke Association. Published by Elsevier Inc. All rights reserved.

Bisphenol A alternatives in thermal paper from the Netherlands, Spain, Sweden and Norway. Screening and potential toxicity.

PubMed

Björnsdotter, Maria K; Jonker, Willem; Legradi, Jessica; Kool, Jeroen; Ballesteros-Gómez, Ana

2017-12-01

Thermal paper contains potentially toxic additives, such as bisphenol A (BPA), as a common color developer. Because of its known endocrine disrupting effects, structural analogues to BPA, such as bisphenol S (BPS), D-8 and Pergafast 201, have been used as alternatives, but little is known about the presence and toxicological effects of alternatives other than BPS. In this study, thermal paper is screened by direct probe ambient mass spectrometry (rapid pre-screening method not requiring sample preparation) and by liquid chromatography (LC) with high resolution time-of flight (TOF-MS) mass spectrometry. Cash receipts and other thermal paper products (cinema tickets, boarding passes and luggage tags) were analyzed. Besides BPA and BPS, other developers only recently reported (Pergafast 201, D-8) or to the best of our knowledge not reported before (D-90, TGSA, BPS-MAE) were frequently found as well as some related unreported impurities (2,4-BPS that is a BPS related impurity and a TGSA related impurity). To gain some insight into the potential estrogenicity of the detected developers, a selection of extracts was further analyzed using a LC-nanofractionation platform in combination with cell-based bioassay testing. These preliminary results seems to indicate very low or absence of estrogenic activity for Pergafast 201, D-8, D-90, TGSA and BPS-MAE in comparison to BPA and BPS, although further dose-response tests with authentic standards are required to confirm these results. Compounds for which standards were available were also tested for developmental toxicity and neurotoxicity using zebrafish (Danio rerio) embryos. TGSA and D-8 induced similar teratogenic effects as BPA in zebrafish embryos. BPS and 2,4-BPS did not induce any developmental effects but 2,4-BPS did alter the locomotor activity at the tested concentration. Our findings suggest that the alternatives used as alternatives to BPA (except BPS) might not be estrogenic. However, TGSA and D-8 showed abnormal developmental effects similar to BPA. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of airborne particulate matter on alternative pre-mRNA splicing in colon cancer cells.

PubMed

Buggiano, Valeria; Petrillo, Ezequiel; Alló, Mariano; Lafaille, Celina; Redal, María Ana; Alghamdi, Mansour A; Khoder, Mamdouh I; Shamy, Magdy; Muñoz, Manuel J; Kornblihtt, Alberto R

2015-07-01

Alternative pre-mRNA splicing plays key roles in determining tissue- and species-specific cell differentiation as well as in the onset of hereditary disease and cancer, being controlled by multiple post- and co-transcriptional regulatory mechanisms. We report here that airborne particulate matter, resulting from industrial pollution, inhibits expression and specifically affects alternative splicing at the 5' untranslated region of the mRNA encoding the bone morphogenetic protein BMP4 in human colon cells in culture. These effects are consistent with a previously reported role for BMP4 in preventing colon cancer development, suggesting that ingestion of particulate matter could contribute to the onset of colon cell proliferation. We also show that the underlying mechanism might involve changes in transcriptional elongation. This is the first study to demonstrate that particulate matter causes non-pleiotropic changes in alternative splicing. Copyright © 2015 Elsevier Inc. All rights reserved.

HIV-1 dynamics revisited: biphasic decay by cytotoxic T lymphocyte killing?

PubMed Central

Arnaout, R A; Nowak, M A; Wodarz, D

2000-01-01

The biphasic decay of blood viraemia in patients being treated for human immunodeficiency virus type 1 (HIV-1) infection has been explained as the decay of two distinct populations of cells: the rapid death of productively infected cells followed by the much slower elimination of a second population the identity of which remains unknown. Here we advance an alternative explanation based on the immune response against a single population of infected cells. We show that the biphasic decay can be explained simply, without invoking multiple compartments: viral load falls quickly while cytotoxic T lymphocytes (CTL) are still abundant, and more slowly as CTL disappear. We propose a method to test this idea, and develop a framework that is readily applicable to treatment of other infections. PMID:10972131

Alternative experiments using the geophysical fluid flow cell

NASA Technical Reports Server (NTRS)

Hart, J. E.

1984-01-01

This study addresses the possibility of doing large scale dynamics experiments using the Geophysical Fluid Flow Cell. In particular, cases where the forcing generates a statically stable stratification almost everywhere in the spherical shell are evaluated. This situation is typical of the Earth's atmosphere and oceans. By calculating the strongest meridional circulation expected in the spacelab experiments, and testing its stability using quasi-geostrophic stability theory, it is shown that strongly nonlinear baroclinic waves on a zonally symmetric modified thermal wind will not occur. The Geophysical Fluid Flow Cell does not have a deep enough fluid layer to permit useful studies of large scale planetary wave processes arising from instability. It is argued, however, that by introducing suitable meridional barriers, a significant contribution to the understanding of the oceanic thermocline problem could be made.

Effects of High-Temperature-Pressure Polymerized Resin-Infiltrated Ceramic Networks on Oral Stem Cells

PubMed Central

Nassif, Ali; Berbar, Tsouria; Le Goff, Stéphane; Berdal, Ariane; Sadoun, Michael; Fournier, Benjamin P. J.

2016-01-01

Objectives The development of CAD—CAM techniques called for new materials suited to this technique and offering a safe and sustainable clinical implementation. The infiltration of resin in a ceramic network under high pressure and high temperature defines a new class of hybrid materials, namely polymer infiltrated ceramics network (PICN), for this purpose which requires to be evaluated biologically. We used oral stem cells (gingival and pulpal) as an in vitro experimental model. Methods Four biomaterials were grinded, immersed in a culture medium and deposed on stem cells from dental pulp (DPSC) and gingiva (GSC): Enamic (VITA®), Experimental Hybrid Material (EHM), EHM with initiator (EHMi) and polymerized Z100™ composite material (3M®). After 7 days of incubation; viability, apoptosis, proliferation, cytoskeleton, inflammatory response and morphology were evaluated in vitro. Results Proliferation was insignificantly delayed by all the tested materials. Significant cytotoxicity was observed in presence of resin based composites (MTT assay), however no detectable apoptosis and some dead cells were detected like in PICN materials. Cell morphology, major cytoskeleton and extracellular matrix components were not altered. An intimate contact appeared between the materials and cells. Clinical Significance The three new tested biomaterials did not exhibit adverse effects on oral stem cells in our experimental conditions and may be an interesting alternative to ceramics or composite based CAD—CAM blocks. PMID:27196425

Stem cells as a good tool to investigate dysregulated biological systems in autism spectrum disorders.

PubMed

Griesi-Oliveira, Karina; Sunaga, Daniele Yumi; Alvizi, Lucas; Vadasz, Estevão; Passos-Bueno, Maria Rita

2013-10-01

Identification of the causes of autism spectrum disorders (ASDs) is hampered by their genetic heterogeneity; however, the different genetic alterations leading to ASD seem to be implicated in the disturbance of common molecular pathways or biological processes. In this scenario, the search for differentially expressed genes (DEGs) between ASD patients and controls is a good alternative to identify the molecular etiology of such disorders. Here, we employed genome-wide expression analysis to compare the transcriptome of stem cells of human exfoliated deciduous teeth (SHEDs) of idiopathic autistic patients (n = 7) and control samples (n = 6). Nearly half of the 683 identified DEGs are expressed in the brain (P = 0.003), and a significant number of them are involved in mechanisms previously associated with ASD such as protein synthesis, cytoskeleton regulation, cellular adhesion and alternative splicing, which validate the use of SHEDs to disentangle the causes of autism. Autistic patients also presented overexpression of genes regulated by androgen receptor (AR), and AR itself, which in turn interacts with CHD8 (chromodomain helicase DNA binding protein 8), a gene recently shown to be associated with the cause of autism and found to be upregulated in some patients tested here. These data provide a rationale for the mechanisms through which CHD8 leads to these diseases. In summary, our results suggest that ASD share deregulated pathways and revealed that SHEDs represent an alternative cell source to be used in the understanding of the biological mechanisms involved in the etiology of ASD. © 2013 International Society for Autism Research, Wiley Periodicals, Inc.

Cost-Benefit Analysis for Alternatives to Aliphatic Isocyanate Polyurethanes

NASA Technical Reports Server (NTRS)

Lewis, Pattie

2007-01-01

NASA and Air Force Space Command (AFSPC) have similar missions and therefore similar facilities and structures in similar environments. The standard practice for protecting metallic substrates in atmospheric environments is the application of an applied coating system. The most common topcoats used in coating systems are polyurethanes that contain isocyanates. Isocyanates are classified as potential human carcinogens and are known to cause cancer in animals. The primary objective of this effort was to demonstrate and validate alternatives to aliphatic isocyanate polyurethanes resulting in one or more isocyanate-free coatings qualified for use at AFSPC and NASA installations participating in this project. This Cost-Benefit Analysis (CBA) quantifies the estimated capital and process costs of coating alternatives and cost savings relative to the current coatings. The estimates in this CBA are to be used for assessing the relative merits of the selected alternatives. The actual economic effects at any specific facility will depend on the alternative material or technology implemented, the number of actual applications converted, future workloads, and other factors . The participants initially considered eighteen (18) alternative coatings as described in the Potential Alternatives Report entitled Potential Alternatives Report for Validation of Alternatives to Aliphatic Isocyanate Polyurethanes, prepared by ITB. Of those, 8 alternatives were selected for testing in accordance with the Joint Test Protocol entitled Joint Test Protocol for Validation of Alternatives to Aliphatic Isocyanate Polyurethanes, and the Field Test Plan entitled Field Evaluations Test Plan for Validation of Alternatives 10 Aliphatic Isocyanate Polyurethanes, both of which were prepared by ITB. A joint Test Report entitled Joint Test Report for Validation of Alternatives to Aliphatic Isocyanate Polyurethanes, prepared by ITB, documents the results of the laboratory and field testing, as well as any test modifications made during the execution of the testing. The coatings selected for evaluation in this CBA are shown in the table below. Only one control coating system is considered in this analysis. These coatings were either downselected for Phase II or performed well enough to be included in the Qualified Products List in the NASA technical standard NASA-STD-5008, Protective Coating of Carbon Steel, Stainless Steel, and Aluminum on Launch Structures, Facilities, and Ground Support Equipment.

F-Test Alternatives to Fisher's Exact Test and to the Chi-Square Test of Homogeneity in 2x2 Tables.

ERIC Educational Resources Information Center

Overall, John E.; Starbuck, Robert R.

1983-01-01

An alternative to Fisher's exact test and the chi-square test for homogeneity in two-by-two tables is developed. The method provides for Type I error rates which are closer to the stated alpha level than either of the alternatives. (JKS)

Integrating non-animal test information into an adaptive testing strategy - skin sensitization proof of concept case.

PubMed

Jaworska, Joanna; Harol, Artsiom; Kern, Petra S; Gerberick, G Frank

2011-01-01

There is an urgent need to develop data integration and testing strategy frameworks allowing interpretation of results from animal alternative test batteries. To this end, we developed a Bayesian Network Integrated Testing Strategy (BN ITS) with the goal to estimate skin sensitization hazard as a test case of previously developed concepts (Jaworska et al., 2010). The BN ITS combines in silico, in chemico, and in vitro data related to skin penetration, peptide reactivity, and dendritic cell activation, and guides testing strategy by Value of Information (VoI). The approach offers novel insights into testing strategies: there is no one best testing strategy, but the optimal sequence of tests depends on information at hand, and is chemical-specific. Thus, a single generic set of tests as a replacement strategy is unlikely to be most effective. BN ITS offers the possibility of evaluating the impact of generating additional data on the target information uncertainty reduction before testing is commenced.

Cytotoxicity and Bioactivity of Calcium Silicate Cements Combined with Niobium Oxide in Different Cell Lines.

PubMed

Mestieri, Leticia Boldrin; Gomes-Cornélio, Ana Lívia; Rodrigues, Elisandra Márcia; Faria, Gisele; Guerreiro-Tanomaru, Juliane Maria; Tanomaru-Filho, Mário

2017-01-01

The aim of this study was to evaluate the cytotoxicity and bioactivity of calcium silicate-based cements combined with niobium oxide (Nb2O5) micro and nanoparticles, comparing the response in different cell lines. This evaluation used four cell lines: two primary cultures (human dental pulp cells - hDPCs and human dental follicle cells - hDFCs) and two immortalized cultures (human osteoblast-like cells - Saos-2 and mouse periodontal ligament cells - mPDL). The tested materials were: White Portland Cement (PC), mineral trioxide aggregate (MTA), white Portland cement combined with microparticles (PC/Nb2O5µ) or nanoparticles (PC/Nb2O5n) of niobium oxide (Nb2O5). Cytotoxicity was evaluated by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) and trypan blue exclusion assays and bioactivity by alkaline phosphatase (ALP) enzyme activity. Results were analyzed by ANOVA and Tukey test (a=0.05). PC/Nb2O5n presented similar or higher cell viability than PC/Nb2O5µ in all cell lines. Moreover, the materials presented similar or higher cell viability than MTA. Saos-2 exhibited high ALP activity, highlighting PC/Nb2O5µ material at 7 days of exposure. In conclusion, calcium silicate cements combined with micro and nanoparticles of Nb2O5 presented cytocompatibility and bioactivity, demonstrating the potential of Nb2O5 as an alternative radiopacifier agent for these cements. The different cell lines had similar response to cytotoxicity evaluation of calcium silicate cements. However, bioactivity was more accurately detected in human osteoblast-like cell line, Saos-2.

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

Detection of fetal cell-free DNA in maternal plasma for Down syndrome, Edward syndrome and Patau syndrome of high risk fetus

PubMed Central

Ke, Wei-Lin; Zhao, Wei-Hua; Wang, Xin-Yu

2015-01-01

Objective: The study aimed to validate the efficacy of detection of fetal cell-free DNA in maternal plasma of trisomy 21, 18 and 13 in a clinical setting. Methods: A total of 2340 women at high risk for Down syndrome based on maternal age, prenatal history or a positive sesum or sonographic screening test were offered prenatal noninvasive aneuploidy test. According to the prenatal noninvasive aneuploidy test, the pregnant women at high risk were offered amniocentesis karyotype analysis and the pregnant at low risk were followed up to make sure the newborn outcome. Results: The prenatal noninvasive aneuploidy test was positive for trisomy 21 in 17 cases, for trisomy 18 in 6 cases and for trisomy 13 in 1 case, which of all were confirmed by karyotype analysis. Newborns of low risk gestational woman detected by prenatal noninvasive aneuploidy for trisomy 21, 18, 13 were followed up and no one was found with trisomy. Conclusions: The prenatal noninvasive aneuploidy test is highly accurate for detection of trisomy 21, 18 and 13, which can be considered as a practical alternative for traditional invasive diagnostic procedures. PMID:26309618

Detection of fetal cell-free DNA in maternal plasma for Down syndrome, Edward syndrome and Patau syndrome of high risk fetus.

PubMed

Ke, Wei-Lin; Zhao, Wei-Hua; Wang, Xin-Yu

2015-01-01

The study aimed to validate the efficacy of detection of fetal cell-free DNA in maternal plasma of trisomy 21, 18 and 13 in a clinical setting. A total of 2340 women at high risk for Down syndrome based on maternal age, prenatal history or a positive sesum or sonographic screening test were offered prenatal noninvasive aneuploidy test. According to the prenatal noninvasive aneuploidy test, the pregnant women at high risk were offered amniocentesis karyotype analysis and the pregnant at low risk were followed up to make sure the newborn outcome. The prenatal noninvasive aneuploidy test was positive for trisomy 21 in 17 cases, for trisomy 18 in 6 cases and for trisomy 13 in 1 case, which of all were confirmed by karyotype analysis. Newborns of low risk gestational woman detected by prenatal noninvasive aneuploidy for trisomy 21, 18, 13 were followed up and no one was found with trisomy. The prenatal noninvasive aneuploidy test is highly accurate for detection of trisomy 21, 18 and 13, which can be considered as a practical alternative for traditional invasive diagnostic procedures.

A study of alternative designs for a system to concentrate carbon dioxide in a hydrogen-depolarized cell

NASA Technical Reports Server (NTRS)

1973-01-01

Experimental results are presented on alternative designs for a hydrogen depolarized cell to concentrate CO2 in spacecraft atmospheric control systems. Data cover technical problems, methods for solving these problems, and the suitability of such a cell for CO2 removal and control of atmospheric humidity during the flight mode.

The Effect of Platinum Electrocatalyst on Membrane Degradation in Polymer Electrolyte Fuel Cells.

PubMed

Bodner, Merit; Cermenek, Bernd; Rami, Mija; Hacker, Viktor

2015-12-08

Membrane degradation is a severe factor limiting the lifetime of polymer electrolyte fuel cells. Therefore, obtaining a deeper knowledge is fundamental in order to establish fuel cells as competitive product. A segmented single cell was operated under open circuit voltage with alternating relative humidity. The influence of the catalyst layer on membrane degradation was evaluated by measuring a membrane without electrodes and a membrane-electrode-assembly under identical conditions. After 100 h of accelerated stress testing the proton conductivity of membrane samples near the anode and cathode was investigated by means of ex situ electrochemical impedance spectroscopy. The membrane sample near the cathode inlet exhibited twofold lower membrane resistance and a resulting twofold higher proton conductivity than the membrane sample near the anode inlet. The results from the fluoride ion analysis have shown that the presence of platinum reduces the fluoride emission rate; which supports conclusions drawn from the literature.

The Effect of Platinum Electrocatalyst on Membrane Degradation in Polymer Electrolyte Fuel Cells

PubMed Central

Bodner, Merit; Cermenek, Bernd; Rami, Mija; Hacker, Viktor

2015-01-01

Membrane degradation is a severe factor limiting the lifetime of polymer electrolyte fuel cells. Therefore, obtaining a deeper knowledge is fundamental in order to establish fuel cells as competitive product. A segmented single cell was operated under open circuit voltage with alternating relative humidity. The influence of the catalyst layer on membrane degradation was evaluated by measuring a membrane without electrodes and a membrane-electrode-assembly under identical conditions. After 100 h of accelerated stress testing the proton conductivity of membrane samples near the anode and cathode was investigated by means of ex situ electrochemical impedance spectroscopy. The membrane sample near the cathode inlet exhibited twofold lower membrane resistance and a resulting twofold higher proton conductivity than the membrane sample near the anode inlet. The results from the fluoride ion analysis have shown that the presence of platinum reduces the fluoride emission rate; which supports conclusions drawn from the literature. PMID:26670258

Alternative Test Method for Olefins in Gasoline

EPA Pesticide Factsheets

This action proposes to allow for an additional alternative test method for olefins in gasoline, ASTM D6550-05. The allowance of this additional alternative test method will provide more flexibility to the regulated industry.

In vitro protein expression: an emerging alternative to cell-based approaches.

PubMed

He, Mingyue

2011-04-30

Protein expression remains a bottleneck in the production of proteins. Owing to several advantages, cell-free translation is emerging as an alternative to cell-based methods for the generation of proteins. Recent advances have led to many novel applications of cell-free systems in biotechnology, proteomics and fundamental biological research. This special issue of New Biotechnology describes recent advances in cell-free protein expression systems and their applications. Copyright © 2010 Elsevier B.V. All rights reserved.

Inhibition of brain tumor cell proliferation by alternating electric fields

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

Jeong, Hyesun; Oh, Seung-ick; Hong, Sunghoi, E-mail: shong21@korea.ac.kr, E-mail: radioyoon@korea.ac.kr

2014-11-17

This study was designed to investigate the mechanism by which electric fields affect cell function, and to determine the optimal conditions for electric field inhibition of cancer cell proliferation. Low-intensity (<2 V/cm) and intermediate-frequency (100–300 kHz) alternating electric fields were applied to glioblastoma cell lines. These electric fields inhibited cell proliferation by inducing cell cycle arrest and abnormal mitosis due to the malformation of microtubules. These effects were significantly dependent on the intensity and frequency of applied electric fields.

Attacking Cancer’s Achilles Heel: Antagonism of Anti-Apoptotic BCL-2 Family Members

PubMed Central

Opferman, Joseph T.

2015-01-01

Malignant cells routinely violate cellular checkpoints that should initiate cell death in normal cells by triggering pro-apoptotic members of the BCL-2 family of proteins. To escape such death inducing signals, cancer cells often select for up regulation of anti-apoptotic BCL-2 family members including BCL-2, BCL-XL, BFL-1, BCL-W, and MCL-1. These family members prevent death by sequestering pro-apoptotic molecules. To counter this resistance mechanism, small molecule inhibitors of anti-apoptotic BCL-2 family members have been under development. These molecules have shown promise in pre-clinical and clinical testing to overcome apoptotic resistance, prompting cancer cells to undergo apoptosis. Alternatively, other strategies have taken advantage of the normal regulatory machinery controlling anti-apoptotic molecules and have used inhibitors of signaling pathways to down-modulate the expression of anti-apoptotic molecules thus tilting the balance in cancer cells to cell death. This review explores recent developments and strategies aimed at antagonizing anti-apoptotic BCL-2 family member action to promote the induction of cell death in cancer therapy. PMID:26293580

Insights on in vitro models for safety and toxicity assessment of cosmetic ingredients.

PubMed

Almeida, Andreia; Sarmento, Bruno; Rodrigues, Francisca

2017-03-15

According to the current European legislation, the safety assessment of each individual cosmetic ingredient of any formulation is the basis for the safety evaluation of a cosmetic product. Also, animal testing in the European Union is prohibited for cosmetic ingredients and products since 2004 and 2009, respectively. Additionally, the commercialization of any cosmetic products containing ingredients tested on animal models was forbidden in 2009. In consequence of these boundaries, the European Centre for the Validation of Alternative Methods (ECVAM) proposes a list of validated cell-based in vitro models for predicting the safety and toxicity of cosmetic ingredients. These models have been demonstrated as valuable and effective tools to overcome the limitations of animal in vivo studies. Although the use of in vitro cell-based models for the evaluation of absorption and permeability of cosmetic ingredients is widespread, a detailed study on the properties of these platforms and the in vitro-in vivo correlation compared with human data are required. Moreover, additional efforts must be taken to develop in vitro models to predict carcinogenicity, repeat dose toxicity and reproductive toxicity, for which no alternative in vitro methods are currently available. This review paper summarizes and characterizes the most relevant in vitro models validated by ECVAM employed to predict the safety and toxicology of cosmetic ingredients. Copyright © 2017 Elsevier B.V. All rights reserved.

High-throughput monitoring of major cell functions by means of lensfree video microscopy

PubMed Central

Kesavan, S. Vinjimore; Momey, F.; Cioni, O.; David-Watine, B.; Dubrulle, N.; Shorte, S.; Sulpice, E.; Freida, D.; Chalmond, B.; Dinten, J. M.; Gidrol, X.; Allier, C.

2014-01-01

Quantification of basic cell functions is a preliminary step to understand complex cellular mechanisms, for e.g., to test compatibility of biomaterials, to assess the effectiveness of drugs and siRNAs, and to control cell behavior. However, commonly used quantification methods are label-dependent, and end-point assays. As an alternative, using our lensfree video microscopy platform to perform high-throughput real-time monitoring of cell culture, we introduce specifically devised metrics that are capable of non-invasive quantification of cell functions such as cell-substrate adhesion, cell spreading, cell division, cell division orientation and cell death. Unlike existing methods, our platform and associated metrics embrace entire population of thousands of cells whilst monitoring the fate of every single cell within the population. This results in a high content description of cell functions that typically contains 25,000 – 900,000 measurements per experiment depending on cell density and period of observation. As proof of concept, we monitored cell-substrate adhesion and spreading kinetics of human Mesenchymal Stem Cells (hMSCs) and primary human fibroblasts, we determined the cell division orientation of hMSCs, and we observed the effect of transfection of siCellDeath (siRNA known to induce cell death) on hMSCs and human Osteo Sarcoma (U2OS) Cells. PMID:25096726

19 CFR 101.9 - Test programs or procedures; alternate requirements.

Code of Federal Regulations, 2014 CFR

2014-04-01

... test and the basis for selecting participants; and, (2) Post publication requirement. Within a... 19 Customs Duties 1 2014-04-01 2014-04-01 false Test programs or procedures; alternate... SECURITY; DEPARTMENT OF THE TREASURY GENERAL PROVISIONS § 101.9 Test programs or procedures; alternate...

Fish Alternatives in Environmental Risk Assessment: Overview of the Current Landscape

EPA Science Inventory

The need for alternative testing strategies has recently expanded into the realm of environmental risk assessment leading to the development of new alternatives to standard aquatic vertebrate testing such as the OECD 203 acute fish toxicity test. The fish embryo test (FET) is one...

A note on Poisson goodness-of-fit tests for ionizing radiation induced chromosomal aberration samples.

PubMed

Higueras, Manuel; González, J E; Di Giorgio, Marina; Barquinero, J F

2018-06-13

To present Poisson exact goodness-of-fit tests as alternatives and complements to the asymptotic u-test, which is the most widely used in cytogenetic biodosimetry, to decide whether a sample of chromosomal aberrations in blood cells comes from an homogeneous or inhomogeneous exposure. Three Poisson exact goodness-of-fit test from the literature are introduced and implemented in the R environment. A Shiny R Studio application, named GOF Poisson, has been updated for the purpose of giving support to this work. The three exact tests and the u-test are applied in chromosomal aberration data from clinical and accidental radiation exposure patients. It is observed how the u-test is not an appropriate approximation in small samples with small yield of chromosomal aberrations. Tools are provided to compute the three exact tests, which is not as trivial as the implementation of the u-test. Poisson exact goodness-of-fit tests should be considered jointly to the u-test for detecting inhomogeneous exposures in the cytogenetic biodosimetry practice.

Guide to detecting epidermal growth factor receptor (EGFR) mutations in ctDNA of patients with advanced non-small-cell lung cancer

PubMed Central

Normanno, Nicola; Denis, Marc G.; Thress, Kenneth S.; Ratcliffe, Marianne; Reck, Martin

2017-01-01

Cancer treatment is evolving towards therapies targeted at specific molecular abnormalities that drive tumor growth. Consequently, to determine which patients are eligible, accurate assessment of molecular aberrations within tumors is required. Obtaining sufficient tumor tissue for molecular testing can present challenges; therefore, circulating free tumor-derived DNA (ctDNA) found in blood plasma has been proposed as an alternative source of tumor DNA. The diagnostic utility of ctDNA for the detection of epidermal growth factor receptor (EGFR) mutations harbored in tumors of patients with advanced non-small-cell lung cancer (NSCLC) is supported by the results of several large studies/meta-analyses. However, recent real-world studies suggest that the performance of ctDNA testing varies between geographic regions/laboratories, demonstrating the need for standardized guidance. In this review, we outline recommendations for obtaining an accurate result using ctDNA, relating to pre-analytical plasma processing, ctDNA extraction, and appropriate EGFR mutation detection methods, based on clinical trial results. We conclude that there are several advantages associated with ctDNA, including the potential for repeated sampling particularly following progression after first-line tyrosine kinase inhibitor (TKI) therapy, as TKIs targeting resistance mutations (eg T790M) are now approved for use in the USA/EU/Japan (at time of writing). However, evidence suggests that ctDNA does not allow detection of EGFR mutations in all patients with known mutation-positive NSCLC. Therefore, although tumor tissue should be the first sample choice for EGFR testing at diagnosis, ctDNA is a promising alternative diagnostic approach. PMID:27980215

Determination of Interference During In Vitro Pyrogen Detection: Development and Characterization of a Cell-Based Assay.

PubMed

Palma, Linda; Rossetti, Francesca; Dominici, Sabrina; Buondelmonte, Costantina; Rocchi, Marco B L; Rizzardi, Gian P; Vallanti, Giuliana; Magnani, Mauro

Contamination of pharmaceutical products and medical devices with pyrogens such as endotoxins is the most common cause of systemic inflammation and, in worst cases, of septic shock. Thus, quantification of pyrogens is crucial. The limulus amebocyte lysate (LAL)-based assays are the reference tests for in vitro endotoxin detection, in association with the in vivo rabbit pyrogen test (RPT), according to European Pharmacopoeia (EP 2.6.14), and U.S. Pharmacopoeia (USP <85>). However, several substances interfere with LAL assay, while RPT is not accurate, not quantitative, and raises ethical limits. Biological assays, as monocyte activation tests, have been developed and included in European Pharmacopoeia (EP 7.0; 04/2010:20630) guidelines as an alternative to RPT and proved relevant to the febrile reaction in vivo. Because this reaction is carried out by endogenous mediators under the transcriptional control of nuclear factor-kappaB (NF-kappaB), we sought to determine whether a NF-kappaB reporter-gene assay, based on MonoMac-6 (MM6) cells, could reconcile the basic mechanism of innate immune response with the relevance of monocytoid cell lines to the organism reaction to endotoxins. This article describes both optimization and characterization of the reporter cells-based assay, which overall proved the linearity, accuracy, and precision of the test, and demonstrated the sensitivity of the assay to 0.24 EU/mL endotoxin, close to the pyrogenic threshold in humans. Moreover, the assay was experimentally compared to the LAL test in the evaluation of selected interfering samples. The good performance of the MM6 reporter test demonstrates the suitability of this assay to evaluate interfering or false-positive samples.

Internalization of rituximab and the efficiency of B Cell depletion in rheumatoid arthritis and systemic lupus erythematosus.

PubMed

Reddy, Venkat; Cambridge, Geraldine; Isenberg, David A; Glennie, Martin J; Cragg, Mark S; Leandro, Maria

2015-05-01

Rituximab, a type I anti-CD20 monoclonal antibody (mAb), induces incomplete B cell depletion in some patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), thus contributing to a poor clinical response. The mechanisms of this resistance remain elusive. The purpose of this study was to determine whether type II mAb are more efficient than type I mAb at depleting B cells from RA and SLE patients, whether internalization influences the efficiency of depletion, and whether Fcγ receptor type IIb (FcγRIIb) and the B cell receptor regulate this internalization process. We used an in vitro whole blood B cell-depletion assay to assess the efficiency of depletion, flow cytometry to study cell surface protein expression, and surface fluorescence-quenching assays to assess rituximab internalization, in samples from patients with RA and patients with SLE. Paired t-test or Mann-Whitney U test was used to compare groups, and Spearman's rank correlation test was used to assess correlation. We found that type II mAb internalized significantly less rituximab than type I mAb and depleted B cells from patients with RA and SLE at least 2-fold more efficiently than type I mAb. Internalization of rituximab was highly variable between patients, was regulated by FcγRIIb, and inversely correlated with cytotoxicity in whole blood B cell-depletion assays. The lowest levels of internalization were seen in IgD- B cells, including postswitched (IgD-CD27+) memory cells. Internalization of type I anti-CD20 mAb was also partially inhibited by anti-IgM stimulation. Variability in internalization of rituximab was observed and was correlated with impaired B cell depletion. Therefore, slower-internalizing type II mAb should be considered as alternative B cell-depleting agents for the treatment of RA and SLE. © 2015 The Authors. Arthritis & Rheumatology is published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology.

Three-dimensional culture of dental pulp stem cells in direct contact to tricalcium silicate cements.

PubMed

Widbiller, M; Lindner, S R; Buchalla, W; Eidt, A; Hiller, K-A; Schmalz, G; Galler, K M

2016-03-01

Calcium silicate cements are biocompatible dental materials applicable in contact with vital tissue. The novel tricalcium silicate cement Biodentine™ offers properties superior to commonly used mineral trioxide aggregate (MTA). Objective of this study was to evaluate its cytocompatibility and ability to induce differentiation and mineralization in three-dimensional cultures of dental pulp stem cells after direct contact with the material. Test materials included a new tricalcium silicate (Biodentine™, Septodont, Saint-Maur-des-Fossés, France), MTA (ProRoot® MTA, DENSPLY Tulsa Dental Specialities, Johnson City, TN, USA), glass ionomer (Ketac™ Molar Aplicap™, 3M ESPE, Seefeld, Germany), human dentin disks and polystyrene. Magnetic activated cell sorting for to the surface antigen STRO-1 was performed to gain a fraction enriched with mesenchymal stem cells. Samples were allowed to set and dental pulp stem cells in collagen carriers were placed on top. Scanning electron microscopy of tricalcium silicate cement surfaces with and without cells was conducted. Cell viability was measured for 14 days by MTT assay. Alkaline phosphatase activity was evaluated (days 3, 7, and 14) and expression of mineralization-associated genes (COL1A1, ALP, DSPP, and RUNX2) was quantified by real-time quantitative PCR. Nonparametric statistical analysis for cell viability and alkaline phosphatase data was performed to compare different materials as well as time points (Mann-Whitney U test, α = 0.05). Cell viability was highest on tricalcium silicate cement, followed by MTA. Viability on glass ionomer cement and dentin disks was significantly lower. Alkaline phosphatase activity was lower in cells on new tricalcium silicate cement compared to MTA, whereas expression patterns of marker genes were alike. Increased cell viability and similar levels of mineralization-associated gene expression in three-dimensional cell cultures on the novel tricalcium silicate cement and mineral trioxide aggregate indicate that the material is cytocompatible and bioactive. The tested new tricalcium silicate cement confirms its suitability as an alternative to MTA in vital pulp therapy.

Engineering cancer microenvironments for in vitro 3-D tumor models

PubMed Central

Asghar, Waseem; El Assal, Rami; Shafiee, Hadi; Pitteri, Sharon; Paulmurugan, Ramasamy; Demirci, Utkan

2017-01-01

The natural microenvironment of tumors is composed of extracellular matrix (ECM), blood vasculature, and supporting stromal cells. The physical characteristics of ECM as well as the cellular components play a vital role in controlling cancer cell proliferation, apoptosis, metabolism, and differentiation. To mimic the tumor microenvironment outside the human body for drug testing, two-dimensional (2-D) and murine tumor models are routinely used. Although these conventional approaches are employed in preclinical studies, they still present challenges. For example, murine tumor models are expensive and difficult to adopt for routine drug screening. On the other hand, 2-D in vitro models are simple to perform, but they do not recapitulate natural tumor microenvironment, because they do not capture important three-dimensional (3-D) cell–cell, cell–matrix signaling pathways, and multi-cellular heterogeneous components of the tumor microenvironment such as stromal and immune cells. The three-dimensional (3-D) in vitro tumor models aim to closely mimic cancer microenvironments and have emerged as an alternative to routinely used methods for drug screening. Herein, we review recent advances in 3-D tumor model generation and highlight directions for future applications in drug testing. PMID:28458612

Economic analysis of human papillomavirus triage, repeat cytology, and immediate colposcopy in management of women with minor cytological abnormalities in Sweden.

PubMed

Ostensson, Ellinor; Fröberg, Maria; Hjerpe, Anders; Zethraeus, Niklas; Andersson, Sonia

2010-10-01

To assess the cost-effectiveness of using human papillomavirus testing (HPV triage) in the management of women with minor cytological abnormalities in Sweden. An economic analysis based on a clinical trial, complemented with data from published meta-analyses on accuracy of HPV triage. The study takes perspective of the Swedish healthcare system. The Swedish population-based cervical cancer screening program. A decision analytic model was constructed to evaluate cost-effectiveness of HPV triage compared to repeat cytology and immediate colposcopy with biopsy, stratifying by index cytology (ASCUS = atypical squamous cells of undetermined significance, and LSIL = low-grade squamous intraepithelial lesion) and age (23-60 years, <30 years and ≥30 years). Costs, incremental cost, incremental effectiveness and incremental cost per additional high-grade lesion (CIN2+) detected. For women with ASCUS ≥30 years, HPV triage is the least costly alternative, whereas immediate colposcopy with biopsy provides the most effective option at an incremental cost-effectiveness ratio (ICER) of SEK 2,056 per additional case of CIN2+ detected. For LSIL (all age groups) and ASCUS (23-60 years and <30 years), HPV triage is dominated by immediate colposcopy and biopsy. Model results were sensitive to HPV test cost changes. With improved HPV testing techniques at lower costs, HPV triage can become a cost-effective alternative for follow-up of minor cytological abnormalities. Today, immediate colposcopy with biopsy is a cost-effective alternative compared to HPV triage and repeat cytology.

Demagnetization Tests Performed on a Linear Alternator for a Stirling Power Convertor

NASA Technical Reports Server (NTRS)

Geng, Steven M.; Niedra, Janis M.; Schwarze, Gene E.

2012-01-01

The NASA Glenn Research Center (GRC) is conducting in-house research on rare-earth permanent magnets and linear alternators to assist in developing free-piston Stirling convertors for radioisotope space power systems and for developing advanced linear alternator technology. This research continues at GRC, but, with the exception of Advanced Stirling Radioisotope Generator references, the work presented in this paper was conducted in 2005. A special arc-magnet characterization fixture was designed and built to measure the M-H characteristics of the magnets used in Technology Demonstration Convertors developed under the 110-W Stirling Radioisotope Generator (SRG110) project. This fixture was used to measure these characteristics of the arc magnets and to predict alternator demagnetization temperatures in the SRG110 application. Demagnetization tests using the TDC alternator on the Alternator Test Rig were conducted for two different magnet grades: Sumitomo Neomax 44AH and 42AH. The purpose of these tests was to determine the demagnetization temperatures of the magnets for the alternator under nominal loads. Measurements made during the tests included the linear alternator terminal voltage, current, average power, magnet temperatures, and stator temperatures. The results of these tests were found to be in good agreement with predictions. Alternator demagnetization temperatures in the Advanced Stirling Convertor (ASC-developed under the Advanced Stirling Radioisotope Generator project) were predicted as well because the prediction method had been validated through the SRG110 alternator tests. These predictions led to a specification for maximum temperatures of the ASC pressure vessel.

TAPAS: tools to assist the targeted protein quantification of human alternative splice variants.

PubMed

Yang, Jae-Seong; Sabidó, Eduard; Serrano, Luis; Kiel, Christina

2014-10-15

In proteomes of higher eukaryotes, many alternative splice variants can only be detected by their shared peptides. This makes it highly challenging to use peptide-centric mass spectrometry to distinguish and to quantify protein isoforms resulting from alternative splicing events. We have developed two complementary algorithms based on linear mathematical models to efficiently compute a minimal set of shared and unique peptides needed to quantify a set of isoforms and splice variants. Further, we developed a statistical method to estimate the splice variant abundances based on stable isotope labeled peptide quantities. The algorithms and databases are integrated in a web-based tool, and we have experimentally tested the limits of our quantification method using spiked proteins and cell extracts. The TAPAS server is available at URL http://davinci.crg.es/tapas/. luis.serrano@crg.eu or christina.kiel@crg.eu Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Induction of cell death in renal cell carcinoma with combination of D-fraction and vitamin C.

PubMed

Alexander, Bobby; Fishman, Andrew I; Eshghi, Majid; Choudhury, Muhammad; Konno, Sensuke

2013-09-01

Although several conventional therapeutic options for advanced renal cell carcinoma (RCC) are currently available, the unsatisfactory outcomes demand establishing more effective interventions. D-fraction (PDF), a bioactive proteoglucan of Maitake mushroom, demonstrates anticancer and immunomodulatory activities, which are also shown to be potentiated by vitamin C (VC). We thus hypothesized that a combination of PDF and VC (PDF + VC) could be an alternative approach to more effectively inhibit the growth of RCC. We examined the dose-dependent effects of PDF + VC on RCC cell viability and also performed biochemical assays to explore the growth regulatory mechanism. Human RCC, ACHN cell line, was employed and exposed to varying concentrations of PDF or VC and their combinations. Cell viability at specified times was determined by MTT assay. Lipid peroxidation assay, cell cycle analysis, and Western blot analysis were also performed. PDF or VC alone led to the significant reduction in cell viability at 72 hours with PDF >500 µg/mL and VC ≥300 µM. When various combinations of PDF and VC were tested, the combination of the ineffective concentrations of PDF (300 µg/mL) and VC (200 µM) resulted in ~90% cell death in 24 hours. Lipid peroxidation assay then indicated significantly (~2.5 fold) elevated oxidative stress with this PDF + VC. Cell cycle analysis also indicated a G1 cell cycle arrest following a 6-hour PDF + VC treatment. Western blots further revealed a downregulation of Bcl2, an upregulation of Bax, and proteolytic activation of PARP (poly[ADP-ribose] polymerase) in PDF + VC-treated cells, indicating induction of apoptosis. The present study demonstrates that the combination of PDF and VC can become highly cytotoxic, inducing severe cell death in ACHN cells. This cytotoxic mechanism appears to be primarily attributed to oxidative stress, accompanied by a G1 cell cycle arrest. Such cell death induced by PDF + VC could be more likely linked to apoptosis, as indicated by the modulation of apoptosis regulators (Bcl2, Bax, and PARP). Therefore, as PDF and VC may work synergistically to induce apoptotic cell death, they may have clinical implications in an alternative, improved therapeutic modality for advanced RCC.

33 CFR 154.2180 - Alternative testing program-Generally.

Code of Federal Regulations, 2014 CFR

2014-07-01

... Control Systems Alternative Analyzer and Pressure Sensor Reliability Testing § 154.2180 Alternative... and pressure sensor safety testing requirements provided by 33 CFR 154.2150(c) and 33 CFR 154.2250(c... 33 CFR 154.2181. (d) All pressure sensors/switches used in a VCS must be tested for safety system...

Rapid Identification of Mycobacterial Whole Cells in Solid and Liquid Culture Media by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry ▿

PubMed Central

Lotz, Aurélie; Ferroni, Agnès; Beretti, Jean-Luc; Dauphin, Brunhilde; Carbonnelle, Etienne; Guet-Revillet, Hélène; Veziris, Nicolas; Heym, Béate; Jarlier, Vincent; Gaillard, Jean-Louis; Pierre-Audigier, Catherine; Frapy, Eric; Berche, Patrick; Nassif, Xavier; Bille, Emmanuelle

2010-01-01

Mycobacterial identification is based on several methods: conventional biochemical tests that require several weeks for accurate identification, and molecular tools that are now routinely used. However, these techniques are expensive and time-consuming. In this study, an alternative method was developed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). This approach allows a characteristic mass spectral fingerprint to be obtained from whole inactivated mycobacterial cells. We engineered a strategy based on specific profiles in order to identify the most clinically relevant species of mycobacteria. To validate the mycobacterial database, a total of 311 strains belonging to 31 distinct species and 4 species complexes grown in Löwenstein-Jensen (LJ) and liquid (mycobacterium growth indicator tube [MGIT]) media were analyzed. No extraction step was required. Correct identifications were obtained for 97% of strains from LJ and 77% from MGIT media. No misidentification was noted. Our results, based on a very simple protocol, suggest that this system may represent a serious alternative for clinical laboratories to identify mycobacterial species. PMID:20943874

The poly(A)-binding protein nuclear 1 suppresses alternative cleavage and polyadenylation sites.

PubMed

Jenal, Mathias; Elkon, Ran; Loayza-Puch, Fabricio; van Haaften, Gijs; Kühn, Uwe; Menzies, Fiona M; Oude Vrielink, Joachim A F; Bos, Arnold J; Drost, Jarno; Rooijers, Koos; Rubinsztein, David C; Agami, Reuven

2012-04-27

Alternative cleavage and polyadenylation (APA) is emerging as an important layer of gene regulation. Factors controlling APA are largely unknown. We developed a reporter-based RNAi screen for APA and identified PABPN1 as a regulator of this process. Genome-wide analysis of APA in human cells showed that loss of PABPN1 resulted in extensive 3' untranslated region shortening. Messenger RNA transcription, stability analyses, and in vitro cleavage assays indicated enhanced usage of proximal cleavage sites (CSs) as the underlying mechanism. Using Cyclin D1 as a test case, we demonstrated that enhanced usage of proximal CSs compromises microRNA-mediated repression. Triplet-repeat expansion in PABPN1 (trePABPN1) causes autosomal-dominant oculopharyngeal muscular dystrophy (OPMD). The expression of trePABPN1 in both a mouse model of OPMD and human cells elicited broad induction of proximal CS usage, linked to binding to endogenous PABPN1 and its sequestration in nuclear aggregates. Our results elucidate a novel function for PABPN1 as a suppressor of APA. Copyright © 2012 Elsevier Inc. All rights reserved.

Cytotoxicity and genotoxicity of natural resin-based experimental endodontic sealers.

PubMed

Silva, Gleyce O; Cavalcanti, Bruno N; Oliveira, Tatiana R; Bin, Claudia V; Camargo, Samira E A; Camargo, Carlos H R

2016-05-01

The development of endodontic sealers based on natural resins seems to be promising, given their improved biological properties. This study evaluated the cytotoxic and genotoxic effects of two experimental root canal sealers, based on extracts from Copaifera multijuga and Ricinus communis (castor oil polymer), comparing them to synthetic resin-based sealers: a single methacrylate-based, a multi-methacrylate-based, and an epoxy resin-based sealers. Sealers were prepared, set, and exposed to cell culture medium for 24 h at 37 °C with CO2. V79 cells were exposed to serial dilutions of the extracts of each sealer for 24 h. Cell viability was measured by the MTT assay and genotoxicity was assessed by the formation of micronuclei. The single methacrylate-based sealer had the most cytotoxic effects, with significant reduction in cell viability in all dilutions of the extract. The castor oil polymer-based sealer was, on the other hand, the most biocompatible sealer, with no cytotoxic effects at any concentration. All tested sealers were not genotoxic, excepting the single methacrylate-based sealer. The tested natural resin-based sealers presented low cytotoxic and no genotoxic effects on cell cultures. These results may suggest a good alternative to develop new endodontic sealers, in order to achieve better biological response and healing, when compared to commercially available sealers.

Phosphoproteomics reveals that glycogen synthase kinase-3 phosphorylates multiple splicing factors and is associated with alternative splicing

PubMed Central

Shinde, Mansi Y.; Sidoli, Simone; Kulej, Katarzyna; Mallory, Michael J.; Radens, Caleb M.; Reicherter, Amanda L.; Myers, Rebecca L.; Barash, Yoseph; Lynch, Kristen W.; Garcia, Benjamin A.; Klein, Peter S.

2017-01-01

Glycogen synthase kinase-3 (GSK-3) is a constitutively active, ubiquitously expressed protein kinase that regulates multiple signaling pathways. In vitro kinase assays and genetic and pharmacological manipulations of GSK-3 have identified more than 100 putative GSK-3 substrates in diverse cell types. Many more have been predicted on the basis of a recurrent GSK-3 consensus motif ((pS/pT)XXX(S/T)), but this prediction has not been tested by analyzing the GSK-3 phosphoproteome. Using stable isotope labeling of amino acids in culture (SILAC) and MS techniques to analyze the repertoire of GSK-3–dependent phosphorylation in mouse embryonic stem cells (ESCs), we found that ∼2.4% of (pS/pT)XXX(S/T) sites are phosphorylated in a GSK-3–dependent manner. A comparison of WT and Gsk3a;Gsk3b knock-out (Gsk3 DKO) ESCs revealed prominent GSK-3–dependent phosphorylation of multiple splicing factors and regulators of RNA biosynthesis as well as proteins that regulate transcription, translation, and cell division. Gsk3 DKO reduced phosphorylation of the splicing factors RBM8A, SRSF9, and PSF as well as the nucleolar proteins NPM1 and PHF6, and recombinant GSK-3β phosphorylated these proteins in vitro. RNA-Seq of WT and Gsk3 DKO ESCs identified ∼190 genes that are alternatively spliced in a GSK-3–dependent manner, supporting a broad role for GSK-3 in regulating alternative splicing. The MS data also identified posttranscriptional regulation of protein abundance by GSK-3, with ∼47 proteins (1.4%) whose levels increased and ∼78 (2.4%) whose levels decreased in the absence of GSK-3. This study provides the first unbiased analysis of the GSK-3 phosphoproteome and strong evidence that GSK-3 broadly regulates alternative splicing. PMID:28916722

Chitin elicits CCL2 from airway epithelial cells and induces CCR2-dependent innate allergic inflammation in the lung

PubMed Central

Roy, René M.; Wüthrich, Marcel; Klein, Bruce S.

2012-01-01

Chitin exposure in the lung induces eosinophilia and alternative activation of macrophages, and is correlated with allergic airway disease. However, the mechanism underlying chitin-induced polarization of macrophages is poorly understood. Here, we show that chitin induces alternative activation of macrophages in vivo, but does not do so directly in vitro. We further show that airway epithelial cells bind chitin in vitro and produce CCL2 in response to chitin both in vitro and in vivo. Supernatants of chitin exposed epithelial cells promoted alternative activation of macrophages in vitro, whereas antibody neutralization of CCL2 in the supernate abolished the alternative activation of macrophages. CCL2 acted redundantly in vivo, but mice lacking the CCL2 receptor, CCR2, showed impaired alternative activation of macrophages in response to chitin, as measured by arginase I, CCL17 and CCL22 expression. Furthermore, CCR2KO mice exposed to chitin had diminished ROS products in the lung, blunted eosinophil and monocyte recruitment, and impaired eosinophil functions as measured by expression of CCL5, IL13 and CCL11. Thus, airway epithelial cells secrete CCL2 in response to chitin and CCR2 signaling mediates chitin-induced alternative activation of macrophages and allergic inflammation in vivo. PMID:22851704

Dynamics of subcellular compartmentalization of steroid receptors in living cells as a strategic screening method to determine the biological impact of suspected endocrine disruptors.

PubMed

Tyagi, Rakesh Kumar

2003-04-01

Although a number of screening methods being used for identifying potential endocrine disruptors have generated a wealth of information, a search for alternative combination of methods is still needed to overcome experimental artefacts. There are no generally accepted or validated screening methods for monitoring and studying impact of environmental endocrine disruptors. Also, no single assay can accurately predict all the deleterious effects of endocrine disruptors. For this reason various environmental protection agencies, mainly European and US, have urged that a battery of tests in current use need to be designed to assess their adequacy in detecting the effects of endocrine disruptors. Some details about endocrine disruptors and screening programs can be found at http://www.epa.gov/scipoly/oscpendo/whatis.htm. Several studies in recent years have used fusion proteins between steroid receptors (estrogen, androgen, progesterone, etc.) and green fluorescent protein (GFP) that can serve as an alternative potent screening method to study intracellular dynamics of receptors in living cells. An approach employing nucleocytoplasmic trafficking of steroid receptors as a parameter in response to potential xenobiotic chemicals in living cells may prove to be promising in terms of being direct, fast, reliable, simple and inexpensive. Copyright 2003 Elsevier Science Ltd.

Identification and validation of nebulized aerosol devices for sputum induction

PubMed Central

Davidson, Warren J; Dennis, John; The, Stephanie; Litoski, Belinda; Pieron, Cora; Leigh, Richard

2014-01-01

Induced sputum cell count measurement has proven reliability for evaluating airway inflammation in patients with asthma and other airway diseases. Although the use of nebulizer devices for sputum induction is commonplace, they are generally labelled as single-patient devices by the manufacturer and, therefore, cannot be used for multiple patients in large clinical sputum induction programs due to infect ion-control requirements. Accordingly, this study investigated the aerosol characteristics of alternative devices that could be used in such programs. BACKGROUND: Induced sputum cell counts are a noninvasive and reliable method for evaluating the presence, type and degree of airway inflammation in patients with asthma. Currently, standard nebulizer devices used for sputum induction in multiple patients are labelled as single-patient devices by the manufacturer, which conflicts with infection prevention and control requirements. As such, these devices cannot feasibly be used in a clinical sputum induction program. Therefore, there is a need to identify alternative nebulizer devices that are either disposable or labelled for multi-patient use. OBJECTIVE: To apply validated rigorous, scientific testing methods to identify and validate commercially available nebulizer devices appropriate for use in a clinical sputum induction program. METHODS: Measurement of nebulized aerosol output and size for the selected nebulizer designs followed robust International Organization for Standardization methods. Sputum induction using two of these nebulizers was successfully performed on 10 healthy adult subjects. The cytotechnologist performing sputum cell counts was blinded to the type of nebulizer used. RESULTS: The studied nebulizers had variable aerosol outputs. The AeroNeb Solo (Aerogen, Ireland), Omron NE-U17 (Omron, Japan) and EASYneb II (Flaem Nuova, Italy) systems were found to have similar measurements of aerosol size. There was no significant difference in induced sputum cell results between the AeroNeb Solo and EASYneb II devices. DISCUSSION: There is a need for rigorous, scientific evaluation of nebulizer devices for clinical applications, including sputum induction, for measurement of cell counts. CONCLUSION: The present study was the most comprehensive analysis of different nebulizer devices for sputum induction to measure cell counts, and provides a framework for appropriate evaluation of nebulizer devices for induced sputum testing. PMID:24288700

Whole-exome sequencing for RH genotyping and alloimmunization risk in children with sickle cell anemia

PubMed Central

Flanagan, Jonathan M.; Vege, Sunitha; Luban, Naomi L. C.; Brown, R. Clark; Ware, Russell E.; Westhoff, Connie M.

2017-01-01

RH genes are highly polymorphic and encode the most complex of the 35 human blood group systems. This genetic diversity contributes to Rh alloimmunization in patients with sickle cell anemia (SCA) and is not avoided by serologic Rh-matched red cell transfusions. Standard serologic testing does not distinguish variant Rh antigens. Single nucleotide polymorphism (SNP)–based DNA arrays detect many RHD and RHCE variants, but the number of alleles tested is limited. We explored a next-generation sequencing (NGS) approach using whole-exome sequencing (WES) in 27 Rh alloimmunized and 27 matched non-alloimmunized patients with SCA who received chronic red cell transfusions and were enrolled in a multicenter study. We demonstrate that WES provides a comprehensive RH genotype, identifies SNPs not interrogated by DNA array, and accurately determines RHD zygosity. Among this multicenter cohort, we demonstrate an association between an altered RH genotype and Rh alloimmunization: 52% of Rh immunized vs 19% of non-immunized patients expressed variant Rh without co-expression of the conventional protein. Our findings suggest that RH allele variation in patients with SCA is clinically relevant, and NGS technology can offer a comprehensive alternative to targeted SNP-based testing. This is particularly relevant as NGS data becomes more widely available and could provide the means for reducing Rh alloimmunization in children with SCA. PMID:29296782

Review of ingot niobium as a material for superconducting radiofrequency accelerating cavities

NASA Astrophysics Data System (ADS)

Kneisel, P.; Ciovati, G.; Dhakal, P.; Saito, K.; Singer, W.; Singer, X.; Myneni, G. R.

2015-02-01

As a result of collaboration between Jefferson Lab and niobium manufacturer Companhia Brasileira de Metalurgia e Mineração (CBMM), ingot niobium was explored as a possible material for superconducting radiofrequency (SRF) cavity fabrication. The first single cell cavity from large-grain high purity niobium was fabricated and successfully tested at Jefferson Lab in 2004. This work triggered research activities in other SRF laboratories around the world. Large-grain (LG) niobium became not only an interesting alternative material for cavity builders, but also material scientists and surface scientists were eager to participate in the development of this technology. Many single cell cavities made from material of different suppliers have been tested successfully and several multi-cell cavities have shown performances comparable to the best cavities made from standard fine-grain niobium. Several 9-cell cavities fabricated by Research Instruments and tested at DESY exceeded the best performing fine grain cavities with a record accelerating gradient of Eacc=45.6 MV/m. The quality factor of those cavities was also higher than that of fine-grain (FG) cavities processed with the same methods. Such performance levels push the state-of-the art of SRF technology and are of great interest for future accelerators. This contribution reviews the development of ingot niobium technology and highlights some of the differences compared to standard FG material and opportunities for further developments.

Discrimination of skin sensitizers from non-sensitizers by interleukin-1α and interleukin-6 production on cultured human keratinocytes.

PubMed

Jung, Daun; Che, Jeong-Hwan; Lim, Kyung-Min; Chun, Young-Jin; Heo, Yong; Seok, Seung Hyeok

2016-09-01

In vitro testing methods for classifying sensitizers could be valuable alternatives to in vivo sensitization testing using animal models, such as the murine local lymph node assay (LLNA) and the guinea pig maximization test (GMT), but there remains a need for in vitro methods that are more accurate and simpler to distinguish skin sensitizers from non-sensitizers. Thus, the aim of our study was to establish an in vitro assay as a screening tool for detecting skin sensitizers using the human keratinocyte cell line, HaCaT. HaCaT cells were exposed to 16 relevant skin sensitizers and 6 skin non-sensitizers. The highest dose used was the dose causing 75% cell viability (CV75) that we determined by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The levels of extracellular production of interleukin-1α (IL-1α) and IL-6 were measured. The sensitivity of IL-1α was 63%, specificity was 83% and accuracy was 68%. In the case of IL-6, sensitivity: 69%, specificity: 83% and accuracy: 73%. Thus, this study suggests that measuring extracellular production of pro-inflammatory cytokines IL-1α and IL-6 by human HaCaT cells may potentially classify skin sensitizers from non-sensitizers. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Review of ingot niobium as a material for superconducting radiofrequency accelerating cavities

DOE PAGES

Kneisel, P.; Ciovati, G.; Dhakal, P.; ...

2014-12-01

As a result of collaboration between Jefferson Lab and niobium manufacturer Companhia Brasileira de Metalurgia e Mineração (CBMM), ingot niobium was explored as a possible material for superconducting radiofrequency (SRF) cavity fabrication. The first single cell cavity from large-grain high purity niobium was fabricated and successfully tested at Jefferson Lab in 2004. This work triggered research activities in other SRF laboratories around the world. The large-grain (LG) niobium became not only an interesting alternative material for cavity builders, but also material scientists and surface scientists were eager to participate in the development of this technology. Many single cell cavities mademore » from material of different suppliers have been tested successfully and several multi-cell cavities have shown performances comparable to the best cavities made from standard fine-grain niobium. Several 9-cell cavities fabricated by Research Instruments and tested at DESY exceeded the best performing fine grain cavities with a record accelerating gradient of E acc=45.6 MV/m. The quality factor of those cavities was also higher than that of fine-grain (FG) cavities processed with the same methods. Such performance levels push the state-of-the art of SRF technology and are of great interest for future accelerators. This contribution reviews the development of ingot niobium technology and highlights some of the differences compared to standard FG material and opportunities for further developments.« less

Development of an in vitro alternative assay method for vaginal irritation.

PubMed

Ayehunie, Seyoum; Cannon, Chris; Larosa, Karen; Pudney, Jeffrey; Anderson, Deborah J; Klausner, Mitchell

2011-01-11

The vaginal mucosa is commonly exposed to chemicals and therapeutic agents that may result in irritation and/or inflammation. In addition to acute effects, vaginal irritation and inflammation can make women more susceptible to infections such as HIV-1 and herpes simplex virus 2 (HSV-2). Hence, the vaginal irritation potential of feminine care formulations and vaginally administered therapeutic agents is a significant public health concern. Traditionally, testing of such materials has been performed using the rabbit vaginal irritation (RVI) assay. In the current study, we investigated whether the organotypic, highly differentiated EpiVaginal™ tissue could be used as a non-animal alternative to the RVI test. The EpiVaginal tissue was exposed to a single application of ingredients commonly found in feminine hygiene products and the effects on tissue viability (MTT assay), barrier disruption (measured by transepithelial electrical resistance, TEER and sodium fluorescein (NaFl) leakage), and inflammatory cytokine release (interleukin (IL)-1α, IL-1β, IL-6, and IL-8) patterns were examined. When compared to untreated controls, two irritating ingredients, nonoxynol 9 and benzalkonium chloride, reduced tissue viability to <40% and TEER to <60% while increasing NaFl leakage by 11-24% and IL-1α and IL-1β release by >100%. Four other non-irritating materials had minimal effects on these parameters. Assay reproducibility was confirmed by testing the chemicals using three different tissue production lots and by using tissues reconstructed from cells obtained from three different donors. Coefficients of variation between tissue lots reconstructed with cells obtained from the same donor or lots reconstructed with cells obtained from different donors were less than 10% and 12%, respectively. In conclusion, decreases in tissue viability and barrier function and increases in IL-1α and IL-1β release appear to be useful endpoints for preclinical screening of topically applied chemicals and formulations for their vaginal irritation potential. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Fish as research tools: alternatives to in vivo experiments.

PubMed

Schaeck, Marlien; Van den Broeck, Wim; Hermans, Katleen; Decostere, Annemie

2013-07-01

The use of fish in scientific research is increasing worldwide, due to both the rapid expansion of the fish farming industry and growing awareness of questions concerning the humane use of mammalian models in basic research and chemical testing. As fish are lower on the evolutionary scale than mammals, they are considered to be less sentient. Fish models are providing researchers, and those concerned with animal welfare, with opportunities for adhering to the Three Rs principles of refinement, reduction and replacement. However, it should be kept in mind that fish should also be covered by the principles of the Three Rs. Indeed, various studies have shown that fish are capable of nociception, and of experiencing pain in a manner analogous to that in mammals. Thus, emphasis needs to be placed on the development of alternatives that replace, as much as possible, the use of all living vertebrate animals, including fish. This review gives the first comprehensive and critical overview of the existing alternatives for live fish experimental studies. The alternative methods described range from cell and tissue cultures, organ and perfusion models, and embryonic models, to in silico computer and mathematical models. This article aspires to guide scientists in the adoption of the correct alternative methods in their research, and, whenever possible, to reduce the use of live fish. 2013 FRAME.

Clinical applications of magnetic nanoparticles for hyperthermia.

PubMed

Thiesen, Burghard; Jordan, Andreas

2008-09-01

Magnetic fluids are increasingly used for clinical applications such as drug delivery, magnetic resonance imaging and magnetic fluid hyperthermia. The latter technique that has been developed as a cancer treatment for several decades comprises the injection of magnetic nanoparticles into tumors and their subsequent heating in an alternating magnetic field. Depending on the applied temperature and the duration of heating this treatment either results in direct tumor cell killing or makes the cells more susceptible to concomitant radio- or chemotherapy. Numerous groups are working in this field worldwide, but only one approach has been tested in clinical trials so far. Here, we summarize the clinical data gained in these studies on magnetic fluid induced hyperthermia.

Prioritizing CD4 Count Monitoring in Response to ART in Resource-Constrained Settings: A Retrospective Application of Prediction-Based Classification

PubMed Central

Liu, Yan; Li, Xiaohong; Johnson, Margaret; Smith, Collette; Kamarulzaman, Adeeba bte; Montaner, Julio; Mounzer, Karam; Saag, Michael; Cahn, Pedro; Cesar, Carina; Krolewiecki, Alejandro; Sanne, Ian; Montaner, Luis J.

2012-01-01

Background Global programs of anti-HIV treatment depend on sustained laboratory capacity to assess treatment initiation thresholds and treatment response over time. Currently, there is no valid alternative to CD4 count testing for monitoring immunologic responses to treatment, but laboratory cost and capacity limit access to CD4 testing in resource-constrained settings. Thus, methods to prioritize patients for CD4 count testing could improve treatment monitoring by optimizing resource allocation. Methods and Findings Using a prospective cohort of HIV-infected patients (n = 1,956) monitored upon antiretroviral therapy initiation in seven clinical sites with distinct geographical and socio-economic settings, we retrospectively apply a novel prediction-based classification (PBC) modeling method. The model uses repeatedly measured biomarkers (white blood cell count and lymphocyte percent) to predict CD4+ T cell outcome through first-stage modeling and subsequent classification based on clinically relevant thresholds (CD4+ T cell count of 200 or 350 cells/µl). The algorithm correctly classified 90% (cross-validation estimate = 91.5%, standard deviation [SD] = 4.5%) of CD4 count measurements <200 cells/µl in the first year of follow-up; if laboratory testing is applied only to patients predicted to be below the 200-cells/µl threshold, we estimate a potential savings of 54.3% (SD = 4.2%) in CD4 testing capacity. A capacity savings of 34% (SD = 3.9%) is predicted using a CD4 threshold of 350 cells/µl. Similar results were obtained over the 3 y of follow-up available (n = 619). Limitations include a need for future economic healthcare outcome analysis, a need for assessment of extensibility beyond the 3-y observation time, and the need to assign a false positive threshold. Conclusions Our results support the use of PBC modeling as a triage point at the laboratory, lessening the need for laboratory-based CD4+ T cell count testing; implementation of this tool could help optimize the use of laboratory resources, directing CD4 testing towards higher-risk patients. However, further prospective studies and economic analyses are needed to demonstrate that the PBC model can be effectively applied in clinical settings. Please see later in the article for the Editors' Summary PMID:22529752

Potential Alternatives Report for Validation of Alternatives to Aliphatic Isocyanate Polyurethanes

NASA Technical Reports Server (NTRS)

Lewis, pattie

2011-01-01

Identifying and selecting alternative materials and technologies that have the potential to reduce the identified HazMats and hazardous air pollutants (HAPs), while incorporating sound corrosion prevention and control technologies, is a complicated task due to the fast pace at which new technologies emerge and rules change. The alternatives are identified through literature searches, electronic database and Internet searches, surveys, and/or personal and professional contacts. Available test data was then compiled on the proposed alternatives to determine if the materials meet the test objectives or if further)laboratory or field-testing will be required. After reviewing technical information documented in the PAR, government representatives, technical representatives from the affected facilities, and other stakeholders involved in the process will select the list of viable alternative coatings for consideration and testing under the project's Joint Test Protocol entitled Joint Test Protocol for Validation of Alternatives to Aliphatic Isocyanate Polyurethanes and Field Test Plan entitled Field Evaluations Test Plan for Validation of Alternatives to Aliphatic Isocyanate Polyurethanes, both prepared by ITB. Test results will be reported in a Joint Test Report upon completion oftesting. The selection rationale and conclusions are documented in this PAR. A cost benefit analysis will be prepared to quantify the estimated capital and process costs of coating alternatives and cost savings relative to the current coating processes, however, some initial cost data has been included in this PAR. For this coatings project, isocyanates, as found in aliphatic isocyanate polyurethanes, were identified as the target HazMat to be eliminated. Table 1-1 lists the target HazMats, the related process and application, current specifications, and affected programs.

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

The Nuclear Option: Evidence Implicating the Cell Nucleus in Mechanotransduction.

PubMed

Szczesny, Spencer E; Mauck, Robert L

2017-02-01

Biophysical stimuli presented to cells via microenvironmental properties (e.g., alignment and stiffness) or external forces have a significant impact on cell function and behavior. Recently, the cell nucleus has been identified as a mechanosensitive organelle that contributes to the perception and response to mechanical stimuli. However, the specific mechanotransduction mechanisms that mediate these effects have not been clearly established. Here, we offer a comprehensive review of the evidence supporting (and refuting) three hypothetical nuclear mechanotransduction mechanisms: physical reorganization of chromatin, signaling at the nuclear envelope, and altered cytoskeletal structure/tension due to nuclear remodeling. Our goal is to provide a reference detailing the progress that has been made and the areas that still require investigation regarding the role of nuclear mechanotransduction in cell biology. Additionally, we will briefly discuss the role that mathematical models of cell mechanics can play in testing these hypotheses and in elucidating how biophysical stimulation of the nucleus drives changes in cell behavior. While force-induced alterations in signaling pathways involving lamina-associated polypeptides (LAPs) (e.g., emerin and histone deacetylase 3 (HDAC3)) and transcription factors (TFs) located at the nuclear envelope currently appear to be the most clearly supported mechanism of nuclear mechanotransduction, additional work is required to examine this process in detail and to more fully test alternative mechanisms. The combination of sophisticated experimental techniques and advanced mathematical models is necessary to enhance our understanding of the role of the nucleus in the mechanotransduction processes driving numerous critical cell functions.

The Nuclear Option: Evidence Implicating the Cell Nucleus in Mechanotransduction

PubMed Central

Szczesny, Spencer E.; Mauck, Robert L.

2017-01-01

Biophysical stimuli presented to cells via microenvironmental properties (e.g., alignment and stiffness) or external forces have a significant impact on cell function and behavior. Recently, the cell nucleus has been identified as a mechanosensitive organelle that contributes to the perception and response to mechanical stimuli. However, the specific mechanotransduction mechanisms that mediate these effects have not been clearly established. Here, we offer a comprehensive review of the evidence supporting (and refuting) three hypothetical nuclear mechanotransduction mechanisms: physical reorganization of chromatin, signaling at the nuclear envelope, and altered cytoskeletal structure/tension due to nuclear remodeling. Our goal is to provide a reference detailing the progress that has been made and the areas that still require investigation regarding the role of nuclear mechanotransduction in cell biology. Additionally, we will briefly discuss the role that mathematical models of cell mechanics can play in testing these hypotheses and in elucidating how biophysical stimulation of the nucleus drives changes in cell behavior. While force-induced alterations in signaling pathways involving lamina-associated polypeptides (LAPs) (e.g., emerin and histone deacetylase 3 (HDAC3)) and transcription factors (TFs) located at the nuclear envelope currently appear to be the most clearly supported mechanism of nuclear mechanotransduction, additional work is required to examine this process in detail and to more fully test alternative mechanisms. The combination of sophisticated experimental techniques and advanced mathematical models is necessary to enhance our understanding of the role of the nucleus in the mechanotransduction processes driving numerous critical cell functions. PMID:27918797

An alternating pressure sequence proposal for an air-cell cushion for preventing pressure ulcers.

PubMed

Arias, Sandra; Cardiel, Eladio; Rogeli, Pablo; Mori, Taketoshi; Nakagami, Gojiro; Noguchi, Hiroshi; Sanada, Hiromi

2014-01-01

The distribution and release of pressure on ischial regions are two important parameters for evaluating the effectiveness of a cushion; especially the release of pressure over time on ischial tuberosities, which is significant for preventing pressure ulcers. The aim of this work is to evaluate the effect on interface pressure through the application of a proposed alternating pressure sequence for an air-cell cushion. Six healthy volunteers were asked to sit on the air cell cushion, in static and alternating modes, as well as on a typical foam cushion for 12 minutes. Interface pressure was monitored with a matrix sensor system. Interface pressure values on ischial tuberosities, user contact area and pressure distribution were analyzed. Results showed that IP on IT tends to increase in both foam and static cushions, while in alternating cushion IP on IT tends to decrease. User contact area was significantly larger in alternating cushion than in static or foam cushions. Moreover, there is a better pressure re-distribution with alternating cushion than with the other cushions. The goal of the alternating sequence is to redistribute pressure and stimulate the ischial regions in order to promote blood flow and prevent pressure occurring in wheelchair users.

Cytotoxic and genotoxic effects of defence secretion of Ulomoides dermestoides on A549 cells.

PubMed

Crespo, Rosana; Villaverde, M Luciana; Girotti, Juan R; Güerci, Alba; Juárez, M Patricia; de Bravo, Margarita G

2011-06-14

Ulomoides dermestoides (Fairmaire, 1893) is a cosmopolitan tenebrionid beetle reared by Argentine people who consume them alive as an alternative medicine in the treatment of different illnesses such as asthma, Parkinson's, diabetes, arthritis, HIV and specially cancer. To evaluate the cytotoxicity and DNA damage of the major volatile components released by Ulomoides dermestoides on human lung carcinoma epithelial cell line A549. The defence compounds of Ulomoides dermestoides were extracted with dichloromethane and analyzed and quantified by capillary gas chromatography. The toxicity effects of the beetle's extract against A549 cell line were evaluated. Cytotoxicity was evaluated by MTT test and Trypan blue assay and genotoxicity was evaluated by the comet assay. The synthetic compounds, individually or combined, were also tested in A549 cells and normal mononuclear human cells. The defence compounds of Ulomoides dermestoides extracted with dichloromethane (methyl-1,4-benzoquinones, ethyl-1,4-benzoquinones and 1-pentadecene as major components) showed cytotoxic activity on A549 cells demonstrated by MTT test and Trypan blue assay, with IC(50) values of 0.26equivalent/ml and 0.34equivalent/ml, respectively (1equivalent=amount of components extracted per beetle). The inhibition of A549 cell proliferation with the synthetic blend (1,4-benzoquinone and 1-pentadecene) or 1,4-benzoquinone alone was similar to that obtained with the insect extract. 1-Pentadecene showed no inhibitory effect. Low doses of insect extract or synthetic blend (0.15equivalent/ml) inhibited mononuclear cell proliferation by 72.2±2.7% and induced significant DNA damage both in tumor and mononuclear cells. Results of this study demonstrated that defence compounds of Ulomoides dermestoides reduced cell viability and induced DNA damage. We also concluded that the insect benzoquinones are primarily responsible for inducing cytotoxicity and genotoxicity in culture cells. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

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

Popp, R.A.; Popp, D.M.; Johnson, F.M.

Mice homozygous for a spontaneous mutation, in which the ..beta..-major globin gene is deleted, have clinical symptoms of ..beta..-thalassemia. These mice have a hypocellular, hypochromic, microcytic anemia that becomes more severe with increasing age. The defective red cell morphology, decreased osmotic fragility of erythrocytes and shortened red cell life span found in ..beta..-thalassemic mice are similar to those observed in human ..beta..-thalassemia. Synthesis of ..beta..-globin is depressed but not as much as might be expected because the expression of the..beta..-minor globin gene is enhanced to encode two to three times more globin than in normal mice. Splenomegaly, an enlarged poolmore » of stem cells for erythropoiesis, and iron overloading occur in older mice. The fact that these mice remain moderately healthy makes them a very suitable animal model in which to develop and test alternative techniques of gene therapy that could be successfully applied to the treatment of human thalassemia. Homozygous ..beta..-thalassemic mice have large deposits of iron in their tissues, which might make these mice also useful for in vivo tests of the effectiveness and possible long-term side effects for newly developed iron chelators.« less

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.

46 CFR 160.027-7 - Pre-approval tests for alternate platform designs.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 6 2014-10-01 2014-10-01 false Pre-approval tests for alternate platform designs. 160... § 160.027-7 Pre-approval tests for alternate platform designs. (a) The tests in this section are for... deformation as a result of this test. (c) The float body must be supported so that the platform is suspended...

46 CFR 160.027-7 - Pre-approval tests for alternate platform designs.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 6 2011-10-01 2011-10-01 false Pre-approval tests for alternate platform designs. 160... § 160.027-7 Pre-approval tests for alternate platform designs. (a) The tests in this section are for... deformation as a result of this test. (c) The float body must be supported so that the platform is suspended...

46 CFR 160.027-7 - Pre-approval tests for alternate platform designs.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 6 2013-10-01 2013-10-01 false Pre-approval tests for alternate platform designs. 160... § 160.027-7 Pre-approval tests for alternate platform designs. (a) The tests in this section are for... deformation as a result of this test. (c) The float body must be supported so that the platform is suspended...

46 CFR 160.027-7 - Pre-approval tests for alternate platform designs.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 6 2010-10-01 2010-10-01 false Pre-approval tests for alternate platform designs. 160... § 160.027-7 Pre-approval tests for alternate platform designs. (a) The tests in this section are for... deformation as a result of this test. (c) The float body must be supported so that the platform is suspended...

46 CFR 160.027-7 - Pre-approval tests for alternate platform designs.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 6 2012-10-01 2012-10-01 false Pre-approval tests for alternate platform designs. 160... § 160.027-7 Pre-approval tests for alternate platform designs. (a) The tests in this section are for... deformation as a result of this test. (c) The float body must be supported so that the platform is suspended...

Conserved functional antagonism of CELF and MBNL proteins controls stem cell-specific alternative splicing in planarians

PubMed Central

Solana, Jordi; Irimia, Manuel; Ayoub, Salah; Orejuela, Marta Rodriguez; Zywitza, Vera; Jens, Marvin; Tapial, Javier; Ray, Debashish; Morris, Quaid; Hughes, Timothy R; Blencowe, Benjamin J; Rajewsky, Nikolaus

2016-01-01

In contrast to transcriptional regulation, the function of alternative splicing (AS) in stem cells is poorly understood. In mammals, MBNL proteins negatively regulate an exon program specific of embryonic stem cells; however, little is known about the in vivo significance of this regulation. We studied AS in a powerful in vivo model for stem cell biology, the planarian Schmidtea mediterranea. We discover a conserved AS program comprising hundreds of alternative exons, microexons and introns that is differentially regulated in planarian stem cells, and comprehensively identify its regulators. We show that functional antagonism between CELF and MBNL factors directly controls stem cell-specific AS in planarians, placing the origin of this regulatory mechanism at the base of Bilaterians. Knockdown of CELF or MBNL factors lead to abnormal regenerative capacities by affecting self-renewal and differentiation sets of genes, respectively. These results highlight the importance of AS interactions in stem cell regulation across metazoans. DOI: http://dx.doi.org/10.7554/eLife.16797.001 PMID:27502555

49 CFR Appendix B to Part 178 - Alternative Leakproofness Test Methods

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Alternative Leakproofness Test Methods B Appendix... FOR PACKAGINGS Pt. 178, App. B Appendix B to Part 178—Alternative Leakproofness Test Methods In addition to the method prescribed in § 178.604 of this subchapter, the following leakproofness test methods...

76 FR 5319 - Regulation of Fuel and Fuel Additives: Alternative Test Method for Olefins in Gasoline

Federal Register 2010, 2011, 2012, 2013, 2014

2011-01-31

... Regulation of Fuel and Fuel Additives: Alternative Test Method for Olefins in Gasoline AGENCY: Environmental... proposing to allow refiners and laboratories to use an alternative test method for olefin content in... test method for compliance measurement while maintaining environmental benefits achieved from our fuels...

26 CFR 53.4942(b)-2 - Alternative tests.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 26 Internal Revenue 17 2012-04-01 2012-04-01 false Alternative tests. 53.4942(b)-2 Section 53.4942....4942(b)-2 Alternative tests. (a) Assets test—(1) In general. A private foundation will satisfy the assets test under the provisions of this paragraph if substantially more than half of the foundation's...

Estrogen receptor-alpha promotes alternative macrophage activation during cutaneous repair.

PubMed

Campbell, Laura; Emmerson, Elaine; Williams, Helen; Saville, Charis R; Krust, Andrée; Chambon, Pierre; Mace, Kimberly A; Hardman, Matthew J

2014-09-01

Efficient local monocyte/macrophage recruitment is critical for tissue repair. Recruited macrophages are polarized toward classical (proinflammatory) or alternative (prohealing) activation in response to cytokines, with tight temporal regulation crucial for efficient wound repair. Estrogen acts as a potent anti-inflammatory regulator of cutaneous healing. However, an understanding of estrogen/estrogen receptor (ER) contribution to macrophage polarization and subsequent local effects on wound healing is lacking. Here we identify, to our knowledge previously unreported, a role whereby estrogen receptor α (ERα) signaling preferentially polarizes macrophages from a range of sources to an alternative phenotype. Cell-specific ER ablation studies confirm an in vivo role for inflammatory cell ERα, but not ERβ, in poor healing associated with an altered cytokine profile and fewer alternatively activated macrophages. Furthermore, we reveal intrinsic changes in ERα-deficient macrophages, which are unable to respond to alternative activation signals in vitro. Collectively, our data reveal that inflammatory cell-expressed ERα promotes alternative macrophage polarization, which is beneficial for timely healing. Given the diverse physiological roles of ERs, these findings will likely be of relevance to many pathologies involving excessive inflammation.

Radiopacity and cytotoxicity of Portland cement associated with niobium oxide micro and nanoparticles

PubMed Central

MESTIERI, Leticia Boldrin; TANOMARU-FILHO, Mário; GOMES-CORNÉLIO, Ana Livia; SALLES, Loise Pedrosa; BERNARDI, Maria Inês Basso; GUERREIRO-TANOMARU, Juliane Maria

2014-01-01

Objective Mineral Trioxide Aggregate (MTA) is composed of Portland Cement (PC) and bismuth oxide (BO). Replacing BO for niobium oxide (NbO) microparticles (Nbµ) or nanoparticles (Nbη) may improve radiopacity and bioactivity. The aim of this study was to evaluate the radiopacity and cytotoxicity of the materials: 1) PC; 2) White MTA; 3) PC+30% Nbµ; 4) PC+30% Nbη. Material and Methods For the radiopacity test, specimens of the different materials were radiographed along an aluminum step-wedge. For cell culture assays, Saos-2 osteoblastic-cells (ATCC HTB-85) were used. Cell viability was evaluated through MTT assay, and bioactivity was assessed by alkaline phosphatase activity assay. Results The results demonstrated higher radiopacity for MTA, followed by Nbµ and Nbη, which had similar values. Cell culture analysis showed that PC and PC+NbO associations promoted greater cell viability than MTA. Conclusions It was concluded that the combination of PC+NbO is a potential alternative for composition of MTA. PMID:25591023

Radiopacity and cytotoxicity of Portland cement associated with niobium oxide micro and nanoparticles.

PubMed

Mestieri, Leticia Boldrin; Tanomaru-Filho, Mário; Gomes-Cornélio, Ana Livia; Salles, Loise Pedrosa; Bernardi, Maria Inês Basso; Guerreiro-Tanomaru, Juliane Maria

2014-01-01

Mineral Trioxide Aggregate (MTA) is composed of Portland Cement (PC) and bismuth oxide (BO). Replacing BO for niobium oxide (NbO) microparticles (Nbµ) or nanoparticles (Nbη) may improve radiopacity and bioactivity. The aim of this study was to evaluate the radiopacity and cytotoxicity of the materials: (1) PC; (2) White MTA; (3) PC+30% Nbµ; (4) PC+30% Nbη. For the radiopacity test, specimens of the different materials were radiographed along an aluminum step-wedge. For cell culture assays, Saos-2 osteoblastic-cells (ATCC HTB-85) were used. Cell viability was evaluated through MTT assay, and bioactivity was assessed by alkaline phosphatase activity assay. The results demonstrated higher radiopacity for MTA, followed by Nbµ and Nbη, which had similar values. Cell culture analysis showed that PC and PC+NbO associations promoted greater cell viability than MTA. It was concluded that the combination of PC+NbO is a potential alternative for composition of MTA.

Can currently available non-animal methods detect pre and pro-haptens relevant for skin sensitization?

PubMed

Patlewicz, Grace; Casati, Silvia; Basketter, David A; Asturiol, David; Roberts, David W; Lepoittevin, Jean-Pierre; Worth, Andrew P; Aschberger, Karin

2016-12-01

Predictive testing to characterize substances for their skin sensitization 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 have provided strong impetus to develop non-animal alternatives. Three test methods have undergone OECD validation: the direct peptide reactivity assay (DPRA), the KeratinoSens™ and the human Cell Line Activation Test (h-CLAT). Whilst these methods perform relatively well in predicting LLNA results, a concern raised is their ability to predict chemicals that need activation to be sensitizing (pre- or pro-haptens). This current study reviewed an EURL ECVAM dataset of 127 substances for which information was available in the LLNA and three non-animal test methods. Twenty eight of the sensitizers needed to be activated, with the majority being pre-haptens. These were correctly identified by 1 or more of the test methods. Six substances were categorized exclusively as pro-haptens, but were correctly identified by at least one of the cell-based assays. The analysis here showed that skin metabolism was not likely to be a major consideration for assessing sensitization potential and that sensitizers requiring activation could be identified correctly using one or more of the current non-animal methods. Published by Elsevier Inc.

Rapid Detection of Cell-Free Mycobacterium tuberculosis DNA in Tuberculous Pleural Effusion.

PubMed

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

2017-05-01

Tuberculous pleurisy is one of the most common types of extrapulmonary tuberculosis, but its diagnosis remains difficult. In this study, we report for the first time on the detection of cell-free Mycobacterium tuberculosis DNA in pleural effusion and an evaluation of a newly developed molecular assay for the detection of cell-free Mycobacterium tuberculosis DNA. A total of 78 patients with pleural effusion, 60 patients with tuberculous pleurisy, and 18 patients with alternative diseases were included in this study. Mycobacterial culture, the Xpert MTB/RIF assay, the adenosine deaminase assay, the T-SPOT.TB assay, and the cell-free Mycobacterium tuberculosis DNA assay were performed on all the pleural effusion samples. The cell-free Mycobacterium tuberculosis DNA assay and adenosine deaminase assay showed significantly higher sensitivities of 75.0% and 68.3%, respectively, than mycobacterial culture and the Xpert MTB/RIF assay, which had sensitivities of 26.7% and 20.0%, respectively ( P < 0.01). All four of these tests showed good specificities: 88.9% for the adenosine deaminase assay and 100% for the remaining three assays. The T-SPOT.TB assay with pleural effusion showed the highest sensitivity of 95.0% but the lowest specificity of 38.9%. The cell-free Mycobacterium tuberculosis DNA assay detected as few as 1.25 copies of IS 6110 per ml of pleural effusion and showed good accordance of the results between repeated tests ( r = 0.978, P = 2.84 × 10 -10 ). These data suggest that the cell-free Mycobacterium tuberculosis DNA assay is a rapid and accurate molecular test which provides direct evidence of Mycobacterium tuberculosis etiology. Copyright © 2017 American Society for Microbiology.

Rapid Detection of Cell-Free Mycobacterium tuberculosis DNA in Tuberculous Pleural Effusion

PubMed Central

Yang, Xinting; Liu, Zichen; Li, Kun

2017-01-01

ABSTRACT Tuberculous pleurisy is one of the most common types of extrapulmonary tuberculosis, but its diagnosis remains difficult. In this study, we report for the first time on the detection of cell-free Mycobacterium tuberculosis DNA in pleural effusion and an evaluation of a newly developed molecular assay for the detection of cell-free Mycobacterium tuberculosis DNA. A total of 78 patients with pleural effusion, 60 patients with tuberculous pleurisy, and 18 patients with alternative diseases were included in this study. Mycobacterial culture, the Xpert MTB/RIF assay, the adenosine deaminase assay, the T-SPOT.TB assay, and the cell-free Mycobacterium tuberculosis DNA assay were performed on all the pleural effusion samples. The cell-free Mycobacterium tuberculosis DNA assay and adenosine deaminase assay showed significantly higher sensitivities of 75.0% and 68.3%, respectively, than mycobacterial culture and the Xpert MTB/RIF assay, which had sensitivities of 26.7% and 20.0%, respectively (P < 0.01). All four of these tests showed good specificities: 88.9% for the adenosine deaminase assay and 100% for the remaining three assays. The T-SPOT.TB assay with pleural effusion showed the highest sensitivity of 95.0% but the lowest specificity of 38.9%. The cell-free Mycobacterium tuberculosis DNA assay detected as few as 1.25 copies of IS6110 per ml of pleural effusion and showed good accordance of the results between repeated tests (r = 0.978, P = 2.84 × 10−10). These data suggest that the cell-free Mycobacterium tuberculosis DNA assay is a rapid and accurate molecular test which provides direct evidence of Mycobacterium tuberculosis etiology. PMID:28275073

PDLLA honeycomb-like scaffolds with a high loading of superhydrophilic graphene/multi-walled carbon nanotubes promote osteoblast in vitro functions and guided in vivo bone regeneration.

PubMed

Silva, Edmundo; Vasconcellos, Luana Marotta Reis de; Rodrigues, Bruno V M; Dos Santos, Danilo Martins; Campana-Filho, Sergio P; Marciano, Fernanda Roberta; Webster, Thomas J; Lobo, Anderson Oliveira

2017-04-01

Herein, we developed honeycomb-like scaffolds by combining poly (d, l-lactic acid) (PDLLA) with a high amount of graphene/multi-walled carbon nanotube oxides (MWCNTO-GO, 50% w/w). From pristine multi-walled carbon nanotubes (MWCNT) powders, we produced MWCNTO-GO via oxygen plasma etching (OPE), which promoted their exfoliation and oxidation. Initially, we evaluated PDLLA and PDLLA/MWCNTO-GO scaffolds for tensile strength tests, cell adhesion and cell viability (with osteoblast-like MG-63 cells), alkaline phosphatase (ALP, a marker of osteoblast differentiation) activity and mineralized nodule formation. In vivo tests were carried out using PDLLA and PDLLA/MWCNTO-GO scaffolds as fillers for critical defects in the tibia of rats. MWCNTO-GO loading was responsible for decreasing the tensile strength and elongation-at-break of PDLLA scaffolds, although the high mechanical performance observed (~600MPa) assures their application in bone tissue regeneration. In vitro results showed that the scaffolds were not cytotoxic and allowed for osteoblast-like cell interactions and the formation of mineralized matrix nodules. Furthermore, MG-63 cells grown on PDLLA/MWCNTO-GO significantly enhanced osteoblast ALP activity compared to controls (cells alone), while the PDLLA group showed similar ALP activity when compared to controls and PDLLA/MWCNTO-GO. Most impressively, in vivo tests suggested that compared to PDLLA scaffolds, PDLLA/MWCNTO-GO had a superior influence on bone cell activity, promoting greater new bone formation. In summary, the results of this study highlighted that this novel scaffold (MWCNTO-GO, 50% w/w) is a promising alternative for bone tissue regeneration and, thus, should be further studied. Copyright © 2016 Elsevier B.V. All rights reserved.

AUTOMOTIVE DIESEL MAINTENANCE 2. UNIT XVII, LEARNING ABOUT AC GENERATOR (ALTERNATOR) PRINCIPLES (PART II).

ERIC Educational Resources Information Center

Minnesota State Dept. of Education, St. Paul. Div. of Vocational and Technical Education.

THIS MODULE OF A 25-MODULE COURSE IS DESIGNED TO DEVELOP AN UNDERSTANDING OF THE OPERATING PRINCIPLES AND THE SERVICING AND TESTING PROCEDURES FOR ALTERNATING CURRENT (AC) GENERATORS AND REGULATORS USED ON DIESEL POWERED EQUIPMENT. TOPICS ARE REVIEW OF ALTERNATOR PRINCIPLES, ALTERNATOR SERVICING AND TESTING, ALTERNATOR REGULATOR OPERATING…

Highly sensitive determination of cadmium and lead using a low-cost electrochemical flow-through cell based on a carbon paste electrode.

PubMed

Wonsawat, Wanida; Dungchai, Wijitar; Motomizu, Shoji; Chuanuwatanakul, Suchada; Chailapakul, Orawon

2012-01-01

A low-cost thin-layer electrochemical flow-through cell based on a carbon paste electrode (CPE), was constructed for the highly sensitive determination of cadmium(II) (Cd(2+)) and lead(II) (Pb(2+)) ions. The sensitivity of the proposed cell for Cd(2+) and Pb(2+) ion detection was improved by using the smallest channel height without the need for any complicated electrode modification. Under the optimum conditions, the detection limits of Cd(2+) and Pb(2+) ions (0.08 and 0.07 µg dm(-3), respectively) were 13.8- and 11.4-fold lower than that of a commercial flow cell (1.1 and 0.8 µg dm(-3), respectively). Moreover, the percentage recoveries of Cd(2+) and Pb(2+) for the in-house designed thin-layer flow cell were higher than those for the commercially available cell in all tested water samples, and within the acceptable range. The proposed flow cell is promising as an inexpensive and alternative one for the highly sensitive monitoring of heavy metal ions. 2012 © The Japan Society for Analytical Chemistry

Regeneration of articular cartilage by adipose tissue derived mesenchymal stem cells: perspectives from stem cell biology and molecular medicine.

PubMed

Wu, Ling; Cai, Xiaoxiao; Zhang, Shu; Karperien, Marcel; Lin, Yunfeng

2013-05-01

Adipose-derived stem cells (ASCs) have been discovered for more than a decade. Due to the large numbers of cells that can be harvested with relatively little donor morbidity, they are considered to be an attractive alternative to bone marrow derived mesenchymal stem cells. Consequently, isolation and differentiation of ASCs draw great attention in the research of tissue engineering and regenerative medicine. Cartilage defects cause big therapeutic problems because of their low self-repair capacity. Application of ASCs in cartilage regeneration gives hope to treat cartilage defects with autologous stem cells. In recent years, a lot of studies have been performed to test the possibility of using ASCs to re-construct damaged cartilage tissue. In this article, we have reviewed the most up-to-date articles utilizing ASCs for cartilage regeneration in basic and translational research. Our topic covers differentiation of adipose tissue derived mesenchymal stem cells into chondrocytes, increased cartilage formation by co-culture of ASCs with chondrocytes and enhancing chondrogenic differentiation of ASCs by gene manipulation. Copyright © 2012 Wiley Periodicals, Inc.

In-vitro analysis of APA microcapsules for oral delivery of live bacterial cells.

PubMed

Chen, H; Ouyang, W; Jones, M; Haque, T; Lawuyi, B; Prakash, S

2005-08-01

Oral administration of microcapsules containing live bacterial cells has potential as an alternative therapy for several diseases. This article evaluates the suitability of the alginate-poly-L-lysine-alginate (APA) microcapsules for oral delivery of live bacterial cells, in-vitro, using a dynamic simulated human gastro-intestinal (GI) model. Results showed that the APA microcapsules were morphologically stable in the simulated stomach conditions, but did not retain their structural integrity after a 3-day exposure in simulated human GI media. The microbial populations of the tested bacterial cells and the activities of the tested enzymes in the simulated human GI suspension were not substantially altered by the presence of the APA microcapsules, suggesting that there were no significant adverse effects of oral administration of the APA microcapsules on the flora of the human gastrointestinal tract. When the APA microcapsules containing Lactobacillus plantarum 80 (LP80) were challenged in the simulated gastric medium (pH = 2.0), 80.0% of the encapsulated cells remained viable after a 5-min incubation; however, the viability decreased considerably (8.3%) after 15 min and dropped to 2.6% after 30 min and lower than 0.2% after 60 min, indicating the limitations of the currently obtainable APA membrane for oral delivery of live bacteria. Further in-vivo studies are required before conclusions can be made concerning the inadequacy of APA microcapsules for oral delivery of live bacterial cells.

Antigen-specific CTLs: to produce autologous cells product for adoptive cellular therapy.

PubMed

Liu, Sai; Shao, Yi; Xu, Jie; Jiang, Na; Dai, Yanchao; Wang, Yu; Sun, Huanqing; Sun, Jianping; Zhang, Yonghong

2017-06-01

As antiretroviral therapy provides long term viral suppression but no cure, alternative therapies such as adoptive cellular therapy have thus been investigated in the anti-AIDS field. This study sought to establish a HLA-A02 specific CTL cell culture method with comparison of the effects of different cytokines used in CTL cultivation to decide the best cultivation environment. In order to produce CTLs with targeted HLA-A02 restricted antigen specificity for adoptive cellular therapy, we evaluated autologous PBMC cultivation in different cytokine environment to select a better expansion condition to produce qualified CTL production. We co-cultivated PBMC and peptides of these patients with HLA-A02 allele with different cytokines. After cultivation, multiple parameters were tested. 1) Cytokines IL-2 alone can effectively amplify HLA-A02 specific CTL cells, and the count of CTLs was >85% all through the process. 2) The HLA-A02 specific cells at the end of the cultivation were mainly CD3+CD8+ cells. 3) The interferon stimulation test had shown that the expanded CTLs secreted more IFN-γ than before cultivation (0.9% -11.70%). This model of CTL cultivation is successful in redirecting the specificity of antigen recognition and safely for HLA-A02+ patients cell adoptive therapy. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Chemotactic cell trapping in controlled alternating gradient fields

PubMed Central

Meier, Börn; Zielinski, Alejandro; Weber, Christoph; Arcizet, Delphine; Youssef, Simon; Franosch, Thomas; Rädler, Joachim O.; Heinrich, Doris

2011-01-01

Directed cell migration toward spatio-temporally varying chemotactic stimuli requires rapid cytoskeletal reorganization. Numerous studies provide evidence that actin reorganization is controlled by intracellular redistribution of signaling molecules, such as the PI4,5P2/PI3,4,5P3 gradient. However, exploring underlying mechanisms is difficult and requires careful spatio-temporal control of external chemotactic stimuli. We designed a microfluidic setup to generate alternating chemotactic gradient fields for simultaneous multicell exposure, greatly facilitating statistical analysis. For a quantitative description of intracellular response dynamics, we apply alternating time sequences of spatially homogeneous concentration gradients across 300 μm, reorienting on timescales down to a few seconds. Dictyostelium discoideum amoebae respond to gradient switching rates below 0.02 Hz by readapting their migration direction. For faster switching, cellular repolarization ceases and is completely stalled at 0.1 Hz. In this “chemotactically trapped” cell state, external stimuli alternate faster than intracellular feedback is capable to respond by onset of directed migration. To investigate intracellular actin cortex rearrangement during gradient switching, we correlate migratory cell response with actin repolymerization dynamics, quantified by a fluorescence distribution moment of the GFP fusion protein LimEΔcc. We find two fundamentally different cell polarization types and we could reveal the role of PI3-Kinase for cellular repolarization. In the early aggregation phase, PI3-Kinase enhances the capability of D. discoideum cells to readjust their polarity in response to spatially alternating gradient fields, whereas in aggregation competent cells the effect of PI3-Kinase perturbation becomes less relevant. PMID:21709255

Human Rhabdomyosarcoma Cell Lines for Rhabdomyosarcoma Research: Utility and Pitfalls

PubMed Central

Hinson, Ashley R. P.; Jones, Rosanne; Crose, Lisa E. S.; Belyea, Brian C.; Barr, Frederic G.; Linardic, Corinne M.

2013-01-01

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. Despite intergroup clinical trials conducted in Europe and North America, outcomes for high risk patients with this disease have not significantly improved in the last several decades, and survival of metastatic or relapsed disease remains extremely poor. Accrual into new clinical trials is slow and difficult, so in vitro cell-line research and in vivo xenograft models present an attractive alternative for preclinical research for this cancer type. Currently, 30 commonly used human RMS cell lines exist, with differing origins, karyotypes, histologies, and methods of validation. Selecting an appropriate cell line for RMS research has important implications for outcomes. There are also potential pitfalls in using certain cell lines including contamination with murine stromal cells, cross-contamination between cell lines, discordance between the cell line and its associated original tumor, imposter cell lines, and nomenclature errors that result in the circulation of two or more presumed unique cell lines that are actually from the same origin. These pitfalls can be avoided by testing for species-specific isoenzymes, microarray analysis, assays for subtype-specific fusion products, and short tandem repeat analysis. PMID:23882450

Capture of circulating tumor cells using photoacoustic flowmetry and two phase flow

NASA Astrophysics Data System (ADS)

O'Brien, Christine M.; Rood, Kyle D.; Bhattacharyya, Kiran; DeSouza, Thiago; Sengupta, Shramik; Gupta, Sagar K.; Mosley, Jeffrey D.; Goldschmidt, Benjamin S.; Sharma, Nikhilesh; Viator, John A.

2012-06-01

Melanoma is the deadliest form of skin cancer, yet current diagnostic methods are unable to detect early onset of metastatic disease. Patients must wait until macroscopic secondary tumors form before malignancy can be diagnosed and treatment prescribed. Detection of cells that have broken off the original tumor and travel through the blood or lymph system can provide data for diagnosing and monitoring metastatic disease. By irradiating enriched blood samples spiked with cultured melanoma cells with nanosecond duration laser light, we induced photoacoustic responses in the pigmented cells. Thus, we can detect and enumerate melanoma cells in blood samples to demonstrate a paradigm for a photoacoustic flow cytometer. Furthermore, we capture the melanoma cells using microfluidic two phase flow, a technique that separates a continuous flow into alternating microslugs of air and blood cell suspension. Each slug of blood cells is tested for the presence of melanoma. Slugs that are positive for melanoma, indicated by photoacoustic waves, are separated from the cytometer for further purification and isolation of the melanoma cell. In this paper, we evaluate the two phase photoacoustic flow cytometer for its ability to detect and capture metastastic melanoma cells in blood.

TCR Signal Strength Regulates Akt Substrate Specificity To Induce Alternate Murine Th and T Regulatory Cell Differentiation Programs.

PubMed

Hawse, William F; Boggess, William C; Morel, Penelope A

2017-07-15

The Akt/mTOR pathway is a key driver of murine CD4 + T cell differentiation, and induction of regulatory T (Treg) cells results from low TCR signal strength and low Akt/mTOR signaling. However, strong TCR signals induce high Akt activity that promotes Th cell induction. Yet, it is unclear how Akt controls alternate T cell fate decisions. We find that the strength of the TCR signal results in differential Akt enzymatic activity. Surprisingly, the Akt substrate networks associated with T cell fate decisions are qualitatively different. Proteomic profiling of Akt signaling networks during Treg versus Th induction demonstrates that Akt differentially regulates RNA processing and splicing factors to drive T cell differentiation. Interestingly, heterogeneous nuclear ribonucleoprotein (hnRNP) L or hnRNP A1 are Akt substrates during Treg induction and have known roles in regulating the stability and splicing of key mRNAs that code for proteins in the canonical TCR signaling pathway, including CD3ζ and CD45. Functionally, inhibition of Akt enzymatic activity results in the dysregulation of splicing during T cell differentiation, and knockdown of hnRNP L or hnRNP A1 results in the lower induction of Treg cells. Together, this work suggests that a switch in substrate specificity coupled to the phosphorylation status of Akt may lead to alternative cell fates and demonstrates that proteins involved with alternative splicing are important factors in T cell fate decisions. Copyright © 2017 by The American Association of Immunologists, Inc.

[Activation of the alternative oxidase of Yarrowia lipolytica by adenosine 5'-monophosphate].

PubMed

Medentsev, A G; Arinbasarova, A Iu; Smirnova, N M; Akimenko, V K

2004-01-01

The study of the effect of nucleoside phosphates on the activity of cyanide-resistant oxidase in the mitochondria and the submitochondrial particles of Yarrowia lipolytica showed that adenosine monophosphate (5'-AMP, AMP) did not stimulate the respiration of the intact mitochondria. The incubation of the mitochondria at room temperature (25 degrees C) for 3-5 h or their treatment with ultrasound, phospholipase A, and detergent Triton X-100 at a low temperature inactivated the cyanide-resistant alternative oxidase. The inactivated alternative oxidase could be reactivated by AMP. The reactivating effect of AMP was enhanced by azolectin. Some other nucleoside phosphates also showed reactivating ability in the following descending order. AMP = GMP > GDP > GTP > XMP > IMP. The apparent reaction rate constant Km for AMP upon the reactivation of the alternative oxidase of mitochondria treated with Triton X-100 or incubated at 25 degrees C was 12.5 and 20 microM, respectively. The Km for AMP upon the reactivation of the alternative oxidase of submitochondrial particles was 15 microM. During the incubation of yeast cells under conditions promoting the development of alternative oxidase, the content of adenine nucleotides (AMP, ADP, and ATP) in the cells and their respiration tended to decrease. The subsequent addition of cyanide to the cells activated their respiration, diminished the intracellular content of ATP three times, and augmented the content of AMP five times. These data suggest that the stimulation of cell respiration by cyanide may be due to the activation of alternative oxidase by AMP.

Coordinated tissue-specific regulation of adjacent alternative 3′ splice sites in C. elegans

PubMed Central

Ragle, James Matthew; Katzman, Sol; Akers, Taylor F.; Barberan-Soler, Sergio; Zahler, Alan M.

2015-01-01

Adjacent alternative 3′ splice sites, those separated by ≤18 nucleotides, provide a unique problem in the study of alternative splicing regulation; there is overlap of the cis-elements that define the adjacent sites. Identification of the intron's 3′ end depends upon sequence elements that define the branchpoint, polypyrimidine tract, and terminal AG dinucleotide. Starting with RNA-seq data from germline-enriched and somatic cell-enriched Caenorhabditis elegans samples, we identify hundreds of introns with adjacent alternative 3′ splice sites. We identify 203 events that undergo tissue-specific alternative splicing. For these, the regulation is monodirectional, with somatic cells preferring to splice at the distal 3′ splice site (furthest from the 5′ end of the intron) and germline cells showing a distinct shift toward usage of the adjacent proximal 3′ splice site (closer to the 5′ end of the intron). Splicing patterns in somatic cells follow C. elegans consensus rules of 3′ splice site definition; a short stretch of pyrimidines preceding an AG dinucleotide. Splicing in germline cells occurs at proximal 3′ splice sites that lack a preceding polypyrimidine tract, and in three instances the germline-specific site lacks the AG dinucleotide. We provide evidence that use of germline-specific proximal 3′ splice sites is conserved across Caenorhabditis species. We propose that there are differences between germline and somatic cells in the way that the basal splicing machinery functions to determine the intron terminus. PMID:25922281

Microtubules as key cytoskeletal elements in cellular transport and shape changes: their expected responses to space environments

NASA Technical Reports Server (NTRS)

Conrad, G. W.; Conrad, A. H.; Spooner, B. S. (Principal Investigator)

1992-01-01

Application of reference standard reagents to alternatively depolymerize or stabilize microtubules in a cell that undergoes very regular cytoskeleton-dependent shape changes provides a model system in which some expected components of the environments of spacecraft and space can be tested on Earth for their effects on the cytoskeleton. The fertilized eggs of Ilyanassa obsoleta undergo polar lobe formation by repeated, dramatic, constriction and relaxation of a microfilamentous band localized in the cortical cytoplasm and activated by microtubules.

Ligand-promoted protein folding by biased kinetic partitioning.

PubMed

Hingorani, Karan S; Metcalf, Matthew C; Deming, Derrick T; Garman, Scott C; Powers, Evan T; Gierasch, Lila M

2017-04-01

Protein folding in cells occurs in the presence of high concentrations of endogenous binding partners, and exogenous binding partners have been exploited as pharmacological chaperones. A combined mathematical modeling and experimental approach shows that a ligand improves the folding of a destabilized protein by biasing the kinetic partitioning between folding and alternative fates (aggregation or degradation). Computationally predicted inhibition of test protein aggregation and degradation as a function of ligand concentration are validated by experiments in two disparate cellular systems.

Ligand-Promoted Protein Folding by Biased Kinetic Partitioning

PubMed Central

Hingorani, Karan S.; Metcalf, Matthew C.; Deming, Derrick T.; Garman, Scott C.; Powers, Evan T.; Gierasch, Lila M.

2017-01-01

Protein folding in cells occurs in the presence of high concentrations of endogenous binding partners, and exogenous binding partners have been exploited as pharmacological chaperones. A combined mathematical modeling and experimental approach shows that a ligand improves the folding of a destabilized protein by biasing the kinetic partitioning between folding and alternative fates (aggregation or degradation). Computationally predicted inhibition of test protein aggregation and degradation as a function of ligand concentration are validated by experiments in two disparate cellular systems. PMID:28218913